- Research article
- Open Access
Complete genome sequence of the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus
BMC Genomics volume 12, Article number: 334 (2011)
Chloroflexus aurantiacus is a thermophilic filamentous anoxygenic phototrophic (FAP) bacterium, and can grow phototrophically under anaerobic conditions or chemotrophically under aerobic and dark conditions. According to 16S rRNA analysis, Chloroflexi species are the earliest branching bacteria capable of photosynthesis, and Cfl. aurantiacus has been long regarded as a key organism to resolve the obscurity of the origin and early evolution of photosynthesis. Cfl. aurantiacus contains a chimeric photosystem that comprises some characters of green sulfur bacteria and purple photosynthetic bacteria, and also has some unique electron transport proteins compared to other photosynthetic bacteria.
The complete genomic sequence of Cfl. aurantiacus has been determined, analyzed and compared to the genomes of other photosynthetic bacteria.
Abundant genomic evidence suggests that there have been numerous gene adaptations/replacements in Cfl. aurantiacus to facilitate life under both anaerobic and aerobic conditions, including duplicate genes and gene clusters for the alternative complex III (ACIII), auracyanin and NADH:quinone oxidoreductase; and several aerobic/anaerobic enzyme pairs in central carbon metabolism and tetrapyrroles and nucleic acids biosynthesis. Overall, genomic information is consistent with a high tolerance for oxygen that has been reported in the growth of Cfl. aurantiacus. Genes for the chimeric photosystem, photosynthetic electron transport chain, the 3-hydroxypropionate autotrophic carbon fixation cycle, CO2-anaplerotic pathways, glyoxylate cycle, and sulfur reduction pathway are present. The central carbon metabolism and sulfur assimilation pathways in Cfl. aurantiacus are discussed. Some features of the Cfl. aurantiacus genome are compared with those of the Roseiflexus castenholzii genome. Roseiflexus castenholzii is a recently characterized FAP bacterium and phylogenetically closely related to Cfl. aurantiacus. According to previous reports and the genomic information, perspectives of Cfl. aurantiacus in the evolution of photosynthesis are also discussed.
The genomic analyses presented in this report, along with previous physiological, ecological and biochemical studies, indicate that the anoxygenic phototroph Cfl. aurantiacus has many interesting and certain unique features in its metabolic pathways. The complete genome may also shed light on possible evolutionary connections of photosynthesis.
The thermophilic bacterium Chloroflexus aurantiacus was the first filamentous anoxygenic phototrophic (FAP) bacterium (also known as the green non-sulfur bacterium or green gliding bacterium) to be discovered . The type strain Cfl. aurantiacus J-10-fl was found in a microbial mat together with cyanobacteria when isolated from a hot spring near Sokokura, Hakone district, Japan. Cfl. aurantiacus can grow phototrophically under anaerobic conditions or chemotrophically under aerobic and dark conditions.
The photosystem of Cfl. aurantiacus includes the peripheral antenna complex known as a chlorosome, the B808-866 light-harvesting core complex, and a quinone-type (or type-II) reaction center [2, 3]. While Cfl. aurantiacus primarily consumes organic carbon sources (i.e. acetate, lactate, propionate, and butyrate) that are released by the associated cyanobacteria in the Chloroflexus/cyanobacterial mats of its natural habitat, it can also assimilate CO2 with the 3-hydroxypropionate (3HOP) autotrophic carbon fixation cycle [4, 5]. Further, studies have reported carbon, nitrogen and sulfur metabolisms of Cfl. aurantiacus.
According to 16S rRNA analysis, Chloroflexi species are the earliest branching bacteria capable of photosynthesis [6–8] (Figure 1) and have long been considered to be critical to understanding the evolution of photosynthesis [9–16]. However, there are also indications that there has been widespread horizontal gene transfer of photosynthesis genes, so the evolutionary history of photosynthesis is still poorly understood .
During the transition from an anaerobic to an aerobic world, organisms needed to adapt to the aerobic environment and to become more oxygen-tolerant. Most of the gene products can function with or without oxygen, whereas several proteins and enzymes are known to be exclusively functional in either aerobic or anaerobic environments. Thus, gene replacements have been found in the evolution of many metabolic processes [18–20]. Some aspects of the genome annotation of Chloroflexi species have been discussed by Bryant, Ward and coworkers [5, 21].
Several genes encoding aerobic and anaerobic enzyme pairs, as well as a number of duplicated gene clusters, have been identified in the Cfl. aurantiacus genome. In this report, we use genomic annotation, together with previous physiological and biochemical studies, to illustrate how Cfl. aurantiacus may be a good model system for understanding the evolution of metabolism during the transition from anaerobic to aerobic conditions. Some of the genomic information is compared with that of the genome of Roseiflexus castenholzii, a recently characterized FAP bacterium that lacks chlorosomes .
Results and Discussion
The genome size of Cfl. aurantiacus J-10-fl (5.3-Mb) (Table 1 and Figure 2) is comparable to that of other phototrophic Chloroflexi species: Chloroflexus sp. Y-400-fl (5.3-Mb), Chloroflexus aggregans (4.7-Mb), Roseiflexus sp. RS-1 (5.8-Mb), and Roseiflexus castenholzii DSM 13941 (5.7-Mb). Here, we summarize several unique features in the Cfl. aurantiacus genome, and compare some of the features with other Chloroflexi species and various photosynthetic and non-photosynthetic microorganisms. The complete genome has been deposited in GenBank with accession number CP000909 (RefSeq entry NC_010175). Further information is available at the Integrated Microbial Genome database (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi?section=TaxonDetail&page=taxonDetail&taxon_oid=641228485). The oriC origin is at twelve o'clock of the circular genome map (Figure 2). Like in many prokaryotes, AT-rich repeated sequence can be found in the origin of replication.
