Identification of DNA homologies among H incompatibility group plasmids by restriction enzyme digestion and Southern transfer hybridization

Abstract

Plasmids belonging to the three HI plasmid incompatibility subgroups were characterized by the use of restriction enzymes and Southern transfer hybridization. A diversity of restriction enzyme patterns was noted among the HI subgroups, and a small amount of DNA homology was observed by probing these digests with a nick-translated HI1 plasmid. Within a single subgroup (HI1 and HI2), similar restriction enzyme patterns were noted. Plasmids of all three HI subgroups and the HII group had a guanine plus cytosine content of 49 to 50 mol%. The IncHII plasmid pHH1508a also showed some homology with the HIt probe. The DNA homology observed is probably responsible for common phenotypic properties encoded by these plasmids.Plasmids have been conveniently classified by incompatibility, the inability of related plasmids to stably coexist within the same host cell. In general, plasmids within a given incompatibility group show a high degree of DNA homology. Plasmids belonging to the H incompatibility group, however, do not necessarily demonstrate this property as determined by DNA-DNA hybridization studies with total plasmid DNA (5, 11). Based on these studies, the H plasmid group has been divided into three subgroups designated Hi, H2, and H3 such that members within a given subgroup show a high degree of DNA homology with one another, but not with members of the other two subgroups. The H3 subgroup to date comprises only a single member, MIP233 (11).Despite the lack of homology between members of the H plasmid group, they do share several common features. They are all large (greater than 100 megadaltons [Mdal]), have a thermosensitive mode of transfer (15), and determine morphologically and serologically related pili (2). These plasmids have played an important role in mediating antibiotic resistance in medically important pathogens (17,18,20 In the present study we investigated the molecular relatedness of these H plasmid subgroups by the use of restriction enzyme analysis and Southern transfer-hybridization techniques. Representative members of each subgroup were isolated, and their buoyant density and corresponding guanine plus cytosine (G+C) contents were determined. They were then digested with restriction enzymes. Although a diversity of fragment patterns was generated, trends were noted in the frequency of cutting by particular restriction enzymes.

Restriction endonuclease mapping of the HI2 incompatibility group plasmid R478

“…On the basis of DNA-DNA hybridization studies, the IncHI group of plasmids is further divided into three subgroups, IncHIl, IncHI2, and IncHI3 (42). Subgroups IncHIl and IncHI2 both have many plasmids, but there is only one plasmid, MIP233, in the IncHI3 subgroup (27).…”

Restriction endonuclease characterization of resistant plasmids in Enterobacteriaceae isolated from children in the Sudan

“…However, the exchange of DNA within plasmids, both by acquisition and loss of transposons, and by gene mutation and deletions (24) results in plasmids of the same incompatibility group having diverse total DNA. Various studies have demonstrated DNA variability within incompatibility groups by restriction endonuclease digests and DNA hybridization (25)(26)(27)(28). While plasmid incompatibility provides evidence for a common origin, a high degree of DNA homology demonstrated by identical or similar digest patterns is a useful indicator of current identity.…”

Genetic and nucleotide sequence analysis of the gene htdA, which regulates conjugal transfer of IncHI plasmids

“…8). (42). The htdA determinant is shown in this study to encode a protein which is involved in the regulation of conjugative transfer of these plasmids.…”

“…In contrast, IncHII plasmids are nonthermosensitive for conjugative transfer but are constitutive for pilus synthesis (8). The IncHI group is further subdivided into subgroups IncHIl, IncHI2, and IncHI3 (26,42). This subdivision is based mainly on DNA homology studies, but the IncHI2 subgroup also encodes several characteristics, not found on IncHIl plasmids, such as resistance to potassium tellurite and inhibition of certain types of DNA phages (31,36).…”