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4th International Meeting on Magnetotactic Bacteria

Meeting Program and Abstracts

4th International Meeting on Magnetotactic Bacteria MTB2014 Rio de Janeiro, September 15th to 18th

2

Chairs

Dr. Daniel Acosta-Avalos Centro Brasileiro de Pesquisas Físicas – CBPF [email protected]

Dr. Fernanda Abreu Universidade Federal do Rio de Janeiro – UFRJ [email protected]

Dr. Ulysses Lins Universidade Federal do Rio de Janeiro – UFRJ [email protected]

Members of the Local Organizing Committee

Ana Carolina Araújo - UFRJ Clarissa Werneck - UFRJ Danielle Moreira - UFRJ

Jefferson Cypriano - UFRJ Lia Teixeira - UFRJ

Marlon Lemos – UFRJ Pedro Leão - UFRJ Sidcley Lira - UFRJ

Special support Adriana Moraes Schoenacher IBMR – AVM [email protected]

September 14th, Sunday, registration

September 15 - 18th, Monday – Thursday, Meeting


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Contents Page

Scientific Program

ORAL PRESENTATIONS

7 – 9

Passive Orientation or Angle-Sensing in Magnetotaxis – R. Frankel

10

Analysis and functional expression of gene clusters controlling magnetosome biosynthesis in magnetotactic bacteria – D. Schuler

10

Genetic tool to study magnetosome membrane formation in Magnetospirillum magneticum AMB-1 – E. Cornejo and A. Komeili

11

Analysis of magnetosome vesicle formation in Magnetospirillum gryphiswaldense – O. Raschdorf et al.

11

Biological control of magnetite crystal morphology by regulating gene expression of mms7 in Magnetospirillum magneticum strain AMB-1 – A. Arakaki et al.

12

Membrane structural regulator protein, MamY, specifically binds to anionic phospholipids – M. Tanaka et al.

12

Magnetofaba australis gen. nov., sp. nov., a magnetotactic bacterium of the phylum Proteobacteria isolated from the Southern Hemisphere – V. Morillo et al.

13

Crystallization and structure of the predicted magnetite interacting associated protein loop from magnetotactic bacteria – H. Nudelman et al.

13

Magnetosome Chains: from structure to mechanics – A. Korning et al.

13

New implication on magnetosome formation by transcriptome of Magnetospirillum gryphiswaldense MSR-1 under aerobic and microaerobic condition – X. Wang et al.

14

Fluorescence live-cell imaging for visualizing the subcellular dynamics of magnetosomes – A. Taoka et al.

14

The Treadmilling Growth of the Actin-Like Protein MamK Drives Magnetosome Chain Positioning and Segregation in M. gryphiswaldense – M. Toro-Nahuelpan et al.

15

Ferritin-like proteins are not required for magnetosome formation – R. Uebe et al.

15

Regulation of OxyR for magnetosome biosynthesis in Magnetospirillum gryphiswaldense MSR-1 – T. Weng et al.

16

Fluorescence lifetime imaging microscopy - Foerster resonance energy transfer for the study of Mam proteins interaction in vivo. – M. Bennet et al.

16

A Genetic Screen Reveals New Magnetosome Genes in Desulfovibrio magnetics RS-1 – L. Rahn-Lee and A. Komeili

17

The interplay between two bacterial actin homologs, MamK and MamK-like, is required for the alignment of magnetosome organelles – N. Abreu et al.

17

Dynamics and diversity of the bacterial actin-like protein, MamK – O. Draper et al.

17

Aerobic Respiration by Sulfate-Reducing Magnetotactic Bacteria – C. T. Lefevre et al.

18

From bacterial to biomimetic magnetosomes – J. Bain and S. S. Staniland

18

Magnetosome magnetite biomineralization - in-vivo analysis – T. Woehl et al.

18

A chemical biosignature of magnetite from magnetotactic bacteria – M. Amor et al. 19


4

Magnetic fingerprint in marine sediments: clues from cultivated Magnetovibrio blakemorei strain MV-1 and recent cores from Brazilian coast – L. Jovane et al.

19

Identifying composition and mapping of magnetosomes and other intracellular compounds in microorganism using Raman spectroscopy – S. Eder et al.

19

Biomineralization process of Magnetospirillum gryphiswaldense by X-ray absorption spectroscopy – M.L. Fdez-Gubieda et al.

20

Towards molecular imaging with high-field MRI and biogenic contrast agents – N. Ginet et al.

20

Structural determination of Mms6 magnetite binding helix and characterization of its mineral binding within the ferritin cage – L. Lewin et al.

20

Effects of crystal shape and structure on the magnetic microstructures of magnetosomes and their chains – A. Kovacs et al.

21

Characterization of the chemistry and magnetism of individual magnetotactic bacterial cells using X-ray spectromicroscopy – X. Zhu et al.

21

Electron microscopy facilities for the study of biominerals in Rio de Janeiro – M. Farina

22

How to combine bacterial division with magnetic polarity for magnetotactic bacteria with a single polar flagellum – C.T. Lefevre et al.

22

Magnetochrome: a c-type cytochrome domain specific to magnetotactic bacteria – P. Arnoux et al.

22

Light and magnetic field influence on Multicellular Magnetotactic Prokaryotes – D. Acosta-Avalos and H. Lins de Barros

23

Diversity and Distribution of Mutilcellular Magnetotactic Prokaryotes in intertidal sediments of China Sea – K. Zhou et al.

23

Peculiar behavior of multicellular magnetotactic prokaryotes – H. Lins de Barros and D. Acosta-Avalos

23

The diversity and distribution of ellipsoidal multicellular magnetotactic prokaryotes – Y. Chen et al.

24

Behavior of the uncultured spherical MMP ‘Candidatus Magnetoglobus multicellularis’ under applied magnetic fields – C. N. Keim et al.

24

Flagella and motility of magnetotactic bacteria – D. Murat et al.

25

Magnetofossil abundance during the Middle Eocene Climatic Optimum (MECO) – J. F. Savian et al.

25

Culture-independent characterization of novel magnetotactic cocci from Antarctica marine sediments – F. Abreu et al.

25

Magneto-aerotaxis – theory and experiment – S. Klumpp et al.

26


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POSTERS PRESENTATIONS

Physicochemistry of magnetite formation in magnetosomes – A. Olszewska et al.

27

Identification of functional domain of crystal growth regulator protein Mms6 from Magnetospirillum magneticum strain AMB-1 by deletion of amino acid sequence – A. Yamagishi et al.

27

Organelle formation and genetics in Desulfovibrio magneticus RS-1 – C. Grant and A. Komeili

27

Magnetotactic bacteria diversity in saline lagoons – C. Werneck et al.

28

Distinct roles for two HtrA proteases in magnetosome biomineralization – D. Hershey and A. Komeili

28

Genome analysis of the freshwater magnetotactic bacterium Magnetospirillum caucasicum SO-1 – D. S. Grouzdev et al.

29

Taxonomic description of three novel freshwater magnetotactic spirillae isolated from distinct geographical points of Russia – M. V. Dziuba et al.

29

Synthesis of magnetic filaments on flagellin-Mms6Cterm fusion proteins as templates – E. Tompa et al.

30

Influence of the mamGFDC operon on magnetosomes functionalization efficiency in Magnetospirillum magneticum AMB-1 - G.Adryanczyk-Perrier et al.

30

Biomimetic and Biokleptic synthesis of magnetic nanoparticles and arrays of magnetic nanoparticles inspired by magnetic bacteria – J. Bain and S. S. Staniland

30

Crystallization and preliminary X-ray diffraction analysis of the Magnetospirillum gryphiswaldense MSR-1 Magnetotaxis protein MtxA – G. Davidov et al.

31

Annual variation of Multicellular magnetotactic prokaryotes and the relationship with biogeochemical parameters in Yuehu Lake – H. Du et al.

31

A novel bacterial magnetic cobalophore for bioremediation applications – J.B. Abbe et al.

31

Transposon mutagenesis as an approach for global genetic analysis of magnetosome biomineralization in M. gryphiswaldense – K. T. Silva and D. Schueler

32

North-seeking magnetotactic bacteria in the Southern Hemisphere – L.C.R.S. Teixeira et al.

32

The effect of light wavelength and magnetic field on the velocity of the MMP Candidatus Magnetoglobus multicellularis – L. V. de Azevedo et al.

33

Structure analysis of the magnetosome associated protein MamB – N. Kerem et al.

33

Regulation of Biomineralization in Magnetotactic Bacteria by the Serine Protease MamE – P. Browne and A. Komeili

33

Five Fur family proteins in Magnetospirillum gryphiswaldense MSR-1 – Q. Wang et al.

34

The isolation of multicellular magnetotactic prokaryotes under different magnetic field intensities – R. D. de Melo et al.

34

RGD-functionalized magnetosomes, a contrast agent with molecular affinity for diagnostic in Magnetic Resonance Imaging – S. Preveral et al.

35

Kinetics Characterization of the Cytosolic Domain of the Magnetosome Associated Protein MamM, WT and Homologous Diseases-Related M – S. Barber-Zucker et al.

35


6

Investigation of lipid compositions and interaction between magnetosomes and plasma membrane in Magnetospirillum gryphiswaldense – S. Kolusheva et al.

36

Reduction of iron in the serum and liver of iron-overloaded mice using the magnetotactic bacterium Magnetospirillum gryphiswaldense – T. Setayesh et al.

36

Studying the formation of elongated magnetosome crystals – T. P. Gonzalez and D. Faivre

37

Global regulator Crp control magnetosomes biosynthesis in Magnetospirillum gryphiswaldense MSR-1 – T. Weng et al.

37

Biogeography of magnetotactic bacteria and its implications for global iron cycle – W. Lin and Y. Pan

38

Characterization and genomic analysis of a newly isolated strain Magnetospirillum sp. XM-1 – Y. Wang et al.

38

Using the high-speed atomic force microscope for imaging MamK cytoskeletal filaments. – Z. Oestreicher et al.

38

Participants list 39


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Scientific Program

Sunday September 14th

15:00 18:00 Reception and Registration

Monday September 15th

Ch

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08:30 08:50 Opening session

08:50 09:20 Opening: Passive Orientation or Angle-Sensing in Magnetotaxis (pg. 10)

R. B. Frankel

09:20 09:50 Invited lecture: Analysis and functional expression of gene clusters controlling magnetosome biosynthesis in magnetotactic bacteria (pg. 10) D. Schüler

09:50 10:10 Genetic tool to study magnetosome membrane formation in Magnetospirillum magneticum AMB-1 (pg. 11) E. Cornejo and A. Komeili

10:10 10:30 Analysis of magnetosome vesicle formation in Magnetospirillum gryphiswaldense (pg. 11)

O. Raschdorf et al.

10:30 10:50 Coffee break

10:50 11:10 Biological control of magnetite crystal morphology by regulating gene expression of mms7 in Magnetospirillum magneticum strain AMB-1 (pg. 12)

A. Arakaki et al.

11:10 11:30 Membrane structural regulator protein, MamY, specifically binds to anionic phospholipids (pg. 12)

M. Tanaka et al.

11:30 11:50 Magnetofaba australis gen. nov., sp. nov., a magnetotactic bacterium of the phylum Proteobacteria isolated from the Southern Hemisphere (pg. 13)

U. Lins

11:50 12:10 Crystallization and structure of the predicted magnetite interacting associated protein loop from magnetotactic bacteria (pg. 13) H. Nudelman et al.

12:10 14:00 Lunch

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De

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14:00 14:30 Invited lecture: Magnetosome Chains: from structure to mechanics (pg. 13)

D. Faivre

14:30 14:50 New implication on magnetosome formation by transcriptome of Magnetospirillum gryphiswaldense MSR-1 under aerobic and microaerobic conditions (pg. 14)

X. Wang et al.

14:50 15:10 Fluorescence live-cell imaging for visualizing the subcellular dynamics of magnetosomes (pg. 14)

A.Taoka et al.

15:10 15:30 The Treadmilling Growth of the Actin-Like Protein MamK Drives Magnetosome Chain Positioning and Segregation in M. gryphiswaldense (pg. 15)

M. Toro-Nahuelpan et al.

15:30 15:50 Ferritin-like proteins are not required for magnetosome formation (pg. 15)

R. Uebe et al.


16:10 16:30 Regulation of OxyR for magnetosome biosynthesis in Magnetospirillum gryphiswaldense MSR-1 (pg .16) T. Wen et al.


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16:30 16:50 Fluorescence lifetime imaging microscopy - Foerster resonance energy transfer for the study of Mam proteins interaction in vivo (pg. 16) M. Bennet et al.

16:50 17:10 A Genetic Screen Reveals New Magnetosome Genes in Desulfovibrio magnetics RS-1 (pg. 17) L. Rahn-Lee and A. Komeili

17:10 17:30 The interplay between two bacterial actin homologs, MamK and MamK-like, is required for the alignment of magnetosome organelles (pg. 17)

N. Abreu et al.

17:30 18:00 Invited lecture: Dynamics and diversity of the bacterial actin-like protein, MamK (pg. 17) A. Komeili

Tuesday September 16th

09:00 09:30 Invited lecture: Aerobic Respiration by Sulfate-Reducing Magnetotactic Bacteria (pg. 18)

D. A. Bazylinski

Ch

air:

Dan

iel A

cost

a-A

valo

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09:30 09:50 From bacterial to biomimetic magnetosomes (pg. 18)

J. Bain and S. Staniland

09:50 10:10 Magnetosome magnetite biomineralization - in-vivo analysis (pg. 18)

T. Woehl et al.


10:30 10:50 A chemical biosignature of magnetite from magnetotactic bacteria (pg. 19)

M. Amor et al.

10:50 11:10 Magnetic fingerprint in marine sediments: clues from cultivated Magnetovibrio blakemorei strain MV-1 and recent cores from Brazilian Coast (pg. 19)

L. Jovane et al.

11:10 11:30 Identifying composition and mapping of magnetosomes and other intracellular compounds in microorganism using Raman spectroscopy (pg. 19)

S.H.K. Eder et al.

11:30 11:50 Biomineralization process of Magnetospirillum gryphiswaldense by X-ray absorption spectroscopy (pg. 20) M.L. Fdez-Gubieda et al.

12:00 14:00 Lunch

Ch

air:

Dir

k Sc

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ler

Bar

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14:00 14:20 Towards molecular imaging with high-field MRI and biogenic contrast agents (pg. 20)

N. Ginet et al.

14:20 14:50 EMBO young scientist lecture: Structural determination of Mms6 magnetite binding helix and characterization of its mineral binding within the ferritin cage (pg. 20)

R. Zarivach

14:50 15:10 Effects of crystal shape and structure on the magnetic microstructures of magnetosomes and their chains (pg. 21)

A. Kovács et al.

15:10 15:30 Characterization of the chemistry and magnetism of individual magnetotactic bacterial cells using X-ray spectromicroscopy (pg. 21) X. Zhu et al.

15:30 15:50 Invited lecture: Peculiar behavior of multicellular magnetotactic prokaryotes (pg. 23)

H. Lins de Barros

15:50 16:10 How to combine bacterial division with magnetic polarity for magnetotactic bacteria with a single polar flagellum (pg. 22) C. T. Lefèvre et al.


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16:10 16:40 Invited lecture: Magnetochrome: a c-type cytochrome domain specific to magnetotactic bacteria (pg. 22)

D. Pignol

16:40 18:30 Poster session

Wednesday September 17th

Free day

Free day

Thursday September 18th

Ch

air:

Uly

sses

Lin

s

09:30 10:00 Invited lecture: Light and magnetic field influence on Multicellular Magnetotactic Prokaryotes (pg. 23)

D. Acosta-Avalos

10:00 10:20 Diversity and Distribution of Muticellular Magnetotactic Prokaryotes in intertidal sediments of China Sea (pg. 23)

K. Zhou et al.


10:50 11:20 Electron microscopy facilities for the study of biominerals in Rio de Janeiro (pg. 22)

M. Farina

11:20 11:40 The diversity and distribution of ellipsoidal multicellular magnetotactic prokaryotes (pg. 24)

Y. Chen et al.

11:40 12:00 Behavior of the uncultured spherical MMP ‘Candidatus Magnetoglobus multicellularis’ under applied magnetic fields (pg. 24) C. N. Keim

12:00 14:00 Lunch

Ch

air:

Raz

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ivac

h 14:00 14:30 Invited lecture: Flagella and motility of magnetotactic bacteria (pg. 25)

Long-Fei Wu

14:30 14:50 Magnetofossil abundance during the Middle Eocene Climatic Optimum (MECO) (pg. 25)

J. F. Savian et al.

14:50 15:10 Culture-independent characterization of novel magnetotactic cocci from Antarctica marine sediments (pg. 25) Ana C. Araújo et al.

15:10 15:30 Magneto-aerotaxis – theory and experiment (pg. 26) S. Klumpp et al.

15:30 16:00 Closing Session


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Oral Presentations

Monday 15th September Invited lecture: Passive Orientation or Angle-Sensing in Magnetotaxis

Richard B. Frankel

California Polytechnic State University, San Luis Obispo

The original hypothesis for magnetotaxis invoked passive magnetic orientation of swimming MTB along the ambient magnetic field B, due to the torque exerted by B on the cellular magnetic dipole M as the cell swam (1). A cell would also be randomly disoriented by thermal fluctuations in the medium. This situation was originally analyzed by P. Langevin, who showed that the time-averaged alignment of a single cell, or the average alignment of a population of identical cells at any given time, was a function of MB/kT (M=magnetic moment; B=magnetic field strength; and kT=thermal energy). This hypothesis was verified first by Kalmijn (2) for undescribed, magnetotactic, cocci, and more recently by Mao et al. (3), for undescribed cocci and for M. bavaricum. In a recent paper, Zhu et al. (4) studied the motion of single cells of Magnetospirillum magneticum, strain AMB-1 and several mutants in magnetic fields. They maintain that for B< 1 mT, magnetic alignment alone is insufficient to orient a cell, and they propose that at biologically relevant B values, the cells are aligned along B by a magnetic sensory system, called ‘angle-sensing’, that involves an interaction between the magnetic torque on the cell and the flagellar motor via a methyl-accepting, chemotaxis, protein amb0995 that is known to interact with MamK. When the cell goes out of alignment with B, the angle between B and the magnetic dipole increases, increasing the torque on MamK. This would result in modulation of the flagellar motor, through the usual chemotaxis cascade, that ultimately reduces the B-M angle. The ‘angle sensing’ hypothesis contains some interesting ideas, but a number of questions remain. My goal here is to illuminate some of the questions. 1. R. Frankel and R. Blakemore,1980, Navigational Compass in Magnetic Bacteria, Journal of Magnetism and Magnetic Materials, 15-18, 1562-1564. 2. A. J. Kalmijn, 1981, Biophysics of Geomagnetic Field Detection, IEEE Transactions on Magnetics, Mag-17, 1113-1124. 3. X. Mao et al., 2014, Magneto-Chemotaxis in Sediments: First Insights, PLOS One. 4. X. Zhu et al., 2014, Angle Sensing in Magnetotaxis of M. magneticum AMB-1, Integrative Biology, DOI: 10.1039/c3ib40259b. Invited lecture: Analysis and functional expression of gene clusters controlling magnetosome biosynthesis in

magnetotactic bacteria

Dirk Schüler

University of Bayreuth, Germany

Biosynthesis of magnetosomes in virtually all magnetotactic bacteria (MTB) is controlled by a set of genes comprised within a large genomic magnetosome island (MAI). Previous genetic analysis of the MAI has been limited to few cultivated species and most abundant members of natural populations. Our targeted genomic analysis of single microsorted cells revealed unknown diversity of MAI-like magnetosome gene clusters of low-abundant uncultivated MTB from marine and freshwater habitats including multicellular representatives as well as those from the Nitrospirae and OP-3 phyla. Whereas we recently demonstrated that respiratory enzymes encoded outside the MAI of Magnetospirillum gryphiswaldense also have accessory roles in magnetite biomineralization by poising the proper redox conditions, we found by genetic analysis that all genes essential for magnetosome biosynthesis are localized within the large mamAB operon. Cloning and chromosomal insertion of this operon along with the entire set of MAI genes resulted in functional expression and reconstitution of magnetosome formation both in the native background as well as in a foreign expression host. Transfer of this gene set into the photosynthetic Rhodospirillum rubrum converted it into a bacterium capable of magnetotaxis.