No special replication patterns can be found in the genome, and genes responsible for DNA replication and repair do not form a cluster (e.g., dnaA (Caur_0001), dnaB (Caur_0951), genes encoding DNA polymerase III (Caur_0523, Caur_0987, Caur_1016, Caur_1621, Caur_1930, Caur_2419, Caur_2639, Caur_2725 and Caur_3069), polA (Caur_0341), genes encoding other DNA polymerases (Caur_1575, Caur_1899, Caur_2099, Caur_2101, Caur_2868, Caur_3077, Caur_3225, Caur_3495, Caur_3509 and Caur_3510), recD (Caur_0824), recF (Caur_3876), recG (Caur_0261), recQ (Caur_2049), rho (Caur_0274), gyrA (Caur_1241), gyrB (Caur_3009), uvrD (Caur_0724) and others).
A. Photosynthetic antenna and reaction center genes
Cfl. aurantiacus has chimeric photosynthetic components, which contain characteristics of green sulfur bacteria (e.g., the chlorosomes) and purple photosynthetic bacteria (e.g., the integral-membrane antenna core complex surrounding a type II (quinone-type) reaction center), As the first FAP bacterium to be discovered, the excitation energy transfer and electron transfer processes in Cfl. aurantiacus have been investigated extensively . During phototrophic growth of Cfl. aurantiacus, the light energy is first absorbed by its peripheral light-harvesting antenna, the chlorosome, which is a self-assembled bacteriochlorophyll complex (the major bacteriochlorophyll in chlorosomes is bacteriochlorophyll c (BChl c)) and encapsulated by a lipid monolayer. Energy is then transferred to the B808-866 light-harvesting core antenna complex, which is a protein-pigment complex associated with two spectral types of bacteriochlorophyll a (BChl a) (B808 and B866), through the baseplate of chlorosomes. The baseplate is a CsmA chlorosome protein-bacteriochlorophyll a (BChl a)-carotenoid complex (i.e. a protein-pigment complex) . Finally, the excitons are transferred to the reaction center (RC), in which photochemical events occur. While both purple photosynthetic proteobacteria and Cfl. aurantiacus have a type II RC , the Cfl. aurantiacus RC is simpler than the purple bacterial RC  and contains only the L- and M-subunits (PufL and PufM), and not the H-subunit [25, 26].
All of the genes encoding the B808-866 core complex (α-subunit (Caur_2090) and β-subunit (Caur_2091)) and RC (pufM (Caur_1051) and pufL (Caur_1052)) are present in the Cfl. aurantiacus genome. The pufL and pufM genes are fused in the Roseiflexus castenholzii genome. The arrangement of genes for the core structural proteins of the photosynthetic complexes is significantly different from that found in purple bacteria, where the puf (photosynthetic unit fixed) operon invariably contains the LH complex genes, the RC genes encoding for the L and M subunits and the tetraheme cytochrome associated with the reaction center (if present) .
Although proteins are not required for BChl c self-assemblies in chlorosomes, various proteins have been identified to be associated with the lipid monolayer of the Cfl. aurantiacus chlorosomes . In addition to the baseplate protein CsmA, the chlorosome proteins CsmM and CsmN have been characterized , and used to be considered as the only two proteins associated with the chlorosome mono-lipid layer. Other chlorosome proteins have also been reported, either through biochemical characterization (CsmP (unpublished results in Blankenship lab from in-solution trypsin digestion of the Cfl. aurantiacus chlorosomes) and AcsF ) or genomic analysis by analogy to green sulfur bacteria (CsmO, CsmP, CsmY) . Among these proteins, AcsF, a protein responsible for chlorophyll biosynthesis under aerobic and semi-aerobic growth conditions, was unexpectedly identified from chlorosome fractions during anaerobic and photoheterotrophic growth of Cfl. aurantiacus. There has been some discussions as to whether AcsF is obligated to be associated with the chlorosomes [21, 30], and the role of AcsF under anaerobic growth condition remains to be addressed, because it is an oxygen-dependent enzyme in other systems [31, 32]. Although more chlorosome proteins have been identified recently, it is clear that CsmA, CsmM and CsmN are the most abundant proteins of the Cfl. aurantiacus chlorosomes. Genes encoding the experimentally identified and proposed chlorosome proteins are csmA (Caur_0126), csmM (Caur_0139), csmN (Caur_0140), csmP (Caur_0142), csmO (Caur_1311), and csmY (Caur_0356).
B. Electron transport complex genes
Figure 3A shows the proposed pathway of photosynthetic electron transport in Cfl. aurantiacus and purple photosynthetic proteobacteria. Similar to the purple photosynthetic proteobacteria, a cyclic electron transport pathway in Cfl. aurantiacus is also proposed. Nevertheless, some protein complexes in the electron transport chain of Cfl. aurantiacus are recognized to be substantially different from those of purple bacteria. Cfl. aurantiacus uses menaquinone as liposoluble electron and proton carrier [33–36], and purple proteobacteria use either ubiquinone [37, 38] or menaquinone  as the mobile carrier in light-induced cyclic electron transport chain. The genetic information, analyses, and possible roles in photosynthesis and respiration for the complexes are described below.
(I) Alternative complex III (ACIII)
Integral membrane oxidoreductase complexes are essential for energy metabolism in all bacteria. In phototrophic bacteria, these almost invariably include the photoreaction center and a variant of respiratory Complex III, either the cytochrome bc1 complex (anoxygenic) or cytochrome b6f complex (oxygenic). No homolog of the Complex III has been identified biochemically in Cfl. aurantiacus, and no genes with significant homology to Complex III are found in the Cfl. aurantiacus genome. Previous experimental evidence indicated that alternative complex III (ACIII) complexes, identified in Cfl. aurantiacus and some non-phototrophic bacteria, function in electron transport [34, 40–44]. Genes encoding an ACIII have also been identified in the genome of Candidatus Chloracidobacterium thermophilum , an aerobic phototrophic Acidobacterium. In the Cfl. aurantiacus genome, two ACIII operons have been identified: one encodes the Cp (subscript p stands for photosynthesis) ACIII complex for anaerobic photosynthesis, and the other encodes the Cr (subscript r stands for respiration) ACIII complex for aerobic respiration (Table 2). The Cp operon is similar to a seven-gene nrf operon in E. coli strain K-12. Hussain et al. suggested that the nrf operon in E. coli is essential for reducing nitrate to ammonia . The Cfl. aurantiacus Cp operon (Caur_0621 to Caur_0627) contains genes encoding two types of cytochrome c; a multi-heme cytochrome c (component A, actA, Caur_0621), which has recently been identified experimentally to be a penta-heme component , and a mono-heme cytochrome c (component E, actE, Caur_0625), which forms a homodimer in the ACIII complex , a putative FeS-cluster-hydrogenase component-like protein (component B, actB, Caur_0622), a polysulfide reductase (component C, actC, Caur_0623), similar to NrfD and likely involved in the transfer of electrons from the quinone pool to cytochrome c, an integral membrane protein (component F, actF, Caur_0626) and two uncharacterized proteins (component D (actD, Caur_0624) and component G (actG, Caur_0627)) (Figure 3B).
