Genomic Organization of <i>C. hydrogenoformans</i>

<p>From the outside inward the circles show: (1, 2) predicted protein-coding regions on the plus and minus strands (colors were assigned according to the color code of functional classes; (3) prophage (orange) and CRISPR (pink) regions; (4) χ²-square score of tri-nucleotide composition; (5) GC skew (blue indicates a positive value and red a negative value); (6) tRNAs (green); (7) rRNAs (blue) and structural RNAs (red).</p

An Electron Micrograph of a <i>C. hydrogenoformans</i> Endospore

<p>The finding of homologs of many genes involved in sporulation in other species led us to test whether <i>C. hydrogenoformans</i> also could form an endospore. Under stressful growth conditions, endospore-like structures form. We note that even though homologs could not be found in the genome for many genes that in other species are involved in protective outer-layer (cortex, coat, and exosporium) formation, those structures seem to be visible and intact.</p

Phylogenetic Tree of CooS Homologs

<p>The figure shows a maximum-likelihood tree of CooS homologs. The tree indicates the five CooS homologs in <i>C. hydrogenoformans</i> are not the result of recent duplications but instead are from distinct subfamilies. The other CooS homologs included in the tree were obtained from the NCBI nr database and include some from incomplete genome sequences generated by United States Department of Energy Joint Genome Institute (<a href="http://www.jgi.doe.gov/" target="_blank">http://www.jgi.doe.gov/</a>).</p

Genome Tree of Representatives of Firmicutes

<p>A maximum likelihood tree was built from concatenated protein sequences of 31 universal housekeeping genes and rooted by two outgroup Actinobacteria (high GC Gram-positives) species: <i>Corynebacterium glutamicum</i> and <i>Streptomyces coelicolor.</i> Bootstrap support values (out of 100 runs) for branches of interest are shown beside them. Each species' ability to sporulate and its number of putative orthologs of the 175 known <i>B. subtilis</i> sporulation genes are also shown.</p

Predicted Complete Acetyl-CoA Pathway of Carbon Fixation in <i>C. hydrogenoformans</i>

<p>Genes predicted to encode each step in the acetyl-CoA pathway of carbon fixation were identified in the genome. The locus numbers are indicated on the figure.</p

Genome Locations of Genes Predicted to Encode Five CODH Complexes

<p>The genome locations of the genes encoding the five CooS homologs (labelled CooS I-V) are shown. Also shown are neighboring genes that are predicted to encode the five distinct CODH complexes (CODH I-V) with each CooS homolog. Possible cellular roles for four of the five CODH complexes are indicated.</p

Alignment of <i>w</i>Mel with a 60 kbp Region of the <i>Wolbachia</i> from <i>B. malayi</i>

<p>The figure shows BLASTN matches (green) and whole-proteome alignments (red) that were generated using the “promer” option of the MUMmer software (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020069#pbio-0020069-Delcher1" target="_blank">Delcher et al. 1999</a>). The B. malayi region is from a BAC clone (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020069#pbio-0020069-Ware1" target="_blank">Ware et al. 2002</a>). Note the regions of alignment broken up by many rearrangements and the presence of repetitive sequences at the regions of the breaks.</p

Genomic Organization and expression of Type IV Secretion Operons in <i>w</i>Mel

<p>(A) Organization of the nine <i>vir</i>-like CDSs (white arrows) and five adjacent CDSs that encode for either putative membrane-spanning proteins (black arrows) or non-<i>vir</i> CDSs (gray arrows) of wMel, R. conorii, and A. tumefaciens. Solid horizontal lines denote RT experiments that have confirmed that adjacent CDSs are expressed as part of a polycistronic transcript. Results of these RT-PCR experiments are presented in (B). Lane 1, <i>virB3</i>-<i>virB4</i>; lane 2, RT control; lane 3, <i>virB6</i>-WD0856; lane 4, RT control; lane 5, WD0856-WD0855; lane 6, RT control; lane 7, WD0854-WD0853; lane 8, RT control; lane 9, <i>virB8</i>-<i>virB9</i>; lane 10, RT control; lane 11, <i>virB9</i>-<i>virB11</i>; lane 12, RT control; lane 13, <i>virB11</i>-<i>virD4</i>; lane 14, RT control; lane 15, <i>virD4</i>-<i>wspB</i>; lane 16, RT control; lane 17, <i>virB4</i>-<i>virB6</i>; lane 18, RT control; lane 19, WD0855-WD0854; lane 20, RT control. Only PCRs that contain reverse transcriptase amplified the desired products. PCR primer sequences are listed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020069#st009" target="_blank">Table S9</a>.</p

Phage Alignments and Neighboring Genes

<p>Conserved gene order between the WO phage in <i>Wolbachia</i> sp. <i>w</i>Kue and prophage regions of <i>w</i>Mel. Putative proteins in <i>w</i>Kue (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020069#pbio-0020069-Masui2" target="_blank">Masui et al. 2001</a>) were searched using TBLASTN against the <i>w</i>Mel genome. Matches with an <i>E</i>-value of less than 1e⁻¹⁵ are linked by connecting lines. CDSs are colored as follows: brown, phage structural or replication genes; light blue, conserved hypotheticals; red, hypotheticals; magenta, transposases or reverse transcriptases; blue, ankyrin repeat genes; light gray, <i>radC</i>; light green, paralogous genes; gold, others. The regions surrounding the phage are shown because they have some unusual features relative to the rest of the genome. For example, WO-A and WO-B are each flanked on one side by clusters of genes in two paralogous families that are distantly related to phage repressors. In each of these clusters, a homolog of the <i>radC</i> gene is found. A third <i>radC</i> homolog (WD1093) in the genome is also flanked by a member of one of these gene families (WD1095). While the connection between <i>radC</i> and the phage is unclear, the multiple copies of the <i>radC</i> gene and the members of these paralogous families may have contributed to the phage rearrangements described above.</p

Long Evolutionary Branches in <i>w</i>Mel

<p>Maximum-likelihood phylogenetic tree constructed on concatenated protein sequences of 285 orthologs shared among <i>w</i>Mel, R. prowazekii, R. conorii, <i>C. crescentus,</i> and E. coli. The location of the most recent common ancestor of the α-Proteobacteria (<i>Caulobacter</i>, <i>Rickettsia</i>, <i>Wolbachia</i>) is defined by the outgroup <i>E. coli.</i> The unit of branch length is the number of changes per amino acid. Overall, the amino acid substitution rate in the <i>w</i>Mel lineage is about 63% higher than that of <i>C. crescentus</i>, a free-living α-Proteobacteria. <i>w</i>Mel has evolved at a slightly higher rate than the <i>Rickettssia</i> spp., close relatives that are also obligate intracellular bacteria that have undergone accelerated evolution themselves. This higher rate is likely in part to be due to an increase in the rate of slightly deleterious mutations, although we have not ruled out the possibility of G+C content effects on the branch lengths.</p