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Genetic tool to study magnetosome membrane formation in Magnetospirillum magneticum AMB-1.

E. Cornejo and A. Komeili

University of California, Berkeley

In Magnetospirillum magneticum AMB-1, the inner cell membrane is deformed to create a unique sub-cellular compartment, the magnetosome, which is capable of synthesizing nanometer-sized magnetic crystals. Despite the growing genetic understanding and structural characterization of magnetosomes, very little is known about how the magnetosome membrane compartment actually forms. Magnetosome membrane biogenesis is difficult to study because a magnetosome chain is always present and the addition of new magnetosomes to the chain occurs very infrequently. However, a subset of genes appears to be required for membrane formation. In this study, magnetosome membrane formation is placed under the control of a genetically inducible system in order to answer questions regarding how and where magnetosome membranes form in the cell. This genetic system offers a unique advantage to studying the early stages of magnetosome formation. Here, we demonstrate that magnetosome membrane formation can be repressed both transcriptionally and post-transcriptionally. Upon induction, a change in the localization of several magnetosome specific proteins is observed. This genetic tool can be utilized to characterize, by fluorescence microscopy, the early and late magnetosome proteins involved in membrane biogenesis. Furthermore, it lends itself to enable the visualization of early stages of membrane biogenesis by electron cryotomography (ECT).

Analysis of magnetosome vesicle formation in Magnetospirillum gryphiswaldense

Oliver Raschdorf 1,2

; Emanuel Katzmann 1,2

; Anna Lohße 1; Rene Uebe

1*; Jürgen Plitzko

2 and Dirk Schüler

1*

1University of Munich, Germany, 2Max-Planck Institute of Biochemistry, Germany

Magnetosome biosynthesis in Magnetospirillum gryphiswaldense and related MTB proceeds in several distinct steps, which are controlled by >30 genes mostly located within a large genomic island. The biosynthesis of magnetosomes starts with the formation of a distinct compartment provided by vesicles of the magnetosome membrane, which serves as a “nanoreactor“ in which physicochemical conditions required for the biomineralization of magnetite crystals are properly controlled. Previous studies revealed that the magnetosome membrane originates via invagination from the cytoplasmic membrane and that magnetosome-associated proteins are specifically targeted to that membrane. However, the key steps of magnetosome membrane formation and protein targeting are poorly understood so far. Previously, in Magnetospirillum magneticum 8 genes were found to be essential for magnetite biomineralization, whereas only mamL, mamQ, mamB and mamI located in the mamAB operon were reported to be essential, but not sufficient for the formation of empty magnetosome compartments (Murat et al., 2010). In some contrast, in M. gryphiswaldense only 6 genes were recently shown to be absolutely essential for the formation of magnetite (Lohße et al., 2014). Surprisingly, in contrast to M. magneticum, deletion of mamI in M. gryphiswaldense did not entirely abolish magnetosome development, but caused the formation of tiny, irregular crystalline hematite particles, suggesting the possible presence of magnetosome membranes to support a rudimentary biomineralization. These recent findings prompted us to reassess the minimal gene set and to analyze the mechanism for magnetosome membrane formation in M. gryphiswaldense.Using cryo-electron tomography, we studied the presence and characteristics of the magnetosome membrane in M. gryphiswaldense wild type and various mutant strains. We found that only deletion of mamB and mamQ caused a virtually complete loss of magnetosome vesicle formation, while mamL, mamM and mamE deletants show intermediate phenotypes with drastically reduced, but not completely abolished vesicle formation. In contrast to that, the non-magnetic mamN and mamI deletion mutants showed dense chains of empty or partly filled vesicles indicating a wild type-like membrane formation process. Fluorescence microscopy of fluorophore-labeled key magnetosome proteins revealed a chain-like localization for most functional fusions, whereas MamQ seemed to be always only localized in patches in the cytoplasmic membrane. This pattern resembles the localization of other magnetosome proteins when expressed in the absence of vesicles. The localization indicates that MamQ, the essential determinant of magnetosome biogenesis, is mainly involved in processes taking place at the cytoplasmic membrane prior to or during magnetosome vesicle invagination. To assess the in-vivo dynamics of magnetosome membrane biogenesis, we currently are establishing a genetic induction system for time resolved studies of de-novo magnetosome vesicle formation. Preliminary results indicate that, after induction of gene expression, magnetosome vesicles form in the time range of minutes to hours. However, the development of the membrane invaginations itself proceeds too rapidly to detect any intermediate stages. Altogether, our experimental data suggest a speculative model in which magnetosome proteins are coordinated in the cytoplasmic membrane by specific “landmark” proteins and are preassembled prior to rapid vesicle formation. Murat, D., Quinlan, A., Vali, H., and Komeili, A. (2010) Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. Proc Natl Acad Sci USA 107: 5593–5598. Anna Lohße, Sarah Borg, Oliver Raschdorf, Isabel Kolinko, Eva Tompa, Mihály Pósfai, Damien Faivre, Jens Baumgartner, Dirk Schüler (2014) Genetic Dissection of the mamAB and mms6 Operons Reveals a Gene Set Essential for Magnetosome Biogenesis in Magnetospirillum gryphiswaldense. J Bacteriol, 196 (4) 2658-69.


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Biological control of magnetite crystal morphology by regulating gene expression of mms7 in Magnetospirillum magneticum

strain AMB-1

Atsushi Arakaki, Ayana Yamagishi, Tadashi Matsunaga

Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, Japan

Mms proteins (Mms5, Mms6, Mms7, and Mms13) are expressed on the surface of cubo-octahedral magnetite crystals in

Magnetospirillum magneticum strain AMB-1. These proteins are shown to be involved in the promotion of crystal growth in

different manners by their coordinated action. This suggested that the morphology of magnetite crystals can be controlled by

regulating the expression levels of the mms gene in bacterial cell. We obtained a spontaneous mutant strain which produces

unique dumbbell-shaped crystals. Genome analysis of this strain revealed that the mms7 gene and 25 kbp region (SID25) were

deficient from the magnetosome island. In this study, we examined to control magnetite crystal morphology by regulating the

expression levels of mms7 gene in the SID 25 and mms7 gene deletion mutant strain (ΔSID25 Δmms7). Tetracycline-inducible

expression vector was used to regulate the mms7 gene expression levels in the cells. In this system, the target gene expression is

induced by the anhydrotetracycline (inducer molecule) which binds to the repressor protein and triggers the dissociation of the

repressor from the promoter region. The mms7 gene inducible strain was established by transformation of this vector into the

ΔSID25 Δmms7 strain. This strain was cultivated under the presence of anhydrotetracycline and observed by the transmission

electron microscope. As a result, morphology of magnetite crystals formed in the mms7 gene inducible strain changed from

dumbbell-shape to spherical shape according to the increasing of the inducer concentration. In addition, the average minor axis of

magnetite crystals in the mms7 gene inducible strain also gradually increased. These results indicated that the growth of magnetite

crystals was promoted to the specific direction by the induction of the mms7 gene expression. Thus, the regulation of the gene

expression of mms7 gene allows for the morphological control of magnetite crystals in magnetotactic bacteria.

Membrane structural regulator protein, MamY, specifically binds to anionic phospholipids Masayoshi Tanaka

1,2, Mayumi Oda

1, Atsushi Arakaki

1, Kanta Oosuga

1, Sarah Staniland

3, Benjamin Johnson

4, Stephen Baldwin

5,

Stephen Evans4 and Tadashi Matsunaga

1*

1. Department of Biotechnology, Tokyo University of Agriculture and Technology, Japan. 2. Department of Chemical Engineering, Tokyo Institute of Technology, Japan. 3. Chemistry Department, University of Sheffield, UK. 4. School of Physics and Astronomy, University of Leeds, UK. 5. School of Biomedical Sciences, University of Leeds, UK

Membrane deformation and structural regulation processes are an ubiquitous event in all organisms. Proteins containing Bin/amphiphysin/Rvs (BAR) protein domains have an important role during membrane deformation process in eukaryotic cells. Interaction between the BAR proteins and phosphatidylinositols is required to produce a pulling force for membrane deformation from flat membrane structure. On the other hand, in prokaryotic cell, the dynamic organization behaviors of lipid and protein localizations for cell division have been investigated. A unique anionic phospholipid of cardiolipin (CL) also has been reported to locate at cell poles and the association with Min proteins revealed an important role for the spatial positioning of the cell division machinery within Escherichia coli cells. Magnetotactic bacteria has been fascinated many scientists due to the presence of prokaryotic membranous organelle, magnetosome, that is comprised of magnetite crystals and the lipid envelope, magnetosome membrane. We have previously identified a magnetosome membrane protein, MamY, which has been preferentially found from magnetosome containing small magnetite crystal (presumably in immature stage). The MamY protein can directly bind to biological membrane (liposome) and causes the deformation of structure from sphere to tubule under in vitro experimental condition. Furthermore, the mamY gene deletion mutant showed the expansion of magnetosome vesicle sizes and the increase of small magnetite crystals. Although the MamY protein was hypothesized to relate for magnetosome membrane formation, the precise function and the binding manner to the membrane are still unclear. In this study, we investigated the interaction of the MamY protein and lipids by (1) binding assay of MamY protein to various phospholipids, (2) profiling of lipids obtained from magnetosome membrane and (3) evaluation of membrane deformation efficiency by MamY protein in the presence of anionic lipids. Lipid-binding assay indicated that MamY specifically binds to anionic phospholipids, including phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], cardiolipin (CL), and sulfatide. Lipid profile analysis of magnetosome membrane by using LC/ESI-TOFMS showed the presence of four classes of phospholipids, including phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phophatidylserine (PS), and CL. Since CL was identified in both lipid-binding and lipid profile analysis in the presence of CL within liposome, the 12rganizati efficiency by MamY protein was evaluated. From this investigation, the increasing of liposome 12rganizati efficiency by increasing CL content in liposome was revealed. These results suggest that not only the direct interaction between MamY and CL contributes to liposome 12rganizati, but also play an important role in the process of magnetosome vesicle formation in magnetotactic bacteria.


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Magnetofaba australis gen. nov., sp. nov., a magnetotactic bacterium of the phylum Proteobacteria isolated from the Southern

Hemisphere

Viviana Morillo 1, Fernanda Abreu

1, Ana C. Araujo

1, Luiz G.P. de Almeida

2, Alex Enrich-Prast

3, Marcos Farina

4, Ana T.R. de

Vasconcelos2, Dennis A. Bazylinski

5 and Ulysses Lins

1*

1Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2Laboratório Nacional de Computação Científica, Departamento de Matemática Aplicada e Computacional, Petrópolis, Brazil, 3Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 4Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 5School of Life Sciences, University of Nevada at Las Vegas, Las Vegas, NV, USA

Magnetotactic bacteria (MTB) are ubiquitous in aquatic habitats are considered fastidious microorganisms with regard to growth and cultivation with only a relatively low number of axenic cultures available to date. Here, we report the first axenic culture of an MTB isolated in the Southern Hemisphere (Itaipu Lagoon in Rio de Janeiro, Brazil). Cells of this new isolate are coccoid to ovoid in morphology and grow microaerophilically in semi-solid medium containing an oxygen concentration gradient either under chemoorganoheterotrophic or chemolithoautotrophic conditions. Each cell contains a single chain of approximately 10 elongated cuboctahedral magnetite (Fe3O4) magnetosomes. Phylogenetic analysis based on the 16S rRNA gene sequence shows that the coccoid MTB isolated in this study represents a new genus in the Alphaproteobacteria; the name Magnetofaba australis strain IT-1 is proposed. Preliminary genomic data obtained by pyrosequencing shows that M. australis strain IT-1 contains a genomic region with genes involved in biomineralization similar to those found in the most closely related magnetotactic cocci Magnetococcus marinus strain MC-1. However, organization of the magnetosome genes differs from M. marinus strain MC-1. According to the preliminary genomic analysis, the genome size of Magnetofaba australis strain IT-1 is approximately 4,98 Mb and the average GC content is 57,95%. Magnetosome related genes are organized in four groups (mamAB; mamCXZ; mamHIE and mms), in a 40.4Kb genomic region that also contains magnetotaxis related genes and several hypothetical proteins. The GC content of this region and the entire genomic sequences are similar.

Crystallization and structure of the predicted magnetite interacting associated protein loop from magnetotactic bacteria.

Hila Nudelman, Geula Davidov and Raz Zarivach

Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben Gurion University of the Negev, Beer Sheva, Israel.

Magnetotactic bacteria (MTB) can navigate through the earth magnetic field. These bacteria synthesize organelles called “magnetosomes”, which are contain magnetic nanoparticle (Fe3O4) or greigite (Fe3S4) and surrounded by lipid membrane. It was shown that magnetosome membrane contains a unique set of proteins that are thought to direct the biomineralization of magnetite crystals. In MTB, most of the magnetosome formation involved genes that are located in genomic magnetosome island (MAI). One of the proteins that involved in biomineralization of magnetite crystals is an integral membrane protein. It is small protein (~15kDa) with two transmemebrane helices. To understand its function we attached its magnetosomal loop (located between H1 to H2) onto The C-terminal of MBP (maltose binding protein). By using X-ray crystallography we determined the MBP-loop structure from magnetotactic bacteria to 2.8A. Based on our results we identify a possible patch which may be important to biomineralization of magnetite and used this structure to predict homologues proteins from other MTB species.

Invited lecture: Magnetosome Chains: from structure to mechanics

André Körnig, Michael Winklhofer, Jiajia Dong, Mathieu Bennet, Frank Müller, Dirk Schüler, Stefan Klumpp and Damien Faivre (presenting author. Damien Faivre)

Max Planck Institute of Colloids and Interfaces and University of Munich

Magnetotactic bacteria are hierarchically organized from the ultrastructure of the magnetite mineral in the magnetosome to the superstructures formed by assembled magnetosomes typically in chains. We have recently focused on this higher level of hierarchy to understand how biological and physical forces interact while the magnetosome chain is forming. Here, we show that we can use 2-dimensional synchrotron-based X-ray diffraction to study the orientation of the magnetite particles in the magnetosome chain with respect to the chain axis. In addition, this technique can be used on living and immobilized bacteria in the presence of an external magnetic field to probe the macromolecular scaffold responsible for the mechanical stabilization of the magnetosome chain. By combining it with in vivo measurements by optical microscopy, TEM and theoretical modelling, we are able to decipher the mechanical properties of the magnetosome chain system encountered in magnetotactic bacteria. In particular, we exploit the magnetic properties of the endogenous intracellular nanoparticles to apply a force on the filament-connector pair involved in the backbone formation and stabilization in Magnetospirillum gryphiswaldense MSR-1. We show that the magnetosome chain can be broken by the application of external field strength higher than 30 mT, and suggest that this originates from the rupture of the magnetosome connector MamJ. In addition, we calculate that the biological determinants can withstand in vivo a force of 25 pN. Actin and actin homologs are essential to micro-organisms since they play an important role in their mechanical stability. Magnetotactic bacteria studied with the presented methodology allow for the mechanical properties of actin-like filaments to be measured artefact-free in situ and in vivo.


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New implication on magnetosome formation by transcriptome of Magnetospirillum gryphiswaldense MSR-1 under aerobic and

microaerobic condition

Xu Wang, Qing Wang, Yang Zhang, Weijia Zhang, Yinjia Wang, Wei Jiang, Jiesheng Tian, Ying Li*, Lei Wang, Jilun Li

State Key Laboratories for Agro-biotechnology and College of Biological Sciences

Hypoxia and high content iron environment is necessary for the magnetotactic bacteria to synthesize magnetosome (14rganizatio oxide type) in laboratorial cultivated condition. Some genes related to oxygen metabolism have important roles in magnetosome formation according to previous reports. However, litter is known about the effects of oxygen on expression profile of genes involved in this biomineralization phenomenon on a global cellular scale. We focus on a type strain Magnetospirillum gryphiswaldense MSR-1 and investigate the difference of transcriptome of cells cultivated in 7.5 liter fermentor under aerobic and microaerobic condition respectively. The results showed that 77 genes were up-reguleted and 95 genes were down-regulated significantly under microaerobic situation. Categories of these corresponding gene productions mainly comprise metabolism, transport, regulation and hypothetical proteins. A temporary model was generated to describe the cellular differences of MSR-1 grown in the different oxygen concentration. Transport and degradation of carbonbydrate, amino acid, fatty lipid are active, and oxygen is served as the terminal electron accepter under aerobic cultivation. While under microaerobic condition, denitrification is actively metabolic and the potential mobility seems to improve. Additionally, genes responding for uptake, storage, and regulation of iron element have notable difference like a fur gene up-regulated and feoB1 gene, two ferritin genes down-regulated under hypoxia. Interestingly, the mam genes considered sharing a close relationship with magnetosome formation apparently have no expression differences, which implicate mam genes maybe not regulated by oxygen. The information of transcriptome reveals the different response of MSR-1 against oxygen with a holistic view and will shed new insights into general principles of magnetosome formation. Acknowledgments This study was supported by the Chinese National Natural Science Foundation (Grant No. 30970041 & 31270093).

Fluorescence live-cell imaging for visualizing the subcellular dynamics of magnetosomes

Azuma Taoka1),2)

, Chika Uesugi1)

, Zachery Oestreicher1)

, Kaori Morii1)

, Ayako Kiyokawa1)

and Yoshihiro Fukumori

1),2)

1) College of Science and Engineering, Kanazawa Univ, Japan. 2) Bio-AFM FRC, Kanazawa Univ, Japan.

Magnetotactic bacteria synthesize nano-sized organelles, magnetosomes, to navigate themselves along the geomagnetic field of the Earth. Although the detailed structure of magnetosomes can be observed in high-resolution using electron microscopy or atomic force microscopy, these techniques cannot visualize the intracellular protein dynamics of living cells. In this study, we developed a live-cell time-lapse fluorescent imaging technique for examining magnetosome dynamics in Magnetospirillum magneticum AMB-1. Using total internal reflection fluorescence microscopy, we imaged two magnetosome-associated proteins fused with GFP, MamC and MamI. These were expressed from plasmids in wild type AMB-1 cells and used as marker proteins for magnetosome vesicles. The cells were imaged for up to 24 hours by securely attaching them to the surface of a poly-L-lysine coated cover slip and keeping the cells incubated in media while imaging. During the time-lapse imaging process we succeeded in imaging the dynamic movements of magnetosomes throughout the cell cycle. We observed that magnetosomes were organized in a stable, chain-like arrangement along the cell axis. We then did the same experiment, but instead used mutant cells with a mamK deletion. Under these circumstances the magnetosomes were scattered throughout the cell and moved randomly or formed large aggregates instead of staying in a straight chain. These results suggest that the MamK cytoskeleton maintains the stability of the magnetosome chain throughout the entire lifecycle of the cell. Consequently, the chain of magnetosomes is propagated to the daughter cells in a stable manner. This technique could be used to investigate other magnetosome-associated proteins in order to understand the dynamics of the proteins during magnetosome synthesis and propagation to daughter cells.


15

The Treadmilling Growth of the Actin-Like Protein MamK Drives Magnetosome Chain Positioning and Segregation in M.

gryphiswaldense

M. Toro-Nahuelpan1,2

, F.D. Müller1, J.M. Plitzko

2 and D. Schüler

1

1Bayreuth University, Dept. of Microbiology, Germany, 2Max Planck Institute of Biochemistry, Dept. of Molecular Structural Biology, Germany.