The proposed Cr ACIII operon contains 12 genes (Caur_2133 to 2144) encoding a putative FAD-dependent oxidase (component K, actK, Caur_2133), D-lactate dehydrogenase (component L, actL, Caur_2134), a Cys-rich protein with Fe-S binding motifs (component M, actM, Caur_2135), components B (actB, Caur_2136), E (actE, Caur_2137), A (actA, Caur_2138), and G (actG, Caur_2139) in the Cp ACIII operon, an electron transport protein SCO1/SenC (Caur_2140), and four subunits of cytochrome c oxidase (component J, Caur_2141 - 2144). The cytochrome c oxidase (COX, or complex IV, EC 18.104.22.168) genes in the Cr operon are part of complex IV, so the Cr ACIII operon clustered with complex IV genes could create a respiratory superoperon (Figure 3B). Additionally, a gene cluster encoding a putative SCO1/SenC electron transport protein (Caur_2423) and two COX subunits (Caur_2425 (subunit II) and Caur_2426 (subunit I)) is 300 genes away from the putative Cr ACIII operon. Note that genes encoding components C (actC), D (actD) and F (actF) in the Cp ACIII operon are absent in the Cr ACIII operon. Whether these three components are required for the formation of the ACIII complex under aerobic respiratory growth will be addressed with biochemical studies.
Two type I blue copper proteins have been isolated and proposed to function as the mobile electron carriers in photosynthetic electron transport of photosynthetic organisms: one is plastocyanin in cyanobacteria, photosynthetic algae and higher plants and the other is auracyanin in Chloroflexus and Roseiflexus. The type I blue copper protein auracyanin, which has a single copper atom coordinated by two histidine, one cysteine and one methionine residues at the active site, is proposed to participate in the electron transfer from ACIII to the reaction center in Cfl. aurantiacus[35, 47–49], and it has also been recently characterized in Roseiflexus castenholzii. Additionally, an auracyanin gene (trd_0373) has been identified in the genome of the non-photosynthetic bacterium Thermomicrobium roseum DSM 5159, which is evolutionally related to Cfl. aurantiacus. Two ACIII operons are proposed in Cfl. aurantiacus, and the two auracyanin proteins of Cfl. aurantiacus, AuraA and AuraB, which share 38% sequence identity, have been suggested to function with the two variant ACIII complexes . AuraA, a water-soluble protein, can only be detected during phototrophic growth, whereas AuraB, a membrane-tethered protein, is synthesized during both phototrophic and dark growth . It has been hypothesized that AuraA transports electron from the Cp ACIII during photosynthesis and AuraB from the Cr ACIII during respiration. The auraA (Caur_3248) and auraB (Caur_1950) genes are distant from the Cp operon (Caur_0621 to Caur_0627) and Cr operon (Caur_2132 to Caur_2144). In addition to auraA and auraB, two more genes encoding auracyanin-like proteins (or type I blue-copper proteins) (Caur_2212 and Caur_2581) have also been found in the Cfl. aurantiacus genome. In contrast, Roseiflexus castenholzii has only one copy of the ACIII operon (a six-gene cluster, Rcas_1462 to Rcas_1467), in which the gene encoding the component G of the Cfl. aurantiacus Cp ACIII complex is missing) (Figure 3B), and one auraA-like gene (Rcas_3112).
(III) NADH:quinone oxidoreductase
Two operons encoding the enzymes for NADH:quinone oxidoreductase (Complex I, EC 22.214.171.124) are present in the genome. Complex I catalyzes electron transport in the oxidative phosphorylation pathway. Many bacteria have 14 genes (nuoA to nuoN) encoding Complex I, and some photosynthetic bacteria, such as the purple photosynthetic proteobacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris, contain two Complex I gene clusters. In Cfl. aurantiacus, two putative Complex I gene clusters have been identified, one with all of the 14 gene subunits arranging in order (nuoA to nuoN, Caur_2896 - 2909), and the other has genes loosely arranged (with nuoE and nuoF 800 genes apart), duplicated nuoM genes, and the lack of nuoG (Table 2). It is possible that either nuoG is shared with the two putative Complex I gene clusters or an alternative gene with less sequence similarity functions as nuoG. For example, two gene loci (Caur_0184 and Caur_2214) encoding gene products that have ~24% sequence identity with NuoG, which is a molybdopterin oxidoreductase. To date, there have been no biochemical studies on the Complex I from Cfl. aurantiacus or any FAP bacteria.
(IV) Other electron transport proteins
In addition to the electron transport proteins described above, the sequence has been determined of cytochrome c554, which is also a subunit of the reaction center of Cfl. aurantiacus[52–54]. The sequence of the cytochrome c subunit in the Roseiflexus castenholzii RC has also been reported . The gene encoding cytochrome c554 (pufC, Caur_2089) is in an operon flanked with two genes encoding the bacteriochlorophyll biosynthesis enzymes, bchP (Caur_2087) and bchG (Caur_2088) at the 5'-end, and two genes encoding the B808-866 complex (Caur_2090 (α-subunit) and Caur_2091 (β-subunit)) at the 3'-end (Figure 4C).
