“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
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