| The GGDEF domain stands for Gly-Gly-Asp-Glu-Phe amino acid residues, which is the conserved sequence of the active center. The majority of GGDEF domain proteins have diguanylate cyclase (DGC) activity, catalyzing the synthesis of cyclic di-GMP (c-di-GMP). Nowsdays, cyclic di-GMP, a novel second messenger. in bacteria, is known to regulate a range of functions in diverse pathogens. Xanthomonas campestris pathovar campestris (Xcc) is a model pathogen for studying molecular microbe-plant interactions. It is the cause of black rot of crucifers. Xcc8004has32genes coding GGDEF domain proteins, some of which contribute to pathogenicity, extracellular enzyme production, biofilm formation, movement ability and so on. XC0831encodes a protein containing GGDEF domain. In previous work, we have constructed the nonpolar insertional mutant of XC0831, NK0831, whose phenotypes are inconsistent with transposon Tn5gusA5insertional mutant. Pathogenicity of both was significantly decreased when compared with that of wild type. However, other phenotypes of the two mutants differed greatly such as biofilm, flocculation, motility, etc. In order to characterize of the function of XC0831, we constructed the deletion mutants DM0831, DMSP and DMGGDEF. The results showed that the deletion of XC0831in wild-type Xcc caused significant decrease in virulence and motility, as well as reduction of its growth on the MMX medium, but increase in the production of extracellular proteases and enhancement of the ability to attach to polystyrene. The deletion of signal peptide domain in XC0831caused the production of extracellular cellulose reduced. The deletion of GGDEF domain in XC0831caused the production of extracellular protease increased. We also have constructed XC0831trans-complementation strains and over-expressed strains (respectively using pLAFR6plasmid and pLAFR3plasmid). Transfering trans-complementation fragments (marked as A, C, D) that contain different structure domains into DM0831to study the function of the domains according to phenotypic analysis. When intracellular c-di-GMP levels are higher than that in wild strain, physiological and biochemical phenotypes were similar with the strains of NK0831, DM0831/pL3-0831, WT8004/pL3-0831, such as virulence, extracellular amylase, extracellular cellulose, EPS production and movement ability reduced, but extracellular protease production increased; in LB medium, the strains grew in an aggregated state, readily forming observable clumps. It was intresting that the virulence of DM0831-pL3/0831was only about5%of wild strain. The results showed that DM0831/pL6-0831could recover the phenotypes of wild-type strain except pathogenicity. Compared with DM083.1, DM0831/pL3-A could recover the motor ability while the motor ability of DM0831/pL3-C reduced. In LB medium, both DM0831/pL3-A and DM0831/pL3-C grew in an aggregated state, forming readily observable clumps. DM0831/pL3-D has no obvious change in the phenotype compared to DM0831. Transcriptome analysis DM2252and Xcc8004results found that in MMX medium XC2252played a negative regulation role of XC0831expression. To identify and analyze XC2252expression regulation relationship with XC0831, this study has constructed XC0831GUS gene expression report and respectively import to WT8004and DM2252, quantitative detection of GUS enzyme activity found GUS0831differently expressed in the two strains, which were mostly consistent with the results of the transcriptome. DM2252/DM0831double mutant also has been constructed. DM2252in the LB medium could produce flocculation while DM2252/DM0831could not. The result showed that over-expression of XC0831maybe played a major role in flocculation production of DM2252. Through identification and analysis of the XC0831functional domains and the relationship between XC0831and vemR, studying the mechanism of biofilm formation, we harbor the expection of providing more evidences to have a better explanation on the mechanism of biofilm formation and dissociation in bacteria. |
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