Functional Characterization Of The C-di-GMP Metabolism Related Genes For The Nitrogen-fixing Pseudomonas Stutzeri

Cyclic diguanylate monophosphate?c-di-GMP?is a general cell second messenger of the bacteria,which involved in a broad spectrum of cellular processes such as in the regulation of metabolic processes,cell differentiation,signal transduction,biofilm formation,virulence factors produced and quorum sensing systems.c-di-GMP is synthesized by the diguanylatecyclases?DGCs?from two GTP molecules,and hydrolyzed by the phosphodiesterases?PDEs?.The protein GTPs that contain ubiquitous conserved GGDEF domain,and the protein PDEs contain ubiquitous conserved EAL domain.In Pseudomonas aeruginosa,SadC is a diguanylatecyclase,and BifA is a phosphodiesterase,these two encoding genes were involved in regulation of the intracellular c-di-GMP concentrations,thereby affecting the motility,biofilm formation and other phenotypes of bacteria.Pseudomonas stutzeri A1501 is an associative nitrogen-fixing bacterium,and the regulatory mechanism of the c-di-GMP is not cleared.The current study,we investigated the roles of sadC and bifA in c-di-GMP metabolic system,meanwhile we determinated the intracellular c-di-GMP concentration level in sadC and bifA mutant strains.The major finding were:1.Genomic analysis showed that the genes which encoding the protein of contain GGDEF domain have 33,and the genes which encoding the protein of contain EAL domain have 17.Wherein the gene PST₃133?named BifA?encoding protein contains a GGDEF domain and a EAL domain,which has an 74% amino acid identity with that of BifA in Pseudomonas aeruginosa.The gene PST₁148?named SadC?encoding protein contains a GGDEF domain,which has an 62% amino acid identity with that of SadC in Pseudomonas aeruginosa.In the study,the sadC and bifA mutant stains,and sadC over-expression and bifA mutant of complementary strains of A1501 were constructed by homologous recombination.2.We determined the intracellularc-di-GMP concentration level and some phenotypes of the sadC mutant.Deletion of sadC has no effect on intracellular c-di-GMP content,biofilm formation and nitrogenase activity of P.stutzeri A1501.The swimming motility of the sadC mutant strain is reduced significantly.Through the transmission electron microscope observation,it is found that there are no changes on the morphology of the flagellum in the sadC mutant strains.Than we determined the intracellularc-di-GMPconcentration level of the sadC over-expression strain,and found an increase of about140% compared with wide type A1501.Meanwhile the biofilm-forming ability also enhance more than about 2-fold.It indicating sadC involved in the c-di-GMP synthesis pathway of A1501.3.Deletion of bif A has an increase of about 2-fold on intracellular c-di-GMP content compared with wide type A1501.Measured the bifA mutant strain phenotype,the results show the biofilm-forming capacity enhanced approximately 2-fold,while the bifA mutant of complementary strain is similar to the wild type A1501.Inferred,the bifA may be a gene of degrading c-di-GMP in A1501.Deletion of the bifA gene resulted in an accumulation of intracellular c-di-GMP,thereby affecting the capability of biofilm formation.4.The real-time PCR results indicate that the higher expression of gene bifA at the initial stage of A1501.Adding different concentration?0,10,20,40?M?extracellular c-di-GMP examined the impact on biofilm formation of A1501,and found that extracellular c-di-GMP did not affect the biofilm formation,speculated that extracellular c-di-GMP was unable transport to intracellular.This study revealed that c-di-GMP as an important second messenger of Pseudomonas stutzeri A1501 involved in regulation of important physiological such as biofilm,motility.