Green Sulfur Bacterium Chlorobium Tepidum Research Articles

New light on ancient enzymes – in vitro CO2 Fixation by Pyruvate Synthase of Desulfovibrio africanus and Sulfolobus acidocaldarius

Two variants of the enzyme family pyruvate:ferredoxin oxidoreductase (PFOR), derived from the anaerobic sulfate-reducing bacterium Desulfovibrio africanus and the extremophilic crenarchaeon Sulfolobus acidocaldarius, respectively, were evaluated for their capacity to fixate CO2 invitro. PFOR reversibly catalyzes the conversion of acetyl-CoA and CO2 to pyruvate using ferredoxin as redox partner. The oxidative decarboxylation of pyruvate is thermodynamically strongly favored, and most previous studies only considered the oxidative direction of the enzyme. To assay the pyruvate synthase function of PFOR during reductive carboxylation of acetyl-CoA is more challenging and requires to maintain the reaction far from equilibrium. For this purpose, a biochemical assay was established where low-potential electrons were introduced by photochemical reduction of EDTA/deazaflavin and the generated pyruvate was trapped by chemical derivatization with semicarbazide. The product of CO2 fixation could be detected as pyruvate semicarbazone by HPLC-MS. In a combinatorial approach, both PFORs were tested with ferredoxins from different sources. The pyruvate semicarbazone product could be detected with low-potential ferredoxins of the green sulfur bacterium Chlorobium tepidum and of S.acidocaldarius whereas CO2 fixation was not supported by the native ferredoxin of D.africanus. Methylviologen as an artificial electron carrier also allowed CO2 fixation. For both enzymes, the results are the first demonstration of CO2 fixation invitro. Both enzymes exhibited high stability in the presence of oxygen during purification and storage. In conclusion, the employed PFOR enzymes in combination with non-native ferredoxin cofactors might be promising candidates for further incorporation in biocatalytic CO2 conversion. ENZYMES: EC1.2.7.1. Pyruvate:Ferredoxin Oxidoreductase.

N-methyl-bacillithiol, a Novel Thiol from Anaerobic Bacteria.

J. Hiras, S. V. Sharma, V. Raman, R. A. J. Tinson, et al. (mBio 9:e01603-18, 2018, https://doi.org/10.1128/mBio.01603-18) report on the identification of a novel thiol, N-methyl-bacillithiol (N-Me-BSH), in the green sulfur bacterium Chlorobium tepidum In N-methyl-bacillithiol, the amine of the cysteine is methylated by a novel S-adenosylmethioneine transferase designated N-methyl-bacillithiol synthase A (NmbA). The Hiras et al. study is significant because it is the first report of the presence of N-Me-BSH in anaerobic bacteria.

Excitonic Energy Landscape of the Y16F Mutant of the Chlorobium tepidum Fenna-Matthews-Olson (FMO) Complex: High Resolution Spectroscopic and Modeling Studies.

We report high-resolution (low-temperature) absorption, emission, and nonresonant/resonant hole-burned (HB) spectra and results of excitonic calculations using a non-Markovian reduced density matrix theory (with an improved algorithm for parameter optimization in heterogeneous samples) obtained for the Y16F mutant of the Fenna-Matthews-Olson (FMO) trimer from the green sulfur bacterium Chlorobium tepidum. We show that the Y16F mutant is a mixture of FMO complexes with three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra. Two of these traps belong to mutated FMO subpopulations characterized by significantly modified low-energy excitonic states. Hamiltonians for the two major subpopulations (Sub821 and Sub817) provide new insight into extensive changes induced by the single-point mutation in the vicinity of BChl 3 (where tyrosine Y16 was replaced with phenylalanine F16). The average decay time(s) from the higher exciton state(s) in the Y16F mutant depends on frequency and occurs on a picosecond time scale.

Two exopolyphosphatases with distinct molecular architectures and substrate specificities from the thermophilic green-sulfur bacterium Chlorobium tepidum TLS.

