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Prokaryotic photosynthesis and phototrophy illuminated
Review
TRENDS in Microbiology
Vol.14 No.11
Prokaryotic photosynthesis and phototrophy illuminated Donald A. Bryant1 and Niels-Ulrik Frigaard2 1 2
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA Institute of Molecular Biology and Physiology, University of Copenhagen, Sølvgade 83H, 1307 Copenhagen K, Denmark
Genome sequencing projects are revealing new information about the distribution and evolution of photosynthesis and phototrophy. Although coverage of the five phyla containing photosynthetic prokaryotes (Chlorobi, Chloroflexi, Cyanobacteria, Proteobacteria and Firmicutes) is limited and uneven, genome sequences are (or soon will be) available for >100 strains from these phyla. Present knowledge of photosynthesis is almost exclusively based on data derived from cultivated species but metagenomic studies can reveal new organisms with novel combinations of photosynthetic and phototrophic components that have not yet been described. Metagenomics has already shown how the relatively simple phototrophy based upon rhodopsins has spread laterally throughout Archaea, Bacteria and eukaryotes. In this review, we present examples that reflect recent advances in phototroph biology as a result of insights from genome and metagenome sequencing. Photosynthesis and phototrophy Photosynthesis is arguably the most important biological process on Earth, and only two mechanisms for collecting light energy and converting it into chemical energy have been described (Box 1). The first mechanism, which is dependent upon photochemical reaction centers (RCs; see Glossary) that contain (bacterio)-chlorophyll [(B)Chl], is found in five bacterial phyla: Cyanobacteria, Proteobacteria, Chlorobi, Chloroflexi and Firmicutes. All currently described Chlorobi and Cyanobacteria strains are photoautotrophs but only some strains of Chloroflexi [filamentous anoxygenic phototrophs (FAPs)], Proteobacteria (purple sulfur and purple non-sulfur bacteria) and Firmicutes (heliobacteria) are phototrophic (Figure 1). The second mechanism employs rhodopsins, retinalbinding proteins that respond to light stimuli [1]. Several homologous types of rhodopsins are known in microbes and include energy-conserving transmembrane proton pumps [bacteriorhodopsin (BR), proteorhodopsin (PR), xanthorhodopsin] (Figure 2), transmembrane chloride pumps (halorhodopsins) and light sensors (sensory rhodopsins) [1,2]. Here, we review how genome and metagenome sequencing studies are providing new insights into the physiology, metabolism and evolution of the organisms that perform these two processes for the capture of light energy. Corresponding author: Bryant, D.A. ([email protected]). Available online 25 September 2006. www.sciencedirect.com
Glossary Anoxygenic photosynthesis: photosynthesis performed by organisms that do not evolve oxygen; it uses electron donors other than water for carbon dioxide reduction. Bacteriorhodopsin (BR): a rhodopsin first identified in haloarchaea; translocates protons to the periplasm after light-induced isomerization of retinal. Chlorobi: bacterial phylum that includes the green-colored and brown-colored green sulfur bacteria; these bacteria have type 1 reaction centers (containing BChl a and Chl a) and chlorosomes containing BChl c, d or e. They fix carbon by the reverse tricarboxylic cycle and oxidize sulfide, sulfur, thiosulfate, Fe2+ or H2. Chloroflexi: bacterial phylum that includes the filamentous anoxygenic phototrophs (FAPs), formerly known as the green gliding or green filamentous bacteria. Cyanobacteria: bacterial phylum that includes all oxygen-evolving photosynthetic bacteria; they have Chl a-containing type 1 and type 2 reaction centers and fix carbon by the reductive pentose-phosphate (Calvin–Benson–Bassham) cycle; most have phycobilisomes as light-harvesting antennae (but see Prochlorophytes). Filamentous anoxygenic phototrophs (FAPs): Chloroflexi that have BChl acontaining, type 2 reaction centers. They might have chlorosomes that contain BChl c and most fix carbon by the 3-hydroxypropionate cycle whereas some oxidize sulfide or H2. Heliobacteria: endospore-producing photoheterotrophic bacteria of the phylum Firmicutes that have type 1 reaction centers and BChl g. Oxygenic photosynthesis: photosynthesis that uses water as the electron donor and leads to oxygen evolution. Photochemical reaction center: a multisubunit protein complex containing chlorophylls or bacteriochlorophylls, in which light energy is transduced into redox chemistry. Photosynthesis: the reduction of carbon dioxide into biomass using energy derived from light. Phototrophy: a metabolic mode in which organisms convert light energy into chemical energy for growth. Prochlorophyte: a cyanobacterium such as Prochlorococcus spp. that synthesizes both divinyl-Chl a and divinyl-Chl b but lacks phycobilisomes. Proteorhodopsin (PR): a rhodopsin first identified in marine proteobacteria, which translocates protons to the periplasm after light-induced isomerization of retinal. Purple bacteria: bacteria of the phylum Proteobacteria that produce BChl a or b under oxic or anoxic conditions. They have type 2 reaction centers and membrane-intrinsic caroteno-BChl antennae; many oxidize sulfide, thiosulfate, or H2 and they fix carbon by the reductive pentose-phosphate (Calvin–Benson– Bassham) cycle. Rhodopsin: a membrane-intrinsic protein characterized by seven transmembrane a-helices and a covalently attached carotenoid, retinal. Type 1 reaction center: RC family found in cyanobacteria, green sulfur bacteria and heliobacteria. They have either homodimeric or heterodimeric cores with [4Fe-4S] clusters as their terminal electron acceptors and produce weak oxidants and strong reductants (reduced ferredoxin). Type 2 reaction center: RC family found in cyanobacteria, purple bacteria and filamentous anoxygenic bacteria; all have heterodimeric cores with quinones as terminal electron acceptors. They produce strong oxidants and weak reductants (hydroquinone).
Genome sequencing projects for photosynthetic prokaryotes Synechocystis sp. PCC6803, a unicellular cyanobacterium, was the third prokaryote and the first photosynthetic organism to have its chromosome completely sequenced [3]. Over the past decade, there has been explosive growth in the
0966-842X/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2006.09.001
Review
TRENDS in Microbiology
Box 1. Photosynthesis and phototrophy Photosynthesis is the reduction of CO2 into biomass using energy derived from light. Biological CO2 reduction requires both ATP and electrons, which can be provided as NADPH or reduced ferredoxin. However, the ultimate electron source is organism-dependent and can be H2O, H2S, H2 or other reduced inorganic compounds. Phototrophy refers to a metabolic mode in which organisms convert light energy into chemical energy for growth. Thus, all photosynthetic bacteria are phototrophic but not all phototrophic bacteria are photosynthetic. Two mechanistically distinct processes empower phototrophy. In the first and simplest case, light energy directly drives proton expulsion from cells through the proteins BR or PR, thereby creating a proton-motive force that can be used either to drive ATP synthesis through ATP synthase or to drive various secondary transport processes [1,47] (Figure 2). Because PRs and BRs do not mediate electron transfer reactions, organisms that use these proteins are phototrophs but they have not yet been shown to be photosynthetic. In the second and more complex type of phototrophy, light initiates electron transfer through oxidation of a chlorophyll and reduction of an electron acceptor; secondary electron transfer reactions that do not require light subsequently lead to the production of proton-motive force that can be coupled to ATP synthesis. This second mechanism is absolutely dependent upon (B)Chl-containing proteins known as photochemical RCs (Figure 1). Type 1 RCs produce weak oxidants and strong reductants through their terminal, electron-accepting [4Fe-4S] clusters; type 2 RCs produce strong oxidants and a weak reductant (a reduced quinone molecule). The two types of RCs have similar structures [57,62–64] and seem to share a common evolutionary origin [57,65]. To date, (B)Chl biosynthesis has not been detected in any archaeal organism, so photosynthesis most probably evolved after the divergence of the archaeal–eukaryal and bacterial lineages. Although most RCcontaining bacteria are autotrophs and are thus photosynthetic, some bacteria (e.g. heliobacteria) that have a single type of RC do not grow autotrophically when provided with CO2 and an electron source [39]; presumably, they only perform cyclic electron transfer for ATP synthesis. Similar to rhodopsin-containing organisms, these bacteria are not photosynthetic but are photoheterotrophs. Photosynthetic organisms produce a variety of light-harvesting antenna structures (the protein components of which do not share a common evolutionary ancestor) to enhance the rate of light-driven electron transport [57,65,66]. Examples include phycobilisomes, chlorosomes and a variety of light-harvesting (B)Chl and caroteno(B)Chl proteins [25,57,66] (Figure 1).
genome sequencing of photosynthetic prokaryotes, and the Genomes On-Line Database (http://www.genomesonline. org/) and other sources currently indicate that 55 Cyanobacteria, 12 Chlorobi, nine Chloroflexi, 24 Proteobacteria and two Firmicutes (heliobacteria) are or soon will be completely sequenced. These data will have a substantial impact on the understanding of the origins and evolution of photosynthesis while providing many exciting new insights into the properties of these ecologically and environmentally important organisms. Cyanobacteria: the oxyphototrophs Cyanobacteria are such an ancient and remarkably diverse group of Bacteria that even data for 55 organisms provide an extremely limited view of their complexity. There are >475 pure strains in the Pasteur Culture Collection of Cyanobacteria, and yet this collection includes only a small number of the several thousand described species. To illustrate the magnitude of this problem, the smallest genomes for photosynthetic bacteria are 1.7 Mb and are found in the marine, unicellular Prochlorococcus spp. [4,5] whereas Nostoc punctiforme has the largest genome www.sciencedirect.com
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(9 Mb) sequenced thus far [6]. N. punctiforme can differentiate into multiple specialized cells (hormogonia, akinetes and heterocysts), establishes cellular patterns of development for heterocysts within its filaments and forms symbioses with fungi and plants. Genome size estimates indicate that some Calothrix sp. genomes are even larger at 12–15 Mb [7]. These prokaryotes have genomes that are as large as those of yeast and gene contents approaching that of Drosophila melanogaster! As additional, morphologically and developmentally distinctive cyanobacteria are studied by genomic methods, new mechanisms that regulate cellular interactions are likely to emerge. Unlike other Gram-negative bacteria, colonial cyanobacteria do not appear to employ autoinducer-2 (a furanosyl borate diester [8]) or acyl-homoserine lactones [9] as quorumsensing and signaling molecules for biofilm development or other cellular interactions. Comparisons of cyanobacterial genome sequences from ecotypes of the same species and from closely related species are already providing new insights into the relationships between ecological niche, gene content and speciation for environments as different as the oligotrophic ocean and dense, nutrient-rich microbial mats. The most detailed studies to date of the closely related marine Synechococcus and Prochlorococcus species have provided important insights into the ecophysiology of these genera [4,5,10–12], and this information has been substantially extended by metagenomic data from the Sargasso Sea [13]. Specific gene-content differences have been correlated with the physiological properties of high-light and low-light adapted ecotypes of these genera. In turn, these properties can be correlated with the chemical and physical differences that are found in the upper and lower portions of the photic zone in the ocean [4,5,10–12]. Delong et al. [14] have recently expanded this concept by examining both organismal and gene-content variation as a function of depth in the planktonic microbial communities in the North Pacific subtropical gyre. Their results show that the distribution of taxonomic groups, functional gene repertoires and metabolic potentials vary with depth in ways that relate to carbon and energy metabolism, adhesion and motility, gene mobility and host–virus interactions. These studies raise interesting questions about how different ecotypes arise and how they persist in the oceans [12]. Ambient temperature and growth temperature optima seem to be important along with light, nutrients and competitor abundances [15]. The observation that cyanophages sometimes carry photosynthesis genes [16– 20] provides one explanation for how genes can be rapidly exchanged throughout these populations. The genome sequencing, metagenomics and ‘metatranscriptomics’ of Ward and coworkers [21] are addressing similar issues in the integrated ‘community metabolism’ and ecophysiology of the cyanobacteria of a different physicochemical environment: the phototrophic mats of the Octopus and Mushroom Springs in Yellowstone National Park. Stenou et al. [21] have recently shown that two populations of thermophilic Synechococcus spp. in these mats perform photosynthesis by day and seem to ferment stored carbohydrates to generate reductant for nitrogen fixation by night.
