This document summarizes a study that used PCR and cloning to analyze the 16S rRNA genes present in a natural marine bacterioplankton population from the Sargasso Sea. Researchers constructed a library of 51 small-subunit rRNA genes and sequenced five unique genes. In addition to genes from known marine Synechococcus and SAR11 lineages, they identified two new classes of genes belonging to alpha- and gamma-proteobacteria, confirming that many planktonic bacteria have not been previously recognized by microbiologists.

1. EVE 161:
Microbial Phylogenomics
Era IV:
Genomes from the Environment
!1

2. Metagenomics History
• Term first use in 1998 paper by Handelsman et al.
Chem Biol. 1998 Oct;5(10):R245-9. Molecular
biological access to the chemistry of unknown soil
microbes: a new frontier for natural products
• Collective genomes of microbes in the soil termed
the “soil metagenome”
• Good review is Metagenomics: Application of
Genomics to Uncultured Microorganisms by
Handelsman Microbiol Mol Biol Rev. 2004
December; 68(4): 669–685.

3. Papers for Today
DOI: 10.1126/science.289.5486.1902
, 1902 (2000);289Science
et al.Oded Béjà,
Phototrophy in the Sea
Bacterial Rhodopsin: Evidence for a New Type of
This copy is for your personal, non-commercial use only.
.clicking herecolleagues, clients, or customers by
, you can order high-quality copies forIf you wish to distribute this article to others
.herefollowing the guidelines
can be obtaPermission to republish or repurpose articles or portions of articles
(this information is current as of May 18, 2010 ):
The following resources related to this article are available online at www.scien
http://www.sciencemag.org/cgi/content/full/289/5486/1902
version of this article at:
including high-resolution figures, can be found inUpdated information and services,
found at:
crelated to this articleA list of selected additional articles on the Science W eb sites
17 GPa. J. Geophys. Res. 102, 12252±12263 (1997).
28. Duffy, T. S. & Vaughan, M. T. Elasticity of enstatite and its relationships to crystal structure. J. Geophys.
Res. 93, 383±391 (1988).
Acknowledgements
We thank C. Karger for participation in the measurements, and M. Ferrero and F. Boudier
for providing the samples from Baldissero and Papua New Guinea, respectively. The
Laboratoire de Tectonophysique's EBSD system was funded by the CNRS/INSU,
Universite of Montpellier II, and NSF project ``Anatomy of an archean craton''. This work
was supported by the CNRS/INSU programme ``Action TheÂmatique Innovante''.
Correspondence should be addressed to A.T.
(e-mail: deia@dstu.univ-montp2.fr).
.................................................................
Proteorhodopsin phototrophy
in the ocean
Oded BeÂjaÁ*², Elena N. Spudich²³, John L. Spudich³, Marion Leclerc*
& Edward F. DeLong*
* Monterey Bay Aquarium Research Institute, Moss Landing, California 95039,
USA
³ Department of Microbiology and Molecular Genetics, The University of
Texas Medical School, Houston, Texas 77030, USA
²
characteristic of
cycle seen with E
proteorhodopsin
Furthermore,
spectrum of the
and same isosbes
dopsin expressed
chemical reactio
generate a red-s
to that of recomb
¯ash-photolysis
existence of pro
retinal molecules
waters.
The photocyc
and, after hydro
adding all-trans
indeed derive fro
–1
0
1
2
orbance(×10–3)
4
http://www.sciencemag.org/content/289/5486/1902.full
http://www.nature.com/nature/journal/v411/n6839/abs/411786a0.html

4. Papers for Today
DOI: 10.1126/science.289.5486.1902
, 1902 (2000);289Science
et al.Oded Béjà,
Phototrophy in the Sea
Bacterial Rhodopsin: Evidence for a New Type of
This copy is for your personal, non-commercial use only.
.clicking herecolleagues, clients, or customers by
, you can order high-quality copies forIf you wish to distribute this article to others
.herefollowing the guidelines
can be obtaPermission to republish or repurpose articles or portions of articles
(this information is current as of May 18, 2010 ):
The following resources related to this article are available online at www.scien
http://www.sciencemag.org/cgi/content/full/289/5486/1902
version of this article at:
including high-resolution figures, can be found inUpdated information and services,
found at:
crelated to this articleA list of selected additional articles on the Science W eb sites
http://www.sciencemag.org/content/289/5486/1902.full

5. Extremely halophilic archaea contain retinal-binding integral membrane pro-
teins called bacteriorhodopsins that function as light-driven proton pumps. So
far, bacteriorhodopsins capable of generating a chemiosmotic membrane
potential in response to light have been demonstrated only in halophilic archaea.
We describe here a type of rhodopsin derived from bacteria that was discovered
through genomic analyses of naturally occuring marine bacterioplankton. The
bacterial rhodopsin was encoded in the genome of an uncultivated -
proteobacterium and shared highest amino acid sequence similarity with
archaeal rhodopsins. The protein was functionally expressed in Escherichia coli
and bound retinal to form an active, light-driven proton pump. The new
rhodopsin ex- hibited a photochemical reaction cycle with intermediates and
kinetics characteristic of archaeal proton-pumping rhodopsins. Our results
demonstrate that archaeal-like rhodopsins are broadly distributed among
different taxa, including members of the domain Bacteria. Our data also
indicate that a previously unsuspected mode of bacterially mediated light-driven
energy generation may commonly occur in oceanic surface waters worldwide.

6. SAR86
Available ge-
cient to iden-
f the proteo-
nments from
al rhodopsins
gly different.
igin, whereas
me halophiles
s greater than
o determine
s retinal, we
herichia coli
n in the pres-
g the protein
on (Fig. 3A).
membranes of
opsin apopro-
520 nm was
ubation (Fig.
e peak at 520
nm half-band-
was generated
proteorhodop-
e presence of
bandwidth is
n absorption
ins. The red-
370 nm in the
onated Schiff
umably to the
Htr (22, 23)]. Although sequence analysis of
proteorhodopsin shows moderate statistical
support for a specific relationship with sen-
cle. The transport rhodopsins (bacteriorho-
dopsins and halorhodopsins) are character-
ized by cyclic photochemical reaction se-
onMay18,2010www.sciencemag.orgDownloadedfrom
!6
Fig. 1. (A) Phylogenetic tree of bacterial 16S rRNA gene sequences,
including that encoded on the 130-kb bacterioplankton BAC clone
(EBAC31A08) (16).

7. The 16S ribosomal RNA neighbor-joining tree was
constructed on the basis of 1256 homologous positions
by the “neighbor” program of the PHYLIP package [J.
Felsenstein, Methods Enzymol. 266, 418 (1996)]. DNA
distances were calculated with the Kimura’s 2-
parameter method.

8. p-
of
is
on
d-
he
ff
he
e-
ll
s-
r-
in
).
as
10
yl
i-
n-
e-
i-
et
o-
of
g-
es
ng
o-
be
Fig. 1. (A) Phylogenetic tree of bacterial 16S rRNA gene sequences, including that encoded on the
130-kb bacterioplankton BAC clone (EBAC31A08) (16). (B) Phylogenetic analysis of proteorhodop-
sin with archaeal (BR, HR, and SR prefixes) and Neurospora crassa (NOP1 prefix) rhodopsins (16).
Nomenclature: Name_Species.abbreviation_Genbank.gi (HR, halorhodopsin; SR, sensory rhodopsin;
BR, bacteriorhodopsin). Halsod, Halorubrum sodomense; Halhal, Halobacterium salinarum (halo-
bium); Halval, Haloarcula vallismortis; Natpha, Natronomonas pharaonis; Halsp, Halobacterium sp;
Neucra, Neurospora crassa.
Fig. 1. (B) Phylogenetic analysis of proteorhodop- sin with archaeal (BR, HR, and SR prefixes) and
Neurospora crassa (NOP1 prefix) rhodopsins (16). Nomenclature: Name_Species.abbreviation_Genbank.gi
(HR, halorhodopsin; SR, sensory rhodopsin; BR, bacteriorhodopsin). Halsod, Halorubrum sodomense;
Halhal, Halobacterium salinarum (halo- bium); Halval, Haloarcula vallismortis; Natpha, Natronomonas
pharaonis; Halsp, Halobacterium sp; Neucra, Neurospora crassa.

9. The proteorhodopsin tree was constructed on the basis of the archaeal
rhodopsin alignment used by Mukohata et al. [Y. Mukohata, K. Ihara, T.
Tamura, Y. Sugiyama, J. Bio- chem. ( Tokyo) 125, 649 (1999)] and the
neighbor- joining method as implemented in the neighbor pro- gram of
the PHYLIP package. The numbers at forks indicate the percent of
bootstrap replications (out of 1000) in which the given branching was
observed. The least-square method (the Kitsch program of PHYLIP) and
the maximum likelihood method (the Puzzle program) produced trees
with essentially identical topologies, whereas the protein parsimony
method (the Protpars program of PHYLIP) placed proteorhodopsin
within the sensory rhodopsin cluster. The Yro/Hsp30 subfamilies
sequences (6) were omitted from the phylogenetic analysis to avoid the
effect of long branch attraction.

10. Cloning of proteorhodopsin. Sequence analysis of a
130-kb genomic fragment that encoded the ribosomal
RNA (rRNA) operon from an uncultivated member of
the marine g-Proteobacteria (that is, the “SAR86”
group) (8, 9) (Fig. 1A) also revealed an open reading
frame (ORF) encoding a putative rhodopsin (referred
to here as proteorhodopsin) (10).

11. Cloning of proteorhodopsin. Sequence analysis of a
130-kb genomic fragment that encoded the ribosomal
RNA (rRNA) operon from an uncultivated member of
the marine g-Proteobacteria (that is, the “SAR86”
group) (8, 9) (Fig. 1A) also revealed an open reading
frame (ORF) encoding a putative rhodopsin (referred
to here as proteorhodopsin) (10).

14. Marine Microbe Background
• rRNA PCR studies of marine microbes have been extensive
• Comparative analysis had revealed many lineages, some very
novel, some less so, that were dominant in many, if not all,
open ocean samples
• Lineages given names based on specific clones: e.g., SAR11,
SAR86, etc
!14

15. http://www.nature.com/nature/journal/v345/n6270/abs/345060a0.html

16. © 1990 Nature

18. Vol. 57, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, p. 1707-1713
0099-2240/91/061707-07$02.00/0
Copyright C) 1991, American Society for Microbiology
Phylogenetic Analysis of a Natural Marine Bacterioplankton
Population by rRNA Gene Cloning and Sequencing
THERESA B. BRITSCHGI AND STEPHEN J. GIOVANNONI*
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331
Received 5 February 1991/Accepted 25 March 1991
The identification of the prokaryotic species which constitute marine bacterioplankton communities has been
a long-standing problem in marine microbiology. To address this question, we used the polymerase chain
reaction to construct and analyze a library of 51 small-subunit (16S) rRNA genes cloned from Sargasso Sea
bacterioplankton genomic DNA. Oligonucleotides complementary to conserved regions in the 16S rDNAs of
eubacteria were used to direct the synthesis of polymerase chain reaction products, which were then cloned by
blunt-end ligation into the phagemid vector pBluescript. Restriction fragment length polymorphisms and
hybridizations to oligonucleotide probes for the SARll and marine Synechococcus phylogenetic groups
indicated the presence of at least seven classes of genes. The sequences of five unique rDNAs were determined
completely. In addition to 16S rRNA genes from the marine Synechococcus cluster and the previously identified
but uncultivated microbial group, the SARll cluster [S. J. Giovannoni, T. B. Britschgi, C. L. Moyer, and
K. G. Field, Nature (London) 345:60-63], two new gene classes were observed. Phylogenetic comparisons
indicated that these belonged to unknown species of a- and -y-proteobacteria. The data confirm the earlier
conclusion that a majority of planktonic bacteria are new species previously unrecognized by bacteriologists.
Within the past decade, it has become evident that bacte-
rioplankton contribute significantly to biomass and bio-
geochemical activity in planktonic systems (4, 36). Until
recently, progress in identifying the microbial species which
constitute these communities was slow, because the major-
ity of the organisms present (as measured by direct counts)
cannot be recovered in cultures (5, 14, 16). The discovery a
decade ago that unicellular cyanobacteria are widely distrib-
uted in the open ocean (34) and the more recent observation
of abundant marine prochlorophytes (3) have underscored
the uncertainty of our knowledge about bacterioplankton
community structure.
Molecular approaches are now providing genetic markers
for the dominant bacterial species in natural microbial pop-
ulations (6, 21, 33). The immediate objectives of these
studies are twofold: (i) to identify species, both known and
novel, by reference to sequence data bases; and (ii) to
construct species-specific probes which can be used in
quantitative ecological studies (7, 29).
Previously, we reported rRNA genetic markers and quan-
titative hybridization data which demonstrated that a novel
lineage belonging to the oa-proteobacteria was abundant in
the Sargasso Sea; we also identified novel lineages which
were very closely related to cultivated marine Synechococ-
cus spp. (6). In a study of a hot-spring ecosystem, Ward and
coworkers examined cloned fragments of 16S rRNA cDNAs
and found numerous novel lineages but no matches to genes
from cultivated hot-spring bacteria (33). These results sug-
gested that natural ecosystems in general may include spe-
cies which are unknown to microbiologists.
Although any gene may be used as a genetic marker,
rRNA genes offer distinct advantages. The extensive use of
16S rRNAs for studies of microbial systematics and evolu-
tion has resulted in large computer data bases, such as the
RNA Data Base Project, which encompass the phylogenetic
diversity found within culture collections. rRNA genes are
highly conserved and therefore can be used to examine
distant phylogenetic relationships with accuracy. The pres-
ence of large numbers of ribosomes within cells and the
regulation of their biosyntheses in proportion to cellular
growth rates make these molecules ideally suited for ecolog-
ical studies with nucleic acid probes.
Here we describe the partial analysis of a 16S rDNA
library prepared from photic zone Sargasso Sea bacterio-
plankton using a general approach for cloning eubacterial
16S rDNAs. The Sargasso Sea, a central oceanic gyre,
typifies the oligotrophic conditions of the open ocean, pro-
viding an example of a microbial community adapted to
extremely low-nutrient conditions. The results extend our
previous observations of high genetic diversity among
closely related lineages within this microbial community and
provide rDNA genetic markers for two novel eubacterial
lineages.
MATERIALS AND METHODS
Collection and amplification of rDNA. A surface (2-m)
bacterioplankton population was collected from hydrosta-
tion S (32°4'N, 64°23'W) in the Sargasso Sea as previously
described (8). The polymerase chain reaction (24) was used
to produce double-stranded rDNA from the mixed-microbi-
al-population genomic DNA prepared from the plankton
sample. The amplification primers were complementary to
conserved regions in the 5' and 3' regions of the 16S rRNA
genes. The amplification primers were the universal 1406R
(ACGGGCGGTGTGTRC) and eubacterial 68F (TNANAC
ATGCAAGTCGAKCG) (17). The reaction conditions were
as follows: 1 ,ug of template DNA, 10 ,ul of 10x reaction
buffer (500 mM KCI, 100 mM Tris HCI [pH 9.0 at 250C], 15
mM MgCl2, 0.1% gelatin, 1% Triton X-100), 1 U of Taq DNA
polymerase (Promega Biological Research Products, Madi-
son, Wis.), 1 ,uM (each) primer, 200 ,uM (each) dATP, dCTP,
dGTP, and dTTP in a 100-,ul total volume; 1 min at 94°C, 1
atUNIVOFCALIFDAVISonMay18,2010aem.asm.orgDownloadedfrom
MARINE BACTERIOPLANKTON 1711
Escherichia coli
Vibrio harveyi
inmm Vibrio anguillarum
- Oceanospirillum linum
- SAR 92
Caulobacter crescentus
SAR83
- Erythrobacter OCh 114
- Hyphomicrobium vulgare
Rhodopseudomonas marina
Rochalimaea quintana
Agrobacterium tumefaciens
SARI
-Es SAR95
SAR1I
CZ
* C)0._
u
10C)
00
.0
0Q
C)
0
0
O)4..
eP.
- Liverwort CP
*Anacystis nidulans
- Prochlorothrix
- SAR6
* SAR7
r SARIOO
SAR139
0
p.0
0
0 0.05 0.10 0.15
FIG. 4. Phylogenetic tree showing relationships of the rDNA clones from the Sargasso Sea to representative, cultivated species (2, 6, 18,
30, 31, 32, 35, 40). Positions of uncertain homology in regions containing insertions and deletions were omitted from the analysis.
Evolutionary distances were calculated by the method of Jukes and Cantor (15), which corrects for the effects of superimposed mutations.
The phylogenetic tree was determined by a distance matrix method (20). The tree was rooted with the sequence of Bacillus subtilis (38).
Sequence data not referenced were provided by C. R. Woese and R. Rossen.
phototroph phyla were conserved in the corresponding 880 (G -
C to C G). We note with caution that some classes
VOL. 57, 1991
I I
atUNIVaem.asm.orgDownloadedfrom

