Research On The Function Of Diguanylate Cyclase Genes In Azorhizobium Caulinodans ORS571

46 Azorhizobium caulinodans ORS571,a stem-nodulating nitrogen-fixing bacterium,can form root and stem nodules in the host plant Sesbania rostrata.This bacterium strain can fix nitrogen in the symbiotic state,and also survive with nitrogen fixation in the self-generated state.As a universal second messenger in bacteria,c-di-GMP(cyclic di-guanosine monophosphate or cyclic diguanylate)plays an important role in cell signal transduction.The premise for c-di-GMP to function in physiological regulation is that diguanylate cyclase(DGC)and phosphodiesterase(PDE)can accept the transmission of signals from the induction domains such as PAS,GAF and REC,and then activate or inhibite the transcription and translation of the related genes,laeding to the modulation of c-di-GMP level in cells.In order to illustrate the regulatory roles of c-di-GMP in the ORS571 strain,a number of candidate DGC genes were identified by bioinformatics tools.Of them,five candidate DGC genes containing different induction domains were selected for the in-depth investigation,including AZC-2411,AZC-2412,AZC-2512,AZC-3566 and AZC-3914.The main contents and findings are as follows:(1)Bioinformatics analysis showed that the fivecandidate DGC proteins all have the GGDEF domain,and thus,it is speculated that they may have the c-di-GMP cyclase activity.The tri-parental conjugation method and the rhizobia knockout technique based on the cre-loxp recombination enzyme system were employed to construct single-gene knockout mutant of these five genes including ?AZC-2411,?AZC-2412,?AZC-2512,?AZC-3566 and ?AZC-3914.All these muntants were verified separately by the gene-specific PCR and electrophoresis.(2)In order to further elucidate the functions of selected DGC genes,the phenotypes of these bacterium mutant strains were examined in detail.The growth conditions were tested in liquid medium and the growth curves were established for the mutant strains.The content of c-di-GMP in the cells was determined by liquid chromatography tandem mass spectrometry(LC-MS)for each mutant.The chemotactic ability of each mutant was examined on an L3 semi-solid plate,and the size of the chemotactic circle of each mutant was recorded.Flocculation experiments were conducted under aerobic conditions to determine the flocculation amount of each mutant strain.The biofilm yield of each mutant strain was measured by crystal violet staining.The content of exopolysaccharide was determined by anthrone-sulfuric acid method for each mutant strain.The interaction ability of each mutant with the host plant was investigated by competitive nodulation experiments.The present results showed that all five mutant strains had no significant difference in the growth phenotype compared to the wild-type strain.Importantly,in comparison with the wild-type strain,two mutants ?AZC-2412 and ?AZC-2512 exhibited reduction in c-di-GMP level,flocculation amount,and extracellular polysaccharide level,but enhancement of chemotactic ability despite the rest three muntans(AZC-2411,AZC-3566 and AZC-3914)displayed no obvious difference in these phenotypes tested.Moreover,?AZC-2412 and ?AZC-2512 strains produced lower level of biofilms than the wild type under both L3-N and L3 + N culture media.The competitive nodulation experiment showed that the nodulation ability of the mutant strains ?AZC-2412 and ?AZC-2512 was significantly decreased while the rest three mutant strains had no change in this regard.The present data demonstrate that DGC genes AZC-2412 and AZC-2512 both functions like a regulator responsible for c-di-GMP level,and consequently,regulate the physiological-biochemical activities(cell motility,flocculation,chemotactic ability,secretion of extracellular polysaccharide and biofilm formation)of A.caulinodans ORS571 strain and its interaction(nitrogen-fixing nodulation)with the host plant.Such findings provide novel insights to understanding of the second messenger c-di-GMP-mediated regulation mechanism in bacteria,and also the scientific references for applications of this excellent nitrogen-fixing bacterium strain in eco-agricultural production.