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Figure 2. Simple scheme for phototrophy based on BR or PR and ATP synthase. Absorption of light by retinal leads to isomerization of retinal causing a conformational change in PR or BR, which in turn leads to the expulsion of a proton to the periplasmic space. Translocation of protons to the cytoplasm is coupled to the synthesis and release of cytoplasmic ATP by ATP synthase. Image of BR molecules (left) reproduced, with permission, from Ref. [58]. ß (2004) Elsevier. ATP synthase image (right) reproduced, with permission, from Ref. [59]. ß (2004) Nature Publishing Group.
Figure 1. Distribution of reaction center types and antenna systems in photosynthetic bacteria, which are found in the Cyanobacteria, Chlorobi, Proteobacteria, Chloroflexi and Firmicutes. Type 1 RCs (left) have [4Fe-4S] clusters (Fe-S) as terminal electron acceptors, whereas type 2 RCs (right) have quinones (Q) as electron acceptors. Colors indicate whether a RC is a homodimer (e.g. heliobacteria and green sulfur bacteria) or a heterodimer [photosystem (PS) I and all type 2 RCs]. Cyanobacteria have Chl-a-containing PS I (left) and PS II (right) and have light-harvesting phycobilisomes that are principally associated with PS II. Dotted lines in the type 1 RC subunits indicate the existence of both a light-harvesting domain, which is structurally related to subunits CP43 and CP47 of PS II, and an electron transfer domain, which is structurally related to the subunits of both the PS II core and other bacterial type 2 RCs. Prochlorophytes lack phycobilisomes and, instead, have light-harvesting PCB proteins, which are structurally related to CP43 and bind to both divinyl-Chl a and divinyl-Chl b. Heliobacteria have homodimeric type 1 RCs with BChl g. Green sulfur bacteria (Chlorobi) have homodimeric type 1 RCs that bind to BChl a and a small amount of Chl a; their chlorosomes contain >200 000 BChl c, d or e molecules and a small amount of BChl a that is bound to the CsmA protein. The BChl-a-binding FMO protein connects chlorosomes to the RCs. Purple bacteria and FAPs are similar and have type 2 bacterial RCs that carry either BChl a (or BChl b in some purple bacteria). The antennae for these RCs are formed by ring-shaped, BChl-a-binding LH1 and LH2 complexes in purple bacteria or chlorosomes containing BChl c and BChl a in some FAPs. The LH1-like complexes of FAPs can also form rings around their type 2 RCs. ‘Red’ FAPs lack the chlorosomes that are found in ‘green’ FAPs. The chlorosomes of FAPs are usually smaller and contain fewer BChl c molecules than those found in the Chlorobi.
Chlorobi: green sulfur bacteria In contrast to the extraordinary richness of cyanobacterial diversity, the phylum Chlorobi (comprising the green sulfur bacteria) is a metabolically limited, physiologically www.sciencedirect.com
well-defined and genetically closely related bacterial group, which shares a common root with the Bacteroidetes. Comparative genomic analyses have enabled the elucidation of their unique BChl and carotenoid biosynthetic pathways. Chlorobi are obligately anaerobic photoautotrophs that (i) oxidize sulfur compounds, H2 or ferrous iron; (ii) fix carbon by the reverse tricarboxylic acid cycle; (iii) synthesize BChl c, d or e along with BChl a and Chl a; and (iv) have a photosynthetic apparatus that comprises a type 1 reaction center, the Fenna-Matthews-Olson (FMO) BChl-a-binding protein and chlorosomes that each contain >200 000 BChl c, d or e molecules (Figure 1) [22–25]. Because of the availability of an efficient natural transformation system and its ability to grow rapidly with thiosulfate as an electron donor, Chlorobium tepidum – the 2.15 Mb genome of which was sequenced by The Institute for Genomic Research [26] – has become the model organism for this group of phototrophs. Insights into the physiology, metabolism and light-harvesting apparatus of this organism have been reviewed elsewhere [22–25]. Although fewer Chlorobi genomes have been sequenced than cyanobacterial genomes, the ten sequenced and two anticipated genomes encompass most of the currently known diversity of this group. The Joint Genome Institute of the Department of Energy (JGI-DOE) has sequenced most of the type strains of the Chlorobi, and these data provide benchmarks for comparisons of future isolates. Complete genomes are already available for two additional strains, Pelodictyon luteolum DSM273 and Chlorobium chlorochromatii (Box 2 and Figure 3), and draft genomes are available for seven additional strains (C. ferrooxidans, C. phaeobacteroides DSM 266, C. limicola DSM245, C. vibrioforme DSM 265, Prosthecochloris aestuarii SK413, Pelodictyon phaeoclathratiforme and an enrichment culture, C. phaeobacteroides BS-1, isolated from 100 m below the surface of the Black Sea [27]). Sequencing of Chloroherpeton thalassium and C. vibrioforme 8327d
Review
TRENDS in Microbiology
Box 2. Phototrophic consortia With the exception of Chloroherpeton thalassium, which exhibits gliding motility, all known green sulfur bacteria are non-motile. Some green sulfur bacteria (‘epibionts’) become motile by forming phototrophic consortia through a specific association with a bproteobacterium, denoted the ‘central rod’ [60,61,67] (Figure 3). Each polarly flagellated central rod carries 20–60 epibiont cells and the entire consortium is phototactic in response to light signals perceived by the epibiont. The nature of any metabolic coupling between the two organisms is not yet known but possibilities include transfer of reduced carbon and/or nitrogen from the epibiont to the central rod and possibly the provision of sulfide from sulfate reduction or H2 from the central rod to the epibiont. At present, the mechanisms of cell-to-cell signaling for phototaxis and coordination of cell division are completely unknown. Genome sequencing of the two partners of ‘Chlorochromatium aggregatum’, which was isolated as an enrichment culture from Lake Dagow [60,67], should help to answer many of these questions. The 2.57 Mb genome of the epibiont, Chlorobium chlorochromatii, which is not an obligate symbiont [67], has already been completely sequenced (http://img.jgi.doe.gov/). Sequencing of the b-proteobacterial central rod, which cannot be grown independently from C. chlorochromatii and is most closely related to Rhodoferax sp. [60], is now in progress at JGI-DOE. C. chlorochromatii encodes a family of calcium-dependent, RTX-toxin-like proteins that might be involved in cellular adhesion to the central rod [68]. The largest gene is predicted to encode a protein of 36 805 amino acids, one of the largest predicted proteins known to date. This gene occurs in an apparent operon with a sequence-related gene of 20 646 codons, producing an operon of >170 kb. Homologs of these genes occur in Magnetococcus sp. MC-1 (15 245 codons) and Synechococcus sp. RS9917 (28 178 codons) but their functions are unknown.
