The Effect Of Glutamine Synthetase For Biofilm Formation In Escherichia Coli And Pseudomonas Putida

Biofilms are communities of bacteria embedded with extracellular matrix which consist of polysaccharide, protein and DNA when attached to biotic and abiotic surface. Majority of bacteria will inhabit in biofilm structure in nature. Bacteria of the matrix can be protected by the structure of biofilm. By forming the biofilm exchanging nutrient and metabolites of bacteria in the biofilm matrix will be more effective while horizontal gene transfer in the biofilm will benefit the evolution. Serious advantages of ecology can be provided by the lifestyle of biofilm.The formation of biofilm can be affected by several factors, such as hydrological condition, surface, nutrient, light and temperature which are environmental factors. It can also be affected by the regulation of bacteria such as quorum sensing. Compared to planktonic growth phase differential gene expression will occur during the biofilm formation, which is related to adhesion processes, quorum sensing, metabolism, stress response and protein folding factor.In the former study, compared to planktonic growth phase several genes of Pseudomonas putida have been detected to be expressed differently by 2-dimensional gel electrophoresis analysis. In this study glutamine synthetase which was one of the differentially expressed proteins was analyzed during the biofilm formation. By studying the overproduction interfering and disruption of glutamine synthetase, the effect of glutamine synthetase during the biofilm formation was analyzed.The strains P. putida and E. coli were incubated in a bioreactor system with Hutner's Minimal Medium at 30℃, medium flow rate 15mL/h, air flow rate 14-16mL/s, and the biofilms were formed on the surface of glass cover slides. The process of biofilm formation was analyzed by microscope. The inncubation time for P. putida to reach attachment phase, microcolonies formation phase and maturation were 0-60h, 60-84h, 84h while for E.coli, 0-72h, 72-96h, 96h, respectively.The full length of E.coli glutamine synthetase gene was obtained from the database. Then the gln gene was amplified by PCR. The gln gene fragment was ligated into vector pBC KS+ with the ligated product pBC KS-glnhb, which was used for the overproduction of glutamine synthetase. The expression of vector pBC KS-glnhb was carried out in E.coli. E.coli-pBC KS-glnhb was incubated in a bioreactor system to form the biofilm, by which the effect of glutamine synthetase was analyzed. In contrast with E.coli at the same inncubation time, the density and depth of the matrix were decreased and the biofilm formation was weakened in E.coli-pBC KS-glnhb.While the antisense RNA of gln gene could interfere the expression of gln gene by hybridizing with the RNA of gln, the atisense strand of gln was ligated into vector pBC KS+ with the ligated product pBC KS-glnbh.Then the vector pBC KS-glnbh was transformed into E. coli DH5a.For analyzing the effect of glutamine synthetase for biofilm formation in P. putida, the vector pBC KS- gsxb with the P. putida gln gene ligating into vector pBC KS+ and vector pBC KS- gsex with the atisense strand of gln ligating into vector pBC KS+ were constructed. The homology region of gln gene which was used for the gene disruption by homologous recombination in the future study was constructed by the fusion PCR.