The genome of the thermophilic green-sulfur bacterium Chlorobium tepidum TLS possesses two genes encoding putative exopolyphosphatases (PPX; EC 3.6.1.11), namely CT0099 (ppx1, 993 bp) and CT1713 (ppx2, 1557 bp). The predicted polypeptides of 330 and 518 aa residues are Ppx-GppA phosphatases of different domain architectures - the largest one has an extra C-terminal HD domain - which may represent ancient paralogues. Both ppx genes were cloned and overexpressed in Escherichia coli BL21(DE3). While CtPPX1 was validated as a monomeric enzyme, CtPPX2 was found to be a homodimer. Both PPX homologues were functional, K(+)-stimulated phosphohydrolases, with an absolute requirement for divalent metal cations and a marked preference for Mg(2+). Nevertheless, they exhibited remarkably different catalytic specificities with regard to substrate classes and chain lengths. Even though both enzymes were able to hydrolyse the medium-size polyphosphate (polyP) P13-18 (polyP mix with mean chain length of 13-18 phosphate residues), CtPPX1 clearly reached its highest catalytic efficiency with tripolyphosphate and showed substantial nucleoside triphosphatase (NTPase) activity, while CtPPX2 preferred long-chain polyPs (>300 Pi residues) and did not show any detectable NTPase activity. These catalytic features, taken together with the distinct domain architectures and molecular phylogenies, indicate that the two PPX homologues of Chl. tepidum belong to different Ppx-GppA phosphatase subfamilies that should play specific biochemical roles in nucleotide and polyP metabolisms. In addition, these results provide an example of the remarkable functional plasticity of the Ppx-GppA phosphatases, a family of proteins with relatively simple structures that are widely distributed in the microbial world.

Isolation and Characterization of Homodimeric Type-I Reaction Center Complex from Candidatus Chloracidobacterium thermophilum, an Aerobic Chlorophototroph

The recently discovered thermophilic acidobacterium Candidatus Chloracidobacterium thermophilum is the first aerobic chlorophototroph that has a type-I, homodimeric reaction center (RC). This organism and its type-I RCs were initially detected by the occurrence of pscA gene sequences, which encode the core subunit of the RC complex, in metagenomic sequence data derived from hot spring microbial mats. Here, we report the isolation and initial biochemical characterization of the type-I RC from Ca. C. thermophilum. After removal of chlorosomes, crude membranes were solubilized with 0.1% (w/v) n-dodecyl β-D-maltoside, and the RC complex was purified by ion-exchange chromatography. The RC complex comprised only two polypeptides: the reaction center core protein PscA and a 22-kDa carotenoid-binding protein denoted CbpC. The absorption spectrum showed a large, broad absorbance band centered at ∼483 nm from carotenoids as well as smaller Q(y) absorption bands at 672 and 812 nm from chlorophyll a and bacteriochlorophyll a, respectively. The light-induced difference spectra of whole cells, membranes, and the isolated RC showed maximal bleaching at 840 nm, which is attributed to the special pair and which we denote as P840. Making it unique among homodimeric type-I RCs, the isolated RC was photoactive in the presence of oxygen. Analyses by optical spectroscopy, chromatography, and mass spectrometry revealed that the RC complex contained 10.3 bacteriochlorophyll a(P), 6.4 chlorophyll a(PD), and 1.6 Zn-bacteriochlorophyll a(P)' molecules per P840 (12.8:8.0:2.0). The possible functions of the Zn-bacteriochlorophyll a(P)' molecules and the carotenoid-binding protein are discussed.

