Screening Of Proteins Interacting With CwlC And Functional Analysis Of YhcZ In Bacillus Thuringiensis

Bacillus thuringiensis?Bt?,one kind of gram-positive bacterium,can produce insecticidal crystal proteins?ICPs?during sporulation,ICPs show specific insecticidal activity to many insects,including Lepidoptera,Coleoptera,Diptera,and so on.Bt has been made into the microbial insecticide applied widely in agriculture with the largest yield due to its broad insecticidal spectrum and environmental friendliness.However,one barrier hindering Bt application in the field is the short duration caused by inactivation of crystal proteins under ultraviolet radiation.It had been shown that Bt with crystal proteins encapsulated with cell wall could effectively prolong the lasting period of pest control under UV.CwlC was determined to be an essential peptidoglycan hydrolase involved in mother cell lysis in our previous studies.Deletion of cwlC completely blocked mother cell lysis without impacting the sporulation frequency,crystal proteins production,as well as insecticidal activity.Based on that this work aimed to study the degradation mechanism of cell wall by secretory CwlC and the regulation mechanism of cwlC gene.The main results are summarized as follows:20 functional genes were screened out by "pull-down" method and mass spectrometry analysis.The proteins encoded by these genes were predicted to interact with the cell wall hydrolase CwlC.Through bioinformatic analysis,pbp?gene number HD73₂479?,which encodes penicillin binding protein,and groEL?gene number HD73₀246?,which encodes chaperone GroEL protein,were selected as the candidate genes for further investigation.PBP and GroEL proteins were purified from Escherichia coli through the optimized heterologous expression system By use of pull-down,the interaction between CwlC and PBP was further verified in vitro.However,the interaction between CwlC and GroEL did not exist.Our above results identified an interacting protein of CwlC through protein-protein interaction.This discovery not only laid the foundation for in-depth study on the secretory pathway of CwlC and further reveal of Bt mother cell lysis mechanism,but also provided a scientific basis for solving the arising practical problems during Bt production and hence initiated a new approach on genetic improvement in developing new Bt pesticides.The transcription factors that regulate cwlC transcription were predicted preliminarily through DBTBS?http://dbtbs.hgc.jp/?.HD73₅824 gene,expressed at the late growth stage,was chosen for functional study based on the gene expression level by RNA-seq data analysis.Through bioinformatics analysis,HD73₅824 gene was defined as yhcZ gene and HD73₅825 gene as yhcY gene.These two genes was speculated to constitute a two-component system.yhcZ gene deletion mutant was constructed by use of homologous recombination.?-galactosidase activity showed that yhcZ did not regulate cwlC expression.Meanwhile,the deletion of the yhcZ gene did not affect mother cell lysis through microscopic analysis.The growth curve showed that AyhcZ was defective significantly in growth.Carbon source metabolism experiments suggested that yhcZ negatively regulated glucose utilization.And Biolog analysis proved that yhcZ in Bt strain also affected other carbon sources utilization.The above results demonstrated that yhcZ remarkably promotes glucose and other carbon sources utilization by Bt strain during growth.Our findings will not only lay a foundation for analyzing the regulatory mechanisms of glucose and carbon sources utilization by yhcZ,but also provide a reference for further research on bacterial growth and fermentation.