ag cypionka / paleomicrobiology · them are members of the alphaproteobacterial phyla rhizobium and...

Ecophysiology of prokaryotes WS 2010/11

AG Cypionka / Paleomicrobiology 28. 02. 2011 – 25.03. 2011

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Seminar planMo 28.02. Tu 01.03. We 02.03. Th 03.03. Fr 04.03.Group Assignment Open questions Introduction A + B Introduction C + D Introduction E + FMo 07.03. Tu 08.03. We 09.03. Th 10.03. Fr 11.03.

Preliminary reports A - C Preliminary reports D - FMo 14.03. Tu 15.03. We 16.03. Th 17.03. Fr 18.03.Literature seminar 1 + 2 Literature seminar 3 + 4 Microbiol. Colloquium Literature seminar 5 + 6 Literature seminar 7 + 8 Mo 21.03. Tu 22.03. We 23.03. Th 24.03. Fr 25.03.Literature seminar 9 + 10 Literature seminar 11 + 12 Final reports D - F Final reports A - C

Projekt A (Judith Lucas) Diversity, abundance and isolation of Roseobacter -affiliated bacteria from marine sedimentsProjekt B (Monika Sahlberg): Abundance of Rhizobium radiobacter and Roseobacter sp. within sediments of the Benguela upwelling systemProjekt C (Maya Soora): Influence of light on the survival of Dinoroseobacter shibae during starvationProjekt D (Tim Engelhardt): Characterization of bacteriophages in sulfate-reducing bacteria (SRB) from deep-subsurface environmentsProjekt E (Bert Engelen): Microbial abundance and diversity within a subterranean gas-storage facilityProjekt F (Heribert Cypionka): Cultivation of sulfate-reducing bacteria from a subterranean gas-storage facility Seminar list

Project A 1. Wietz M, Gram L, Jorgensen B, Schramm A (2010) Latitudinal patterns in the abundance

of major marine bacterioplankton groups. Aquat Microb Ecol (61) 179-189 2. Giebel HA, Kalhoefer D, Lemke A, Thole S, Gahl-Janssen R, Simon M, Brinkhoff T

(2010) Distribution of Roseobacter RCA and SAR11 lineages in the North Sea and characteristics of an abundant RCA isolate. The ISME Journal 1-12

Project B 3. Schäfer H, Ferdelman TG, Fossing H, Muyzer G (2007) Microbial diversity in deep

sediments of the Benguela Upwelling System. Aquatic Microbial Ecology 50: 1-9. 4. Mollenhauer G, Schneider RR, Müller PJ, Spieß V, Wefer G (2002) Glacial/Interglacial

variability in the Benguela upwelling system: Spatial distribution and budgets of organic carbon accumulation. Global Biogeochemical Cycles 16: 81-1 - 81-15.

Project C 5. Holert, J., Hahnke, S. and Cypionka, H. (2011) Influence of light and anoxia on

chemiosmotic energy conservation in Dinoroseobacter shibae. Environmental Microbiology Reports, 3: 136–141. doi: 10.1111/j.1758-2229.2010.00199.x

6. Gómez-Consarnau L, Akram N, Lindell K, Pedersen A, Neutze R, et al. (2010)

Proteorhodopsin Phototrophy Promotes Survival of Marine Bacteria during Starvation. PLoS Biol 8(4): e1000358. doi:10.1371/journal.pbio.1000358

Project D 7. Williamson S J, Cary S C, Williamson K E, Helton R R, Bench S R, Winget D, Wommack

K E (2008) Lysogenic virus–host interactions predominate at deep-sea diffuse-flow hydrothermal vents. ISME J 2:1112-1121.

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8. Eydal H SC, Jägevall S,Hermansson M, Pederson K (2009) Bacteriophage lytic to Desulvovibrio aespoeensis isolated from deep groundwater. ISME J 3:1139-1147.

Project E 9. Orphan, V.J., et al. (2000) Culture-dependent and culture-independent characterization of

microbial assemblages associated with high-temperature petroleum reservoirs. Applied and Environmental Microbiology 66(2): p. 700-711.

10. Mochimaru H, Yoshioka H, Tamaki H, Nakamura K, Kaneko N, Sakata S, Imachi H,

Sekiguchi Y, Uchiyama H, Kamagata Y (2007) Microbial diversity and methanogenic potential in a high temperature natural gas field in Japan. Extremophiles 11:453–461

Project F 11. Sass H, Cypionka H (2004) Isolation of sulfate-reducing bacteria from the terrestrial deep

subsurface and description of Desulfovibrio cavernae sp. nov. System Appl Microbiol 27:541-548

12. Rozanova, E.P., et al. (2001) Desulfacinum subterraneum sp nov., a new thermophilic

sulfate- reducing bacterium isolated from a high-temperature oil field. Microbiology, 70(4): p. 466-471.

