Regulatory Networks Underlying Nitrogen-Fixing Biofilm Formation In Pseudomonas Stutzeri A1501

In the natural environment,bacteria often adapt to the environment by forming biofilm.The regulatory system of biofilm formation is very complicated,usually transcriptional and post-transcriptional regulations are the main ways.Lots of research indicate that the exopolysaccharides as the main biofilm matrix componenet has a great impact on the biofilm formation.In addition,the second messenger cyclic-dimeric-guanosine monophosphate?c-di-GMP?has been shown to regulate the exopolysaccharides production.In Pseudomonas,RNA binding protein RsmA binds with the mRNAs of genes related with the exopolysaccharides production and c-di-GMP synthesis and represses the translation of them.Non-coding RNA RsmX,RsmY and RsmZ could repress the activity of RsmA by binding with it.Furthermore,the sigma factors RpoN and RpoS have been reported to be involved in the biofilm formation,the clear mechanism of regulation has not yet been studied.In this thesis,rice root-associated azotobacter Pseudomonas stutzeri A1501 was chosen to study the regulation system of nitoren-fixing biofilm.The main purpose of research and their respective results are as follows:1.The impacts of carbon and nitrogen on the biofilm formation were investigated.Under sufficient carbon and limited nitrogen conditions,A1501 could form biofilm and fix nitrogen at the highest level.When the concentration of NH₄⁺increased up to 0.5 mM,the nitrogenase activity was repressed completely for the planktonic cells,however,biofilm cells still had 70%nitrogenase activity,in addition,the nif genes were expressed at high level when A1501 formed biofilm.In summary,the biofilm formation could enhance the nitrogenase genes expression when nitrogen fixation was under NH₄⁺repression.2.RsmA negatively regulated the biofilm formation through post-transcriptional repressing the expression of exopolysaccharides synthesis regulator algU and c-di-GMP synthetase sadC.The protein level of AlgU and SadC increased due to the RsmA mutation.C-di-GMP level and exopolysaccharides production increased significantly because of the up-regulated expression of AlgU and SadC,as a result,the biofilm formation was enhanced in RsmA mutant.Gel Mobility-Shift?EMSA?assay indicated RsmA protein could specifically bind algU and sadC mRNA,this interaction could repress the gene expression level.3.EMSA assay indicated ncRNA RsmX,RsmY and RsmZ could specifically bind RsmA protein,in that case,RsmX,RsmY and RsmZ could relieve the repression of the RsmA on biofilm formation.RsmY and RsmZ mutants formed less biofilm and RsmY and RsmZ overexpression increased biofilm formation.The expression of RsmX,RsmY and RsmZ were down-regulated in the transcriptional regulator GacA mutant,implying that promoters of them were GacA-dependent.RpoS mutation enhanced the biofilm formation and RsmZ level was up-regulated in RpoS mutant,indicating that the RpoS repressed biofilm formation in an unclear way.4.RpoN mutation caused the loss of ability to form biofilm and fix nitrogen,indicating that RpoN played an important role in nitrogen-fixing biofilm formation.The exopolysaccharides concentration of glycosylphosphotransferase PslA mutant reduced upto 34%compared with wild type A1501.The PslA mutant could not form biofilm indicating that exopolysaccharide has an essential role on biofilm formation.The nitrogen fixation regulator nifA and pslA reduced their expression by more than 50 times and 10 times in RpoN mutant.As shown in EMSA assay,RpoN protein could bind promoters of nifA and pslA,in addition,RpoN binding site(GGN₁₀GC)were found in the promoter region of them.RpoN transcriptional regulated the nitrogen-fixing biofilm through activating the expression of pslA and nifA directly.5.The regulation system model of nitrogen-fixing biofilm formation was presented.At transcriptional level,RpoN could respond to nitrogen starvation,enhance exopolysaccharides production and biofilm formation by activating the transcription of pslA,at the same time,nitrogenase activity of the biofilm was positively regulated by RpoN through increasing the expression of nifA.At post-transcriptional level,GacA activated the transcription of ncRNAs RsmX,RsmY and RsmZ that positively regulated biofilm formation.RsmX,RsmY and RsmZ could sequester the RsmA protein from its targets mRNAs AlgU and SadC and relieved the repression of RsmA on biofilm formation.