Metabolic flexibility revealed in the genome of the cyst-forming alpha-1 proteobacterium Rhodospirillum centenum

Abstract

Background: Rhodospirillum centenum is a photosynthetic non-sulfur purple bacterium that favors growth in an anoxygenic, photosynthetic N2-fixing environment. It is emerging as a genetically amenable model organism for molecular genetic analysis of cyst formation, photosynthesis, phototaxis, and cellular development. Here, we present an analysis of the genome of this bacterium.

Results: R. centenum contains a singular circular chromosome of 4,355,548 base pairs in size harboring 4,105 genes. It has an intact Calvin cycle with two forms of Rubisco, as well as a gene encoding phosphoenolpyruvate carboxylase (PEPC) for mixotrophic CO2 fixation. This dual carbon-fixation system may be required for regulating internal carbon flux to facilitate bacterial nitrogen assimilation. Enzymatic reactions associated with arsenate and mercuric detoxification are rare or unique compared to other purple bacteria. Among numerous newly identified signal transduction proteins, of particular interest is a putative bacteriophytochrome that is phylogenetically distinct from a previously characterized R. centenum phytochrome, Ppr. Genes encoding proteins involved in chemotaxis as well as a sophisticated dual flagellar system have also been mapped.

Conclusions: Remarkable metabolic versatility and a superior capability for photoautotrophic carbon assimilation is evident in R. centenum.

Figure 1

Circular representation of the R. centenum chromosome. (a) Circular representation of the R. centenum chromosome. The different rings represent (from outer to inner) all genes and insertion elements, color-coded by functional category (rings 1 and 2), BLASTx results (E-value = 0.0001) comparing translated R. centenum DNA to R. rubrum (NC_007643) proteins scaled according to percent identity (ring 3), BLASTn results (E-value = 0.0001) comparing R. centenum DNA to R. rubrum DNA (ring 4), deviation from average G+C content (ring 5), and deviation from average GC skew [(C-G)/(C+G); ring 6]. Color codes for gene functional categories are as follows: energy and central intermediary metabolism, green; fatty acid and phospholipid metabolism, turquoise; purine/pyrimidine/nucleoside/nucleotide biosynthesis, salmon; protein synthesis and fate, yellow; cofactor biosynthesis, pink; amino acid synthesis, orange; cellular processes and envelope, light green; DNA metabolism, red; transcription, dark blue; mobile and extra-chromosomal elements, dark green; cell division and chromosomal partitioning, light blue; general function prediction only, brown; unknown function and hypothetical proteins, dark gray. (b) Scanning electron micrograph of a R. centenum mature colony undergoing cyst formation, showing a heterogeneous array of vibrioid-shaped vegetative cells and clusters of spherical cyst cells.

Figure 2

Genomic organization and phylogenetic analysis of the two forms of Rubisco of R. centenum. (A) The structural gene arrangement for Form IAq and IC of Rubisco found in R. centenum and Form II of Rubsico in R. rubrum are shown. Yellow indicates those genes encoding both large and small subunits of Rubisco and the Calvin cycle enzymes. Red indicates the cbb operon transcriptional regulator. (B) Maximum likelihood unrooted tree of Rubisco Form IAq, IAc, IB, and IC based on a multiple protein alignment. The two forms of Rubisco found in R. centenum are indicated red.

Figure 3

A schematic of putative carbon flux in R. centenum. Aerobic reactions are designated with solid lines, micro- or anaerobic reactions with dotted lines. The participating enzymes in the major reactions are numbered: 1. pyruvate dikinase (RC1_1667); 2. pyruvate kinase (RC1_2135 and RC1_2401); 3. phosphoenolpyruvate synthase (RC1_3562); 4. phosphoenolpyruvate carboxykinase (RC1_2822); 5. phosphoenolpyruvate carboxylase (RC1_2446); 6. malic enzyme (RC1_0405 and RC1_3260); 7. malate dehydrogenase (RC1_4080); 8. malate synthase (RC1_2688); 9. fumarase (RC1_1865); 10. succinate dehydrogenase (RC1_3941). Inter-converison of fumarate and succinate (SUC) can oxidize electron carriers such as quinone (purple labeled) for anaerobic respiration. Alternatively, both pyruvate and acetyl CoA can be used in fermentation. Putatively, Glycerate-3-phosphate (3PG) produced by fixing CO₂ into Ribulose-1,5-bipohsphate (RUBP) by Rubisco may be shuffled between the Calvin cycle and glycolysis (hollow arrowhead with question mark).

Figure 4

Photosynthesis gene cluster arrangement in the purple bacteria Bradyrhizobium sp, R. centenum, R. rubrum, and Rhodobacter capsulatus. Genes are presented as arrows indicating their direction of transcription. Genes are colored as listed: chlorophyll biosynthesis (bch) in green, carotenoid biosynthesis (crt) in yellow, reaction centers (puf) and light-harvesting complexes (puh) in red, regulatory proteins (aerR, ppsR, and crtJ) in purple, other functional proteins in light blue, and uncharacterized genes in white. Brbphp in orange is a bacteriophytochrome unique in Bradyrhizobium sp. Lines illustrate gene rearrangement, and arrows illustrate the inversion of large superoperonal clusters.

Figure 5

Flagellar gene cluster (FGC) arrangement. A linear representation of the R. centenum genome with the relative positions of the five flagellar gene clusters is shown at the top. Beneath, genes in individual clusters are presented as arrows indicating the direction of transcription and colored as follows: flg genes, red; fli genes, yellow; fla and flb genes, blue; flh genes, green; regulatory and other functional proteins, pink; and uncharacterized genes, white.

Figure 6

Maximum likelihood tree based on conserved flagellar proteins depicting the position of R. centenum flagellar systems among other flagellated bacteria. The tree is based on a concatenated alignment of 11 flagellar proteins (Flg) that are present in all primary and secondary flagellar systems [36]. A blue "1" or "2" after the species name indicates primary and secondary flagella systems, respectively. Alpha I (alphaproteobacteria group I) and Alpha II (alphaproteobacteria group II) are boxed. The primary (polar) and secondary (lateral) flagella of R. centenum are in red.