Mobilization of the gonococcal 5.2kb β-lactamase plasmid pSJ5.2 into Escherichia coli by cointegration with several gram-conjugative plasmids

Abstract

We report the mobilization by cointegration of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 in an Escherichia coli background. Transfer of pSJ5.2 was measured by filter mating assays with five different conjugative plasmids from Enterobacteriaceae and the gonococcal 41 kb tet(M). Plasmid pSJ5.2 was mobilized to E. coli at frequencies of 1.7x10(-6), 9.3x10(-8) and 2.7x10(-5) by the tet(M), R64 drd-33 and N3 conjugative plasmids, respectively. Mobilization of pSJ5.2 by the 41 kb tet(M) conjugative plasmid resulted in stable Amp(R) E. coli transconjugants consisting of pSJ5.2 plasmid with an insertion located in the 2.4 kb BamHI-BamHI fragment. Mobilization of pSJ5.2 by R64drd-33 and N3 conjugative plasmids involved stable cointegrates as detected by Southern Blot with a DIG-labelled PstI-digested pSJ5.2 probe. Restriction analysis of the R64::pSJ5.2 and N3::pSJ5.2 cointegrates and Southern Blot with the pSJ5.2 probe showed that cointegrates formed by deletion of DNA regions within the 1.8 kb BamHI-HindIII fragment of pSJ5.2. The plasmid thus appears to use multiple recombination mechanisms for cointegration with different conjugative plasmids. The complete nucleotide sequence of pSJ5.2 was determined, and will be a useful tool to further investigate the molecular mechanisms leading to its cointegrative transfer.

Genomic analyses of Neisseria gonorrhoeae reveal an association of the gonococcal genetic island with antimicrobial resistance

“…Sequences from the beta-lactamase plasmid conferring resistance to beta-lactams were retrieved from plasmid pSJ5.2 containing bla TEM1 , and defined as NEIS2357–NEIS2360 (DQ355980) with NEIS2357 designating the bla TEM gene (Table 1). 21 …”

Four different integrative recombination events involved in the mobilization of the gonococcal 5.2kb beta-lactamase plasmid pSJ5.2 in Escherichia coli

“…Previously published physical maps of the N3::pSJ5.2 and R64::pSJ5.2 cointegrate DNA of transconjugants with BamH1 and HindIII revealed that a region within the 1.8 kb BamH1-HindIII fragment of pSJ5.2 is deleted during fusion with the R64 and N3 plasmids (Scharbaai-Vázquez et al ., 2007; Region B in Fig. 1).…”

“…The 5.2 kb beta-lactamase plasmid from Neisseria gonorrhoeae (the gonococcus) is one of the three small broad-host range and mobilizable resistant (R)-plasmids associated with the worldwide spread and prevalence of Pennicillinase-Producing Neisseria gonorrhoeae (PPNG) infections (Phillips, 1976; Ray et al, 2005; Scharbaai-Vázquez et al, 2007). The 5.2 kb R-plasmid is a mob/oriT deletion derivative of the parental 7.4 kb R-plasmid (Dillon et al, 1991; Pagotto et al ., 2000; Candelas, 2000).…”

“…We previously demonstrated the cointegration of the 5.2 kb R-plasmid isolated in San Juan, Puerto Rico (pSJ5.2) with the genomes of the Enterobacteria-derived R64 and N3 and the gonococcal 41 kb tet(M) conjugative plasmids in recA - E. coli (Scharbaai-Vázquez, et al ., 2007). Stable R64::pSJ5.2 and N3::pSJ5.2 plasmid cointegrates lost pSJ5.2-derived DNA within the 1.8 kb BamH1-HindIII fragment of pSJ5.2 indicating that cointegrates formed by recombination within that fragment (Scharbaai-Vazquez, et al ., 2007).…”

“…Stable R64::pSJ5.2 and N3::pSJ5.2 plasmid cointegrates lost pSJ5.2-derived DNA within the 1.8 kb BamH1-HindIII fragment of pSJ5.2 indicating that cointegrates formed by recombination within that fragment (Scharbaai-Vazquez, et al ., 2007). E. coli -to- E. coli mobilization of pSJ5.2 using N. gonorrhoeae providing the tet(M) conjugative plasmid was observed to occur by transposomal insertion of IS 1 in the 2.4 kb BamH1-BamH1 fragment of pSJ5.2 (Scharbaai-Vázquez, et al ., 2007). In addition, the 1.0 kb BamH1-HindIII fragment of pSJ5.2 was capable of mobilizing a pUC19 vector (pUC1.0) at frequencies of 1.9 × 10 -7 and 2.2 × 10 -7 transconjugants per donor cell in the presence of R64 and N3, respectively (unpubl.…”