Review TRENDS in Microbiology Vol.14 No.11

Prokaryotic and phototrophy illuminated

Donald A. Bryant1 and Niels-Ulrik Frigaard2

1 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA 2 Institute of Molecular Biology and Physiology, University of Copenhagen, Sølvgade 83H, 1307 Copenhagen K, Denmark

Genome sequencing projects are revealing new Glossary information about the distribution and evolution of photosynthesis and phototrophy. Although coverage Anoxygenic photosynthesis: photosynthesis performed by organisms that do of the five phyla containing photosynthetic prokaryotes not evolve oxygen; it uses electron donors other than water for carbon dioxide reduction. (Chlorobi, Chloroflexi, Cyanobacteria, Proteobacteria Bacteriorhodopsin (BR): a first identified in ; translo- and Firmicutes) is limited and uneven, genome cates to the periplasm after light-induced isomerization of . Chlorobi: bacterial phylum that includes the -colored and brown-colored sequences are (or soon will be) available for >100 strains green sulfur bacteria; these bacteria have type 1 reaction centers (containing from these phyla. Present knowledge of photosynthesis BChl a and Chl a) and chlorosomes containing BChl c, d or e. They fix carbon by 2+ is almost exclusively based on data derived from culti- the reverse tricarboxylic cycle and oxidize sulfide, sulfur, thiosulfate, Fe or H2. Chloroflexi: bacterial phylum that includes the filamentous anoxygenic vated species but metagenomic studies can reveal new (FAPs), formerly known as the green gliding or green filamentous organisms with novel combinations of photosynthetic bacteria. and phototrophic components that have not yet been Cyanobacteria: bacterial phylum that includes all oxygen-evolving photosyn- thetic bacteria; they have Chl a-containing type 1 and type 2 reaction centers described. Metagenomics has already shown how the and fix carbon by the reductive pentose-phosphate (Calvin–Benson–Bassham) relatively simple phototrophy based upon cycle; most have phycobilisomes as light-harvesting antennae (but see has spread laterally throughout , Bacteria and Prochlorophytes). Filamentous anoxygenic phototrophs (FAPs): Chloroflexi that have BChl a- eukaryotes. In this review, we present examples that containing, type 2 reaction centers. They might have chlorosomes that contain reflect recent advances in biology as a result BChl c and most fix carbon by the 3-hydroxypropionate cycle whereas some of insights from genome and metagenome sequencing. oxidize sulfide or H2. : endospore-producing photoheterotrophic bacteria of the phy- lum Firmicutes that have type 1 reaction centers and BChl g. Photosynthesis and phototrophy Oxygenic photosynthesis: photosynthesis that uses water as the electron Photosynthesis is arguably the most important biological donor and leads to oxygen evolution. Photochemical reaction center: a multisubunit complex containing process on Earth, and only two mechanisms for collecting or bacteriochlorophylls, in which light energy is transduced into light energy and converting it into chemical energy have redox chemistry. been described (Box 1). The first mechanism, which is Photosynthesis: the reduction of carbon dioxide into biomass using energy derived from light. dependent upon photochemical reaction centers (RCs; Phototrophy: a metabolic mode in which organisms convert light energy into see Glossary) that contain (bacterio)- chemical energy for growth. [(B)Chl], is found in five bacterial phyla: Cyanobacteria, Prochlorophyte: a cyanobacterium such as Prochlorococcus spp. that synthe- sizes both divinyl-Chl a and divinyl-Chl b but lacks phycobilisomes. Proteobacteria, Chlorobi, Chloroflexi and Firmicutes. All (PR): a rhodopsin first identified in marine proteobacteria, currently described Chlorobi and Cyanobacteria strains which translocates protons to the periplasm after light-induced isomerization are photoautotrophs but only some strains of Chloroflexi of retinal. Purple bacteria: bacteria of the phylum Proteobacteria that produce BChl a or b [filamentous anoxygenic phototrophs (FAPs)], Proteobac- under oxic or anoxic conditions. They have type 2 reaction centers and teria (purple sulfur and purple non-sulfur bacteria) and membrane-intrinsic caroteno-BChl antennae; many oxidize sulfide, thiosulfate, or H2 and they fix carbon by the reductive pentose-phosphate (Calvin–Benson– Firmicutes (heliobacteria) are phototrophic (Figure 1). Bassham) cycle. The second mechanism employs rhodopsins, retinal- Rhodopsin: a membrane-intrinsic protein characterized by seven transmem- binding that respond to light stimuli [1].Several brane a-helices and a covalently attached carotenoid, retinal. Type 1 reaction center: RC family found in cyanobacteria, green sulfur bacteria homologous types of rhodopsins are known in microbes and heliobacteria. They have either homodimeric or heterodimeric cores with and include energy-conserving transmembrane [4Fe-4S] clusters as their terminal electron acceptors and produce weak pumps [bacteriorhodopsin (BR), proteorhodopsin (PR), oxidants and strong reductants (reduced ferredoxin). Type 2 reaction center: RC family found in cyanobacteria, purple bacteria and xanthorhodopsin] (Figure 2), transmembrane chloride filamentous anoxygenic bacteria; all