Effects of homologous phosphoenolpyruvate-carbohydrate phosphotransferase system proteins on carbohydrate uptake and poly(3-Hydroxybutyrate) accumulation in Ralstonia eutropha H16.

Abstract

Seven gene loci encoding putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified in the genome of Ralstonia eutropha H16 by in silico analysis. Except the N-acetylglucosamine-specific PEP-PTS, an additional complete PEP-PTS is lacking in strain H16. Based on these findings, we generated single and multiple deletion mutants defective mainly in the PEP-PTS genes to investigate their influence on carbon source utilization, growth behavior, and poly(3-hydroxybutyrate) (PHB) accumulation. As supposed, the H16 ΔfrcACB and H16 ΔnagFEC mutants exhibited no growth when cultivated on fructose and N-acetylglucosamine, respectively. Furthermore, a transposon mutant with a ptsM-ptsH insertion site did not grow on both carbon sources. The observed phenotype was not complemented, suggesting that it results from an interaction of genes or a polar effect caused by the Tn5::mob insertion. ptsM, ptsH, and ptsI single, double, and triple mutants stored much less PHB than the wild type (about 10 to 39% [wt/wt] of cell dry weight) and caused reduced PHB production in mutants lacking the H16_A2203, H16_A0384, frcACB, or nagFEC genes. In contrast, mutant H16 ΔH16_A0384 accumulated 11.5% (wt/wt) more PHB than the wild type when grown on gluconate and suppressed partially the negative effect of the ptsMHI deletion on PHB synthesis. Based on our experimental data, we discussed whether the PEP-PTS homologous proteins in R. eutropha H16 are exclusively involved in the complex sugar transport system or whether they are also involved in cellular regulatory functions of carbon and PHB metabolism.