Studies On PA2072,a Gene Involved In C-di-GMP Metabolism,in Pseudomonas Aeruginosa PAO1

Objective Pseudomonas aeruginosa（Pa）,a Gram-negative bacterium,is one of the most challenging pathogenic bacteria in hospital infection.cyclic di-guanosine monophosphate（c-di-GMP）is a common small molecule second messenger in bacteria,which can regulate the physiological and biochemical functions of bacteria.PAO1 contains highly complex c-di-GMP metabolism genes,among which PA2072 gene contains GGDEF/EAL domain involved in c-di-GMP metabolism,and this gene profoundly affects a series of physiological and biochemical functions of bacteria.The effects of PA2072 on bacterial phenotype,intracellular c-di-GMP content,and its physiological and biochemical roles in response to extracellular chemical signals were further analyzed by studying the role of PA2072 in det alcting environmental factors.Methods The recombinant vectors of PA2072 gene and its domains were constructed using Polymerase Chain Reaction（PCR）and gene recombination techniques.Three knockout mutant strains and complement strains of PA2072gene were obtained by gene knockout method.Through swimming,swarming,twitching and biofilm quantitative experiments,the phenotypes of the bacteria were preliminatively analyzed.The content of Pyocyanin（Pyocyanin）produced by bacterial metabolism was analyzed by photometry.The intracellular Siderophore content of bacteria was analyzed by CAS ferrophilin detection method,and the intracellular PVD（Pyoverdine）content was analyzed by fluorescence intensity.Intracellular c-di-GMP content was quantitatively determined by GFP reporter fusion method.Furthermore,Congo red plate staining was used to analyze the intracellular c-di-GMP content of bacterial strains,and 12 compounds were selected to further explore the influence of environmental stimulation on the intracellular c-di-GMP content of bacterial strains.Results1.The recombinant vector of PA2072 gene was successfully constructed by PCR and molecular cloning techniques,and verified by enzyme digestion;PA2072gene knockout mutant strain and complement strain were successfully constructed by gene knockout method and verified by PCR and sequencing.2.Quantitative results of biofilm showed that compared with the wild-type PAO1 strain,the biofilm content of the three knockout mutant strains was lower than that of the wild type at 12h culture,and the biofilm content of the three knockout mutant strains was higher than that of the wild type at 24h culture.After the complement plasmid was transferred into the knockout mutant strain,the biofilm formation of the three complement strains basically recovered to the level of the wild type after 12-48h culture compared with the wild type PAO1 strain.Compared with wild-type PAO1,the motility and swimming ability of the colonies of the three knockout mutant strains were enhanced（P<0.05）,and the motion ability of rubbing was decreased（P<0.05）,the movement levels of the three complement strains were basically restored to the wild type after the complement plasmid was transferred into the knockout mutant strains.3.The content of Pyocyanin produced by bacterial metabolism was analyzed by photometry.The results showed that the content of pyocyanin in 3 knockout mutant strains was higher than that of wild type after 24h culture（P<0.05）;After the complement plasmid was transferred into the knockout mutant strain,the content of pyocyanin in the three complement strains partially recovered to the level of wild type.Ferriophilic solid plate results of CAS showed that ferriophilic content of wild-type PAO1,3 knockout mutant strains and 3 complement strains was higher than that of the 3 knockout mutant strains after 48h culture.The ferriophilic content of ferriophilic strain PAO1 was higher than that of the 3knockout mutant strains.After the complement plasmid was transferred into the knockout mutant strain,Three replacement strains partially recovered to the level of wild type.Bacterial PVD（Pyoverdine）content was analyzed by fluorescence intensity.The results showed that the PVD content of three knockout mutant strains was lower than that of wild type（P<0.05）,the PVD content of the three complement strains basically recovered to the wild-type level after the complement plasmid was transferred into the knockout mutant strain.4.The results of GFP reporter fusion(RFU fluorescence ratio to OD₆₀₀)showed that the intracellular c-di-GMP content of the three knockout mutant strains was higher than that of the wild type（P<0.05）;After the complement plasmid was transferred into the knockout mutant strain,the content of c-di-GMP in the three complement strains basically recovered to the wild-type level,suggesting that PA2072 gene had PDE activity.Congo red plate test showed that the three knocked out mutant strains were all red,and c-di-GMP content increased.After the complement plasmid was transferred to the knocked out mutant strain,the color of the three knocked out mutant strains became lighter,the level of c-di-GMP decreased,and partially recovered to the level of wild type.It was further confirmed that PA2072 gene had PDE activity.5.12 compounds were selected to detect the intracellular c-di-GMP content of bacteria.The results showed that saccharide compounds increased the intracellular c-di-GMP content of bacteria strains（P<0.05）,high concentration of maltose decreased the intracellular c-di-GMP content（P<0.05）;Zn SO₄and（NH₄）₂SO₄decreased the content of c-di-GMP（P<0.05）;Mn SO₄increased intracellular c-di-GMP content（P<0.05）;Low concentration of Cu SO₄,Fe SO₄and Ca Cl₂increased the intracellular c-di-GMP content,while high concentration decreased the intracellular c-di-GMP content（P<0.05）.It is concluded that PA2072 gene is sensitive to environmental stimuli through the N-terminal induction domain and thus affects intracellular c-di-GMP level.Conclusion In P.aeruginosa PAO1,different domains of PA2072 had different physiological functions on the biofilm and locomotor ability of the bacteria.PA2072 gene can regulate derivatives produced by intracellular metabolism,such as regulating the content of pyocyanin,ferrophilin and intracellular water-soluble fluorescent iron carrier PVD.The results of GFP gene fusion and Congo red plate staining suggested that PA2072 protein might have PDE activity,and the phenotypic changes caused by PA2072 gene might be caused by changes in the intracellular c-di-GMP level of Pseudomonas aeruginosa.The stimulation of external environmental factors such as extracellular polysaccharides and metal ions can also regulate the intracellular c-di-GMP level through the induction domain contained in the N-terminal.Our work lays a foundation for further study of the biological function of gene PA2072.