Role Of A GntR-Family Transcriptional Regulator In Biofilm Formation And Environmental Tolorence Of Lysobacter Capsici Strain X2-3

Lysobacter capsici strain X2-3 was isolated from the rhizosphere of wheat,which has strong antimicrobial activity against many plant pathogenic fungi and oomycetes.This genus of bacteria has been described as showed the distinctive trait of slick colony on media,gliding motility and high G+C content.In this study,the mutant library of L.capsici X2-3 was constructed by using Tn5 transposon.The mutants showing different morphology in colony were selected and the inserted genes were cloned.Functions of the genes involved in these variation of morphology were analysised.The main results are as follows:1.Construction of mutant libraryThe mutant library of L.capsici X2-3 was successfully constructed by using Tn5 transposon and a total of 4800 mutants were obtained.Seven mutants showing changed colony morphology were selected and named from MT1 to MT7 respectively.The transposon insertion sites were identified by plasmid rescue.Among them,3 mutant were disrupted in the same gene named LC0409.The other mutants were inseted in genes of LC3417,LC3753,LC4356 and LC0306.2.Genes in MT4 and its functionIn mutant strain MT4,the insertion position was located at gene of LC4356 which was identified as a GntR family transcription factor in the genome of X2-3.The similarity of LC4356 amino acid sequence of L.capsici X2-3 and those of L.capsici strain?accession no: ALN83455?,L.enzymogenes strain?accession no: WP₀57949624?were 100%,96%.The biofilm formation ability of MT4 was significantly decreased compared to the X2-3 strain.Antimicrobial activity of MT4 anginst four pathogens was as well as X2-3,displayed similar antagonistic activities.This indicating that the change of colony morphology was not involved in the antimicrobial activity.3.Complementation analysis of the mutant strain MT4The gene of LC4356 was amplified by PCR from X2-3,and inserted between the Hind ? and BamH I sites of pBBR1MCS-5 plasmid.The resulting plasmid was used to transform the mutant MT4 strain by electroporation,and the complementated strain MCS-4356 was selected that varied in colony morphology.4.GntR family gene LC4356 regulates its downstream genesReal-time quantitative PCR?qRT-PCR?was performed to measure the transcription level of the LC4356 gene and downstream gene LC4357,LC4358 and LC4359 in strain X2-3,MT4 and MCS-4356.qRT-PCR showed that LC4356 gene and downstream genes in MT4 and MCS-4356 were both changed.5.Characteristics of mutant and wild-type strainsThe bacterial growth was measured and no significant changes in bacterial growth were detected among the X2-3,MT4 and MCS-4356 strains.The biofilm formation ability,EPS production and gliding motility of MT4 were significantly decreased compared to the X2-3 strain.6.Stress tolerance of mutant and wild-type strainsBacterial strains were test under five environmental stresses,including UV radiation,saline stress,potential of hydrogen?pH?,SDS stress and temperature stress.No significant differences in saline stress,pH and temperature stress were observed between the MT4,X2-3 and MCS-4356,but in contrast,the MT4 was more sensitive in UV radiation and SDS for difference concentration,showed significantly decreased than X2-3 and MCS-4356.These experiments revealed that MT4 was more sensitive than the wild-type strain to some stresses tested.