should be completed later this year. The sequenced Chlorobi have 2–3 Mb genomes that encode 1750– 2800 genes (http://img.jgi.doe.gov/) and pairwise comparisons show that Chlorobi strains share a common core-set of 1400–1500 genes. Chlorobi genomes encode only a few
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predicted transporters for organic molecules, have a small number of predicted transcription regulators, and are largely devoid of two-component histidine kinases and response regulators. These observations suggest that Chlorobi live in relatively constant (and energy-limited) conditions and that they probably have a limited capacity to respond to changes in their physicochemical environment [22,26]. This is a trait that is shared by members of the cyanobacteria with reduced genomes, such as Prochlorococcus and marine Synechococcus [4,5,10]. Pigment biosynthetic pathways in Chlorobi The availability of multiple Chlorobi genome sequences and of a highly efficient natural transformation system for C. tepidum has facilitated the identification of genes that encode enzymes for pigment biosynthesis and other physiological processes in green sulfur bacteria. C. tepidum synthesizes three chlorophylls: BChl c, BChl a and Chl a [24]. Before the completion of its genome sequence, no enzyme specifically involved in BChl c, d or e biosynthesis had been identified, and now all steps but one in the pathway leading from chlorophyllide a to BChl c are known. Only the enzyme responsible for the removal of the C-132 methylcarboxyl group has not yet been identified. Moreover, the erroneous identification of the 8-vinyl reductase as BchJ has recently been corrected [24]. Similarly, although nothing was known about the pathway for carotenoid biosynthesis in Chlorobi before the availability of the genome sequence, all of the enzymes required for synthesis of chlorobactene and isorenieratene have been identified [28,29]. Through these analyses, it is now clear that carotenoid biosynthesis in Chlorobi is more similar to the pathway in Cyanobacteria than to that in other bacteria [28]. Although no member of the three known families of
Figure 3. Phototrophic consortia. (a) Transmission electron micrograph of a thin section of the phototrophic consortium ‘Chlorochromatium aggregatum’. The central rod (CR; a b proteobacterium with a putative genome size of 4–5 Mb) and the epibiont cells (EB; the green sulfur bacterium Chl. Chlorochromatii with a genome size of 2 572 079 bp) are indicated. (b) Light micrograph of ‘Chlorochromatium aggregatum’. Scale bar = 5 mm. Scanning electron micrographs of ‘Pelochromatium roseum’ before (c) and after (d) division of the EB. Scale bar = 1 mm. (e) Thin-section electron micrograph of the junction of an EB cell with the central rod. Note that chlorosomes, the electron-dense ellipsoids on the inner surface of the cytoplasmic membrane of the EB cells, do not occur at the junction with the CR and that an additional wall layer between the cells can be seen at this junction [60]. Additionally, a paracrystalline structure is seen at the cell junctions at the inner surface of the CR (boxed region). Scale bar = 0.5 mm. Parts (b), (c) and (d) reproduced, with permission, from Ref. [61]. ß (2002) Springer. www.sciencedirect.com
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lycopene cyclases could be identified in the C. tepidum genome, phylogenetic profiling (using data from multiple photosynthetic bacteria) and complementation of a lycopene-producing strain of Escherichia coli both identified a fourth type of lycopene cyclase encoded by the C. tepidum open reading frame CT0456 [29] (J.A. Maresca et al., unpublished). The identification of the ‘missing’ lycopene cyclase in C. tepidum also enabled the identification of ‘missing’ lycopene cyclases in several cyanobacteria. Interestingly, many cyanobacteria have an ortholog and a paralog of CT0456, and both of these enzymes seem to be involved in cyclase reactions with lycopene (J.E. Graham and D.A. Bryant, unpublished). Synechococcus sp. PCC7942 has lycopene cyclases that belong to two of the four families, which demonstrates the mosaic nature of carotenoid biosynthesis and is a likely example of lateral gene transfer. The sequenced Chlorobi strains are metabolically similar but can be separated according to particular phenotypes (e.g. green-colored strains that contain BChl c and chlorobactene versus brown-colored strains that contain BChl e and isorenieratene, or strains that oxidize sulfur compounds versus ferrous iron). Thus, whole-genome comparisons can identify candidate genes that are responsible for defined physiological differences among these strains. This approach was recently used to search for candidate gene(s), the product(s) of which could convert BChl c into BChl e. This analysis identified a radical SAM enzyme and an adjacent dehydrogenase as the most likely candidates for this transformation (J.A. Maresca and D.A. Bryant, unpublished). Interestingly, these two genes occur adjacent to the gene encoding g-carotene cyclase, which produces b-carotene, the precursor of isorenieratene [29] (J.A. Maresca et al., unpublished). The proximity of these genes on a 6 kb segment of the chromosome provides a possible explanation for the polyphyletic nature of the ‘brown’ phenotype among green sulfur bacteria. This gene proximity would greatly facilitate their lateral transfer among the Chlorobi, and such transfer would immediately confer the ability to populate a new environmental niche. The synthesis of BChl e is another example of a reaction for which both oxygen-independent and oxygen-dependent enzymes occur in nature [23,30]. The C-7 formyl group introduced during BChl e biosynthesis must be derived from water because this reaction occurs under anoxic conditions; however, the C-7 formyl group of Chl b in Prochlorococcus sp., green algae and higher plants is derived from oxygen [31]. Chloroflexi: filamentous anoxygenic phototrophs Because of their diverse metabolic and physiological properties, genomic analyses of diverse strains of Chloroflexi are likely to produce novel insights into the evolution of photosynthesis. The phylum Chloroflexi is one of the earliest diverging lineages of the Bacteria, and it contains several genera of filamentous, gliding bacteria that perform anoxygenic photosynthesis (FAPs) [32]. The phylum contains two orders, the ‘Chloroflexales’ and ‘Herpetosiphonales’. Herpetosiphon aurantiacus, the type strain of the latter order, has recently been sequenced by JGI-DOE. The 6.6 Mb genome of this heterotrophic bacterium www.sciencedirect.com
encodes the enzymes for carotenoid biosynthesis but has no genes for the enzymes of BChl biosynthesis or components of the photosynthetic apparatus. Pierson and Castenholz first isolated Chloroflexus aurantiacus, the type strain of the Chloroflexi and ‘Chloroflexales’, in the early 1970s from Yellowstone National Park and other thermal features [32]. Cfx. aurantiacus synthesizes BChl a and BChl c and has type 2 RCs and chlorosomes but lacks the FMO protein (Figure 1). The ‘red/orange’ FAPs of the genera Roseiflexus and Heliothrix do not synthesize BChl c and lack chlorosomes [32,33] (Figure 1). Until recently, the incomplete 5.2 Mb draft sequence of Cfx. aurantiacus was the only genomic information available for any photosynthetic member of the Chloroflexi. However, JGI-DOE will soon release the draft genomes of four additional Chloroflexi: Cfx. aggregans (4.5 Mb), Roseiflexus sp. strain RS-1 (5.8 Mb; from Octopus Springs, Yellowstone National Park), Roseiflexus castenholzii (5.6 Mb; from Nakabusa Hot Springs, Japan) and the heterotroph Herpetosiphon aurantiacus (6.6 Mb). In addition, three sulfide-oxidizing FAPs (Chlorothrix halophila, Oscillochloris sp. strain UdG 9002 and Chloronema giganteum strain UdG 9001) are scheduled for sequencing by JGI-DOE later this year. A consortium of Russian scientists is sequencing the genome of Oscillochloris trichoides (R. Ivanovskii and B. Kuznetsov, personal communication). O. trichoides produces a type I ribulose-1,5-bisphosphate carboxylase–oxygenase (RubisCO) and fixes carbon dioxide by the Calvin cycle rather than by the 3-hydroxypropionate cycle [34]. However, it is not known whether other FAPs have RubisCO and use the Calvin cycle. It will be interesting to see whether the sulfide-oxidizing FAPs have type 1 RCs like the Chlorobi or have type 2 RCs (and reverse electron transport) like Cfx. aurantiacus and purple bacteria. Roseiflexus sp. and O. trichoides have nitrogenase genes, and other FAPs also probably fix dinitrogen. Much more information will soon be available for these poorly characterized phototrophs. Like purple non-sulfur bacteria, Cfx. aurantiacus exhibits considerable metabolic diversity and it can grow as an aerobic chemoheterotroph or as an anaerobic photoheterotroph. Using electrons derived from H2 or H2S under anoxic or microaerophilic conditions, some strains of Chloroflexus sp. grow photoautotrophically by fixing CO2 through the 3-hydroxypropionate pathway [32]. In nature, Cfx. aurantiacus probably grows under microaerophilic or alternating oxic and anoxic conditions but it only fully develops its photosynthetic apparatus under anoxic conditions. As a result, Cfx. aurantiacus encodes some enzymes that can function under either oxic or anoxic conditions [23,30]. For example, both bchE and acsF genes are found in the Cfx. aurantiacus genome; these genes encode the oxygen-independent isocyclic ring cyclase and the oxygen-dependent isocyclic ring cyclase, respectively. Both Roseiflexus sp. strains and Cfx. aggregans also produce both of these enzymes. Interestingly, the same pattern is not found for hemF and hemN, which encode oxygen-dependent and oxygen-independent coproporphyrinogen oxidases, respectively. The two Chloroflexus strains have both genes, whereas the Roseiflexus sp. strains do not encode hemF.
Review
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Proteobacteria: purple non-sulfur and purple sulfur bacteria Photosynthesis is a trait that is widespread but not universal among members of the Proteobacteria and is found in morphologically and metabolically diverse species. Genome sequence information has largely confirmed the physiological versatility and corresponding large genome sizes of these organisms. Some of the photosynthetic proteobacteria are well suited for studies of global gene regulation because many members are facultatively phototrophic or photosynthetic under anoxic conditions. Photosynthetic Proteobacteria can be found in the a, b and g subdivisions but, to date, almost all available genome sequences are from members of the a subdivision. Several genomes have been determined (http://www.genomesonline.org), including those of Rhodopseudomonas palustris (5.5 Mb) [35], two Rhodobacter sphaeroides strains (4.6 Mb) [36] (http:// www.ncbi.nlm.nih. gov/), Rhodospirillum rubrum (4.4 Mb) (http://www.ncbi. nlm.nih.gov/), Roseobacter denitrificans (http://genomes. tgen.org/rhodobacter.html), Bradyrhizobium sp. (9.1 Mb) and Roseobacter sp. (4.1 Mb) (http:// www.genomesonline. org). Only two g-subdivision members have been studied and no photosynthetic b-proteobacterium has been sequenced. Thus, the environmentally important purple sulfur bacteria, all of which belong to the g subdivision, along with purple bacteria of the b subdivision (many of which can also use sulfur or H2 as electron donors), represent groups about which little genome sequence information is available. Finally, a large number of mostly a-proteobacteria in diverse freshwater, saline, marine, soil and hot-spring environments seem to have photosynthesis gene clusters but, in many cases, the function of the relatively low levels of BChl a produced under aerobic conditions is unknown [37,38]. Projected sequencing projects and comparative analyses will help to define the genetic, physiological and metabolic differences among these aerobic anoxygenic phototrophs, photoheterotrophs like Rhodobacter sp., and the photolithoautotrophic purple sulfur bacteria. Genome sequence data, in combination with the physiological and metabolic versatility of these organisms, should lead to engineered strains for diverse applications in biotechnology, including bioremediation, lignin degradation and biofuels and hydrogen production [35]. Heliobacteria Heliobacteria, first described by Gest and Favinger in 1983 [39], are the most recently discovered group of bacteria containing RCs and they remain the most poorly characterized overall. Heliobacteria are members of the phylum Firmicutes and are closely related to clostridia. Like Bacillus or Clostridium sp., heliobacteria produce heatresistant endospores and, to date, no characterized member of this group is known to grow photoautotrophically. Studies with Heliobacillus mobilis have shown that many of the genes for synthesis of BChl g and the RC are located in a 30 kb cluster similar to the photosynthesis gene clusters found in purple bacteria [40,41]. Thus, it is possible that the heliobacteria obtained their photosystem components through a lateral gene transfer event. However, a recent detailed analysis of RC and www.sciencedirect.com