19. JOURNAL OF BACTERIOLOGY, JUlY 1991, p. 4371-4378
0021-9193/91/144371-08$02.00/0
Copyright C) 1991, American Society for Microbiology
Vol. 173, No. 14
Analysis of a Marine Picoplankton Community by 16S rRNA
Gene Cloning and Sequencing
THOMAS M. SCHMIDT,t EDWARD F. DELONG,t AND NORMAN R. PACE*
Department ofBiology and Institute for Molecular and Cellular Biology, Indiana University,
Bloomington, Indiana 47405
Received 7 January 1991/Accepted 13 May 1991
The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing
the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident
microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific
Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned
into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2
x 104 recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and
sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with
an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including
four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was
identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to
sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the
Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the y subdivision
of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were
obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences
from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the
jb.asmDownloadedfrom

20. INSIGHT REVIEW NATURE|Vol 437|15 September 2005
Crenarchaeota
Group I Archaea
Euryarchaeota
Group II Archaea
Group III Archaea
Group IV Archaea
α-Proteobacteria
* SAR11 - Pelagibacter ubique
* Roseobacter clade
OCS116
ß-Proteobacteria
* OM43
µ-Proteobacteria
SAR86
* OMG Clade
* Vibrionaeceae
* Pseudoalteromonas
* Marinomonas
* Halomonadacae
* Colwellia
* Oceanospirillum
δ-Proteobacteria
Cyanobacteria
* Marine Cluster A
(Synechococcus)
* Prochlorococcus sp.
Lentisphaerae
* Lentisphaera araneosa
Bacteroidetes
Marine Actinobacteria
Fibrobacter
SAR406
Planctobacteria
Chloroflexi
SAR202
Archaea
Bacteria
Figure 1| Schematic illustration of the phylogeny of
the major plankton clades.Black letters indicate
microbial groups that seem to be ubiquitous in
seawater. Gold indicates groups found in the photic
zone. Blue indicates groups confined to the
mesopelagic and surface waters during polar winters.
Green indicates microbial groups associated with
coastal ocean ecosystems.
%&'())%#*+,-###./*/!0##*1""#23##4(56#,!
343
Molecular diversity and ecology
of microbial plankton
Stephen J. Giovannoni1
& Ulrich Stingl1
The history of microbial evolution in the oceans is probably as old as the history of life itself. In contrast to
terrestrial ecosystems, microorganisms are the main form of biomass in the oceans, and form some of the
largest populations on the planet. Theory predicts that selection should act more efficiently in large
populations. But whether microbial plankton populations harbour organisms that are models of adaptive
sophistication remains to be seen. Genome sequence data are piling up, but most of the key microbial
plankton clades have no cultivated representatives, and information about their ecological activities is sparse.
cultivation of key organisms, metagenomics and ongoing biogeo-
chemical studies. It seems very likely that the biology of the dominant
microbial plankton groups will be unravelled in the years ahead.
Here we review current knowledge about marine bacterial and
archaeal diversity, as inferred from phylogenies of genes recovered
from the ocean water column, and consider the implications of micro-
bial diversity for understanding the ecology of the oceans. Although
we leave protists out of the discussion, many of the same issues apply
to them. Some of the studies we refer to extend to the abyssal ocean,
but we focus principally on the surface layer (0–300 m) — the region
of highest biological activity.
Phylogenetic diversity in the ocean
Small-subunit ribosomal (RNA) genes have become universal phylo-
genetic markers and are the main criteria by which microbial plank-
ton groups are identified and named9
. Most of the marine microbial
groups were first identified by sequencing rRNA genes cloned from
seawater10–14
, and remain uncultured today. Soon after the first reports
came in, it became apparent that less than 20 microbial clades
accounted for most of the genes recovered15
. Figure 1 is a schematic
illustration of the phylogeny of these major plankton clades. The taxon
names marked with asterisks represent groups for which cultured iso-
lates are available.
The recent large-scale shotgun sequencing of seawater DNA is pro-
viding much higher resolution 16S rRNA gene phylogenies and bio-
geographical distributions for marine microbial plankton. Although
the main purpose of Venter’sSorcerer IIexpedition is to gather whole-
genome shotgun sequence (WGS) data from planktonic microorgan-
isms16
, thousands of water-column rRNA genes are part of the
by-catch. The first set of collections, from the Sargasso Sea, have
yielded 1,184 16S rRNA gene fragments. These data are shown in
Fig. 2, organized by clade structure. Such data are a rich scientific
resource for two reasons. First, they are not tainted by polymerase
chain reaction (PCR) artefacts; PCR artefacts rarely interfere with the
correct placement of genes in phylogenetic categories, but they are a
major problem for reconstructing evolutionary patterns at the popu-
lation level17
. Second, the enormous number of genes provided by the
Sorcerer II expedition is revealing the distribution patterns and abun-
dance of microbial groups that compose only a small fraction of the
Certain characteristics of the ocean environment — the prevailing
low-nutrient state of the ocean surface, in particular — mean it is
sometimes regarded as an extreme ecosystem. Fixed forms of nitrogen,
phosphorus and iron are often at very low or undetectable levels in the
ocean’s circulatory gyres, which occur in about 70% of the oceans1
.
Photosynthesis is the main source of metabolic energy and the basis of
the food chain; ocean phytoplankton account for nearly 50% of global
carbon fixation, and half of the carbon fixed into organic matter is
rapidly respired by heterotrophic microorganisms. Most cells are freely
suspended in the mainly oxic water column, but some attach to aggre-
gates. In general, these cells survive either by photosynthesizing or by
oxidizing dissolved organic matter (DOM) or inorganic compounds,
using oxygen as an electron acceptor.
Microbial cell concentrations are typically about 105
cells mlǁ1
in
the ocean surface layer (0–300 m) — thymidine uptake into microbial
DNA indicates average growth rates of about 0.15 divisions per day
(ref. 2). Efficient nutrient recycling, in which there is intense competi-
tion for scarce resources, sustains this growth, with predation by
viruses and protozoa keeping populations in check and driving high
turnover rates3
. Despite this competition, steady-state dissolved
organic carbon (DOC) concentrations are many times higher than
carbon sequestered in living microbial biomass4
. However, the average
age of the DOC pool in the deep ocean, of about 5,000 years5
(deter-
mined by isotopic dating), suggests that much of the DOM is refrac-
tory to degradation. Although DOM is a huge resource, rivalling
atmospheric CO2 as a carbon pool6
, chemists have been thwarted by
the complexity of DOM and have characterized it only in broad terms7
.
The paragraphs above capture prominent features of the ocean
environment, but leave out the complex patterns of physical, chemical
and biological variation that drive the evolution and diversification of
microorganisms. For example, members of the genusVibrio — which
include some of the most common planktonic bacteria that can be iso-
lated on nutrient agar plates — readily grow anaerobically by fermen-
tation. The life cycles of some Vibrio species have been shown to
include anoxic stages in association with animal hosts, but the broad
picture of their ecology in the oceans has barely been characterized8
.
The story is similar for most of the microbial groups described below:
the phylogenetic map is detailed, but the ecological panorama is thinly
sketched. New information is rapidly flowing into the field from the
1
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA.
Natu rePu blishin g Gro u p©2 0 0 5
!20

21. The ecoty
many micro
tions in the w
best exampl
entiated by
adapted (hig
Phylogeneti
gests that th
cally distinct
Cluster A S
of which ca
characterist
urobilin)37,3
ample suppo
teristics that
SS120 has a
ammonium
extreme, Syn
nitrate, cyan
interesting t
seem to pro
whereby nu
conditions.
seasonal spe
lular cyanob
The obse
libraries prepared using PCR methods that reduced sequence arte-
facts17
. They concluded that most sequence variation was clustered
SAR11 subgroups Ia
+
Ib
SAR86
(γ-Proteobacteria)
SAR11 subgroup
II
M
arine
Picophytoplankton
Uncultured
α-Proteobacteria
Clade
SAR406
(Fibrobacter)
Bacteroidetes
SAR324
(δ-Proteobacteria)
M
arine
Actinobacteria
Alterom
onas/Pseudoalterom
onas
%of16SrRNAsequences
0
5
10
15
20
25
30
35
SAR116
(α-Proteobacteria)
Rheinheim
era
Roseobacter clade
SAR202
(Chloroflexi)
Phylogenetic clade !21

22. DeLong Lab
• Studying Sar86 and other marine plankton
• Note - published one of first genomic studies of
uncultured microbes - in 1996
JOURNAL OF BACTERIOLOGY, Feb. 1996, p. 591–599 Vol. 178, No. 3
0021-9193/96/$04.00 0
Copyright 1996, American Society for Microbiology
Characterization of Uncultivated Prokaryotes: Isolation and
Analysis of a 40-Kilobase-Pair Genome Fragment
from a Planktonic Marine Archaeon
JEFFEREY L. STEIN,1
* TERENCE L. MARSH,2
KE YING WU,3
HIROAKI SHIZUYA,4
AND EDWARD F. DELONG3
*
Recombinant BioCatalysis, Inc., La Jolla, California 920371
; Microbiology Department, University
of Illinois, Urbana, Illinois 618012
; Department of Ecology, Evolution and Marine Biology,
University of California, Santa Barbara, California 931063
; and Division of Biology,
California Institute of Technology, Pasadena, California 911254
Received 14 July 1995/Accepted 14 November 1995
One potential approach for characterizing uncultivated prokaryotes from natural assemblages involves
genomic analysis of DNA fragments retrieved directly from naturally occurring microbial biomass. In this
study, we sought to isolate large genomic fragments from a widely distributed and relatively abundant but as
yet uncultivated group of prokaryotes, the planktonic marine Archaea. A fosmid DNA library was prepared
from a marine picoplankton assemblage collected at a depth of 200 m in the eastern North Pacific. We
identified a 38.5-kbp recombinant fosmid clone which contained an archaeal small subunit ribosomal DNA
gene. Phylogenetic analyses of the small subunit rRNA sequence demonstrated its close relationship to that of
previously described planktonic archaea, which form a coherent group rooted deeply within the Crenarchaeota
Downloadedfrom

23. Delong Lab
isolated from fosmid clones
arious restriction endonucle-
, the F-factor-based vector
osmid subfragments. Partial
striction enzyme to 1 ⇥g of
re. The reaction mixture was
oved at 10, 40, and 60 min.
1 ⇥l of 0.5 M EDTA to the
e partially digested DNA was
ribed above except using a 1-
es of the separated fragments
dards. The distances of the
promoter sites on the excised
of T7- or SP6-specific oligo-
d hybridizing with Southern
d and pBAC clones digested
ed with labeled T7 and SP6
ubclones and PCR fragments
sequencing described above.
mates from the partial diges-
fosmids and their subclones.
DeSoete distance (9) analyses
ng GDE 2.2 and Treetool 1.0,
P) (23). DeSoete least squares
rwise evolutionary distances,
count for empirical base fre-
d from the RDP, version 4.0
A sequences were performed
DP. For distance analyses of
ary distances were estimated
topology was inferred by the
tion and global branch swap-
n sequences, the Phylip pro-
ion and ordinary parsimony
sequences reported in Table
ollowing accession numbers:
3, U40244, and U40245. The
FIG. 1. Flowchart depicting the construction and screening of an environ-
mental library from a mixed picoplankton sample. MW, molecular weight;
PFGE, pulsed-field gel electrophoresis.
GENOMIC FRAGMENTS FROM PLANKTONIC MARINE ARCHAEA 593
atUNjb.asm.orgDownloadedfrom
!23

24. Delong Lab
FIG. 4. High-density filter replica of 2,304 fosmid clones containing approx-
imately 92 million bp of DNA cloned from the mixed picoplankton community.
The filter was probed with the labeled insert from clone 4B7 (dark spot). The
lack of other hybridizing clones suggests that contigs of 4B7 are absent from this
portion of the library. Similar experiments with the remainder of the library
yielded similar results.
J. BACTERIOL.
Downl
!24

