Preliminary Reseach Of Regulation Mechanism On Flagellar Biosynthisis In Bacillus Thuringiensis And Insertion Sequence Analysis Of Arsenite Oxidizing Strain Acidovorax Sp. NO1

This study mainly concentrated on regulation of flagellar biosynthesis in Bacillus thuringiensis and the bioinformatics analysis of insertion sequences in acidovorax, of which the achieved results would be displayed respectively.Flagella play an important role on bacterial identification and classification, pathogenicity, antigenicity, is the result of bacterial evolutionary process during the long-term adaptation. At present, the study of flagellar regulatory mechanism is common in Gram-negative pathogens of plants and animals domestic and overseas, but for Bacillus thuringiensis, flagellar expression regulation is not clear. Bacillus thuringiensis serovar. chinensis CT-43which produces insecticidal crystal proteins and thuringiensin cannot form flagella. Through bioinformatics analysis, our group has found that in CT-43genome, genes related to flagellar biosynthesis almost all exist, but the genome lacks a pair of flagellar switch genes motP-motS upstream of flagellar gene cluster and two-component genes cheY-cheA associated with the bacterial chemotaxis. Therefore, this article has studied the genes related to flagellar formation in CT-43primarily.1) Through nucleotide and amino acid sequence alignment of flagellar genes in the genomes of YBT-1520, BMB171, CT-43by BLAST, it revealed that flagellar synthesis related genes in these three strains have high homology;2) We have constructed vectors of pHTlK-PlipcheYATS and pHT1K-PlipmotPSTS. However, as CT-43contains so many endogenous plasmids that the expression vectors are failing to be transformed into CT-43cells.3) It has revealed that flagella genes which we selected in this study all could be detected corresponding transcription products by RT-PCR, such as some structural genes and genes related to the type three secretion system.4) We have used antiserum of flagellin to examine expression of flagellin in CT-43by Western blotting. Although the FliC and Hag can be detected among the extracellular proteins of CT-43, but the concentration detected is far less than B. thuringiensis serovar thuringiensis HD-2, of which the biochemical reaction is same as CT-43’s and the flagellar filament sequences of the two strains have100%similarityUnder natural conditions, flagellin FliC and Hag would be expressed in great concentration during the logarithmic phase and then secreted to the outside of cells to participate flagellar assembly. It could be guessed that the quantity of flagellin expression may be so low, or has a large degradation that could not be accumulated, which cannot meet the needs of flagellar assembly.Insertion sequences (ISs) are simple and transposable elements that widely exist in prokaryotic genomes. ISs tend to influence the expression of genes related to environmental adaptability and they are very important to genomic evolution. Acidvorax sp. NO1is an arsenite oxidizing strain which was isolated from surface soil of a gold mine. The analysis of NO1genome identified64insertion sequences including30complete ISs and some truncated elements, which distributed into15families. The study had a careful analysis of IS copies, location and insertion into functional genes. It revealed that the insertion sequences which belonged to IS3family and IS5family most widely distributed in the genome of strain NOl. We also found that there were two typical Tn3transposons which carried arsenate reduction gene cluster ars and mercury resistant gene cluster mer respectively, and the insertion sequences of IS256family in NO1were surrounding at CzcCBA efflux pump. All these results indicated that Acidvorax sp. NO1obtained some new features to adapt to the environment by insertion sequences. However, all the insertion sequences were inserted into the non-coding regions in NO1genome.Moreover, we also have done a comparison analysis of the completed Acidovorax genomes to explore the connection between ISs and strain features. Acidovorax strains have two different sources. Some were isolated from water, sludge and the other environment; however some are pathogens which can cause spot on plant leaves and fruits. Whole genome sequences and genome annotation of nine Acidovorax strains have been published in NCBI at present.1) A total of18families insertion sequences distributed into genomes of the nine strains in this study. The ISs elements in every strain have many copies (more than5copies), which suggested that the Acidovorax strains had happened recently mass transfer events in their evolutionary histories. Compared to the environmental strains which had18families ISs, there were only5families in plant pathogenic bacteria, this indicated that insertion sequences were diversity in environmental strains of Acidovrax.2) In the genomes of the environmental strains, there were ISs elements surrounding the gene clusters associated with environmental adaptabilitys such as metal resistance, however these phenomenons didn’t exist in plant pathogenic bacteria. Moreover, and unique genes of plant pathogenic bacteria such as pectinase, type III secretion system were not surrounded by insertion sequences, perhaps because these genes were ancient, and environmental strains lost these features during the course of evolution.3) The phylogenetic analysis of IS familes which widely distributed in the genomes of all Acidovorax strains was extended surveyed. It showed none one to one correlation between MLSA phylogenetic relatedness and distribution of IS families, this indicated that f ISs of the same family differentiated before the differentiation of strains. However part of the ISs from environmental strains clustered together, it showed that the evolution of the insertion sequences was related to the environment the host strains existed.Although this study was unable to overcome the transformation bottleneck and has failed to get better experimental materials for further studies, but it has proved that the absence of motPS and cheYA didn’t affect the expression the flagellar structure of CT-43. Moreover, through the analysis of the insertion sequences, it has preliminary confirmed that the insertion sequences have played a promoting role in bacteria function differentiation of Acidovorax.