Magnetosomes of Magnetospirillum gryphiswaldense are composed of membrane-enclosed magnetite crystals. To maximize the magnetic orientation, the magnetosomes are aligned into a linear chain inside the cell. Magnetosome chain biosynthesis and assembly is an orchestrated mechanism under strict control of more than 30 genes encoded within a large genomic magnetosome island. The magnetosome chain is positioned at midcell, in which during cytokinesis it becomes split at the cellular division site. Subsequently, the chain is equipartitioned and separated against magnetostatic forces into daughter cells. Previous studies have demonstrated that the actin-like protein MamK polymerizes into a cytoskeletal filament from pole to pole throughout the entire cell. The magnetosomes are bound to the MamK filament via the interacting MamJ protein, thus assisting the chain assembly. Furthermore, deletion of mamK caused shorter and fragmented magnetosome chains, which no longer were recruited to the cell division plane, thereby, suggesting a mamK-dependent assembly and positioning of the chain. However, the mechanism of magnetosome concatenation into a chain, the recruitment at the cell division site, and the further pole-to-midcell repositioning into nascent cells remains unclear. In this work, we studied the MamK filament dynamics and its role in the magnetosome chain assembly and positioning during the cell cycle. The MamK filament has a rapid turnover (halt-time recovery of the fluorescence: T½ ≈ 1 min), and exhibits a treadmilling growth that nucleates at the cell poles and moves towards midcell as indicated by Fluorescence Recovery After Photobleaching and Photo-conversion studies. In contrast, a generated mamK D161A point mutant, affected in ATPase activity, displayed significantly impaired filament turnover (T½ ≈ 25 min) suggesting that forms a very stable and rigid filamentous structure. Moreover, the mamK D161A mutant exhibited an intermediate phenotype between the WT and ∆mamK with respect to magnetosome chain organization. Therefore, the mamK D161A strain displayed both WT-like magnetosome chains, and also fragmented chains resembling that of the mamK deletion. Strikingly, the mamK D161A strain displayed an accentuated impairment of magnetosome concatenation into a linear chain. In addition, in vivo time-lapse fluorescence microscopy of the magnetosome chain in mamK D161A cells revealed an uneven partitioning and inheritance, as well as a pole-to-midcell repositioning defect of the chain into daughter cells. In conclusion, we suggest a model in which the MamK filament dynamic turnover and treadmilling growth are fundamental properties for the magnetosome chain concatenation, for the proper partitioning during segregation, and for driving the pole-to-midcell repositioning of the chain into daughter cells.

Ferritin-like proteins are not required for magnetosome formation

René Uebe1,2

, Stephanie Bauer1,2

, Katharina Jäger3, Jean Eberlein

2, Berthold Matzanke

3, and Dirk Schüler

1,2

1University of Bayreuth, Germany; 2Ludwig Maximilian University Munich, Germany; 3University of Lübeck, Germany

Magnetite biomineralization within magnetosome vesicles requires the uptake of large amounts of iron, its intracellular sequestration, and crystallization. Although of central interest, only few studies have addressed the connection of magnetite biomineralization with general iron metabolism of magnetotactic bacteria. For instance, it was shown that ferritin-like iron-storage proteins might play an important role for iron homeostasis and might be involved in magnetite formation in Magnetospirillum species. Using biophysical methods it was observed that a highly disordered, phosphate-rich ferric hydroxide phase consistent with prokaryotic ferritins transforms to magnetite. Thus, ferritin-like iron-storage proteins were recently proposed to participate in the magnetite biomineralization pathway of Magnetospirillum species. However, genetic evidence for the proposed pathway is missing so far. To elucidate their roles for magnetosome formation in vivo, we started to investigate ferritin-like proteins in M. gryphiswaldense by genetic and physiological approaches. Genome analysis revealed the absence of genes encoding genuine prokaryotic non-heme ferritins FtnA, while genes encoding ferritin-like iron storage proteins Dps (DNA protecting protein under starved conditions) and the heme-containing Bfr (bacterioferritin) are present. Deletion mutants of either dps or bfr were indistinguishable from the wildtype with respect to magnetite crystal size, morphology as well as particle numbers. To test for functional redundancy between Bfr and Dps a double mutant, Δdps/bfr, was constructed. The double mutant, which could be isolated under anaerobic conditions only, showed a reduced magnetic response under microaerobic but not under anaerobic growth conditions compared to the wildtype and the single mutants. Magnetite formation, however, was not affected under either condition as magnetite crystals were virtually indistinguishable from the wildtype with resepct to number, size, and morphology. Using whole cell transmission Mössbauer spectroscopy no ferritin-like ferrihydrite component was detectable in the Δdps/bfr strain excluding the presence a so far unrecognized ferritin-like protein. In summary, our results suggest that the identified ferritin-like proteins are not required for magnetite formation but might have a role in protection against oxidative stress.


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Regulation of OxyR for magnetosome biosynthesis in Magnetospirillum gryphiswaldense MSR-1

Tong Wen, Fang Fang Guo, Yun Peng Zhang, Wei Jiang*, Jiesheng Tian, Ying Li, JiLun Li

State Key Laboratory of Agricultural Biotechnology, China Agriculture University

The consist of magnetosomes synthesized by magnetotactic bacteria (MTB) were conventionally believed to be magnetic magnetite (Fe3O4) or iron sulfide greigite (Fe3S4) in most MTB. Although numerous genes were involved in magnetosome biosynthesis, the precisely biomineralizational mechanism of these particles remains mostly unknown. In this study, we introduced the disruption of oxyR in Magnetospirillum gryphiswaldense MSR-1, defined as a hydrogen peroxide-inducible genes activator in Escherichia coli and we found that compared with MSR-1 wild type and the oxyR complementary strain, the ferromagnetism and the iron content of the oxyR-deficient mutant decrease dramatically, suggesting that the OxyR regulate of iron uptake and magnetosome formation process in MSR-1. To explain why OxyR regulate magnetosomes formation in MSR-1, we focus on which genes can be affected after the disruption of oxyR in MSR-1. Comparative expression profiling was used to detect the difference of gene expression level between wild type and the oxyR mutant. The total mRNA of MSR-1 wild type and oxyR mutant were extracted and inverse transcripted into cDNA, then the cDNA were sequenced by high-throughput sequencing. The result of GO (gene ontology) functional enrichment analysis reveals that most of the affected genes were related to cell communication, signal transduction and stimulus response in oxyR-deficient mutant. KEGG (16rgan encyclopedia of genes and genomes) enrichment result indicates that 214 differentially expressed genes were identified between wild type and oxyR mutant among those genes with a KEGG pathway annotation, specific enrichment of genes was observed for pathways involved in two-component system, carbon fixation and oxidative phosphorylation. These results suggested that OxyR as a global regulator regulate functional genes directly or through other regulators indirectly involved in energy metabolism, stimulus response and cell communication. Realtime fluorescence quantitative PCR (Q-PCR) was then performed to confirm if the consequence from expression profile are credible. All the selected differentially expressed genes tested by Q-PCR were consistent with the expression profile findings on their expression level changes, suggesting that the expression profile provide reliable data for the analysis of differentially expressed genes. References: 1. Jogler C., Schüler D. Genomics, genetics, and cell biology of magnetosome formation. Annu. Rev. Microbiol. 63, 501-521 (2009). 2. Geng R., Yuan C., Chen Y. Exploring differentially expressed genes by RNA-Seq in cashmere goat (Capra hircus) skin during hair follicle development and cycling. PloS one. 8, e62704 (2013). Acknowledgment: This work was supported by National Natural Science Foundation of China (Grant Nos. 31170089 and 31270093).

Fluorescence lifetime imaging microscopy – Foerster resonance energy transfer for the study of Mam proteins interaction in

vivo.

Mathieu Bennet; Maria A. Carillo; Damien Faivre

Max-Planck Institute for Colloids and Interfaces, Biomaterials, Potsdam, Germany

Although the resolution of light microscope is in theory limited by the diffraction limit theorem, which imped the study of many biological structures with features smaller than ca. 250 nm, light microscopists are constantly finding ways around this limit in order to study quantitatively sub-diffraction limited structures. FRET is one of these techniques. It is based on the non-radiative energy transfer between an excited fluorophore (donor) and a neighboring molecule (acceptor). Because FRET can only be detected when the donor and acceptor are separated by less than ca. 10 nm, it can be used to question the interactions of proteins. A paradigm in bacterial protein assemblies is the sub-cellular organization forming the magnetic chain found in magnetotactic bacteria. Although the role of individual magnetosomes-associated proteins has started to be unraveled, their interaction has not been addressed with current state-of-the-art optical microscopy techniques, effectively leaving models of the magnetotactic bacteria protein assembly arguable. Here we report on the use of FLIM-FRET to assess the interaction of MamK and MamJ. We used a host organism (E. coli) to express eGFP_MamJ and MamK_mCherry, the latest expectedly forming a filament in vivo. We found that in the presence of MamK, the fluorescence of eGFP_MamJ is distributed along the MamK filament but is distributed in the whole cytosol in its absence. FRET analysis using the fluorescence lifetime of the donor, eGFP, revealed a spatial proximity of MamK_mCherry and eGFP_MamJ typical of a stable physical interaction between two proteins.


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A Genetic Screen Reveals New Magnetosome Genes in Desulfovibrio magnetics RS-1

Lilah Rahn-Lee and Arash Komeili

University of California Berkeley

With members across the Proteobacteria and the Nitrospira that construct cubo-octohedral or bullet shaped or needle shaped crystals, the magnetotactic bacteria encompass an enormous amount of phylogentic and phenotypic diversity. However, our molecular and mechanistic understandings of how magnetosomes are constructed are based only on a small group of closely related Alphaproteobacteria. Here, we extend our knowledge of the genetic players in magnetosome formation into the Deltaproteobacteria by performing a genetic screen for non-magnetic mutants of Desulfovibrio magneticus RS-1. With its bullet-shaped magnetite crystals, RS-1 is a representative of a group of Deltaproteobacteria that comparative genomic studies have suggested represent an ancestral form of magnetosome synthesis. Our findings provide a lens through which we can both understand the magnetotactic Deltaproteobacteria and develop a broader model of magnetosome synthesis in all magnetotactic bacteria.

The interplay between two bacterial actin homologs, MamK and MamK-like, is required for the alignment of magnetosome

organelles

Nicole Abreu, Soumaya Mannoubi, Ertan Ozyamak, David Pignol, Nicolas Ginet and Arash Komeili

UC Berkeley USA, Aix-Marseille Universite France

Background: Many bacterial species contain multiple actin-like proteins tasked with the execution of crucial cell biological functions. MamK, an actin-like protein found in magnetotactic bacteria, is important in organizing magnetosome organelles into chains that are used for navigation along geomagnetic fields. MamK is encoded by a genetic island termed the Magnetosome Island. Unlike most magnetotactic bacteria, Magnetospirillum magneticum AMB-1 (AMB-1) contains a second island of magnetosome related genes. A homologous copy of mamK, called mamK-like, resides within this region and encodes for a protein capable of filament formation in vitro but has unknown function in vivo. We therefore sought to evaluate the potential in vivo function of mamK-like in AMB-1.Methods: Genetic deletions of mamK-like were made in wild-type and ∆mamK backgrounds. RT-qPCR was performed on these two strains (∆mamK-like and ∆mamK∆mamK-like) in addition to wild-type and ∆mamK strains to determine transcript levels. Organelle alignment was determined in all four strains using a fluorescent marker for magnetosome membranes. Interaction between MamK and MamK-like were assessed using a yeast two-hybrid assay. Phosphate release assays were performed to measure ATPase activity. Finally, Fluorescence Recovery After Photobleaching (FRAP) was conducted.Results: RT-qPCR determined that both mamK and mamK-like were expressed, with mamK expressed approximately 2-fold higher. Organelle alignment was most disrupted in cells lacking both mamK and mamK-like, followed by cells lacking only mamK, indicating a dominant role. MamK-like and MamK interact in a yeast two-hybrid assay and the presence of MamK-like assists in MamK filament turnover when endogenous protein is absent. Conclusions: Our results indicate that MamK-like has a similar function to MamK, a previously studied bacterial actin. In our model organism, both bacterial actins serve to align magnetosome organelles, though MamK plays a more dominant role. Taken together, these experiments suggest that direct interactions between MamK and MamK-like contribute to magnetosome alignment in AMB-1.

Dynamics and diversity of the bacterial actin-like protein, MamK

Olga Draper, Meghan Byrne, Ertan Ozyamak, Sepehr Keyhani, Nancy Hom, Andy Greenstein, Justin Kollman, Arash Komeili


Magnetotactic bacteria have become an attractive system to understand the biogenesis and organization of lipid-bounded organelles in bacteria. MamK, a bacterial actin-like protein, is at the center of the magnetosome chain formation process. In its absence magnetosome chains appear fragmented and disorganized. We have been studying the in vivo and in vitro properties of MamK in Magnetospirillum magneticum AMB-1 in order to uncover the mechanisms of magnetosome chain formation. Additionally, we believe that understanding the function of MamK can contribute to a broader understanding of cytoskeletal proteins and cellular organization in bacteria. Using a fluorescence recovery after photobleaching (FRAP) assay we have previously shown that MamK forms dynamic filaments in vivo that require its ATPase activity and the action of other magnetosome proteins. We have also characterized the polymerization kinetics and filament architecture of MamK in vitro. In order to understand MamK in more detail, we have conducted a targeted mutagenesis of its putative surface exposed amino acids. These mutants display a wide range of changes in filament organization, filament dynamics as assessed by FRAP and magnetosome chain architecture. Interestingly, we find that the rate of MamK filament dynamics is not correlated to the chain formation activity of the protein. Through in vitro analysis of these variants, we find that changes in MamK filament dynamics are due to inherent changes in its polymerization and depolymerization kinetics. Finally, we show that the bundling of MamK filaments under physiological potassium conditions is an important element in its dynamic behavior in vivo and in vitro. We are now examining the biochemical and


18

biophysical properties of MamK homolog in diverse bacteria in order to correlate specific sequence changes in the protein to distinct alterations in its activity and structure.

Tuesday 16th September

Invited lecture: Aerobic Respiration by Sulfate-Reducing Magnetotactic Bacteria

Christopher T. Lefèvre, Paul A. Howse, George W. Luther 3rd

, Dennis A. Bazylinski CEA Cadarache/CNRS/Aix-Marseille Université, France. University of Nevada at Las Vegas, USA. University of Delaware, USA.

(Presentation by Dennis A. Bazylinski, [email protected])

In general, dissimilatory sulfate-reducing bacteria (dSRB) are considered to be strict anaerobes regarding growth. These include members of the genus Desulfovibrio which were described as strictly anaerobic bacteria since their discovery more than 100 years ago. In the last two decades, mainly based on the fact that many dSRB have been discovered to be metabolically active in microaerobic environments, several reports have shown that these bacteria are quite resistant to oxygen; some not only surviving oxygen exposure for at least days, but some even reducing oxygen to water. One report describes growth of a Desulfovibrio species although growth was poor or meager at best if even observed. To our knowledge, consistent, reproducible aerobic growth of dSRB has never been achieved. Among the diverse magnetotactic bacteria, some are representatives of the dSRB. For example, the species Desulfovibrio magneticus strain RS-1 and several magnetotactic alkaliphilic bacteria of the genus Desulfonatronum have been clearly shown to be dSRB. In characterizing magnetotactic dSRB, we found that some strains utilized O2 as a terminal electron acceptor for growth. To prove this we grew magnetotactic dSRB in several different oxygen concentration growth media under air or N2 gas. Cells grew as a microaerophilic band at the oxic-anoxic interface (OAI) in media under air lacking sulfate (cellular sulfur was supplied as yeast extract or casamino acids and cysteine). Sulfide was not produced in these tubes. Cells did not grow under anaerobic conditions (under N2) in this medium unless sulfate was present. When sulfate was present in the growth medium under air, initial growth was also as a microaerophilic band of cells at the OAI. However as time went on, the band split into two. Our results show that some magnetotactic dSRB are capable of aerobic growth with O2 as a terminal electron acceptor. This is the first report of consistent, reproducible aerobic growth of dSRB with O2 as the terminal electron acceptor. This finding is critical in determining the important ecological roles dSRB play in the environment, particularly those characterized as microaerobic containing an OAI.

From Bacterial to Biomimetic magnetosomes Jennifer Bain and Sarah Staniland

University of Sheffield, UK

We have been working with several magnetic bacterial proteins and utilising them as an additive to chemical precipitations, to

control the formation of magnetite nanoparticle in vitro. Here we will present the results of our most recent candidate native

protein (MmsF) as well as several artificial proteins and scaffolds to control magnetite precipitation. We will discuss how we are

then utilising these proteins in the creation of a synthetic magnetosome for biomedical application and biomineralised

magnetic nanoparticle nanoarrays for data storage.

Magnetosome magnetite biomineralization – in-vivo analysis

Taylor Woehl, Emre Firlar, Sanjay Kashyap (US DOE Ames Laboratory) Teresa Perez Gonzalez, Damien Faivre (Maxx Plank Institute

for Colloids and Interfaces) Denis Trubitsyn, Dennis A. Bazylinski (UNLV) Tanya Prozorov (US DOE Ames Laboratory) US DOE Ames Laboratory

Magnetotactic bacteria can serve as a model system for the study of molecular mechanisms of magnetite biomineralization. We present results on the magnetosome magnetite biomineralization in magnetotactic bacteria in-vivo by using a fluid cell TEM holder platform. Our approach can be expanded to the in vivo characterization of a wide range of inorganic structures biomineralized by various microorganisms, and as such it is expected to have a direct impact on the understanding of biological processes.

mailto:dennis.bazylinski@unlv


19

A chemical biosignature of magnetite from magnetotactic bacteria

M. Amor, V. Busigny, M. Durand-Dubieff, M. Tharaud, G. Ona-Nguema, A. Gélabert, E. Alphandéry, N. Menguy, I. Chebbi, F. Guyot Institut de Physique du Globe de Paris & Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie

Magnetite is a widespread iron oxide found in various geological sedimentary deposits such as banded iron formations, carbonate platforms or paleosols. Magnetite can be produced through either abiotic or biological processes. Magnetotactic bacteria (MTB) and dissimilatory iron-reducing bacteria are known to synthetize magnetite nanoparticles. Although MTB are ubiquitous in modern natural environments, their identification in the geological record remains challenging. Because of the selective iron uptake by MTB for magnetite precipitation within the cell, the chemical composition of magnetite from MTB is expected to be pure (i. e. low incorporation of elements other than iron into magnetite). Although chemical purity has been suggested as a biosignature, which could help to identify fossil magnetites from MTB in ancient sedimentary deposits, it has never been calibrated quantitatively and systematically. In this work, we determined the incorporation of 34 elements in magnetite in both cases of abiotic aqueous precipitation and of production by the magnetotactic bacterium Magnetospirillum magneticum strain AMB-1. We showed that, for most elements, elemental incorporation is 100 times lower in magnetite from AMB-1 than that in abiogenic magnetite. Furthermore, the 19rganiza of some elements such as strontium appeared to be dramatically different between abiogenic and biogenic magnetites. The chemical purity of magnetite from MTB as well as the incorporation patterns of elements other than iron into magnetite can thus be used as reliable biosignatures relevant for searching magnetites from MTB in the geological record.

Magnetic fingerprint in marine sediments: clues from cultivated Magnetovibrio blakemorei strain MV-1 and recent cores from Brazilian coast

L. Jovane1*, V.H. Pellizari

1, F.P. Brandini

1, D. A. Bazylinski

2 and U. Lins

3

1 Instituto Oceanográfico, Universidade de São Paulo, São Paulo, Brazil. 2 School of Life Sciences, University of Nevada at Las Vegas, Las Vegas, NV, USA. 3 Universidade Federal do Rio de Janeiro, Instituto de Microbiologia, Rio de Janeiro, Brazil.

The magnetic properties (first order reversal curves, ferromagnetic resonance and decomposition of saturation remanent magnetization acquisition) of Magnetovibrio blakemorei strain MV-1, a marine magnetotactic vibrio, differ from those of other magnetotactic species from sediments deposited in lakes and marine habitats previously studied. This finding suggests that magnetite produced by some magnetotactic bacteria retains magnetic properties in relation to the crystallographic structure of the magnetic phase produced and thus might represent a “magnetic fingerprint” for a specific magnetotactic bacterium. The use of this fingerprint is a non-destructive, new technology that might allow for the identification and presence of specific species or types of magnetotactic bacteria in certain environments such as sediment. We will also show some preliminary results on the biogeochemical factors that control magnetotactic bacterial populations, documenting the environment and the preservation of bacterial magnetite, which dominates the palaeomagnetic signal throughout recent sediments from Brazilian Coast. We searched for magnetotactic bacteria in order to understand the ecosystems and environmental change related to their presence in sediments. We studied magnetotactic bacterial populations in marine settings measuring crucially nutrients availability in the water column and in sediments, on particulate delivery to the seafloor, to understand the environmental condition that allow the presence of magnetotactic bacteria and magnetosomes in sediments.

Identifying composition and mapping of magnetosomes and other intracellular compounds in microorganism using Raman

spectroscopy.

