Date Published: May 21, 2019
Publisher: Public Library of Science
Author(s): Amanda R. M. Campbell, Benjamin R. Titus, Madeline R. Kuenzi, Fernando Rodriguez-Perez, Alysha D. L. Brunsch, Monica M. Schroll, Matthew C. Owen, Jeff D. Cronk, Kirk R. Anders, Jennifer N. Shepherd, Joel H. Weiner.
The lipophilic electron-transport cofactor rhodoquinone (RQ) facilitates anaerobic metabolism in a variety of bacteria and selected eukaryotic organisms in hypoxic environments. We have shown that an intact rquA gene in Rhodospirillum rubrum is required for RQ production and efficient growth of the bacterium under anoxic conditions. While the explicit details of RQ biosynthesis have yet to be fully delineated, ubiquinone (Q) is a required precursor to RQ in R. rubrum, and the RquA gene product is homologous to a class I methyltransferase. In order to identify any additional requirements for RQ biosynthesis or factors influencing RQ production in R. rubrum, we performed transcriptome analysis to identify differentially expressed genes in anoxic, illuminated R. rubrum cultures, compared with those aerobically grown in the dark. To further select target genes, we employed a bioinformatics approach to assess the likelihood that a given differentially expressed gene under anoxic conditions may also have a direct role in RQ production or regulation of its levels in vivo. Having thus compiled a list of candidate genes, nine were chosen for further study by generation of knockout strains. RQ and Q levels were quantified using liquid chromatography-mass spectrometry, and rquA gene expression was measured using the real-time quantitative polymerase chain reaction. In one case, Q and RQ levels were decreased relative to wild type; in another case, the opposite effect was observed. These results comport with the crucial roles of rquA and Q in RQ biosynthesis, and reveal the existence of potential modulators of RQ levels in R. rubrum.
Bacteria and simple eukaryotic organisms that have adapted to anoxic or hypoxic conditions for all or part of their life-cycle employ a variety of metabolic strategies to cope with such environments [1,2]. One such strategy relies upon fumarate (E°′ = +30 mV) as an electron acceptor in a reversal of the succinate dehydrogenase (SDH) reaction of the citric acid cycle that comprises a fundamental component of aerobic metabolism. While ubiquinone (Coenzyme Q or Q, Fig 1, compound 1) is the electron acceptor in the SDH reaction, a quinone with a lower standard reduction potential is required to make fumarate reduction more favorable. Rhodoquinone (RQ) (Fig 1, compound 2) or menaquinone (MK) (Fig 1, compound 3) are naturally occurring compounds that meet this requirement .
To search for additional genes that may be involved in RQ biosynthesis in R. rubrum, we used a candidate gene approach. Candidate genes were deleted from R. rubrum, and then we assayed for effects on anaerobic growth and the quantity of RQ and Q in the mutant R. rubrum strains. We reasoned that genes involved in RQ biosynthesis would be expressed at a higher level during anaerobic growth, when RQ production is essential, than during aerobic growth, when RQ is not necessary. We also anticipated that such genes would be more closely related to genes in bacteria that produce RQ than to genes in bacteria that do not produce RQ. We further reasoned that since RQ contains an amino group where Q has a methoxy group, genes involved in RQ synthesis might encode proteins with transferase activities, or perhaps proteins with no known function. We chose to delete the Rru_A2553 gene since it was annotated in the NCBI genome database as a ubiquinone/menaquinone methyltransferase. We predicted that if Q levels were reduced in this mutant, a similar effect would be observed on RQ levels, since Q has been proposed to be a precursor to RQ . It is known that in Saccharomyces cerevisiae, Q biosynthesis requires a complex of at least eleven proteins , and therefore we previously hypothesized that RquA may similarly function in conjunction with other enzymes, such as an amido- or aminotransferase, as part of a multicomponent complex for RQ biosynthesis . The deletion of genes required for a biosynthetic complex would be predicted to affect the synthesis of RQ and possibly the expression of rquA.