Preliminary Research Of DidR, An Essential Gene For Fruiting Body Development In Myxococcus Xanthus

Myxobacteria are a group of Gram-negative bacteria which own complicated social behaviors. Myxobacterial cells crawl on solid surface when environment is full of nutrient, and when nutrient is limited they form multicellular fruiting bodies and develop into resistant myxospores. The complex social behavior of myxobacteria is associated with its complicated gene-regulation systems.The fruiting body morphogenesis of myxobacteria include cellular starvation recognitions, popular starvation recognition and contact-dependent signaling three main steps. Two component systems which act as transducer of extracellular signals play a significant function during the genesis of fruiting body.We found that the deletion of MXAN1093result in altered fruiting body morphogenesis and decreasing sporulation ability, and so we named the gene MXAN1093didR (defective in developmental regulator). As DidR is composed of REC domain, we define it to be response regulator or signal transduction protein and both the two kind of protein belong to two component system. Which position does DidR play in the signal transduction process of fruiting body morphogenesis9We answer the question by analyzing the phenotype of mutation and the organization of didR.We analyses the motility, fruiting body and myxospore formation and extracellular polysaccharide production of the knockout strain and site-directed mutagenesis strain. We found that the knockout strain AMXAN1093with normal A motility, normal S motility, increased EPS production, normal aggregation, altered fruiting body formation and decreased sporulation ability compared with wild type strain DK1622. In order to know what function does the phosphorylated DidR play, we generate a phosphorylated site-directed mutagenesis strain D128N and it is reported that the substitution of the conserved phosphorylated site aspartic acid with asparagine can be the analog of the non phosphorelylation protein. The site-directed mutagenesis strain D128N obtains a different phenotype with both wild type strain and knockout strain. It has decreased A motility, decreased S motility, decreased EPS production, decreased aggregation, altered fruiting body formation and decreased sporulation ability. Compared the phenotype of the wild type strain, the knock-out strain and the site-mutated strain, We made the conclusion that the deletion of DidR causes a loss of myxospores and the non-phosphorylated DidR suppresses the motility, the production of EPS and the aggregation of myxobacteria.The genes of prokaryote with related functions always organized in the same operon. Reverse transcription PCR is used to know whether didR and the upstream genes locate in the same operon. We found that didR is cotranscribed with the upstream genes MXAN1096-1094and the three upstream genes all relate to the synthesis and activation of liposaccharide component KDO. We further delete the gene MXAN1094and analyze the phenotype of this knockout strain. And the knockout strain△MXAN1094have normal A motility, decreased S motility but increased EPS production, impaired LPS. The phenotype of△MXAN1094reveals that liposaccharide component KDO play an important role in the social motility. As didR may also related to LPS, we make an analysis of LPS in the three strain DK1622、△AMXAN1093, D128N. And through the analysis, we found that the deletion of didR result in a different LPS pattern compared with DK1622and the site-directed mutation of didR form the same LPS pattern compared with DK1622. The result show that the LPS in the knockout strain△MXAN1093is impaired. We proposed that the different aggregation state of AMXAN1093and D128N may be related to their different LPS patterns.In order to know whether DidR can bind to DNA, we analyze whether DidR can bind to DNA by band shift experiment. We found that DidR can bind the DNA but until now we did not obtain the precise DNA.