Transcript delong_lecture_july24 - C-MORE

“Non-oxygenic microbial
photophysiologies in the ocean:
rhodopsin and bacteriochlorophyll
based systems”
Agouron Microbial Oceanography
Summer Course 2007
Photophysiology in the sea
Solar energy
Photosynthesis
Plants Algae,
photosynthetic bacteria
CO2
+
carbon
dioxide
H2O
water
Chemical
energy or heat
N,P,S,Fe….
Respiration
Animals
Bacteria
C6H12O6 + O2
organic
carbon
oxygen
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
O2
Winogradsky column
H 2S
OXYGENIC PAs
b
a
LOTS OF DIVERSITY IN BACTERIAL ANOXYGENIC PHOTOTROPHS !
General features of anaerobic photosynthetic bacteria
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
1% to 6 % of isolates from sand, seaweed, seawater, sediments Tokyo Bay
Erythrobacter longus
Erythrobacter sp. OCh114. (Roseobacter denitrificans)
Roseobacter litoralis
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 Bacteria
MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Sept. 1998, p. 695ﾐ724. Yurkov and Beatty
“Pump and probe”
or
Fast repetition rate fluorometry
IRFRR
Instrument
Vent
photosyn !!!
Bcll-containing bacteria may contribute 2- 5 % photosynthetic electron transport in the upper ocean
Kolber et al. 2000. Nature 407:178
“Photosynthetically competent anoxygenic phototrophic
bacteria comprise at least 11% ofthe total microbial community”
Kolber et al. Science 292:2492
P/I curves and CO2 fixation in NAP-1 isolate
(a little more controversial…)
Kolber et al. Science 292:2492
“Daily cellular rates of CO2 fixation about or 3% of the cellular carbon content...”
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 phylogeny
Béjà, Suzuki, et al. 2002.
Nature 415:630-633
BACTERIOCHLOROPHYLL BIOSYNTHETIC GENES in
BACTERIOPLANKTON from MONTEREY BAY
BEJA et al, 2002 NATURE
Matching Environmental DNA Sequence to Cultured Cell Proteomes:
A protein profile of the photosynthetic reaction center of HTCC2080
Courtesy Steve Giovannoni
Unpublished:
Jang Cho
Martha Degan
Doug Barofsky
Steve Giovannoni
HTC Lab (LIONS)/
EHSC Mass Spec Lab
Oregon State Univ.
Cho et al.
OM60 and Congregibacter littoralis
HALOARCHAEA
light
H+
Halobacterium salinarum
H+
(electron microscope image)9 0.5-1.2 um x
1.0-6.0 um in size10
Purple membrane = 2-D crystalline
bacteriorhodopsin lattice
ADP
ATP
Sensor rhodopsins
SR I and SR II
ATP-synthase
H+
flagellae
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (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 base
to 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
Genome sequence of Halobacterium species NRC-1
Wailap Victor N et al., PNAS | October 24, 2000 | vol. 97 | no. 22 | 12176-12181
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Microbial rhodopsins fall into two different functional classes
• Light-driven ion pumps
•
Sensory rhodopsins
LIBRARY
CONSTRUCTION
AND
SCREENING
SAR86 130 kbp BAC
1
1
“SAR86” 130kb GENOME FRAGMENT
LIGHT-DRIVEN PROTON PUMPING IN E. COLI
(via Т SAR86У PROTEORHODOPSIN )
Oded Beja
OFF
+
+
Expression of proteorhodopsin in E. coli
Retinal
ON
+
+
5 min
pH
0.02
Proteorhodopsin
ON
OFF
Béjà et al. Science 289: 1902-1906 (2000)
Fast photcycle kinetics
Phylogenetic distribution of proteorhodopsin variants
SAR86 SUBGROUPS from the COASTAL and OPEN OCEANS
Sabehi et al., Environ. Microbiol. 6:903(2004)
Monterey
Red Sea
Hawaii
Do different SAR86 phylotypes encode proteorhodopsins ?