Coherent Control Protocol for Separating Energy-Transfer Pathways in Photosynthetic Complexes by Chiral Multidimensional Signals

Adaptive optimizations performed using a genetic algorithm are employed to construct optimal laser pulse configurations that separate spectroscopic features associated with the two main energy-transfer pathways in the third-order nonlinear optical response simulated for the Fenna-Matthews-Olson (FMO) photosynthetic complex from the green sulfur bacterium Chlorobium tepidum. Superpositions of chirality-induced tensor components in both collinear and noncollinear pulse configurations are analyzed. The optimal signals obtained by manipulating the ratios of various 2D spectral peaks reveal detailed information about the excitation dynamics.

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

In green sulfur photosynthetic bacteria, the cytochrome cz (cyt cz) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt cz subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt cz subunit (C-cyt cz) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt cz consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt cz supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt cz.

Sulfur oxidation in mutants of the photosynthetic green sulfur bacterium Chlorobium tepidum devoid of cytochrome c-554 and SoxB

A mutant devoid of cytochrome c-554 (CT0075) in Chlorobium tepidum (syn. Chlorobaculum tepidum) exhibited a decreased growth rate but normal growth yield when compared to the wild type. From quantitative determinations of sulfur compounds in media, the mutant was found to oxidize thiosulfate more slowly than the wild type but completely to sulfate as the wild type. This indicates that cytochrome c-554 would increase the rate of thiosulfate oxidation by serving as an efficient electron carrier but is not indispensable for thiosulfate oxidation itself. On the other hand, mutants in which a portion of the soxB gene (CT1021) was replaced with the aacC1 cassette did not grow at all in a medium containing only thiosulfate as an electron source. They exhibited partial growth yields in media containing only sulfide when compared to the wild type. This indicates that SoxB is not only essential for thiosulfate oxidation but also responsible for sulfide oxidation. An alternative electron carrier or electron transfer path would thus be operating between the Sox system and the reaction center in the mutant devoid of cytochrome c-554. Cytochrome c-554 might function in any other pathway(s) as well as the thiosulfate oxidation one, since even green sulfur bacteria that cannot oxidize thiosulfate contain a cycA gene encoding this electron carrier.

Type I reaction center from the green sulfur bacterium Chlorobium tepidum: is Chl a a primary electron acceptor?

The green sulfur bacterium Chlorobium tepidum has one of the simplest type I reaction center (RC) complexes. While its structure is still unknown, biochemical and protein sequence analyses suggest that it is similar to photosystem I (PS I), with two BChl a forming a special pair P840, four Chl a serving as pairs of accessory and primary electron acceptor (A0) pigments and 14 BChl a constituting as an immediate RC antenna. This is a dramatic simplification compared to PS I RC, where 90 Chl a antenna pigments serve as antenna and 6 additional Chl a molecules function as electron transfer cofactors. The resulting spectral congestion has prevented direct visualization of ultrafast electron transfer processes within PS I RC and even the sequence of primary electron transfer processes in PS I is still under debate. The suggested presence of two types of pigments in RC from Chlorobium tepidum removes spectral congestion and opens a way to directly visualize electron transfer steps in type I RC using ultrafast spectroscopy, since the Chl a and BChl a pigments absorb at ∼670 nm and ∼800 nm, respectively. To confirm the proposed functional role of Chl a as electron transfer cofactor we performed extensive ultrafast optical pump-probe experiments on different preparations of RC complexes from Chlorobium tepidum, revealing energy/electron transfer rates between different groups of pigments. Surprisingly, we found that ∼3 out of 4 Chl a pigments do not transfer excitation energy to the BChl a antenna or to P840, which indicates that these pigments must be >20A away from any other BChl a pigment and thus argues against the suggested presence of 4 Chl a in the reaction center core complex.

