| The Arginine deiminase(ADI)pathway is widely found in bacteria,archaea,and a few lower eukaryotes,and its mainly involved in the metabolism of arginine in anaerobic and facultative anaerobic organisms.At present,no ADI pathway has been found in aerobic microorganisms such as Bacillus subtilis.It is generally believed that the physiological functions of the ADI pathway are mainly to provide energy for microbial growth under anaerobic conditions and to maintain the stability of the intracellular environment.Although the ADI pathway is widely distributed in prokaryotes,many studies have shown that the genetic composition of the operon encoding the ADI pathway and its regulatory methods are diverse.At present,the role and regulation of ADI pathway in Bacillus cereus has not been reported.The study of the ADI pathway of Bacillus cereus not only helps to understand the arginine metabolic pathway of the bacteria,but also has an important value in revealing the relationship between arginine metabolism and the environmental adaptation mechanism of bacteria.In the study,the genomes of B.cereus 0-9 were first re-sequenced,and the resulting 0-9 genomic sequences were analyzed by using biological software.It was found that there existed ADI pathways in the 0-9 genome.Genes,which use related bioinformatics methods and homologous recombination principles,knock out the gene argG associated with arginine synthesis and the gene arcA,which is commonly used in bacteria for the ADI protein that anaerobicly metabolizes arginine.At the same time,double knockout strains of arcA and argG genes were obtained.The traits of the gene-deficient strain under salt stress,growth under anaerobic conditions,biofilm formation,extracellular protease and extracellular amylase production ability,and bacterial colonization ability were compared with the 0-9 wild type strain.As a result,it was found that the deletion of the gene arcA attenuates the production of extracellular proteases,extracellular amylase,and the production of membranes from 0-9.The loss of the gene argG results in a decrease in the ability of the strain to produce a film and an increase in extracellular amylase production.The colony growth ability of 0-9 and its derivative strains under high salt and anaerobic conditions was determined.It was found that in the low salt conditions(LB medium containing 1%NaCl),several gene deletion strains and 0-9 wild type were not obvious In contrast,under high salt conditions(4%NaCl in LB medium),the growth status of several gene-deleted strains was significantly worse than that of the 0-9 wild type,indicating that deletion of the arcA and argG genes resulted in sensitivity of 0-9 to salt sensitivity.In order to further confirm this conclusion,the expression of arcA gene was determined by RT-PCR and the arcA gene was found under high salt conditions by extracting 0-9 total RNA in different periods under low salt and high salt conditions.The increase in expression level indicates that the arcA gene influences the salt stress response of 0-9.The population growth of the strains under aerobic and anaerobic conditions was determined.It was found that under aerobic conditions,the deleted strains and double knockout strains of the arcA gene and the argG gene were not significantly different from the wild-type 0-9 strain,and the abominable Under oxygen conditions,the growth status of the gene-deficient strains was significantly worse than that of the 0-9 wild type.After addition of arginine,the double knockout strains of the AarcA gene knockout strain,the arcA gene,and the argG gene were in logarithmic phase.The growth was restored,but the biomass of 0-9 was still low in the stationary phase,while the argG knockout strain almost recovered to be consistent with the 0-9 wild type,indicating that the arcA gene and the argG gene were involved in 0-9.The growth of strains under anaerobic conditions,in order to further determine the effect of gene arcA gene 0-9 anaerobic growth,extract 0-9 total RNA in different periods of normal anaerobic ventilation,using RT-PCR technology to determine the gene of arcA gene.The amount of expression was found to increase in the expression of the arcA gene under anaerobic conditions.This result further indicates that the arcA gene influences the growth of 0-9 under anaerobic conditions.At the same time,this conclusion is indicated in B.cereus 0-9,ADI pathway involved in 0-9 anaerobic.The colonization abilities of AarcA,AargG and double-knockout and 0-9 wild-type strains in the wheat root were determined.It was found that the colonization abilities of AarcA,ΔargG and AarcAΔargG in wheat were significantly lower than 0-9,indicating that the arcA gene and The argG is involved in the colonization of wheat in soil by 0-9.Using protein expression and purification and ADI enzyme activity assay,it was verified that the protein encoded by arcA gene has ADI protein activity.The phenotypes of ΔarcA and its complementary strains were determined and their complementary strains were found to be essentially complementary to each other in terms of biofilm formation ability,protease production ability,and anaerobic growth ability,further demonstrating that the expression of the arcA gene gene affects 0-9.Biofilm formation ability,extracellular protease production ability,extracellular amylase production ability,growth ability under anaerobic conditions.In this study,the effects of the gene arcA and argG initiation on the physiological function of B.cereus 0-9 were determined,and the roles of arcA and argG in B.cereus 0-9 were preliminarily understood to reveal the synthesis and metabolism of arginine.It lays a certain theoretical foundation for the role of bacterial environmental adaptation mechanisms. |
