The Role Of ClpX/Hsp100 Protein Family Components In Bacillus Cereus

Protein quality contol system is a conserved protein degradation mechanism existed in both prokaryotic and eukaryotic cells which participates in protein unfolding and remodeling associated with many cellular functions including DNA replication,tolerance to heat stress,and control of gene expression.Bacillus cereus 0-9,an endophytic bacteria isolated from healthy wheat root in our laborary previously,has biological control potentials against several soil-borne diseases such as sharp eyespot and root rot of wheat.This strain could produce biofilm and endospore,have strong swimming motility and colonization ability.In recent years,several reports were shown that protein quality control system existed in model microorganisms such as Escherichia coli and Bacillus subtilis.there was no report about protein quality control system existed in B.cereus.the aim of this work was to find existence of protein quality control system in B.cereus and to analysize physiological functions of protein quality control system compontents in B.cereus 0-9.Bioinformatics methods were used to analyze the existence of Clpx/Hsp 100 protein families in B.cereus 0-9 genome.The result showed total of 11 genes coded putative protein quality control system components existed in genome.Eleven gene deletion mutants,included 0-9(?clpB),0-9(?clpC),0-9(?clpPl),0-9(?clpP2),0-9(?ftsH),0-9(?clpX),0-9(?tepA),0-9(?lonN),0-9(?lonB),0-9(?hsIV)and 0-9(?hsIU),were constructed after the procedures of knockout vector construction,transformation and homologous double crossover.Phenotypes including growth,endospore formation,swimming motility,biofilm formation,tolerance to oxidation and colony morphology among 11 gene deletion mutants and its wild type B.cereus were investigated.The results revealed that deletion of clpx and ftsH leaded to several phenotype changes.Compared with the wild type 0-9,the clpX and ftsH gene deletion mutants display reduction in motility and biofilm formation ability,lack of endospore formation and sensitive to high osmotic stress or oxidizing agent treatment.The clpX and ftsH mutants also displayed less antifungal substances production and filamentary cell phenotype respectively compared to the wild type.Other nine gene deletion display some phenotype changes such as reduction in extracellular enzymes synthesis,motility and tolerance to high temperature insults.The observation above suggested that components of protein quality control system affected many cellular processes in B.cereus.To analyse gene functions of clpX and ftsH in B.cereus,coding sequence of clpX andy ftsH with its native promoter respectively were PCR amplified from wild type genomic DNA and then inserted into shuttle plasmid pAD123-1-pgal to construct complementation vectors of the two genes.Trans-complementation of mutant strains were done by transforming the complementation vectors of clpX and ftsH into mutants of ?clpX and ?ftsH to acquired complementation strains of 0-9(?clpX::clpX)and 0-9(?ftsH::ftsH).Phenotypes of B.cereus wild type and its derivative gene deletion mutants and complementation strains of the two genes were determined.The results showed altered phenotypes of mutants could restore after gene complementation.This data demonstrated both clpX and ftsH involved in endospore formation,biofilm production,motility and stress tolerance to high osmotic.conditions.The data also indicated clpX and ftsH played individual role in antifungal substance production and cell division.To further assess the role of ClpX in maintain cell protein homeostasis,gene transcription of clpX in B.cereus was determined with qPCR under several stress conditions.The results showed clpX expression increased under acid and high salt concentration compared to normal growth condition.Fusion protein expression vector with GFP and protein degradation tag ssrA(GFP-ssrA)were constructed.The vector was transformed into B.cereus wild type and clpX mutant respectively.Fluorescence intensities of GFP-ssrA were measured so as to evaluate the effects of ClpX on protein degradation in bacterial cell.The results showed fluorescence intensity of GFP-ssrA in clpX mutants was lower than that in wild type significantly,which indicated ClpX participated in abnormal protein degradation in B.cereus.