Transcriptional responses and transcriptional regulators of Gluconobacter oxydans 621H

For strain development of the acetic acid bacterium Gluconobacter oxydans with regard to further industrial applications studies concerning transcriptional regulation were performed. This included genome-wide transcriptional analyses of cells cultivated on the two carbon sources mannitol and fructose, and studies with selected transcriptional regulators (TRs) of G. oxydans. Growth on mannitol is characterised by periplasmatic oxidation which leads to high growth rates and high O2 consumption rates. In contrast, growth on fructose is characterised by half the growth rate and high CO2 production. Transcriptional analyses of cells grown on fructose versus cells grown on mannitol revealed that fructose as carbon source lead to higher expression of pentose phosphate pathway genes. Growth on mannitol resulted in higher expression levels of Entner-Doudoroff pathway and tricarboxylic acid cycle genes. Additionally, 3 candidate TRs for further studies were identified. The annotation of the genome sequence in 2005 revealed the presence of 77 TRs. In this work in silico research using diverse bioinformatical tools uncovered a TR repertoire of 117 putative regulators that were assigned to 38 different regulator families. 10 TRs are encoded on 4 plasmids of G. oxydans, 7 proteins encode sigma factors and 17 genes encode response regulators of two-component systems. Out of this repertoire another 3 TRs were chosen for further analysis aiming at an identification of TRs participating in redox and energy metabolism. The decisions based on the findings of homologies, literature research and location in the genome. Transcriptome analyses of the six chosen TR deletion strains versus the reference strain were performed. 4 TRs displayed a regulon with interesting target genes, and for more detailed analysis, Fnr-like regulator GOX0974 was chosen. It was assumed that GOX0974 might have a function differing from the Escherichia coli Fnr protein, as G. oxydans is strictly aerobic and not able to use alternative electron acceptors as far as is derivable from the genome. Transcriptome analysis of G. oxydans mutant Δ0974 versus wild type displayed a regulon consisting of genes encoding Pntα1α2β transhydrogenase, two NAD-dependent oxidoreductases, cytochrome bd oxidase, thioredoxin, a sugar kinase, genes for flagellae and chemotaxis, and prophage function geness. It was shown that Pntα1α2β transhydrogenase and the sugar kinase genes were directly activated by the Fnr-like regulator, whereas cytochrome bd oxidase expression was repressed by GOX0974. Proceeded studies revealed a putative binding motif for GOX0974 which resembles the E. coli motif and the transcriptional start sites of pntA1 (transhydrogenase) and cydA (bd oxidase). Studies with reporter assays proved the oxygen-dependent activity of GOX0974, and protein studies with this TR showed the same brownish colour of E. coli Fnr and displayed the same features in the wavelength spectrum. GOX0974 was identified to be a protein with structural features of its E. coli homologue, but interestingly showing a different function in G. oxydans. It is active under oxygen-limited conditions and involved in redox and energy metabolism. Therefore, the protein was named Gluconobacter redox regulator, GoxR.