Accumulation of chlorophyllous pigments esterified with the geranylgeranyl group and photosynthetic competence in the CT2256-deleted mutant of the green sulfur bacterium Chlorobium tepidum

Green sulfur bacteria contain chlorophyllous pigments, chlorophyll (Chl) aPD and bacteriochlorophyll (BChl) aP, esterified with Delta2,6-phytadienol and phytol, respectively, which would be produced by reduction of the geranylgeranyl group at the C-17 propionate residue. In the genome of Chlorobium tepidum, two paralogous genes presumably encoding geranylgeranyl reductase, CT1232 and CT2256, are found. The deletion mutants of the CT1232 and CT2256 genes were constructed using an insertional inactivation method in order to clarify the biosynthetic process of the Delta2,6-phytadienyl and phytyl groups in green sulfur bacteria. The compositions of chlorophyllous pigments in the two mutants were determined by LC-MS analysis. The CT2256-deleted mutant accumulated Chl aGG and BChl aGG esterified with geranylgeraniol, indicating that CT2256 was involved in the production of both Delta2,6-phytadienyl and phytyl groups. The relatively high fluorescence emission from chlorosomes in the mutant also suggested some hindrance of the energy transfer from chlorosomes to the reaction center complex. However, the CT1232-deleted mutant almost showed no apparent phenotype compared to the wild type. Furthermore, the purple bacterium Rhodobacter capsulatus mutant defective in the bchP gene was partially complemented with the CT2256 gene; BChl aP was synthesized in the mutant in addition to accumulating other intermediates.

Spectroscopic properties and bacteriochlorophyll c isomer composition of extramembranous light-harvesting complexes in the green sulfur photosynthetic bacterium Chlorobium tepidum and its CT0388-deleted mutant under vitamin B12-limited conditions

The effects of exogenous vitamin B12 on the green sulfur photosynthetic bacterium Chlorobium (Chl.) tepidum were examined. Wild-type cells and mutant cells lacking a gene CT0388 (denoted as VB0388) of Chl.tepidum were grown in liquid cultures containing different concentrations of vitamin B12. The VB0388 cells hardly grew in vitamin B12-limited media, indicating that the product of CT0388 actually played an important role in vitamin B12 biosynthesis in Chl. tepidum. Both wild-type and VB0388 cells in vitamin B12-limited media exhibited absorption bands and CD signals at the Qy region that were shifted to a shorter wavelength than those of cells grown in normal media. BChl c isomers that had S-stereochemistry at the 3(1)-position tended to increase in Chl. tepidum grown in vitamin B12-limited media.

Unifying principles in homodimeric type I photosynthetic reaction centers: Properties of PscB and the F A, F B and F X iron–sulfur clusters in green sulfur bacteria

The photosynthetic reaction center from the green sulfur bacterium Chlorobium tepidum (CbRC) was solubilized from membranes using Triton X-100 and isolated by sucrose density ultra-centrifugation. The CbRC complexes were subsequently treated with 0.5 M NaCl and ultrafiltered over a 100 kDa cutoff membrane. The resulting CbRC cores did not exhibit the low-temperature EPR resonances from F A − and F B − and were unable to reduce NADP +. SDS-PAGE and mass spectrometric analysis showed that the PscB subunit, which harbors the F A and F B clusters, had become dissociated, and was now present in the filtrate. Attempts to rebind PscB onto CbRC cores were unsuccessful. Mössbauer spectroscopy showed that recombinant PscB contains a heterogeneous mixture of [4Fe–4S] 2+,1+ and other types of Fe/S clusters tentatively identified as [2Fe–2S] 2+,1+ clusters and rubredoxin-like Fe 3+,2+ centers, and that the [4Fe–4S] 2+,1+ clusters which were present were degraded at high ionic strength. Quantitative analysis confirmed that the amount of iron and sulfide in the recombinant protein was sub-stoichiometric. A heme-staining assay indicated that cytochrome c 551 remained firmly attached to the CbRC cores. Low-temperature EPR spectroscopy of photoaccumulated CbRC complexes and CbRC cores showed resonances between g = 5.4 and 4.4 assigned to a S = 3/2 ground spin state [4Fe–4S] 1+ cluster and at g = 1.77 assigned to a S = 1/2 ground spin state [4Fe–4S] 1+ cluster, both from F X −. These results unify the properties of the acceptor side of the Type I homodimeric reaction centers found in green sulfur bacteria and heliobacteria: in both, the F A and F B iron–sulfur clusters are present on a salt-dissociable subunit, and F X is present as an interpolypeptide [4Fe–4S] 2+,1+ cluster with a significant population in a S = 3/2 ground spin state.