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Project A (Judith Lucas) Diversity, abundance and isolation of Roseobacter-affiliated bacteria from marine sediments Background The Roseobacter clade is a major lineage of the family Rhodobacteraceae of theAlphaproteobacteria. Its members form one of the most abundant and successful groups in the marine environment. No other phylogenetically coherent clade occurs in such a variety of marine habitats and ecosystems of different geographic regions and has such a diverse physiology. Members of the Roseobacter clade have been detected in the water column, in biofilms, oxic and anoxic sediments and in association with other organisms (Buchan et al 2005, Wagner-Döbler & Biebl 2006). Although this phylogenetic lineage exhibits a broad metabolic versatility, members of the Roseobacter clade are generally considered to be obligate aerobes. Nevertheless, there are some exceptions with a facultatively anaerobe metabolism (e.g. Roseobacter denitrificans, Roseovarius crassostreae, Dinoroseobacter shibae) and others are even capable of performing aerobic anoxgenic photosynthesis (AAP).

Sediments have been identified as the third-most important habitat for the Roseobacter-clade (Buchan et al., 2005). Molecular surveys showed that this group contributes with an average of 3% to the microbial communities thriving in marine surface sediments (e.g. Li et al. 1999, Madrid et al. 2001, Mills et al. 2003), but they were also detected in deep anoxic layers. However, the abundance, distribution and metabolic potential of sediment-dwelling Roseobacter have not been studied systematically so far.

To elucidate their role in marine sediments in comparison to other surfaces, algal and sediment samples were taken from Helgoland island and analyzed on different phylogenetic levels. DGGE was performed using primers specific for Bacteria, Rhodobacteraceae and Phaeobacter sp.. All phylogenetic groups were analyzed by quantitative PCR. To confirm the results, numbers of Bacteria and Rhodobacteraceae were determined by CARD-FISH.

During another extended cultivation study (Köpke et al., 2005), five facultatively anaerob Roseobacter-affiliated strains were isolated from tidal-flat sediments. One strain NB43 seems to form a new genus. Some physiological analyses like determination of the optimal growth temperature and ph still have to be done. Aim of the project: While investigated numbers of Roseobacter-affiliated bacteria within the Helgoland samples are unlikely high (CARD-FISH: ~ 20% qPCR: 40-60%), Phaeobacter sp. could hardly be found by applying specific PCR. Questions and tasks which will be addressed are: • Does the DNA-concentration influence the quantification via qPCR? • And if so, is there a correlation between DNA-concentration and cell-quantification? • Verification of qPCR results via CARD-FISH • Detection of Phaeobacter sp. with new specific primers. • Determination of temperature- and pH-Optimum of the tidal-flat isolate NB43.

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Methods/ working program DNA extraction and quantification DNA will be extracted from Helgoland sediments by using the MolBio-Kit for soil and quantified afterwards via PicoGreen on a microtiter plate reader. General primers for the bacterial 16S rRNA gene will be used to make sure that the extracted DNA is amplifiable. PCR A PCR using primers PHA-16S-129f and PHA-ITS-111r specific for Phaeobacter will be applied to detect Phaeobacter sp. in the Helgoland samples. Real-time PCR The extracted and quantified DNA serves as a template to determine the abundance of Rhodobacteraceae. and Phaeobacter sp. by qPCR. The template DNA will be diluted (1:100, 1:200, 1:300, 1:400) to analyze the influence of different template concentrations on the cell quantification. CARD-FISH To verify and prove the qPCRs results, CARD-FISH will be performed using a Rhodonacteraceae specific probe (HRP-Ros536). Determination of growth parameters In order to determine optimal growth temperature and pH for NB43 growth curves should be recorded. Growth of NB43 will be monitored through cell counting after staining with SybrGreen I as direct detection and quantification method. In addition the turbidity will be measured photometrically (OD436). Time schedule: First week: Monday: General introduction Tuesday: DNA-extraction/Quantification of DNA-amount Wednesday: PCR/Preparation of qPCR-standard Thursday: Preparation dilution series and qPCR Roseobacter Friday: evaluation qPCR/2nd qPCR Roseobacter Second week: CARD-FISH, SybrGreenI-cell counts, Phaeobacter PCR Third week: Preparation of medium/ Growth curves temperature-optimum Fourth week: Preparation of buffers and media/Growth curves pH-optimum Literature: Buchan A, González JM, Moran MA (2005) Overview of the marine Roseobacter lineage. Appl Environ

Microbiol 71: 5665-5677 Köpke B, Wilms R, Engelen B, Cypionka H, Sass H (2005) Microbial diversity in coastal subsurface sediments -

a cultivation approach using various electron acceptors and substrate gradients. Appl Environ Microbiol 71:7819-7830

Li L, Guenzennec J, Nichols P, Henry P, Yanagibayashi M, and Kato C (1999) Microbial diversity in Nankai Trough sediments at a depth of 3,843 m. J. Oceanogr. 55:635-642.