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light-harvesting protein sequences argues that lateral gene transfer is not required to explain the current distribution of RC sequences [42]. No complete heliobacterial genome sequence is yet available, although the 3.1 Mb draft genome of Heliobacterium modesticaldum has recently been made available for searches (http://genomes. tgen.org/helio.html). The H. modesticaldum genome sequence will help to clarify the possible lateral acquisition of photosynthesis genes and it will also help to identify genes that are required for carotenoid, BChl, and RC synthesis and function. The relatively small genome of H. modesticaldum is likely to provide new insights into genes that are functionally important in sporulation and regulation of this process. Bacteriorhodopsin-based phototrophy in halophilic prokaryotes Comparative analyses of sequenced genomes and metagenomic data from the ocean have shown the great diversity in the structure and function of rhodopsins and have demonstrated how easily lateral gene transfer can occur among unrelated organisms. Bacteriorhodopsin (BR) in the so-called ‘purple membrane’ of halophilic archaea has been studied for three decades and the structure and function of BR is known in great detail [1] (Figure 2). The halophilic archaea grow well in the dark as aerobic chemoheterotrophs; however, strains that synthesize BR exhibit light-enhanced growth under anoxic conditions and are, therefore, facultatively phototrophic [43]. Among these haloarchaea, multiple rhodopsins with diversified functions can exist within a single cell. The genome of the haloarchaeon Haloarcula marismortui encodes six homologous rhodopsins: one proton-pumping BR, one chloride-pumping halorhodopsin, two sensory rhodopsins and two opsins of unknown function [44]. The importance of these rhodopsins for environmental adaptation was recently and elegantly illustrated through the genome sequence of a halophilic bacterium, Salinibacter ruber, which taxonomically belongs to the Cytophaga–Flexibacter–Bacteroides group [45]. Genomewide analyses showed that S. ruber has evolved convergently towards halophilic archaea at both the physiological level (different genes producing a similar overall phenotype) and at the molecular level (independent mutations yielding proteins with similar sequences or structures). Although the identity of the donor organism (or organisms) is uncertain, the genome of S. ruber encodes four rhodopsins – the obvious result of lateral gene exchange with the haloarchaea. One rhodopsin resembles a chloride pump and two resemble sensory rhodopsins. The fourth is a proton-pumping rhodopsin, xanthorhodopsin, which is related to proteorhodopsins but contains a salinixanthin carotenoid chromophore in addition to retinal [46]. Proteorhodopsin in marine prokaryotes Other than those in haloarchaea, the first example of a prokaryotic rhodopsin was one found in a marine proteobacterium and, thus, it was named proteorhodopsin (PR) [47,48]. This identification was based on a metagenomics approach in which large fragments of genomic DNA isolated from marine picoplankton were cloned and
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sequenced. Like BR, PR has been shown by heterologous expression to function as a light-driven, transmembrane proton pump in E. coli [47] and, thus, it could contribute substantially to the energy budget in cells living in the photic zone of oceans. Therefore, it is not surprising that sequencing of both short and long fragments of environmental DNA has shown that a wide range of PRs exist in highly different types of marine prokaryotic plankton [13,49–51]. For example, PR is present in marine euryarchaeotes in the photic zone of the North Pacific subtropical gyre, whereas the marine euryarchaeotes living below the photic zone do not have PR, clearly as a consequence of environmental adaptation [51]. Recently, Pelagibacter ubique, a representative of the ubiquitous SAR11 marine proteobacteria, was axenically cultivated and its genome was sequenced [52]. This genome contains one gene encoding PR [52,53] but has no genes that are characteristic for CO2 fixation. The genomes of other marine proteobacteria (Photobacterium sp. SKA34 and Vibrio angustum S14) and marine Bacteroidetes (Polaribacter irgensii, Cellulophaga sp. MED134, Tenacibaculum sp. MED152 and Psychroflexus torquis ATCC700755), which are currently being sequenced by the J. Craig Venter Institute (https:// research.venterinstitute.org/moore/), also contain PRs. Although laboratory growth experiments with P. ubique have not yet demonstrated that this bacterium grows faster in the light than in the dark [53], it seems likely that PR enables the bacterium to benefit from light even though the experimental conditions to show this have not yet been identified. Although the structure of this PR is consistent with a function in proton pumping, it remains possible that this molecule could instead transport another substrate or function as a sensory molecule. To put it briefly, it seems that many, if not most, of the planktonic prokaryotes in the photic zone of the oceans that do not contain photosynthetic reaction centers nevertheless exploit light by having acquired PR through lateral gene transfer. The exact physiology of the marine prokaryotes that harbor these PRs is not always clear. For example, a recently characterized 95 kb genomic fragment from a marine proteobacterium that encodes a PR also encodes a putative reverse dissimilatory sulfite reductase operon, which could provide reducing equivalents for autotrophic growth by oxidizing a reduced sulfur compound [54]. The ‘cosmopolitan’ rhodopsins versus the ‘refined’ reaction centers Shotgun sequencing of DNA from the Sargasso Sea illustrated how PRs are widespread in oceanic microorganisms [13]. Although the exact functions of the rhodopsins are often not known, genome sequencing projects of organisms in pure culture have also confirmed that rhodopsins are much more widely distributed among different organismal lineages than first anticipated. For example, rhodopsins of unknown function have been found in the genomes of organisms as diverse as halophilic archaea and bacteria, marine proteobacteria, marine Bacteroidetes, marine euryarchaeotes, g-radiation-resistant actinobacteria (Rubrobacter xylanophilus and Kineococcus radiotolerans) www.sciencedirect.com
and the Gram-positive Exiguobacterium sp. 255–15, in addition to certain cyanobacteria, fungi, green algae and a dinoflagellate (Pyrocystis lunula). The single rhodopsins encoded by the genomes of the cyanobacteria Nostoc sp. PCC7120 (Alr3165) and of the fungus Leptosphaeria maculans have recently been shown by experimental characterization to be a sensory rhodopsin [55] and a proton-pumping rhodopsin [56], respectively. As a last example, the early-diverging cyanobacterium, Gloeobacter violaceus, encodes a rhodopsin (Gll0198) that seems to be most similar to the proton-pumping xanthorhodopsin of S. ruber. If rhodopsin-based phototrophy is transferred so easily among organisms, why do only a few groups of RC-based phototrophs dominate phototrophic niches? Part of the answer might be that rhodopsin-based phototrophy uses light energy relatively inefficiently. First, to produce the proton-motive force required to produce one ATP, three to four BR molecules must each absorb a photon and release a proton to the periplasmic space. When a proteobacterial RC absorbs four photons, two ubiquinol molecules are produced and their dark re-oxidation by the cytochrome bc1 complex enables the production of two ATP molecules [57]. Second, the single retinal chromophore in the photochemical units of most rhodopsin-based phototrophs has a much smaller absorption cross-section than the photochemical units of RC-based phototrophs, which can have hundreds to thousands of chromophores [57]. Thus, a rhodopsin-based phototroph would have to synthesize many more energetically expensive BR or PR molecules to absorb the same amount of light energy. The highly efficient and extensive lightharvesting antenna systems of photosynthetic bacteria
Box 3. Are there more phototrophs out there? Our current knowledge of photosynthesis is based almost exclusively on cultivated strains but, given the vast diversity of microorganisms on Earth, these organisms are unlikely to represent the full spectrum of light utilization. Continued genome sequencing of cultivated and uncultivated organisms will undoubtedly reveal many more microbial groups that harbor rhodopsins. It will be interesting to see if there are any organisms that combine rhodopsin-based phototrophy with CO2 fixation because such organisms could then be classified as Chl-independent photosynthetic organisms. A priori, there seems to be no reason why the carbon fixation reactions of the Calvin cycle could not be driven by the combination of PR and sulfide:quinone oxidoreductase coupled to a type I NADH dehydrogenase for reverse electron flow. Alternatively, a rhodopsin and hydrogenase could be coupled to provide the energy and reducing power for photolithoautotrophic growth. As more environments are sampled, metagenomics are likely to reveal photosynthetic organisms with combinations of components that have not yet been observed in cultivated species (e.g. an anoxygenic organism with two types of RCs), novel solutions to the problem of light harvesting or the existence of new phyla that have not previously been shown to contain phototrophic or photosynthetic members. This latter prediction is not simply an idle speculation: a new phylum-level, RC-containing phototroph has recently been discovered in this manner and will be described in detail elsewhere (D.A. Bryant et al., unpublished). The rapidly increasing body of genome sequence and metagenomic data will help to answer questions about the origin and evolution of photosynthesis [42,69].
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enable the RCs to function at maximal efficiency even at relatively low light intensities. If RC-based phototrophy is so much more efficient than rhodopsin-based systems, why does this type of phototrophy not spread laterally? Perhaps it does on rare occasions, but because even the simplest chlorophyll-based photosystem requires 30 unique genes, this capability probably cannot be laterally transferred as easily as rhodopsinbased phototrophy. Lateral transfer of rhodopsin-based photosystems requires only the genes encoding the rhodopsin apoprotein and a carotenoid oxygenase–lyase that produces retinal [54] if the recipient already has the capability to produce an appropriate carotenoid. If it does not, retinal biosynthesis can be performed with just four genes (crtBIY–blh) [54]. Therefore, rhodopsin-based systems might be much more prone to lateral transfer and, driven by the selection of light, could thus distribute themselves among distantly related but sympatric organisms. Metagenomic analyses of photic environments are likely to identify many new examples in the near future (Box 3). Concluding remarks and future perspectives Genome sequencing of cultured organisms and metagenome sequencing of DNA from uncultured organisms of photic environments has already greatly accelerated the pace of new discoveries for the Cyanobacteria and Chlorobi. As more genomic sequence data become available for other phototrophic organisms, comparative bioinformatics will certainly catalyze advances in knowledge of the metabolism, gene regulation and physiology of these groups and might answer many outstanding questions about these organisms (Box 4). Metagenomic studies will ultimately lead to insights that transcend the level of individual organisms and will facilitate a broader and deeper understanding of the community-level dynamics among phototrophs (and non-phototrophs). This approach has the potential to Box 4. Outstanding questions (i) Because photosynthetic reaction centers evolved only once, which came first: type 1 or type 2 reaction centers? (ii) How did ancestors of cyanobacteria acquire type 1 and type 2 reaction centers, and how did oxygenic photosynthesis evolve? (iii) Did bacteria that principally synthesize BChl a arise before or after those that principally synthesize Chl a? (iv) Beyond the five known examples, how many additional bacterial phyla contain members that can synthesize (B)Chl and photochemical reaction centers? (v) Are there organisms that combine a rhodopsin-based ATP generation system with enzymes for the oxidation of an inorganic electron donor (e.g. sulfide or H2) to drive autotrophic carbon dioxide reduction? (vi) What is the physiological role of the low amounts of BChl a found in aerobic anoxygenic phototrophs that are common in the oceans? (vii) Nature has evolved multiple, independent solutions to the problem of harvesting solar energy. Are there novel types of light-harvesting antenna complexes yet to be discovered, and can metagenomics help to identify them? (viii) What are the principal drivers behind the evolution and distribution of phototrophy? How do nutrients, temperature, light and other parameters interact with genomic content to produce niche partitioning and ecotype distributions in photic environments? www.sciencedirect.com
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explain how gene contents map onto taxonomic compositions, physiological and metabolic capabilities and gene expression patterns of phototrophs in diverse photic environments – all of which together lead to the primary energy input that ultimately drives life and its evolution on Earth. Acknowledgements The authors would like to thank Julia A. Maresca for critical reading of the manuscript and many helpful comments. We also thank Joachim Weber (Texas Tech University) for use of the ATP synthase image in Figure 2 and Jo¨ rg Overmann (Ludwig Maximilians Universita¨t, Mu¨nchen) for providing Figure 3 parts (a) and (e), and for use of parts (b), (c) and (d). D.A.B. gratefully acknowledges support for genomics studies from the National Science Foundation (MCB-MCB-0519743 and MCB-0523100) and from the Department of Energy (DE-FG02– 94ER20137). N.-U.F gratefully acknowledges support from The Danish Natural Science Research Council (grant 21–04–0463).