C. Aerobic/anaerobic enzyme pairs
In the biosynthesis of heme and chlorophyll (Chl), three aerobic/anaerobic enzyme pairs participate: coproporphyrinogen III decarboxylase (aerobic, HemF (EC 126.96.36.199); anaerobic, HemN (EC 188.8.131.52)) and protoporphyrinogen IX oxidase (anaerobic and aerobic) in heme biosynthesis, and Mg-protoporphyrin IX monomethyl ester cyclase (aerobic, AcsF; anaerobic, BchE) in chlorophyll (Chl) biosynthesis. Both aerobic and anaerobic gene pairs can be found in Cfl. aurantiacus, for example, acsF (Caur_2590) and bchE (Caur_3676)  as well as hemF (Caur_2599) and hemN (Caur_0209 and/or Caur_0644) gene pairs. The acsF and hemF genes cannot be found in the green sulfur bacterium Chlorobaculum tepidum and other strictly anaerobic bacteria. The genes involved in the biosynthesis of tetrapyrroles, as well as proposed gene replacements, are further elaborated below.
The gene replacements during the anaerobic to aerobic transitions are best known in the biosynthesis of cobalamin, in which the genes in the anaerobic pathway up to cobalt insertion into the corrin ring are completely replaced in the aerobic pathway . Different strategies are used to generate cobyrinate diamide, the end product of both anaerobic and aerobic pathways, in which cobalt is introduced into the corrin ring at the dihydroisobacteriochlorin stage (early stage) of the anaerobic pathway and at the late stage of the aerobic pathway. The genomic information of Cfl. aurantiacus reveals a large cobalamin biosynthesis and cobalt transporter operon (Caur_2560 - 2580), containing genes in both aerobic and anaerobic biosynthesis pathways, suggesting that Cfl. aurantiacus can synthesize cobalamin under various growth conditions. Genes encoding anaerobic cobalt chelatase (EC 184.108.40.206) (cbiK, Caur_2572) and aerobic cobalt chelatase (EC 220.127.116.11) (cobNST, cobN (Caur_2579), cobS (Caur_1198) and cobT (Caur_2578)) have been identified (Tables 3 and 4). The aerobic cobalt chelatase (EC 18.104.22.168), containing three subunits (CobN, CobS and CobT), is a close analog to Mg-chelatase (also containing three subunits, BchH, BchI and BchD) that catalyzes the Mg-insertion in the chlorin ring in chlorophyll biosynthesis. It is known that aerobic cobalt chelatase subunits CobN and CobS are homologous to Mg-chelatase subunits BchH and BchI, respectively, and that CobT has also been found to be remotely related to the third subunit of Mg-chelatase, BchD. Compared to other strictly aerobic and anaerobic photosynthetic bacteria, the aerobic anoxygenic phototrophic proteobacterium Roseobacter denitrificans only carries the cobNST genes , and the strictly anaerobic bacterium Heliobacterium modesticaldum has only the cbiK gene . The presence of cobNST and cbiK gene pairs in the Cfl. aurantiacus genome suggests a gene replacement in cobalamin biosynthesis by Cfl. aurantiacus under different growth conditions.
The heme operon (Caur_2593 to Caur_2599: hemA, hemC, hemD, hemB, hemE, hemL and hemF) is downstream of the cobalamin operon (Caur_2560 to Caur_2580). Except for hemF, other genes in the heme operon are utilized for synthesizing heme under both aerobic and anaerobic conditions. Furthermore, Caur_0209 and Caur_0644, encoding the putative O2-independent coproporphyrinogen III decarboxylase/oxidase (HemN, EC 22.214.171.124), and Caur_0645, encoding O2-dependent protoporphyrinogen oxidase (HemG/HemY, EC 126.96.36.199), are ~ 2000 genes away from the heme gene cluster. It is interesting to note that two hemN genes have been identified in the Cfl. aurantiacus genome, while no gene encoding the O2-independent protoporphyrinogen oxidase in Cfl. aurantiacus has been characterized. Because heme can be synthesized by Cfl. aurantiacus in both aerobic and anaerobic environments, it is possible that one of the hemN genes in Cfl. aurantiacus may encode an O2-independent protoporphyrinogen oxidase. Finally, in addition to protoheme (heme b), the genome predicts that heme o and heme a can be synthesized respectively by the gene products of Caur_0029 (encoding protoheme IX farnesyltransferase) and Caur_1010 (encoding a cytochrome aa 3 biosynthesis protein), consistent with the spectral evidence provided by Pierson that protoheme and heme derivatives can be identified .
The anaerobic to aerobic transitions are particularly intriguing in chlorophyll (Chl) biosynthesis and photosynthesis, in which molecular oxygen is lethal for photosynthetic anaerobes but is required for the life of aerobic phototrophs. Contrary to the cobalamin and heme biosynthesis, no gene cluster for (B)Chl biosynthesis is recognized in the Cfl. aurantiacus genome, whereas photosynthesis gene clusters are present in purple photosynthetic proteobacteria [27, 60, 61] (Figure 4B) and heliobacteria . The photosynthetic genes of Cfl. aurantiacus are rather spread out in the chromosome, similar to the distribution of photosynthetic genes in Cba. tepidum (Figure 4A and Additional file 1: Table S1). Both aerobic and anaerobic genes, acsF (Caur_2590) and bchE (Caur_3676), have been identified in the Cfl. aurantiacus genome (Table 3), as well as in the genome of other phototrophic Chloroflexi species, including Roseiflexus castenholzii. AcsF and BchE catalyze the isocyclic ring (or the E-ring) formation of Chl under aerobic and anaerobic growth conditions, respectively. AcsF (aerobic cyclase) catalyzes the formation of the isocyclic ring and reduces O2 into H2O. BchE requires cobalamin for catalytic activity, and has putative cobalamin and [4Fe-4S] cluster/S-adenosyl-L-methionine binding motifs [28, 31, 32, 62–64].
