Functional Characterization And Regulation Of The RNA Chaperone Hfq In The Nitrogen-fixing Pseudomonas Stutzeri A1501

RNA chaperone Hfq which encoded by hfq gene is involved in many intracellular metabolic processes by promoting the interaction between protein or non-coding RNA and target m RNA, regulating the stability of m RNAs. Previous studies indicated that Hfq could affect nitrogen fixation efficiency and the interaction between nitrogen-fixing microbes and plants. Pseudomonas stutzeri A1501 has a Hfq homologous protein, a hfq mutant was constructed and used to detect physiological and biochemical traits, express and purify the protein and the electrophoresis mobility shift assay. The major findings from this study were:1. P. stutzeri A1501 has a highly conserved Hfq homologous protein. A hfq mutant was constructed in order to study its function. The hfq mutant had no influence on the growth of P. stutzeri A1501 in LB and Medium K. However, the mutant showed a decrease resistance to oxidative stress(exposure to 18 mmol/L H2O2 for 10 min) and osmotic stress(exposure to 0.3 mol/L sorbitol for 1 h). Real-time RT-PCR revealed significant decreased expression of the catalase encoding genes kat A/B/E/G, alkyl hydroperoxide reductase encoding genes ahp F/C and glutathione peroxidase encoding genes. The phenotype was similar to that of most bacteria. In addition, the hfq mutant showed an enhanced biofilm formation and decreased denitrification ability with nitrite as electron acceptor. The expression of nir B, nir S and nos R which encode key cytochrome C-552, cytochrome cd1 nitrite reductase precursor and regulatory protein Nos R were reduced 80%, 80% and 50%, respectively. The above results showed that the Hfq involved in various physiological processes in P. stutzeri A1501.2. We measured the nitrogenase activity of these strains in order to analysis the function of Hfq in nitrogen regulation. The result showed that the nitrogenase activity of the hfq mutant was reduced to 30% compared to the wild-type. Real-time RT-PCR revealed that the expression of nif A, nif K, gln A, gln K and ntr C which involved in regulation of nitrogen metabolism and the RNA polymerase σ54 factor rpo N were reduced in hfq mutant. Furthermore, the Western blot revealed the expression of Nif D and Nif K were obviously reduced. These results indicated that Hfq played an important regulatory function in the nitrogen fixation of P. stutzeri A1501.3. Rpo S( σS or σ38), a global regulatory protein of the general stress response in bacterial species, regulated the expression of various stress related genes in stationary phase. Studies have reported the Rpo S regulated the expression of hfq. We found a Rpo S conservative binding site overlapping the promoter region of hfq: TCTATTCTCA. The loss of rpo S also reduced the expression of hfq. It is possible that hfq is regulated by the Rpo S. Electrophoresis mobility shift assay(EMSA) showed that the promoter region of hfq could specific bind to Rpo S.In conclusion, P. stutzeri A1501 has a highly conserved Hfq homologous protein, and the expression of hfq is Rpo S dependent. Hfq involved in large amount of physiological processes by phenotypic characterization in P. stutzeri A1501, such as the resistance of oxidative stress and osmotic stress, the ability of denitrification and energy metabolism. It regulated the nitrogen fixation in an indirect or direct way. These work laid a theoretical foundation for the metabolic regulation network in P. stutzeri A1501.