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Prokaryotic photosynthesis and phototrophy illuminated Donald A. Bryant1 and Niels-Ulrik Frigaard2 1 2
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA Institute of Molecular Biology and Physiology, University of Copenhagen, Sølvgade 83H, 1307 Copenhagen K, Denmark
Genome sequencing projects are revealing new information about the distribution and evolution of photosynthesis and phototrophy. Although coverage of the five phyla containing photosynthetic prokaryotes (Chlorobi, Chloroflexi, Cyanobacteria, Proteobacteria and Firmicutes) is limited and uneven, genome sequences are (or soon will be) available for >100 strains from these phyla. Present knowledge of photosynthesis is almost exclusively based on data derived from cultivated species but metagenomic studies can reveal new organisms with novel combinations of photosynthetic and phototrophic components that have not yet been described. Metagenomics has already shown how the relatively simple phototrophy based upon rhodopsins has spread laterally throughout Archaea, Bacteria and eukaryotes. In this review, we present examples that reflect recent advances in phototroph biology as a result of insights from genome and metagenome sequencing. Photosynthesis and phototrophy Photosynthesis is arguably the most important biological process on Earth, and only two mechanisms for collecting light energy and converting it into chemical energy have been described (Box 1). The first mechanism, which is dependent upon photochemical reaction centers (RCs; see Glossary) that contain (bacterio)-chlorophyll [(B)Chl], is found in five bacterial phyla: Cyanobacteria, Proteobacteria, Chlorobi, Chloroflexi and Firmicutes. All currently described Chlorobi and Cyanobacteria strains are photoautotrophs but only some strains of Chloroflexi [filamentous anoxygenic phototrophs (FAPs)], Proteobacteria (purple sulfur and purple non-sulfur bacteria) and Firmicutes (heliobacteria) are phototrophic (Figure 1). The second mechanism employs rhodopsins, retinalbinding proteins that respond to light stimuli [1]. Several homologous types of rhodopsins are known in microbes and include energy-conserving transmembrane proton pumps [bacteriorhodopsin (BR), proteorhodopsin (PR), xanthorhodopsin] (Figure 2), transmembrane chloride pumps (halorhodopsins) and light sensors (sensory rhodopsins) [1,2]. Here, we review how genome and metagenome sequencing studies are providing new insights into the physiology, metabolism and evolution of the organisms that perform these two processes for the capture of light energy. Corresponding author: Bryant, D.A. ([email protected]). Available online 25 September 2006. www.sciencedirect.com
Glossary Anoxygenic photosynthesis: photosynthesis performed by organisms that do not evolve oxygen; it uses electron donors other than water for carbon dioxide reduction. Bacteriorhodopsin (BR): a rhodopsin first identified in haloarchaea; translocates protons to the periplasm after light-induced isomerization of retinal. Chlorobi: bacterial phylum that includes the green-colored and brown-colored green sulfur bacteria; these bacteria have type 1 reaction centers (containing BChl a and Chl a) and chlorosomes containing BChl c, d or e. They fix carbon by the reverse tricarboxylic cycle and oxidize sulfide, sulfur, thiosulfate, Fe2+ or H2. Chloroflexi: bacterial phylum that includes the filamentous anoxygenic phototrophs (FAPs), formerly known as the green gliding or green filamentous bacteria. Cyanobacteria: bacterial phylum that includes all oxygen-evolving photosynthetic bacteria; they have Chl a-containing type 1 and type 2 reaction centers and fix carbon by the reductive pentose-phosphate (Calvin–Benson–Bassham) cycle; most have phycobilisomes as light-harvesting antennae (but see Prochlorophytes). Filamentous anoxygenic phototrophs (FAPs): Chloroflexi that have BChl acontaining, type 2 reaction centers. They might have chlorosomes that contain BChl c and most fix carbon by the 3-hydroxypropionate cycle whereas some oxidize sulfide or H2. Heliobacteria: endospore-producing photoheterotrophic bacteria of the phylum Firmicutes that have type 1 reaction centers and BChl g. Oxygenic photosynthesis: photosynthesis that uses water as the electron donor and leads to oxygen evolution. Photochemical reaction center: a multisubunit protein complex containing chlorophylls or bacteriochlorophylls, in which light energy is transduced into redox chemistry. Photosynthesis: the reduction of carbon dioxide into biomass using energy derived from light. Phototrophy: a metabolic mode in which organisms convert light energy into chemical energy for growth. Prochlorophyte: a cyanobacterium such as Prochlorococcus spp. that synthesizes both divinyl-Chl a and divinyl-Chl b but lacks phycobilisomes. Proteorhodopsin (PR): a rhodopsin first identified in marine proteobacteria, which translocates protons to the periplasm after light-induced isomerization of retinal. Purple bacteria: bacteria of the phylum Proteobacteria that produce BChl a or b under oxic or anoxic conditions. They have type 2 reaction centers and membrane-intrinsic caroteno-BChl antennae; many oxidize sulfide, thiosulfate, or H2 and they fix carbon by the reductive pentose-phosphate (Calvin–Benson– Bassham) cycle. Rhodopsin: a membrane-intrinsic protein characterized by seven transmembrane a-helices and a covalently attached carotenoid, retinal. Type 1 reaction center: RC family found in cyanobacteria, green sulfur bacteria and heliobacteria. They have either homodimeric or heterodimeric cores with [4Fe-4S] clusters as their terminal electron acceptors and produce weak oxidants and strong reductants (reduced ferredoxin). Type 2 reaction center: RC family found in cyanobacteria, purple bacteria and filamentous anoxygenic bacteria; all have heterodimeric cores with quinones as terminal electron acceptors. They produce strong oxidants and weak reductants (hydroquinone).
Genome sequencing projects for photosynthetic prokaryotes Synechocystis sp. PCC6803, a unicellular cyanobacterium, was the third prokaryote and the first photosynthetic organism to have its chromosome completely sequenced [3]. Over the past decade, there has been explosive growth in the
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Box 1. Photosynthesis and phototrophy Photosynthesis is the reduction of CO2 into biomass using energy derived from light. Biological CO2 reduction requires both ATP and electrons, which can be provided as NADPH or reduced ferredoxin. However, the ultimate electron source is organism-dependent and can be H2O, H2S, H2 or other reduced inorganic compounds. Phototrophy refers to a metabolic mode in which organisms convert light energy into chemical energy for growth. Thus, all photosynthetic bacteria are phototrophic but not all phototrophic bacteria are photosynthetic. Two mechanistically distinct processes empower phototrophy. In the first and simplest case, light energy directly drives proton expulsion from cells through the proteins BR or PR, thereby creating a proton-motive force that can be used either to drive ATP synthesis through ATP synthase or to drive various secondary transport processes [1,47] (Figure 2). Because PRs and BRs do not mediate electron transfer reactions, organisms that use these proteins are phototrophs but they have not yet been shown to be photosynthetic. In the second and more complex type of phototrophy, light initiates electron transfer through oxidation of a chlorophyll and reduction of an electron acceptor; secondary electron transfer reactions that do not require light subsequently lead to the production of proton-motive force that can be coupled to ATP synthesis. This second mechanism is absolutely dependent upon (B)Chl-containing proteins known as photochemical RCs (Figure 1). Type 1 RCs produce weak oxidants and strong reductants through their terminal, electron-accepting [4Fe-4S] clusters; type 2 RCs produce strong oxidants and a weak reductant (a reduced quinone molecule). The two types of RCs have similar structures [57,62–64] and seem to share a common evolutionary origin [57,65]. To date, (B)Chl biosynthesis has not been detected in any archaeal organism, so photosynthesis most probably evolved after the divergence of the archaeal–eukaryal and bacterial lineages. Although most RCcontaining bacteria are autotrophs and are thus photosynthetic, some bacteria (e.g. heliobacteria) that have a single type of RC do not grow autotrophically when provided with CO2 and an electron source [39]; presumably, they only perform cyclic electron transfer for ATP synthesis. Similar to rhodopsin-containing organisms, these bacteria are not photosynthetic but are photoheterotrophs. Photosynthetic organisms produce a variety of light-harvesting antenna structures (the protein components of which do not share a common evolutionary ancestor) to enhance the rate of light-driven electron transport [57,65,66]. Examples include phycobilisomes, chlorosomes and a variety of light-harvesting (B)Chl and caroteno(B)Chl proteins [25,57,66] (Figure 1).
genome sequencing of photosynthetic prokaryotes, and the Genomes On-Line Database (http://www.genomesonline. org/) and other sources currently indicate that 55 Cyanobacteria, 12 Chlorobi, nine Chloroflexi, 24 Proteobacteria and two Firmicutes (heliobacteria) are or soon will be completely sequenced. These data will have a substantial impact on the understanding of the origins and evolution of photosynthesis while providing many exciting new insights into the properties of these ecologically and environmentally important organisms. Cyanobacteria: the oxyphototrophs Cyanobacteria are such an ancient and remarkably diverse group of Bacteria that even data for 55 organisms provide an extremely limited view of their complexity. There are >475 pure strains in the Pasteur Culture Collection of Cyanobacteria, and yet this collection includes only a small number of the several thousand described species. To illustrate the magnitude of this problem, the smallest genomes for photosynthetic bacteria are 1.7 Mb and are found in the marine, unicellular Prochlorococcus spp. [4,5] whereas Nostoc punctiforme has the largest genome www.sciencedirect.com
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(9 Mb) sequenced thus far [6]. N. punctiforme can differentiate into multiple specialized cells (hormogonia, akinetes and heterocysts), establishes cellular patterns of development for heterocysts within its filaments and forms symbioses with fungi and plants. Genome size estimates indicate that some Calothrix sp. genomes are even larger at 12–15 Mb [7]. These prokaryotes have genomes that are as large as those of yeast and gene contents approaching that of Drosophila melanogaster! As additional, morphologically and developmentally distinctive cyanobacteria are studied by genomic methods, new mechanisms that regulate cellular interactions are likely to emerge. Unlike other Gram-negative bacteria, colonial cyanobacteria do not appear to employ autoinducer-2 (a furanosyl borate diester [8]) or acyl-homoserine lactones [9] as quorumsensing and signaling molecules for biofilm development or other cellular interactions. Comparisons of cyanobacterial genome sequences from ecotypes of the same species and from closely related species are already providing new insights into the relationships between ecological niche, gene content and speciation for environments as different as the oligotrophic ocean and dense, nutrient-rich microbial mats. The most detailed studies to date of the closely related marine Synechococcus and Prochlorococcus species have provided important insights into the ecophysiology of these genera [4,5,10–12], and this information has been substantially extended by metagenomic data from the Sargasso Sea [13]. Specific gene-content differences have been correlated with the physiological properties of high-light and low-light adapted ecotypes of these genera. In turn, these properties can be correlated with the chemical and physical differences that are found in the upper and lower portions of the photic zone in the ocean [4,5,10–12]. Delong et al. [14] have recently expanded this concept by examining both organismal and gene-content variation as a function of depth in the planktonic microbial communities in the North Pacific subtropical gyre. Their results show that the distribution of taxonomic groups, functional gene repertoires and metabolic potentials vary with depth in ways that relate to carbon and energy metabolism, adhesion and motility, gene mobility and host–virus interactions. These studies raise interesting questions about how different ecotypes arise and how they persist in the oceans [12]. Ambient temperature and growth temperature optima seem to be important along with light, nutrients and competitor abundances [15]. The observation that cyanophages sometimes carry photosynthesis genes [16– 20] provides one explanation for how genes can be rapidly exchanged throughout these populations. The genome sequencing, metagenomics and ‘metatranscriptomics’ of Ward and coworkers [21] are addressing similar issues in the integrated ‘community metabolism’ and ecophysiology of the cyanobacteria of a different physicochemical environment: the phototrophic mats of the Octopus and Mushroom Springs in Yellowstone National Park. Stenou et al. [21] have recently shown that two populations of thermophilic Synechococcus spp. in these mats perform photosynthesis by day and seem to ferment stored carbohydrates to generate reductant for nitrogen fixation by night.
