Essential Genome of the Metabolically Versatile Alphaproteobacterium Rhodopseudomonas palustris

Abstract

Rhodopseudomonas palustris is an alphaproteobacterium that has served as a model organism for studies of photophosphorylation, regulation of nitrogen fixation, production of hydrogen as a biofuel, and anaerobic degradation of aromatic compounds. This bacterium is able to transition between anaerobic photoautotrophic growth, anaerobic photoheterotrophic growth, and aerobic heterotrophic growth. As a starting point to explore the genetic basis for the metabolic versatility of R. palustris, we used transposon mutagenesis and Tn-seq to identify 552 genes as essential for viability in cells growing aerobically on semirich medium. Of these, 323 have essential gene homologs in the alphaproteobacterium Caulobacter crescentus, and 187 have essential gene homologs in Escherichia coli. There were 24 R. palustris genes that were essential for viability under aerobic growth conditions that have low sequence identity but are likely to be functionally homologous to essential E. coli genes. As expected, certain functional categories of essential genes were highly conserved among the three organisms, including translation, ribosome structure and biogenesis, secretion, and lipid metabolism. R. palustris cells divide by budding in which a sessile cell gives rise to a motile swarmer cell. Conserved cell cycle genes required for this developmental process were essential in both C. crescentus and R. palustris. Our results suggest that despite vast differences in lifestyles, members of the alphaproteobacteria have a common set of essential genes that is specific to this group and distinct from that of gammaproteobacteria like E. coli. IMPORTANCEEssential genes in bacteria and other organisms are those absolutely required for viability. Rhodopseudomonas palustris has served as a model organism for studies of anaerobic aromatic compound degradation, hydrogen gas production, nitrogen fixation, and photosynthesis. We used the technique of Tn-seq to determine the essential genes of R. palustris grown under heterotrophic aerobic conditions. The transposon library generated in this study will be useful for future studies to identify R. palustris genes essential for viability under specialized growth conditions and also for survival under conditions of stress.

Essential Genes Predicted in the Genome of Rubrivivax gelatinosus

“…At least two, if not more, genes can be found encoding a R. palustris fumarase, C. crescentus citrate synthase, B. subvibrioides citrate synthase, A. tumefaciens aconitase and malate dehydrogenase, and P. aeruginosa aconitase, isocitrate dehydrogenase, and malate dehydrogenase. There is no redundancy to explain the nonessentiality of succinyl coenzyme A (succinyl-CoA) synthetase in R. palustris, but the authors proposed that succinate may be generated by the glyoxylate bypass and excess succinyl-CoA may be shuttled into heme synthesis (21). Similarly, there is no redundancy to explain the succinyl-CoA synthetase nonessentiality in P. aeruginosa, but that organism does possess a succinyl-CoA:acetate CoA transferase.…”

“…Other COG categories with large numbers of essential genes include H (coenzyme transport and metabolism) (36 genes), I (lipid transport and metabolism) (20 genes), and M (cell wall/membrane/envelope biogenesis) (35 genes). It is unsurprising that there are many essential genes in the last category, as it contains genes for peptidoglycan synthesis as well as assembly, which tend to be essential in other organisms (14,15,21). Essential genes in category I not only include genes for fatty acid and phospholipid biosynthesis but several genes involved in isoprenoid biosynthesis as well.…”

“…This result likely reflects the alphaproteobacterial heritage of R. palustris, where many organisms use two-component signal transduction proteins in complex networks to regulate the cell cycle as exemplified by the developmental cell cycle of C. crescentus (reviewed in reference 22). Indeed, many of the essential signal transduction genes in R. palustris encode two-component proteins homologous to known C. crescentus developmental regulators (21). The lone essential signal transduction gene in R. gelatinosus is a putative homolog of dksA.…”

“…Similarly, the metabolic flexibility of the organism can contribute to lower essential gene content, as a blockage in one type of metabolic pathway may be bypassed by adopting a different type of metabolism. However, Rhodopseudomonas palustris, a purple photosynthetic and metabolically versatile alphaproteobacterium, has a similar genome size to R. gelatinosus with 4,838 protein-encoding ORFs, and TnSeq analysis, using a rich medium that was also supplemented with light (thereby providing conditions for more types of metabolism than were provided for R. gelatinosus), found 552 essential genes, which covers 11.2% of the genome (21). Therefore, genome size and metabolic flexibility cannot be the sole determinants of essential gene content.…”

“…Included in Table 1 is a COG category breakdown of the essential genes of R. palustris (21), the most comparable organism to R. gelatinosus on which TnSeq analysis has been performed. Similar to R. gelatinosus, the COG category with the most genes in R. palustris was J (translation, ribosomal structure, and biogenesis) with 91 essential genes.…”