have heterodimeric cores with quinones pumps () and light sensors (sensory rho- as terminal electron acceptors. They produce strong oxidants and weak dopsins) [1,2]. Here, we review how genome and metagen- reductants (hydroquinone). ome sequencing studies are providing new insights into the physiology, metabolism and evolution of the organ- Genome sequencing projects for photosynthetic isms that perform these two processes for the capture of prokaryotes light energy. Synechocystis sp. PCC6803, a unicellular cyanobacterium, was the third prokaryote and the first photosynthetic organ- ism to have its chromosome completely sequenced [3].Over Corresponding author: Bryant, D.A. ([email protected]). Available online 25 September 2006. the past decade, there has been explosive growth in the www.sciencedirect.com 0966-842X/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2006.09.001 Review TRENDS in Microbiology Vol.14 No.11 489

Box 1. Photosynthesis and phototrophy (9 Mb) sequenced thus far [6]. N. punctiforme can differ- entiate into multiple specialized cells (hormogonia, aki- Photosynthesis is the reduction of CO into biomass using energy 2 netes and heterocysts), establishes cellular patterns of derived from light. Biological CO2 reduction requires both ATP and electrons, which can be provided as NADPH or reduced ferredoxin. development for heterocysts within its filaments and forms However, the ultimate electron source is organism-dependent and symbioses with fungi and plants. Genome size estimates can be H2O, H2S, H2 or other reduced inorganic compounds. indicate that some Calothrix sp. genomes are even larger at Phototrophy refers to a metabolic mode in which organisms convert light energy into chemical energy for growth. Thus, all photosyn- 12–15 Mb [7]. These prokaryotes have genomes that are as thetic bacteria are phototrophic but not all phototrophic bacteria are large as those of yeast and gene contents approaching that photosynthetic. of Drosophila melanogaster! As additional, morphologi- Two mechanistically distinct processes empower phototrophy. In cally and developmentally distinctive cyanobacteria are the first and simplest case, light energy directly drives proton studied by genomic methods, new mechanisms that reg- expulsion from cells through the proteins BR or PR, thereby creating a proton-motive force that can be used either to drive ATP synthesis ulate cellular interactions are likely to emerge. Unlike through ATP synthase or to drive various secondary transport other Gram-negative bacteria, colonial cyanobacteria do processes [1,47] (Figure 2). Because PRs and BRs do not mediate not appear to employ autoinducer-2 (a furanosyl borate electron transfer reactions, organisms that use these proteins are diester [8]) or acyl-homoserine lactones [9] as quorum- phototrophs but they have not yet been shown to be photosynthetic. sensing and signaling molecules for biofilm development In the second and more complex type of phototrophy, light initiates electron transfer through oxidation of a chlorophyll and or other cellular interactions. reduction of an electron acceptor; secondary electron transfer Comparisons of cyanobacterial genome sequences from reactions that do not require light subsequently lead to the ecotypes of the same species and from closely related production of proton-motive force that can be coupled to ATP species are already providing new insights into the rela- synthesis. This second mechanism is absolutely dependent upon tionships between ecological niche, gene content and spe- (B)Chl-containing proteins known as photochemical RCs (Figure 1). Type 1 RCs produce weak oxidants and strong reductants through ciation for environments as different as the oligotrophic their terminal, electron-accepting [4Fe-4S] clusters; type 2 RCs ocean and dense, nutrient-rich microbial mats. The most produce strong oxidants and a weak reductant (a reduced quinone detailed studies to date of the closely related marine molecule). The two types of RCs have similar structures [57,62–64] Synechococcus and Prochlorococcus species have provided and seem to share a common evolutionary origin [57,65]. To date, important insights into the ecophysiology of these genera (B)Chl biosynthesis has not been detected in any archaeal organism, so photosynthesis most probably evolved after the divergence of [4,5,10–12], and this information has been substantially the archaeal–eukaryal and bacterial lineages. Although most RC- extended by metagenomic data from the Sargasso Sea [13]. containing bacteria are autotrophs and are thus photosynthetic, Specific gene-content differences have been correlated with some bacteria (e.g. heliobacteria) that have a single type of RC do the physiological properties of high-light and low-light not grow autotrophically when provided with CO2 and an electron source [39]; presumably, they only perform cyclic electron transfer adapted ecotypes of these genera. In turn, these properties for ATP synthesis. Similar to rhodopsin-containing organisms, these can be correlated with the chemical and physical differ- bacteria are not photosynthetic but are . Photo- ences that are found in the upper and lower portions of the synthetic organisms produce a variety of light-harvesting antenna photic