Research On Extracytoplasmic Function Sigma Factors Involved In Thermal Stress Response In Myxococcus Xanthus DZ2

The extracytoplasmic function sigma factors?ECF-??is a simple and varied alternative ? factors,which involved in cell perception and response to environmental signals.It is a kind of basic signal transduction and transcriptional regulation mechanism commonly used by prokaryotes.Myxobacteria,known for its complex multicellular behavior and secondary metabolites,is broadly distributed in the environment and can tolerate a wide range of environmental stimulation such as nutritional,temperature,pH,osmotic pressure and oxidation.However,little is known about the molecular mechanisms underlying its ecological adaptation.A total of 35 ECF-? and 14 anti-? factors genes were respectively deleted in M.xanthus DZ2 by master Meilin Shi,according to Myxococcus xanthus DK1622 genome reference sequences.All the mutans' phenotypes about growth and development under various abiotic environmental signals stimulation were subsequently analyzed.On the basis of previous work,the following research is carried out in this paper.Two specific temperature signal response ECF-? factor MXAN₃959 and MXAN₇214,and one global signal responsECF-? factor MXAN₄147 which response to the environmental signals are used as a target to study their respective signals transduction pathways.First,mutant strains regained their ability to respond environmental signals stimulation by introduction of the full length complementary gene,indicating that the mutant phenotype was indeed caused by the deletion of the related gene.Then,the differences of gene expression under specific environmental signals stimulation were represented by RT-qPCR.Except for the expression of the global signal response ECF-? factor MXAN₄147 that decreased under the condition of Na+ stress,the expression of other ECF-? factor were all up-regulated in different ranges,which was consistent with the growth phenotype of their respective mutants.Considering that most ECF-? factors are inactive resulting from combination with their homologous anti-? factor in the absence of environmental signals stimulation.We analyzed the potential anti-? factors that could bind to the above mentioned ECF-?factor utilizing co-transcription assay and bacterial two-hybrid systems.The results showed that MXAN₃960,MXAN₇215 and MXAN₄148 might be the anti-? factors corresponding to MXAN₃959,MXAN₇214 and MXAN₄147,respectively.In order to predict their targeted genes,we determined their transcription start sites by 5'RACE after determining their respective operon,and obtained the promoter sequence of MXAN₃959 and MXAN₇214.Then use the promoter sequence as a probe to predict their target genes that may be involved in regulation.The above results preliminary clarified the signal transduction pathways of MXAN₃959 and MXAN₇214 involved in high temperature response in M.xanthus DZ2.In addition,the above mentioned ECF-?factor was also heterologous expressed and purified.Experiments about identification of the target genes regulated by ECF-? factors in vivo and in vitro is ongoing.The above results not only provide a basis for elucidating the mechanism of ECF-? factors responding to temperature regulation in M.xanthus,but also provide technical accumulation for related research.The ECF-? factors involved in M.xanthus DZ2 biological response was screened.Three ECF-? factors involved in antibiotic TA synthesis were found according to the zone of inhibition against Acinetobacter baumannii 19606 COLR and E.coli K12 MG1655.Among which,MXAN₀203 and MXAN₄148 played a positive role in the synthesis of antibiotics TA,while MXAN₆173 played a negative role.Our results laid the foundation for the strategy to analyze and improve secondary metabolites biosynthesis regulation mechanism in M.xanthus.