The Analysis Of Major Metabolic Pathways In Bacillus Thuringiensis Under Alkaline Stress

Bacillus thuringiensis belongs to B. cereus group. Bt has become one of the most widely used bio-pesticides because of its specific toxicity against many types of insects. It can produce parasporal crystal during sporulation. The parasporal crystal is mainly composed of insecticidal crystal proteins(ICPs). There is an alkaline and low-oxygen environment in the midgut cavity of most Lepidoptera larvae. The alkaline condition in the midgut is very important for the interaction between Bt and insects.Therefore, it is necessary to analyze the adaptation mechanism of Bt under the alkaline condition. Here,we analyzed the gene expression profiles and primary metabolic pathways of wild-type strain HD73,using the microarray data which was obtained under the alkaline condition. We constructed the deletion mutant ldh2 and crp to explore their functions under alkaline condition.There were 1401 differential expressed genes in the microarray data, including 662 significantly up-regulated and 739 down-regulated genes. A total of 48 differential expressed genes were selected to conduct the Real Time PCR experiments. q RT-PCR result showed that most of them(31) had expression patterns in accordance with microarray data, implying the high reliability of the microarray data. The GO and COG analyses were performed for the upregulated genes in the microarray data under alkaline condition. We noticed that the activities of some primary metabolic pathways of Bt were enhanced.Up-regulated genes were mainly distributed in 21 pathways of COG database, including carbohydrate metabolism, amino acid transport and metabolism, synthesis of lipid. These phenomena were also found in the GO enrichment analysis. 19 genes in the glycolytic pathway and 12 genes in citric acid cycle were induced. In addition, many genes encoding proteins involved in the synthesis and transportation of amino acid, nucleic acid and the cell surface were significantly induced. Specially, the gene ldh2 encoding a lactate dehydrogenase 2 was significantly induced(35 fold changes) in the microarray data.Using the homologous recombination method, deletion mutant of ldh2 were obtained and the corresponding growth status was determined and compared with the wild type strain. However, we did not find any significant difference between them in LB medium with both the 24mmol/L and 30mmol/L Na OH, respectively. The expression of a transcription factor Crp, which was predicted to regulate the expression of ldh2, was 21 times more than control. The deletion mutant of Crp was also constructed. The result showed that HD(△crp) need more time to recover its growth compared withthe wild type strain under Na OH LB medium with the 28 mmol/L.At last, we selected out four genes from glycolytic pathway to analyze whether their transcription are regulated by Crp, including lactate dehydrogenase gene ldh2, 6-glucosamine phosphate aminotransferase gene glu F, transcription factor crp, phosphate mutase gene phg. The promoters of genes were connected with lac Z vector to construct corresponding recombinant plasmids. These recombinant plasmids were transmitted into B. thuringiensis strain HD73 and HD(△crp). Theβ-galactosidase assay showed that the transcription of ldh2 is regulated by Crp in the exponetial phase,but not glu F, crp, phg.