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

Lysine2,3-aminomutase （KAM, EC5.4.3.2.） catalyzes the interconversion of L-lysine and L-β-lysine. It was reported that the kam locus related to lysine degradation was under the genetic control ofSigmaEin B. subtilis. We found that the kam locus were very conserved in B. cereus groups, and thepromoter region of kamA gene in these strains were found to contain conservative recognition sequencefor Sigma54, which suggested that regulation mechanism of the kamA gene in B. cereus group may bedifferent from that in some other strains. Sigma54is quite distinct both structurally and functionally fromthe Sigma70family, it directs its RNAP to promoters characterized by conserved sequences located at-24and-12bp upstream from the transcriptional start site. Sigma54is involved in the regulation of manymetabolic pathways, but little is known about the metabolic pathways controlled by Sigma54in B.cereus.Bacillus thuringiensis belongs to the family of B. cereus group, is the most widely used microbialpesticides in the world. Eight54-dependent transcriptional activators were found in Bacillusthuringiensis subsp. kurstaki strain HD73according to our study. The eight54-dependenttranscriptional activators may be involved in different metabolic pathways, including lysine degradationpathway. Studies about this pathway make it possible to analyze the forming mechanism of spores andcrystals from the perspective of metabolic regulation, and improve the production of spores and crystalsin Bacillus thuringiensis through the effective use of carbon and nitrogen.The structure of the gene cluster was analyzed. Nine open reading frames （ORFs） are localized atthe kam locus （8571bp） of B. thuringiensis HD73, and they are respectively annotated asaminotransferase （yodT, HD73₂534）,3-keto-6-acetamidohexanoate cleavage enzyme （yodS, yodR,HD73₂535-HD73₂536）, deacetylase （yodQ, HD73₂537）, beta-lysine acetyltransferase （yodP,HD73₂538）, L-lysine aminomutase regulator （kamR, HD73₂539）, lysine2,3-aminomutase （kamA,HD73₂540） and hypothetical protein （yokU-yozE, HD73₂541-HD73₂542）. Conserved domainanalysis showed that the KamR protein consists of three typical domains: a central AAA+domain thatinteracts with Sigma54, a helix-turn-helix DNA-binding domain, and a PAS-sensing domain. Itsuggested that the KamR protein is a Sigma54-dependent transcriptional activator.Transcriptional units of the kam locus and the transcriptional start sites （TSSs） of the kamA genewere determined. The results indicated that kamR and5upstream genes （yodT-yodS-yodR-yodQ-yodP）formed a transcriptional unit, while kamA, yokU and yozE formed a transcriptional unit.5RACEanalysis revealed that the transcriptional start sites （TSSs） was located in the kamA gene at27bpupstream from the ATG start codon in HD（sigK） mutant, while at143bp upstream from the ATG startcodon in HD（sigL） mutant. They were consistent with the Sigma54and SigmaKputative binding sites,respectively.The transcriptional mechanism of the kam locus was clarified. The kamR and kamA mutants wereprepared by homologous recombination to examine the role of the kam locus. yodT and kamA promoter fusions with lacZ were constructed to analyze the transcription activity of PyodTand PkamA.Therecombinant plasmids were introduced into B. thuringiensis strain HD73, HD（kamR）, HD（sigE）,HD（sigL）, HD（sigH）, and HD（sigK）. The β-galactosidase assay showed that PkamAis controlled bySigmaHand SigmaL, and regulated by the KamR protein, while controlled by SigmaKand regulated bythe GerE protein in the late stage of sporulation. And PyodTis controlled by SigmaH. The results of ourelectrophoretic mobility shift assay showed that SigK and GerE could bind to the kamA promoter.The growth curve and protein quantitation analysis showed that the deletion of kamR and kamA-yokU-yozE effect neither the growth nor Cry1Ac protein production in Bt HD73. However, it wassurprising that the sporulation rate of HD（kamR） was slightly decreased compared to that of HD73. Itmeans that KamR maybe control some genes controlled by Sigma54to affect on spore formation.