To further confirm that single point mutation only causing Sepantronium in vitro non-synonymous mutation was truly involved in the serotype shift, we performed the opposite experiment, i. e. induction of Inaba serotype in Ogawa strains. We created the T472C substitution on the chromosomal rfbT gene of Ogawa strain 7743 through homologous recombination. As expected, this substitution caused serotype shift from Ogawa to Inaba in strain 7743. Subsequent introducing the recombinant plasmid pBR-rfbT carrying intact the rfbT gene induced the seroconversion from Inaba to Ogawa phenotype. Taken together, our study experimentally demonstrated T472C substitution is truly involved in the serotype shift. Discussion In this study,

we presented the descriptive data regarding cholera serotype-cycling in China over a 48-year (1961–2008) period, Selleck Linsitinib and also noted the multiplicity of rfbT sequence variations in V. cholerae O1 isolates. Three single nucleotide substitutions and deletion mutations of rfbT have been reported which caused serotype switching due to a frameshift or crucial amino acid residue change in RfbT [22, 41, 42]. In our study much more mutations are found in the Inaba

serotype strains, including single amino acid residue substitutions, frameshifts caused by single nucleotide and short fragment insertions/deletions, and transposition events. These mutations occurred randomly over the entire open selleck chemicals reading frame of rfbT, which may suggest the mutations occurred frequently and differently under pressures from environment and human immunity, as well as spontaneous mutation. With the complementation of the intact rfbT gene, these Inaba strains were

converted to the Ogawa serotype, which validate the mutations on the Inaba serotype conversion. Our study provides the first evidence that mobile genetic elements, including the transposase OrfAB and ISVch5 transposase, are involved in inactivating the rfbT of V. cholerae, thus contributing to nearly the serotype interconversion. The insertion of the two kinds of transposases both led to duplication of the inserted sequence. Although there is difference in terms of the insertion position and orientation, the target sequence (AAAC) of the transposase OrfAB elements in different strains was the same. We further surveyed the distribution of transposase OrfAB copies in several strains which genome sequences are available. The copy number and the distribution of transposase OrfAB on chromosomes I and/or II vary in strains from different regions and years. Strain N16961 contains six copies, each chromosome harbors three copies. In IEC224, in addition to the three copies on each chromosome, there is an additional transposase OrfAB subunit B on chromosome I. In strain MJ-1236, all four copies are located on chromosome II. All these and our data suggest that the transposase OrfAB is quite active in transposition in V.