Functional Characterization Of Non-coding RNAs RsmX/Y/Z Involved In Nitrogen Fixation And Biofilm Formation In Pseudomonas Stutzeri A1501

Rsm system(Repressor of secondary metabolites)is a class of widespread secondary metabolite regulatory system in Pseudomonas.Rsm A and non-coding RNAs Rsm X/Y/Z as members of this system play important roles in carbon metabolism,motility,quorum sensing,pathogenicity and biofilm formation.The Gac S/Gac A system promotes the transcription of Rsm X/Y/Z,which sequester the small RNA-binding protein Rsm A.Pseudomonas stutzeri A1501 is an associative nitrogen fixing bacterium isolated from the rice rhizosphere.The genome analysis indicated that there are four Rsm X/Y/Z-coding genes(rsm X1,rsm X2,rsm Y,rsm Z)existed in A1501 chromosome,however,the functions of them related to nitrogen fixation and biofilm formation are unclear.In this research,the expressional characterization of Rsm X/Y/Z was studied,and the rsm X/Y/Z mutants were constructed which were used to identify the functions of Rsm X/Y/Z involved in nitrogen fixation and biofilm formation.The major results of this study were described as follows:1.Northern blot confirmed that Rsm X/Y/Z are four non-coding RNAs(Rsm X1/X2/Y/Z)(100 bp-200 bp in length),further,the transcription start sites and direction of rsm X/Y/Z were determined by 5'Race.A binding site of Gac A(TGTN12ACA)was found in the promoter regions of rsm X/Y/Z.The results of q RT-PCR showed that the transcriptional levels of rsm X1//X2/Y/Z were down-regulated in the gac A mutant indicating that the transcription of rsm X/Y/Z were affected by Gac A.2.?rsm X,?rsm Y and ?rsm Zwere constructed,and no significant differences were observed in growth of these three mutants,compared to wild type A15 01.Biolog analysis results showed that mutants of rsm X1/Y/Z improved the utilization ability of several carbon sources.Compared to the wild type,?rsm X1 improved the utilization ability of thirteen carbon sources,?rsm Y improved the utilization ability of nine carbon sources,and ?rsm Z improved the utilization ability of twelve carbon sources implying the Rsm X/Y/Z might participate in the carbon metabolism of A1501.3.The nitrogenase activities of ?rsm Y and ?rsm Z were reduced 50% and transcriptional levels of the nitrogen fixation regulator nif A and the nitrogenase coding genes nif H were down-regulated significantly in ?rsm Y and ?rsm Zdetected by q RT-PCR.This is the first discovered non-coding RNAs of Rsm system involved in the nitrogen fixation.The biofilm formation results indicated that the capacity of biofilm formation of ?rsm X1 were enhanced compared with the wild type,however,the ability of biofilm formation was decreased in the mutants of rsm Y and rsm Z,which implied that rsm X/Y/Z might be function in the biofilm formation.4.The cooperation mechanism of Rsm X/Y/Z in P.Stutzeri A1501.The q RT-PCR results indicated that,compared with A1501,the transcriptional levels ofrsm X2,rsm Y and rsm Z were increased by100-folds,18-folds and 5-folds respectively in ?rsm X;the transcriptional levels of rsm X1 and rsm Z did not show any significant change in ?rsm Y;the transcriptional level of rsm X was increased by 2-folds in ?rsm Z.We suspect that there is a cooperation among them,which can explain why there are many non-coding RNAs of Rsm system in P.stutzeri A1501.In summary,there are four non-coding RNAs of Rsm system in P.stutzeri A1501.The transcriptional levels of rsm X/Y/Z were affected by Gac A.Rsm X/Y/Z could participatein the carbon source u tilization,nitrogen fixation and biofilm formation in A1501.The expression of Rsm X/Y/Z could affect each other and formed the cooperation mechanism in P.stutzeri A1501.