Madrid VM, Aller JY, Aller RC, and Chistoserdov AY (2001) High prokaryote diversity and analysis of community structure in mobile mud deposits off French Guiana: identification of two new bacterial candidate divisions. FEMS Microbiol. Ecol. 37:197-209

Mills HJ, Hodges C, Wilson K, MacDonald IR, and Sobecky PA (2003) Microbial diversity in sediments associated with surface-breaching gas hydrate mounds in the Gulf of Mexico. FEMS Microbiol. Ecol. 46:39-52.

Wagner-Döbler I, Biebl H (2006) Environmental biology of the marine Roseobacter lineage. Ann Rev Microbiol 60: 255-280

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Project B (Monika Sahlberg) Abundance of Rhizobium radiobacter and Roseobacter sp. within sediments of the Benguela upwelling system Background The marine deep biosphere is known to be the largest biotope on Earth. Estimates of prokaryotic cells indicate that it harbours 55-85% of the Earth´s prokaryotic biomass and 30% of the total living biomass (Jørgensen and D´Hondt, 2006). One goal of the Integrated Ocean Drilling Program (IODP) is to investigate these microorganisms and to understand the nature of the complex microbial ecosystem that inhabits the Earth´s subseafloor. Within the frame of IODP several studies have shown that there are certain phyla which seem to dominate the subseafloor communities. However, so far it is largely unknown how indigenous microorganisms migrate into the subsurface and establish as populations. Therfore, we have chosen four model organisms to trace their abundance along different sediment cores. Two of them are members of the alphaproteobacterial phyla Rhizobium and Roseobacter.

1) Rhizobium radiobacter: This species represents the most often cultured deep-biosphere bacterium, worldwide (Süß et al. 2006). Members of this species are facultative anaerobes which have been detected in shallow and deep sediments. Further, this group shows a relative increase with depth. Therefore, it is hypothesized that this group is able to adapt and to grow in the deep subsurface.

2) Roseobacter: The Roseobacter clade is distributed worldwide, contributing

significantly to the communities in the water column (Selje et al. 2004). Some of them have also been isolated from the upper layers of sediment. Furthermore, this species was found to have certain capabilities of anaerobic growth. It is suggested that this group enters the sediments only by the sedimentation process. They are buried, may survive for some time, but will not thrive in deeper and older layers.

The Benguela upwelling area – origin of the samples Generally, upwelling areas are characterized by a high productivity and the Benguela upwelling area constitutes one of the four major coastal upwelling systems in the world. Primary production is highly stimulated due to the circulation-driven upwelling of nutrient-rich waters from the deep sea. The extremely high contents of organic carbon of up to 20% dry weight (Mollenhauer et al. 2002) provoke elevated microbial activities and metabolic rates (Glud et al. 1994; Niewöhner et al. 1998; Emeis et al. 2004).

A steep slope of the continental margin with decreasing sedimentation rates at increasing water depths allows studying a variety of sedimentary systems within a relatively narrow area. By the use of molecular techniques we want to get a hint to the question, to which extend the model organisms respond to increasing water depths and varying content of organic matter. Highest organic matter contents have been observed in shelf sediments, indicating that the maximum of primary production takes place in waters overlying the continental shelf. A second maximum in organic carbon content is located at the upper slope, where a high input of organic matter occurs due to an elevated lateral transport (Mollenhauer et al. 2002).

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Dep

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mbs

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104 105 106 107 108 109 1010

128 02-1 -3 790 m128 03-3 -1 1940 m

128 06-2 -8 130 m128 07-1 -2 300 m

128 08-1 -2 3800 m

MUC GC water depth

Dep

th[c

mbs

f]

Total cell counts [cm-3]

0

100

200

300

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104 105 106 107 108 109 1010

128 02-1 -3 790 m128 03-3 -1 1940 m

128 06-2 -8 130 m128 07-1 -2 300 m

128 08-1 -2 3800 m

MUC GC water depth

128 02-1 -3 790 m128 03-3 -1 1940 m

128 06-2 -8 130 m128 07-1 -2 300 m

128 08-1 -2 3800 m

MUC GC water depth

Figure 2: Total cell numbers counted onboard the RV Meteor. The site-specific decrease with sediment depth is a response to geochemical settings within the sediments of the transect.