Other anaerobic to aerobic transitions may also be found in the biosynthesis of BChls in Cfl. aurantiacus. For example, Mg-insertion to the porphyrin ring is the first committed step of BChl biosynthesis, and three bchH (Caur_2591, Caur_3151, and Caur_3371), three bchI (Caur_0117, Caur_0419 and Caur_1255) and one bchD (Caur_0420) have been annotated for the Mg-chelatase (BchHID) of Cfl. aurantiacus, whereas one each for Hbt. modesticaldum, Rsb. denitrificans and several strictly anaerobic and aerobic bacteria. On the other hand, three bchH, and one copy of bchD and bchI, have been identified in the green sulfur bacterium Cba. tepidum (Additional file 1: Table S1), and Eisen et al. proposed  that different BchH gene products may contribute to synthesize different isoforms of BChl (BChl a, BChl c, and Chl a can be synthesized in Cba. tepidum). In comparison, two types of BChls, BChl a and BChl c can be synthesized by Cfl. aurantiacus. It is also possible that different bchH and bchI genes catalyze Mg-chelation to the BChl in various growth conditions of Cfl. aurantiacus.
Two bchG-like genes (bchG and bchK) encoding chlorophyll synthases that attach the tail into (bacterio)chlorophylls are present in the genome, as shown in Additional file 1: Table S1. Because the tails of BChl a (mostly phytyl- or geranylgeranyl-substituted) and BChl c (mainly stearyl-substituted) are rather distinct, it was suggested that one bchG-like gene encodes the enzyme synthesizing BChl a and the other homolog synthesizes BChl c. The bchG gene sequence reported by Lopez et al.  was proposed to be BChl a synthase, since the encoding protein sequence is analogous to the sequence of chlorophyll synthase from Rhodobacter capsulatus. The proposed gene function was later verified . The bchK gene encoding BChl c synthase was later confirmed with the bchK-knockout Cba. tepidum mutant . Thus, bchG (Caur_2088) and bchK (Caur_0138) encode enzymes synthesizing BChl a and BChl c, respectively, in Cfl. aurantiacus. Although genes responsible for chlorophyll biosynthesis are rather spread out in the Cfl. aurantiacus genome, two genes responsible for BChl c biosynthesis, bchU, encoding C-20 methyltransferase , and bchK, are clustered with the genes encoding chlorosome proteins, and two BChl a biosynthesis genes, bchP, encoding geranylgeranyl hydrogenase , and bchG, are in the operon containing genes encoding cytochrome c554 (pufC) and the B808-866 light-harvesting complex (Figure 4C).
(II) Nucleic acids
The level of oxygen tolerance in Cfl. aurantiacus may be suggested from the presence of genes encoding ribonucleotide reductase (RNR), which is essential for DNA synthesis. Three classes of RNR have been reported, in which the class I is a diiron oxygen-dependent (NrdB, EC 188.8.131.52), class II is coenzyme B12-dependent (NrdJ, EC 184.108.40.206), and class III is S-adenosyl-L-methionine/[4Fe-4S] cluster-dependent (NrdG, EC 220.127.116.11). It has been suggested that biosynthesis of dNTP is catalyzed by NrdB, NrdJ, and NrdG in aerobic, aerobic and anaerobic, and strictly anaerobic environments, respectively. The activity of NrdJ in Cfl. aurantiacus has been reported . Genes encoding NrdB and NrdJ, but not NrdG, have been found in the Cfl. aurantiacus genome, suggesting that NrdG and NrdJ produce dNTP for Cfl. aurantiacus in response to the oxygen level (Table 3). Moreover, in the fourth step of the biosynthesis of pyrimidine, the conversion of dihydroorotoate to orotate, dihydroorotate oxidase (EC 18.104.22.168, aerobic) versus dihydroorotate dehydrogenase (EC 22.214.171.124, anaerobic) are expressed in aerobic versus anaerobic conditions and genes encoding these enzymes have been identified (Table 3). Together, different classes of RNR and dihydroorotate oxidoreductase in the nucleic acid biosynthesis also suggest adaptation or evolution from anaerobic to aerobic conditions.
(III) Central carbon metabolism
Genes encoding several aerobic/anaerobic enzyme pairs in the central carbon metabolism, such as genes encoding pyruvate dehydrogenase (PDH, EC 126.96.36.199) and α-ketoglutarate dehydrogenase (KDH, EC 188.8.131.52), as well as pyruvate:ferredoxin oxidoreductase (or pyruvate synthase) (PFOR, EC 184.108.40.206) and α-ketoglutarate:ferredoxin oxidoreductase (or α-ketoglutarate synthase) (KFOR, EC 220.127.116.11) are present in the genome (Table 3). PFOR and KFOR, which are essential for pyruvate metabolism and energy metabolism through the TCA cycle, are commonly found in anaerobic organisms, whereas PDH and KDH are more widely spread and have been found in all aerobic organisms.
D. CO2 assimilation and carbohydrate, nitrogen and sulfur metabolisms
(I) Carbon fixation and metabolism
Genes encoding carbon monoxide dehydrogenase (coxG and coxSML) have been found in the genome, suggesting that Cfl. aurantiacus can use CO as an electron source during aerobic or semi-aerobic growth. A similar mechanism has been suggested in the aerobic anoxygenic phototrophic proteobacterium Rsb. denitrificans. CO2 generated from CO oxidation can be assimilated by Cfl. aurantiacus via the autotrophic carbon fixation cycle and/or the CO2-anaplerotic pathways. Under autotrophic growth conditions Cfl. aurantiacus is known to use a unique carbon fixation pathway: the 3-hydroxypropionate (3HOP) autotrophic cycle [4, 5, 71–74]. Three inorganic carbon molecules are assimilated into the 3HOP cycle to produce one molecule of pyruvate (Figure 5). A similar carbon fixation pathway called 3-hydroxypropionate/4-hydroxybutyrate (3HOP/4HOB) cycle was reported recently in archaea (Crenarchaeota) [75–78]. Several enzymes responsible for the 3HOP and 3HOP/4HOB cycles, including CO2-fixing enzymes (e.g., acetyl-CoA carboxylase and propionyl-CoA carboxylase), are common to the two pathways.