The three-dimensional structure of CsmA: A small antenna protein from the green sulfur bacterium Chlorobium tepidum

The structure of the chlorosome baseplate protein CsmA from Chlorobium tepidum in a 1:1 chloroform:methanol solution was determined using liquid-state NMR spectroscopy. The data reveal that the 59-residue protein is predominantly α-helical with a long helical domain extending from residues V6 to L36, containing a putative bacteriochlorophyll a binding domain, and a short helix in the C-terminal part extending from residues M41 to G49. These elements are compatible with a model of CsmA having the long N-terminal α-helical stretch immersed into the lipid monolayer confining the chlorosome and the short C-terminal helix protruding outwards, thus available for interaction with the Fenna–Matthews–Olson antenna protein.

SoxAX Binding Protein, a Novel Component of the Thiosulfate-Oxidizing Multienzyme System in the Green Sulfur Bacterium Chlorobium tepidum

From the photosynthetic green sulfur bacterium Chlorobium tepidum (pro synon. Chlorobaculum tepidum), we have purified three factors indispensable for the thiosulfate-dependent reduction of the small, monoheme cytochrome c(554). These are homologues of sulfur-oxidizing (Sox) system factors found in various thiosulfate-oxidizing bacteria. The first factor is SoxYZ that serves as the acceptor for the reaction intermediates. The second factor is monomeric SoxB that is proposed to catalyze the hydrolytic cleavage of sulfate from the SoxYZ-bound oxidized product of thiosulfate. The third factor is the trimeric cytochrome c(551), composed of the monoheme cytochrome SoxA, the monoheme cytochrome SoxX, and the product of the hypothetical open reading frame CT1020. The last three components were expressed separately in Escherichia coli cells and purified to homogeneity. In the presence of the other two Sox factors, the recombinant SoxA and SoxX showed a low but discernible thiosulfate-dependent cytochrome c(554) reduction activity. The further addition of the recombinant CT1020 protein greatly increased the activity, and the total activity was as high as that of the native SoxAX-CT1020 protein complex. The recombinant CT1020 protein participated in the formation of a tight complex with SoxA and SoxX and will be referred to as SAXB (SoxAX binding protein). Homologues of the SAXB gene are found in many strains, comprising roughly about one-third of the thiosulfate-oxidizing bacteria whose sox gene cluster sequences have been deposited so far and ranging over the Chlorobiaciae, Chromatiaceae, Hydrogenophilaceae, Oceanospirillaceae, etc. Each of the deduced SoxA and SoxX proteins of these bacteria constitute groups that are distinct from those found in bacteria that apparently lack SAXB gene homologues.

Parallel electron donation pathways to cytochrome cz in the type I homodimeric photosynthetic reaction center complex of Chlorobium tepidum

We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome c z bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome c z. It was also shown that the rereduction rate of cytochrome c z by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center.

Unravelling Coherent Dynamics and Energy Dissipation in Photosynthetic Complexes by 2D Spectroscopy