Figure 1: Sampling station located along a transect perpendicular to the coast of Namibia within the Benguela upwelling system. S: sampling station.

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128 07-MUC-1 25°20.65´S 13°46.47É 299 S, 30128 07-GC-2 25°20.65´S 13°46.47É 299 150, 250, 350

128 02-MUC-1 25°30.00´S 13°27.01É 792 S, 30128 02-GC-3 25°30.00´S 13°27.00É 787 150, 340, 430

128 03-MUC-3 25°45.60´S 13°04.20É 1938 S, 20128 03-GC-1 25°45.06´S 13°04.20É 1942 150, 390, 450

128 08-MUC-1 26°22.13´S 11°53.46É 3794 S, 20128 08-GC-2 26°22.13´S 11°53.46É 3794 150, 390, 450

Water Sedimentdepth [m] depth [cmbsf]

Site/device Latitude Longitude

128 06-MUC-2 25°00.00´S 14°23.36É 133 S, 40128 06-GC-8 25°00.00´S 14°23.36É 133 150, 245

Water was sampled at all sites at 5m and directly above the seafloorS = sediment surface (0-5 cm)

128 07-MUC-1 25°20.65´S 13°46.47É 299 S, 30128 07-GC-2 25°20.65´S 13°46.47É 299 150, 250, 350128 07-MUC-1 25°20.65´S 13°46.47É 299 S, 30128 07-GC-2 25°20.65´S 13°46.47É 299 150, 250, 350




Water Sedimentdepth [m] depth [cmbsf]

Site/device Latitude Longitude

128 06-MUC-2 25°00.00´S 14°23.36É 133 S, 40128 06-GC-8 25°00.00´S 14°23.36É 133 150, 245

Water was sampled at all sites at 5m and directly above the seafloorS = sediment surface (0-5 cm)

Aim of this project In this project we will determine the abundance of R. radiobacter and Roseobacter sp. within sediments from different sampling sites of the Benguela upwelling area in order to investigate their distribution from shallow to deep sediments. Samples were taken along a transect off the coast of Namibia during Meteor-expedition M76/1 in 2008 (Figure 1). Since total cell numbers were determined onboard the RV Meteor (Figure 2), we will analyse the sediment samples for comparison by the application of quantitative (real-time) PCR as a molecular biological tool. Methods

• DNA extraction from sediments • DNA quantification with PicoGreen • PCR in order to create a standard for the quantitative PCR • Quantitative (real-time) PCR • DGGE

Time schedule: First week: Monday: General introduction Tuesday: DNA-extraction Wednesday: DNA-extraction/Quantification of DNA-amount Thursday: PCR (Bacteria) Friday: PCR/Preparation of qPCR-standard Second week: qPCR Bacteria, Preparation of qPCR-standards for Roseobacter and R.

radiobacter Third week: qPCR Roseobacter, R. radiobacter and Alphaproteobacteria Fourth week: DGGE Alphaproteobacteria

Table 1: Origin of sediment samples from the Namibian coast sampled during RV Meteor cruise M76/1.

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Literature Emeis KC, Brüchert V, Currie B, Endler R, Ferdelman T, Kiessling A, Leipe T, Noli-Peard

K, Struck U, and Vogt T (2004): Shallow gas in shelf sediments of the Namibian coastal upwelling ecosystem. Cont. Shelf Res., 24: 627-642.

Glud RN, Gundersen JK, Jørgensen BB, Revsbech NP and Schulz HD (1994): Diffusive and total oxygen uptake of deep-sea sediments in the eastern South Atlantic Ocean: in situ and laboratory measurements. Deep-Sea Res. I, 41: 1767-1788.

Jørgensen BB, D´Hondt S (2006): A starving majority deep beneath the seafloor. Science 314: 932-934.

Mollenhauer G, Schneider RR, Muller PJ, Spiess V, and Wefer G (2002): Glacial/interglacial variability in the Benguela upwelling system: Spatial distribution and budgets of organic carbon accumulation. Global Biogeochemical Cycles 16.

Niewöhner C, Hensen C, Kasten S, Zabel M, and Schulz HD (1998): Deep sulfate reduction completely mediated by anaerobic methane oxidation in sediments of the upwelling area off Namibia. Geochimica et Cosmochimica Acta 62: 455-464.

