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Mesorhizobium Tianshanense Exopolysaccharide Gene PssP Is Regulated By ActS And Required For Root-hair Adherence Of Glycyrrhiza And Biofilm Formation

Posted on:2008-10-12Degree:MasterType:Thesis
Country:ChinaCandidate:J ZhouFull Text:PDF
GTID:2120360242965450Subject:Microbiology
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Mesorhizobium tianshanense is a nitrogen-fixing bacterium which can establishsymbiotic associations with Glycyrrhiza uralensis. The formation of its nodules needsvarious carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides(EPS). In this study, seven M.tianshanense transposon insertion mutants including two pssNmutants, three pssP mutants and two exo5 mutants were isolated and found to producemuch lower EPS than the wild-type. A simple method was used to screen the regulatorygene ofpssP gene based on a promoterless lacZ in the transposon which used to screenedexopolysaccharides deficient mutants, and ActS was found to regulate thepssP positively.In order to study the expression of the exopolysaccharides gene pssP, an pssP:: lacZtranscriptional fusion was constructed by cloningpssP internal fragment into pVIK 112 andintergrated into the chromosome of a wild-type strain by homologous recombianation.Thistranscriptional fusion strain has no exopolysaccharides but itsβ-galactosidase activity isabout 200 miller units.To investigate whether actS regulated by ActR in M. tianshanense, actR in-framedeletion were constructed by overlapping PCR of flanking regions and cloned into the sacBsuicide vector pJQ200SK to select for double-crossover events.The deletion ofactR in wildtype or pssP:: lacZ fusion strain has no effect on its EPS production orβ-galactosidaseactivity.EPS deficient mutants do not affect their autoinducer production and auterinducer orroot exudates can not enhance the expression of the genes pssN, pssP, exo5.We assayed theroot-hair attachment and the biofilm formation of the mutants and wild type, the resultssuggest that EPS in M.tianshanense can enhance the root-hair attachment and biofilmformation.
Keywords/Search Tags:Mesorhizobium tianshanense, exopolysaccharides, biofilm, root-hair adherence
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