Phylogentic relationships of naturally
occurring SAR86 ribotypes
env . clone MB11B0, AY 033326
env . clone MB11E0, AY 033304
env . clone MB12D0, AY 033314
env . clone MB11G0, AY 033311
89
BAC c lo ne eB ACRe d2 0E09
BAC c lo ne eB ACHOT4 E0 7
env . cloneOM10, U70693
env . clone OCS44, AF001650
env . clone KTc0917, AF173974
BAC clone EBAC 31A08 AF279106
env . clone ZD 0108, AJ400345
env . clone NAC11-19, AF245642
100
97
env . clone MB12G1, AY 033317
env . clone CHAB-III-1, AJ240912
env . clone MB12G0, AY 033328
env . cloneKTc1112, AF241654
82
env . cloneKTc1121, AF241653
env . cloneKTc1107, AF173975
100
env . clone AR CTIC97A-18, AF354613
env . cloneOCS5, AF001651
100
marine bact erium ZD 0107, AJ400344
BAC clone EBAC 27G05, AF268217
100
EB750-02H09, AY458632
marine bact erium ZD 0433, AJ400356
BAC Clone EB000-65A11
**
**
***
100
0.10
Sar86 - I
SSU rRNA
Sar86 - II
Sar86 - IIIa
Sar86 - IIIb
de la Torre et al. PNAS 2003
(Pelagibacter)
Venter et al., Environmental Genome Shotgun
Sequencing of the Sargasso Sea,
Science 394:66-74 (2004)
ARCHAEA
CRENARCHAEOTA
EURYARCHAEOTA
Methanobacteriales
Thermoplasmatales
Methanococcales
ТMarine Group IУ
Thermococcales
Archaeoglobales
ТMarine Group IIIУ
ТMarine Group IIУ
Methanopyrales
ТMarine Group IVУ
pSL12
Haloarchaea
Thermoproteales
Sulfolobales
pJP33
Methanomicrobiales
To Eucarya, Bacteria
Depth-specific differences in proteorhodopsin variants
Béjà et al. Nature 411:786-789 (2001)
Leu105 -> Gln105
Man et al. EMBO J. 2003
Man et al. EMBO J. 2003
Sabehi et al. ISME J. 2007
Glu96 (cytoplasmic H+ donor)
Asp85 (periplasmic H+ acceptor)
Leu105 -> Gln105
Sensory rhodopsins
lack the cytoplasmic
proton donor - 22 of
Sargasso Sea PR variants
have either Thr (18),
Ile (3), or Lys (1). Each appears
linked in an operon to a putative
sensory rhodopsin.
Sensory rhodopsins in bacteria
R
ST
R
ST
R
ST
R
ST
R, rhodopsin
ST, Signal transducer (histidine kinase domain)
Sharma,et al.
TRENDS in Microbiology Vol.14
p. 463, 2006
Jay McCarren
GENOMES
& BACS
PR-1 PR-2
MB_41B09
Betaproteobacterium
“Typical” carotenoid (retinal) biosynthesis genes
co-associated with PRs
blh
Exceptions:
GII Archaea, Pelagibacter, a few others, PR unlinked to retinal biosynthetic operon
CFB = PR-blh linkage (Pinhassi and colleagues)
Proteorhodopsin photosystem gene organization
PR
crtE
crtI
crtB
crtY
blh
Ipp
moaE
Jay McCarren/Chon Martinez
PR,carotenoid and retinal biosynthetic gene co-evolution ?
Jay
McCarren
crtB
crtI
crtB
*
*
*
*
Gram -
crtE
*
crtY
*
CFB
Gram -
Chon Martinez
X X
X
X
A single genetic event can confer phototrophy
~ 1e5 ATP/cell/min
Distribution of PR photosystems among marine bacteria
Life on Earth Today: The Foundation
Solar energy
Photosynthesis
Plants Algae,
photosynthetic bacteria
CO2
+
carbon
dioxide
H2O
water
Chemical
energy or heat
N,P,S,Fe….
Respiration
Animals
Bacteria
C6H12O6 + O2
organic
carbon
oxygen
Dave Karl, Nature, 2002