Selje N, Simon M, Brinkhoff T (2004): A newly discovered Roseobacter cluster in temperate and polar oceans. Nature 427: 445-448.

Süß J, Schubert K, Sass H, Cypionka H, Overmann J, Engelen B (2006) Widespread distribution and high abundance of Rhizobium radiobacter within Mediterranean subsurface sediments. Environ Microbiol 8:1753-1763

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Project C (Maya Soora) Influence of light on the survival of Dinoroseobacter shibae during starvation Background Dinoroseobacter shibae was isolated from a culture of the marine dinoflagellate Prorocentrum lima (Biebl et al., 2005). The strains belong to the aerobic anoxygenic phototrophic bacteria (AAPB), which were discovered in 1979 by Shiba et al. and have been studied with high efforts since then. AAPB are related to the facultative anaerobic purple-non-sulfur bacteria, but developed some major differences during their evolution: AAPB are able to perform light-driven energy conservation only under oxic conditions and without the production of elemental oxygen (anoxygenic). Like the purple-non-sulfur bacteria, AAPB contain bacteriochlorophyll a (BChl a) as light-absorbing pigment, but in significantly lower cellular content, while an unusually high amount of carotenoids can be found, which mainly determine the color of the cells and play an important role in preventing photo-oxidative damage to the cells. Pigment production in AAPB is only active under oxic conditions and, even more important, influenced strongly by light: Even low light intensities inhibit the production of pigments completely (Yurkov and Beatty, 1998). AAPs are capable of using light as a source of energy under oxic conditions without the generation of oxygen. Light was shown to induce ATP formation and proton translocation by the cells (Holert et al., 2010). However, the cells do not grow by light energy alone. Instead there is only a certain level of light-dependent increase in the amount of biomass, protein and pigment concentrations (Biebl et al., 2006).

In this project, the major focus would be under which conditions does light have the maximum competitive advantages for the bacteria. Accordingly, we test that the role of light energy for the cellular metabolism is proportional to the degree of starvation. Batch cultures of D.shibae will be maintained for several days under starvation in a) the dark, b) under continuous illumination and c) under dark-light cycles. The physiological fitness will be recorded by analysing respiration, chemiosmotic proton translocation and the adenylate energy charge. Aim of the project By analyzing the physiological response to light we could answer:

Whether the cells stay viable and how fast various components of the biomass are degraded?

How the cells survive and keep the capability of rapid recovery during prolonged periods of starvation in the dark or under light-dark cycles?

What activities does the cells show when substrate or light are supplied again?

Is light used for the build-up of reserve material or is it used for biosynthesis of protein and cell division?

Methods • Preparation of sea water medium

The essential part of the project is the cultivation of D. shibae in chemically defined medium which is similar to sea water and finally added with succinate as substrate.

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• Analysis of biomass parameters The physiological experiments are based on the analyses of several biomass-related parameters that will be used by standard methods. These are substrate consumption, cell number, dry mass, contents of protein, bacteriochlorphyll a.

• Determination of metabolic activities The metabolic activities to be measured include substrate-stimulated and endogenous respiration in the light and in the dark, and electron-transport driven proton translocation under the same conditions.

• Assessment of the energetic state The energy charge will be measured by the luciferin-luciferase test [ATP bioluminescence Assay Kit CLS II (Boehringer Mannheim)] and a luminometer. As the adenylates showed intracellular concentration changes of more than 10 mM per min, it is important to stop the metabolism instantaneously. This is achieved by adding ice-cold perchloric acid to the assays. The principle behind is that the luciferin reacts with oxygen to emit light. The luciferase acts as a catalyst to speed up the reaction, which is mediated by ATP. The so generated light is determined using luminometer. A recorder at the outlet of the luminometer collects measured values in mV.

Luciferin + ATP + O2 ------------------> Oxyluciferin + AMP + PPi + CO2 + Light

Time schedule First week: First and second day: General introduction and handling of instruments;

preparation of media and buffers. 3rd day: Inoculation and incubate under different conditions

Second week: Analysis of biomass and energetic parameters Third week: Analysis of biomass and energetic parameters Fourth week: Analysis of biomass and energetic parameters and present the results Literature Biebl, H., Allgaier, M., Tindall, B. J., Koblizek, M., Lunsdorf, H., Pukall, R. and Wagner-Döbler, I. (2005).

Dinoroseobacter shibae gen. nov., sp. nov., a new aerobic phototrophic bacterium isolated from dinoflagellates. International Jornal of Systematic and Evolutionary Microbiology 55 (Pt 3): 1089-96.