Spectroscopic studies of light harvesting and the subsequent energy conversion in photosynthesis can track quantum dynamics happening on the microscopic level. The Fenna-Matthews-Olson complex of the photosynthetic green sulfur bacteria Chlorobium tepidum is a prototype efficient light-harvesting antenna: it stores the captured photon energy in the form of excitons (collective excitations), which are subsequently converted to chemical energy with almost 100% efficiency. These excitons show an elaborate relaxation pattern involving coherent and incoherent pathways. We make use of the complex chirality and fundamental symmetries of multidimensional optical signals to design new sequences of ultrashort laser pulses that can distinguish between coherent quantum oscillations and incoherent energy dissipation during the exciton relaxation. The cooperative dynamical features, which reflect the coherent nature of excitations, are amplified. The extent of quantum oscillations and their timescales in photosynthesis can be readily extracted from the designed signals, showing that cooperativity is maintained during energy transport in the Fenna-Matthews-Olson complex. The proposed pulse sequences may also be applied to reveal information on the robustness of quantum states in the presence of fluctuating environments in other nanoscopic complexes and devices.

Crystallization and preliminary X-ray studies of ferredoxin-NAD(P)+ reductase from Chlorobium tepidum.

Ferredoxin-NAD(P)(+) reductase (FNR) is a key enzyme that catalyzes the photoreduction of NAD(P)(+) to generate NAD(P)H during the final step of the photosynthetic electron-transport chain. FNR from the green sulfur bacterium Chlorobium tepidum is a homodimeric enzyme with a molecular weight of 90 kDa; it shares a high level of amino-acid sequence identity to thioredoxin reductase rather than to conventional plant-type FNRs. In order to understand the structural basis of the ferredoxin-dependency of this unique photosynthetic FNR, C. tepidum FNR has been heterologously expressed, purified and crystallized in two forms. Form I crystals belong to space group C222(1) and contain one dimer in the asymmetric unit, while form II crystals belong to space group P4(1)22 or P4(3)22. Diffraction data were collected from a form I crystal to 2.4 A resolution on the synchrotron-radiation beamline NW12 at the Photon Factory.

Chlorosome lipids from Chlorobium tepidum: characterization and quantification of polar lipids and wax esters

We have extracted polar lipids and waxes from isolated chlorosomes from the green sulfur bacterium Chlorobium tepidum and determined the fatty acid composition of each lipid class. Polar lipids amounted to 4.8 mol per 100 mol bacteriochlorophyll in the chlorosomes, while non-polar lipids (waxes) were present at a ratio of 5.9 mol per 100 mol bacteriochlorophyll. Glycolipids constitute 60 % of the polar lipids while phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and an aminoglycosphingolipid make up respectively 15, 3, 8 and 12 %. A novel glycolipid was identified as a rhamnose derivative of monogalactosyldiacylglycerol, while the other major glycolipid was monogalactosyldiacylglycerol. Tetradecanoic acid was the major fatty acid in the aminoglycosphingolipid, while the other polar lipids contained predominantly hexandecanoic acid. The chlorosome waxes are esters of unbranched fatty acids and fatty alcohols with 14 or 16 carbon atoms, joined to form molecules with between 28 and 32 carbon atoms. The stoichiometry between lipids and bacteriochlorophyll suggests that much of the chlorosome surface is covered by protein.

Intact Protein Profiling of Chlorobium tepidum by Capillary Isoelectric Focusing, Reversed-Phase Liquid Chromatography, and Mass Spectrometry

Capillary isoelectric focusing (CIEF) coupled with reversed-phase liquid chromatography (RPLC) and electrospray ionization (ESI) mass spectrometry (MS) is shown to provide a liquid-based alternative to 2D-PAGE for intact protein profiling. This combination exhibits high resolution, sensitivity and throughput for protein profiling based on pI vs MW. The CIEF-RPLC-MS system described here facilitates the use of IEF markers for internal calibration of pI. It also provides a high dynamic range as evidenced by the detection of 100 pg (3 fmol) of a test protein spiked into 1 microg of a complex protein mixture. About 1200 individual proteins/polypeptides were detected from lysates of the green sulfur bacterium Chlorobium tepidum in a single <8 h run. The pI vs MW profile obtained from CIEF-RPLC-MS compares favorably with theoretical data derived from the C. tepidum genome and experimental data obtained from 2D-PAGE.

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria

A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria.