Expression And Regulation Of Gene Cluster Related To Sarcosine Metabolism In Bacillus Thuringiensis

Sarcosine oxidase （SOX, EC1.5.3.1） catalyzes the oxidative demethylation of sarcosine to yieldglycine, formaldehyde, and hydrogen peroxide. The sarcosine oxidase gene （sox） locus related tosarcosine metabolism in different bacteria were reported to be regulated by70factor, and their differentorganizations reveal the utilization of sarcosine in different metabolic pathways. The organization of thesox locus is similar in Bacillus cereus group and contains a54-dependent transcriptional regulator gene,suggesting that the regulation of the sox locus in the B. cereus group occurs through the54factor,which is different from the well-studied regulatory mechanisms of sox locus in other bacteria.54factorrecognized the conserved-12/-24promoter is a class of factor. The Lencoded by sigL gene is a σ54factor and plays an important role to regulate many metabolic pathways in bacteria. At present, little isknown about the metabolic pathways controlled through the54factor in B. cereus. Bacillusthuringiensis （Bt） is a species of Bacillus cereus group, and is widely-used as a insecticidalmicroorganism, which is friendly for human and environment. In this report, the expression andregulation of the sox locus in Bt HD73were studied.The bioinformatic analysis indicated that the hd73₃147-hd73₃138genes of the sox locus in BtHD73were separately designated as soxI （sodium/alanine symporter family protein）, soxH （aldehydedehydrogenase）, soxG （dihydrodipicolinate synthase）, soxF （hypothetical protein）, soxE （prolineracemase）, soxB （sarcosine oxidase beta subunit）, soxR （sigma-54-dependent transcriptional activator）,soxC （hypothetical protein）, soxD （hypothetical protein）, and soxA （sarcosine oxidase alpha subunit）according to their annotations. Conserved domain analysis showed that the SoxR protein containedthree domains, an AAA structure for interacting with the54, a helix-turn-helix for DNA binding and aPAS domain for accepting signals. These characteristics indicated that SoxR protein is a54-dependenttranscriptional activator.RT-PCR analysis revealed that this locus formed two opposite operons, soxB （soxB/E/F/G/H/I） andsoxR （soxR/C/D/A）. DNA microarray data showed that there exists the third operon soxC/D/A. Thetranscriptional start sites （TSSs） of the soxR, soxB and soxC genes were determined by the5RACEanalysis. There were two TSSs in the soxR gene, located at nucleotide positions17bp and18bpupstream from the ATG start codon respectively. The TSSs of soxB and soxC gene were located atnucleotide position28bp and29bp respectively, upstream from their start codon. Two typical-12/-2454binding sites were identified in the promoter of the soxB gene and the soxC gene respectively. Itindicates that the sox locus of Bt HD73is dependent on σ54factor.The soxR mutant was obtained through the method of homologous recombination technology. Thepromoters of the three operons, PsoxB, PsoxR and PsoxC, were fused with the lacZ gene. And thenthese recombination plasmids were transformed into HD73strain, soxR mutant and sigL mutant. The-galactosidase assay demonstrated that PsoxB and PsoxC were controlled through the54factor andactivated through the54-dependent transcriptional regulator SoxR. The SoxR regulator was found tonegatively autoregulate the PsoxR promoter. The activities of the5’ end truncated promoters of soxB and soxC genes indicated that SoxR bind to the same sequence to activate the transcription of soxB andsoxC genes.The growth curve and protein quantitation analysis showed that the soxR gene deletion did noteffect the growth and Cry1Ac protein production of B. thuringiensis. The soxB and soxI mutants wereobtained through the method of homologous recombination technology, respectively. Growth assay withsarcosine as sole nitrogen source indicated that the sox locus is responsible for the utilization ofsarcosine in B. thuringiensis. SDS-PAGE showed that the soxR gene of1662bp from Bt HD73encoding a protein with63.4kDa was expressed in E. coli BL21strain. It will be helpful for performingthe molecular regulatory mechanisms of sox locus. This study provides a foundation for further studyingthe metabolic pathways that the sox locus involved in B. thuringiensis.