Biebl, H. and Wagner-Döbler, I. (2006). Growth and bacteriochlorophyll a formation in taxonomically diverse aerobic anoxygenic phototrophic bacteria in chemostat culture: Influence of light regimen and starvation. Process Biochemistry 41 (10): 2153-2159.

Holert, J., Hahnke, S. and Cypionka, H. (2011). Influence of light and anoxia on chemiosmotic energy conservation in Dinoroseobacter shibae. Environmental Microbiology Reports, 3: 136–141. doi: 10.1111/j.1758-2229.2010.00199.x

Shiba, T., Simidu, U. and Taga, N. (1979). Distribution of aerobic bacteria which contain bacteriochlorophyll a. Applied Environmental Microbiology 38 (1): 43-45.

Yurkov, V. V. and Beatty, J. T. (1998). Aerobic anoxygenic phototrophic bacteria. Microbiology and Molecular Biology Review 62 (3): 695-724.

Luciferase

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Project D (Tim Engelhardt) Characterization of bacteriophages in sulfate-reducing bacteria (SRB) from deep-subsurface environments Background Viruses are the most abundant biological entities on earth and are known to greatly affect microbial ecology in all environments. Bacterioplankton is approximately on the order of 106 to 107 cells per milliliter of seawater and their number increases up to 109 cells per gram in nutrient-rich sediments layers. The viral population in respective habitats is typically one order of magnitude higher1. Viruses have an influence on bacterial and archaeal mortality and subsequently shaping the microbial communities. According to the “killing the winner”-theory, viruses diversify indigenous microorganisms by providing niches for slow growing or less adapted organisms. The lysis of host organisms due to viral infections and the release of cell compounds affect biogeochemical cycles and may provide nutrients for other inhabitants2,3,4 (viral shunt). In addition, viruses contribute to genetic adaptation of microorganisms to the given environmental conditions via horizontal gene transfer and may increase the survivability of lysogenized host cells5.

Deep subsurface environment are typically characterized by anoxic conditions and nutrient limitation. Indigenous microorganisms are often prone to starvation6. The viral impact on these communities is less understood, as viral activity is basically linked to microbial activity. Considering the viral reproduction cycles, the lysogeny appears to be a better strategy and is favored under oligotrophic and anoxic conditions. Furthermore, viruses become an more important mortality factor in this environments due to the absence of grazers7,8

. In the frame of the practical course we will examine the characteristics of lysogenic

and lytic phage-host interactions with a selection of subsurface SRB isolates. Desulfovibrio aespoeensis strain P20, D. indonesiensis strain P23 and Desulfotignum balticum strain P18 were isolated from the sediment column of IODP Site U13019. The sampling site is located at the eastern flank of the Juan de Fuca Ridge, Northeast Pacific, which exhibits a deep sulfate-containing zone due to crustal-fluid diffusion from below. The type strain of Desulfovibrio aespoeensis was isolated from deep granitic groundwater sampled at depth of 600m10. This organism was found to be sensitive to lytic infection of the indigenous podovirus HEy211. In addition, prophage-like sequences was found in its genome. This organism will provide a reference for the analysis of lysogenic phage-host interactions and will be subject for a comparison of Desulfovibrio-affiliated SRBs from different subsurface environments. Aim of the study During the practical course we will perform induction experiments under anoxic conditions with Desulfovibrio aespoeensisT and strain P20 to test, if the isolates harbor inducible prophages. This approach will give informations about the latent period and the burst size of the respective phage. Examination of the morphology and the genome sizes of the obtained phages will further contribute to the characteristics of the phage-host system.

In a second experiment we will test if the different strains of Desulfovibrio spp. and Desulfotignum balticum are sensitive to lytic infection. Phage extract from corresponding sediment depths of the isolates will be prepared. Plaque assays will be done under anoxic conditions to detect and isolate possible lytic phages. In addition, morphology and genome sizes will be analysed.

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Work schedule

Time schedule First week: Preparation of materials, Phage extraction, Plaque assays Second week: Induction experiment + VLP-/cell counts, Phage purification + preparation of

phage concentrate Third week: Puls-field gel electrophoresis (PFGE) Fourth week: Transmission electron microscopy (TEM) Literature 1. Wommack KE, Colwell RR (2000) Virioplankton: Viruses in Aquatic Ecosystems. Microbiology and

Molecular Biology Reviews 64: 69–114. 2. Fuhrman J A (1999) Marine viruses and their biogeochemical and ecological effects. Nature 399: 541. 3. Suttle C A (2005) Viruses in the sea. Nature 437: 356. 4. Wilhelm S W, Suttle C A (1999) Viruses and Nutrient Cycles in the Sea. Bioscience 49: 781. 5. Weinbauer MG, Rassoulzadegan F (2004) Are viruses driving microbial diversification and diversity?

