| C.botulinum is a heterogeneous, Gram-positive, anaerobic species that comprises four genetically distinct groups of bacteria. Group I includes proteolytic type A, B, F strains, Group II, non-proteolytic type B, E, F strains, Group III includes type C, D strains and Group IV includes type G strains. The four groups are located at different branches on the Clostridial phylogenetic tree, so they are usually referred to as different species.Previous research on C.botulinum is mainly about the structure and function of botulinum toxin, which is considered the most poisonous toxin in the world. Studies on the genome of C.botulinum, pathogenesis and regulatory mechanisms for gene expression seem limited.The pathogenecity of C.botulinum mainly lies in its ability to produce the most poisonous toxin, botulinum toxin. The gene coding for botulinum locates at a 15 kb toxin gene cluster in chromosome or plasmid, which harbors 6-7 genes including botulinum coding gene bont, regulator botR etc. Little was known about the evolutional source of the 15 kb toxin gene cluster, and its insertion sites at genomes, insertion inclination, mechanisms for structural diversity as well.Some studies figured out the binding sites of botR at corresponding DNA, and the relationship between BotR and RNA polymerase II, to conclude that botR is a regulator for bont expression. However, there is no complete research on the regulation of botR to bont under practical physiological condition and on the relationship between fluctuation of botR and bont relative expression.In order to further learn about the pathogenicity and regulation of expression of C.botulinum, we determined the complete genome sequence of type A and F C.botulinum strains. The structure of genomes, toxin gene clusters, virulence factors and evolutional relationship were analyzed in this study. In order to further study the effect of regulatory gene botR on the toxin gene bont, both of which locate at the toxin gene cluster (15 kb or so), the type A strain (comparatively more poisonous one), was selected in further studies on the effect of pH condition on the growth of bacteria, toxin gene expression, secretion of toxin protein and regulation of gene expression.Part1. Analysis on genome sequence of type A and F C.botulinum, and botulinum toxin gene clusterWe explored the complete genome sequence of type A and F C.botulinum and carried out bioinformatics analysis. The results show that:The genome of type A C.botulinum (str. 230611) includes a chromosome (3 885 231 bp), which was predicted to have 3772 CDSs. The genome of type F C.botulinum (str. 230613) includes a chromosome (3 993 083 bp) and a plasmid (17 531 bp), which were predicted to have 3502 and 25 CDSs, respectively.The structure of type A botulinum toxin gene cluster was highly similar to that of C.botulinum A str. 19397 from the NCBI database. The type F botulinum toxin gene cluster was most similar to that of type A2 botulinum toxin gene cluster. The alignment analysis on botulinum genome sequence and Clostridium sporogenes (genetically close species which doesn't produce botulinum toxin) genome sequence, plus analysis on GC content indicate that during the evolution, an exotic fragment including the botulinum toxin gene cluster was inserted at one site (the current site of botulinum toxin gene cluster), hence the ability of bacteria to produce botulinum toxin.Through alignment analysis on sporogenes genome sequence and type A1, A2, F C.botulinum genome sequence, we found that the botulinum toxin gene cluster of the three types of strain were inserted at three different sites in chromosomes.Through genome alignments, we located the insertion sites of botulinum toxin gene cluster from 9 different C.botulinum strains at the genome of F str. 230613, which indicated that the toxin gene clusters were inserted at one of three certain sites in the chromosome regularly instead of randomly. Type A1 and B strains incline to insert their botulinum toxin gene clusters at site 3, type A2, site 1, and type F, site 2.The comparison analysis on different type of botulinum toxin gene clusters indicated that the locations of non-toxin part of the cluster were site-specific, and the locations of toxin gene bont were comparatively variable. We found the recombination event between bontF toxin gene cluster at site 2 and bontA1 toxin gene cluster at site 3, and this recombination resulted in a novel arrangement of bontA1 toxin gene cluster, which is a mechanism for structural diversity. In this part, we analyzed the structure of different types of botulinum toxin gene cluster (15 kb or so), the evolutional source, insertion sites of different toxin gene clusters at chromosome and insertion inclination, and the recombination event between different toxin gene clusters as well.Part2. Effect of pH condition on the expression of toxin gene bontA and regulation of the regulator botR to bontAIn order to further learn the effect of regulator botR on the toxin coding gene bontA among the toxin gene cluster, type A C.botulinum (str. 230611) was cultivated under different pH condition (5.0-9.0). The growth curve, relative expression of bontA and botR, concentration of secreted toxin protein were determined during the whole growth period. The results showed:The relative expression of toxin gene bontA and regulator botR varied under different pH condition. Under pH 7.0 condition, the peaks of the two genes'expression were the highest. Under acid or alkaline condition, the expressions were lower, and under pH 9.0 condition, the expressions were the lowest.This study firstly detected the relative expression of bontA and botR under different pH condition (6.0, 7.0, 8.0, and 9.0) during the whole growth period and analyzed the relationship between fluctuation of bontA and botR expressions. The result showed that the expression of bontA was positively correlated with the expression of botR under different pH condition, with the pearson correlation coefficient between 0.7 and 1.0, which indicated that botR and bontA were co-expressed during the growth period. Our study proved that botR is a positive regulator of bontA, and the bacteria inhibited the expression of bontA through inhibiting expression of botR during acid and alkaline growth condition. Concentrations of toxin protein secreted in the cultures were detected under different pH condition, which indicated that secretion of toxin protein was lowered under pH 6.0 and 8.0 conditions in contrast to pH 7.0 condition, however, secretion of toxin protein under pH 9.0 condition was not lowered. The reason that toxin protein secretion didn't descend due to inhibition of bontA expression might be that although the transcript of bontA was inhibited under pH 9.0, the subsequent translation and transportation of protein were improved, or the stability of toxin protein under pH 9.0 condition was better.In this part, we determined the relative expression of bontA and botR under different pH condition during the whole growth period and analyzed the relationship between fluctuation of bontA and botR expressions, which further indicated the positive regulation of the regulator botR to toxin gene bontA. In a word, our genomic study facilitated our understanding on the genome structure of C.botulinum, insertion sites of botulinum toxin gene cluster and insertion inclination, and the mechanisms for structural diversity of toxin gene clusters. Study on the effect of pH condition on the expression of toxin gene and regulator further proved the positive regulation of botR to bontA in terms of correlation of expression. |
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