zone in the ocean [4,5,10–12]. structures (the protein components of which do not share a Delong et al. [14] have recently expanded this concept by common evolutionary ancestor) to enhance the rate of light-driven electron transport [57,65,66]. Examples include phycobilisomes, examining both organismal and gene-content variation as chlorosomes and a variety of light-harvesting (B)Chl and caroteno- a function of depth in the planktonic microbial commu- (B)Chl proteins [25,57,66] (Figure 1). nities in the North Pacific subtropical gyre. Their results show that the distribution of taxonomic groups, functional genome sequencing of photosynthetic prokaryotes, and the gene repertoires and metabolic potentials vary with depth Genomes On-Line Database (http://www.genomesonline. in ways that relate to carbon and energy metabolism, org/) and other sources currently indicate that 55 Cyano- adhesion and motility, gene mobility and host–virus inter- bacteria, 12 Chlorobi, nine Chloroflexi, 24 Proteobacteria actions. These studies raise interesting questions about and two Firmicutes (heliobacteria) are or soon will be com- how different ecotypes arise and how they persist in the pletely sequenced. These data will have a substantial oceans [12]. Ambient temperature and growth tempera- impact on the understanding of the origins and evolution ture optima seem to be important along with light, nutri- of photosynthesis while providing many exciting new ents and competitor abundances [15]. The observation that insights into the properties of these ecologically and envir- cyanophages sometimes carry photosynthesis genes [16– onmentally important organisms. 20] provides one explanation for how genes can be rapidly exchanged throughout these populations. The genome Cyanobacteria: the oxyphototrophs sequencing, metagenomics and ‘metatranscriptomics’ of Cyanobacteria are such an ancient and remarkably diverse Ward and coworkers [21] are addressing similar issues group of Bacteria that even data for 55 organisms provide in the integrated ‘community metabolism’ and ecophysiol- an extremely limited view of their complexity. There are ogy of the cyanobacteria of a different physicochemical >475 pure strains in the Pasteur Culture Collection of environment: the phototrophic mats of the Octopus and Cyanobacteria, and yet this collection includes only a small Mushroom Springs in Yellowstone National Park. Stenou number of the several thousand described species. To et al. [21] have recently shown that two populations of illustrate the magnitude of this problem, the smallest thermophilic Synechococcus spp. in these mats perform genomes for photosynthetic bacteria are 1.7 Mb and photosynthesis by day and seem to ferment stored carbo- are found in the marine, unicellular Prochlorococcus spp. hydrates to generate reductant for nitrogen fixation by [4,5] whereas Nostoc punctiforme has the largest genome night. www.sciencedirect.com 490 Review TRENDS in Microbiology Vol.14 No.11

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.

well-defined and genetically closely related bacterial group, which shares a common root with the Bacteroidetes. Comparative genomic analyses have enabled the elucida- tion of their unique BChl and carotenoid biosynthetic pathways. Chlorobi are obligately anaerobic photoauto- trophs 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 transfor- Figure 1. Distribution of reaction center types and antenna systems in mation system and its ability to grow rapidly with thio- photosynthetic bacteria, which are found in the Cyanobacteria, Chlorobi, sulfate as an electron donor, Chlorobium tepidum – the 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) 2.15 Mb genome of which was sequenced by The Institute as electron acceptors. Colors indicate whether a RC is a homodimer (e.g. for Genomic Research [26] – has become the model organ- heliobacteria and green sulfur bacteria) or a heterodimer [photosystem (PS) I and ism for this group of phototrophs. Insights into the phy- 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. siology, metabolism and light-harvesting apparatus of this Dotted lines in the type 1 RC subunits indicate the existence of both a light-harvesting organism have been reviewed elsewhere [22–25]. domain, which is structurally related to subunits CP43 and CP47 of PS II, and an Although fewer Chlorobi genomes have been sequenced 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, than cyanobacterial genomes, the ten sequenced and two instead, have light-harvesting PCB proteins, which are structurally related to CP43 anticipated genomes encompass most of the currently andbindtobothdivinyl-Chla and divinyl-Chl b. Heliobacteria have homodimeric known diversity of this group. The Joint Genome Institute 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 of the Department of Energy (JGI-DOE) has sequenced >200 000 BChl c, d or e molecules and a small amount of BChl a that is bound to the most of the type strains of the Chlorobi, and these data 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 provide benchmarks for comparisons of future isolates. BChl a (or BChl b in some purple bacteria). The antennae for these RCs are formed by Complete genomes are already available for two additional