Stephan H. K. Eder (1), Alexander M. Gigler (1), Marianne Hanzlik (2), Michael Winklhofer (1)

(1) Department of Earth and Environmental Sciences, Geophysics, Ludwig-Maximilians-University, Munich, Theresienstr. 41, 80333 Munich, Germany (2) Department of Chemistry, Elektronenmikroskopie, Technical University Munich, Lichtenbergstr. 4, 85748 Garching, Germany

Bio-genic magnetite (Fe3O4) is a major compound in magnetotactic bacteria, and can also be found in many eukaryotic organisms. Here we demonstrate that such magnetic inclusions can be detected and mapped with confocal Raman microscopy, even though the magnetosomes occur in sub-100 nm size range, well below the optical diffraction limit. By using low laser-powers (< 0.25 mW) in combination with long integration times, the sample is prevented from laser induced heating, and therefore from dehydration of organics or even from oxidizing magnetite to hematite (Fe2O3). Secondly, this setting reduces the volume of confocal light-excitation and, by filtering for specific Raman spectra, it enhances the detectability of small particles. Here we identified magnetite in magnetotactic bacteria by its characteristic Raman spectrum (303, 535, 665 cm-1), well distinguishable from greigite (cubic Fe3S4; Raman lines: 253, 351 cm-1), which could be found in the Deltaproteobacteria class. Sulfur globuli in the Candidatus Magnetobacterium bavaricum (Nitrospirae) could be determined to consist solely of cyclo-octasulfur (S8: 151, 219, 467 cm-1),


20

while the detected spectra of the phosphorus-rich inclusions in magnetic vibrio are pointing to orthophosphate, and those in magnetic cocci to polyphosphate. Interestingly, the heme-based molecule cytochrome c, which occurs in the periplasmatic space, provides a strong resonant Raman signal, and can therefore be used as a label-free marker to visualize bacterial cytoplasmic membranes, and further more to identify the redox-state in the bacterial cells.

Biomineralization process of Magnetospirillum gryphiswaldense by X-ray absorption spectroscopy

M. Luisa Fdez-Gubieda, A. Muela, J. Alonso, A. García Prieto, J.M. Barandiaran

Universidad del País Vasco & BCMaterials. Spain

We have studied the biomineralization process of the magnetotactic bacteria Magnetospirillum gryphiswaldense strain MSR-1 by the combination of magnetic and structural techniques in a time-resolved study. For these purpose,we used different techniques like inductively coupled plasma-atomic emission spectroscopy (ICP-AES), magnetic analysis, transmission electron microscopy (TEM), and Fe K-edge X-ray absorption near edge structure (XANES). XANES is an element specific technique, sensitive to the oxidation state and to the local structure of the absorber element, in our case the Fe atom. Additionally, this technique is a powerful tool not only to identify but also quantify the different Fe inorganic phases present in the cell regardless of their magnetic state. The bacterium M. gryphiswaldense MSR-1 was grown microaerobically at 28ºC in low iron medium. For induction of magnetite biomineralization, iron starved cells in logarithmic growth phase were harvested by centrifugation and transferred to fresh medium supplemented with 100 μM Fe(III)-citrate. At specific times between 20 and 360 min the cells have been imaged by Transmission Electron Microscopy (TEM), the magnetic properties have been studied by means of room-temperature hysteresis loops, and finally Fe K-edge X-ray Absorption Fine Spectroscopy studies have been performed at the Elettra Synchrotron in Trieste (Italy)These techniques have allowed identification of two Fe phases during the process, magnetite and ferrihydrite, and, more importantly, measurement of the mass of each phase during the mineralization process. Additionally, we have distinguished two steps in the biomineralization process: the first in which ferrihydrite is accumulated, and the second in which magnetite is biomineralized.

Towards molecular imaging with high-field MRI and biogenic contrast agents

Nicolas Ginet, Sébastien Mériaux, Marianne Boucher, Benjamin Marty, Yoann Lalatonne,Sandra Préveral, Laurence Motte, Christopher T. Lefèvre, Françoise Geffroy, Franck Lethimonnier, Michel Péan, Daniel Garcia, Géraldine Adryanczyk, David Pignol

CNRS / CEA / Aix – Marseille University

Development of personalized, targeted treatments requires innovative technologies for molecular diagnosis. MRI is routinely used for diagnostics purposes to reveal physiological features, often in conjunction with injectable contrast agents. We achieved the sensitivity required for in-vivo molecular detection by combining high-field (17.2T) MRI protocols with magnetic iron-oxide nanoparticles shelled by a lipid bilayer, the magnetosomes, naturally produced by magnetotactic bacteria. Following biophysical characterization, we acquired MRI brain angiograms of living mice after injection of this contrast agent with a five-fold increase in sensitivity compared to commercial USPIO. We evidenced that magnetosomes can be detected in the picomolar range with high-field MRI, providing a promising and versatile tool to evolve molecular diagnostics in the field of medical research on human pathologies. Invited lecture and EMBO Young Scientist Lecture:

Structural determination of Mms6 magnetite binding helix and characterization of its mineral binding within the ferritin cage

Limor Lewin1, Radoul Marina

2, Batya Cohen

2, Stanislav Popov

1, Geula Davidov

1, Ronit Bitton

3 Michal Neeman

2 and

Raz Zarivach

1

1 Department of Life Sciences and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, POB 653, 84105 Beer-Sheva, Israel. 2 Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100 Israel. 3 Department of Chemical Engineering, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel & Ilse Katz Institute for Nanoscale Science and Technology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.

In nature, many organisms have evolved proteins that participate in production of iron oxides in forms of paramagnetic ferrihydrite (Fe2O3*nH2O) and ferromagnetic magnetite (Fe3O4), but the mechanisms by which these biomineralization proteins function are generally poorly understood. For instance, ferrihydrite is found in the core of ferritin, the main iron storage and controlled-release intracellular protein. Ferrihydrite can be converted to magnetite within the ferritin, thereby creating a magnetoferritin. Magnetite can be generated biologically as occurs in magnetotactic bacteria which synthesize cellular organelles called “magnetosomes” composed of magnetic nanoparticle surrounded by a lipid membrane. It has been shown that the nucleation and growth of magnetite crystals involve putative iron-binding proteins including Mms6. Mms6 is a magnetosome associated protein that was shown to interact with magnetite and affect its size and shape. To better understand Mms6 role in biomineralization and its involvement in the formation of magnetite nanocrystals of uniform size and shape, we studied its structure–function relationships. Here we studied the structural property of Mms6 magnetite binding helix and characterize its mineral binding within the ferritin cage. Because the C-terminus of Mms6 was shown to have a key role in the regulation of crystal growth, we designed a recombinant fusion protein, where M6A peptide (12 amino acids from the C-terminal Mms6 unit that is able to bind magnetite) is attached to the C-terminal of the inner surface of mouse H-ferritin to generate ferritin-M6A.


21

We have showed that M6A peptide addition into the ferritin did not affected the ferritin structure via X-ray crystallography, SAXS and TEM studies. These experiments showed that purified ferritin-M6A assembles into a stable ferritin protein cage while the M6A protruding into the cage core enabling magnetite biomineralization. We managed to obtain high-resolution (up to 2.5 Å) structure of mouse H-ferritin-M6A. As expected, the X-ray structure of ferritin-M6A adopted the ferritin fold with fold axis pore channels and ferroxidase canters with detectable metal ions. Our ability to use such construct as a contrast agent for hypoxia regions in tumors indicate for the importance of such approach.

Effects of crystal shape and structure on the magnetic microstructures of magnetosomes and their chains

András Kovács, René Uebe, Dirk Schüler, Christopher T. Lefèvre, Dennis A. Bazylinski, Richard B. Frankel, Éva Tompa, Mihály Pósfai, Rafal E. Dunin-Borkowski

Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons

The cells of magnetotactic bacteria can be regarded as model systems for studying the magnetic properties of 21rganization21 nanocrystals. Since each bacterial strain produces magnetosomes that have distinct sizes, shapes, crystallographic orientations and spatial arrangements, magnetotactic bacteria can be used to provide magnetic nanocrystal systems with a variety of distinct physical properties that is unmatched by synthetic samples. By studying the magnetic behavior of magnetosomes in appropriately chosen strains, the competing effects that influence the magnetic moments and microstructures of individual crystals and their assemblages can be assessed and understood separately. The effect of crystal size on magnetic domain character, the influence of shape and magnetocrystalline anisotropy on the direction of the magnetic induction within and between crystals, the changes that are imposed on the magnetic behavior of magnetosomes by their interactions and the effects of structural imperfections on magnetic moments can all be studied. We have used off-axis electron holography (EH), an advanced transmission electron microscopy (TEM) technique, to study the magnetic properties of individual and closely-spaced magnetite magnetosomes. The EH experiments were combined with high-resolution TEM, selected-area electron diffraction and tilting experiments in the TEM, in order to obtain information about the structures, orientations and morphologies of the magnetosomes. We studied cells from the strains LO-1, HSMV-1 and an unidentified strain, that each produce magnetite magnetosomes that have elongated bullet-like shapes and different crystallographic axes of magnetosome elongation ([100], [110] and [111], respectively). We also studied magnetosomes that were isolated from two strains of Magnetospirillum gryphiswaldense, including the wild-type strain and a strain carrying mutations in different magnetosome biosynthesis genes. Both strains were found to produce isometric magnetite magnetosomes of approximately equal size. Whereas the wild-type strain produced mostly single-crystal magnetosomes, the mutant strain contained a large fraction of composite magnetosomes that were either twinned or composed of several crystals. By comparing EH results from the two strains, the effect of structural imperfections on the magnetic properties of individual magnetosomes could be studied. EH results from the bullet-shaped magnetosomes show that each magnetosome contains a single magnetic domain. In isolated magnetosomes, the magnetic induction is strictly confined to be parallel to the elongation axes of the crystals, irrespective of the crystallographic direction that is parallel to the direction of elongation. Even though [111] is the magnetic easy axis in magnetite at room temperature, the magnetic induction was found to be parallel to [100] and [110] in LO-1 and HSMV-1, respectively. The shape anisotropy of each particle therefore overrides the effect of crystal structure-related anisotropy. In some disordered chains, bullet-shaped crystals occur side by side, with their long axes parallel. In such cases, magnetostatic interactions between the particles result in some of the bullet-shaped magnetosomes being magnetized perpendicular to their direction of elongation, parallel to the general chain direction. In both strains of M. gryph. The magnetosomes comprise single-magnetic domains, with the direction of the magnetic field inside them governed primarily by their shapes and by magnetostatic interactions between the magnetosomes; the chain configuration is the most important constraint on the direction of magnetic induction. The magnetic moments of composite (structurally imperfect) magnetosomes in the mutant are only about 2/3 those of single crystal magnetosomes in the same strain and in the wild-type strain. A notable difference between the isolated magnetosomes in the wild-type and mutant strains is that whereas the magnetosomes from the wild-type strain self-organize into chains on the supporting surface of the TEM specimen, the magnetosomes from the mutant are either scattered or only form short chain segments. The lack of chain formation could be a result of weaker magnetic moments in aggregated crystals than those in single crystals. This research was supported by the EU FP7 projects Bio2MaN4MRI and ESTEEM2.

Characterization of the chemistry and magnetism of individual magnetotactic bacterial cells using X-ray spectromicroscopy

Xiaohui ZHU 1, Adam Hitchcock

1, Tolek Tyliszczak

2, Pedro E. Leão

3, Dennis A. Bazylinski

4, Ulysses Lins

3

1 McMaster University; 2 Advanced Light Source;3 UFRJ; 4 University of Nevada

Magnetotactic bacteria (MTB) in the northern hemisphere migrate preferentially towards the north magnetic pole (north-seeking, NS), whereas those in the southern hemisphere swim towards the south magnetic pole (south-seeking, SS). Torres de Araujo et al. (1990) proposed a model to explain NS and SS behavior which postulates that the magnetic vector of magnetosome chains relative to the motile flagellum is reversed in NS and SS MTB cells [1]. However, this hypothesis has never been verified experimentally. We are using X-ray magnetic circular dichroism (XMCD) measured in a Scanning Transmission X-ray Microscope (STXM) to study magnetism of individual cells of MTB [2-4]. Recently we observed that, in some cases, the magnetosome chain in a single cell of Magnetovibrio blakemorei strain MV-1 is divided into sub-chains which are magnetically polarized opposite to that of other sub-chains in the same cell [4]. These results have provided insights and stimulated further STXM-XMCD measurements relating to the


22

origin of NS and SS behavior. The XMCD spectra of magnetosome chains in several MV-1 cells from both the northern hemisphere and southern hemisphere have been measured. Our results show that different MV-1 cells from each hemisphere exhibit opposite magnetic polarities, which is consistent with Torres’ hypothesis that some NS (SS) MTB cells are produced in the southern (northern) hemisphere. We will present a revised model to explain the origins of NS and SS behavior through a feature of cell division in strain MV-1 (which needs verification). Other applications of STXM to MTB will be described, including 2D and potentially 3D Fe 2p spectromicroscopy measurements of dry and hydrated MTB cells, which hopefully will provide insights into the mechanism of magnetosome biomineralization. [5] References: 1.F.F. Torres de Araujo et al., Biophys J. 58 (1990) 549. 2.K.P. Lam et al. , Chemical Geology 270 (2010) 110-116 3.S.S. Kalirai, et al, Chemical Geology 300-301 (2012) 14-23 4.S.S. Kalirai, D.A. Bazylinski, and A.P. Hitchcock, Plos ONE 8 (2013) e53368. 5.Research performed at CLS 10ID1 STXM (supported by CFI and U. Saskatchewan), and at ALS 11.0.2 STXM (supported by BES, DoE).

Electron microscopy facilities for the study of biominerals in Rio de Janeiro Marcos Farina

Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

How to combine bacterial division with magnetic polarity for magnetotactic bacteria with a single polar flagellum

Christopher T. Lefèvre, Mathieu Bennet, Stefan Klumpp, Damien Faivre CEA/CNRS/Aix-Marseille Université

Cellular complexity has long been recognized in eukaryotes in term of organization and cellular processes including division. However, it is only in the last decade that such complexity has started to be documented for bacteria. One example for such organization is cellular polarity with concomitant anisotropic cell division, which has specifically been observed for monotrichous bacteria, cells with one polar flagellum. These microorganisms are known to divide following a scheme where the newly synthesized flagellum emerges from the old pole devoid of flagellum and opposite to the flagellated pole. In this context, dividing monotrichous magnetotactic bacteria face the challenge to form daughter cells combining two polarities at once: the magnetic polarity set by the chain of oriented magnetic nanoparticles that passively aligned cells with the Earth’s magnetic field and the flagellar polarity that enables the cells to actively swim towards desired destinations. Indeed, if these bacteria follow the known division mechanism, the flagellum of the daughter cells would emerge at the old pole devoid of flagellum, and the two daughter-cells would have opposite magnetic polarity with respect to the position of their flagellum. Using the diversity of cultivated magnetotactic bacteria, we investigated the dividing mechanisms allowing monotrichous magnetotactic bacteria to efficiently divide and transfer both mother-cell polarities to the next generation.

Invited lecture: Magnetochrome: a c-type cytochrome domain specific to magnetotactic bacteria Pascal Arnoux, Marina Siponen, Damien Faivre, Michèle Chang, Christopher Lefèvre, Nicolas Ginet, David Pignol

Amongst the Mam proteins, a series of predicted redox proteins exhibit a c-type cytochromes motif endemic in MTB and potentially play a role in the iron biocrystallization process that takes place inside the magnetosome (Siponen et al., 2012). The magnetochrome (MCR) domain contains a CXXCH motif that forms a c-type heme-binding site, which is only found in four proteins associated with the magnetosome (MamP, E, T, X). It is usually present as a tandem repeat, rarely alone or in more repeats, and in all cases the MCR-containing proteins are predicted to be associated to the magnetosome membrane through a single membrane spanning α-helix. In a recent study focused on the biochemistry of MamP and its structural characterization, it was found that MamP displays ferroxidase activity with an original wrapping of c-type cytochromes that contributes to the formation of a crucible in which iron could be stabilized (Siponen et al., 2013). Thus, MCR-containing MamP would be involved in the control of the Fe(II) and Fe(III) ratio required for magnetite biomineralization. Recent functional studies on MamE and MamX were also published, hinting at potential functions for MCR domains during magnetosome biogenesis link to the original wrapping of their c-type cytochromes. Whether it concerns bioenergetics to drive iron import, manage the redox balance of the iron pool or any other molecular mechanisms requiring electron transport, the bioinformatics, structural and functional data available on MCR-containing Mam proteins suggest a key role in iron redox chemistry to ensure the proper mineralization of magnetosomes.

http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00117/full#B32

http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00117/full#B33


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Thursday 18th September

Light and magnetic field influence on Multicellular Magnetotactic Prokaryotes

Daniel Acosta-Avalos and Henrique Lins de Barros

Centro Brasileiro de Pesquisas Fisicas – CBPF

Multicellular magnetotactic prokaryotes (MMP) are sensible to light. For some circumstances it was observed a “photomagnetic” behavior, characterized by the inversion of the MMP magnetic polarity to avoid the region illuminated by UV or blue light. That behavior has been observed in our laboratory but it is rare, happening after a long time of illumination with laser light of 405 nm. We choose to study the motility because for magnetotactic organisms some characteristics of the trajectory depend on the ambient magnetic field, as is the case for the U-turn. We observed that the U-turn time changes with the light wavelength, indicating that some “light dependent magnetotaxis” could be present during illumination. Some other phenomena studied in our CBPF group and related to MMP magnetotaxis will be discussed in this opportunity. Financial support: CNPq and FAPERJ.

Diversity and Distribution of Mutilcellular Magnetotactic Prokaryotes in intertidal sediments of China Sea

Ke Zhou, Yiran Chen, Haijian Du, Rui Zhang,Wenyan Zhang, Hongmiao Pan, Long-Fei Wu1 and Tian Xiao

IOCAS and LIA-BioMNSL

Multicellular magnetotactic prokaryotes (MMPs) are a group of magnetotactic microorganisms composed of 10-100 Gram-negative cells. Although they are thought to be globally distributed, MMPs have been observed only in marine environment in America, Europe and Oceania. Additionally, most identified MMPs show a spherical mulberry-like morphology and synthesize mainly greigite magnetosomes. Here, we report the diversity of a ellipsoidal MMP with pineapple-like morphology in intertidal sediments of China Sea. This type of MMPs was observed from four different places. Genetic analysis revealed that the ellipsoidal MMPs belonged to six novel species representing five new genera of Deltaproteobacteria. In particular, five species belonging to four new genera were detected from Sanya. However, only one species was indentified from Qingdao, Yuehu Lake and Drummond Island, respectively. Investigation on the distribution and relationship to various biogeochemical parameters indicated that spherical MMPs from Qingdao were concentrated near the oxic–anoxic transition zone (OATZ) with high proportion of fine sand and total organic carbon, rich in leachable iron but poor in nitrates. And ellipsoidal MMPs from lake Yuehu were mainly distributed in gray sediment with high sulfur content, where was mainly composed of fine sand (0.05 ≤ particle size < 0.25 mm, 15.5%) and medium sand (0.25 ≤ particle size < 0.5 mm, 56.7%). We also found that the annual variations of two types of MMPs in lake Yuehu were significant. In summer and autumn, the abundances of two types of MMPs were high, but low in winter and spring. These annual variation characterizations were consistent with changes of some biogeochemical parameters such as salinity, temperature, total oganic carbon, total sulfur, sulphate, phosphate and silicate. These findings provide new insights into the diversity of MMPs in general, and contribute to our understanding of the adaptive evolution of MMPs to the marine intertidal sediment habitat.