Environmental Microbiology 6: 1–11. 6. Jorgensen B B, D'Hondt S (2006) A starving majority deep beneath the seafloor. Science 314: 932. 7. Chibani-Chennoufi S, Bruttin A, Dillmann M L, Brüssow H (2004) Phage-host interaction: an ecological

perspective. Journal of Bacteriology 186: 3677. 8. Weinbauer M G, Höfle M G (1998) Significance of viral lysis and flagellate grazing as factors controlling

bacterioplankton production in a eutrophic lake. Applied and Environmental Microbiology 64: 431-438. 9. Fichtel K, Mathes F, Könneke M, Cypionka H, Engelen B (unpublished) Sulfate-reducing bacteria thrive in

deep sediment layers that are affected by crustal fluid diffusion. 10. Motamedi M, Pederson K (1998) Desulfovibrio aespoeensis sp. Nov.,a mesophilic sulfate-reducing

bacterium from deep groundwater at Äspö hard rock laboratory, Sweden. Int J Sys Bacteriol 48:311-315. 11. Eydal H SC, Jägevall S,Hermansson M, Pederson K (2009) Bacteriophage lytic to Desulvovibrio aespoeensis

isolated from deep groundwater. ISME J 3:1139-1147-

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Project E (Bert Engelen) Microbial abundance and diversity within a subterranean gas-storage facility Background Subterranean gas and oilfields are hotspots of the terrestrial deep-biosphere. Especially the disturbance of the system by human activities can lead to a stimmulation of indigenous microorganisms. Contamination by the exploration process can additionally introduce surface bacteria that form growing populations within the subsurface.

As these reservoirs represent anoxic environments, fermenting bacteria are growing on the degradation of hydrocarbons. Their fermentation products are further metabolized by other anaerobic microorganisms. Therefore, the reservoirs are characterized by complex microbial communities building up an anaerobic foodweb. One important group of bacteria that causes immense technical and financial problems for the oil and gas industry are sulfate reducers. On the one hand, the microbially mediated formation of H2S can result in a bad quality of the gas itself. On the other hand, the chemical reaction with iron or other heavy metals might clogg the pore space within the reservoir or industrial gas filters. The described issues are not only problematic for natural gas-fields but also for subterranean gas-storage facilities. In this case, gas from other areas (e.g. Sibiria) is pumped via pipelines to exploited gas-fields where it is stored until further usage. One week before the start of the course, we had the opportunity to sample one of these storage facilities, located in southern Germany. The company has asked for a detailed microbial community analysis to understand which processes are going on in their gas-field. The trigger to commence this investigation was a gas filter that showed ironsulfide precipitates. We have a strong indication that microbial sulfate reduction is the reason for this. The samples will be analysed during the practical course in projects E and F. While project F focuses on the cultivation of sulfate-reducing bacteria, in this project, we will quantify Archaea, Bacteria and sulfate reducers by qPCR and will determine their diversity by denaturing gradient gel electrophoresis (DGGE). Aim of this study Analyzing the above-described material, we want to answer the questions: • How many Archaea and Bacteria are present within the subterranean gas-storage facility? • How abundant are sulfate-reducing bacteria? • What is the diversity of the different phylogenetic and metabolic groups? Methods / Working program DNA extraction and quantification and control of purity DNA will be extracted from different material (water and pump-pit samples) by using the FastDNA®Spin® Kit for soil and quantified afterwards via PicoGreen on a microtiter plate reader. General primers for the bacterial 16S rRNA gene will be used to make sure that the extracted DNA is amplifiable. Quantitative PCR The extracted and quantified DNA serves as a template to determine the abundance of Bacteria, Archaea and sulfate reducers (dsrA-gene) by qPCR. The results will be compared to direct counts performed in project F.

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Molecular screening by DGGE The DNA will be amplified by specific PCR for the above described phylogenetic and metabolic groups by using primers that are feasible for DGGE analysis. DGGE will be performed to determine their diversity. Several bands will be extracted, reamplified and send for commercial sequence analysis. Time schedule: First week: Monday: general introduction Tuesday: DNA extraction Wednesday: Further DNA extraction and quantification Thursday + Friday: 16S rRNA gene amplification Second week: qPCR Third week: specific DGGE-PCR and DGGE Fourth week: DGGE and reamplification of bands, sequence analysis (if possible) Literature: Mochimaru H, Yoshioka H, Tamaki H, Nakamura K, Kaneko N, Sakata S, Imachi H, Sekiguchi Y, Uchiyama H,

Kamagata Y (2007) Microbial diversity and methanogenic potential in a high temperature natural gas field in Japan. Extremophiles (2007) 11:453–461 DOI 10.1007/s00792-006-0056-8

Orphan, V.J., et al., (2000) Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs. Applied and Environmental Microbiology. 66(2): p. 700-711.