ring-shaped, BChl-a-binding LH1 and LH2 complexes in purple bacteria or strains, Pelodictyon luteolum DSM273 and Chlorobium 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 chlorochromatii (Box 2 and Figure 3), and draft genomes that are found in ‘green’ FAPs. The chlorosomes of FAPs are usually smaller and are available for seven additional strains (C. ferrooxidans, contain fewer BChl c molecules than those found in the Chlorobi. C. phaeobacteroides DSM 266, C. limicola DSM245, C. vibrioforme DSM 265, Prosthecochloris aestuarii SK413, Chlorobi: green sulfur bacteria Pelodictyon phaeoclathratiforme and an enrichment In contrast to the extraordinary richness of cyanobacterial culture, C. phaeobacteroides BS-1, isolated from 100 m diversity, the phylum Chlorobi (comprising the green sul- below the surface of the Black Sea [27]). Sequencing of fur bacteria) is a metabolically limited, physiologically Chloroherpeton thalassium and C. vibrioforme 8327d www.sciencedirect.com Review TRENDS in Microbiology Vol.14 No.11 491

predicted transporters for organic molecules, have a small Box 2. Phototrophic consortia number of predicted transcription regulators, and are With the exception of Chloroherpeton thalassium, which exhibits largely devoid of two-component histidine kinases and gliding motility, all known green sulfur bacteria are non-motile. response regulators. These observations suggest that Some green sulfur bacteria (‘epibionts’) become motile by forming phototrophic consortia through a specific association with a b- Chlorobi live in relatively constant (and energy-limited) proteobacterium, denoted the ‘central rod’ [60,61,67] (Figure 3). conditions and that they probably have a limited capacity Each polarly flagellated central rod carries 20–60 epibiont cells and to respond to changes in their physicochemical environ- the entire consortium is phototactic in response to light signals ment [22,26]. This is a trait that is shared by members of perceived by the epibiont. The nature of any metabolic coupling the cyanobacteria with reduced genomes, such as Prochlor- between the two organisms is not yet known but possibilities include transfer of reduced carbon and/or nitrogen from the epibiont ococcus and marine Synechococcus [4,5,10]. 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 Pigment biosynthetic pathways in Chlorobi mechanisms of cell-to-cell signaling for and coordination The availability of multiple Chlorobi genome sequences of cell division are completely unknown. Genome sequencing of the two partners of ‘Chlorochromatium and of a highly efficient natural transformation system for aggregatum’, which was isolated as an enrichment culture from C. tepidum has facilitated the identification of genes that Lake Dagow [60,67], should help to answer many of these questions. encode enzymes for pigment biosynthesis and other phy- The 2.57 Mb genome of the epibiont, Chlorobium chlorochromatii, siological processes in green sulfur bacteria. C. tepidum which is not an obligate symbiont [67], has already been completely synthesizes three chlorophylls: BChl c, BChl a and Chl a sequenced (http://img.jgi.doe.gov/). Sequencing of the b-proteobac- terial central rod, which cannot be grown independently from C. [24]. Before the completion of its genome sequence, no chlorochromatii and is most closely related to Rhodoferax sp. [60],is enzyme specifically involved in BChl c, d or e biosynthesis now in progress at JGI-DOE. C. chlorochromatii encodes a family of had been identified, and now all steps but one in the calcium-dependent, RTX-toxin-like proteins that might be involved pathway leading from chlorophyllide a to BChl c are in cellular adhesion to the central rod [68]. The largest gene is known. Only the enzyme responsible for the removal of predicted to encode a protein of 36 805 amino acids, one of the 2 largest predicted proteins known to date. This gene occurs in an the C-13 methylcarboxyl group has not yet been identi- apparent operon with a sequence-related gene of 20 646 codons, fied. Moreover, the erroneous identification of the 8-vinyl producing an operon of >170 kb. Homologs of these genes occur in reductase as BchJ has recently been corrected [24]. Simi- Magnetococcus sp. MC-1 (15 245 codons) and Synechococcus sp. larly, although nothing was known about the pathway for RS9917 (28 178 codons) but their functions are unknown. carotenoid biosynthesis in Chlorobi before the availability of the genome sequence, all of the enzymes required for should be completed later this year. The sequenced synthesis of chlorobactene and isorenieratene have been Chlorobi have 2–3 Mb genomes that encode 1750– identified [28,29]. Through these analyses, it is now clear 2800 genes (http://img.jgi.doe.gov/) and pairwise compar- that carotenoid biosynthesis in Chlorobi is more similar to isons show that Chlorobi strains share a common core-set the pathway in Cyanobacteria than to that in other bacteria of 1400–1500 genes. Chlorobi genomes encode only a few [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 492 Review TRENDS in Microbiology Vol.14 No.11 lycopene cyclases could be identified in the C. tepidum encodes the enzymes for carotenoid biosynthesis but has genome, phylogenetic profiling (using data from multiple no genes for