When the genome was first available, some genes required for the 3HOP cycle could not be found , whereas some of the missing genes/enzymes for the 3HOP cycle were later characterized experimentally : (1) acetyl-CoA carboxylase (accC (Caur_1378 and Caur_3421, biotin carboxylase), accA (Caur_1647, α-subunit), accD (Caur_1648, β-subunit) and accB (Caur_3739) , (2) malonyl-CoA reductase (mcr, Caur_2614) , (3) propionyl-CoA synthase (pcs, Caur_0613) , (4) propionyl-CoA carboxylase (pccB, Caur_2034, Caur_3435), (5) methylmalonyl-CoA epimerase (mcee, Caur_3037), (6) L-methylmalonyl-CoA mutase (MCM) (mut, Caur_1844, Caur_2508, Caur_2509), (7) succinyl-CoA:(S)-malyl-CoA transferase (smtA (Caur_0179), smtB (Caur_0178)), (8) succinate dehydrogenase (sdhBAC, Caur_1880 to Caur_1882), (9) fumarate hydratase (fh, Caur_1443), (10) (S)-malyl-CoA/β-methylmalyl-CoA/(S)-citramalyl-CoA (MMC) lyase (mcl, Caur_0174), (11) β-methylmalyl-CoA dehydratase (mch, Caur_0173), (12) mesaconyl-C1-CoA-C4-CoA transferase (mct, Caur_0174) and (13) mesaconyl-C4-CoA hydratase (meh, Caur_0180) (Figure 5). The enzymes responsible for the 4th step to the 6th step of the 3HOP cycle are involved in fatty acid oxidation and amino acid metabolism. Finally, no genes encoding ribulose 1,5-bisphosphate carboxylase (RuBisCO) (the Calvin-Benson cycle), ATP citrate lyase (the reductive TCA cycle) and acetyl-CoA synthase (the Wood-Ljungdahl pathway) are present, strongly suggesting that these autotrophic carbon fixation pathways are not present in Cfl. aurantiacus.
Additionally, genes encoding malic enzyme (tme), phosphoenolpyruvate (PEP) carboxykinase (pckA) and PEP carboxylase (ppc) have been identified, suggesting that Cfl. aurantiacus can assimilate some CO2 and replenish the metabolites in the TCA cycle through the CO2-anaplerotic pathways. The active CO2-anaplerotic pathways have been identified experimentally in other anoxygenic phototrophs during autotrophic, mixotrophic and heterotrophic growth [82–87], and the activities of PEP carboxylase and malic enzyme have also been detected in cell extracts during photoheterotrophic growth of Cfl. aurantiacus (Tang and Blankenship, unpublished results). Moreover, all of the genes in the TCA cycle are present in the Cfl. aurantiacus genome.
In central carbon metabolism, all of the genes in the TCA cycle as well as the glyoxylate cycle have been identified. The glyoxylate cycle is one of the anaplerotic pathways for assimilating acetyl-CoA, thus lipids can be converted to carbohydrates. Glyoxylate is synthesized and also assimilated in the 3HOP cycle (Figure 5), and is also produced by isocitrate lyase (EC 18.104.22.168) (icl, Caur_3889) and consumed by malate synthase (EC 22.214.171.124) (mas, Caur_2969) in the glyoxylate cycle. With acetate as the sole organic carbon source to support the photoheterotrophic growth of Cfl. aurantiacus, higher activities of isocitrate lyase and malate synthase have been reported . Further, some FAP bacteria have been shown to assimilate glycolate from their habitat . As glycolate can be converted to glyoxylate by glycolate oxidase (glcDEF, EC 126.96.36.199) and glyoxylate reductase (glyr, EC 188.8.131.52), the glyoxylate shunt and the 3HOP cycle can be employed by Cfl. aurantiacus for assimilating glycolate. Together, genes encoding central carbon metabolism, 3HOP cycle, glycolate assimilation, the glyoxylate shunt and CO oxidation are listed in Table 4.
(II) Carbohydrate metabolism
Three carbohydrate metabolism pathways are utilized by various bacteria: the Embden-Meyerhof-Parnas (EMP) pathway (glycolysis), the Entner-Doudoroff (ED) pathway, and the pentose phosphate (PP) pathway. Cfl. aurantiacus does not have genes in the ED pathway, but has genes in the oxidative PP pathway, in agreement with the activities reported for the essential enzymes (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) in the oxidative PP pathway . Genes in the non-oxidative pathway have also been found. The gene encoding fructose-1,6-bisphosphate (FBP) aldolase (EC 184.108.40.206), catalyzing the reaction of D-fructose-1,6-bisphosphate (FBP) ↔ glyceraldehyde-3-phosphate (GAP) + dihydroxyacetone phosphate (DHAP) in the EMP/gluconeogenic pathway, is missing in the genomes of Chloroflexi species (e.g., Cfl. aurantiacus, Chloroflexus sp. Y-400-fl and Chloroflexus aggregans). If Cfl. aurantiacus were unable to synthesize FBA, an active pentose phosphate pathway would be required for the interconversion of D-glucose-6-phosphate and GAP, so glucose and other sugars can be converted to pyruvate and other energy-rich species, and vice versa. However, Cfl. aurantiacus has been reported to grow well in glucose and a number of sugars during aerobic respiration , and uses the EMP pathway for carbohydrate catabolism . Also, higher activities of phosphofructokinase and FBP aldolase have been found in the cells grown with glucose than with acetate [1, 92, 93]. Note that Roseiflexi species (e.g., Roseiflexus sp. RS-1 and Roseiflexus castenholzii DSM 13941), which are closely related to Chloroflexi species, have a putative bifunctional FBP aldolase/phosphatase gene identified , and genes encoding various types of aldolase have been found in the Cfl. aurantiacus genome. Taken together, Cfl. aurantiacus and other Chloroflexi species may employ either a novel FBP aldolase or more than one carbohydrate metabolic pathway. Further efforts will be needed to clarify this picture.