Peculiar behavior of multicellular magnetotactic prokaryotes

Henrique Lins de Barros and Daniel Acosta-Avalos Centro Brasileiro de Pesquisas Fisicas – CBPF

Some peculiar movements have been observed in the multicellular magnetotactic prokaryote Candidatus Magnetoglobus multicellularis (CMM). The organisms were collected in hyper saline coastal lagoons in the Rio de Janeiro state, Brazil, in cities as Araruama (22o 50’ S, 42o 13’ W) or Itaipu (22o 57’ S, 43o 02’ W). The observations were made in an optical microscope (Nikon Eclipse TS100, objectives 10X to 40X) putting a drop of the sample on a slide and concentrated with a magnetic field produced by a pair of Helmholtz coils (circa 5 Oe). It was observed that most of the organisms are South seeking but a few was North seeking. It was possible to get populations over thousands of CMM organisms in the sample. In some occasions, when the organisms were concentrated in the edge of the drop, a part of the population presents a coordinated group movement, like an “explosion”, that starts a chaotic dance, and then that group makes escape motilities directed to the center of the drop and swimming in the opposite direction of their natural migration direction. In the presence of magnetic sand grains it was observed that the organisms swim following the magnetic field lines and stay concentrated only in the grain magnetic North pole. This observation has ecological implications, because in natural sediments magnetotactic organisms must concentrated around any magnetic grain and not necessarily following the oxygen gradient. Another peculiar behavior is observed in the last stage of the CMM division. These organisms present, in the first stage of its life cycle, around twenty cells. During their life cycle they double the number of component cells and, after this event, they change their shape to a near ellipsoid. In this stage the organisms swim parallel to the large axis of the ellipsoid. Then a constriction appears in the middle and they assume a peanut or “w” shape and swim perpendicular to the large axis before they complete the division process, giving rise to two new CMMs. This implies in a different


24

magnetosome distribution during the division stages. All these peculiar behaviors show that CMM organisms are very complex and that new studies must be done to understand their environmental adaptation involving magnetotaxis and other taxis. Financial support: CNPq.

The diversity and distribution of ellipsoidal multicellular magnetotactic prokaryotes

Yiran Chen1,2

, Haijian Du1,3

, Rui Zhang1,3

, Hongmiao Pan1,2

, Wenyan Zhang1,2

, Ke zhou4, Long-Fei Wu

2,5* and Tian Xiao

1,2*

1 Key Laboratory of Marine Ecology & Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. 2Laboratoire International Associé de la Bio-Minéralisation et Nano-Structures (LIA-BioMNSL), CNRS, F13402 Marseille cedex 20, France. 3 University of Chinese Academy of Sciences, Beijing 100864, China. 3 Laboratoire de Chimie Bactérienne, Université de la Méditerranée Aix-Marseille II, CNRS, F-13402 Marseille cedex 20, France. 4 College of Resource and Environment, Qingdao Agricultural University, Qingdao 266109, China. 5 Aix Marseille Université, CNRS, LCB UMR 7257, Institut de Microbiologie de la Méditerranée, 31, chemin Joseph Aiguier, F-13402, Marseille, France. * Email: [email protected]，Telephone; 33-4-91164157. **Email: [email protected], Telephone: 86-532-82898586, Fax: 86-532-82898586. Multicellular magnetotactic prokaryotes (MMPs) are a peculiar group of magnetotactic bacteria assembled from 10 ~ 100 cells with the same phylotype. Currently two morphotypes of MMP have been identified, including several species of spherical mulberry-like MMPs globally distributed and two species of ellipsoidal pineapple-like MMPs from Qingdao and Rongcheng city in Yellow Sea of China. Recently, two more kinds of ellipsoidal MMPs were discovered from sediments of Marseille in the Mediterranean and Drummond Island in the South China Sea, respectively. Phylogenetic analysis revealed that all ellipsoidal MMPs from the four sampling sites belong to three species. The MMPs from Marseille and the ellipsoidal MMP Candidatus Magnetananas rongchenensis belong to one species with 98.5% sequence similarity, while the MMPs from Drummond Island affiliate to a novel genus with more than 7.1% sequence divergence compared to the most closely related ellipsoidal MMP Candidatus Magnetananas tsingtaoensis. Despite significant geographic distances between Marseille and Rongcheng city, the MMPs from the two habits belonged to the same species. This was speculated to be related with the physical and chemical parameters (grain size of sand, and content of nitrite and ammonium salt) of the two marine intertidal sediment habitats. These findings reveal a great diversity and wide distribution of ellipsoidal MMPs, and provide insights into our understanding of the adaptive evolution of MMPs.

Behavior of the uncultured spherical MMP ‘Candidatus Magnetoglobus multicellularis’ under applied magnetic fields

Carolina N. Keim1*

, Roger Duarte de Melo2, Amanda A. Moreira

1, Lukas Bolini

1, Felipe Pitzer

1, Mariana S. J. Oliveira

1, Daniel Acosta-

Avalos2, Henrique G. P. Lins de Barros

2

1 Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2 Centro Brasileiro de Pesquisas Físicas – CBPF – Rio de Janeiro, Brazil.

Magnetotactic bacteria are able to align to magnetic field lines while swimming propelled by flagella, due to the presence of magnetosomes, which are intracytoplasmic membrane-bound magnetic crystals. Some magnetotactic bacteria consist of multicellular ensembles known as multicellular magnetotactic prokaryotes or MMPs. The uncultured microorganism ‘Candidatus Magnetoglobus multicellularis’ (CMM) is presently the best known MMP. CMM swim in helical trajectories, and both the trajectory and the body rotate clockwise. At the Earth’s magnetic field, they swim in complex, looping trajectories (walking), but at higher fields their trajectories are parallel to the applied magnetic field (free motion). When they find an obstacle, they remain rotating (rotation) and eventually undergo backward excursions followed by a forward movement (escape motility) [2]. Because no pure cultures of MMPs are available, it is not possible to do controlled experiments to investigate cell-to-cell communication in these microorganisms, and thus studies on motility could be used to put on evidence behavioral aspects that would imply in cell-to-cell communication. In this work, we investigate the movements of CMM under magnetic fields 2.5 to 85 Gauss, applied parallel or perpendicular to the optical axis of a light microscope, and observed the effects of the magnetic field intensity on motility when they approach an obstacle. To obtain samples of CMM, water and sediment were collected in Araruama Lagoon (RJ, Brazil) and maintained in the lab for a few weeks. CMMs were magnetically separated and enriched samples were placed between slide and coverslip separated by an O-ring. Samples were observed with a light microscope equipped with a pair of coils arranged to produce magnetic fields oriented either parallel or perpendicular to the optical axis of the microscope. Digital video records were obtained, and analyzed to produce a frame by frame trajectory of individual microorganisms. When we used the applied magnetic field parallel to the optical axis of the microscope with intensities 2.5, 5.0, or 11 Gauss, we observed that all CMM individuals performed helical movements, most of them apparently not aligned to the applied magnetic field (walking), whereas a few underwent circular trajectories, parallel to the applied magnetic field (rotation). Most microorganisms remained in focus, which means that they were close to the coverslip as a result of magnetotaxis. The proportion of CMm performing “rotation” increased at 21.5 and 43.5 Gauss and, at 85 Gauss, virtually all CMm performed “rotation”. “Free motion” was observed when the magnetic field was inverted. All microorganisms rotated clockwise during “free motion” and “rotation”, and also in most looping trajectories (walking). These results agreed well with the results obtained with the magnetic field applied perpendicular to the optical axis of the microscope. The ability to swim parallel to the lines of the local magnetic field is the hallmark of magnetotactic bacteria. The alignment of magnetotactic bacteria to the magnetic field lines depends on the interaction of magnetosomes and local magnetic field relatively to thermal energy (MB/kT). Our results suggest that CMM is able to actively change direction under magnetic fields smaller than 11


25

Gauss. Increasing the strength of the magnetic field increases the magnetic interaction between the external magnetic field and the magnetosomes, and thus the velocity of re-alignment to the magnetic field lines after active direction changes, up to the level that active changes in direction are not distinguished because re-orientation is too fast. Thus, the movements of CMM depend on the interaction between the local magnetic field and magnetosomes, on thermal energy and also on active direction change. In conclusion, active changes in direction implies in some sort of cell-to-cell communication and puts on evidence the high degree of integration between the cells of CMM. Financial support: CNPq.

Invited lecture: Flagella and motility of magnetotactic bacteria

D. Murat1,8

, C.-L. Santini1,8

, M. Hérisse1, A. Bossa

1, F. Alberto

1,8, W.-J. Zhang

2,8, S.-D. Zhang

2,8, J. Ruan

3, T. Kato

3, K. Namba

3, T.

Song4,8

, Y. Li5,8

, N. Petersen6, A.-M. Gué

7,8 and L.-F. Wu

1,8

1.LCB, UMR7283, AMU-CNRS, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France; 2.Sanya Institute of Deep Sea Science and Engineering, CAS, Fenghuang Road 62, 572000 Sanya, China; 3.Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan; 4.Institute of Electrical Engineering, CAS, Beijing 100190, China; 5.State Key Laboratories for Agro-Biotechnology, China Agricultural University, Beijing 100193, China; 6.Ludwig Maximilians University, 82152 Munich, Germany; 7.Nano Ingénierie et Intégration des Systèmes, LAAS, 7 avenue du Colonel Roche, CNRS, F-31077 Toulouse Cedex 4, France; 8.French-Chinese Laboratory of biomineralization and nanostructure (LIA-BioMNSL)

How bacteria could improve their food supply in aquatic habitats with pervasive and changing chemical and physical gradients? Magnetotactic bacteria (MTB) exploit the unique capacity of swimming along the geomagnetic field lines to find and remain at the oxic-anoxic transition zoon in chemically stratified water column or sediments, where the redox potential is optimal for their growth. MTB are morphologically diverse and propelled by various kinds of flagellar apparatus. Using genetic engineering and molecular fluorescence labeling we found that Magnetospirillum magneticum AMB-1 is driven by asymmetric rotation of its amphitrichous flagella. It reverses the swimming direction by coordinated switch of the flagellar rotation directions while keeping aligned along the magnetic field lines. Magnetotactic ovoid strain MO-1 synthesizes an exquisite propulsion apparatus composed of 7 flagella and 24 fibrils within a sheath. MO-1 cells rotate around and translate along their short body axis and swim forward constantly without apparent stops. When encountering obstacles, they squeezed through or swam southward to circumvent the obstacles. The distances of the MO-1 swimming southward were inversely proportional to the field strength. The robust, powerful flagellar apparatus of MO-1 could sustain the swimming in marine sediments in order to derive energy via micro-aerophil respiration. We provide the first experimental evidence for the pivotal role of magnetotaxis for the growth of MO-1. Ecological implication of magnetotaxis will be discussed.

Magnetofossil abundance during the Middle Eocene Climatic Optimum (MECO)

Jairo F. Savian[1]

, Luigi Jovane[2]

, Ricardo I.F. Trindade[3]

, Andrew P. Roberts[4]

, Fabio Florindo[5]

, Fabrizio Frontalini[6]

, Rodolfo Coccioni

[6]

[1] Universidade Federal do Rio Grande do Sul, Brazil; [2,3] Universidade de São Paulo, Brasil; [4] The Australian National University, Australia; [5] Istituto Nazionale di Geofisica e Vulcanologia, Italy; [6] Università degli Studi di Urbino “Carlo Bo”, Urbino, Italy

Magnetotactic bacteria produce intracellular crystals magnetic minerals with precisely controlled size, morphology, and stoichiometry, specifically magnetite or greigite. These magnetotactic bacteria are widely observed in aquatic environments and remains preserved in the sediments as magnetofossils that record ancient geomagnetic field and paleoenvironmental variations. Although the globally importance of the biogeochemical remanent magnetisations and the useful information of the magnetofossils provide about paleoproductivity or paleoenvironmental is very limited. Magnetofossils have been reported from equatorial Indian and Neo-Tethys Oceans during the Middle Eocene Climatic Optimum (MECO, ~40 Ma). The MECO event is a strong transient warming event (global temperature increase 4-5°C) that has recognized in the oceanic records. Here, using environmental magnetism and first order reversal curves (FORC), we present evidence for abundant magnetofossils during the MECO event from Neo-Tethys and Indian Oceans sections. Moreover, our results integrated with biostratigraphy and geochemistry shows a period of high primary productivity in the Ocean. Enhanced global weathering during MECO, and expanded suboxic diagenetic environments, probably provided more bioavailable iron that enabled biomineralization of magnetofossils. Our observations suggest that during the MECO event the oceans provided ideal conditions for magnetofossils, and that these organisms were globally distributed. However, much more work is needed to understand the interplay between magnetofossil morphology, climate, nutrient availability, and environmental variability.

Culture-independent characterization of novel magnetotactic cocci from Antarctica marine sediments

Fernanda Abreu, Ana Carolina V. Araujo*, Karen Tavares Silva, Marcos Farina, Vivian H. Pellizari and Ulysses Lins *Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro.

Magnetotactic bacteria (MTB) are a heterogeneous group of ubiquitous microorganisms capable of producing nano-sized, magnetic iron mineral, membrane-bound particles called magnetosomes. MTB are found in stratified aquatic sediments and/or water with a wide range of salinities, moderate to high temperatures, and pH varying from neutral to alkaline. Here, we characterized MTB from the low temperature Antarctic maritime region. Sediment samples were collected at nine sampling sites within Admiralty Bay, King George Island (62°23’S 58°27’W) from 2009 to 2013. Five sites were positive for MTB and cocci were the predominant morphology.


26

Rod-shaped bacteria and spirilla were rarely found (two sites), but disappeared from samples after storage. Two sampling sites (Machu Picchu and Ullman Point) containing large number of cocci were chosen for analysis with electron microscopy and molecular techniques. Three morphotypes of magnetotactic cocci were identified based on magnetosome size and chain organization. Most cocci contained two or four chains of magnetosomes, but some cells contained magnetosomes clustered at one cell pole. The crystalline habit and composition of all magnetosomes analyzed with high-resolution transmission electron microscopy and energy dispersive X-ray microanalysis were consistent with elongated prismatic magnetite (Fe3O4) particles. Magnetosomes organized in four chains or as clustered particles were smaller than magnetosomes arranged in two chains. Average length, width and shape factor of magnetosomes were respectively 102 nm ± 21, 72 nm ± 17, 0.6 ± 0.09 for cocci with four chains, 87 nm ± 13, 47 nm ± 7, 0.5 ± 0.06 for cocci with clustered particles, and 115 nm ± 35, 70 nm ± 20, 0.6 ± 0.1 for cocci with two chains. Consistently, the majority of retrieved 16S rRNA gene sequences from enriched magnetotactic cocci in Machu Picchu site were affiliated with three distinct clusters in the Alphaproteobacteria class. The new members of each phylogenetic cluster (named group I, II, and III) were confirmed with fluorescent in situ hybridization (FISH) microscopy. Group I was closely related to sequences from samples geographically distant and environmentally distinct from Antarctica sediments. Sequence similarity values ranged from 96 to 100% among clones within group I. Group II clones showed 94-95 % 16S rRNA gene sequence similarity with the closest MTB sequence available in GenBank. Sequence similarity was relatively low between clones within group III (94%) and between group III and other MTB (6% minimum sequence divergence). Because magnetotactic cocci from Itaipu Lagoon (Brazil) containing clustered magnetosomes in one pole of the cell are closely related to group I and both strains produce magnetosomes with similar aspect ratio, we can reasonably assume group I contains cocci with clustered magnetosomes. It is unclear to which group cocci containing two or four magnetosome chains belong. Interestingly, FISH probes designed for each group also hybridized with cocci enriched from Ullman Point site, indicating these groups of MTB are dispersed in Admiralty Bay sediments. Low sediment temperature (always below 1°C) suggests magnetotactic cocci from maritime Antarctica consist of psychrophilic strains. Moreover, because similar sequences were retrieved from samples collected along different years, these new strains are probably indigenous members of Antarctic microbiota. Financial support: Brazilian CNPq, CAPES, FAPERJ.

Magneto-aerotaxis – theory and experiment

S. Klumpp, C. Lefevre, M. Bennet, L. Landau, D. Faivre Max Planck Institute of Colloids and Interfaces

It is generally believed that the physiological role of the magnetotactic apparatus is to help guide aquatic bacteria to their preferred habitat in the oxic-anoxic transition zone. The corresponding tactic behavior is thus called magneto-aerotaxis. We have characterized magneto-aerotactic motility in 12 different strains of cultured magnetotatic bacteria with various morphologies, phylogenies, physiologies and flagellar apparatus under controlled magnetic fields and oxygen concentration gradients. We observed six different magneto-aerotactic behaviors that can be described as a combination of three distinct mechanisms, including the previously reported (di)polar and axial mechanisms and a previously undescribed unipolar mechanism. In addition, we implemented a mathematical model, which suggests that these mechanisms can be interpreted as different ways how the bacteria obtain directional information from the two salient vectorial parameters, the magnetic field and the oxygen gradient. The results suggest that sensing of the oxygen gradient can be replaced by following the direction given by the magnetic field. Moreover, there may be separate mechanisms for sensing oxygen concentration gradients employed under high- and low-oxygen conditions that can be replaced individually.


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Poster Presentations

(Alphabetical order by first author´s name)

Physicochemistry of magnetite formation in magnetosomes

Agata Olszewska, Katharina Tomschek, Damien Faivre

Department of Biomaterials, Max-Planck-Institute of Colloids and Interfaces, Potsdam, Germany

Physicochemical parameters of the bacterial growth medium such as the pH value and the redox potential (Eh) as well as the cellular iron uptake kinetics may play crucial roles in the formation of magnetite in magnetosomes (Faivre am mineral, 2008). Because the thermodynamical stability of magnetite is limited to a narrow redox potential and pH range, we investigate how external parameters can influence the formation of magnetosomes and crystal growth in the Magnetospirillum magneticum (AMB-1) strain. Temperature, pH, Eh and the concentration of dissolved oxygen can be controlled within a fermentation process. We used a 16 liter fermentor to investigate the influence of the pH ranging from 5 to 8 on iron uptake, final size and morphology of magnetosomes. We measured iron concentration in medium during the fermentation process via inductively coupled plasma optical emission spectrometry (ICP-OES), in order to compare iron uptake at different pH values over time. Bacterial growth was determined by measuring the optical density (OD) at 565nm. Furthermore, we studied morphology and of both bacteria and magnetosomes by transmission and scanning electron microscopy. References: Faivre, D., & Schüler, D. (2008). Magnetotactic bacteria and magnetosomes. Chemical reviews, 108(11), 4875–98. Doi:10.1021/cr078258w

Identification of functional domain of crystal growth regulator protein Mms6 from Magnetospirillum magneticum strain AMB-1

by deletion of amino acid sequence

Ayana Yamagishi1, Kaori Narumiya

1, Masayoshi Tanaka

2 Atsushi Arakaki

1 and Tadashi Matsunaga

1

1. Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, Japan 2. Department of Chemical Engineering, Tokyo Institute of Technology, Japan

The group of proteins, called Mms (Mms5, Mms6, Mms7, and Mms13), was isolated from the surface of cubo-octahedral magnetite

crystals in Magnetospirillum magneticum strain AMB-1. The proteins contain well-conserved N-terminal hydrophobic and C-

terminal hydrophilic regions. Based on the phenotypic characterization of the gene deletion mutants, all Mms proteins were shown

to be involved in crystal growth and defining the surface structures of the magnetite crystals. An in vitro experiment of magnetite

synthesis using Mms6 also indicated that the protein influences crystal size and morphology. Moreover, Mms6 protein showed

iron-binding ability in the C-terminal region and self-assembling ability in the N-terminal region. The observed characteristics are

likely to be associated with the crystal formation. In order to understand the role of the conserved regions of Mms6 protein in

magnetite formation, we constructed a series of gene deletion mutants that contain deletions in different region of the mms6 gene

sequence. TEM analysis of the gene deletion mutants revealed that the deletions in both N-terminal and C-terminal regions

significantly affect to the crystal shape and size. The deletion mutant produced relatively small elongated crystals as observed in

the mms6 whole deletion mutant. These results suggested that both C-terminal and N-terminal regions plays important role for

iron oxide formation in magnetotactic bacteria. The morphology control mechanism of cubo-octahedral magnetite by Mms6 will be

discussed in this presentation.

