Streptococcal R plasmid pIP501: endonuclease site map, resistance determinant location, and construction of novel derivatives

Abstract

The streptococcal resistance plasmid pIP501 (30 kilobase pairs [kb]) encodes resistance to chloramphenicol (Cmr) and erythromycin (Emr) and is capable of conjugative transfer among numerous streptococcal species. By using a streptococcal host-vector recombinant DNA system, the Cmr and Emr determinants of pIP501 were localized to 6.3-kb HindlIl and 2.1-kb HindIII-AvaI fragments, respectively. pIP501 was lost at a frequency of 22% in Streptococcus sanguis cells grown at 42°C but was stable in cells grown at 37°C (<1% frequency of loss). Sequences from a cryptic multicopy plasmid, pVA380-1, were substituted for the pIP501 Emr determinant in vitro, and the resulting recombinant plasmid, designated pVA797, was recoverd in transformed S. sanguis cells. The replication of pVA797 was governed by the pVA380-1 sequences based on temperature-stable replication and incompatibility with pVA380-1-derived replicons. The self-ligation of partially cleaved HindIII pIP501 DNA fragments allowed the localization of a pIP501 region involved in autonomous plasmid replication. A small pIP501 derivative (pVA798) obtained from this experiment had a greatly increased copy number but was unstably inherited. Our data indicate that the sequences encoding the resistance determinants and some of the plasmid replication machinery are relatively clustered on the pIP501 molecule. The properties of pVA797 and pVA798 indicate that these molecules will enhance current streptococcal genetic systems from the standpoint of conjugative mobilization (pVA797) and gene amplification (pVA798).

Evidence for the conjugal transfer of the broad host range plasmid pIP501 into strains of Lactobacillus helveticus

“…faecalis strain CIT294. The fragment sizes confirmed the published data for plasmid pIP501 (Evans & Macrina 1983). This is good evidence for the physical transfer of an intact pIP50l entity.…”

“…There is evidence that the broad host range plasmids PAM81 and/or pIP501 can undergo conjugal transfer from streptococci into enteric lactobacilli (Vescovo et al 1983), L p t . casei (Gibson et al 1979), Pediococcus spd(Gonza1ez & Kunka 1983) and Lact. plantarum (West & Warner 1985;Shrago et al 1986).…”

A new vector, pGID052, for genetic transfer in

“…The pVA797 was found at about four copies in L. lactis while pIP501 at about three copies in E. faecalis. These results were similar to those previously obtained by Evans and Macrina in Streptococcus sanguis [26].…”

“…L. lactis DG52 harboring the conjugative plasmid pVA797 was chosen as donor since the pIP501 could not be used for mobilization because it possesses the same antibiotic resistance marker as pGID052. On the contrary, pVA797 plasmid is useful because it only contains a chloramphenicol resistance gene [26]. This strain was used as the donor for the mating experiments with two O. oeni strains, named 8413 and 8417.…”

Inhibition of a naturally occurring rolling‐circle replicon in derivatives of the theta‐replicating plasmid pIP501

“…Plasmid pIP501 belongs to a family of thermosensitive -replicating genomes including pAM␤1 and pSM19035 (Evans and Macrina, 1983;Janniè re et al, 1990; see Janniè re et al, 1993 as review). Its replication region is represented in Fig.…”