31. 1 ttgttatatc agtaatggct attgctccaa taacttaata ctaatatata attagtttat
61 gaataaattt tatatatttg ggttattgtt ttttacacta aatgcatttt cttgctcaga
121 tcttctagat acagacatga gagttcttga ttccgctgag tcaagaaacc tttgcgagtt
181 tgaaggaaaa gctttactag ttgtgaatgt tgcaagtaga tgtggttaca cttatcaata
241 tgctggcctt caaaagttat atgaaagtta taaagatgaa gattttctag taattgggat
301 cccatctaga gattttcttc aagaatactc tgatgaaagc gatgttgcag aattttgttc
361 tacagaatac ggtgttgaat ttcctatgtt ctcaactgct aaagtcaaag gaaaaaaagc
421 acacccattt tataaaaaac ttattgcaga atcaggtttt actccctcat ggaactttaa
481 taaatactta atctcaaaag agggcaaggt tgtatccaca tatggatcaa aggtaaagcc
541 tgattcaaaa gagcttatat cagctataga aggcttgctg taaaattatt acttagaaac
601 taatacagtt ttaggcttgt ttgctgcaaa tattccatta tctacaactc caggaatatt
661 attaatcaaa gcttccattt cagtggggtt tgaaatatcc atattagaga tatctaaaat
721 gtgattacct tggtctgtta taaatccagt tctatatgtg ggtattccac cgatcgagat
781 tatttttctt gcaacaaggc tcctactttc aggtatcacc tctataggca gtggaaaagc
841 tcccaaaaga ttaaccatct ttgactgatc aactatacat ataaactcgt tagaggcaga
901 agcaactatc ttttctctag tatgtgcgcc accaccacct ttaataagac aattttcagg
961 agacacctca tctgcaccat ctatgtaata agctatatca actacatcat taaggctaaa
1021 gacctctatc ccattttcat ttaataattt tgatgaagca tctgaactag aaacagctcc
1081 agcaaatttg tgcctatgct cctttagttc ttctataaaa aaattaactg ttgagccggt
1141 tccaatacct aaaatcatct caggatgaag attatttttg atatattcta tagcttgttt
1201 agcaacattt atctttgagc cactcataga gttataatac aagaaaatat aggtagttaa
1261 ttattttgag actaaaaatt aaaaaaacag gttcttttaa gaattcccag aagtacctaa
1321 agcttattcc taatgcatct gtttatgacg tagcattaaa atcacctata acatttgctc
1381 taaatatttc ttcaaagctg gggaataaag ttttcctaaa aagagaggat ctgcaaccta
1441 tattttcttt taaaaacaga ggagcgtata acaagattgt aaatttatcc gatgccgaaa
1501 agaagagggg ggttattgct gcatcagcag gaaatcatgc tcaaggggta gccagtgcat
1561 gtaagaaatt aaaaattaat tgcttgatag ttatgccaat aacaactcca gaaataaaaa
1621 taaaagatgt aaaaagattt ggagccaaaa tactccaaca tggggacaac gtagatgcag
1681 cattaaaaga ggcactgttt attgcaaaga aaaaaaaatt gtcttttgtt catccttttg
1741 acgaccctct aacaattgct ggccaaggga ctataggaca agaaattctt gaagataaaa
1801 ataattttga tgttgtcttt gttccggtgg gaggaggagg tattctagct ggtgtatctg
1861 cctggatagc acagaataat aagaaaataa aaattgttgg tgttgaggtt gaggattccg
1921 cttgtcttgc tgaggccgta aaagctaata aaagagttat tttaaaagaa gtgggcctct
1981 ttgctgatgg ggtggcagta tcaagggttg gaaaaaataa ttttgatgtt attaaagagt
2041 gcgtagatga agtcattaca gttagcgttg atgaggtctg caccgctgta aaagatatct
2101 ttgaagatac aagggttcta tcagaacctg ctggggcatt agcacttgca gggttaaaag
2161 cctacgcaag gaaagttaaa aataaaaaac ttattgctat aagttctggc gctaatgtaa
2221 atttccaaag acttaatttt attgttgagc gatcagagat tggtgaaaat agagaaaaaa
2281 tattaagtat caaaatccca gagatacctg gaagttttct taagctttca aggatgtttg
2341 gcagctctca agttacagag tttaactaca ggaaatctag cttaagcgat gcatatgttt
2401 tagttggtgt tagaactaaa actgaaaaat catttgaaat cttaaagtcc aaattaaaaa
2461 aagcaggctt cacctttagc gactttactc gaaatgaaat atccaatgat catctgaggc
2521 atatggttgg tggcagaaat agtgactcag gctctcataa caatgaaaga atatttaggg
2581 gagagtttcc tgagaagccg ggcgcgctgt taaattttct agagaaattt ggaaataaat
2641 ggmatatttc cttatttcat tacaggaacc taggttcagc ttttggaaag atattaattg
2701 gcatcgagag taaggataaa gacaagctaa taaatcattt aaataagtca ggnactattt
2761 ttacagaaga aacctctaac aaggcataca aagatttttt aaaatgaaag gttaatactt
2821 taatctaaat ttaattgaaa aaagctcatc gctagggttt tcccacggct ctttgaacaa
2881 ctcggattga gatctatcat cctcctcgtc gtaaattctc ccacctttag aatagaccaa
2941 aaatagatat gacaaaggag cgagctcata tttatatcta atttggaacg aagctacgcc
3001 agtattaaat tcattaacta tattatttcc tttataaagg tatccattag catctgaaaa
3061 aatactaata ggattttttg ctttcaaagc aacaaattga ctcttaagtc tgatctcatg
3121 tttattattt ttaaaccaat ttagatcaaa agataaggta tcttgtcttg aatcatatga
3181 ggcaagatta ttattatcct gccatatcag ccattcattt tcctttctta ttctatattg
3241 cgcattgatt cttaagttat catttggaaa tattgaacct gctatcttgt aaaattttct
3301 accataccca ttcgaatccc accgattatc tttctctccc ttaaagaagc taactctcca
3361 gtcatatgtc cagaatgaat agttctttgc ctcaaagtct gctgtaatac ctattcttct
3421 ttttgatttg ataaaggggt aggcttcatt ttttcttgtg atagttgtat ttttcccaga
3481 agatctaaag ttaaaatcta attgaaattt agagttgtcc ttaaaactaa aagaattttt
3541 ttgatcgatg cctattggat ttgaattacc agctgtgtca gcatcataat ttagatcaat
3601 tccataatct atttgtttta atatcgagct attatcaaat tcatttattt ttcgatttcc
3661 tccaatacca gcatgaatcc agtctcttct ttgcagataa ccaaagtcat ttaactcgaa
3721 gtcgtcttca aaataaagaa ggcttccact tatgttagat agtttatttg gaagatatgt
3781 aaactgagtc ctatacccaa gcccattttt accatctttt tctgaggcta acagatctga
3841 atatgtaatt aatttttttg aacgaatatt gatgtaatca ataacattga ctgttgatga
3901 ttcgcctgtc atttcattct caacattcgt taccatgaag ccaagcgttt tatttccaag
3961 ctttgttcga gatcgaagag cataatagtc tcttccaact gaaaaggctt catcagcctc
4021 acttgctaca aatactccaa attcattatt attacttttt tgagtaagtc ttaatgcaaa
4081 atcaatatca gaatagtttt ttttagctgc ctcgcaacct tcttcattac tttcttctga
4141 gcaattatag ctgggggcag ctccaatcct ccgcgtattt ataaccgagt atctatcata
4201 attactaata tcaaatagtg attggttttc attgaaaaat gctctttttt ctgagtaaaa
4261 agtttcttga gcagaaaagt taataaccac atcatcactc tcagcttgtc cgaaatctgg
4321 attaatagct aaatttattt gacgaccttt tccagtgcta taaaagattt cagccccaat
4381 atctgaccct tcttggtttg taactgaatt tttatttgaa gatatatatg gaaaaaaagt
4441 aagctttgat tttgtatagt tttgtatttc taagctatct aactcttgaa agtagtcatt
4501 tctactagcc attgttccgg cactgctaac ccatgactca ttttcggcac tataacgtaa
4561 tgcggtgtaa ttaatttttc ttatatcacc atcaggctgt ttcattaacg ttacatccca
4621 aggaataaaa aactcagaga cccaataccc atcaaatttt tgtgtttttg caatccaatc
4681 tccatcccag tctgtcttaa agtctcctgc ttgcgttttt atggcatcga aaagcgagtt
4741 cccaagattt atagcaagaa tgaaagcttt gttaccatca ccatcaaagt ctatatttat
4801 agagttttta tcgctaagtg aatttatttg atctctaagc gtcttcctgg agaacataga
4861 atcattactt tgaaaattct taaatccaac atatatacca tccttatttg agaaaattaa
4921 agccgttgta agaagttcat ttttcttaag agtaaaagga gatgtttcat aaaaatctgt
4981 aatttcaaat gcattattcc actcaggctc atcaagagag ccatcaataa caattgagtt
5041 tgaccaaatc agcacagagg taagtaaaag tgataatgag gctaaagtta ttttcataga
5101 tagatttaat tcagagtatt ttaatctatg aagagtagga aatcatccga tcattctcag
5161 aaaaaacata atcatattta aggtcatgtt tttctccaaa atgcatatta caaatctggt
5221 attgataaca cagtccaacc aataaaggtc ttgatacctc ctcgtttata gagccaataa
5281 ttctatcgaa atatccagag ccatagccaa gcctgtatcc atttaaatca actcctgtca
5341 taggaataaa cattaaatca atttcattta tgttgacata atcttcactt ttaacctctt

32. cat /Users/jeisen/Desktop/sequence.gb.txt | grep product
/product="predicted threonine dehydratase"
/product="predicted secreted protein"
/product="predicted 5-formyltetrahydrofolate cyclo-ligase"
/product="predicted Xaa-Pro aminopeptidase"
/product="predicted quinone biosynthesis monooxygenase"
/product="predicted 50S ribosomal protein L28"
/product="predicted deoxyuridine 5'-triphosphate
/product="predicted primosomal protein N' (replication
/product="predicted N-acetylmuramoyl-L-alanine amidase"
/product="predicted kinase of the phosphomethylpyrimidine
/product="predicted kinase of P-loop ATPase superfamily"
/product="predicted eukaryote-type paraoxonase"
/product="predicted alpha/beta hydrolase"
/product="predicted MutT superfamily hydrolase"
/product="predicted metallobeta lactamase fold protein"
/product="predicted acetyl-coenzyme A synthetase"
/product="predicted deoxypurine kinase"
/product="predicted poly(A) polymerase"
/product="predicted DskA family protein with conserved
/product="predicted ribokinase family sugar kinase"
/product="predicted glutamate-1-semialdehyde
/product="predicted enzyme with conserved cysteine"
/product="predicted metallopeptidase of the G-G peptidase
/product="predicted proline dehydrogenase"
/product="predicted tyrosyl-tRNA synthetase"
/product="predicted prolyl endopeptidase"
/product="tRNA-Ile"
/product="predicted unknown protein"
/product="predicted cation efflux pump"
/product="predicted TonB-dependent receptor protein"
/product="predicted conserved membrane protein"
/product="predicted LicA, choline kinase involved in LPS
/product="predicted NAD-dependent formate dehydrogenase"
/product="predicted 2-nitropropane dioxygenase"
/product="predicted oxidoreductase"
/product="predicted CsgA, Rossman fold oxidoreductase"
/product="predicted acid--CoA ligase fadD13"
/product="proteorhodopsin"
/product="predicted phosphopantetheine
/product="predicted formamidopyrimidine-DNA glycosylase"
/product="predicted outer membrane protein TolC"
/product="predicted topoisomerase IV subunit B"
/product="predicted topoisomerase IV subunit A"
/product="predicted succinylornithine aminotransferase"
/product="predicted 6-phosphofructokinase"
/product="predicted
/product="predicted ORF"
/product="predicted penicillin-binding protein 2"
/product="predicted membrane-bound lytic transglycosylase"
/product="predicted penicillin-binding protein 6
/product="predicted D-alanine aminotransferase"
/product="predicted DNA polymerase III, delta subunit"
/product="predicted leucyl-tRNA synthetase (leuS)"
/product="predicted acetyl-CoA carboxylase, biotin
/product="predicted esterase of alpha/beta hydrolase fold"
/product="predicted ABC transporter ATPase subunit"
/product="predicted dihydroxyacid dehydratase"
/product="predicted porphobilinogen synthase"
/product="predicted transcription termination factor Rho"
/product="predicted porphobilinogen deaminase"
/product="predicted transporter (ACRA/ACRE type)"
/product="predicted cation efflux system (AcrB/AcrD/AcrF
/product="predicted secreted lipoprotein"
/product="predicted ribosomal large subunit pseudouridine
/product="predicted acetolactate synthase III large chain"
/product="predicted ketol-acid reductoisomerase"
/product="16S ribosomal RNA"
/product="23S ribosomal RNA"
/product="predicted YacE family of P-loop kinases"
/product="predicted preprotein translocase secA subunit"

34. The inferred amino acid sequence of the proteorho-
dopsin showed statistically significant similarity to
archaeal rhodopsins (11).

35. The inferred amino acid sequence of the proteorho-
dopsin showed statistically significant similarity to
archaeal rhodopsins (11).

36. p-
of
is
on
d-
he
ff
he
e-
ll
s-
r-
in
).
as
10
yl
i-
n-
e-
i-
et
o-
of
g-
es
ng
o-
be
Fig. 1. (A) Phylogenetic tree of bacterial 16S rRNA gene sequences, including that encoded on the
130-kb bacterioplankton BAC clone (EBAC31A08) (16). (B) Phylogenetic analysis of proteorhodop-
sin with archaeal (BR, HR, and SR prefixes) and Neurospora crassa (NOP1 prefix) rhodopsins (16).
Nomenclature: Name_Species.abbreviation_Genbank.gi (HR, halorhodopsin; SR, sensory rhodopsin;
BR, bacteriorhodopsin). Halsod, Halorubrum sodomense; Halhal, Halobacterium salinarum (halo-
bium); Halval, Haloarcula vallismortis; Natpha, Natronomonas pharaonis; Halsp, Halobacterium sp;
Neucra, Neurospora crassa.
Fig. 1. (B) Phylogenetic analysis of proteorhodop- sin with archaeal (BR, HR, and SR prefixes) and
Neurospora crassa (NOP1 prefix) rhodopsins (16). Nomenclature: Name_Species.abbreviation_Genbank.gi
(HR, halorhodopsin; SR, sensory rhodopsin; BR, bacteriorhodopsin). Halsod, Halorubrum sodomense;
Halhal, Halobacterium salinarum (halo- bium); Halval, Haloarcula vallismortis; Natpha, Natronomonas
pharaonis; Halsp, Halobacterium sp; Neucra, Neurospora crassa.

37. Halophiles …
!37

38. Bacteriorhodopsin and its relatives
Figure 3. Phylogenetic tree based on the amino acid sequences of 25 archaeal rhodopsins. (a) NJ-tree. The numbers at each node are clustering probabilities generated by
bootstrap resampling 1000 times. D1 and D2 represent gene duplication points. The four shaded rectangles indicate the speciation dates when halobacteria speciation occurred at
the genus level. (b) ML-tree. Log likelihood value for ML-tree was −6579.02 (best score) and that for topology of the NJ-tree was −6583.43. The stippled bars indicate the 95%
confidence limits. Both trees were tentatively rooted at the mid-point of the longest distance, although true root positions are unknown.
From Ihara et al. 1999
!38

39. What Does It Do?

40. Proteorhodopsin Predicted Secondary Structure
Fig. 2. Secondary
structure of proteo-
rhodopsin. Single-
letter amino acid
codes are used (33),
and the numbering
is as in bacteriorho-
dopsin. Predicted
retinal binding pock-
et residues are
marked in red.
R E S E A R C H A R T I C L E S
!40

41. The amino acid residues that form a retinal binding pocket in archaeal
rhodopsins are also highly conserved in proteorhodopsin (Fig. 2). In
particular, the critical lysine residue in helix G, which forms the Schiff
base linkage with retinal in archaeal rhodopsins, is present in
proteorhodopsin. Analysis of a structural model of proteorhodopsin (14 ),
in conjunction with multiple sequence alignments, indicates that the
majority of active site residues are well conserved between
proteorhodopsin and archaeal bacteriorhodopsins (15).