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Figure 2. Simple scheme for phototrophy based on BR or PR and ATP synthase. Absorption of light by retinal leads to isomerization of retinal causing a conformational change in PR or BR, which in turn leads to the expulsion of a proton to the periplasmic space. Translocation of protons to the cytoplasm is coupled to the synthesis and release of cytoplasmic ATP by ATP synthase. Image of BR molecules (left) reproduced, with permission, from Ref. [58]. ß (2004) Elsevier. ATP synthase image (right) reproduced, with permission, from Ref. [59]. ß (2004) Nature Publishing Group.
Figure 1. Distribution of reaction center types and antenna systems in photosynthetic bacteria, which are found in the Cyanobacteria, Chlorobi, Proteobacteria, Chloroflexi and Firmicutes. Type 1 RCs (left) have [4Fe-4S] clusters (Fe-S) as terminal electron acceptors, whereas type 2 RCs (right) have quinones (Q) as electron acceptors. Colors indicate whether a RC is a homodimer (e.g. heliobacteria and green sulfur bacteria) or a heterodimer [photosystem (PS) I and all type 2 RCs]. Cyanobacteria have Chl-a-containing PS I (left) and PS II (right) and have light-harvesting phycobilisomes that are principally associated with PS II. Dotted lines in the type 1 RC subunits indicate the existence of both a light-harvesting domain, which is structurally related to subunits CP43 and CP47 of PS II, and an electron transfer domain, which is structurally related to the subunits of both the PS II core and other bacterial type 2 RCs. Prochlorophytes lack phycobilisomes and, instead, have light-harvesting PCB proteins, which are structurally related to CP43 and bind to both divinyl-Chl a and divinyl-Chl b. Heliobacteria have homodimeric type 1 RCs with BChl g. Green sulfur bacteria (Chlorobi) have homodimeric type 1 RCs that bind to BChl a and a small amount of Chl a; their chlorosomes contain >200 000 BChl c, d or e molecules and a small amount of BChl a that is bound to the CsmA protein. The BChl-a-binding FMO protein connects chlorosomes to the RCs. Purple bacteria and FAPs are similar and have type 2 bacterial RCs that carry either BChl a (or BChl b in some purple bacteria). The antennae for these RCs are formed by ring-shaped, BChl-a-binding LH1 and LH2 complexes in purple bacteria or chlorosomes containing BChl c and BChl a in some FAPs. The LH1-like complexes of FAPs can also form rings around their type 2 RCs. ‘Red’ FAPs lack the chlorosomes that are found in ‘green’ FAPs. The chlorosomes of FAPs are usually smaller and contain fewer BChl c molecules than those found in the Chlorobi.
Chlorobi: green sulfur bacteria In contrast to the extraordinary richness of cyanobacterial diversity, the phylum Chlorobi (comprising the green sulfur bacteria) is a metabolically limited, physiologically www.sciencedirect.com
well-defined and genetically closely related bacterial group, which shares a common root with the Bacteroidetes. Comparative genomic analyses have enabled the elucidation of their unique BChl and carotenoid biosynthetic pathways. Chlorobi are obligately anaerobic photoautotrophs that (i) oxidize sulfur compounds, H2 or ferrous iron; (ii) fix carbon by the reverse tricarboxylic acid cycle; (iii) synthesize BChl c, d or e along with BChl a and Chl a; and (iv) have a photosynthetic apparatus that comprises a type 1 reaction center, the Fenna-Matthews-Olson (FMO) BChl-a-binding protein and chlorosomes that each contain >200 000 BChl c, d or e molecules (Figure 1) [22–25]. Because of the availability of an efficient natural transformation system and its ability to grow rapidly with thiosulfate as an electron donor, Chlorobium tepidum – the 2.15 Mb genome of which was sequenced by The Institute for Genomic Research [26] – has become the model organism for this group of phototrophs. Insights into the physiology, metabolism and light-harvesting apparatus of this organism have been reviewed elsewhere [22–25]. Although fewer Chlorobi genomes have been sequenced than cyanobacterial genomes, the ten sequenced and two anticipated genomes encompass most of the currently known diversity of this group. The Joint Genome Institute of the Department of Energy (JGI-DOE) has sequenced most of the type strains of the Chlorobi, and these data provide benchmarks for comparisons of future isolates. Complete genomes are already available for two additional strains, Pelodictyon luteolum DSM273 and Chlorobium chlorochromatii (Box 2 and Figure 3), and draft genomes are available for seven additional strains (C. ferrooxidans, C. phaeobacteroides DSM 266, C. limicola DSM245, C. vibrioforme DSM 265, Prosthecochloris aestuarii SK413, Pelodictyon phaeoclathratiforme and an enrichment culture, C. phaeobacteroides BS-1, isolated from 100 m below the surface of the Black Sea [27]). Sequencing of Chloroherpeton thalassium and C. vibrioforme 8327d
Review
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Box 2. Phototrophic consortia With the exception of Chloroherpeton thalassium, which exhibits gliding motility, all known green sulfur bacteria are non-motile. Some green sulfur bacteria (‘epibionts’) become motile by forming phototrophic consortia through a specific association with a bproteobacterium, denoted the ‘central rod’ [60,61,67] (Figure 3). Each polarly flagellated central rod carries 20–60 epibiont cells and the entire consortium is phototactic in response to light signals perceived by the epibiont. The nature of any metabolic coupling between the two organisms is not yet known but possibilities include transfer of reduced carbon and/or nitrogen from the epibiont to the central rod and possibly the provision of sulfide from sulfate reduction or H2 from the central rod to the epibiont. At present, the mechanisms of cell-to-cell signaling for phototaxis and coordination of cell division are completely unknown. Genome sequencing of the two partners of ‘Chlorochromatium aggregatum’, which was isolated as an enrichment culture from Lake Dagow [60,67], should help to answer many of these questions. The 2.57 Mb genome of the epibiont, Chlorobium chlorochromatii, which is not an obligate symbiont [67], has already been completely sequenced (http://img.jgi.doe.gov/). Sequencing of the b-proteobacterial central rod, which cannot be grown independently from C. chlorochromatii and is most closely related to Rhodoferax sp. [60], is now in progress at JGI-DOE. C. chlorochromatii encodes a family of calcium-dependent, RTX-toxin-like proteins that might be involved in cellular adhesion to the central rod [68]. The largest gene is predicted to encode a protein of 36 805 amino acids, one of the largest predicted proteins known to date. This gene occurs in an apparent operon with a sequence-related gene of 20 646 codons, producing an operon of >170 kb. Homologs of these genes occur in Magnetococcus sp. MC-1 (15 245 codons) and Synechococcus sp. RS9917 (28 178 codons) but their functions are unknown.
