Proteomic and genetic analyses of heat-resistant Rhodobacter capsulatus mutants with modified hydrogen production capacity

Hydrogen is a clean energy carrier whose importance in energy systems has been increasing, especially for the last two decades. The biological hydrogen production by using photoheterotrophic microorganisms is one of the cleanest methods of hydrogen production. To overcome efficiency losses due to overheating in outdoor photobioreactors, heat-resistant microorganisms are desirable. In this study, a combination of the comparative whole genome sequencing, comparative proteomic, and transcriptomic analyses of two previously obtained Rhodobacter capsulatus heat-resistant mutants; A52 and B41, with decreased and elevated hydrogen production capacities, respectively, have been performed to identify mutations and expression differences related to desired higher hydrogen production capacity and heat resistance. Uncovering the molecular mechanisms of resistance to high temperature besides hydrogen production by R.capsulatus will be a reference for the development of strains resistant to heat stress. Also the characterization of mechanisms affecting the production of hydrogen in R.capsulatus allowed the determination of target genes and metabolic pathways for the creation of high-efficiency hydrogen producing mutants through metabolic engineering. Genomic analyses showed a small group of mutations which was directly related to hydrogen production metabolism. In the mutant B41 genome, mutations were characterized in nifD, nifJ, glnD, nifB1, ccpA, hupD, dmsA and cbbR1 genes, on the other hand; in the A52 mutant, mutations were characterized on the nifB2, rnfF, nifJ, cbbO, anfH, amt, moeA, and hupD genes. Proteomic analyses revealed possible regulatory functions of the hypothetical proteins RCAP_rcc03215, RCAP_rcc03277, and RCAP_rcc00730 in hydrogen production metabolism of R.capsulatus. The effects of nitrogen metabolism-related mutations were tested by quantitative real-time polymerase chain reaction (qRT-PCR) on the expression levels of nifA gene (the activator of other nitrogen fixation genes), nitrogenase structural gene nifH, nitrogenase regulator ntrC gene, RubisCO structural genes cbbL, cbbS and cbbO, nitrogenase iron protein encoding gene anfH, hydrogenase maturation protease encoding hupD, molybdopterin biosynthesis protein encoding moeA, ammonium transporter gene amt, and dimethyl sulfoxide reductase, A subunit encoding dmsA genes. The most promising mutation was the one found on the glnD gene of B41 strain which is the highest hydrogen-producing strain among the heat-resistant mutants. This gene encodes PII uridylyltransferase and the effect of this mutation was confirmed by quantitative polymerase chain reaction (qRT-PCR) analyses.