(III) Nitrogen metabolism and amino acid biosynthesis
Cfl. aurantiacus is known to use ammonia as the sole nitrogen source, and several amino acids (nitrogenous compounds), but not nitrate, can enhance the growth. Neither nitrogenase nor nitrogen fixation has been reported in Cfl. aurantiacus. Consistent with the physiological studies, genes encoding enzymes in ammonia production, such as histidine ammonia lyase (hal), tyrosine phenol-lyase (tpl), asparaginase (aspg), glutamate dehydrogenase (glud1), and glutamate ammonia-lyase (glul), but not nitrogen metabolism (nifDHK) and nitrate reduction, are present in the genome. Note that Cfl. aurantiacus has genes encoding a copper-containing nitrite reductase (EC 220.127.116.11) (Caur_1570) and the α-subunit (narG, Caur_3201), but not other subunits (narHIJ) and the catalytic subunit (nasA), of nitrate reductase. Two threonine/serine dehydratases (EC 18.104.22.168), one of which is inhibited by isoleucine may be related to the isoleucine biosynthesis, and other key enzymes in isoleucine biosynthesis have been reported . Consistent with the biochemical studies, two ilvA genes (Caur_2585 and Caur_3892) encoding threonine dehydratases, and genes in the isoleucine/leucine/valine biosynthesis pathway have been identified (Table 4). The biosynthesis of isoleucine has recently been discovered through the citramalate pathway in several microbes [97, 98], while no gene encoded citramalate synthase (CimA, EC 22.214.171.124), required for the citramalate pathway, has been found in the Cfl. aurantiacus genome.
(IV) Sulfur assimilation and sulfate reduction
Cfl. aurantiacus can use a variety of compounds as sulfur sources, including cysteine, glutathione, methionine, sulfide and sulfate, during photoheterotrophic or photoautotrophic growth [99, 100]. When Cfl. aurantiacus uses sulfate as the sulfur source, high activity of ATP sulfurylase has been reported . Sulfate is reduced to sulfide during photoautotrophic and photoheterotrophic growth for synthesizing cysteine and cofactors. Consistent with the experimental data, a complete sulfur reduction pathway with a sulfur reduction operon (Caur_0686 - 0692) has been identified (Table 4). Genes encoding two ATP sulfurylases (ATP + sulfate → adenosine 5'-phosphosulfate (APS) + PPi) can be identified: sulfate adenylyltransferase (EC 126.96.36.199, Caur_0690) and a bifunctional sulfate adenylyltransferase/adenylylsulfate kinase (Caur_2113). Pyrophosphate (PPi) produced in the reaction of ATP sulfurylase is hydrolyzed to inorganic phosphate (Pi) via inorganic diphosphatase (EC 188.8.131.52) (Caur_3321). The bifunctional enzyme or/and adenylylsulfate kinase (EC 184.108.40.206, Caur_0692) converts APS to 3'-phosphoadenosine 5'-phosphosulfate (PAPS), which is reduced to sulfite and PAP (adenosine 3',5'-diphosphate) by PAPS reductase (EC 220.127.116.11, cysH, Caur_0691). While many organisms reduce APS instead of PAPS to sulfite, it is unknown if Cfl. aurantiacus carries out this reaction as genes encoding APS reductase (EC 18.104.22.168) have not been identified in the genome. In addition to the proposed pathway, sulfotransferase (Caur_2114) can also transfer the sulfate group from PAPS, which serves as a sulfur donor, to an alcohol or amine acceptor for generating various cellular sulfate compounds. PAP is generated as a by-product in the reactions catalyzed by PAPS reductase and sulfotransferase, and has no known functions in metabolism and is likely hydrolyzed to AMP and Pi via PAP phosphatase (unidentified yet). Sulfite reductase (EC 22.214.171.124, Caur_0686) reduces sulfite to sulfide, which is incorporated into cysteine by cysteine synthase A (cysK, Caur_1341) or cysteine synthase B (cysM, Caur_3489). The overall proposed sulfur reduction and assimilation pathways are illustrated in Figure 6.
Other than using sulfide as the sulfur source during photoheterophic growth, Cfl. aurantiacus grows photoautotrophically in the presence of sufficient sulfide [100–102]. Under these circumstances, sulfide likely functions as electron donor by replacing organic carbon sources contributed from cyanobacteria. In agreement with these physiological and ecological studies, the gene encoding a type II sulfide:quinone oxidoreductase (SQR) (sqr, Caur_3894), has been found in the genome. SQRs belong to the members of the disulfide oxidoreductase flavoprotein superfamily. Other than type II SQRs, type I and type III SQRs with distinct sequences and structures and cofactor requirements have also been reported . Although all of the characterized SQRs catalyze oxidization of sulfide to elemental sulfur (E(ox) + H2S → EH2(red) + S°), different types of SQR have been identified in various classes of photosynthetic bacteria : type I, purple non-sulfur anoxygenic photosynthetic proteobacteria (such as Rhodobacter capsulatus ) ; type II, Cfl. aurantiacus and cyanobacteria (such as Synechocystis PCC 6803) ; and type III, green sulfur bacteria . In addition to being characterized in phototrophic microbes, type II SQRs have also been identified in various non-photosynthetic bacteria as well as in the mitochondria of animals, and are suggested to be involved in sulfide detoxification, heavy metal tolerance, sulfide signaling, and other essential cellular processes .
E. Evolution perspectives
Our paper reports numerous aerobic/anaerobic gene pairs and oxygenic/anoxygenic metabolic pathways in the Cfl. aurantiacus genome. As suggested by phylogenetic analyses [6–8] and comparisons to the genome and reports of other photosynthetic bacteria, one can propose lateral or horizontal gene transfers between Cfl. aurantiacus and other photosynthetic bacteria. Some proposed lateral gene transfers are listed below and also illustrated in Figure 7. Note that horizontal/lateral gene transfers suggested below are important in the evolution of photosynthesis. It is important to remember that it has not yet been generally accepted which organisms were donors and which were acceptors during gene transfers. The proposed gene transfers below remain to be verified with more sequenced genomes and biochemical studies in photosynthetic organisms.
(I) Photosynthetic components
Chlorosomes were transferred between Cfl. aurantiacus and the green sulfur bacteria (GSBs). The GSBs have larger chlorosomes and more genes encoding chlorosome proteins [21, 29]. The integral membrane core antenna complex and a type II (quinone-type) reaction center were transferred either to or from the purple photosynthetic bacteria.
