The genome of Heliobacterium modesticaldum, a phototrophic representative of the Firmicutes containing the simplest photosynthetic apparatus

Abstract

Despite the fact that heliobacteria are the only phototrophic representatives of the bacterial phylum Firmicutes, genomic analyses of these organisms have yet to be reported. Here we describe the complete sequence and analysis of the genome of Heliobacterium modesticaldum, a thermophilic species belonging to this unique group of phototrophs. The genome is a single 3.1-Mb circular chromosome containing 3,138 open reading frames. As suspected from physiological studies of heliobacteria that have failed to show photoautotrophic growth, genes encoding enzymes for known autotrophic pathways in other phototrophic organisms, including ribulose bisphosphate carboxylase (Calvin cycle), citrate lyase (reverse citric acid cycle), and malyl coenzyme A lyase (3-hydroxypropionate pathway), are not present in the H. modesticaldum genome. Thus, heliobacteria appear to be the only known anaerobic anoxygenic phototrophs that are not capable of autotrophy. Although for some cellular activities, such as nitrogen fixation, there is a full complement of genes in H. modesticaldum, other processes, including carbon metabolism and endosporulation, are more genetically streamlined than they are in most other low-G+C gram-positive bacteria. Moreover, several genes encoding photosynthetic functions in phototrophic purple bacteria are not present in the heliobacteria. In contrast to the nutritional flexibility of many anoxygenic phototrophs, the complete genome sequence of H. modesticaldum reveals an organism with a notable degree of metabolic specialization and genomic reduction.

FIG. 1.

Circular genome map of the 3.1-Mb H. modesticaldum chromosome. The rings indicate (from outside to inside) all the genes and insertion elements, color coded by functional category (rings 1 and 2), the deviation from the average G+C content (ring 3), and the GC skew (ring 4). The approximate location of the origin of replication is at the beginning of the dnaA gene. The colors indicate the following: turquoise, small-molecule biosynthesis; yellow, central or intermediary metabolism; orange, energy metabolism; red, signal transduction; light blue, DNA metabolism; blue, transcription; purple, protein synthesis/fate; dark green, surface-associated features; gray, miscellaneous features; pink, phage and insertion elements; light green, unknown function; dark gray, conserved hypothetical proteins; black, hypothetical proteins; brown, pseudogenes.

FIG. 2.

Putative pathway of carbon metabolism in H. modesticaldum (adapted from reference 44). A partial reverse citric acid cycle with CO₂ incorporation via PEP carboxykinase is shown. The enzymes involved in the reduction of acetate to pyruvate putatively function in an oxidative direction during chemotrophic (dark) growth on pyruvate. The oxidation of pyruvate to acetyl-CoA is likely accompanied by hydrogen evolution via an [FeFe] hydrogenase. The numbers indicate the following enzymes: 1, pyruvate:ferredoxin oxidoreductase; 2, pyruvate-phosphate dikinase; 3, PEP carboxykinase; 4, oxaloacetate decarboxylase; 5, malate dehydrogenase; 6, fumarase; 7, fumarate reductase; 8, succinyl-CoA synthetase; 9, 2-oxoglutarate:ferredoxin oxidoreductase; 10, NADP-dependent isocitrate dehydrogenase; 11, aconitate hydratase; 12, AMP-forming acetyl-CoA synthetase; 13, lactate dehydrogenase.

FIG. 3.

Schematic representation of hydrogenase and photosynthesis gene clusters in H. modesticaldum. Arrows represent individual genes and indicate the direction of transcription. (A) Putative [FeFe] hydrogenase genes in H. modesticaldum and the close phylogenetic relative P. thermopropionicum SI. As in Heliobacillus mobilis, nuoE and nuoF are fused in H. modesticaldum, suggesting that this feature may be universal in heliobacteria. Colors indicate the following: gold, NADH dehydrogenase subunits; orange, [FeFe] hydrogenase structural genes. (B) Uptake [NiFe] hydrogenase genes in related Firmicutes. The genes are located in a single operon in H. modesticaldum, whereas they are dispersed in different regions of the D. hafniense Y51 chromosome. Colors indicate the following: blue, [NiFe] hydrogenase structural genes; purple, hydrogenase expression/formation; red, hydrogenase assembly/maturation. (C) Photosynthesis gene clusters from H. modesticaldum and the purple bacterium R. capsulatus. Shared genes are outlined with bold lines. Lines indicate gene synteny, as follows: black lines, single gene rearrangements; red lines, inverted genes; blue lines, inverted genes with a gene insertion. Dashed boxes indicate R. capsulatus photosynthesis genes absent from H. modesticaldum. The colors of the arrows indicate the following: green, Bchl biosynthesis (bch); orange, carotenoid biosynthesis (crt); pink, proteobacterial reaction centers (puf) and light harvesting complexes (puh); olive, heliobacterial reaction center (psh); teal, regulatory proteins; light green, electron transport (pet); red, cofactor biosynthesis; purple, cell division and sporulation; light blue, nitrogen fixation; gray, transcription; light gray, other nonphotosynthetic genes; white, uncharacterized genes.

FIG. 4.

Phylogenetic tree showing the relationship of concatenated bchXYZ, bchLNB, and nifHDK genes from H. modesticaldum to those of other organisms containing these genes. Although not present in H. modesticaldum, alternative nitrogenase genes, anfHDK and vnfHDK, are also included to balance the tree. Organisms containing more than one set of genes used in the comparison appear multiple times in the tree. The following organisms were included in the analysis: Anabaena siamensis strain TISTR8012, Azospirillum brasilense, Azotobacter vinelandii, Bradyrhizobium sp. strain BTAi1, Bradyrhizobium sp. strain ORS278, Chlorobaculum tepidum, Chloroflexus aurantiacus, Clostridium acetobutylicum, Clostridium pasteurianum, Clostridium kluyveri, Desulfitobacterium hafniense strain Y51, Geobacter sulfurreducens, Heliobacterium modesticaldum, Jannaschia sp. strain CCS1, Klebsiella pneumoniae, Methanothermobacter thermoautotrophicus, Methanococcus maripaludis, Methanosarcina acetivorans strain C2A, Nostoc sp. strain PCC7120, Prosthecochloris aestuarii, Rhodobacter capsulatus, Rhodopseudomonas palustris, Roseiflexus castenholzii, Roseobacter denitrificans, Synechococcus elongatus strain PCC6301, and Synechocystis sp. strain PCC6803.

FIG. 5.

Proposed pathway of later steps in Bchl g biosynthesis in H. modesticaldum. Divinyl protochlorophyllide a is reduced to 8-vinyl chlorophyllide a by the activity of the bchLNB gene products. This is followed by the reduction of the C-7—C-8 double bond via the bchXYZ gene products, which yields C-8 vinyl bacteriochlorophyllide a. Bacteriochlorophyllide g is produced by the isomerization of the 8¹-vinyl group to an ethylidene group. Bchl synthetase (BchG) then catalyzes the addition of a farnesyl group, which yields the completed Bchl g.

FIG. 6.

Diagram showing a putative pathway of electron transfer based on genetic components present in H. modesticaldum. Cyclic electron transfer has not been confirmed in heliobacteria. In addition, the reduction of NAD⁺ by cytoplasmic ferredoxin has not been confirmed, as a gene encoding FNR was not identified in the genome. Despite this, genes encoding all 14 subunits of NADH:quinone oxidoreductase (nuoA to nuoN) were putatively identified.