“Non-oxygenic microbial photophysiologies in the ocean:

rhodopsin and bacteriochlorophyll based systems”

Agouron Microbial Oceanography Summer Course 2007

Photophysiology in the seaPhotophysiology in the sea

COCO22 + H + H22OO

carbon watercarbon waterdioxidedioxide

CC66HH1212OO66 + O + O2 2

organic oxygenorganic oxygencarboncarbon

Plants Algae, Plants Algae, photosynthetic bacteriaphotosynthetic bacteria

PhotosynthesisPhotosynthesisSolar energySolar energy

Animals Animals BacteriaBacteria

Chemical Chemical energy or heatenergy or heat

RespirationRespiration

N,P,S,Fe….N,P,S,Fe….

Dave Karl, Nature, 2002

OTHER SORTS of PHOTOTROPHY

Type Electron donor C source

Photolithoautotroph H2O, H2S, S0, H2 CO2

Photolithoheterotroph H2O, H2S, S0, H2 Organic substrate

Photoorganoautotroph Organic substrate CO2

Photoorganoheterotroph Organic substrate Organic substrate

Photomixotroph Mixed inorganic/organic

Mixed inorganic/organic

http://helios.bto.ed.ac.uk/bto/microbes/winograd.htm http://ecosystems.mbl.edu/SES/MicrobialMethods/Winogradsky/default.htm

Winogradsky columnO2 H2S

OXYGENIC PAs

LOTS OF DIVERSITY IN BACTERIAL ANOXYGENIC PHOTOTROPHS !

Many grow photoorganotrophically in the absence of oxygen

When growing phototrophically, derive most of their ATP from light

Carbon sources used predominantly for reducing power, biosynthesis

Many are capable of photoautotrophic growth

General features of anaerobic photosynthetic bacteria

Erythrobacter longus Erythrobacter sp. OCh114. (Roseobacter denitrificans) Roseobacter litoralis

1% to 6 % of isolates from sand, seaweed, seawater, sediments Tokyo Bay

Shimada coined the term in 1995 :“Aerobic anoxygenic phototrophs ”

Not capable of anaerobic phototrophic growth; most strict aerobes

Wide variety and large amounts of carotenoids

Relatively low amounts of bacteriochlorophyll a

Appear not able use light as sole source of energy

Light-induced oxid./reduct. of photosynthetic apparatus demonstrated

Mostly organotrophic (carbon used for energy and as carbon source)

Aerobic Anoxygenic Phototrophic BacteriaMICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Sept. 1998, p. 695 ﾐ 724. Yurkov and Beatty

“Pump and probe”or

Fast repetition rate fluorometry

Bcll-containing bacteria may contribute 2- 5 % photosynthetic electron transport in the upper ocean

IRFRRInstrument

Vent photosyn !!!

Kolber et al. 2000. Nature 407:178

Kolber et al. Science 292:2492

“Photosynthetically competent anoxygenic phototrophicbacteria comprise at least 11% ofthe total microbial community”

P/I curves and CO2 fixation in NAP-1 isolate(a little more controversial…)

“Daily cellular rates of CO2 fixation about or 3% of the cellular carbon content...”

Kolber et al. Science 292:2492

Anapleurotic reactions !• TCA cycle intermediates are

used to provide carbon skeletons for other biomolecules. Cycle would halt if OAA is not replaced.

• Anapleurotic reactions produce TCA cycle intermediates from pyruvate or PEP.

Oceanic puf M/L phylogenyOceanic puf M/L phylogenyBéjà, Suzuki, et al. 2002.

Nature 415:630-633

BACTERIOCHLOROPHYLL BIOSYNTHETIC GENES in BACTERIOCHLOROPHYLL BIOSYNTHETIC GENES in BACTERIOPLANKTON from MONTEREY BAYBACTERIOPLANKTON from MONTEREY BAYBEJA et al, 2002 NATURE

Matching Environmental DNA Sequence to Cultured Cell Proteomes:

A protein profile of the photosynthetic reaction center of HTCC2080

Unpublished: Jang ChoMartha DeganDoug BarofskySteve GiovannoniHTC Lab (LIONS)/EHSC Mass Spec Lab

Oregon State Univ.

Courtesy Steve Giovannoni

Cho et al.

OM60 and Congregibacter littoralis

HALOARCHAEAHALOARCHAEA

Halobacterium salinarum (electron microscope image)9 0.5-1.2 um x 1.0-6.0 um in size10

light

H+

H+

ADP ATP

H+

Sensor rhodopsins SR I and SR II

Purple membrane = 2-D crystalline bacteriorhodopsin lattice

ATP-synthase

flagellae

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

The cycle can be formally described in terms of 6 steps :isomerization (I), ion transport (T), accessibility change (switch S). Retinal first photo-isomerizes from an all-trans to a 13-cis configuration followed by a proton transfer from the Schiff baseto the proton acceptor Asp-85. To allow vectoriality, reprotonation of the Schiff base from Asp-85 must be excluded. Thus, its accessibility is switched from extracellular to intracellular. The Schiff base is then reprotonated from Asp-96 in the cytoplasmic channel. After reprotonation of Asp-96 from the cytoplasmic surface, retinal reisomerizes thermally and the accessibility of the Schiff base switches back to extracellular to reestablish

the initial state.