43. The amino acid residues that form a retinal binding pocket in archaeal
rhodopsins are also highly conserved in proteorhodopsin (Fig. 2). In
particular, the critical lysine residue in helix G, which forms the Schiff
base linkage with retinal in archaeal rhodopsins, is present in
proteorhodopsin. Analysis of a structural model of proteorhodopsin (14 ),
in conjunction with multiple sequence alignments, indicates that the
majority of active site residues are well conserved between
proteorhodopsin and archaeal bacteriorhodopsins (15).

45. Proteorhodopsin in E. coli
duce
in th
occu
pigm
and
ed at
tiona
sorp
in 0.
sorp
botto
nated
retin
ms d
deca
shift
appe
term
cay o
step
singl
upw
amp
gene
with
ms p
reco
phot
prod
Fig. 3. (A) Proteorhodopsin-expressing E. coli cell suspension (ϩ ) compared to control cells (Ϫ ),
both with all-trans retinal. (B) Absorption spectra of retinal-reconstituted proteorhodopsin in E. coli
membranes (17). A time series of spectra is shown for reconstituted proteorhodopsin membranes
(red) and a negative control (black). Time points for spectra after retinal addition, progressing from
low to high absorbance values, are 10, 20, 30, and 40 min.
!45

46. Fig. 3. (A) Proteorhodopsin-expressing E. coli cell suspension (+)
compared to control cells (-), both with all-trans retinal. (B) Absorption
spectra of retinal-reconstituted proteorhodopsin in E. coli membranes
(17). A time series of spectra is shown for reconstituted proteorhodopsin
membranes (red) and a negative control (black). Time points for spectra
after retinal addition, progressing from low to high absorbance values,
are 10, 20, 30, and 40 min.

47. Proteorhodopsin was amplified from the 130-kb bac- terioplankton BAC
clone 31A08 by polymerase chain reaction (PCR), using the primers 5 -
ACCATGGGTA- AAT TAT TACTGATAT TAGG-3 and 5 -AGCAT TA-
GAAGAT TCT T TAACAGC-3 . The amplified fragment was cloned
with the pBAD TOPO TA Cloning Kit (Invitrogen). The protein was
cloned with its native signal sequence and included an addition of the V5
epitope and a polyhistidine tail in the COOH-terminus. The same PCR
product in the opposite orientation was used as a negative control. The
protein was expressed in the E. coli outer membrane protease- deficient
strain UT5600 [M. E. Elish, J. R. Pierce, C. F. Earhart, J. Gen. Microbiol.
134, 1355 (1988)] and induced with 0.2% arabinose for 3 hours.
Membranes were prepared according to Shimono et al. [K. Shi- mono,
M. Iwamoto, M. Sumi, N. Kamo, FEBS Lett. 420, 54 (1997)] and
resuspended in 50 mM tris-Cl (pH 8.0) and 5 mM MgCl2. The
absorbance spectrum was measured according to Bieszke et al. (7) in the
presence of 10 M all-trans retinal.

48. Proteorhodopsin function
geneity in
with 87% o
ms photocy
recovery. A
photocycle
produces a b
this alternat
well as a tw
component,
the total am
with a 45-m
amplitude).
cycle rate, w
teristic of
strong evid
tions as a tr
rhodopsin.
Implicat
harbor the
tributed in
bacteria hav
ture-indepen
oceanic reg
Oceans, as
(8, 25–29).
tribution, pr
this ␥-prote
both with all-trans retinal. (B) Absorption spectra of retinal-reconstituted proteorhodopsin in E. coli
membranes (17). A time series of spectra is shown for reconstituted proteorhodopsin membranes
(red) and a negative control (black). Time points for spectra after retinal addition, progressing from
low to high absorbance values, are 10, 20, 30, and 40 min.
Fig. 4. (A) Light-driven transport of protons by a
proteorhodopsin-expressing E. coli cell suspension.
The beginning and cessation of illumination (with
yellow light Ͼ485 nm) is indicated by arrows labeled ON and OFF, respectively. The cells were
suspended in 10 mM NaCl, 10 mM MgSO4⅐7H2O, and 100 ␮M CaCl2. (B) Transport of 3
Hϩ
-labeled
tetraphenylphosphonium ([3
Hϩ
]TPP) in E. coli right-side-out vesicles containing expressed proteorho-
dopsin, reconstituted with (squares) or without (circles) 10 ␮M retinal in the presence of light (open
symbols) or in the dark (solid symbols) (20).
15 SEPTEMBER 2000 VOL 289 SCIENCE www.sciencemag.org1904

49. 20. Right-side-out membrane vesicles were prepared according to
Kaback [H. R. Kaback, Methods Enzymol. 22, 99 (1971)]. Membrane
electrical potential was measured with the lipophilic cation TPP by
means of rapid filtration [E. Prossnitz, A. Gee, G. F. Ames, J. Biol. Chem.
264, 5006 (1989)] and was calculated according to Robertson et al. [D.
E. Robertson, G. J. Kaczorowski, M. L. Garcia, H. R. Kaback,
Biochemistry 19, 5692 (1980)]. No energy sources other than light were
added to the vesicles, and the experiments were conducted at room
temperature.

50. Figure 5
Laser flash-induced absorbance
changes in suspensions of E. coli
membranes containing
proteorhodopsin. A 532-nm pulse
(6 ns duration, 40 mJ) was delivered
at time 0, and absorption changes
were monitored at various
wavelengths in the visible range in
a lab-constructed flash photolysis
system as described (34). Sixty-
four transients were collected for
each wavelength. (A) Transients at
the three wavelengths exhibiting
maximal amplitudes. (B) Absorption
difference spectra calculated from
amplitudes at 0.5 ms (blue) and
between 0.5 ms and 5.0 ms (red).
http://www.sciencemag.org/content/289/5486/1902/F5.expansion.html

52. Papers for Today
Correspondence should be addressed to A.T.
(e-mail: deia@dstu.univ-montp2.fr).
.................................................................
Proteorhodopsin phototrophy
in the ocean
Oded BeÂjaÁ*², Elena N. Spudich²³, John L. Spudich³, Marion Leclerc*
& Edward F. DeLong*
* Monterey Bay Aquarium Research Institute, Moss Landing, California 95039,
USA
³ Department of Microbiology and Molecular Genetics, The University of
Texas Medical School, Houston, Texas 77030, USA
² These authors contributed equally to this work
..............................................................................................................................................
Proteorhodopsin1
, a retinal-containing integral membrane pro-
tein that functions as a light-driven proton pump, was discovered
in the genome of an uncultivated marine bacterium; however, the
prevalence, expression and genetic variability of this protein in
native marine microbial populations remain unknown. Here we
report that photoactive proteorhodopsin is present in oceanic
surface waters. We also provide evidence of an extensive family of
to that of recomb
¯ash-photolysis
existence of pro
retinal molecules
waters.
The photocyc
and, after hydro
adding all-trans
indeed derive fro
–4
–3
–2
–1
0
1
2
ΔAbsorbance(×10–3)0–3)
0
4
2
3
http://www.nature.com/nature/journal/v411/n6839/abs/411786a0.html

53. NATURE |VOL 411 |14 JUNE 2001 |www.nature.com
hn L. Spudich³, Marion Leclerc*
ute, Moss Landing, California 95039,
lar Genetics, The University of
30, USA
work
...................................................................
ining integral membrane pro-
n proton pump, was discovered
marine bacterium; however, the
c variability of this protein in
ons remain unknown. Here we
odopsin is present in oceanic
dence of an extensive family of
psin variants. The protein pig-
family seem to be spectrally
orbing light at different wave-
available in the environment.
oteorhodopsin-based phototro-
ic microbial process.
taining membrane protein that
n pump, was discovered nearly
Halobacterium salinarum2
. The
on contain a high concentration
cked in an ordered two-dimen-
tion of light, bacteriorhodopsin
al shifts (a photocycle), causing a
the membrane. The resulting
al drives ATP synthesis, through
indeed derive from retinylidene pigmentation. We conclude that
–4
–3
–2
–1
0
1
2
∆Absorbance(×10–3)∆Absorbance(×10–3)
806040200
Time (ms)
580 nm
500 nm
400 nm
Wavelength (nm)
400 450 500 550 600 650 700
–3
–2
–1
0
1
2
3
Monterey Bay environmental membranes
Proteorhodopsin in E. coli membranes
Figure 1 Laser ¯ash-induced absorbance changes in suspensions of membranes
prepared from the prokaryotic fraction of Monterey Bay surface waters. Top, membrane
absorption was monitored at the indicated wavelengths and the ¯ash was at time 0 at
532 nm. Bottom, absorption difference spectrum at 5 ms after the ¯ash for the
environmental sample (black) and for E. coli-expressed proteorhodopsin (red).
© 2001 Macmillan Magazines Ltd

54. NATURE |VOL 411 |14 JUNE 2001 |www.nature.com
strongly suggests that this protein has a signi®cant role in the
Laser
flash
Untreated membranes
Hydroxylamine-treated membranes
Retinal-reconstituted membranes
10–3
AU
10–1
s
Figure 2 Laser ¯ash-induced transients at 500 nm of a Monterey Bay bacterioplankton
membrane preparation. Top, before addition of hydroxylamine; middle, after 0.2 M
hydroxylamine treatment at pH 7.0, 18 8C, with 500-nm illumination for 30 min; bottom,
after centrifuging twice with resuspension in 100 mM phosphate buffer, pH 7.0, followed
by addition of 5 mM all-trans retinal and incubation for 1 h.
M
M
MB
MB
HOT
HOT
0.01
HO
PAL
PAL B
PAL B
PAL E
PAL
PAL
PAL
PAL
M
M
M
M
MB
BA
PAL E
B
HOT
Figure 3 Phylogenetic analysis of the inferred
proteorhodopsin genes. Distance analysis of 22
by neighbour-joining using the PaupSearch pr
10.0 (Genetics Computer Group; Madison, Wis
was used as an outgroup, and is not shown. Sc
per site. Bold names indicate the proteorhodop
this study.
© 2001 Macmillan Magazines Ltd

55. of the surface of a single cell3
. This
nt to produce substantial amounts of
efore, the high density of proteorho-
rane indicated by our calculations
otein has a signi®cant role in the
in amino-acid sequences were not restricted to the hydrophilic
ed membranes
e-treated membranes
stituted membranes
10–3
AU
10–1
s
at 500 nm of a Monterey Bay bacterioplankton
tion of hydroxylamine; middle, after 0.2 M
C, with 500-nm illumination for 30 min; bottom,
n in 100 mM phosphate buffer, pH 7.0, followed
incubation for 1 h.
MB 0m2
MB 0m1
MB 20m2
MB 20m5
MB 40m12
MB 100m9
HOT 75m3
HOT 75m8
0.01
MontereyBayandshallowHOT
AntarcticaanddeepHOT
HOT 75m1
PAL B1
PAL B5
PAL B6
PAL E7
PAL E1
PAL B7
PAL B2
PAL B8
MB 100m10
MB 100m5
MB 100m7
MB 20m12
MB 40m1
MB 40m5
BAC 40E8
BAC 31A8
PAL E6
BAC 64A5
HOT 0m1
HOT 75m4
Figure 3 Phylogenetic analysis of the inferred amino-acid sequence of cloned
proteorhodopsin genes. Distance analysis of 220 positions was used to calculate the tree
by neighbour-joining using the PaupSearch program of the Wisconsin Package version
10.0 (Genetics Computer Group; Madison, Wisconsin). H. salinarum bacteriorhodopsin
was used as an outgroup, and is not shown. Scale bar represents number of substitutions
per site. Bold names indicate the proteorhodopsins that were spectrally characterized in
this study. !55

56. mechanisms of wavelength regu-
hodopsins. Furthermore, palE6
larly to its Monterey Bay
diated transport of protons in
. Spudich et al., manuscript in
rom Antarctica (E. F. DeLong,
dopsin primers, and sequenced
minary sequence analyses based
indicate that, despite differences
orption spectra, the Antarctic
a bacterium highly related to
AR86 bacteria of Monterey Bay10
nes from the HOT station were
m samples. Most proteorhodop-
surface waters belonged to the
(on the amino-acid level) to the
40E8. In contrast, most of the
) fell within the Antarctic clade
es from the HOT station, HOT
d in E. coli and their absorption
d from their primary sequences,
ng with the Antarctic clade gave
um, whereas the proteorhodop-
clade gave a green (527-nm)
central North Paci®c gyre, most
ge, with maximal intensity near
ak is maintained over depth,
At the surface, the energy peak
th between 400 and 650 nm. In
narrows and the half bandwidth
00 nm (Fig. 5). Considering the
members of the two different
dopsin genetic variants in mixed populations with a selective
advantage at different points along the depth-dependent light
0.06
0.05
0.04
0.03
0.02
0.01
1.0
0.8
0.6
0.4
0.2
0
350 400
75 m
5 m
Monterey
Bay
HOT 0 m
HOT 75 m
Antarctica
450 500
Wavelength (nm)
RelativeirradianceAbsorbance
550 600 650
Figure 5 Absorption spectra of retinal-reconstituted proteorhodopsins in E. coli
membranes. All-trans retinal (2.5 mM) was added to membrane suspensions in 100 mM
phosphate buffer, pH 7.0, and absorption spectra were recorded. Top, four spectra for
palE6 (Antarctica), HOT 75m4, HOT 0m1, and BAC 31A8 (Monterey Bay) at 1 h after
retinal addition. Bottom, downwelling irradiance from HOT station measured at six
wavelengths (412, 443, 490, 510, 555 and 665 nm) and at two depths, for the same
depths and date that the HOT samples were collected (0 and 75 m). Irradiance is plotted
relative to irradiance at 490 nm.
!56

57. 788 NATURE |VOL 411 |14 JUNE 2001 |www.nature.com
whereas the total energy decreases. At the surface, the energy peak
is very broad with a half bandwidth between 400 and 650 nm. In
deeper water below 50 m, the peak narrows and the half bandwidth
is restricted to between 450 and 500 nm (Fig. 5). Considering the
different wavelengths absorbed by members of the two different
Figure 4 Multiple alignment of proteorhodopsin amino-acid sequences. The secondary structure shown (boxes for transmembrane helices) is derived from hydropathy plots. Residues
predicted to form the retinal-binding pocket are marked in red.
palE6 (Antarctica), HOT 75m4, HOT 0m1, and BAC 31A8 (Monterey Bay) at 1 h after
retinal addition. Bottom, downwelling irradiance from HOT station measured at six
wavelengths (412, 443, 490, 510, 555 and 665 nm) and at two depths, for the same
depths and date that the HOT samples were collected (0 and 75 m). Irradiance is plotted
relative to irradiance at 490 nm.
© 2001 Macmillan Magazines Ltd
!57

