Essential Genes Predicted in the Genome of Rubrivivax gelatinosus

Abstract

Rubrivivax gelatinosus is a betaproteobacterium with impressive metabolic diversity. It is capable of phototrophy, chemotrophy, two different mechanisms of sugar metabolism, fermentation, and H 2 gas production. To identify core essential genes, R. gelatinosus was subjected to saturating transposon mutagenesis and high-throughput sequencing (TnSeq) analysis using nutrient-rich, aerobic conditions. Results revealed that virtually no primary metabolic genes are essential to the organism and that genomic redundancy only explains a portion of the nonessentiality, but some biosynthetic pathways are still essential under nutrient-rich conditions. Different essentialities of different portions of the Pho regulatory pathway suggest that overexpression of the regulon is toxic and hint at a larger connection between phosphate regulation and cellular health. Lastly, various essentialities of different tRNAs hint at a more complex situation than would be expected for such a core process. These results expand upon research regarding cross-organism gene essentiality and further enrich the study of purple nonsulfur bacteria. IMPORTANCEMicrobial genomic data are increasing at a tremendous rate, but physiological characterization of those data lags far behind. One mechanism of high-throughput physiological characterization is TnSeq, which uses high-volume transposon mutagenesis and highthroughput sequencing to identify all of the essential genes in a given organism's genome. Here TnSeq was used to identify essential genes in the metabolically versatile betaproteobacterium Rubrivivax gelatinosus. The results presented here add to the growing TnSeq field and also reveal important aspects of R. gelatinosus physiology, which are applicable to researchers working on metabolically flexible organisms. R ubrivivax gelatinosus (formerly Rhodocyclus gelatinosus) is apurple nonsulfur bacterium in the Betaproteobacteria clade. Its physiology has principally been studied for its photosynthetic capability (1-8), but the organism is very metabolically versatile; it is capable of growing phototrophically (including photoheterotrophically) in anaerobic light conditions and chemoheterotrophically under aerobic conditions in light or dark (9, 10). It has the necessary components for the Embden-Meyerhof-Parnas pathway and the Entner-Doudoroff pathway as well as for the pentose phosphate pathway (11). It is capable of carbon and nitrogen fixation, fermentation, and H 2 gas production, making it of interest in biofuel production. Interestingly, while 16S rRNA gene sequencing places the organism in the Betaproteobacteria, the phylogenetic history of the photosynthesis genes strongly suggests that they were horizontally transferred from purple photosynthetic Alphaproteobacteria (12).To identify a minimal set of essential genes in R. gelatinosus IL-144, saturating transposon mutagenesis and high-throughput sequencing (TnSeq) was performed on the organism using aerobic, dark, nutrient-rich conditions. TnSeq is a method of identifying essential ge...

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