http://www.biochem.mpg.de/oesterhelt/photobiology/br.html

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Genome sequence of Halobacterium species NRC-1Wailap Victor N et al., PNAS | October 24, 2000 | vol. 97 | no. 22 | 12176-12181

Microbial rhodopsins fall into two different functional classes• Light-driven ion pumps

• Sensory rhodopsins

LIBRARYLIBRARYCONSTRUCTION CONSTRUCTION

AND AND SCREENINGSCREENING

1

SAR86 130 kbp BAC

1

“SAR86” 130kb GENOME FRAGMENT

Fast photcycle kinetics

Expression of proteorhodopsin in E. coli

ON

OFF

5 min

OFF

ON

Retinal Proteorhodopsin

+

-

+

+

-+ -

-

pH

0.02

LIGHT-DRIVEN PROTON PUMPING IN E. COLI(via “ SAR86” PROTEORHODOPSIN)

Oded Beja

Béjà et al. Science 289: 1902-1906 (2000)

Phylogenetic distribution of proteorhodopsin variants

SAR86 SUBGROUPS from the COASTAL and OPEN OCEANS

SSU rRNA

Monterey

Red Sea

Hawaii

**

0.10

env. clone MB11B0, AY033326env. clone MB11E0, AY033304env. clone MB12D0, AY033314

env. clone MB11G0, AY033311

BAC clone eBACRed20E09BAC clone eBACHOT4E07env. clone OM10, U70693

env. clone OCS44, AF001650env. clone KTc0917, AF173974

BAC clone EBAC31A08 AF279106env. clone ZD0108, AJ400345

env. clone NAC11-19, AF245642env. clone MB12G1, AY033317

env. clone CHAB-III-1, AJ240912env. clone MB12G0, AY033328env. clone KTc1112, AF241654env. clone KTc1121, AF241653

env. clone KTc1107, AF173975env. clone ARCTIC97A-18, AF354613

env. clone OCS5, AF001651marine bacterium ZD0107, AJ400344BAC clone EBAC27G05, AF268217

EB750-02H09,AY458632marine bacterium ZD0433, AJ400356

BAC Clone EB000-65A11

Sar86 - II

Sar86 - I

Sar86 - IIIa

Sar86 - IIIb100

100

100

100

82

100

89

97

***

Phylogentic relationships of naturally occurring SAR86 ribotypes

Sabehi et al., Environ. Microbiol. 6:903(2004)

**

Do different SAR86 phylotypes encode proteorhodopsins ?

de la Torre et al. PNAS 2003

(Pelagibacter)

Venter et al., Environmental Genome ShotgunSequencing of the Sargasso Sea,

Science 394:66-74 (2004)

Haloarchaea

“Marine Group IV”

“Marine Group II”“Marine Group III”

ThermoplasmatalesMethanococcales

Thermococcales

Archaeoglobales

pSL12

“Marine Group I”

Thermoproteales

pJP33

To Eucarya, Bacteria

EURYARCHAEOTACRENARCHAEOTA

ARCHAEA

Methanobacteriales

Sulfolobales

Methanopyrales

Methanomicrobiales

Depth-specific differences in proteorhodopsin variants

Béjà et al. Nature 411:786-789 (2001)

Man et al. EMBO J. 2003

Leu105 -> Gln105

Man et al. EMBO J. 2003

Sabehi et al. ISME J. 2007

Leu105 -> Gln105

Glu96 (cytoplasmic H+ donor)Asp85 (periplasmic H+ acceptor)

Sensory rhodopsinslack the cytoplasmicproton donor - 22 ofSargasso Sea PR variantshave either Thr (18),Ile (3), or Lys (1). Each appears linked in an operon to a putative sensory rhodopsin.

Sensory rhodopsins in bacteria

R

R

R

R

ST

ST

ST

ST

R, rhodopsinST, Signal transducer (histidine kinase domain)

Sharma,et al.TRENDS in Microbiology Vol.14p. 463, 2006

Jay McCarren

MB_41B09 PR-1 PR-2

Betaproteobacterium

GENOMES

& BACS

“Typical” carotenoid (retinal) biosynthesis genesco-associated with PRs

blh

Exceptions:GII Archaea, Pelagibacter, a few others, PR unlinked to retinal biosynthetic operonCFB = PR-blh linkage (Pinhassi and colleagues)

PR crtE crtI crtB crtY blh moaEIpp

Jay McCarren/Chon Martinez

Proteorhodopsin photosystem gene organization

crtB

PR,carotenoid and retinal biosynthetic gene co-evolution ?

crtI crtB

Gram -

CFB

crtI

crtE crtY

Gram -

**

**

**

**

****

Jay McCarren

X

X

X

X

Chon Martinez

A single genetic event can confer phototrophy

~ 1e5 ATP/cell/min

Distribution of PR photosystems among marine bacteria

Life on Earth Today: The Life on Earth Today: The FoundationFoundation

COCO22 + H + H22OO

carbon watercarbon waterdioxidedioxide

CC66HH1212OO66 + O + O2 2

organic oxygenorganic oxygencarboncarbon

Plants Algae, Plants Algae, photosynthetic bacteriaphotosynthetic bacteria

PhotosynthesisPhotosynthesisSolar energySolar energy

Animals Animals BacteriaBacteria

Chemical Chemical energy or heatenergy or heat

RespirationRespiration

N,P,S,Fe….N,P,S,Fe….

Dave Karl, Nature, 2002