the enzymes of BChl biosynthesis or photosynthetic bacteria) and complementation of a components of the photosynthetic apparatus. Pierson lycopene-producing strain of Escherichia coli both identified and Castenholz first isolated Chloroflexus aurantiacus, a fourth type of lycopene cyclase encoded by the C. tepidum the type strain of the Chloroflexi and ‘Chloroflexales’, in open reading frame CT0456 [29] (J.A. Maresca et al., the early 1970s from Yellowstone National Park and other unpublished). The identification of the ‘missing’ lycopene thermal features [32]. Cfx. aurantiacus synthesizes BChl a cyclase in C. tepidum also enabled the identification of and BChl c and has type 2 RCs and chlorosomes but lacks ‘missing’ lycopene cyclases in several cyanobacteria. the FMO protein (Figure 1). The ‘red/orange’ FAPs of the Interestingly, many cyanobacteria have an ortholog and a genera Roseiflexus and Heliothrix do not synthesize BChl c paralog of CT0456, and both of these enzymes seem to be and lack chlorosomes [32,33] (Figure 1). involved in cyclase reactions with lycopene (J.E. Graham Until recently, the incomplete 5.2 Mb draft sequence of and D.A. Bryant, unpublished). Synechococcus sp. PCC7942 Cfx. aurantiacus was the only genomic information avail- has lycopene cyclases that belong to two of the four families, able for any photosynthetic member of the Chloroflexi. which demonstrates the mosaic nature of carotenoid However, JGI-DOE will soon release the draft genomes biosynthesis and is a likely example of lateral gene of four additional Chloroflexi: Cfx. aggregans (4.5 Mb), transfer. Roseiflexus sp. strain RS-1 (5.8 Mb; from Octopus Springs, The sequenced Chlorobi strains are metabolically similar Yellowstone National Park), Roseiflexus castenholzii but can be separated according to particular phenotypes (5.6 Mb; from Nakabusa Hot Springs, Japan) and the (e.g. green-colored strains that contain BChl c and chloro- heterotroph Herpetosiphon aurantiacus (6.6 Mb). In addi- bactene versus brown-colored strains that contain BChl e tion, three sulfide-oxidizing FAPs (Chlorothrix halophila, and isorenieratene, or strains that oxidize sulfur compounds Oscillochloris sp. strain UdG 9002 and Chloronema gigan- versus ferrous iron). Thus, whole-genome comparisons can teum strain UdG 9001) are scheduled for sequencing by identify candidate genes that are responsible for defined JGI-DOE later this year. A consortium of Russian scien- physiological differences among these strains. This tists is sequencing the genome of Oscillochloris trichoides approach was recently used to search for candidate gene(s), (R. Ivanovskii and B. Kuznetsov, personal communica- the product(s) of which could convert BChl c into BChl e.This tion). O. trichoides produces a type I ribulose-1,5-bispho- analysis identified a radical SAM enzyme and an adjacent sphate carboxylase–oxygenase (RubisCO) and fixes carbon dehydrogenase as the most likely candidates for this trans- dioxide by the Calvin cycle rather than by the 3-hydro- formation (J.A. Maresca and D.A. Bryant, unpublished). xypropionate cycle [34]. However, it is not known whether Interestingly, these two genes occur adjacent to the gene other FAPs have RubisCO and use the Calvin cycle. It will encoding g-carotene cyclase, which produces b-carotene, be interesting to see whether the sulfide-oxidizing FAPs the precursor of isorenieratene [29] (J.A. Maresca et al., have type 1 RCs like the Chlorobi or have type 2 RCs (and unpublished). The proximity of these genes on a 6 kb reverse electron transport) like Cfx. aurantiacus and pur- segment of the chromosome provides a possible explana- ple bacteria. Roseiflexus sp. and O. trichoides have nitro- tion for the polyphyletic nature of the ‘brown’ phenotype genase genes, and other FAPs also probably fix dinitrogen. among green sulfur bacteria. This gene proximity would Much more information will soon be available for these greatly facilitate their lateral transfer among the Chlor- poorly characterized phototrophs. obi, and such transfer would immediately confer the Like purple non-sulfur bacteria, Cfx. aurantiacus ability to populate a new environmental niche. The exhibits considerable metabolic diversity and it can grow synthesis of BChl e is another example of a reaction for as an aerobic chemoheterotroph or as an anaerobic photo- which both oxygen-independent and oxygen-dependent heterotroph. Using electrons derived from H2 or H2S under enzymes occur in nature [23,30].TheC-7formylgroup anoxic or microaerophilic conditions, some strains of Chlor- introduced during BChl e biosynthesis must be derived oflexus sp. grow photoautotrophically by fixing CO2 from water because this reaction occurs under anoxic through the 3-hydroxypropionate pathway [32]. In nature, conditions; however, the C-7 formyl group of Chl b in Cfx. aurantiacus probably grows under microaerophilic or Prochlorococcus sp., green algae and higher plants is alternating oxic and anoxic conditions but it only fully derived from oxygen [31]. develops its photosynthetic apparatus under anoxic con- ditions. As a result, Cfx. aurantiacus encodes some Chloroflexi: filamentous anoxygenic phototrophs enzymes that can function under either oxic or anoxic Because of their diverse metabolic and physiological