Organelle formation and genetics in Desulfovibrio magneticus RS-1

Carly Grant, Arash Komeili


Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing bacterium that was originally isolated and cultured due to its magnetotactic capabilities. The magnetosomes formed by this deltaproteobacterium are irregular bullet-shaped crystals of magnetite. This magnetosome phenotype has been observed in the magnetotactic deltaproteobacteria and gammaproteobacteria and is unlike the magnetosomes formed by the well-studied magnetotactic alphaproteobacteria, such as Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Additionally, the magnetosome gene island, which has been found in all magnetotactic bacteria to date, has significant differences when comparing alphaproteobacteria with deltaproteobacteria. The differences in phenotype and genotype suggest that magnetosome formation is regulated differently between alphaproteobacteria and RS-1. In addition to forming magnetosomes, RS-1 forms iron- and phosphorus-rich (FeP) granules that are membrane-bounded. We hypothesize that the formation of these granules is a regulated process controlled by


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distinct genes in the RS-1 genome. We have begun to study these granules using biochemical analyses. In addition to biochemical analysis, genetic tools would be useful to study magnetosome and FeP granule formation in RS-1. Currently, there are no tools for targeted gene deletion in RS-1 and, while plasmids can be transferred into RS-1, the native restriction system makes working with foreign DNA difficult. Here, I describe a method for genetic manipulation in RS-1 that employs a conditionally replicative plasmid. Biochemical analysis and genetic tools will enable RS-1 to be an important model organism for organelle formation in bacteria.

Magnetotactic bacteria diversity in saline lagoons

Clarissa Werneck, Ulysses Lins and Fernanda Abreu

Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Magnetotactic bacteria (MTB) are a diverse group of prokaryotes capable of producing magnetosomes, which are intracellular magnetic nanocrystals surrounded by a biological membrane. Magnetosomes are usually organized in one or more chains within the cell, functioning as a biological compass, which is used for guidance in a magnetic field lines (Bazylinski and Frankel, 2004). The ability to produce magnetosomes rely on a set of genes that were acquired by lateral gene transfer (Jogler et al., 2009) or/and by vertical gene transfer in Proteobacteria phylum (Lefèvre et al., 2013). Recently, several MTB species have been described and a few isolated in pure culture (Lefèvre and Long-Fei, 2013). However, based on the mechanisms of transfer we believe that our knowledge of the total MTB diversity is still limited. Here we use light microscopy to observe the morphological diversity of MTB in environmental samples from saline lagoons and transmission electron microscopy (TEM) to characterize magnetosome structure and composition. Amplification, sequencing and analysis of the 16S rRNA gene will be used to access molecular diversity and phylogeny. Water and sediment were collected in Maricá and Itaipu Lagoons in Rio de Janeiro state. MTB were magnetically enriched as described in Lins et al. (2003) and observed in a Zeiss Axioimager microscope. Whole cells were deposited on formvar-coated grids and observed with a Morgagni TEM operating at 80 kV. Magnetosome length, width and shape factor will measured using iTEM software and results were analyzed with Graphpad InStat version 3.0. The 16S rRNA gene will be PCR amplified with bacterial specific primers 8bF and 1512uR; PCR products will be cloned into pGEM-T Easy vector and sequenced. In Itaipu lagoon environmental samples magnetic cocci, rods, spirilla and multicellular magnetotactic prokaryotes were present. Small rods and cocci were the dominant morphotype. Based on cell size and magnetosome chain organization we expect that the magnetic cocci represent different species. Although MTB from Itaipu lagoon have been studied (Lins and Farina, 1998; Spring et al., 1998; Morillo et al., 2013), the magnetic spirilla and rods have never been observed in this environment. In Maricá lagoon we could observed magnetotactic cocci, vibrio and magnetotactic multicellular prokaryotes, which was the dominant morphotype. TEM observation, magnetosome characterization and phylogenetic analysis are under development. Bazylinski, D.A. and Frankel, R.B. (2004). Nat. Rev. Microbiol. 2, 217–230. Jogler, C., Kube, M., Schübbe, S., Ullrich, S., Teellng, H., Bazylinski, D.A. et al. (2009). Environ. Microbiol. 11, 1267–1277. Lefèvre, C.T. and Long-Fei, W. (2013). Trends Microbiol. 21, 534–543. Lins, U., Farina, M. (1998). Micros. Res. Tech., 42, 459-464. Lins, U., Freitas, F., Neumann, C.K., Barros, H.L., Esquivel, D.M. and Farina, M. (2003). Braz. J. Microbiol. 34, 111–116. Morillo, V., Abreu, F., Araujo, A. C., De Almeida, L.G.P., Enrich-Prast, A., Farina, M., Vasconcelos, A.T.R., Bazylinski, D.A., Lins, U. (2014). Frontiers in Microbiology (Online), v. 5, p. 1. Spring, S., Lins, U., Amann, R., Schleifer, K.H., Ferreira, L.C., Esquivel, D.M. and Farina, M. (1998). Arch. Microbiol 169: 136–147.

Distinct roles for two HtrA proteases in magnetosome biomineralization

David Hershey and Arash Komeili

University of California – Berkeley

Magnetotactic bacteria produce nanometer-sized crystals of magnetite within their cells. While the genetic factors required for magnetite formation are known in Magnetospirillum magneticum AMB-1, the biochemical properties of these gene products remain elusive. We have focused our attention on two such factors, MamE and MamO, which show homology to the HtrA family of serine proteases. Using a combination of genetic and biochemical approaches, both in vivo and in vitro, we have characterized MamE as the main proteolytic factor in magnetosome maturation. Surprisingly, our analyses strongly indicate that MamO is a degenerate serine protease that plays distinct roles in magnetite crystal initiation and maturation. The specificity of proteolytic targets in vivo and in vitro is consistent with a model where MamE activates other late biomineralization factors by releasing them from the magnetosome membrane.


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Genome analysis of the freshwater magnetotactic bacterium Magnetospirillum caucasicum SO-1

Denis S. Grouzdev, Marina V. Dziuba, Marina V. Sukhacheva, Veronika V. Kozyaeva, Boris B. Kuznetsov Lomonosov Moscow State University, Centre Bioengineering RAS

Here, we present genome analysis of Magnetospirillum caucasicum SO-1, a freshwater magnetotactic spirillum isolated from the sediments of the Ol’khovka river. Phylogenetic 16S rRNA gene analysis revealed that the nearest to M. caucasicum SO-1 organism was M. magneticum AMB-1 (99.4%). However DNA-DNA hybridization scores between SO-1 and AMB-1 as well as with Magnetospirillum type strains (M. magnetotacticum MS-1T and M. gryphiswaldense MSR-1T) were below 70% indicating that SO-1 represents a novel species in genus Magnetospirillum. SO-1 genome contained 3 rRNA genes (5S-23S-16S) and 52 aminoacyl-tRNA synthetase genes, coding density was 89.98% with an average gene length of 941 bp. A total of 4717 ORFs were found in the genome, where 3573 (75.75%) were functionally annotated using Rapid Annotation Subsystem Technology (RAST) on-line service.The MAI of SO-1 exhibit several characteristic features of a typical genomic island, such as the presence of direct repeats (DRs), a lower GC content, an abundance of transposases, and many hypothetical genes. The estimated size of the MAI is approximately 100 kb.. From the 97 genes annotated within the 100 kb region from the M. caucasicum SO-1, 38 showed homology to genes implicated in magnetotaxis in Magnetospirillum species (mam and mms genes). Mam and mms were organized in five clusters within MAI (mamGFDC, mms6, mamAB, mamEOQRB, mamXY) as it was shown for Magnetospirillum magneticum AMB-1. A distinctive feature of the genome SO-1 is the presence of the gene mamG-like. Another feature of the SO-1 genome is the absence of 1133 bp flanking direct repeat on the right boundary of the MAI. As previously shown (Fukuda et.al., 2006), the MAI of M. magneticum AMB-1 can be deleted via the XerC integrase and the two 1133-bp DR. One of the DRs in the genome SO-1 is followed by XerC and flanking the left boundary of the MAI, but second one is in contig those with no relationship to the island. In addition, a number of essential genes involved in nitrogen metabolism are located downstream of the MAI. Thus we hypothesized that excision of the genomic region flanked by DR from chromosome of SO-1 leads to bacterial death. Indeed, we have never observed the loss of magnetic phenotype of SO-1 during 3 years of lab cultivation.

Taxonomic description of three novel freshwater magnetotactic spirillae isolated from distinct geographical points of Russia

Dziuba M.V., Koziaieva V.V., Grouzdev D.S., Kuznetsov B.B.

Centre Bioengineering RAS; Lomonosov Moscow State University

Members of the genus Magnetospirillum are the most studied MTB in terms of their morphology, physiology and magnetosome formation. Currently within this genus three magnetotactic species (M. gryphiswaldense, M. magnetotacticum, M. magneticum), and several non-magnetotactic (M. aberrantis and M. bellicus) have been described. Being a relatively easy cultivated MTB, magnetotactic Magnetospirillum are of great importance as model organisms for studying magnetite biomineralization, and as potential producers of biogenic magnetic nanoparticles. However, only two Magnetospirillum species – M. gryphiswaldense MSR-1T and M. magnetotacticum MS-1T have been characterized according to the bacterial classification requirements and validly taxonomically described. The absence of the appropriate taxonomic species description and the relatively small number of Magnetospirillum in pure culture, especially magnetotactic representatives, limit studying of their phylogeny and evolution of genes involved in magnetotaxis. Recently 13 novel Magnetospirillum-like isolates were described by Lefévre et al., but these strains were not completely characterized (Lefévre et al., 2011). In this study, three novel strains of magnetotactic spirillum were isolated from three rivers located in distinct areas of the European Russia: the Ol’khovka (Kislovodsk, Caucasus), the Pshada (Krasnodar region), and the Moskva (Moscow). The axenic cultures were obtained using a capillary race-track method and subsequent inoculation the semi-solid Magnetospirillum media (№380 DSMZ) with serial dilution. The isolated strains were designated SO-1(Ol’khovka), SP-1 (Pshada) and BB-1 (Moskva). All isolated strains were motile, helical in shape, bipolar flagellated and synthesized a single chain of cubo-octahedral magnetosomes, typical to those found in Magnetospirillum species. Phylogenetic analysis of the 16S rRNA gene sequences indicated that the strains belong to the genus Magnetospirillum. The most closely related to SO-1 was M. magneticum AMB-1 (99.4% 16S rRNA sequence similarity) and the next close organism was M. magnetotacticum MS-1T (99.3% 16S rRNA sequence similarity). The closest organisms to SP-1 and BB-1 were M. magnetotacticum MS-1T (97.9%) and M. gryphiswaldense MSR-1T (97.1%) respectively. Due to the high degree of 16S rRNA gene similarity (˃97%) we carried out a DNA-DNA hybridization to conclude whether new isolates belong to novel species. DNA-DNA hybridization between SO-1, SP-1, BB-1 and M. magnetotacticum MS-1T, M. gryphiswaldense MSR-1T, M. magneticum AMB-1, as well as among SO-1, SP-1, BB-1 themselves, showed similarities well below 70%, a threshold required to distinguish two strains as separate species. Cultivation experiments revealed traits shared among the isolates and other known Magnetospirillum species, as well as distinguishable features for each strain. SO-1 and BB-1 exhibited NaCl tolerance similar to that previously reported to M. magnetotacticum (<1%). SP-1 demonstrated the lowest tolerance to NaCl concentration among known Magnetospirillum (<0.1%). Like other Magnetospirillum species, SO-1, SP-1 and BB-1 were chemoorganoheterotrophic and oxidized a range of fermentation end products, mainly short-chained carbonic acids: acetate, lactate, fumarate, succinate and others. Unlike M. magnetotacticum and M.gryphiswaldense, SO-1and BB-1 were able to utilize glycerol. All strains used oxygen as terminal electron acceptor. They also utilized nitrate as electron acceptor, demonstrating ability to facultative anaerobic growth. In contrast to M. bellicus, SO-1, SP-1 and BB-1 did not use perchlorate and nitrite as electron acceptors. All isolates were microaerophilic, however the degree of aerotolerance varied among the strains. SP-1 was found to be less aerotolerant than others (≤5% O2 in the gas phase). SO-1 and BB-1 could grow aerobically in


30

presence of thioglycolate; however under these conditions they did not produce magnetosomes. Thus, based on the phylogenetic analysis, DNA-DNA hybridization and phenotypic characteristics, the strains SO-1, SP-1 and BB-1 should be considered as type strains of the novel species and we propose them the names Magnetospirillum caucasicum, Magnetospirillum meridiorum and Magnetospirillum moscoviense respectively.

Synthesis of magnetic filaments on flagellin-Mms6Cterm fusion proteins as templates

Éva Tompa, Ilona Nyirő-Kósa, Mihály Pósfai, Balázs Tóth, Ferenc Vonderviszt

Department of Earth and Environmental Sciences, University of Pannonia, Veszpré

Studies of genetically-engineered bacteria revealed the roles of distinct proteins in the nucleation and growth of magnetite magnetosome crystals. This information is being used to design biomimetic synthesis pathways for the laboratory production of magnetite nanoparticles with special properties. Our approach is to combine two known bacterial functions, filament formation and magnetosome synthesis, in order to produce one-dimensional magnetic nanostructures. Flagellar filaments are composed of thousands of flagellin subunits (FliC). Our strategy is to construct recombinantly expressed flagellin variants containing iron-binding motifs. The flagellins are then utilized for the construction of filaments displaying periodically repeated recognition sites on their surfaces that have strong affinity to bind iron from solution. These filaments may serve as templates for the formation of magnetite nanofibers. We have chosen to use C-terminal part of the Mms6 magnetosome protein as the iron-binding motif to be inserted into the flagellin protein, since Mms6 is known to be involved in magnetite synthesis in magnetotactic bacteria, and was shown to bind iron from solution. Three types of flagellin-based fusion proteins were created by inserting MmsCterm in place of the highly variable D3 domain of flagellin from Salmonella. Mms6Cterm was ligated into ΔD3_FliC plasmid i) without any linkers; ii) with small linkers, and iii) that had only part of the D3 domain cut. In all three cases the successful construction of the fusion protein was confirmed by SDS-PAGE electrophoresis. The motility of cells having the mutagenized flagella was preserved (as checked using dark-field optical microscopy), indicating that the folding of the flagellin protein was not affected. Moreover, the mutagenized flagellin proteins were still able to self-organize in vitro. The amount and morphology of flagella made of the fusion proteins were also analyzed using transmission electron microscopy (TEM). The mutagenized filaments were used as templates for the formation of one-dimensional iron oxide nanostructures, by nucleating magnetite on the filaments directly from iron-containing solutions. Iron concentration, rate of iron addition, pH and temperature were varied, and the solid products of the crystal nucleation experiments were studied using TEM. Our initial experiments brought mixed success: conditions under which the filaments were preserved in the solution did not typically favor the formation of magnetite. Instead, other iron oxides such as ferrihydrite nucleated on the mutagenized filaments. In order to check whether Mms6Cterm indeed binds iron from solution, we also performed isothermal titration microcalorimetry experiments. However, the results were inconclusive. Further experiments are under progress (i) to fine-tune the conditions for magnetite nucleation on flagellin-Mms6Cterm filaments, (ii) to insert magnetosome protein fragments other than Mms6Cterm into the flagellin, and (iii) to try to attach magnetite nanoparticles to the mutagenized filaments. This research was supported by the EU FP7 grant “Bio2MaN4MRI”.

Influence of the mamGFDC operon on magnetosomes functionalization efficiency in Magnetospirillum magneticum AMB-1

G.Adryanczyk-Perrier, S.Prévéral, C.Lefevre, N.Ginet and D.Pignol

CNRS/CEA/Aix Marseille University

Magnetospirillum magneticum AMB-1 is a magnetotactic bacterium that forms magnetosomes. These membrane-embedded nanocrystals of magnetite are of interest for Magnetic Resonance Imaging as efficient contrast agents. This functionalized contrast agent with the RGD peptide on its surface is able to target the integrin anb3 receptor which is overexpressed in human gliobastoma cells. (cf. poster of S. Prévéral). In our laboratory we functionalized magnetosomes with a fluorescent fusion protein Venus-RGD fused with MamC, a major protein of the magnetosome membrane. Thanks to bacterial growth studies, magnetotaxis measurements, quantification of Venus-RGD functionalization by immunochemical techniques, we show that paradoxically this functionalization is much more efficient in M. magneticum WT strain than in a M. magneticum strain devoid of the mamFDC cluster (coll. Arash Komeili).

Biomimetic and Biokleptic synthesis of magnetic nanoparticles and arrays inspired by magnetic bacteria Jennifer Bain and Sarah Staniland

University of Sheffield, UK We have been working with several magnetic bacterial proteins and utilizing them as an additive to chemical precipitations, to control the formation of magnetite nanoparticles in vitro. Here we will present the results of our most recet candidate native protein (MmsF) as well as several artificial proteins and scaffolds to control magnetite precipitation. We wil discuss how we are then utilizing these proteins in the creation of a synthetic magnetosome for biomedical application and biomineralised magnetic nanoparticle nanoarrays for data storage.


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Crystallization and preliminary X-ray diffraction analysis of the Magnetospirillum gryphiswaldense MSR-1 Magnetotaxis protein

MtxA

Geula Davidov1, Jens Baumgartner

2, Damien Faivre

2 and Raz Zarivach

1

1Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer Sheva, Israel 2Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Science Park Golm, 14424 Potsdam, Germany

Magnetotactic bacteria (MTB) are a very diverse group of marine bacteria that have the ability to navigate and align themselves toward the geomagnetic field lines. This special navigation is the result of a unique linear assembly of specialized organelle, the magnetosome. The magnetosome is defined by a biomineralized magnetic nanocrystal enveloped by a cytoplasmic membrane. By using the magnetosome chain, the MTB has an intricate phenotype of magnetotaxis, the ability to sense and coordinate movement to a microoxic zone on the bottom of chemically stratified natural water. The Magnetospirillum gryphiswaldense MSR-1 MGR0208 protein (MtxA) was identified in, or attached to, the magnetosome membrane and it has been suggested to play a role in the bacterial magnetotaxis due to its gene location in an operon encoding signal transduction genes. To shed light on MTBs magnetotaxis we initiated structural and functional studies of MtxA via X-ray crystallography. Here, we present the successful expression, purification, crystallization and preliminary X-ray analysis of MtxAΔ1-24, a truncated polypeptide lacking the signal peptide.

Annual variation of Multicellular magnetotactic prokaryotes and the relationship with biogeochemical parameters in Yuehu Lake Haijian Du

1,2, 3, Yiran Chen

1,3, Rui Zhang

1,3, Long-Fei Wu

3,4and Tian Xiao

1,3*

1Key Laboratory of Marine Ecology & Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. 2University of Chinese Academy of Sciences, Beijing,China. 3Laboratoire International Associé de la Bio-Minéralisation et Nano-Structures (LIA-BioMNSL), CNRS, F13402 Marseille cedex 20, France. 4Aix Marseille Université, CNRS, LCB UMR 7257, Institut de Microbiologie de la Méditerranée, 31, chemin Joseph Aiguier, F-13402, Marseille, France.

Two morphotypes (spherical and ellipsoidal) of multicellular magnetotactic prokaryotes (MMPs) have been observed in the sediments of a coastal lagoon, lake Yuehu, in the Yellow Sea. Here we report the annual variation of two types of MMPs and the relationship with biogeochemical parameters. Samples from two different sampling sites (A and B) were collected at about two-week intervals from September 2012 to December 2013. At both sampling sites, the annual variations were significant. We found that the abundances of the MMPs were high in summer and autumn, but low in winter and spring. The spherical MMPs were dominant with a mean abundance of 65% at site A. However, at site B, the dominant MMPs were the ellipsoidal with a mean abundance of 63%. A peak abundance (12.22 ind./cm

3) of the spherical MMPs occurred in September at site A, while peak in

abundance of the ellipsoidal MMPs (14.50 ind./cm3) occurred in October at site B. The change of amount of MTB showed the

similar trend with spherical MMPs. Investigation on various biogeochemical parameters indicated that salinity, nitrate, phosphate and silicate exhibited significant difference between the two sampling sites, which may cause different abundance of MMPs. Pearson correlation analyses showed that the abundance of the spherical MMPs was positively correlated with variation of total sulfur, phosphate and silicate, and negatively correlated with variation of salinity and sulphate. Nevertheless, the abundance of the ellipsoidal MMPs was positively correlated with variation of temperature and pH, and negatively correlated with variation of sulphate. Our findings provide new insights into the adaptive evolution of MMPs to the different marine intertidal sediment habitats.

