Functional Characterization Of The GacS/GacA Two-Componentregulatory System Involved In Nitrogen Fixation And Biofilm Formation In Pseudomonas Stutzeri A1501

The response regulator Gac A and the sensor kinase Gac S are members of two-component system that is present in a wide variety of gram-negative bacteria and has been studied mainly in enteric bacteria and pseudomonads.In the case of the model bacterium Pseudomonas aeruginosa,the Gac S/Gac A system acts as a global regulator to control various physiological processes such as the synthesis of secondary metabolites,biofilm formation,ecological fitness,or tolerance to stresses.Pseudomonas stutzeri A1501,which was isolated from the rhizosphere of rice,is one of the few reported Pseudomonas strains with nitrogen-fixing ability.The bacterium is able to colonize near rice roots and form nitrogen-fixing biofilm.The A1501 genome encodes the Gac S-Gac A regulatory system,but the roles of the Gac S/Gac A system involved in biofilm formation in P.stutzeri A1501 are not clear.In this study,we studied the expression characteristics of both gac S and gac A genes,their roles in biofilm formation and nitrogen fixation,and obtained the following results:1.The expression patterns of gac S and gac A genes of Pseudomonas stutzeri under different abiotic stress and biofilm formation conditions were analyzed.The results showed that the expression levels of gac S and gac A genes were affected by environmental signals such as external temperature,nitrogen source,salt concentration and oxygen concentration.The expression levels of gac S and gac A genes were highly induced in the environment with strong biofilm formation ability.The gac A gene was highly expressed under microaerobic conditions(oxygen concentration 0.5%),which was significantly higher than that under normal culture conditions by more than 30%.Further,the function of a Fnr-type transcription factor Anr which was reported to be involved in oxygen induction regulation was studied.It was showed that the expression of anr gene specifically responded to external oxygen signal,and its mutation resulted in a decrease of nitrogenase activity by more than 80%,a decrease in biofilm formation ability of about 25%,and a significant down-regulation of nitrogen and biofilm-related genes.2.The deletion mutants of gac S and gac A genes were constructed and the effects of gene deletion on cell growth,nitrogen fixation and biofilm formation were determined.The results showed that the mutations of gac S and gac A genes did not affect the growth of cells,but the nitrogenase activity of gac A mutant decreased by more than 40%,and enzyme activity of the gac S mutant decreased by more than 30% when compared with the wild type.Real-time quantitative analysis showed that expression levels of nif gene(nif A,nif H,nif D & nif K)were significantly down-regulated in the gac A mutant strain,while the expression level of nif gene in gac S mutant strain was up-regulated.The biofilm formation of the gac A mutant was significantly reduced(up to 69%),while the biofilm formation of the gac S mutant did not change significantly.In addition,both gene mutations resulted in significant down-regulation of small RNA(rsm X1,rsm X2,rsm Y,& rsm Z)involved in biofilm synthesis and the rpo S gene involved in the general stress response.3.Transcriptome analysis of the A1501 wild-type and gac A mutants at different growth stages(log phase and stationary phase)was performed.The results showed that in the logarithmic phase,the gac A mutation resulted in a significant down-regulation of 64 genes,and 29 genes were up-regulated;in the stationary phase,246 genes were down-regulated and 211 genes were up-regulated.The main functions of these genes may be related to carbon and nitrogen metabolism,biofilm formation,exercise and chemotaxis,substance transport,and stress response.Promoter analysis shows that the promoter region of a total of 41 gene contains a conserved Gac A recognition sequence,suggesting that Gac A may directly interact with the promoter of these genes to regulate the transcription.In summary,the gac S and gac A genes of Pseudomonas stutzeri A1501 can participate in the regulation of nitrogen and biofilm formation related genes in response to environmental signals such as external oxygen and temperature.This study lays an important theoretical foundation for a comprehensive understanding of the biofilm molecular regulation of nitrogen-fixing bacteria and the coordinated regulation of biological nitrogen fixation and environmental adaptation mechanisms.