Studies Of C-di-GMP Metabolic Genes In Pseudomonas Aeruginosa PAO1

Subjective Metabolic genes of Cyclic di-guanylat monophosphate, the newly identified secondary messenger, were studied and analyzed, to deepen the understandings for the molecular mechanism of chronic infections, which are caused by Pseudomonas aeruginosa PAO1.Methods PAO1 wild-type bacteria and a number of transposon insertion mutant strains, related to C-di-GMP metabolic genes, which contain GGDEF or/and EAL domain, were requested and maintained in our lab. First, polymerase chain reactions were applied to confirm the individual transposon insertion positions. Afterwards, biofilm and motility analysis were applied to screen genes of biological importance. Then, a number of the catalytic domains from gene of interest were amplified and cloned into the prokaryotic expression vectors. The expression constructs were verified by sequencing. The recombinant proteins were expressed in BL-21 cells, and separated through SDS-PAGE. Catalytic activity of the recombinant proteins from gene PA0847, which shows good solubility, was analyzed through High Performance Liquid Chromatography.Results(1) Using transposon specific primers and gene-specific primers, bright clear bands are obtained in PCR reactions, thus confirming the transposon insertion inactivation of specific genes.(2) Biofilm analysis of 4 GGDEF-EAL domain containing protein shows significant differences between PA0285, PA0575, PA5442 mutants and wild type PAO1 were identified(Z0285=3.584,Z0575=3.587,Z5442=3.584;P=0.000,P < 0.05), while PA5295 mutant shows no statistical significance to PAO1(Z5295=1.195,P=0.232,P > 0.05).(3)Similarly, swimming motility analysis demonstrates that PA0285, PA0575, PA5442 mutants differs significantly to wild type PAO1(Z0285=3.645,Z0575=3.662,Z5442=3.610;P=0.000, P < 0.05), while PA5295 does not(Z5295=1.735,P=0.083,P>0.05).(4)C-di-GMP metabolic genes from PAO1, in addition to those characterized and reported in literature, were classified into three groups: containing GGDEF domains only; containing EAL domains only; containing both GGDEF and EAL domains. Many catalytic domains were cloned into p ET-32 a(+)vector. The expression constructs were verified, and sequenced. The recombinant proteins were expressed in BL-21 cells, and analyzed through SDS-PAGE.(5)DGC activity of GGDEF domain from PA0847, were detected through High Performance Liquid Chromatography analysis.Conclusion Researchers have demonstrated GGDEF/EAL domains are involved in bacterial second messenger C-di-GMP metabolism, which has profound effects on various physiological processes of bacteria. To further study these genes, phenotypic and biochemical analysis were carried out systematically. A number of recombinant proteins containing GGDEF、EAL or GGDEF+EAL domains were expressed in vitro. Finally, we demonstrated experimentally the DGC activity of PA0847 from Pseudomonas aeruginosa PAO1.