should be completed later this year. The sequenced Chlorobi have 2–3 Mb genomes that encode 1750– 2800 genes (http://img.jgi.doe.gov/) and pairwise comparisons show that Chlorobi strains share a common core-set of 1400–1500 genes. Chlorobi genomes encode only a few
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predicted transporters for organic molecules, have a small number of predicted transcription regulators, and are largely devoid of two-component histidine kinases and response regulators. These observations suggest that Chlorobi live in relatively constant (and energy-limited) conditions and that they probably have a limited capacity to respond to changes in their physicochemical environment [22,26]. This is a trait that is shared by members of the cyanobacteria with reduced genomes, such as Prochlorococcus and marine Synechococcus [4,5,10]. Pigment biosynthetic pathways in Chlorobi The availability of multiple Chlorobi genome sequences and of a highly efficient natural transformation system for C. tepidum has facilitated the identification of genes that encode enzymes for pigment biosynthesis and other physiological processes in green sulfur bacteria. C. tepidum synthesizes three chlorophylls: BChl c, BChl a and Chl a [24]. Before the completion of its genome sequence, no enzyme specifically involved in BChl c, d or e biosynthesis had been identified, and now all steps but one in the pathway leading from chlorophyllide a to BChl c are known. Only the enzyme responsible for the removal of the C-132 methylcarboxyl group has not yet been identified. Moreover, the erroneous identification of the 8-vinyl reductase as BchJ has recently been corrected [24]. Similarly, although nothing was known about the pathway for carotenoid biosynthesis in Chlorobi before the availability of the genome sequence, all of the enzymes required for synthesis of chlorobactene and isorenieratene have been identified [28,29]. Through these analyses, it is now clear that carotenoid biosynthesis in Chlorobi is more similar to the pathway in Cyanobacteria than to that in other bacteria [28]. Although no member of the three known families of
Figure 3. Phototrophic consortia. (a) Transmission electron micrograph of a thin section of the phototrophic consortium ‘Chlorochromatium aggregatum’. The central rod (CR; a b proteobacterium with a putative genome size of 4–5 Mb) and the epibiont cells (EB; the green sulfur bacterium Chl. Chlorochromatii with a genome size of 2 572 079 bp) are indicated. (b) Light micrograph of ‘Chlorochromatium aggregatum’. Scale bar = 5 mm. Scanning electron micrographs of ‘Pelochromatium roseum’ before (c) and after (d) division of the EB. Scale bar = 1 mm. (e) Thin-section electron micrograph of the junction of an EB cell with the central rod. Note that chlorosomes, the electron-dense ellipsoids on the inner surface of the cytoplasmic membrane of the EB cells, do not occur at the junction with the CR and that an additional wall layer between the cells can be seen at this junction [60]. Additionally, a paracrystalline structure is seen at the cell junctions at the inner surface of the CR (boxed region). Scale bar = 0.5 mm. Parts (b), (c) and (d) reproduced, with permission, from Ref. [61]. ß (2002) Springer. www.sciencedirect.com
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lycopene cyclases could be identified in the C. tepidum genome, phylogenetic profiling (using data from multiple photosynthetic bacteria) and complementation of a lycopene-producing strain of Escherichia coli both identified a fourth type of lycopene cyclase encoded by the C. tepidum open reading frame CT0456 [29] (J.A. Maresca et al., unpublished). The identification of the ‘missing’ lycopene cyclase in C. tepidum also enabled the identification of ‘missing’ lycopene cyclases in several cyanobacteria. Interestingly, many cyanobacteria have an ortholog and a paralog of CT0456, and both of these enzymes seem to be involved in cyclase reactions with lycopene (J.E. Graham and D.A. Bryant, unpublished). Synechococcus sp. PCC7942 has lycopene cyclases that belong to two of the four families, which demonstrates the mosaic nature of carotenoid biosynthesis and is a likely example of lateral gene transfer. The sequenced Chlorobi strains are metabolically similar but can be separated according to particular phenotypes (e.g. green-colored strains that contain BChl c and chlorobactene versus brown-colored strains that contain BChl e and isorenieratene, or strains that oxidize sulfur compounds versus ferrous iron). Thus, whole-genome comparisons can identify candidate genes that are responsible for defined physiological differences among these strains. This approach was recently used to search for candidate gene(s), the product(s) of which could convert BChl c into BChl e. This analysis identified a radical SAM enzyme and an adjacent dehydrogenase as the most likely candidates for this transformation (J.A. Maresca and D.A. Bryant, unpublished). Interestingly, these two genes occur adjacent to the gene encoding g-carotene cyclase, which produces b-carotene, the precursor of isorenieratene [29] (J.A. Maresca et al., unpublished). The proximity of these genes on a 6 kb segment of the chromosome provides a possible explanation for the polyphyletic nature of the ‘brown’ phenotype among green sulfur bacteria. This gene proximity would greatly facilitate their lateral transfer among the Chlorobi, and such transfer would immediately confer the ability to populate a new environmental niche. The synthesis of BChl e is another example of a reaction for which both oxygen-independent and oxygen-dependent enzymes occur in nature [23,30]. The C-7 formyl group introduced during BChl e biosynthesis must be derived from water because this reaction occurs under anoxic conditions; however, the C-7 formyl group of Chl b in Prochlorococcus sp., green algae and higher plants is derived from oxygen [31]. Chloroflexi: filamentous anoxygenic phototrophs Because of their diverse metabolic and physiological properties, genomic analyses of diverse strains of Chloroflexi are likely to produce novel insights into the evolution of photosynthesis. The phylum Chloroflexi is one of the earliest diverging lineages of the Bacteria, and it contains several genera of filamentous, gliding bacteria that perform anoxygenic photosynthesis (FAPs) [32]. The phylum contains two orders, the ‘Chloroflexales’ and ‘Herpetosiphonales’. Herpetosiphon aurantiacus, the type strain of the latter order, has recently been sequenced by JGI-DOE. The 6.6 Mb genome of this heterotrophic bacterium www.sciencedirect.com
encodes the enzymes for carotenoid biosynthesis but has no genes for the enzymes of BChl biosynthesis or components of the photosynthetic apparatus. Pierson and Castenholz first isolated Chloroflexus aurantiacus, the type strain of the Chloroflexi and ‘Chloroflexales’, in the early 1970s from Yellowstone National Park and other thermal features [32]. Cfx. aurantiacus synthesizes BChl a and BChl c and has type 2 RCs and chlorosomes but lacks the FMO protein (Figure 1). The ‘red/orange’ FAPs of the genera Roseiflexus and Heliothrix do not synthesize BChl c and lack chlorosomes [32,33] (Figure 1). Until recently, the incomplete 5.2 Mb draft sequence of Cfx. aurantiacus was the only genomic information available for any photosynthetic member of the Chloroflexi. However, JGI-DOE will soon release the draft genomes of four additional Chloroflexi: Cfx. aggregans (4.5 Mb), Roseiflexus sp. strain RS-1 (5.8 Mb; from Octopus Springs, Yellowstone National Park), Roseiflexus castenholzii (5.6 Mb; from Nakabusa Hot Springs, Japan) and the heterotroph Herpetosiphon aurantiacus (6.6 Mb). In addition, three sulfide-oxidizing FAPs (Chlorothrix halophila, Oscillochloris sp. strain UdG 9002 and Chloronema giganteum strain UdG 9001) are scheduled for sequencing by JGI-DOE later this year. A consortium of Russian scientists is sequencing the genome of Oscillochloris trichoides (R. Ivanovskii and B. Kuznetsov, personal communication). O. trichoides produces a type I ribulose-1,5-bisphosphate carboxylase–oxygenase (RubisCO) and fixes carbon dioxide by the Calvin cycle rather than by the 3-hydroxypropionate cycle [34]. However, it is not known whether other FAPs have RubisCO and use the Calvin cycle. It will be interesting to see whether the sulfide-oxidizing FAPs have type 1 RCs like the Chlorobi or have type 2 RCs (and reverse electron transport) like Cfx. aurantiacus and purple bacteria. Roseiflexus sp. and O. trichoides have nitrogenase genes, and other FAPs also probably fix dinitrogen. Much more information will soon be available for these poorly characterized phototrophs. Like purple non-sulfur bacteria, Cfx. aurantiacus exhibits considerable metabolic diversity and it can grow as an aerobic chemoheterotroph or as an anaerobic photoheterotroph. Using electrons derived from H2 or H2S under anoxic or microaerophilic conditions, some strains of Chloroflexus sp. grow photoautotrophically by fixing CO2 through the 3-hydroxypropionate pathway [32]. In nature, Cfx. aurantiacus probably grows under microaerophilic or alternating oxic and anoxic conditions but it only fully develops its photosynthetic apparatus under anoxic conditions. As a result, Cfx. aurantiacus encodes some enzymes that can function under either oxic or anoxic conditions [23,30]. For example, both bchE and acsF genes are found in the Cfx. aurantiacus genome; these genes encode the oxygen-independent isocyclic ring cyclase and the oxygen-dependent isocyclic ring cyclase, respectively. Both Roseiflexus sp. strains and Cfx. aggregans also produce both of these enzymes. Interestingly, the same pattern is not found for hemF and hemN, which encode oxygen-dependent and oxygen-independent coproporphyrinogen oxidases, respectively. The two Chloroflexus strains have both genes, whereas the Roseiflexus sp. strains do not encode hemF.
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Proteobacteria: purple non-sulfur and purple sulfur bacteria Photosynthesis is a trait that is widespread but not universal among members of the Proteobacteria and is found in morphologically and metabolically diverse species. Genome sequence information has largely confirmed the physiological versatility and corresponding large genome sizes of these organisms. Some of the photosynthetic proteobacteria are well suited for studies of global gene regulation because many members are facultatively phototrophic or photosynthetic under anoxic conditions. Photosynthetic Proteobacteria can be found in the a, b and g subdivisions but, to date, almost all available genome sequences are from members of the a subdivision. Several genomes have been determined (http://www.genomesonline.org), including those of Rhodopseudomonas palustris (5.5 Mb) [35], two Rhodobacter sphaeroides strains (4.6 Mb) [36] (http:// www.ncbi.nlm.nih. gov/), Rhodospirillum rubrum (4.4 Mb) (http://www.ncbi. nlm.nih.gov/), Roseobacter denitrificans (http://genomes. tgen.org/rhodobacter.html), Bradyrhizobium sp. (9.1 Mb) and Roseobacter sp. (4.1 Mb) (http:// www.genomesonline. org). Only two g-subdivision members have been studied and no photosynthetic b-proteobacterium has been sequenced. Thus, the environmentally important purple sulfur bacteria, all of which belong to the g subdivision, along with purple bacteria of the b subdivision (many of which can also use sulfur or H2 as electron donors), represent groups about which little genome sequence information is available. Finally, a large number of mostly a-proteobacteria in diverse freshwater, saline, marine, soil and hot-spring environments seem to have photosynthesis gene clusters but, in many cases, the function of the relatively low levels of BChl a produced under aerobic conditions is unknown [37,38]. Projected sequencing projects and comparative analyses will help to define the genetic, physiological and metabolic differences among these aerobic anoxygenic phototrophs, photoheterotrophs like Rhodobacter sp., and the photolithoautotrophic purple sulfur bacteria. Genome sequence data, in combination with the physiological and metabolic versatility of these organisms, should lead to engineered strains for diverse applications in biotechnology, including bioremediation, lignin degradation and biofuels and hydrogen production [35]. Heliobacteria Heliobacteria, first described by Gest and Favinger in 1983 [39], are the most recently discovered group of bacteria containing RCs and they remain the most poorly characterized overall. Heliobacteria are members of the phylum Firmicutes and are closely related to clostridia. Like Bacillus or Clostridium sp., heliobacteria produce heatresistant endospores and, to date, no characterized member of this group is known to grow photoautotrophically. Studies with Heliobacillus mobilis have shown that many of the genes for synthesis of BChl g and the RC are located in a 30 kb cluster similar to the photosynthesis gene clusters found in purple bacteria [40,41]. Thus, it is possible that the heliobacteria obtained their photosystem components through a lateral gene transfer event. However, a recent detailed analysis of RC and www.sciencedirect.com