A novel bacterial magnetic cobalophore for bioremediation applications

Jean-Baptiste ABBÉ, Pascal ARNOUX, Catherine BRUTESCO, Nicolas GINET, David PIGNOL & Monique SABATY CEA/CNRS/Cadarache (France)

Since their discovery, magnetotactic bacteria (MTB) have raised interest for biotechnological applications. Their magnetic abilities coupled with their capacity to deal with high concentrations of metals make them useful tools to operate in metal-polluted area. Our project consists in engineering new strains of magnetotactic bacteria to accumulate metal ions for decontamination of polluted effluents. In order to fulfil those objectives, we chose to heterogenous expression in bacteria of prokaryotes homologues of nicotianamine synthase (NAS), an enzyme that plays a key role in metal homeostasis in plants. Indeed, NAS is responsible for the synthesis of nicotianamine (NA), a well-known 303 Da molecule that can bind a large variety of metal ions with high affinity, for their transport in the different organs of the plant. Moreover, recent works have evidenced that different species of archea and bacteria possess NAS-like genes and are able to synthesize NA analogues. We first used E. coli strains to express NAS-like and associated proteins from Pseudomonas aeruginosa and Staphylococcus aureus in order to produce bacterial NA analogues. Transformant strains have shown enhanced resistance toward cobalt and nickel as well as a two-fold increase in cobalt accumulation. The genetic constructions have been transferred into the model magnetic bacterium Magnetospirillum gryphiswaldense MSR-1 and we are currently assessing both resistance and accumulation of cobalt in these strains. This poster will describe the phenotype of these strains expressing a NAS homologue.


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Transposon mutagenesis as an approach for global genetic analysis of magnetosome biomineralization in M. gryphiswaldense

Karen Tavares Silva, Dirk Schüler

University of Bayreuth, Germany.

Magnetosome biomineralization in magnetotactic bacteria requires a specific set of genes clustered within a genomic island, called magnetosome island (MAI). However, there is growing evidence that in addition to the MAI, genes located outside of this region contribute with accessory functions and might even have essential roles in magnetosome formation and magnetotaxis. So far, the role of non-MAI factors was mostly ignored and no comprehensive forward genetic analysis was performed to address their function in detail. Transposon mutagenesis is an approach for near-random mutation of bacterial genomes and an ideal forward genetic strategy to identify unknown genetic determinants of complex pathways. We developed a highly efficient system for random transposon mutagenesis in Magnetospirillum gryphiswaldense. We chose an engineered Tn5-based transposon vector, which are known to function in broad range of diverse bacteria. The initial frequency of Tn insertion upon conjugational transfer was only 7.9 x 10-6 even after optimization of mating conditions. This prompted us to adapt the system specifically for use in magnetotactic bacteria. First, the native transposase gene was replaced by a synthetic allele optimized for codon usage of M. gryphiswaldense. Additionally, the optimized transposase gene was set under the control of the strong native PmamDC45 promoter. This approach significantly and consistently increased the transposition efficiency to 4.4 x 10-4. Importantly, the random and unique nature of Tn5 insertions was confirmed by sequencing of the insertion location of random 70 mutants, which were distributed over the entire genome. Furthermore we sought to establish a protocol for screening and identification of clones which displayed different defects of magnetite biomineralization. While entirely non-magnetic clones could be easily recognized by their light colony color compared to the dark brown color of the WT, more subtle phenotypes were more difficult to discern. The growth conditions in solid plate were improved by increasing iron concentration, medium volume and the cultivation time. The visual screening of non/weakly magnetic colonies was validated using known mutants with variable impairments in magnetosome biomineralization. The screening could discriminate between mutants and wild-type with more than 90% of accuracy. In a first approach, the improved transposon system in conjunction with the optimized screening procedure were used to create and screen a library of 3000 Tn5 insertants: 8 non-magnetic and 22 weakly magnetic clones were retrieved and identified. As expected, all non magnetic mutants were mapped within the mamAB operon, known to be essential for magnetosome biomineralization. Transmission electron microscopy revealed that the weakly magnetic mutants displayed different magnetosome phenotypes including shorter chains with WT crystals, nearly regular chain with two distinct crystals morphologies, irregularly aligned aberrantly shaped magnetosomes and small crystals lacking any chain configuration. Insertion sites of these mutants are currently being identified and further studies are being conducted to elucidate the function of the affected genes in biomineralization. In conclusion, the results obtained up to date suggest that transposon mutagenesis is a reliable forward strategy for identification of genetic determinants of magnetosome biosynthesis and other metabolic pathways in M. gryphiswaldense.

North-seeking magnetotactic bacteria in the Southern Hemisphere

Lia C. R. S. Teixeira¹, Ana Carolina V. Araujo¹, Pedro E. Leão¹, Alex Enrich-Prast², Marcos Farina³, Ulysses Lins¹

¹Instituto de Microbiologia Paulo de Góes, ²Instituto de Biologia, ³Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

Magnetotactic bacteria (MTB) consist of a phylogenetically diverse group of prokaryotes capable of orienting along local magnetic fields lines and swimming propelled by flagella because of nano-sized, iron-rich, magnetic crystals enveloped by a lipid membrane, called magnetosomes. Under oxic conditions, in the Northern Hemisphere, MTB show north-seeking (NS) polarity, i.e., they swim parallel to a magnetic field. In this case, MTB would swim northward in the geomagnetic field. In the Southern Hemisphere, MTB swim antiparallel to the magnetic field and show south-seeking (SS) polarity. It is assumed that the inclination of the geomagnetic field lines guides MTB in both Hemispheres in the water column, helping them to swim downwards to locate and maintain an optimal position in vertical redox gradients. Here we report magnetotactic vibrios with NS polarity from a brackish lagoon in Brazilian Southwestern coast coexisting with magnetotactic cocci with SS polarity. Sediment was collected at Piripiri Lagoon located in Parque Nacional da Restinga de Jurubatiba (22o 12.461 S; 41o 28.352´N) and stored in two-liter bottles kept at room temperate for three weeks before checking for MTB. North-seeking and south-seeking MTB populations were monitored for four months. MTB populations were characterized with light and electron microscopy associated with molecular biology techniques. Oxygen concentration profiles along the sediment were measured using a microelectrode system. Population density varied along this period, but overall north-seeking bacteria was more abundant than south-seeking populations. Oxygen disappeared 0.5 cm above water-sediment interface. Both north-seeking and south-seeking MTB were located below the oxic-anoxic interface (OAI) suggesting anaerobic metabolism for both populations. Each MTB polarity was detected at a different sediment depth, indicating that chemical gradients other than oxygen could be modulating bacteria position and behavior. South-seeking MTB were more abundant at 2 cm below the water-sediment interface whereas north-seeking population were more abundant at 3.5 cm. High-resolution transmission electron microscopy and energy dispersive X-ray microanalysis indicated magnetosome crystalline habit and composition is consistent with elongated prismatic magnetite (Fe3O4) particles for north-seeking MTB. Average size [(length + width)/2] and shape factor for magnetosomes were respectively 117.52 ± 22.9 and 0.84 ± 0.07 for north-seeking vibrio and 111.81


33

± 23.40 and 0.80 ± 0.08 for south-seeking cocci. Phylogenetic analysis based on 16S rRNA gene sequencing is under development. Phylogenic identity will allow FISH probes to be developed for MTB identification. The coexistence of MTB with north and south polarity in the same environment has been reported in the Northern Hemisphere. The presence of north seeking MTB in the Southern Hemisphere corroborates to the contradiction of the currently accepted model of magnetotaxis, which states that MTB in the Southern Hemisphere swim south and downward to reach their suitable habitats when exposed to oxic conditions. We propose that magnetotaxis is not dependent only on oxygen and potential redox gradients, but other factors could modulate the bacteria behavior in the sediment column. Support: CNPq, CAPES, FAPERJ.

The effect of light wavelength and magnetic field on the velocity of the MMP Candidatus Magnetoglobus multicellularis

Lyvia Vidinho de Azevedo, Henrique Lins de Barros and Daniel Acosta-Avalos Centro Brasileiro de Pesquisas Fisicas – CBPF

It has been reported that multicellular magnetic prokaryotes (MMPs) are sensible to UV and blue light, showing a change in magnetotactic polarity response during illumination. Recently we reported the effect of monochromatic light on the velocity of the MMP Candidatus Magnetoglobus multicellularis, showing that green light decrease the velocity and red light increase it, when compared to blue light and white light. Now, we continue that study changing the light intensity and the magnetic field amplitude. For that, MMPs were observed in an inverted microscope. The white light source was replaced by led lamps with different wavelength emissions. A coil was adapted to a glass slide to generate magnetic fields up to 1.8 Oe. The light power was varied from 28 to 289 microW for every wavelength used ( 469 nm – blue, 517 nm – green and 628 nm – red). We try to obtain the Langevin curve to estimate the magnetic moment with our experimental data, but it was no possible to get trustable numerical results. The velocity showed the lower values for green light and the higher values for red light as expected. The dependence with the light intensity and magnetic field amplitude is very complex. Combined values of both parameters create regions with lower or higher velocity values for the same light wavelength. The ecological implications of these findings will be discussed. Finantial support: CNPq and FAPERJ.

Structure analysis of the magnetosome associated protein MamB

Noa Keren, Natalie Zeytuni, Geula Davidov and Raz Zarivach Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben Gurion University of the Negev, Beer Sheva, Israel

The magnetosome is a subcellular organelle in magnetotactic bacteria that consists of a linear-chain assembly of lipid vesicles. Each magnetosome is able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. MamB is abundant magnetosomal membrane protein that share similarity to cation diffusion facilitator (CDF) protein family. MamB is a multifunctional protein that was shown to be essential for iron transport and magnetosome membrane formation. Recent studies have demonstrated that deletion of MamB leads to the abolishment of the magnetosome. To understand the differences between MamB and other CDFs we started a structural work using X-ray crystallography. Here we present the crystallization and the structure of the MamB cytosolic domain from Marine magnetic spirillum QH-2 and Candidatus Desulfamplus magnetomortis BW-1 Greigite forming protein. Additionally, we were able to obtain a zinc bound state for QH-2 MamB that allowed us to locate the iron binding sites. Together these results shed light on the structure conservation of CDFs while pointing on unique divalent binding sites.

Regulation of Biomineralization in Magnetotactic Bacteria by the Serine Protease MamE

Patrick Browne, Arash Komeili

UC Berkeley, Department of Plant and Microbial Biology

In recent years, several genes crucial to the precise biomineralization of magnetosome crystals in magnetotactic bacteria have been determined. However, how the proteins coded by many of these genes function remains poorly understood. One of these key genes involved in biomineralization is the serine protease degP homolog mamE. While the protease function of MamE has been previously demonstrated to be necessary for complete biomineralization in Magnetospirillum magneticum AMB-1, actual targets of MamE proteolysis have not been identified. Using a candidate gene approach we will present evidence of processing of several potential targets of MamE. In some cases additional mutation analysis of these targets can directly link MamE-dependent proteolysis to the correct function of these proteins. Furthermore, using proteomic analysis of magnetosomes from wild type and protease inactive mamE cells, we can observe differences between proteomes of completely and incorrectly biomineralized magnetosomes. Together, these paired approaches have greatly revealed how the protease function of MamE leads to correctly mineralized crystals.


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Five Fur family proteins in Magnetospirillum gryphiswaldense MSR-1

Qing Wang, Meiwen Wang, Xu Wang, Wei Jiang, Jiesheng Tian, Ying Li*, Jilun Li

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China

Based on analysis of the complete genomic sequence of Magnetospirillum gryphiswaldense MSR-1, it was revealed that five genes (MGMSRv2_3137, MGMSRv2_1721, MGMSRv2_3149, MGMSRv2_3660, MGMSRv2_2136) encoded products belonging to the ferric uptake regulator (Fur) protein family. Amino acid sequence analysis showed that Fur3137 had a HHDH motif for binding metal which was conserved in real Fur proteins. Fur1721, Fur3660 and Fur3149 had three consecutive histidine residues (HHH motif) for binding ferrous haem, which was conserved in most iron response regulators (Irr). The amino acid sequence of Fur2136 had low homology compared with other four Furs in MSR-1 and it belonged to zinc uptake regulator (Zur). Flask culturing MSR-1 cells in the presence vs. absence of 20 μM ferric citrate (termed as high-iron and low-iron condition) and performing transcriptome analysis of two kinds of cells which synthesized magnetosomes or not, and abundant RNA-seq data was obtained. The results showed that there was obvious difference of furs expression between early stage (8 h) and mature stage (18 h) in the magnetosome formation. In the early stage, genes encoding Fur3137 and Fur2136 belonged to the differentially expressed genes (DEG), but fur3137 and fur2136 had up-regulated and down-regulated expression in high-iron condition respectively. Previous studies proved that Fur3137 regulated genes related iron and oxygen metabolism, therefore, up-regulated of fur3137 might be helpful for the function of Fur3137 in high-iron condition and it plays an important role on maintain the ferrous iron homeostasis in the early stage of magnetosome formation. In the mature stage, only fur3149 encoding Fur3149 belonged to DEG, and showed down-regulated expression in high-iron condition. Because of possessing the characteristic of Irr, Fur3149 may be stable in the low-iron condition. Down-regulated of fur3149 lead to haem proteins synthesis such as catalases and peroxidases which detoxify H2O2 and peroxides respectively. Genes encoding Fur3660 and Fur1721 both had indistinctively expressed difference under the condition with or without iron. However, Fur3660 performed the function for peroxide stress in the previous study and gene encoding Fur1721 showed up-regulated expression in the low-oxygen condition, therefore, these two proteins may be responsible for regulation of oxygen balance. Further, the expression levers of other furs were up-regulated in the different fur mutants using RT-qPCR. Consequently, Furs in MSR-1 had different effects but also the complementary function for controlling the balance of iron and oxygen metabolism. It made sense of coexisting Furs for MSR-1. We established the Furs and other regulatory factors (such as Crp, CytR, NarL, SigH and GerE) coupling regulatory network jointly and the foundation for revealing the iron and oxygen metabolism of MTB. Acknowledgments: This study was supported by the Chinese National Natural Science Foundation (Grant No. 31270093).

The isolation of multicellular magnetotactic prokaryotes under different magnetic field intensities

Roger Duarte de Melo, Henrique Lins de Barros, Daniel Acosta-Avalos Centro Brasileiro de Pesquisas Fisicas – CBPF

Several reports show that is common to find North seeking (NS) and South seeking (SS) magnetotactic organisms in the same sediments. The same had been observed when magnetotactic organisms are isolated using strong magnets. For example, in Rio de Janeiro, Brazil, the prevalence of magnetotactic microorganisms in SS (they swim toward the north pole of a magnet), but some few NS organisms sometimes appear in the same population. In this report we analyzed the proportion of NS and SS organisms as a function of the magnetic field from a strong magnet. The organism chosen for this study was the multicellular magnetotactic prokaryote Candidatus Magnetoglobus multicellularis (CMM) because it is bigger than a bacteria and it is easy to quantify the number of CMM present in a single drop when the isolation is not rich. The isolation was done using a traditional glass cuvette with a capillary end. Sediments were put inside the cuvette and a strong magnet was facing the capillary end. To change the magnetic field at the capillary end, the distance among the magnet and the capillary end was changed. Magnetic fields from 15 Oe to 480 Oe were obtained. Ten different isolations were done for every magnetic field used. A drop was obtained from the capillary end and it was observed in a digital optical microscope. The drop border was recorded and the film was analyzed frame to frame to quantify the number of NS and SS CMMs present in the drop. The proportion of NS and SS CMMS was calculated for every drop and the average proportion for each magnetic field intensity. It was observed that the proportion of NS CMM increased with the magnetic field intensity, being almost zero at 15 Oe. We hypothesize that the presence of the NS population is related to the sudden change from strong to weak magnetic field intensities. More studies are in progress to verify that hypothesis. This study was supported by CNPq.


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RGD-functionalized magnetosomes, a contrast agent with molecular affinity for diagnostic in Magnetic Resonance Imaging.

S. Prévéral2, G. Adryanczyk-Perrier

2, F. Geffroy

1, M. Pean

2, M. Boucher

1, C. T. Lefèvre

2, D. Garcia

2, S. Mériaux

1, D. Pignol

2 and N.

Ginet2

1UNIRS, CEA / DSV / I2BM / NeuroSpin, Gif-sur-Yvette, France 2CEA/CNRS/Aix-Marseille Université, UMR7265 Biologie Végétale et Microbiologie Environnementales, Laboratoire de Bioénergétique Cellulaire, Saint Paul lez Durance, France.

Magnetosomes are organite synthetized by magnetotactic bacteria (MTB) and constituted by a ferric magnetite core coated by a bilayer lipidic membrane. We recently characterized magnetosomes as efficient contrast agents for in vivo high field MR-based molecular imaging. We were able to acquire brain angiograms in the living mouse with very low dose of iron injected. Here, after genetic modification of Magnetospirillum magneticum AMB-1, we purified and characterized magnetosomes decorated with the RGD peptide in fusion with the fluorescent protein Venus and anchored to the membrane with the MamC protein. The RGD peptide (Arg-Gly-Asp) interacts specifically with the integrin anb3 receptors, which is overexpressed at the surface of cancer cells and is a specific marker of tumor vascularization. We present here the process of fabrication of this contrast agent (see also the poster of G. Adryanczyk). We obtained the biomass of modified MTB from a controlled culture in bioreactor and we purified the magnetosomes by magnetic separation. Then, we characterized the preparation with several biochemical parameters that attested of the validity of the contrast agent. Finally, fluorescence imaging with cancer cells line U87 showed a specific interaction with the RGD-functionalized magnetosomes and its internalization.These results open the door to the first IRM assays with functionalized contrast agent on a model of human glioblastoma implanted in living mouse.

Kinetics Characterization of the Cytosolic Domain of the Magnetosome Associated Protein MamM, WT and Homologous

Diseases-Related M

Shiran Barber-Zucker1, Sofiya Kolusheva

2, Geula Davidov

1 and Raz Zarivach

1

1Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer Sheva, Israel, 2Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer Sheva, Israel.

Magnetotactic bacteria (MTB) are group of gram negative microorganisms that have the unique ability of orienting themselves along geomagnetic fields. This ability is achieved by the Magnetosome, a sub-cellular organelle in MTB that is able to biomineralize a ~50 nm crystals of magnetite or gregite. The Magnetosome Associated Protein MamM involves in regulation of several important steps in the magnetosome formation. It was recently proposed that MamM acts as a magnetosome-directed iron transporter, as its deletion or single point mutations within the c-terminal domain (CTD) of the protein abolish magnetite biomineralization. Additionally, mutations within the conserved trans-membrane domain metal binding site and within the CTD cause alterations in magnetite crystals size and morphology. Recent structural and functional studies of this protein confirmed that MamM is a member of the Cation Diffusion Facilitator (CDF) protein family, a highly conserved protein family that maintains divalent cation homeostasis in all organisms. In these studies, MamM was also used as a homologous model for other CDF protein, SLC30A8 (ZnT-8), a human-CDF related Type-II Diabetes. It was shown that addition of divalent cation to MamM CTD solutions, wild type and homologous diseases-related mutations, cause conformational changes from a dimer-apo form to a closed state, suggesting that the binding of the cation is in the V-shape internal surface. In this work, we use MamM from the Magnetospirillum gryphiswaldense MSR-1 strain to examine MamM CTD WT and ZnT-8 homologous diseases-related mutations interactions with Iron(II) and Zinc(II), MamM’s and ZnT-8’s ligands, for their kinetic parameters. For that, we use the stopped-flow technique, which enables us to measure the chemical kinetics of the reaction between MamM and metal cations in short time scales (milliseconds). These results help to understand the divalent cation affinity of both MamM CTD WT and mutations and constitute another step in the understanding of the biomineralization mechanism in MTB and the ZnT-8 related diseases. References: [1] Zeytuni N, Uebe R, Maes M, Davidov G, Baram M, et al. (2014) Bacterial Magnetosome Biomineralization – A Novel Platform to Study Molecular Mechanisms of Human CDF-Related Type-II Diabetes. PloS ONE 9(5): e97154. Doi:10.1371/35rgani.pone.0097154 [2] Zeytuni N, Uebe R, Maes M, Davidov G, Baram M, et al. (2014) Cation Diffusion Facilitators Transport Initiation and Regulation Is Mediated by Cation Induced Conformational Changes of the Cytoplasmic Domain. PloS ONE 9(3): e92141.doi:10.1371/journal.pone.0092141 [3] Uebe R, Junge K, Henn V, Poxleitner G, Katzmann E, et al. (2011) The cation diffusion facilitator proteins MamB and MamM of Magnetospirillum gryphiswaldense have distinct and complex functions, and are involved in magnetite biomineralization and magnetosome membrane assembly. Mol Microbiol 82: 818–835


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Investigation of lipid compositions and interaction between magnetosomes and plasma membrane in Magnetospirillum

gryphiswaldense

Sofiya Kolusheva, Mathieu Bennet, Natalie Zeytuni, Oliver Raschdorf, Dirk Schüler, Damien Faivre, Raz Zarivach Ben Gurion University of the Negev, Beer-Sheva, Israel; Max Planck Institute of Colloids and Interfaces, Golm, Germany; Ludwig Maximillian University of Munich, Martinsried, Germany

The magnetosome, a biomineralizing organelle within magnetotactic bacteria, allows their navigation along geomagnetic fields. Magnetosomes are membrane-bound compartments containing magnetic nanoparticles and organized into a chain within the cell. Their assembly and biomineralization are controlled by magnetosome-associated proteins. Recent research has shown that magnetosomes are invaginations of the inner membrane and not free standing vesicles. All parameters (type of generation, composition, place and orientation in bacteria matrix) demonstrate the dependence of magnetosomes membrane on cell membrane. However there is currently no evidence of the existence or absence of links between the cell and the magnetosome membranes. These membranes may be connected by proteins belonging to the two membranes or joints lipids or they may form a completely non-contact system. To investigate these aspects of magnetosome – cell membrane interactions, we have developed two microscopic assays, based on fluorescence measurements of membrane lipids. Also were analyzed a lipid composition of plasma and magnetosomes membranes. Accordingly, on the results from all of the microscopic experiments and lipid compositions with high probability can indicate about independency of two membranes: plasma membrane and magnetosomes membrane. This indicates that once the magnetosome membrane is formed out of the inner membrane it become detached or separated by proteins that hinder lipid diffusion between the membranes.