Rozanova, E.P., et al., (2001) Desulfacinum subterraneum sp nov., a new thermophilic sulfate- reducing bacterium isolated from a high-temperature oil field. Microbiology 70(4): p. 466-471.

Sass H, Cypionka H (2004) Isolation of sulfate-reducing bacteria from the terrestrial deep subsurface and description of Desulfovibrio cavernae sp. nov. System Appl Microbiol 27:541-548

Wilms, R., Sass H, Köpke B, Cypionka H, Engelen B (2007) Methane and sulfate profiles within the subsurface of a tidal flat are reflected by the distribution of sulfate-reducing bacteria and methanogenic archaea. FEMS Microbiol. Ecol. 59:611-621.

Wilms, R., Sass H, Köpke B, Köster J, Cypionka H, Engelen B (2006) Specific bacterial, archaeal, and eukaryotic communities in tidal-flat sediments along a vertical profile of several meters. Appl. Environ. Microbiol. 72:2756-2764.

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Project F (Heribert Cypionka) Cultivation of sulfate-reducing bacteria from a subterranean gas-storage facility Background As described for project E, subterranean gas-fields are hotspots of the terrestrial deep-biosphere, characterized by complex anaerobic foodwebs. While project E focuses on the molecular microbial community analysis of a subterranean gas-storage facility, project F will use the same material to cultivate sulfate-reducing bacteria (SRB). This group of bacteria causes immense technical and financial problems for the gas industry. On the one hand, the microbially mediated formation of H2S can result in a bad quality of the gas itself. On the other hand, the chemical reaction with iron or other heavy metals might clogg the pore space within the reservoir or industrial gas filters. The latter was the trigger to commence this investigation. The samples that were taken one week before the start of the course will be used to stimmulate growth of sulfate-reducing bacteria. Aim of this study Analyzing the above-described material, we want to answer the questions: • What is the total cell number within samples of the subterranean gas-storage facility? • Which sulfate-reducing bacteria can be stimmulated to grow and what is their proportion? • Can we simulate the precipitation of iron-sulfur complexes by specific cultivation

techniques? Methods / Working program Total cell counting Total cell numbers will be determined by flurescence microscopy using SybrGreen as nucleic acid dye (Lunau et al. 2005). Cell counting will be performed by the application of the program CountThem. Most probable number technique The fraction of SRB that can be stimmulated to grow will be determined by anoxic incubations in serial dillutions. Incubation experiments The in-situ sulfur precipitation will be simulated in H2S-oxidant gradient cultures. Fe(III)-salts and O2 will be used as oxidants in agar and the gaseous phase, respectively. H2S will be produced by pure cultures of SRB or enrichments from the gas-storage facility. Time schedule: First week: Monday: General introduction and desing of the experiments Tuesday: Preparation of culture media Wednesday: Inoculation of MPN-cultures Thursday + Friday: Set up of precipitation expetiment Second week: Total cell counting, monitoring of growth Third week: Monitoring of growth, (additional precipitation set up) Fourth week: Analysis of MPN-cultures

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Literature: Lunau M, Lemke A, Walther K, Martens-Habbena W, Simon M (2005) An improved method for counting

bacteria from sediments and turbid environments by epifluorescence microscopy. Environ Microbiol 7:961-968

Mochimaru H, Yoshioka H, Tamaki H, Nakamura K, Kaneko N, Sakata S, Imachi H, Sekiguchi Y, Uchiyama H, Kamagata Y (2007) Microbial diversity and methanogenic potential in a high temperature natural gas field in Japan. Extremophiles (2007) 11:453–461 DOI 10.1007/s00792-006-0056-8

Orphan, V.J., et al., (2000) Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs. Applied and Environmental Microbiology. 66(2): p. 700-711.

Rozanova, E.P., et al., (2001) Desulfacinum subterraneum sp nov., a new thermophilic sulfate- reducing bacterium isolated from a high-temperature oil field. Microbiology 70(4): p. 466-471.

Sass H, Cypionka H (2004) Isolation of sulfate-reducing bacteria from the terrestrial deep subsurface and description of Desulfovibrio cavernae sp. nov. System Appl Microbiol 27:541-548

ag cypionka / paleomicrobiology · them are members of the alphaproteobacterial phyla rhizobium and...

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