prop- conditions [23,30]. For example, both bchE and acsF genes erties, genomic analyses of diverse strains of Chloroflexi are found in the Cfx. aurantiacus genome; these genes are likely to produce novel insights into the evolution of encode the oxygen-independent isocyclic ring cyclase and photosynthesis. The phylum Chloroflexi is one of the ear- the oxygen-dependent isocyclic ring cyclase, respectively. liest diverging lineages of the Bacteria, and it contains Both Roseiflexus sp. strains and Cfx. aggregans also pro- several genera of filamentous, gliding bacteria that per- duce both of these enzymes. Interestingly, the same pat- form anoxygenic photosynthesis (FAPs) [32]. The phylum tern is not found for hemF and hemN, which encode contains two orders, the ‘Chloroflexales’ and ‘Herpetosi- oxygen-dependent and oxygen-independent coproporphyr- phonales’. Herpetosiphon aurantiacus, the type strain of inogen oxidases, respectively. The two Chloroflexus strains the latter order, has recently been sequenced by JGI-DOE. have both genes, whereas the Roseiflexus sp. strains do not The 6.6 Mb genome of this heterotrophic bacterium encode hemF. www.sciencedirect.com Review TRENDS in Microbiology Vol.14 No.11 493

Proteobacteria: purple non-sulfur and purple sulfur light-harvesting protein sequences argues that lateral bacteria gene transfer is not required to explain the current dis- Photosynthesis is a trait that is widespread but not tribution of RC sequences [42]. No complete heliobacterial universal among members of the Proteobacteria and is genome sequence is yet available, although the 3.1 Mb found in morphologically and metabolically diverse species. draft genome of Heliobacterium modesticaldum has Genome sequence information has largely confirmed the recently been made available for searches (http://genomes. physiological versatility and corresponding large genome tgen.org/helio.html). The H. modesticaldum genome sizes of these organisms. Some of the photosynthetic pro- sequence will help to clarify the possible lateral acquisition teobacteria are well suited for studies of global gene regula- of photosynthesis genes and it will also help to identify tion because many members are facultatively phototrophic genes that are required for carotenoid, BChl, and RC or photosynthetic under anoxic conditions. Photosynthetic synthesis and function. The relatively small genome of Proteobacteria can be found in the a, b and g subdivisions H. modesticaldum is likely to provide new insights into but, to date, almost all available genome sequences are from genes that are functionally important in sporulation and members of the a subdivision. Several genomes have been regulation of this process. determined (http://www.genomesonline.org), including those of Rhodopseudomonas palustris (5.5 Mb) [35],two Bacteriorhodopsin-based phototrophy in halophilic strains (4.6 Mb) [36] (http:// prokaryotes www.ncbi.nlm.nih. gov/), Rhodospirillum rubrum (4.4 Mb) Comparative analyses of sequenced genomes and (http://www.ncbi. nlm.nih.gov/), Roseobacter denitrificans metagenomic data from the ocean have shown the great (http://genomes. tgen.org/rhodobacter.html), Bradyrhizo- diversity in the structure and function of rhodopsins and bium sp. (9.1 Mb) and Roseobacter sp. (4.1 Mb) (http:// have demonstrated how easily lateral gene transfer can www.genomesonline. org). occur among unrelated organisms. Bacteriorhodopsin (BR) Only two g-subdivision members have been studied and in the so-called ‘purple membrane’ of halophilic archaea no photosynthetic b-proteobacterium has been sequenced. has been studied for three decades and the structure and Thus, the environmentally important purple sulfur bac- function of BR is known in great detail [1] (Figure 2). The teria, all of which belong to the g subdivision, along with halophilic archaea grow well in the dark as aerobic che- purple bacteria of the b subdivision (many of which can moheterotrophs; however, strains that synthesize BR exhi- also use sulfur or H2 as electron donors), represent groups bit light-enhanced growth under anoxic conditions and are, about which little genome sequence information is avail- therefore, facultatively phototrophic [43]. able. Finally, a large number of mostly a-proteobacteria in Among these haloarchaea, multiple rhodopsins with diverse freshwater, saline, marine, soil and hot-spring diversified functions can exist within a single cell. The environments seem to have photosynthesis gene clusters genome of the haloarchaeon Haloarcula marismortui but, in many cases, the function of the relatively low levels encodes six homologous rhodopsins: one proton-pumping of BChl a produced under aerobic conditions is unknown BR, one chloride-pumping , two sensory [37,38]. Projected sequencing projects and comparative rhodopsins and two of unknown function [44]. analyses will help to define the genetic, physiological The importance of these rhodopsins for environmental and metabolic differences among these aerobic anoxygenic adaptation was recently and elegantly illustrated phototrophs, photoheterotrophs like Rhodobacter sp., and through the genome sequence of a halophilic bacterium, the photolithoautotrophic purple sulfur bacteria. Genome Salinibacter ruber, which taxonomically