58. More on Large Insert Metagenomics
(e-mail: denning@atmos.colostate.edu).
.................................................................
Unsuspected diversity among marine
aerobic anoxygenic phototrophs
Oded BeÂjaÁ*², Marcelino T. Suzuki*, John F. Heidelberg³,
William C. Nelson³, Christina M. Preston*, Tohru Hamada§²,
Jonathan A. Eisen³, Claire M. Fraser³ & Edward F. DeLong*
* Monterey Bay Aquarium Research Institute, Moss Landing,
California 95039-0628, USA
³ The Institute for Genomic Research, Rockville, Maryland 20850, USA
§ Marine Biotechnology Institute, Kamaishi Laboratories, Kamaishi City,
Iwate 026-0001, Japan
..............................................................................................................................................
Aerobic, anoxygenic, phototrophic bacteria containing bacterio-
chlorophyll a (Bchla) require oxygen for both growth and Bchla
synthesis1±6
. Recent reports suggest that these bacteria are widely
distributed in marine plankton, and that they may account for up
to 5% of surface ocean photosynthetic electron transport7
and
8
b
98
6
http://www.nature.com/nature/journal/v415/n6872/full/415630a.html

59. Erythrobacter litoralis*
MBIC3019*
Erythrobacter longus*
Erythromicrobium ramosum
cDNA 0m1
cDNA 20m21
Roseobacter denitrificans*
Roseobacter litoralis*
Rhodovulum sulfidophilum*
100
BAC 39B11
BAC 29C02
cDNA 20m10
BAC 52B02
BAC 65D09
BAC 24D02
99
100
100
54
Rhodoferax fermentans
Allochromatium vinosum
Roseateles depolymerans
Rubrivivax gelatinosus
cDNA 0m20
Rhodospirillum rubrum
56
96
52
67
Thiocystisge latinosa
Rhodomicrobium vannielii
Rhodocyclus tenuis
Rhodopseudomonas palustris
Chloroflexus aurantiacus
Sphingomonas natatoria
Rhodobacter sphaeroides
Rhodobacter capsulatus
BAC 56B12a
b
BAC 60D04
BAC 30G07
R2A84*
R2A163*
cDNA 20m22
cDNA 0m13
cDNA 20m8
R2A62*
cDNA 20m11
0.1
98
72
99
100
76
61
73
100
82
96
β
γ
α-3
α-4
α–1
α-2
env20m1
env20m5
env0m2
MBIC3951*
env0m1
envHOT1
envHOT2
envHOT3
100
99
100
100
100
Roseobacter denitrificans*
Roseobacter litoralis*
R2A84*
0.1
100
52
91
esearch, Rockville, Maryland 20850, USA
ute, Kamaishi Laboratories, Kamaishi City,
........................................................................................
ototrophic bacteria containing bacterio-
quire oxygen for both growth and Bchla
rts suggest that these bacteria are widely
ankton, and that they may account for up
photosynthetic electron transport7
and
obial community8
. Known planktonic
belong to only a few restricted groups
ia a-subclass. Here we report genomic
thetic gene content and operon organiza-
ng marine bacteria. These photosynthetic
ome that most closely resembled those of
b-subclass, which have never before been
ronments. Furthermore, these photosyn-
ly distributed in marine plankton, and
itic bacterioplankton assemblages, indi-
nti®ed phototrophs were photosyntheti-
ta demonstrate that planktonic bacterial
ply composed of one uniform, widespread
otrophs, as previously proposed8
; rather,
ain multiple, distantly related, photo-
erial groups, including some unrelated
types.
uired for the formation of bacteriochloro-
ystems in aerobic, anoxygenic, photo-
re clustered in a contiguous, 45-kilobase
n (superoperon)6
. These include bch and crt
nzymes of the bacteriochlorophyll and
pathways, and the puf genes coding for
harvesting complex (pufB and pufA) and
ex (pufL and pufM). To better describe the
planktonic, anoxygenic, photosynthetic
surface-water bacterial arti®cial chromo-
BAC 39B11
BAC 29C02
cDNA 20m10
BAC 52B02
BAC 65D09
BAC 24D02
99
100
100
54
67
Chloroflexus aurantiacus
b
envHOT2
envHOT3
100
Roseobacter denitrificans*
Roseobacter litoralis*
MBIC3951*
R2A84*
R2A62*
Rhodovulum sulfidophilum*
Rhodobacter sphaeroides
Rhodobacter capsulatus
Erythromicrobium ramosum
Erythrobacter litoralis*
MBIC3019*
Sphingomonas natatoria
Erythrobacter longus*
Rhodomicrobium vannielii
Rhodopseudomonas palustris
Rhodospirillum rubrum
Roseateles depolymerans
Rubrivivax gelatinosus
Rhodoferax fermentans
Rhodocyclus tenuis
Allochromatium vinosum
Thiocystis gelatinosa
Chloroflexus aurantiacus
0.1
α-3
α-4
α-2
α-1
β
γ
100
100
100
100
100
100
69
98
61
65
100
82
95
69
100
52
91
100
95
Figure 1 Phylogenetic relationships of pufM gene (a) and rRNA (b) sequences of AAP
bacteria. a, b, Evolutionary distances for the pufM genes (a) were determined from an
alignment of 600 nucleotide positions, and for rRNA genes (b) from an alignment of 860
nucleotide sequence positions. Evolutionary relationships were determined by neighbour-
joining analysis (see Methods). The green non-sulphur bacterium Chloro¯exus
aurantiacus was used as an outgroup. pufM genes that were ampli®ed by PCR in this
study are indicated by the env pre®x, with `m' indicating Monterey, and HOT indicating
Hawaii ocean time series. Cultivated aerobes are marked in light blue, bacteria cultured
from sea water are marked with an asterisk, and environmental cDNAs are marked in red.
Photosynthetic a-, b- and g-proteobacterial groups are indicated by the vertical bars to
rRNA PUF
a, b, Evolutionary distances for the
pufM genes (a) were determined from
an alignment of 600 nucleotide
positions, and for rRNA genes (b) from
an alignment of 860 nucleotide
sequence positions. Evolutionary
relationships were determined by
neighbour-joining analysis (see
Methods). The green non-sulphur
bacterium Chloroflexus aurantiacus
was used as an outgroup. pufM genes
that were amplified by PCR in this
study are indicated by the env prefix,
with 'm' indicating Monterey, and HOT
indicating Hawaii ocean time series.
Cultivated aerobes are marked in light
blue, bacteria cultured from sea water
are marked with an asterisk, and
environmental cDNAs are marked in
red. Photosynthetic -, - and -
proteobacterial groups are indicated
by the vertical bars to the right of the
tree. Bootstrap values greater than
50% are indicated above the
branches. The scale bar represents
number of substitutions per site.

60. NATURE |VOL 415 |7 FEBRUARY 2002 |www.nature.com 631
complementary DNA. However, a different primer targeting a from which the BACs originated are functional.
BAC 65D09
crt bch puf
hemN
orf276
orf154
orf227
puhA
lhaA
bch
ppsR
bch
Rubrivivax
gelatinosus
Rhodobacter
sphaeroides
AB L M C G PIH B N FM LBCDAE F C X Y Z
C X Y Z L M C ABB C EI F H B N FM L G P
HBNF MLGP J EBE IF D A I DCXYZLM BAX
orf358
orf440
H B N FM LGPB C EI FDAID C X Y Z L MB AQO
BAC 60D04
cycA
Q
orf641
OC
tspO
ppsR
ppa
bchpufbchcrt bchbch
puhA
lhaA
orf213
orf128
orf277
orf292
idi
idi
Figure 2 Schematic comparison of photosynthetic operons from R. gelatinosus (b-
proteobacteria), R. sphaeroides (a-proteobacteria) and uncultured environmental BACs.
ORF abbreviations use the nomenclature de®ned in refs 13, 14 and 24. Predicted ORFs
are coloured according to biological category: green, bacteriochloropyll biosynthesis
genes; orange, carotenoid biosynthesis genes; red, light-harvesting and reaction centre
genes; and blue, cytochrome c2. White boxes indicate non-photosynthetic and
hypothetical proteins with no known function. Homologous regions and genes are
connected by shaded vertical areas and lines, respectively.
© 2002 Macmillan Magazines Ltd

61. letters to nature
We compared several photosynthetic genes found on the oceanic
bacteriochlorophyll superoperons with characterized homologues
from cultured photosynthetic bacteria. The relationships among
Bchla biosynthetic proteins BchB (a subunit of light-independent
protochlorophyllide reductase) and BchH (magnesium chelatase)
were determined18
(Fig. 3). Similar to pufM relationships, BchB and
operon) to cultivated Erythrobacter species.
Recently, it has been suggested that cultivated Erythrobacter
species (a-4 subclass of proteobacteria) may represent the pre-
dominant AAPs in the upper ocean8,20
. Surprisingly, we were not
able to retrieve photosynthetic operon genes belonging to this group
in any of the samples analysed. Furthermore, very few sequences
BAC 60D04
BAC 65D09
BAC 29C02
ba BchH
100/96
100/100
100/99
100/100
100/100
100/100
100/100
BAC 60D04
BAC 65D09
BAC 29C02
100/66
100/100
100/94
100/100
100/100
100/100
BchB
0.1
0.1
Acidiphilium rubrum
Rhodobacter capsulatus
Rhodobacter sphaeroides
Rhodopseudomonas palustris
Rubrivivax gelatinosus
Chloroflexus aurantiacus
Chlorobium tepidum
Heliobacillus mobilis
Rhodopseudomonas palustris
Rhodobacter capsulatus
Rhodobacter sphaeroides
Acidiphilium rubrum
Heliobacillus mobilis
Chlorobium tepidum
Chloroflexus aurantiacus
Rubrivivax gelatinosus
Figure 3 Phylogenetic analyses of BchB and BchH proteins. a, Phylogenetic tree for the
BchB protein. b, Phylogenetic tree for the BchH protein. The BchH sequences from
Chlorobium vibrioforme25
and BchH2 and BchH3 from C. tepidum18
were omitted from the
tree because these genes potentially encode an enzyme for bacteriochlorophyll c
biosynthesis and are probably of distinct origin (J. Xiong, personal communication).
Bootstrap values (neighbour-joining/parsimony method) greater than 50% are indicated
next to the branches. The scale bar represents number of substitutions per site. The
position of Acidiphilium rubrum (bold branch) was not well resolved by both methods.

62. opsins. Furthermore, palE6
to its Monterey Bay
d transport of protons in
udich et al., manuscript in
Antarctica (E. F. DeLong,
in primers, and sequenced
ary sequence analyses based
ate that, despite differences
on spectra, the Antarctic
cterium highly related to
bacteria of Monterey Bay10
om the HOT station were
mples. Most proteorhodop-
ce waters belonged to the
he amino-acid level) to the
. In contrast, most of the
within the Antarctic clade
om the HOT station, HOT
E. coli and their absorption
m their primary sequences,
th the Antarctic clade gave
whereas the proteorhodop-
e gave a green (527-nm)
al North Paci®c gyre, most
with maximal intensity near
s maintained over depth,
he surface, the energy peak
tween 400 and 650 nm. In
ows and the half bandwidth
m (Fig. 5). Considering the
mbers of the two different
advantage at different points along the depth-dependent light
0.06
0.05
0.04
0.03
0.02
0.01
1.0
0.8
0.6
0.4
0.2
0
350 400
75 m
5 m
Monterey
Bay
HOT 0 m
HOT 75 m
Antarctica
450 500
Wavelength (nm)
RelativeirradianceAbsorbance
550 600 650
Figure 5 Absorption spectra of retinal-reconstituted proteorhodopsins in E. coli
membranes. All-trans retinal (2.5 mM) was added to membrane suspensions in 100 mM
phosphate buffer, pH 7.0, and absorption spectra were recorded. Top, four spectra for
palE6 (Antarctica), HOT 75m4, HOT 0m1, and BAC 31A8 (Monterey Bay) at 1 h after
retinal addition. Bottom, downwelling irradiance from HOT station measured at six
wavelengths (412, 443, 490, 510, 555 and 665 nm) and at two depths, for the same
depths and date that the HOT samples were collected (0 and 75 m). Irradiance is plotted
relative to irradiance at 490 nm.

63. Community structure and metabolism
through reconstruction of microbial
genomes from the environment
Gene W. Tyson1
, Jarrod Chapman3,4
, Philip Hugenholtz1
, Eric E. Allen1
, Rachna J. Ram1
, Paul M. Richardson4
, Victor V. Solovyev4
,
Edward M. Rubin4
, Daniel S. Rokhsar3,4
& Jillian F. Banfield1,2
1
Department of Environmental Science, Policy and Management, 2
Department of Earth and Planetary Sciences, and 3
Department of Physics, University of California,
Berkeley, California 94720, USA
4
Joint Genome Institute, Walnut Creek, California 94598, USA
...........................................................................................................................................................................................................................
Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their
roles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report
reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other
genomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency of
genomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a different
individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level.
The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance
variants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous
recombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed the
pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme
environment.
The study of microbial evolution and ecology has been revolutio-
nized by DNA sequencing and analysis1–3
. However, isolates have
been the main source of sequence data, and only a small fraction of
microorganisms have been cultivated4–6
. Consequently, focus has
shifted towards the analysis of uncultivated microorganisms via
cloning of conserved genes5
and genome fragments directly from
the environment7–9
. To date, only a small fraction of genes have been
recovered from individual environments, limiting the analysis of
fluorescence in situ hybridization (FISH) revealed that all biofilms
contained mixtures of bacteria (Leptospirillum, Sulfobacillus and, in
a few cases, Acidimicrobium) and archaea (Ferroplasma and other
members of the Thermoplasmatales). The genome of one of these
archaea, Ferroplasma acidarmanus fer1, isolated from the Richmond
mine, has been sequenced previously (http://www.jgi.doe.gov/JGI_
microbial/html/ferroplasma/ferro_homepage.html).
A pink biofilm (Fig. 1a) typical of AMD communities was
articles
!63
Environmental Genome Shotgun
Sequencing of the Sargasso Sea
J. Craig Venter,1
* Karin Remington,1
John F. Heidelberg,3
Aaron L. Halpern,2
Doug Rusch,2
Jonathan A. Eisen,3
Dongying Wu,3
Ian Paulsen,3
Karen E. Nelson,3
William Nelson,3
Derrick E. Fouts,3
Samuel Levy,2
Anthony H. Knap,6
Michael W. Lomas,6
Ken Nealson,5
Owen White,3
Jeremy Peterson,3
Jeff Hoffman,1
Rachel Parsons,6
Holly Baden-Tillson,1
Cynthia Pfannkoch,1
Yu-Hui Rogers,4
Hamilton O. Smith1
chlorococcus, tha
photosynthetic bio
Surface water
were collected ab
from three sites o
February 2003. A
lected aboard the S
station S” in May
are indicated on F
S1; sampling prot
one expedition to
was extracted from
genomic libraries w
2 to 6 kb were m
prepared plasmid
RESEARCH ARTICLE

