Study On The Integration Of PMT1 Plasmid Into Chromosome In Two Yersinia Pestis Strains

Objective Genome sequencing and bioinformatics analysis were performed on two Yersinia pestis strains whose pMT1 plasmids were not detected by plasmid electrop Horesis but whose F1 antigens were positive,to determine whether their pMT1 plasmids were integrated into chromosomes,their integration positions and possible integration mechanisms,and whether such integration had an impact on F1 antigen expression and biochemical characteristics.Methods The whole genome DNA of O-1 and ST strain were extracted and sequenced to obtain the whole genome sequence and circle map.The general characteristics of the genome were described by comparison with the reference strains;The integration sites were identified by sequence alignment(Blast / Mauve),and then verified by primer design and PCR amplification.Results The genome of the O-1 strain consist of a single circular chromosome(4.79Mb)and two plasmids(70.3Kb,9.6Kb).The number of chromosomal genes of O-1 strain was different from that of CO92 strain,but more than that of CO92 strain.The insertion sequence of chromosome(especially IS100 and IS1541)was more than that of CO92strain;The plasmid of O-1 strain were similar to CO92 strain(p CD1 and p PCP1).However,there was no independent pMT1 plasmid in O-1 strain.The pMT1 plasmid sequence was located in the chromosome of the O-1 strain,ranging from1429315 to 1526240,with a length of 96926 bp.The integration site is confirmed by PCR amplification.It was similar to the pMT1 plasmid characteristics of the CO92 strain and contained five IS sequences:2 IS100,2 IS1541 A,1 IS285.The insertion site of the pMT1 sequence is in the non-coding region of the O-1 chromosome.The genome of the ST strain consist of a single circular chromosome(4.67Mb)and two plasmids(70.3Kb,9.6K b).The number of chromosomal genes in ST strain was similar to that of CO92 strain.The insertion sequence of chromosome(especially IS1541)was more than that of CO92 strain;The plasmids of ST strain were similar to CO92strain(p CD1 and p PCP1).However,there was no independent pMT1 plasmid in ST strain.The pMT1 plasmid sequence was located in the chromosome of the ST strain,ranging from 960704 to 1058866,with a length of 98163 bp.The integration site is confirmed by PCR amplification.It was similar to the pMT1 plasmid characteristics of the CO92 strain and contained five IS sequences:3 IS100,1 IS1541 A,1 IS285.Overall comparison of the number of chromosome insertion sequences of 7 strains showed no statistically significant difference(2=13.227,P=0.778>0.05).The mean positive titer of F1 antigen test of the four strains was significantly different by one-way ANOVA(F=6.698,P<0.05).Pantwise comparison results showed that the mean positive titer values of ST,O-1,1409 and EV76 strains were statistically significant(P<0.05),while the mean positive titer values of other strains were not statistically significant(P=1.000).Biochemical identification results showed that O-1 and ST strain could decompose maltose and arabinose,without decomposing glycerol,molasses and rhamnose,and could reduce nitrate to nitrite.Nitric acid could be formed by oxidation process.Methyl red test was positive,VP test was negative,H2 S test was positive,and One of the virulence factors,P factor test was negative;pHenylalanine dependence.Conclusions This study determined that the pMT1 plasmid of the O-1 and ST strains integrated into the chromosome and was a complete sequence integration.The chromosomal characteristics of O-1 and ST strains were different from CO92 strain,and the insertion sequence was more.IS1541 A and IS100-mediated homologous recombination may be responsible for the integration of the pMT1 plasmid into the chromosomes.And integration has little effect on F1 antigen expression and biochemical properties.