belongs to the sequence data, in combination with the physiological and Cytophaga–Flexibacter–Bacteroides group [45]. Genome- metabolic versatility of these organisms, should lead to wide analyses showed that S. ruber has evolved conver- engineered strains for diverse applications in biotechnol- gently towards halophilic archaea at both the physiological ogy, including bioremediation, lignin degradation and bio- level (different genes producing a similar overall pheno- fuels and hydrogen production [35]. type) and at the molecular level (independent mutations yielding proteins with similar sequences or structures). Heliobacteria Although the identity of the donor organism (or organisms) Heliobacteria, first described by Gest and Favinger in 1983 is uncertain, the genome of S. ruber encodes four rhodop- [39], are the most recently discovered group of bacteria sins – the obvious result of lateral gene exchange with the containing RCs and they remain the most poorly charac- haloarchaea. One rhodopsin resembles a chloride pump terized overall. Heliobacteria are members of the phylum and two resemble sensory rhodopsins. The fourth is a Firmicutes and are closely related to clostridia. Like proton-pumping rhodopsin, xanthorhodopsin, which is Bacillus or Clostridium sp., heliobacteria produce heat- related to but contains a salinixanthin resistant endospores and, to date, no characterized mem- carotenoid chromophore in addition to retinal [46]. ber of this group is known to grow photoautotrophically. Studies with Heliobacillus mobilis have shown that Proteorhodopsin in many of the genes for synthesis of BChl g and the RC Other than those in haloarchaea, the first example of a are located in a 30 kb cluster similar to the photosynth- prokaryotic rhodopsin was one found in a marine proteo- esis gene clusters found in purple bacteria [40,41]. Thus, bacterium and, thus, it was named proteorhodopsin (PR) it is possible that the heliobacteria obtained their [47,48]. This identification was based on a metagenomics photosystem components through a lateral gene transfer approach in which large fragments of genomic DNA event. However, a recent detailed analysis of RC and isolated from marine picoplankton were cloned and www.sciencedirect.com 494 Review TRENDS in Microbiology Vol.14 No.11 sequenced. Like BR, PR has been shown by heterologous and the Gram-positive Exiguobacterium sp. 255–15, in expression to function as a light-driven, transmembrane addition to certain cyanobacteria, fungi, green algae and in E. coli [47] and, thus, it could contribute a dinoflagellate (Pyrocystis lunula). The single rhodopsins substantially to the energy budget in cells living in the encoded by the genomes of the cyanobacteria Nostoc photic zone of oceans. Therefore, it is not surprising that sp. PCC7120 (Alr3165) and of the fungus Leptosphaeria sequencing of both short and long fragments of environ- maculans have recently been shown by experimental mental DNA has shown that a wide range of PRs exist in characterization to be a sensory rhodopsin [55] and a highly different types of marine prokaryotic plankton proton-pumping rhodopsin [56], respectively. As a last [13,49–51]. For example, PR is present in marine euryarch- example, the early-diverging cyanobacterium, Gloeobacter aeotes in the photic zone of the North Pacific subtropical violaceus, encodes a rhodopsin (Gll0198) that seems to be gyre, whereas the marine euryarchaeotes living below the most similar to the proton-pumping xanthorhodopsin of photic zone do not have PR, clearly as a consequence of S. ruber. environmental adaptation [51]. If rhodopsin-based phototrophy is transferred so Recently, Pelagibacter ubique, a representative of the easily among organisms, why do only a few groups of ubiquitous SAR11 marine proteobacteria, was axenically RC-based phototrophs dominate phototrophic niches? cultivated and its genome was sequenced [52]. This Part of the answer might be that rhodopsin-based photo- genome contains one gene encoding PR [52,53] but has trophy uses light energy relatively inefficiently. First, to no genes that are characteristic for CO2 fixation. The produce the proton-motive force required to produce one genomes of other marine proteobacteria (Photobacterium ATP, three to four BR molecules must each absorb a sp. SKA34 and Vibrio angustum S14) and marine and release a proton to the periplasmic space. Bacteroidetes (Polaribacter irgensii, Cellulophaga sp. When a proteobacterial RC absorbs four , two MED134, Tenacibaculum sp. MED152 and Psychroflexus ubiquinol molecules are produced and their dark re-oxida- torquis ATCC700755), which are currently being tion by the cytochrome bc1 complex enables the production sequenced by the J. Craig Venter Institute (https:// of two ATP molecules [57]. Second, the single retinal research.venterinstitute.org/moore/), also contain PRs. chromophore in the photochemical units of most rhodop- Although laboratory growth experiments with P. ubique sin-based phototrophs has a much smaller absorption have not yet demonstrated that this bacterium grows cross-section than the photochemical units of RC-based faster in the light than in the dark [53], it seems likely phototrophs, which can have hundreds to thousands of that PR enables the bacterium