Biological Mechanism Underlying The Biofilm Formation Of The Nitrogen-fixing Bacterium Pseudomonas Stutzei A1501

Bacterial biofilm is a kind of distinct functional structure,in that bacterial cells attaches to the surface of solid medium during its growth and thus helps to adopt with the natural environmental changes.The ability of biofilm formation is a decisive factor for developing of specific physiological function.Up to date,a larger number of reports have found that biofilm play critical roles for many processes of bacteria,such as stresses and drugs resistance host colonization,infection and pathogenicity.In bacteria,the components of biofilm are in different types,and also the environmental factors and regulatory mechanisms affecting the biofilm formation are varied.It is of great significance to study on the detailed knowledge of bacterial society behaviors and its involvement on the metabolic functions.Pseudomonas stutzeri A1501 is a nitrogen-fixing strain that can colonize rice roots.A large amount of investigations have been explored on the expression and regulation of the nitrogen fixation gene,together with the mechanism of environmental adaptation in A1501.However,it is still unclear on the biological mechanism of biofilm formation and the response mechanism of biofilm formation to the external signal.In this research,the ingredients of A1501 biofilm,the environmental factors and nutrition conditions that influence the biofilm formation were determined.And the optimum conditions of A1501 biofilm formation were defined.In the present research,the ingredients of A1501 biofilm and the environmental factors and nutrition conditions that influence biofilm formation were determined,And the optimum conditions of A1501 biofilm formation were defined.Additionally,the induction mechanism of the putative external glucose signal in the involvement of biofilm formation was explored by using qRT-PCR.The main findings are as follow:(1)To validate the main affecting factors of A1501 biofilm formation,four kinds of enzymes,Protease K,DNase?,cellulose and alginate lyase,were added to the bacterium solution both in the early stage of biofilm formation and mature period.The crystal violet staining was used to detect the effect of these enzymes against biofilm formation.It was observed that additions of four kinds of enzymes can inhibit or degrade the biofilm no matter in the early stage of biofilm formation or the mature period,implying that Protein,DNA,cellulose and alginate are important effective factors.It is speculated that they are the important components of A1501 biofilm.A standard reaction system for Pseudomonas stutzeri A1501 biofilm was established by setting altered concentrations and different incubation time treated with different enzymes.(2)We measured the variation patterns of growth and the biological mechanism of biofilm formation in A1501 under different NaCl concentrations,pH and temperatures.The results revealed that these three environmental factors have great influence on both the growth and the biofilm formation of A1501 with certain extent of regularity.The optimal growth condition for A1501 with those factors was 2 mmol/L NaCl,pH6.8,and 30 ?,respectively.However,the maximum biofilm formation ability was reached at the stress conditions of 0.6 mol/L NaCl,pH6.0,and 40 ?.When compared to the optimal growth condition,a 7.8-,7.0-or 2.0-fold increment was observed,respectively.Further,the biofilm formation of A1501 was measured when the bacteria were cultured in different carbon sources and nitrogen sources.Compared to the advantage carbon source of sodium lactate condition,the biofilm formation of A1501 was increased by 2.3-fold in the present of glucose which is the disadvantage carbon source.The influence of different concentrations of nitrogen source on the biofilm formation ability in A1501 was also investigated.It was observed that the the biofilm formation of A1501 was improved by 2 times to 6 times through limiting the concentration of nitrogen source.(3)The optimal condition of biofilm formation for A1501 with those factors was 0.6 mol/L NaCl,pH6.0,40 ?,glucose and nitrogen-limiting condition,respectively.The results revealed that these three environmental factors and two nutrition conditions have great influence on the biofilm formation of A1501 with certain extent of regularity.In a conclusion,under the tested conditions,the biofilm formation of A1501 strain showed a negative correlation with the growth,i.e.,high salt,weak acid,high temperature,the present of glucose and the nitrogen-limiting were the unfavorable conditions for the growth while it could stimulate the biofilm formation.(4)It was observed that addition of exogenous glucose could intensely induce the biofilm formation of A1501.We thus speculated that glucose might be an important signal molecular inducer for the biofilm formation in A1501.The biofilm related genes were focused and their expression patterns were further analyzed by qRT-PCR.It is found that addition of glucose could significantly induce the expression of the homologous genes in A1501 that has been previously reported to be involved in the biofilm formation in other strains.Those genes are composed of non-coding RNA rsmX1,RNA polymerase gene algU and LPS synthetic genes lpxB,kdsC,rmlD,wbpM.However,no expression changes were found on the expression of the second messenger c-di-GMP regulatory genes sadC,bifA and the cellulose synthesis genes bcsA,bcsE.Base on the results above,we hypothesized that the induction of glucose in stimulating the biofilm formation of A1501 may preform through the RsmA and AlgU regulatory systems and the LPS synthesis pathway.In a summary,this research identified the composition of A1501 biofilm,illustrated the environmental factors and nutrition conditions that influences on the biofilm formation,additionally,the mechanism of the biofilm formation of A1501 was analyzed.Screened and identified glucose as a signal molecule for A1501 biofilm formation.This work supplied a fundamental theoretical data on the biofilm formation of A1501,and may helpful for deep exploration on the molecular regulation of A1501 biofilm,and the cooperative relation between root colonization and nitrogen fixation.