Reduction of iron in the serum and liver of iron-overloaded mice using the magnetotactic bacterium Magnetospirillum

gryphiswaldense

Tahereh Setayesh , Seyed Fazlollah Mousavi , Seyed Davar Siadat, Bahar Shaghaghi

1. Microbiology Research Center & Department of Bacteriology, Pasteur Institute of Iran, Tehran, Iran, 2. Department of 36rganization36 toxicology, Institute of cancer research, medical

Magnetotactic bacteria (MTB), such as Magnetospirillum gryphiswaldense (MSR-1), are gram-negative and have a highly controlled biomineralization process to synthesize well ordered nano-sized particles with perfect magnetic and crystalline properties. Each crystal is surrounded by a membrane and this crystal-membrane unit is called a magnetosome. MSR-1 accumulates iron from the environment (up to 4% of the dry weight) and uses it to biomineralize up to 60 crystals of cub octahedral magnetite (Fe3O4) per cell. This amount indicates that MTB have very efficient biological systems for uptake, transport, and precipitation of iron. On the basis of Mössbauer spectroscopy and biochemical analyses, it was suggested that for bacterial magnetite formation, ferritin and Fe2+ are essential. The magnetite biomineralization pathway begins with iron uptake from the environment either as Fe2+ or Fe3+, followed by a biochemical pool of iron composed of ferritin and Fe2+ within the cell. Iron is a mineral element that is of great importance for microbial growth. In addition, iron is a biologically important trace element required for many metabolic pathways; an increase in the level of iron in the body will cause many iron-overloaded disorder such as, haemochromatosis, is a disease in which too much iron builds up in your body. Aim: The focus of this research was to determine whether MSR-1 bacteria have an effect on the concentration of iron levels in mammalian cells in vivo. Material and method: To examine this effect, two different conditions were examined, mice overloaded with iron and mice without iron overloading. For the iron overload condition, female BALB/c mice were given i.p. injections of iron-dextran for 4 consecutive weeks; after which the injections were ended and the iron in the mice was allowed to equilibrate for 15 days. The MRS-1 bacteria was injected into iron-overloaded mice. The animals were then sacrificed the spleen, liver and lymph nodes were removed. The viable bacterial number was determined in these organs by measuring the colony-forming units (CFUs) for at least 96 hours. Serum iron levels were tested using commercial kits and the total iron levels in the liver were measured by wet ashing and analyzed for total iron excreted using flame atomic absorption spectrophotometer for 10 days. Results and Conclusion: After i.p administration of iron dextran in mice, serum iron and total liver iron were increased. According to CFU measurements, after 96 hours mice can clear MSR-1 from its body. We have also shown that MSR-1 bacteria can effect on the blood iron level in iron- overloaded mice. The serum iron levels have been decreased compare with control level to 10 days (P< 0.05), the total liver iron levels in liver have been significant decreased compare with control level during the first 3 days (P< 0.05) and total iron excretion have been significant increased compare with control level to 8 days after MSR-1 i.v injection. Significance and Impact of the Study: This study provides the base on another study that it is offered new application for MSR-1 in iron-overloaded disease. MSR-1 cells have the ability to decrease iron values in iron-overloaded mice, and therefore inhibit the possible damage to different body organs caused by iron overloading. Our research on using optimizing the biological magnetic system is still continuing. Key words: Magnetospirillum gryphiswaldense; Iron Level Reduction; Animal Iron Overload


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Studying the formation of elongated magnetosome crystals

Teresa Perez Gonzalez and Damien Faivre Department of Biomaterials, Max-Planck-Institut of Colloids and Interfaces, Potsdam, Germany

Magnetic nanoparticles are used for different applications. A great variety of methods exist to produce them, and some are able to control the size of the resulting particles. However, controlling other factors that could help to tailor the magnetic properties of the particles, like morphology and elongation, remains a challenge. In order to design new synthesis methods we look for inspiration to magnetotactic bacteria that can produce different crystal morphologies. Magnetite belongs to the Fd3m crystallographic system so that anisotropic crystals are not expected. Some strains of MTB however are able to cross this rule and grow magnetosome longer in some axis. . Two main theories had been proposed: First an equal growth in the different directions of space and then, after it reached a critical size, the anisotropic grow was supposed to start in one direction (Isambert et al., 2007). Alternatively, an aggregation formation process from multiple nucleation sites was also speculated as another formation mechanism after the observation of crystals from AV-1, which magnetosomes are known to be formed by multiple magnetite crystal with different orientation (Lefevre et al., 2011). We have studied the formation of magnetosomes from two different species, Desulfovibrio magneticus (RS-1), which produces magnetite crystal elongated along the 100 axis with bullet shape morphology and Magnetococcus marinus (MC-1) that produces elongated hexaoctahedral crystals along the 111 axis. Both strains were grown without detectable Fe for many generations to obtain magnetosome free cells. Fe was then supplied to them and the formation of the magnetosome was observed with high resolution electron transmission microscopy (HRTEM) at several time points. We will present our main results in our contribution. Isambert et al., 2007. Am. Mineral. 92(4). Lefevre et al., 2014. Earth Planet. Sci. Let. 312,194-200.

Global regulator Crp control magnetosomes biosynthesis in Magnetospirillum gryphiswaldense MSR-1

Tong Wen, Fang Fang Guo, Yun Peng Zhang, Wei Jiang*, Jie Sheng Tian, Ying Li, JiLun Li

State Key Laboratory of Agricultural Biotechnology, China Agriculture University

Cyclic AMP receptor protein (CRP) is a kind of important transcriptional regulator widely distributed in many microorganisms. Though the bioprocess regulated by this regulator is highly diverse among different microorganisms, the Crp control energy metabolism in most species and proteins belonging to this family have a similar regulation mechanism: the protein first activated by cAMP, then binding to the promoter region of target gene to change its conformation and promote its transcription. Recent reports show that ATPase involved in ferrous ion uptake in magnetotactic bacteria and magnetosome membrane protein MamK also works as ATPase and GTPase, these results suggested that magnetosome formation is probability relate to energy metabolism. To confirm this speculation, crp gene (locus tag: MGR_1896) was disrupted in magnetotactic bacteria Magnetospirillum gryphiswaldense MSR-1, The phenotype of the mutant and the regulatory mechanism of Crp protein were then analyzed. To detect magnetosome synthesis in crp mutant, the strain were incubated in SLM (supplemented with 60 μM ferric citrate) medium until it reached the stationary phase. No ferromagnetism was detected by Cmag value detection and no magnetosome was found by transmission electron microscope (TEM) observation in the crp mutant. Intracellular iron content also decreased dramatically in crp mutant compared with wild type under the same condition. Expression profiling results between MSR-1 wild type and crp mutant was analyzed to reveal the regulation mechanism of Crp protein in MSR-1. The enrichment of differentially expressed genes by GO (gene ontology) functional enrichment and KEGG (37rgan encyclopedia of genes and genomes) pathway enrichment show that genes related to pentose and glucuronate interconversions, D-glutamine and D-glutamate metabolism, oxidative phosphorylation, peptidoglycan biosynthesis and ribosome were affected most after the deletion of crp. Interestingly, a lot of MAI (magnetosome island) genes were also down regulated after the deletion of crp. These findings confirm that the Crp protein in MSR-1 regulate energy metabolism like in other microbes and control the expression of MAI genes which have close relationship with magnetosome synthesis in Magnetospirillum gryphiswaldense MSR-

1.Key words: Magnetospirillum gryphiswaldense, Crp, magnetosome，regulatory factor, expression profile. References: 1. Bazylinski D. A., Frankel R. B. Magnetosome formation in prokaryotes. Nat. Rev. Microbiol. 2, 217-230 (2004). 2. Suzuki T., Okamura Y., Arakaki A., Takeyama H., Matsunaga T. Cytoplasmic ATPase involved in ferrous ion uptake from magnetotactic bacterium Magnetospirillum magneticum AMB-1. FEBS 37rg. 581, 3443-3448 (2007). 3. Sonkaria S., Fuentes G., Verma C., Narang R., Khare V., Fischer A., Faivre D. Insight into the assembly properties and functional 37rganization of the magnetotactic bacterial actin-like homolog, MamK. PloS one 7, e34189 (2012).

Acknowledgment：This work was supported by National Natural Science Foundation of China (Grant Nos. 31170089 and

31270093).


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Biogeography of magnetotactic bacteria and its implications for global iron cycle

Wei Lin, Yongxin Pan 1 Biogeomagnetism Group, Key Laboratory of Earth and Planetary Physics, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing 100029, China; 2 France-China Bio-Mineralization and Nano-Structures Laboratory, Chinese Academy of Sciences, Beijing 100029, China

A number of microorganisms biomineralize intracellular or extracellular iron minerals and play essential roles in biogeochemical cycling of iron. One of the most interesting examples of these types of organisms are the magnetotactic bacteria (MTB), a polyphyletic group of prokaryotes that uptake iron from environments and form intracellular nano-sized iron minerals of magnetite and/or greigite, known as magnetosomes. However, knowledge on their potential contributions to the iron cycling remains unknown because the diversity and biogeography of environmental MTB are still not fully understood. We have investigated the diversity and distribution of MTB communities from freshwater to saline habitats in China and US. By comparing our results with publicly available dataset, we revealed that the composition of MTB communities represents a biogeographic distribution across globally heterogeneous environments, and both environmental heterogeneity and geographic distance play significant roles in shaping their community composition. Building on the knowledge on MTB diversity and biogeography, we estimated that the annual yield of magnetite mineral by global MTB is no less than 10^8 kg. These results suggest that MTB communities play important roles in the present-day iron cycling and the deposition of iron formation through the geological history. Moreover, since magnetosomes are species-specific and could be preserved in sediments or rocks as magnetofossils, biogeography of MTB communities will open new avenue to paleoenvironmental studies. More research involving the ecology and biogeography of MTB will help us better understand the conditions under which and to what extent MTB affect the biogeochemical cycle of iron.

Characterization and genomic analysis of a newly isolated strain Magnetospirillum sp. XM-1

Yinzhao Wang, Wei Lin, Tongwei Zhang, Jinhua Li and Yongxin Pan

Biogeomagnetism Group, Key Laboratory of Earth and Planetary Physics, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing, China, and France-China Bio-Mineralization and Nano-Structures Laboratory, Chinese Academy of Sciences, Beijing, China

A new magnetotactic bacteria strain belonging to the Alphaproteobacteria was recently isolated and cultivated, named as Magnetospirillum sp. XM-1. Transmission electron microscope observation showed that it contains a single chain of approximately 10 magnetite magnetosomes on average which are morphologically similar to that of Magnetospirillum magneticum strain AMB-1. Rock magnetic measurements confirmed the single-domain magnetite magnetosomes. The coercivity and the ratio of saturation remanence and saturation magnetism of magnetosomes are ~30 mT and ~0.5, respectively. Low-temperature and ferromagnetic resonance also detected the chain arrangement signatures of magnetite for the whole-cell sample of magnetotactic bacteria. Response surface methodology combined with rock magnetic measurements were used to optimize the production of magnetite magnetosome synthesized by XM-1. The complete genome of XM-1 was sequenced. It is composed of 4,825,187-bp chromosome and one 167,290-bp plasmid. The GC content of chromosome and the plasmid are 65.67%. The genome of Magnetospirillum sp. XM-1 contains ~ 4,550 predicted coding sequences (CDSs), 53 tRNA genes, and 6 rRNA genes, which corresponds to 89.98% of the genome being coding sequences.

Using the high-speed atomic force microscope for imaging MamK cytoskeletal filaments

Zachery Oestreicher, Azuma Taoka, Syou Shimajiri, and Yoshihiro Fukumori

Kanazawa University

Magnetotactic bacteria (MTB) synthesize magnetosomes, which are organelles containing biomineralized crystals of magnetite or greigite. These organelles are organized within the cells using several different proteins, one of which is MamK, a bacterial actin-like protein, which is conserved in MTB. To investigate MamK polymerization, we first tested different parameters in which to assemble MamK filaments. We examined different concentrations of magnesium, potassium, and sodium to determine the optimal parameters to assemble MamK. We also examined which nucleotide was best for the polymerization of MamK. For this we tested the assembly kinetics of different nucleotides, ATP, GTP, UTP, CTP, and found that ATP lead to the most efficient polymerization. Finally, we determined that the ATP analogues, ATP-gamma-S and AMP-PNP prevented MamK polymerization. The goal of our present work is to image MamK dynamics at high resolution in real time. For this task we will use the high-speed atomic force microscope (HS-AFM). This technique is capable of imaging molecular dynamics of proteins at nanoscale spatial resolution and millisecond temporal resolution. The HS-AFM achieves high resolution images by using a cantilever with a sharp carbon tip to gently tap over the surface of molecules to produce a three dimensional image of molecules. The first part of our work is to determine the ideal biological and microscope parameters in order to observe MamK filaments using the HS-AFM. Once we establish the best imaging conditions, we can use the HS-AFM to investigate the dynamic properties of the cytoskeleton.


39

Participants list

NAME E-MAIL INSTITUTION

Agata Olszewska [email protected] MPIKG (Germany)

Alicia Muela [email protected] Universidad del Pais Vasco (Spain)

Ana Carolina Araujo [email protected] UFRJ (Brazil)

Arash Komeili [email protected] U. C. Berkeley (USA)

Atsushi Arakaki [email protected] Tokyo Univ. Agric. And Tech. (Japan)

Ayana Yamagishi [email protected] Tokyo Univ. Agric. And Tech. (Japan)

Azuma Taoka [email protected] Kanazawa Univ. (Japan)

Carlos Pinheiro [email protected] UFRRJ (Brazil)

Carly Grant [email protected] U.C. Berkeley (USA)

Carolina Neumann Keim [email protected] UFRJ (Brazil)

Caroline Monteil [email protected] Swansea Univ. (UK)

Christopher Lefevre [email protected] CEA/CNRS/Cadarache (France)

Clarissa Werneck Ribeiro [email protected] UFRJ (Brazil)

Damien Faivre [email protected] MPIKG (Germany)

Daniel Acosta-Avalos [email protected] CBPF (Brazil)

Daniel Garcia [email protected] CEA/CNRS/Cadarache (France)

David Hershey [email protected] U.C. Berkeley (USA)

David Pignol [email protected] CEA/CNRS/Cadarache (France)

Denis Grouzdev [email protected] LMSU (Russia)

Dennis Bazylinski [email protected] UNLV (USA)

Dirk Schuler [email protected] Univ. of Bayreuth (Germany)

Elias Cornejo [email protected] U.C. Berkeley (USA)

Fernanda Abreu [email protected] UFRJ (Brazil)

Geraldine Adryanczyk Perrier

[email protected] IBEB CEA/Cadarache (France)

Geula Davidov [email protected] BGU (Israel)

Henrique Lins de Barros [email protected] CBPF (Brazil)

Hila Nudelman [email protected] BGU (Israel)

Hongmiao Pan [email protected] IOCAS (China)

Jairo F. Savian [email protected] UFRGS (Brazil)

Jean Baptiste Abbe [email protected] CEA/CNRS/Cadarache (France)

Karen Tavares Silva [email protected]

U. Bayreuth (Germany)

Lia Teixeira [email protected] UFRJ (Brazil)

Lilah Rahn-Lee [email protected] U.C. Berkeley (USA)

Liliam C. Agudelo Morimitsu [email protected] U. Nacional de Colombia (Colombia)

Long-Fei Wu [email protected] LCB, CNRS-AMU (France)

Ludiane Silva Lima [email protected] UERJ (Brazil)

Luigi Jovane [email protected] USP (Brazil)

Lyvia Vidinho de Azevedo [email protected] CBPF (Brazil)

M. Luisa Fdez-Gubieda [email protected] U. del Pais Vasco (Spain)

Marcos Farina [email protected] UFRJ (Brazil)

Marina Dziuba [email protected] RAS (Russia)

Masayoshi Tanaka [email protected] Tokyo Inst. Technology (Japan)

Mathieu Bennet [email protected] MPIKG (Germany)

Matthieu Amor [email protected] IPGP (France)

Mauricio Toro-Nahuelpan [email protected] U. Bayreuth (Germany)

mailto:[email protected]













































40

Melissa L. E. Gutarra [email protected] UFRJ (Brazil)

Michelle Pang [email protected] U.C. Berkeley (USA)

Mihaly Posfai [email protected] U. Pannonia (Hungary)

Monique Sabaty [email protected] CEA / CNRS / Cadarache (France)

Nicolas Ginet [email protected] CNRS / CEA / Marseille (France)

Nicole Abreu [email protected] U.C. Berkeley (USA)

Noa Keren [email protected] BGU (Israel)

Oliver Raschdorf [email protected] LMU (Germany)

Patrick Browne [email protected] U.C. Berkeley (USA)

Pedro E. L. Leão [email protected] UFRJ (Brazil)

Raz Zarivach [email protected] BGU (Israel)

René Uebe [email protected] U. Bayreuth (Germany)

Richard B. Frankel [email protected] CalPoly St. U. (USA)

Roger Duarte de Melo [email protected] CBPF (Brazil)

Sandra Preveral [email protected] CNRS / CEA / AMU (France)

Jennifer Bain [email protected] U. Sheffield (UK)

Shiran Barber-Zucker [email protected] BGU (Israel)

Sidcley Silva de Lyra [email protected] UFRJ (Brazil)

Sofiya Kolusheva [email protected] BGU (Israel)

Stefan Klumpp [email protected] MPIKG (Germany)

Stephan Eder [email protected]

LMU (Germany)

Tahereh Setayesh [email protected] MUW (Austria)

Tania Prozorov [email protected] US DOE Ames Lab. (USA)

Teresa Perez-Gonzalez [email protected]

MPIKG (Germany)

Tian Xiao [email protected] IOCAS (China)

Tong Wen [email protected] China Agriculture Univ. (China)

Ulysses Lins [email protected] UFRJ (Brazil)

Xiaohui Zhu [email protected] McMaster U. (Canada)

Xu Wang [email protected] China Agriculture Univ. (China)

Yinzhao Wang [email protected] IGG CAS (China)

Yiran Chen [email protected] IOCAS (China)

Zach Oestreicher [email protected] Kanazawa Univ. (Japan)


































magnetotactic bacteria program and... · 2014. 9. 11. · 4th international meeting on...

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