64. Shotgun metagenomics
shotgun
sequence
!64

65. Acid Mine Drainage 2004
clones).
The first step in assignment of scaffolds to organism types was to
represent a nearly complete genome of
uncultured Ferroplasma species distinct
this as Ferroplasma type II. The dominan
was unexpected before the genomic analy
We assigned the roughly 3£ coverage
Leptospirillum group III on the basis of rRN
up to 31 kb, totalling 2.66 Mb). Comparis
those assigned to Leptospirillum group
sequence divergence and only locally co
firming that the scaffolds belong to a rel
Leptospirillum group II. A partial 16S rR
Sulfobacillus thermosulfidooxidans was
assembled reads, suggesting very low cov
any Sulfobacillus scaffolds .2 kb were a
grouped with the Leptospirillum group II
We compared the 3£ coverage, low Gþ
4.12 Mb) to the fer1 genome in order to
types (Supplementary Fig. S6). Scaffold
identity to fer1 were assigned to an enviro
I genome (170 scaffolds up to 47 kb in
1.48 Mb of sequence). The remaining
scaffolds are tentatively assigned to G-pla
in this bin (62 kb) contains the G-plasma
scaffolds assigned to G-plasma comprise
partial 16S rRNA gene sequence from A-pl
unassembled reads, suggesting low covera
scaffolds from A-plasma .2 kb would be
bin. Although eukaryotes are present in th
in low abundance in the biofilm studied.
eukaryotes have been detected.
As independent evidence that the Lep
Ferroplasma type II genomes are nearly co
complement of transfer RNA synthetases
An almost complete set of these genes
Leptospirillum group III. The G-plasma bin
set of tRNA synthetases, consistent with in
scaffolds. In addition, we established
group II, Leptospirillum group III, Ferrop
type II and G-plasma bins contained onl
Figure 1 The pink biofilm. a, Photograph of the biofilm in the Richmond mine (hand
included for scale). b, FISH image of a. Probes targeting bacteria (EUBmix; fluorescein
isothiocyanate (green)) and archaea (ARC915; Cy5 (blue)) were used in combination with a
probe targeting the Leptospirillum genus (LF655; Cy3 (red)). Overlap of red and green
(yellow) indicates Leptospirillum cells and shows the dominance of Leptospirillum.
c, Relative microbial abundances determined using quantitative FISH counts.
NATURE | doi:10.1038/na2 ©2004 NaturePublishing Group !65

66. Bassham cycle (using type II ribulose 1,5-bisphosphate carboxy-
lase–oxygenase). All genomes recovered from the AMD system
by some, or all, organisms. Given the large number of ABC-type
sugar and amino acid transporters encoded in the Ferroplasma type
Figure 4 Cell metabolic cartoons constructed from the annotation of 2,180 ORFs
identified in the Leptospirillum group II genome (63% with putative assigned function) and
1,931 ORFs in the Ferroplasma type II genome (58% with assigned function). The cell
drainage stream (viewed in cross-section). Tight coupling between ferrous iron oxidation,
pyrite dissolution and acid generation is indicated. Rubisco, ribulose 1,5-bisphosphate
carboxylase–oxygenase. THF, tetrahydrofolate.
!66

67. Environmental Genome Shotgun
Sequencing of the Sargasso Sea
J. Craig Venter,1
* Karin Remington,1
John F. Heidelberg,3
Aaron L. Halpern,2
Doug Rusch,2
Jonathan A. Eisen,3
Dongying Wu,3
Ian Paulsen,3
Karen E. Nelson,3
William Nelson,3
Derrick E. Fouts,3
Samuel Levy,2
Anthony H. Knap,6
Michael W. Lomas,6
Ken Nealson,5
Owen White,3
Jeremy Peterson,3
Jeff Hoffman,1
Rachel Parsons,6
Holly Baden-Tillson,1
Cynthia Pfannkoch,1
Yu-Hui Rogers,4
Hamilton O. Smith1
We have applied “whole-genome shotgun sequencing” to microbial populations
collected en masse on tangential flow and impact filters from seawater samples
collected from the Sargasso Sea near Bermuda. A total of 1.045 billion base pairs
of nonredundant sequence was generated, annotated, and analyzed to elucidate
the gene content, diversity, and relative abundance of the organisms within
these environmental samples. These data are estimated to derive from at least
1800 genomic species based on sequence relatedness, including 148 previously
unknown bacterial phylotypes. We have identified over 1.2 million previously
unknown genes represented in these samples, including more than 782 new
chlorococcus, th
photosynthetic bi
Surface wate
were collected a
from three sites
February 2003. A
lected aboard the
station S” in Ma
are indicated on
S1; sampling pro
one expedition to
was extracted fro
genomic libraries
2 to 6 kb were
prepared plasmid
both ends to prov
Craig Venter Sc
nology Center on
ers (Applied Bi
Whole-genome ra
the Weatherbird II
4) produced 1.66
in length, for a tota
microbial DNA se
sequences were g
RESEARCH ARTICLE
!67
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68. Sargasso Sea
two groups of scaffolds representing two dis-
tinct strains closely related to the published
at depths ranging from 4ϫ to 36ϫ (indicated
with shading in table S3 with nine depicted in
Fig. 1. MODIS-Aqua satellite image of
ocean chlorophyll in the Sargasso Sea grid
about the BATS site from 22 February
2003. The station locations are overlain
with their respective identifications. Note
the elevated levels of chlorophyll (green
color shades) around station 3, which are
not present around stations 11 and 13.
Fig. 2. Gene conser-
vation among closely !68
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69. frames (5). A total of 69,901 novel genes be-
longing to 15,601 single link clusters were iden-
tified. The predicted genes were categorized
Fig. 5. Prochlorococcus-related scaffold 2223290 illustrates the assembly of a broad commu-
nity of closely related organisms, distinctly nonpunctate in nature. The image represents (A)
global structure of Scaffold 2223290 with respect to assembly and (B) a sample of the multiple
sequence alignment. Blue segments, contigs; green segments, fragments; and yellow segments,
stages of the assembly of fragments into the resulting contigs. The yellow bars indicate that
fragments were initially assembled in several different pieces, which in places collapsed to
form the final contig structure. The multiple sequence alignment for this region shows a
homogenous blend of haplotypes, none with sufficient depth of coverage to provide a
separate assembly.
Table 1. Gene count breakdown by TIGR role
category. Gene set includes those found on as-
semblies from samples 1 to 4 and fragment reads
from samples 5 to 7. A more detailed table, sep-
arating Weatherbird II samples from the Sorcerer II
samples is presented in the SOM (table S4). Note
that there are 28,023 genes which were classified
in more than one role category.
TIGR role category
Total
genes
Amino acid biosynthesis 37,118
Biosynthesis of cofactors,
prosthetic groups, and carriers
25,905
Cell envelope 27,883
Cellular processes 17,260
Central intermediary metabolism 13,639
DNA metabolism 25,346
Energy metabolism 69,718
Fatty acid and phospholipid
metabolism
18,558
Mobile and extrachromosomal
element functions
1,061
Protein fate 28,768
Protein synthesis 48,012
Purines, pyrimidines, nucleosides,
and nucleotides
19,912
Regulatory functions 8,392
Signal transduction 4,817
Transcription 12,756
Transport and binding proteins 49,185
Unknown function 38,067
Miscellaneous 1,864
Conserved hypothetical 794,061
Total number of roles assigned 1,242,230
Total number of genes 1,214,207
www.sciencemag.org SCIENCE VOL 304 2 APRIL 2004 69
!69
We closely examined the multiple sequence
alignments of the contigs with high SNP rates
and were able to classify these into two fairly
distinct classes: regions where several closely
related haplotypes have been collapsed, in-
creasing the depth of coverage accordingly
(10), and regions that appear to be a relatively
homogenous blend of discrepancies from the
consensus without any apparent separation into
haplotypes, such as the Prochlorococcus scaf-
fold region (Fig. 5). Indeed, the Prochlorococ-
cus scaffolds display considerable heterogene-
ity not only at the nucleotide sequence level
(Fig. 5) but also at the genomic level, where
multiple scaffolds align with the same region of
the MED4 (11) genome but differ due to gene
or genomic island insertion, deletion, rearrange-
ment events. This observation is consistent with
previous findings (12). For instance, scaffolds
2221918 and 2223700 share gene synteny with
each other and MED4 but differ by the insertion
of 15 genes of probable phage origin, likely
representing an integrated bacteriophage. These
genomic differences are displayed graphically
in Fig. 2, where it is evident that up to four
conflicting scaffolds can align with the same
region of the MED4 genome. More than 85%
of the Prochlorococcus MED4 genome can be
aligned with Sargasso Sea scaffolds greater
than 10 kb; however, there appear to be a
couple of regions of MED4 that are not repre-
sented in the 10-kb scaffolds (Fig. 2). The
larger of these two regions (PMM1187 to
PMM1277) consists primarily of a gene cluster
coding for surface polysaccharide biosynthesis,
which may represent a MED4-specific polysac-
charide absent or highly diverged in our Sar-
gasso Sea Prochlorococcus bacteria. The heter-
ogeneity of the Prochlorococcusscaffolds suggest
that the scaffolds are not derived from a single
discrete strain, but instead probably represent a
conglomerate assembled from a population of
closely related Prochlorococcus biotypes.
The gene complement of the Sargasso.
The heterogeneity of the Sargasso sequences
complicates the identification of microbial
genes. The typical approach for microbial an-
notation, model-based gene finding, relies en-
tirely on training with a subset of manually
eate scaffold borders.
Fig. 4. Circular diagrams of nine complete megaplasmids. Genes encoded in the forward direction
are shown in the outer concentric circle; reverse coding genes are shown in the inner concentric
circle. The genes have been given role category assignment and colored accordingly: amino acid
biosynthesis, violet; biosynthesis of cofactors, prosthetic groups, and carriers, light blue; cell
envelope, light green; cellular processes, red; central intermediary metabolism, brown; DNA
metabolism, gold; energy metabolism, light gray; fatty acid and phospholipid metabolism, magenta;
protein fate and protein synthesis, pink; purines, pyrimidines, nucleosides, and nucleotides, orange;
regulatory functions and signal transduction, olive; transcription, dark green; transport and binding
proteins, blue-green; genes with no known homology to other proteins and genes with homology
to genes with no known function, white; genes of unknown function, gray; Tick marks are placed
on 10-kb intervals.
2 APRIL 2004 VOL 304 SCIENCE www.sciencemag.org68
assembly to identify a set of large, deeply as-
sembling nonrepetitive contigs. This was used to
set the expected coverage in unique regions (to
23ϫ) for a final run of the assembler. This al-
lowed the deep contigs to be treated as unique
sequence when they would otherwise be labeled
as repetitive. We evaluated our final assembly
results in a tiered fashion, looking at well-sampled
genomic regions separately from those barely
sampled at our current level of sequencing.
The 1.66 million sequences from the
Weatherbird II samples (table S1; samples 1 to
4; stations 3, 11, and 13), were pooled and
assembled to provide a single master assembly
for comparative purposes. The assembly gener-
ated 64,398 scaffolds ranging in size from 826
bp to 2.1 Mbp, containing 256 Mbp of unique
sequence and spanning 400 Mbp. After assem-
bly, there remained 217,015 paired-end reads,
or “mini-scaffolds,” spanning 820.7 Mbp as
well as an additional 215,038 unassembled sin-
gleton reads covering 169.9 Mbp (table S2,
column 1). The Sorcerer II samples provided
almost no assembly, so we consider for these
samples only the 153,458 mini-scaffolds, span-
ning 518.4 Mbp, and the remaining 18,692
singleton reads (table S2, column 2). In total,
1.045 Gbp of nonredundant sequence was gen-
erated. The lack of overlapping reads within the
unassembled set indicates that lack of addition-
al assembly was not due to algorithmic limita-
tions but to the relatively limited depth of se-
quencing coverage given the level of diversity
within the sample.
The whole-genome shotgun (WGS) assembly
has been deposited at DDBJ/EMBL/GenBank
under the project accession AACY00000000,
and all traces have been deposited in a corre-
sponding TraceDB trace archive. The version
described in this paper is the first version,
AACY01000000. Unlike a conventional WGS
entry, we have deposited not just contigs and
scaffolds but the unassembled paired singletons
and individual singletons in order to accurate-
ly reflect the diversity in the sample and
allow searches across the entire sample with-
in a single database.
Genomes and large assemblies. Our
analysis first focused on the well-sampled ge-
nomes by characterizing scaffolds with at least
3ϫ coverage depth. There were 333 scaffolds
comprising 2226 contigs and spanning 30.9
Mbp that met this criterion (table S3), account-
ing for roughly 410,000 reads, or 25% of the
pooled assembly data set. From this set of well-
sampled material, we were able to cluster and
classify assemblies by organism; from the rare
species in our sample, we used sequence similar-
ity based methods together with computational
gene finding to obtain both qualitative and quan-
titative estimates of genomic and functional diver-
sity within this particular marine environment.
We employed several criteria to sort the
major assembly pieces into tentative organism
“bins”; these include depth of coverage, oligo-
nucleotide frequencies (7 ), and similarity to
previously sequenced genomes (5). With these
techniques, the majority of sequence assigned
to the most abundant species (16.5 Mbp of the
30.9 Mb in the main scaffolds) could be sepa-
rated based on several corroborating indicators.
In particular, we identified a distinct group of
scaffolds representing an abundant population
clearly related to Burkholderia (fig. S2) and
two groups of scaffolds representing two dis-
tinct strains closely related to the published
Shewanella oneidensis genome (8) (fig. S3).
There is a group of scaffolds assembling at over
6ϫ coverage that appears to represent the ge-
nome of a SAR86 (table S3). Scaffold sets
representing a conglomerate of Prochlorococ-
cus strains (Fig. 2), as well as an uncultured
marine archaeon, were also identified (table S3;
Fig. 3). Additionally, 10 putative mega plasmids
were found in the main scaffold set, covered
at depths ranging from 4ϫ to 36ϫ (indicated
with shading in table S3 with nine depicted in
Fig. 1. MODIS-Aqua satellite image of
ocean chlorophyll in the Sargasso Sea grid
about the BATS site from 22 February
2003. The station locations are overlain
with their respective identifications. Note
the elevated levels of chlorophyll (green
color shades) around station 3, which are
not present around stations 11 and 13.
Fig. 2. Gene conser-
vation among closely
related Prochlorococ-
cus. The outermost
concentric circle of
the diagram depicts
the competed genom-
ic sequence of Pro-
chlorococcus marinus
MED4 (11). Fragments
from environmental
sequencing were com-
pared to this complet-
ed Prochlorococcus ge-
nome and are shown in
the inner concentric
circles and were given
boxed outlines. Genes
for the outermost cir-
cle have been as-
signed psuedospec-
trum colors based on
the position of those
genes along the chro-
mosome, where genes
nearer to the start of
the genome are col-
ored in red, and genes
nearer to the end of the genome are colored in blue. Fragments from environmental sequencing
were subjected to an analysis that identifies conserved gene order between those fragments and
the completed Prochlorococcus MED4 genome. Genes on the environmental genome segments
that exhibited conserved gene order are colored with the same color assignments as the
Prochlorococcus MED4 chromosome. Colored regions on the environmental segments exhibiting
color differences from the adjacent outermost concentric circle are the result of conserved gene
order with other MED4 regions and probably represent chromosomal rearrangements. Genes that
did not exhibit conserved gene order are colored in black.
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70. rRNA phylotyping from metagenomics
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71. Shotgun Sequencing Allows Alternative Anchors (e.g., RecA)
!71
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72. nomic group using the phylogenetic analysis
described for rRNA. For example, our data set
marker genes, is roughly comparable to the
97% cutoff traditionally used for rRNA. Thus
Fig. 6. Phylogenetic diversity of Sargasso Sea sequences using multiple phylogenetic markers. The
relative contribution of organisms from different major phylogenetic groups (phylotypes) was
measured using multiple phylogenetic markers that have been used previously in phylogenetic
studies of prokaryotes: 16S rRNA, RecA, EF-Tu, EF-G, HSP70, and RNA polymerase B (RpoB). The
relative proportion of different phylotypes for each sequence (weighted by the depth of coverage
of the contigs from which those sequences came) is shown. The phylotype distribution was
determined as follows: (i) Sequences in the Sargasso data set corresponding to each of these genes
were identified using HMM and BLAST searches. (ii) Phylogenetic analysis was performed for each
phylogenetic marker identified in the Sargasso data separately compared with all members of that
gene family in all complete genome sequences (only complete genomes were used to control for
the differential sampling of these markers in GenBank). (iii) The phylogenetic affinity of each
sequence was assigned based on the classification of the nearest neighbor in the phylogenetic tree.
RIL 2004 VOL 304 SCIENCE www.sciencemag.org !72
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73. Functional Inference from Metagenomics
• Can work well for individual genes
!73