to benefit from light even chromophores [57]. Thus, a rhodopsin-based phototroph though the experimental conditions to show this have not wouldhavetosynthesizemanymoreenergetically yet been identified. Although the structure of this PR is expensive BR or PR molecules to absorb the same amount consistent with a function in proton pumping, it remains of light energy. The highly efficient and extensive light- possible that this molecule could instead transport another harvesting antenna systems of photosynthetic bacteria 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 Box 3. Are there more phototrophs out there? that do not contain photosynthetic reaction centers never- Our current knowledge of photosynthesis is based almost theless exploit light by having acquired PR through lateral exclusively on cultivated strains but, given the vast diversity of gene transfer. The exact physiology of the marine prokar- microorganisms on Earth, these organisms are unlikely to represent yotes that harbor these PRs is not always clear. For the full spectrum of light utilization. Continued genome sequencing example, a recently characterized 95 kb genomic fragment of cultivated and uncultivated organisms will undoubtedly reveal many more microbial groups that harbor rhodopsins. It will be from a marine proteobacterium that encodes a PR also interesting to see if there are any organisms that combine encodes a putative reverse dissimilatory sulfite reductase rhodopsin-based phototrophy with CO2 fixation because such operon, which could provide reducing equivalents for auto- organisms could then be classified as Chl-independent photosyn- trophic growth by oxidizing a reduced sulfur compound thetic organisms. A priori, there seems to be no reason why the [54]. 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. The ‘cosmopolitan’ rhodopsins versus the ‘refined’ Alternatively, a rhodopsin and hydrogenase could be coupled to reaction centers provide the energy and reducing power for photolithoautotrophic Shotgun sequencing of DNA from the Sargasso Sea growth. illustrated how PRs are widespread in oceanic microorgan- As more environments are sampled, metagenomics are likely to reveal photosynthetic organisms with combinations of components isms [13]. Although the exact functions of the rhodopsins are that have not yet been observed in cultivated species (e.g. an often not known, genome sequencing projects of organisms anoxygenic organism with two types of RCs), novel solutions to the in pure culture have also confirmed that rhodopsins are problem of light harvesting or the existence of new phyla that have much more widely distributed among different organismal not previously been shown to contain phototrophic or photosyn- lineages than first anticipated. For example, rhodopsins of thetic members. This latter prediction is not simply an idle speculation: a new phylum-level, RC-containing phototroph has unknown function have been found in the genomes of recently been discovered in this manner and will be described in organisms as diverse as halophilic archaea and bacteria, detail elsewhere (D.A. Bryant et al., unpublished). The rapidly marine proteobacteria, marine Bacteroidetes, marine increasing body of genome sequence and metagenomic data will euryarchaeotes, g-radiation-resistant actinobacteria help to answer questions about the origin and evolution of photosynthesis [42,69]. (Rubrobacter xylanophilus and Kineococcus radiotolerans) www.sciencedirect.com Review TRENDS in Microbiology Vol.14 No.11 495 enable the RCs to function at maximal efficiency even at explain how gene contents map onto taxonomic composi- relatively low light intensities. tions, physiological and metabolic capabilities and gene If RC-based phototrophy is so much more efficient than expression patterns of phototrophs in diverse photic envir- rhodopsin-based systems, why does this type of phototro- onments – all of which together lead to the primary energy phy not spread laterally? Perhaps it does on rare occasions, input that ultimately drives life and its evolution on Earth. but because even the simplest chlorophyll-based photosys- tem requires 30 unique genes, this capability probably Acknowledgements cannot be laterally transferred as easily as rhodopsin- The authors would like to thank Julia A. Maresca for critical reading of based phototrophy. Lateral transfer of rhodopsin-based the manuscript and many helpful comments. We also thank Joachim Weber (Texas Tech University) for use of the ATP synthase image in photosystems requires only the genes encoding the rho- Figure 2 and Jo¨rg Overmann (Ludwig Maximilians Universita¨ t, dopsin apoprotein and a carotenoid oxygenase–lyase that Mu¨ nchen) for providing Figure 3 parts (a) and (e), and for use of parts produces retinal [54] if the recipient already has the cap- (b), (c) and (d). D.A.B. gratefully acknowledges support for genomics ability to produce an appropriate carotenoid. If it does not, studies from the National Science Foundation (MCB-MCB-0519743 and retinal biosynthesis can be performed with just four genes MCB-0523100) and from the Department of Energy (DE-FG02– 94ER20137). N.-U.F gratefully acknowledges support from The Danish (crtBIY–blh) [54]. 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