| Vibrio cholerae is a gastrointestinal pathogen that causes "cholera", one of the most serious enteric diseases belonging to class A infection disease in our country. Of the more than 200 O-antigen serogroups identified, only serogroups O1 and O139 are the major causative agent of cholera. The world has experienced seven major pandemics of cholera since 1871. The 6th pandemics ended in1923 were caused by toxigenic serogroup Ol classical strains, while the 71t pandemic was caused by those belonging to El Tor biotype, a newly emerged O1 biotype that originated in 1961. In 1992, a novel serogroup,0139, emerged and caused epidemic cholera in Bangladesh and India.Chromosomal integron (CI) arrays in Vibrio spp. are generally large and display great variation. Here we report the complete sequence of the CI in the toxigenic V. cholerae 0139 strain JX20062026 for the first time. This was compared to the CI arrays from the strains O1 El Tor strain N16961,O1 El Tor hybrid strain MJ1236 and 01 classical strain 0395 available in public databases. The CI arrays in JX20062026, N16961 and MJ1236 were similar but also notable in that four indels were present in all three strains were compared. According to the CI in the reference toxigenic 0139 strain JX20062026,75 overlapping primers were designed. Then PCR scanning was used to determine the CI array variations in 83 epidemic 0139 strains. A total of nine distinct PCR CI patterns were identified in 79 toxigenic 0139 strains and these were designated Group 4_patterns 4A-41. In addition to the toxigenic strains, four V. cholerae 0139 strains identified as being nontoxigenic based on the absence of a ctxAB PCR amplicon, were also subjected to CI gene cassette profiling. For three of these strains, only eight amplicons were obtained from 225 reactions. The overall failure to recover amplicons from three nontoxigenic strains based on primers designed to the JX20062026 array suggested the presence of a large number of differences in these nontoxigenic strains, possibly resulting from cassette content or cassette order. Regardless, the failure to generate numerous amplicons from these three nontoxigenic strains suggests they are distinct lineages from toxigenic strains. The fourth nontoxigenic 0139 strain, GD20022001, based on the absence of a ctxAB PCR product, was notable in that it was indistinguishable from toxigenic strains conforming to Group 4_pattern 4A. We speculate that this strain is derived from the toxigenic lineage by loss of the ctx phage or without lysogenic infection. Subsequently the variations in all the 79 toxigenic epidemic 0139 strains were compared with that found in toxigenic epidemic O1 El Tor strains in our previous work. Few differences were observed in the cohort of toxigenic 0139 strains compared to the toxigenic 01 El Tor strains. On the basis of CI arrays, the toxigenic O1 El Tor and 0139 strains isolated concurrently in recent years appear to be more similar to each other than to the 01 strains isolated in previous decades, suggesting a closer evolutionary relationship between them. Comparison of CI arrays in toxigenic epidemic 01 El Tor and 0139 V. cholerae strains isolated between 1961 and 2009 revealed a purifying trend with lower CI complexity and smaller CI size in the CI arrays in the chronological order during the seventh pandemic.N16961 contains an integron with at least an attendant array of 178 gene cassettes. This array having been previously annotated enabled a detailed examination of the differential expression of cassette-associated genes. In order to explore the ORF expression in large CI arrays, we used N16961 as a model system. In order to select good reference genes for normalization, a set of most commonly used reference genes (thyA, recA, rpoA, gyrB,16SrRNA, and VCA0862) in Vibrio cholerae was explored in this study. The gene expression stabilities of the six reference genes were evaluated using the freely distributed Mcrosoft Excel application geNorm and normFinder. The results showed that the gene expression level of six candidate reference genes was different under different culture conditions. The gyrB turned out to be the most suitable reference gene under different culture condations. The qRT-PCR was used to detect the gene expression level in CI in N16961 under eight of physiological and environrment imitate-culturing conditions. The results have shown that the transcripts in CI array are regulated by regional control. Compared with the transcription of the ORFs in CI in N16961 under the reference condition of 37℃ pH8.0 LB, the results indicated that the ORFs’transcription changed little in the conditions of 30℃,37℃ transfer to 4℃ LB,30℃ transfer to 4℃ ASW and 0.4%bile salt media. However, most of the ORFs’transcriptions were down-regulated with significantly changes when the strain was cultured in the conditions of pH 5.5, pH 10.0 and 6%NaCl. Most of the ORFs are hypothetical protein. A small number of ORFs with known functions mainly are the metabolism, DNA repairing, mobile and extrachromosomal element. In order to find the possible cassette promoters, common PCR with primers designed associated to the different regulations were performed. Four positions of possible cassette promoters were found. In all, in response to environmental stressors, the large cassette arrays may produce diverse and complex phenotypes that are reactive to different environmental changes. This provides the important clue to the knowledge about the adaptive capabilities of the integron/gene cassette system in Vibrio cholerae. |
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