74. Functional Diversity of Proteorhodopsins?
!74
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75. ARTICLE
Received 3 Nov 2010 | Accepted 11 Jan 2011 | Published 8 Feb 2011 DOI: 10.1038/ncomms1188
Proteorhodopsins are light-driven proton pumps involved in widespread phototrophy.
Discovered in marine proteobacteria just 10 years ago, proteorhodopsins are now known
to have been spread by lateral gene transfer across diverse prokaryotes, but are curiously
absent from eukaryotes. In this study, we show that proteorhodopsins have been acquired
by horizontal gene transfer from bacteria at least twice independently in dinoflagellate
protists. We find that in the marine predator Oxyrrhis marina, proteorhodopsin is indeed the
most abundantly expressed nuclear gene and its product localizes to discrete cytoplasmic
structures suggestive of the endomembrane system. To date, photosystems I and II have
been the only known mechanism for transducing solar energy in eukaryotes; however, it now
appears that some abundant zooplankton use this alternative pathway to harness light to
A bacterial proteorhodopsin proton pump
in marine eukaryotes
Claudio H. Slamovits1,†
, Noriko Okamoto1
, Lena Burri1,†
, Erick R. James1
& Patrick J. Keeling1
http://www.nature.com/ncomms/journal/v2/n2/abs/ncomms1188.html

76. ARTICLENATURE COMMUNICATIONS | DOI: 10.1038/ncomms1188
NATURE COMMUNICATIONS | 2:183 | DOI: 10.1038/ncomms1188 | www.nature.com/naturecommunications
© 2011 Macmillan Publishers Limited. All rights reserved.
50
100
Neurospora crassa
51 Aspergillus fumigatus
Gibberella zeae
90
‘Oryza_sativa’
95 Leptosphaeria maculans
97 Pyrenophora tritici
Bipolaris oryzae
100 Oxyrrhis marina OM2197
Dinoflagellate 3
Dinoflagellate 1
Dinoflagellate 2
O. marina OM1497
Cyanophora paradoxa
80
Guillardia theta 1490
99
100 Rhodomonas salina 987
G. theta t309
62
Cryptomonas sp.
G. tetha 2135
100 G. theta 2
G. theta 1712
99 Haloterrigena sp.
68 Haloarcula argentinensis
Haloquadratum walsbyi
100
Nostoc sp.
64
Natronomonas pharaonis
Halorubrum sodomense
62 Haloterrigena sp.
Halobacterium salinarum
100
89
58
Salinibacter ruber (Xanthorhodopsin)
uncult. marine picoplankton (Hawaii)
Thermus aquaticus
Gloeobacter violaceus
Roseiflexus sp.
52
100 Env. SS AACY01492695
Env. SS AACY01598379
99
GS012-3
73 GS020-43
98 GS020-39
GS020-70
99
GS033-3
56
100 GS012-21
53
MWH-Dar4
MWH-Dar1
87
GS012-37
98
GS020-82
GS012-39
EgelM2-3.D
GS012-14
86
100 uncult. marine bacterium EB0_41B09 (Monterey Bay)
79 Methylophilales bacterium
Beta proteobacterium KB1
59 Marinobacter sp.
Alpha proteobacterium BAL199
Octadecabacter antarcticus
92
100 uncult. GOS 7929496
uncult. GOS ECY53283
100
Alexandrium catenella
53
Pyrocystis lunula
‘Amphioxus’
83
O. marina 27
91 O. marina 344
O. marina 5001
91
O. marina 173
O. marina 72
55
uncult. marine picoplankton 2 (Hawaii)
65
75 Exiguobacterium sibiricum
100 GS013_1
GS033_122
91
100 Env. SS AACY01002357
98 Tenacibaculum sp.
Psychroflexus torquis
95
Env. SS AACY01054069
76
uncult. marine group II euryarchaeote HF70_39H11
69
52
uncult. gamma proteobacterium eBACHOT4E07
Env. SS AACY01043960
86 uncult. marine bacterium HF130_81H07
Env. SS AACY01120795
100
Env. SS AACY01118955
79
uncult. marine proteobacterium ANT32C12
77
uncult. bacterium MedeBAC35C06
99 uncult. bacterium
uncult. marine gamma proteobacterium EBAC31A08
51 uncult. marine alpha proteobacterium HOT2C01
100 uncult. marine bacterium EB0_41B09
Photobacterium sp.
50
Env. SS AACY01499050
70
uncult. marine gamma proteobacterium HTCC2207
uncult. bacterium eBACred22E04
100 Karlodinium micrum 02
K. micrum 01
100 uncult. bacterium eBACmed86H08
Pelagibacter ubique
50
Env. SS AACY01015111
uncult. marine bacterium 66A03
80
100 GS033_85
Vibrio harveyi
76
Rhodobacterales bacterium
uncult. bacterium MedeBAC82F10
55
uncult. marine bacterium 66A03 (2)
uncult. bacterium MEDPR46A6
Fungal ( H+ / ?)
Halorhodopsins
(H+
/ Cl
–
)
Algal opsins
(sensory)
Proteorhodopsins
(H+)
Figure 1 | Phylogenetic distribution of dinoflagellate rhodopsins. Protein sequences of 96 rhodopsins encompassing the known diversity of microbial
(type I) rhodopsins from the three domains of life10
were used to generate a maximum likelihood phylogenetic tree (See Supplementary Table S1 for accession
numbers). Grey boxes distinguish the recognized groupings of type 1 rhodopsin, and the prevalent function in each group is shown: proton pumps (H+
), sensory,
chlorine pumps (Cl−
) and unknown (?). Numbers indicate bootstrap support when 50% (over 300 replicates). Black boxes highlight dinoflagellate genes:
Dinoflagellate 1 is a large group of proteorhodopsins present in diverse dinoflagellates; dinoflagellate 2 includes two K. micrum proteorhodopsin genes
of independent origin; dinoflagellate 3 includes two O. marina genes related to algal sensory rhodopsins, probably of endosymbiotic origin.

77. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1188
NATURE COMMUNICATIONS | 2:183 | DOI: 10.1038/ncomms1188 | www.nature.com/naturecommunications
© 2011 Macmillan Publishers Limited. All rights reserved.
SAR-86 31A08
Karlodinium micrum
Oxyrrhis 27
Oxyrrhis 5001
Alexandrium
Pyrocystis lunula
‘Amphioxus’
Oxyrrhis 2197
XR Salinibacter ruber
1
1
1
1
1
1
1
1
1
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71
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62
62
62
62
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62
M K L L L I L G SV I A L P T F A A G G G DL DA SDY T GV SF WL V T A A L L A S TV F F FV ER DR V S AK WK T S LT V S GL V T G I A F WH
- - - - M GA PM S S TK PV DN P A DA F L QP ND GV A I S F WI I S I A M I A A T A F F FA EA ST VK AHWK T T LH V G A L V T LV A G VH
- - - - - - - - - - - M A P LA Q DWT YA EW SA V Y NA L S F- G I - A GM G S AT I F FW LQ L P NV T K NY RT A L T I T G I V T L I A T Y H
- - - - - - - - - - - M A P LA GD F S YG EW NA V Y NA L S F- G I - A AM G S AT V F FW LQ L P NV T R SY RT A L T I T G I V TW I A T Y H
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L V M L V FG YM G E AG - - I M A - - - AW PA F I I G CL AW VY M I Y E LWA G EG K S AC NT A S P- A V Q S AY NT MM Y I I I F G WA I Y
V VM L L FG Y L GE I A - - V V P- - - K L I G F I L G MC GW F F I L N E I F LG EA G G TA K D C S E- A I S S A F SNMR L I V T VG WA I Y
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A VM V A L G YP GE IQ DN L A V - - - RW G WWA L AM I P F F YV V YS L L SG LG EA - T A R QP E- SV GG LV SA A RY LT A V SW LT Y
A LM V A L G YP GE IQ DD L T V- - - RW VWWG LA M I P F C YV V YE LV V GL ND A - T K RQ A S AT V S S LI S S AR Y L T V I S WC TY
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A LM V A L G YP GE IQ D D N S Q- - - RW I WWA L A M I P F C YV V NT L L V G L S GA - T ER QP A - A A KG L I V K AR Y L T A I S WL T Y
M LM I T A G Y I GA ST EQ FV - - - - - WQG FG V S MV F F I L V L GY L- - GD GV L- A L D E DS - K NT GT A R NL FW LT V L I W CT Y
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P LG YV LG M- - - - - M I G S- - E GD V F LN V T YN I A D F VN K I A F V L AC - W SC AK T D SA SK T D A L L P - - - - - - - - - - - - -
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P FV Y I V K D- - - - - I G L SG PT A T MY EQ VG Y S VA EV V AK G- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
P GV Y I I K S- - - - - M G L AG N I A T T YE QV GY SV A D V V AK AV FG V L I - WA I A A GK SD E E EK NG L L G- - - - - - - - - - - -
P FV Y I I K M- - - - - VG L S GA FA T C A E Q I GY S I SD V T A K AV FG V L I - WA I A A A K S ED E- - - - - - - - - - - - - - - - - - -
P LY FV L E HT - - - - M G L S- - - - T F Q E I L CY G I SD V L AK VV FA L V LV YN FD D D DE PA V YQ Q Q MV VMQ Q Q QP MV T T I G
P I A Y M I P MA FP EA FP SN T P GT I V A L QV GY T I A D V L AK AG YG V L I - Y N I A K A K S E E EG FN V S EM V E PA T A SA - - - -
A B
C D
E F
G
SAR-86 31A08
Karlodinium micrum
Oxyrrhis 27
Oxyrrhis 5001
Alexandrium
Pyrocystis lunula
‘Amphioxus’
Oxyrrhis 2197
XR Salinibacter ruber
SAR-86 31A08
Karlodinium micrum
Oxyrrhis 27
Oxyrrhis 5001
Alexandrium
Pyrocystis lunula
‘Amphioxus’
Oxyrrhis 2197
XR Salinibacter ruber
SAR-86 31A08
Karlodinium micrum
Oxyrrhis 27
Oxyrrhis 5001
Alexandrium
Pyrocystis lunula
‘Amphioxus’
Oxyrrhis 2197
XR Salinibacter ruber
Exterior
Interior
N
Y
V
A
S
W
E
A
Y
T
W
D
QA
L
P
A
M
A L S
F G I
A G M
G S A
T I F
F W L
Q
L
P N V T K N Y R
T
A
L T I
T G I
V T L
I A T
Y H Y
F R I
F
N
S
W
V
A
A F
N
V
G
L
G
V
N
G
A
Y
E
V
T
V
S
G
T P
F
N
D
A Y
R
Y V
D W L L
T V P
L L L
V E L
I L V
M K L
P A K E
T V C L
A W T
L G I
A S A
V M V
A L G
Y
P
G E I Q D D L S V
R
W
F W W
A C A
M V P
F V Y
V V G
T L V
V
G
L
G
A
A
T A K Q P E G V V
D
L
V
S
A
A
R Y L T
V V S
W L T
Y P F
V Y I
V K N
I
G
L A G S T A T M Y
E
Q
I G Y S
A A D
V T A
K A V
F G V
L I W
A
I
A
N
A
K
S
R
L
E
E
E
G K
L
R
A
101/97
112/108
A B C D E F G
109/105
Helix
Membrane 237/231
Figure 2 | Structural and comparative analysis of a dinoflagellate proteorhodopsin. (a) Amino-acid alignment of various rhodopsins from bacteria and
dinoflagellates: SAR86-31A08 is a functionally characterized proteorhodopsin1
; K. micrum is a ‘Dinoflagellate group 2’ in Figure 1; Oxyrrhis 2197 represents
‘Dinoflagellate 3’; XR S. ruber is a Xanthorhodopsin; the remaining sequences belong to ‘Dinoflagellate group 1’. Numbers on the right indicate residue
number. Black rectangles show predicted transmembrane segments. Functional sites (coloured triangles): blue, proton acceptor and donor; green, spectral
tuning (L109/105
for green and Q for blue); red, lysine linked to the cofactor retinal. Epitope for antibody preparation is indicated with an orange rectangle.
(b) Secondary structure of the O. marina proteorhodopsin predicted using PHD (http://www.predictprotein.org/). Single-letter amino-acid codes are
used, and numbers correspond to the positions in O. marina and 31A081
, respectively. Functional residues are highlighted as follows. Blue: proton acceptor
(D101/97
) and proton donor (E112/108
); green: spectral tuning; red: retinal pocket; red filled: lysine linked to retinal; orange: epitope for antibody.

78. ARTICLENATURE COMMUNICATIONS | DOI: 10.1038/ncomms1188
Rhodopsin function requires the prosthetic group retinal, sug-
gesting that retinal biosynthesis should also be present in O. marina.
a distinctive transit peptide19
. There is no evidence of such a pep-
tide on any O. marina rhodopsin gene, arguing against a location
DIC Rhodopsin MitoTracker DAPI Merge
260
60
40
30
20
15
Figure 3 | Cellular localization of proteorhodopsin in O. marina cells. (a) Western blot of total O. marina protein probed with an antibody raised against
the C-terminal peptide of the proteorhodopsin OM27 from O. marina. Expected protein size is 28kDa. (b) Localization of proteorhodopsin in O. marina
cells using immunofluorescence assay with the same antibody. Antibody signal forms small irregular and ring-like structures independent of mitochondria
in O. marina. Three independent cells are shown, each showing (left to right) differential interference contrast (DIC) light micrograph, anti-OM27
proteorhodopsin, MitoTracker staining, Hoechst 33258 staining for DNA and a merge of all four. White bar=10 m. See Supplementary Movies 1–4 for a
360° video rendering. DAPI, 4,6-diamidino-2-phenylindole.