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light-harvesting protein sequences argues that lateral gene transfer is not required to explain the current distribution of RC sequences [42]. No complete heliobacterial genome sequence is yet available, although the 3.1 Mb draft genome of Heliobacterium modesticaldum has recently been made available for searches (http://genomes. tgen.org/helio.html). The H. modesticaldum genome sequence will help to clarify the possible lateral acquisition of photosynthesis genes and it will also help to identify genes that are required for carotenoid, BChl, and RC synthesis and function. The relatively small genome of H. modesticaldum is likely to provide new insights into genes that are functionally important in sporulation and regulation of this process. Bacteriorhodopsin-based phototrophy in halophilic prokaryotes Comparative analyses of sequenced genomes and metagenomic data from the ocean have shown the great diversity in the structure and function of rhodopsins and have demonstrated how easily lateral gene transfer can occur among unrelated organisms. Bacteriorhodopsin (BR) in the so-called ‘purple membrane’ of halophilic archaea has been studied for three decades and the structure and function of BR is known in great detail [1] (Figure 2). The halophilic archaea grow well in the dark as aerobic chemoheterotrophs; however, strains that synthesize BR exhibit light-enhanced growth under anoxic conditions and are, therefore, facultatively phototrophic [43]. Among these haloarchaea, multiple rhodopsins with diversified functions can exist within a single cell. The genome of the haloarchaeon Haloarcula marismortui encodes six homologous rhodopsins: one proton-pumping BR, one chloride-pumping halorhodopsin, two sensory rhodopsins and two opsins of unknown function [44]. The importance of these rhodopsins for environmental adaptation was recently and elegantly illustrated through the genome sequence of a halophilic bacterium, Salinibacter ruber, which taxonomically belongs to the Cytophaga–Flexibacter–Bacteroides group [45]. Genomewide analyses showed that S. ruber has evolved convergently towards halophilic archaea at both the physiological level (different genes producing a similar overall phenotype) and at the molecular level (independent mutations yielding proteins with similar sequences or structures). Although the identity of the donor organism (or organisms) is uncertain, the genome of S. ruber encodes four rhodopsins – the obvious result of lateral gene exchange with the haloarchaea. One rhodopsin resembles a chloride pump and two resemble sensory rhodopsins. The fourth is a proton-pumping rhodopsin, xanthorhodopsin, which is related to proteorhodopsins but contains a salinixanthin carotenoid chromophore in addition to retinal [46]. Proteorhodopsin in marine prokaryotes Other than those in haloarchaea, the first example of a prokaryotic rhodopsin was one found in a marine proteobacterium and, thus, it was named proteorhodopsin (PR) [47,48]. This identification was based on a metagenomics approach in which large fragments of genomic DNA isolated from marine picoplankton were cloned and
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sequenced. Like BR, PR has been shown by heterologous expression to function as a light-driven, transmembrane proton pump in E. coli [47] and, thus, it could contribute substantially to the energy budget in cells living in the photic zone of oceans. Therefore, it is not surprising that sequencing of both short and long fragments of environmental DNA has shown that a wide range of PRs exist in highly different types of marine prokaryotic plankton [13,49–51]. For example, PR is present in marine euryarchaeotes in the photic zone of the North Pacific subtropical gyre, whereas the marine euryarchaeotes living below the photic zone do not have PR, clearly as a consequence of environmental adaptation [51]. Recently, Pelagibacter ubique, a representative of the ubiquitous SAR11 marine proteobacteria, was axenically cultivated and its genome was sequenced [52]. This genome contains one gene encoding PR [52,53] but has no genes that are characteristic for CO2 fixation. The genomes of other marine proteobacteria (Photobacterium sp. SKA34 and Vibrio angustum S14) and marine Bacteroidetes (Polaribacter irgensii, Cellulophaga sp. MED134, Tenacibaculum sp. MED152 and Psychroflexus torquis ATCC700755), which are currently being sequenced by the J. Craig Venter Institute (https:// research.venterinstitute.org/moore/), also contain PRs. Although laboratory growth experiments with P. ubique have not yet demonstrated that this bacterium grows faster in the light than in the dark [53], it seems likely that PR enables the bacterium to benefit from light even though the experimental conditions to show this have not yet been identified. Although the structure of this PR is consistent with a function in proton pumping, it remains possible that this molecule could instead transport another substrate or function as a sensory molecule. To put it briefly, it seems that many, if not most, of the planktonic prokaryotes in the photic zone of the oceans that do not contain photosynthetic reaction centers nevertheless exploit light by having acquired PR through lateral gene transfer. The exact physiology of the marine prokaryotes that harbor these PRs is not always clear. For example, a recently characterized 95 kb genomic fragment from a marine proteobacterium that encodes a PR also encodes a putative reverse dissimilatory sulfite reductase operon, which could provide reducing equivalents for autotrophic growth by oxidizing a reduced sulfur compound [54]. The ‘cosmopolitan’ rhodopsins versus the ‘refined’ reaction centers Shotgun sequencing of DNA from the Sargasso Sea illustrated how PRs are widespread in oceanic microorganisms [13]. Although the exact functions of the rhodopsins are often not known, genome sequencing projects of organisms in pure culture have also confirmed that rhodopsins are much more widely distributed among different organismal lineages than first anticipated. For example, rhodopsins of unknown function have been found in the genomes of organisms as diverse as halophilic archaea and bacteria, marine proteobacteria, marine Bacteroidetes, marine euryarchaeotes, g-radiation-resistant actinobacteria (Rubrobacter xylanophilus and Kineococcus radiotolerans) www.sciencedirect.com
and the Gram-positive Exiguobacterium sp. 255–15, in addition to certain cyanobacteria, fungi, green algae and a dinoflagellate (Pyrocystis lunula). The single rhodopsins encoded by the genomes of the cyanobacteria Nostoc sp. PCC7120 (Alr3165) and of the fungus Leptosphaeria maculans have recently been shown by experimental characterization to be a sensory rhodopsin [55] and a proton-pumping rhodopsin [56], respectively. As a last example, the early-diverging cyanobacterium, Gloeobacter violaceus, encodes a rhodopsin (Gll0198) that seems to be most similar to the proton-pumping xanthorhodopsin of S. ruber. If rhodopsin-based phototrophy is transferred so easily among organisms, why do only a few groups of RC-based phototrophs dominate phototrophic niches? Part of the answer might be that rhodopsin-based phototrophy uses light energy relatively inefficiently. First, to produce the proton-motive force required to produce one ATP, three to four BR molecules must each absorb a photon and release a proton to the periplasmic space. When a proteobacterial RC absorbs four photons, two ubiquinol molecules are produced and their dark re-oxidation by the cytochrome bc1 complex enables the production of two ATP molecules [57]. Second, the single retinal chromophore in the photochemical units of most rhodopsin-based phototrophs has a much smaller absorption cross-section than the photochemical units of RC-based phototrophs, which can have hundreds to thousands of chromophores [57]. Thus, a rhodopsin-based phototroph would have to synthesize many more energetically expensive BR or PR molecules to absorb the same amount of light energy. The highly efficient and extensive lightharvesting antenna systems of photosynthetic bacteria
Box 3. Are there more phototrophs out there? Our current knowledge of photosynthesis is based almost exclusively on cultivated strains but, given the vast diversity of microorganisms on Earth, these organisms are unlikely to represent the full spectrum of light utilization. Continued genome sequencing of cultivated and uncultivated organisms will undoubtedly reveal many more microbial groups that harbor rhodopsins. It will be interesting to see if there are any organisms that combine rhodopsin-based phototrophy with CO2 fixation because such organisms could then be classified as Chl-independent photosynthetic organisms. A priori, there seems to be no reason why the carbon fixation reactions of the Calvin cycle could not be driven by the combination of PR and sulfide:quinone oxidoreductase coupled to a type I NADH dehydrogenase for reverse electron flow. Alternatively, a rhodopsin and hydrogenase could be coupled to provide the energy and reducing power for photolithoautotrophic growth. As more environments are sampled, metagenomics are likely to reveal photosynthetic organisms with combinations of components that have not yet been observed in cultivated species (e.g. an anoxygenic organism with two types of RCs), novel solutions to the problem of light harvesting or the existence of new phyla that have not previously been shown to contain phototrophic or photosynthetic members. This latter prediction is not simply an idle speculation: a new phylum-level, RC-containing phototroph has recently been discovered in this manner and will be described in detail elsewhere (D.A. Bryant et al., unpublished). The rapidly increasing body of genome sequence and metagenomic data will help to answer questions about the origin and evolution of photosynthesis [42,69].
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enable the RCs to function at maximal efficiency even at relatively low light intensities. If RC-based phototrophy is so much more efficient than rhodopsin-based systems, why does this type of phototrophy not spread laterally? Perhaps it does on rare occasions, but because even the simplest chlorophyll-based photosystem requires 30 unique genes, this capability probably cannot be laterally transferred as easily as rhodopsinbased phototrophy. Lateral transfer of rhodopsin-based photosystems requires only the genes encoding the rhodopsin apoprotein and a carotenoid oxygenase–lyase that produces retinal [54] if the recipient already has the capability to produce an appropriate carotenoid. If it does not, retinal biosynthesis can be performed with just four genes (crtBIY–blh) [54]. Therefore, rhodopsin-based systems might be much more prone to lateral transfer and, driven by the selection of light, could thus distribute themselves among distantly related but sympatric organisms. Metagenomic analyses of photic environments are likely to identify many new examples in the near future (Box 3). Concluding remarks and future perspectives Genome sequencing of cultured organisms and metagenome sequencing of DNA from uncultured organisms of photic environments has already greatly accelerated the pace of new discoveries for the Cyanobacteria and Chlorobi. As more genomic sequence data become available for other phototrophic organisms, comparative bioinformatics will certainly catalyze advances in knowledge of the metabolism, gene regulation and physiology of these groups and might answer many outstanding questions about these organisms (Box 4). Metagenomic studies will ultimately lead to insights that transcend the level of individual organisms and will facilitate a broader and deeper understanding of the community-level dynamics among phototrophs (and non-phototrophs). This approach has the potential to Box 4. Outstanding questions (i) Because photosynthetic reaction centers evolved only once, which came first: type 1 or type 2 reaction centers? (ii) How did ancestors of cyanobacteria acquire type 1 and type 2 reaction centers, and how did oxygenic photosynthesis evolve? (iii) Did bacteria that principally synthesize BChl a arise before or after those that principally synthesize Chl a? (iv) Beyond the five known examples, how many additional bacterial phyla contain members that can synthesize (B)Chl and photochemical reaction centers? (v) Are there organisms that combine a rhodopsin-based ATP generation system with enzymes for the oxidation of an inorganic electron donor (e.g. sulfide or H2) to drive autotrophic carbon dioxide reduction? (vi) What is the physiological role of the low amounts of BChl a found in aerobic anoxygenic phototrophs that are common in the oceans? (vii) Nature has evolved multiple, independent solutions to the problem of harvesting solar energy. Are there novel types of light-harvesting antenna complexes yet to be discovered, and can metagenomics help to identify them? (viii) What are the principal drivers behind the evolution and distribution of phototrophy? How do nutrients, temperature, light and other parameters interact with genomic content to produce niche partitioning and ecotype distributions in photic environments? www.sciencedirect.com
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explain how gene contents map onto taxonomic compositions, physiological and metabolic capabilities and gene expression patterns of phototrophs in diverse photic environments – all of which together lead to the primary energy input that ultimately drives life and its evolution on Earth. Acknowledgements The authors would like to thank Julia A. Maresca for critical reading of the manuscript and many helpful comments. We also thank Joachim Weber (Texas Tech University) for use of the ATP synthase image in Figure 2 and Jo¨ rg Overmann (Ludwig Maximilians Universita¨t, Mu¨nchen) for providing Figure 3 parts (a) and (e), and for use of parts (b), (c) and (d). D.A.B. gratefully acknowledges support for genomics studies from the National Science Foundation (MCB-MCB-0519743 and MCB-0523100) and from the Department of Energy (DE-FG02– 94ER20137). N.-U.F gratefully acknowledges support from The Danish Natural Science Research Council (grant 21–04–0463).
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