(II) (Bacterio)chlorophyll biosynthesis
AcsF and BchE are proposed to be responsible for biosynthesis of the isocyclic ring of (bacterio)chlorophylls under aerobic and anaerobic growth conditions, respectively [28, 31, 32, 62–64]. The acsF gene was transferred either to or from purple bacteria and cyanobacteria, and the bchE gene either to or from heliobacteria, purple bacteria and GSBs.
(III) Electron transfer complexes
Two gene clusters of the complex I genes were transferred to (some) purple bacteria. Genes encoding auracyanin may have been transferred either to or from cyanobacteria where the type I blue copper protein plastocyanin is found. Alternative complex III (ACIII) may have evolved from or to the cytochrome bc 1 or b 6 /f complex.
(IV) Central carbon metabolism
Genes encoding pyruvate dehydrogenase and α-keto-glutarate dehydrogenase were transferred either to or from purple bacteria and cyanobacteria, and genes encoding PFOR (or pyruvate synthase) and KFOR (or α-ketoglutarate synthase) to or from heliobacteria and GSBs. GSBs may have acquired the ATP citrate lyase gene to complete the reductive TCA (RTCA) cycle for CO2 fixation, and heliobacteria obtained the gene encoding (Re)-citrate synthase for synthesizing citrate and operating the partial oxidative TCA (OTCA) cycle . Since Cfl. aurantiacus operates the OTCA cycle, the RTCA cycle in GSBs may have evolved from the OTCA cycle .
The filamentous anoxygenic phototrophic (FAP) bacteria (or the green non-sulfur bacteria) have been suggested to be a critical group in the evolution of photosynthesis. As the first characterized FAP bacterium, the thermophilic bacterium Chloroflexus aurantiacus is an amazing organism. It has a chimerical photosystem that comprises characteristic types of green sulfur bacteria and purple photosynthetic bacteria. It is metabolically versatile, and can grow photoautotrophically and photoheterotrophically under anaerobic growth conditions, and chemotrophically under aerobic growth conditions. Consistent with these physiological and ecological studies, the Cfl. aurantiacus genome has duplicated genes and aerobic/anaerobic enzyme pairs in (photosynthetic) electron transport chain, central carbon metabolism, and biosynthesis of tetrapyrroles and nucleic acids. In particular, duplicate genes and gene clusters for two unique proteins and protein complexes in Cfl. aurantiacus and several FAP bacteria, the alternative complex III (ACIII) and type I blue copper protein auracyanin, have been identified in Cfl. aurantiacus genome. The genomic information and previous biochemical studies also suggest that Cfl. aurantiacus operates diverse carbon assimilation pathways. In contrast to the purple photosynthetic bacteria, the photosynthetic genes are rather spread out in the Cfl. aurantiacus chromosome. Overall, the genomic analyses presented in this report, along with previous physiological, ecological and biochemical studies, indicate that Cfl. aurantiacus has many interesting and certain unique features in its metabolic pathways.
The genome of Chloroflexus aurantiacus J-10-fl was sequenced at the Joint Genome Institute (JGI) using a combination of 8-kb and 14-kb DNA libraries. All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov/. Draft assemblies were based on 58246 total reads. Both libraries provided 10x coverage of the genome. The Phred/Phrap/Consed software package (http://www.phrap.com) was used for sequence assembly and quality assessment [107–109]. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher  or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification (Roche Applied Science, Indianapolis, IN). A total of 1893 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The completed Cfl. aurantiacus genome sequence contains 61248 reads, achieving an average of 11-fold sequence coverage per base with an error rate less than 1 in 100,000.
The genome of Chloroflexus aurantiacus J-10-fl has been annotated by the default JGI annotation pipeline. Genes were identified using two gene modeling programs, Glimmer  and Critica  as part of the Oak Ridge National Laboratory genome annotation pipeline. The two sets of gene calls were combined using Critica as the preferred start call for genes with the same stop codon. Briefly, structural RNAs were predicted using BLASTn and tRNAscan-SE  with default prokaryotic settings. Protein-coding genes were identified using gene modeling program Prodigal . Genes with less than 80 amino acids which were predicted by only one of the gene callers and had no Blast hit in the KEGG database at 1e-05 were deleted. Predicted gene models were analyzed using GenePRIMP pipeline , and erroneous gene models were manually curated. The revised gene-protein set was searched by BLASTp against the KEGG GENES database  and GenBank NR using e-value of 1.0e-05, a minimum of 50% identity and alignment length of at least 80% of both the query and subject protein. These BLASTp hits were used to perform the initial automated functional assignments. In addition, protein sequences were searched against Pfam  and TIGRFAM  databases using HMMER2 package and trusted cutoffs for each model. Protein sequences were also searched against COG database  using RPS-BLAST search with e-value of 1.0e-05 and retaining the best hit. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE , TMHMM , and signalP . The annotated genome sequence was submitted to GenBank and loaded into the Integrated Microbial Genomes (IMG) database .
The 16S rRNA gene sequences of various photosynthetic bacteria were obtained from NCBI. The sequences of 16S rRNA genes were aligned using the program Bioedit , and the phylogenetic tree was constructed using the program MEGA 4.1 . The tree is an unrooted neighbor joining tree.
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Acknowledgements and Funding
The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The authors thank the contribution of Alex Copeland, Susan Lucas, Tijana Glavina del Rio, Nancy Hammon, Hope N. Tice, Jeremy Schmutz, Thomas S. Brettin, David Bruce, Chris Detter, Nikos C. Kyrpides, and Paul Richardson for gene sequencing, assembling and annotation.
The authors declare that they have no competing interests.
BKP and REB designed research and provided DNA; KB, OC, ED, CSH, LJH, MLL, AL, FWL, NM and SP conducted sequencing, assembly and automated annotation; KHT, BMH, LEK and REB analyzed data; and KHT and REB wrote the paper. All authors read and approved the final manuscript.
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Tang, KH., Barry, K., Chertkov, O. et al. Complete genome sequence of the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus. BMC Genomics 12, 334 (2011). https://doi.org/10.1186/1471-2164-12-334
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