The Role Of Alkaline Tolerance Genes ClpP And LytS In Bacillus Thuringiensis

Bacillus thuringiensis(Bt) is worldwidely used as insecticidal microbe due to its characteristicsof high insecticidal activity, safety and environment-friendliness. Insecticidal crystal protein(ICPs) issynthesized during sporulation and accumulates in the mother cell as crystal inclusion and is releasedwith spores. B. thuringiensis can produce ICPs up to25%of dry weight of the sporulated cells.Because the alkaine condition of insect midgut impact on the interaction of insect and bacteria, evenresulted in lethal effect, therefore, research on gene expression and regulation mechanism after alkalineshock has a very important significance in Bacillus thuringiensis. We compared growth curves among B.group strains after alkaline shock. The clpP and lytS mutants were constucted by homologousrecombination, respectively and their functions were studied.In this study, we measured growth curves in the standard bacteria B. subtitle168and Bacilluscereus ATCC14579, Bacillus thuringiensis HD73,YBT-1520, HBF-18,Bt22in addition to differentconcentrations of NaOH. We found that Bt and Bc can recover growth when the pH value in themedium was8.9-9.1while that was8.2-8.4for Bs after alkaline shock. It suggests that Bt and Bc havemore alkaline tolerance than Bs.The B. thuringiensis HD73mutant with the deletion of clpP gene HD△clpP was constructed byhomologous recombination. We found that the clpP mutant grew slower than HD73in the LB mediumin addition to30mmol/L NaOH. It indicates that the ClpP plays an important role in the alkalinetolerance of Bt strain. Assay of sporulation and germintion rate showed that the deletion of clpP genehad no influence on sporulation and germination. t The mutant HD△clpP grew slower than HD73inthe LB medium with6%NaCl, which means that the ClpP plays an important role in the salt tolerance.DNA microarray data showed that a large number of genes were induced in B. thuringiensisafter alkaline shock, which include transpoter and regulator genes. Among them, expression of24genes was increased with10folds,136genes with4folds and320genes with2folds, andexpression of360genes was decreased with2folds, which were mostly related to growth. Theexpression lrgAB gene of them was increased over70folds.The B. thuringiensis HD73mutant with the deletion of lytS gene which may control lrgA gene,HD lytS, was constructed by homologous recombination. Measurement of growth indicated thedeletion of lytS gene didn’t increase the sensitivity of alkaline.We constructed expression vectors withlrgAB gene promoter-lacZ fusions. β-galactosidase assay showed that that lrgAB gene is regulated by the lytSR two-Component Signal transduction system. RealTime PCR was used to analysisexpression of lrgAB after alkaline shock. The result was similar to DNA microarray data. Thesporulation rate of HD lytS mutant was lower than that of wild strain. The analysis of autolysis andexcellular protein, indicated that lytS gene deletion decreased the autolysis of the strain, which resultedin the decreasing sporulation.