Synergistic Effect On The RNA Chaperone Hfq And The Catabolitic Repression Protein Crc From Pseudomonas Stutzeri A1501 In Carbon Metabolic Pathways

In the case of microorganisms present in a variety of carbon, they choose more carbon resource which have advantages than those have disadvantages,this phenomenon known as carbon metabolism inhibition. In Pseudomonas, Crc is a global regulator of carbon metabolism inhibition which is considered to be the core protein involved in the regulation of metabolic inhibition, it can interact with the mRNA which belongs the target gene and thus play a regulatory role. In recent years, some studies show that RNA chaperone Hfq involved the interaction of Crc and molecular target mRNA, Hfq by interaction with mRNA molecules thereby regulating the stability of the Crc.Currently, two different academic perspectives remains controversial, while the study of Hfq and Crc in nitrogen-fixing has not been reported in bacteria. Pseudomonas stutzeri A1501 was isolated from the rhizosphere of rice, it is important to how to efficiently use rhizosphere complex carbon source for the bacteria rhizosphere colonization and adaptation to the environment is crucial, but the Synergistic effect on Crc and Hfq in the use of carbon source to regulate the processes which also lack of synergy molecular evidence. The research in our laboratory has been hfq mutants and crc mutants basis, by constructing hfq and crc double mutants, and analyzes the mixing carbon utilization of physiological and biochemical phenotype and preliminary study the mechanism of Hfq and Crc in the regulation of carbon metabolism, achieved the following results:Studied the function of Hfq and Crc protein in biological carbon metabolism pathway. We measured the ability of hfq and crc single mutants and double mutants in single and mixed carbon resource(mixed carbon resource which are advantage and disadvantage carbon) conditions found in the advantages of a carbon source(sodium lactate and succinate) is under the sole carbon source, mutant and wild-type growth capacity were not significantly different, but in both the non-dominant carbon sources(benzoic acid and glucose) as a sole carbon source, crc and hfq single mutants and the ability to grow significantly decreased, while the double mutant strain can not grow substantially. Advantages in carbon lactate acid mixed with carbon nondominant cultureconditions, crc and hfq single mutant ability to enhance growth, and double mutants grow weaker. These results suggest that, in the case of non-dominant presence of a carbon source under(whether single or mixed carbon source of carbon), and phenotypic changes in the level of crc and hfq double mutant strains were significantly stronger than the single mutants, suggesting that there may be both functional superimposed on.Benzoic acid metabolism studied by real-time quantitative RT-PCR analysis of the expression under(single, mixed carbon source) the presence of benzoic acid degradation of key genes, the results show that compared with wild-type and single mutant crc and hfq double mutant acid metabolism genes benA, benR, benK expression was significantly reduced. Based on these results, suggesting that Hfq and Crc play a synergistic effect in carbon metabolic pathway in A1501.To further investigate Crc and Hfq protein target mechanism of action the in carbon metabolism inhibition regulatory process in A1501 bacteria, the use of prokaryotic expression system were expressed and purified Crc and Hfq protein, and by microcalorimetry surge test(MST) detected Crc and Hfq protein target molecule BenR(gene of benzoic acid degradation regulation) mRNA in vitro interaction. The results show, Hfq can specific bind with benR gene of benzoic acid degradation regulation.The study found hfq and crc gene plays an important role in the regulation of biological nitrogen fixation, oxidative stress adaptation and chemotactic movement.Determined the nitrogenase activity of hfq and crc single mutants and double mutants showed that hfq and crc single mutants and double mutants significant effect on nitrogenase activity, including double mutants compared to wild type nitrogenase activity decreased by about 80%. Real-time quantitative RT-PCR showed the expression levels of nitrogen fixation gene nifH, nifA, nifK, glnA, glnK and ntrC down significantly of hfq and crc double mutant strains.The study also found that, oxidative stress(10 mM H2O2 treatment 8 min) of hfq and crc gene mutations significantly reduced, and real-time quantitative RT-PCR results showed that hydrogen stress enzymes which participation peroxide(catalase gene encoding katABEG, alkyl catalase gene encoding ahpFC and Superoxide dismutase sodBC) significantly reduced encoding geneexpression. We also found that hfq and crc gene double mutation lose chemotaxis. These results suggest that, in addition to participation in the regulation of carbon metabolism outside, Hfq and Crc also possibly participate in the regulation of nitrogen fixation,hydrogen stress and other physiological adaptation process, in some unknown way, it is worth further study.In summary, Pseudomonas stutzeri A1501 regulatory protein Crc and Hfq synergistic effects in inhibiting the regulation of carbon metabolism, simultaneously with Hfq and Crc protein may synergistically involved in nitrogen fixation bacteria,hydrogen stress,sports and other physiological processes in some unknown regulation.The research of carbon and nitrogen metabolism regulatory network laid an important theoretical basis to study stutzeri A1501.