Isolation and location on the R27 map of two replicons and an incompatibility determinant specific for IncHI1 plasmids

Abstract

Two replicons were isolated independently from different IncHI1 plasmids. One was isolated from R27, and a second was isolated from pIP522. We demonstrate, by DNA-DNA hybridization experiments, that these maintenance regions are different and that they are specific to, and carried by, all IncHI1 plasmids tested. In view of this specificity we decided to designate the replicon isolated from R27 as RepHI1A and the replicon isolated from pIP522 as RepHI1B. These two autoreplicative regions are not related to a third replicon present in all IncHI1 plasmids that bears homology with RepFIA and that expresses the characteristic incompatibility of IncHI1 subgroup plasmids toward F factor (D. Saul, D. Lane, and P. L. Bergquist, Mol. Microbiol. 2:219-225, 1988; D. E. Taylor, R. W. Hedges, and P. L. Bergquist, J. Gen. Microbiol. 131:1523-1530, 1985). These results demonstrate that all IncHI1 plasmids tested contain at least three replicons. An incompatibility (Inc) region that hybridizes specifically to all the IncHI1 plasmids was previously isolated (M. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Although this Inc locus is not located in an autoreplicative region of IncHI1 plasmids, we observed that this locus stabilizes a low-copy-number replicon. This Inc locus is probably a component of an active partition locus involved in the maintenance of IncHI1 plasmids. The nucleotide sequence of the Inc region contains direct repeats of 31 bp. In addition, this incompatibility determinant hybridizes specifically with IncHI1 plasmids but expresses incompatibility toward plasmids of both IncHI subgroups (IncHI1 and IncHI2). In this communication, we present the mapping of these maintenance elements on the R27 genome.

Characterization of Multidrug-Resistant Typhoid Outbreaks in Kenya

“…Plasmids isolated from both the donor serovar Typhi, and their transconjugants were subjected to PCR to determine whether they belonged to the IncHI1 incompatibility group. The RepHI1A replicon, present in IncHI plasmids, was amplified by using PCR with the primers 5Ј-GGTCCAACCCATTGCTTTAC-3Ј and 5Ј-CACGGAAAGAA ATCACAAC-3Ј as previously reported (8,14) on a Techgene MiniCycler (Techne, Inc., Princeton, N.J.). Reaction conditions consisted of 50 ng of plasmid DNA and 100 nM concentrations of each primer in a buffer composed of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , a 200 M concentration of deoxynucleoside triphosphate mixture, and 1 U of Taq polymerase in a final volume of 100 l. Amplification conditions were 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension step of 72°C for 10 min.…”

Emergence of Multidrug-Resistant Salmonella enterica Serovar Typhi in Korea

“…The alleles for bla TEM and the class 1 integron were detected by methods reported previously (4,13). A 365-bp region of the RepHI1A replicon was amplified by PCR, which was based on previous studies (7,26), with primer HI1A-F (5Ј-GGT CCA ACC CAT TGC TTT AC-3Ј) and primer HI1A-R (5Ј-CAC GGA AAG AAA TCA CAA C-3Ј). A 285-bp oriT region was detected with primer F (5Ј-ATA TGG TAC CGG TTA TTG CTA CTT AAT GCC GA-3Ј) and primer R (5Ј-ATA TAA GCT TAT CTG ATT CTG ACA TGT GCG-3Ј) (12).…”

Molecular characterization of antibiotic resistance in clinical Salmonella typhi isolated in Ghana

“…The RepHI1A replicon, present in IncHI plasmids, was ampli¢ed via the polymerase chain reaction using the primers 5P-GGTCCAACCCATTGCTTTAC-3P and 5P-CACGGAA-AGAAATCACAAC-3P as previously reported [22] …”