79. 1
Primer
The Light-Driven Proton Pump Proteorhodopsin
Enhances Bacterial Survival during Tough Times
Edward F. DeLong1,2
*, Oded Be´ja`3
*
1 Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 2 Department of
Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 3 Faculty of Biology, Technion - Israel Institute of
Technology, Technion City, Haifa, Israel
Some microorganisms contain proteins that can interact with
light and convert it into energy for growth and survival, or into
sensory information that guides cells towards or away from light.
The simplest energy-harvesting photoproteins are the rhodopsins,
which consist of a single, membrane-embedded protein covalently
bound to the chromophore retinal (a light-sensitive pigment) [1].
One class of archaeal photoproteins (called bacteriorhodopsin) was
shown to function as a light-driven proton pump, generating
biochemical energy from light [2,3]. For many years, these light-
driven proton pumps were thought to be found only in relatively
obscure Archaea living in high salinity.
Ten years ago, a new type of microbial rhodopsin (proteorho-
dopsin) was discovered in marine planktonic bacterial assemblages
[4]. This proteorhodopsin was co-localized on a large genome
fragment containing the small subunit ribosomal RNA gene,
which identified its genomic source—an uncultured gammapro-
teobacterium (of the SAR86 bacterial lineage). Further work
showed that proteorhodopsin could be expressed in Escherichia coli,
Cultivation-independent genomic surveys (e.g., ‘‘metage-
nomics’’) revealed proteorhodopsin presence and diversity, and
heterologous expression in E. coli demonstrated many of its
functional properties. Access to cultivable marine bacteria that
contain proteorhodopsin, however, would be very useful to further
characterize its native function in diverse physiological contexts.
Whole-genome sequencing then came to the rescue. The
Gordon and Betty Moore Foundation (GBMF) Microbial Genome
Sequencing Project (http://www.moore.org/microgenome) unex-
pectedly revealed that many culturable marine bacteria submitted
for sequencing (including Pelagibacter spp., Vibrio spp., and
Flavobacteria isolates) in fact possessed proteorhodopsin genes
(Table 1). What can these proteorhodopsin-containing isolates tell
us? Experiments with the proteorhodopsin-containing isolate
‘Cand. P. ubique’ (a member of the SAR11 bacterial lineage, the
most abundant bacterial group in the ocean [19,20]) showed no
significant light enhancement of growth rate or yield [11]. Later,
http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1000359#pbio-1000359-g002

80. Table 1. Marine bacterial isolates and genome fragments containing proteorhodopsins.
Organism Strain General Group Reference
Genomes
Methylophilales HTCC2181 Betaproteobacteria GBMF
Rhodobacterales sp. HTCC2255 Alphaproteobacteria GBMF
Vibrio angustum S14 Gammaproteobacteria GBMF
Photobacterium SKA34 Gammaproteobacteria GBMF
Vibrio harveyi ATCC BAA-1116 Gammaproteobacteria GenBank # CP000789
Marine gamma HTCC2143 Gammaproteobacteria GBMF
Marine gamma HTCC2207 Gammaproteobacteria GBMF
Cand. P. ubique HTCC1002 Alphaproteobacteria GBMF
Cand. P. ubique HTCC1062 Alphaproteobacteria [26]
Rhodospirillales BAL199 Alphaproteobacteria GBMF
Marinobacter ELB17 Gammaproteobacteria GBMF
Vibrio campbelli AND4 Gammaproteobacteria GBMF
Vibrio angustum S14 Gammaproteobacteria GBMF
Dokdonia donghaensis MED134 Flavobacteria GBMF
Polaribacter dokdonensis MED152 Flavobacteria GBMF
Psychroflexus ATCC700755 Flavobacteria GBMF
Polaribacter irgensii 23-P Flavobacteria GBMF
Flavobacteria bacterium BAL38 Flavobacteria GBMF
BACs and fosmids
HF10_05C07 Proteobacteria [24]
HF10_45G01 Proteobacteria [24]
HF130_81H07 Gammaproteobacteria [24]
EB0_39F01 Alphaproteobacteria [24]
EB0_39H12 Proteobacteria [24]
EB80_69G07 Alphaproteobacteria [24]
EB80_02D08 Gammaproteobacteria [24]
EB0_35D03 Proteobacteria [24]
EB0_49D07 Proteobacteria [24]
EBO_50A10 Gammaproteobacteria [24]
EB0_55B11f Alphaproteobacteria [24]
EBO_41B09 Betaproteobacteria [24]
HF10_19P19 Proteobacteria [17]
HF10_25F10 Proteobacteria [17]
HF10_49E08 Planctomycetes [24]
HF10_12C08 Alphaproteobacteria [24]
HF10_29C11 Euryarchaea [24]
MED13K09 unknown [10]
MED18B02 unknown [10]
MED35C06 unknown [10]
MED42A11 unknown [10]
MED46A06 unknown [10]
MED49C08 unknown [10]
MED66A03 unknown [10]
MED82F10 unknown [10]
MED86H08 unknown [10]
RED17H08 unknown [10]
RED22E04 unknown [10]
eBACHOT4E07 Gammaproteobacteria [25]
EBAC20E09 Gammaproteobacteria [25]
PLoS Biology | www.plosbiology.org 2 April 2010 | Volume 8 | Issue 4 | e1000359
strategy on marine bacterioplankton could be substantial given the
‘‘feast or famine’’ existence experienced by many of these
microbes. The Vibrio/proteorhodopsin model system is likely to
reveal further secrets on the nature and function of proteorho-
dopsin photosytems in bacteria that are usually (but erroneously)
considered as strict heterotrophs not capable of utilizing light at all.
While this study [16] adds an important new result, it certainly
does not solve the whole puzzle of proteorhodopsin photophysiol-
ogy. Considering the staggering variety of genetic, physiological
and environmental contexts in which proteorhodopsin and related
photoproteins are found, a great variety of light-dependent
adaptive strategies are likely to occur in the natural microbial
world. For example, in 2005, a new type of bacterial rhodopsin
(xanthorhodopsin) was discovered in the salt-loving bacterium
Salinibacter ruber [28]. Xanthorhodopsin is a proton-pumping
retinal protein/carotenoid complex in which the carotenoid
Box 1. A Decade of Proteorhodopsin
Milestones
2000 NFirst proteorhodopsin gene found in uncultured
SAR86 using metagenomics; proteorhodopsin
light-driven proton pump activity confirmed in
heterologous E. coli cells [4].
2001 NProteorhodopsin presence confirmed directly in
the ocean using laser flash photolysis [5].
2003 NProteorhodopsin genes also found in other
bacterial groups [8].
2004 NEnormous diversity of proteorhodopsin genes
found in the Sargasso Sea using metagenomics [9].
2005 NRetinal biosynthesis pathways found in metage-
nomic data and confirmed using E. coli cells [10].
NProteorhodopsin genes are found in ‘Canditatus
Pelagibacter ubique’ (SAR11), the most abundant
bacterium on earth; environmental SAR11 proteor-
hodopsin presence confirmed using metaproteo-
mics [11].
2006 NProteorhodopsin genes found in uncultured
marine Archaea [12].
2007 NFirst indication of proteorhodopsin light-depen-
dent growth in cultured Flavobacteria [13] (see
Figure 1 for colony morphologies and pigmenta-
tion).
2008 NProteorhodopsin genes found in non-marine
environments [14,15].
2010 NProteorhodopsin phototrophy directly confirmed
using a genetic system in marine Vibrio sp. [16]
Organism Strain General Group Reference
HOT2C01 unknown [8]
EBAC31A08 Gammaproteobacteria [4]
ANT32C12 unknown [8]
HF70_39H11_ArchHighGC unknown [12]
HF10_3D09_mediumGC unknown [12]
HF70_19B12_highGC unknown [12]
HF70_59C08 unknown [12]
Marine microbial isolates and large genome fragments from the environment GBMF, microbial genomes sequenced as part of the Gordon and Betty Moore Foundation
microbial genome sequencing project (http://www.moore.org/microgenome), found to encode proteorhodopsin genes. The list includes whole genome sequences
from a wide array of cultivated marine microorganisms (Genomes), as well as cloned large DNA fragments (BACs and fosmids) recovered directly from the environment.
doi:10.1371/journal.pbio.1000359.t001
Table 1. Cont.
Figure 1. Various colony morphologies and coloration of
different proteorhodopsin-containing bacteria used to study
proteorhodopsin phototrophy. From top to bottom, the flavobac-
terium Polaribacter dokdonensis strain MED152 used to show proteor-
hodopsin light stimulated growth [13]; the flavobacterium Dokdonia
donghaensis strain MED134 used to show proteorhodopsin light
stimulated CO2-fixation [23]; and Vibrio strain AND4 used to show
proteorhodopsin phototrophy [16]; note the lack of detectable
pigments in Vibrio strain AND4. However, when these vibrio cells are
pelleted, they do show a pale reddish color, which is the result of
proteorhodopsin pigments presence in their membranes. Photos are
courtesy of Jarone Pinhassi.
doi:10.1371/journal.pbio.1000359.g001
PLoS Biology | www.plosbiology.org 3 April 2010 | Volume 8 | Issue 4 | e1000359

81. Box 1. A Decade of Proteorhodopsin
Milestones
2000 NFirst proteorhodopsin gene found in uncultured
SAR86 using metagenomics; proteorhodopsin
light-driven proton pump activity confirmed in
heterologous E. coli cells [4].
2001 NProteorhodopsin presence confirmed directly in
the ocean using laser flash photolysis [5].
2003 NProteorhodopsin genes also found in other
bacterial groups [8].
2004 NEnormous diversity of proteorhodopsin genes
found in the Sargasso Sea using metagenomics [9].
2005 NRetinal biosynthesis pathways found in metage-
nomic data and confirmed using E. coli cells [10].
NProteorhodopsin genes are found in ‘Canditatus
Pelagibacter ubique’ (SAR11), the most abundant
bacterium on earth; environmental SAR11 proteor-
hodopsin presence confirmed using metaproteo-
mics [11].
2006 NProteorhodopsin genes found in uncultured
marine Archaea [12].
2007 NFirst indication of proteorhodopsin light-depen-
dent growth in cultured Flavobacteria [13] (see
Figure 1 for colony morphologies and pigmenta-
tion).
2008 NProteorhodopsin genes found in non-marine
environments [14,15].
2010 NProteorhodopsin phototrophy directly confirmed
using a genetic system in marine Vibrio sp. [16]
Figure 1. Various col
different proteorhodo
proteorhodopsin phot
terium Polaribacter dokdo
hodopsin light stimulated
donghaensis strain MED
stimulated CO2-fixation [
proteorhodopsin photot
pigments in Vibrio strain
pelleted, they do show
proteorhodopsin pigmen
courtesy of Jarone Pinhas
doi:10.1371/journal.pbio.1

82. Pretty proteorhodopsin
marine bacterioplankton could be substantial given the
famine’’ existence experienced by many of these
The Vibrio/proteorhodopsin model system is likely to
her secrets on the nature and function of proteorho-
tosytems in bacteria that are usually (but erroneously)
as strict heterotrophs not capable of utilizing light at all.
is study [16] adds an important new result, it certainly
lve the whole puzzle of proteorhodopsin photophysiol-
dering the staggering variety of genetic, physiological
nmental contexts in which proteorhodopsin and related
ns are found, a great variety of light-dependent
rategies are likely to occur in the natural microbial
example, in 2005, a new type of bacterial rhodopsin
(xanthorhodopsin) was discovered in the salt-loving bacterium
Salinibacter ruber [28]. Xanthorhodopsin is a proton-pumping
retinal protein/carotenoid complex in which the carotenoid
A Decade of Proteorhodopsin
ones
First proteorhodopsin gene found in uncultured
SAR86 using metagenomics; proteorhodopsin
ight-driven proton pump activity confirmed in
heterologous E. coli cells [4].
Proteorhodopsin presence confirmed directly in
he ocean using laser flash photolysis [5].
Proteorhodopsin genes also found in other
bacterial groups [8].
Enormous diversity of proteorhodopsin genes
ound in the Sargasso Sea using metagenomics [9].
Retinal biosynthesis pathways found in metage-
nomic data and confirmed using E. coli cells [10].
Proteorhodopsin genes are found in ‘Canditatus
Pelagibacter ubique’ (SAR11), the most abundant
bacterium on earth; environmental SAR11 proteor-
hodopsin presence confirmed using metaproteo-
mics [11].
Proteorhodopsin genes found in uncultured
marine Archaea [12].
First indication of proteorhodopsin light-depen-
dent growth in cultured Flavobacteria [13] (see
Figure 1 for colony morphologies and pigmenta-
ion).
Proteorhodopsin genes found in non-marine
environments [14,15].
Proteorhodopsin phototrophy directly confirmed
using a genetic system in marine Vibrio sp. [16]
Figure 1. Various colony morphologies and coloration of
different proteorhodopsin-containing bacteria used to study
proteorhodopsin phototrophy. From top to bottom, the flavobac-
terium Polaribacter dokdonensis strain MED152 used to show proteor-
hodopsin light stimulated growth [13]; the flavobacterium Dokdonia
donghaensis strain MED134 used to show proteorhodopsin light
stimulated CO2-fixation [23]; and Vibrio strain AND4 used to show
proteorhodopsin phototrophy [16]; note the lack of detectable
pigments in Vibrio strain AND4. However, when these vibrio cells are
pelleted, they do show a pale reddish color, which is the result of
proteorhodopsin pigments presence in their membranes. Photos are
courtesy of Jarone Pinhassi.
doi:10.1371/journal.pbio.1000359.g001
Biology | www.plosbiology.org 3 April 2010 | Volume 8 | Issue 4 | e1000359

83. Figure 2. An artist’s rendition of the fundamental arrangement of proteorhodopsin in the cell membrane. Left panel: a cartoon (not to
scale) of planktonic bacteria in the ocean water column. Right panel: a simple view of one potential proteorhodopsin energy circuit. (1)
Proteorhodopsin – uses light energy to translocate protons across the cell membrane. (2) Extracellular protons – the excess extracellular protons
create a proton motive force, that can energetically drive flagellar motility, transport processes, or ATP synthesis in the cell. (3) Proton-translocating
ATPase – a multi-protein membrane-bound complex that can utilize the proton motive force to synthesize 5. Adenosine triphosphate (ATP, a central
high energy biochemical intermediate for the cell) from 4. Adenosine triphosphate (ADP, a lower energy biochemical intermediate). Illustration by
Kirsten Carlson, ß MBARI 2001.
doi:10.1371/journal.pbio.1000359.g002