The 79,370-bp conjugative plasmid pB4 consists of an IncP-1beta backbone loaded with a chromate resistance transposon,the strA-strB streptomycin resistance gene pair,the oxacillinase gene bla(NPS-1), and a tripartite antibiotic efflux system of the resistance-nodulation-division family

Plasmid pB4 is a conjugative antibiotic resistance plasmid, originally isolated from a microbial community growing in activated sludge, by means of an exogenous isolation method with Pseudomonas sp. B13 as recipient. We have determined the complete nucleotide sequence of pB4. The plasmid is 79,370 bp long and contains at least 81 complete coding regions. A suite of coding regions predicted to be involved in plasmid replication, plasmid maintenance, and conjugative transfer revealed significant similarity to the IncP-1beta backbone of R751. Four resistance gene regions comprising mobile genetic elements are inserted in the IncP-1beta backbone of pB4. The modular 'gene load' of pB4 includes (1) the novel transposon Tn 5719 containing genes characteristic of chromate resistance determinants, (2) the transposon Tn 5393c carrying the widespread streptomycin resistance gene pair strA-strB, (3) the beta-lactam antibiotic resistance gene bla(NPS-1) flanked by highly conserved sequences characteristic of integrons, and (4) a tripartite antibiotic resistance determinant comprising an efflux protein of the resistance-nodulation-division (RND) family, a periplasmic membrane fusion protein (MFP), and an outer membrane factor (OMF). The components of the RND-MFP-OMF efflux system showed the highest similarity to the products of the mexCD-oprJ determinant from the Pseudomonas aeruginosa chromosome. Functional analysis of the cloned resistance region from pB4 in Pseudomonas sp. B13 indicated that the RND-MFP-OMF efflux system conferred high-level resistance to erythromycin and roxithromycin resistance on the host strain. This is the first example of an RND-MFP-OMF-type antibiotic resistance determinant to be found in a plasmid genome. The global genetic organization of pB4 implies that its gene load might be disseminated between bacteria in different habitats by the combined action of the conjugation apparatus and the mobility of its component elements.     

Facilitated transfer of IncP beta R751 derivatives from the chromosome of Bacteroides uniformis to Escherichia coli recipients by a conjugative Bacteroides tetracycline resistance element.

The broad-host-range IncP beta plasmid R751 can mobilize itself from Escherichia coli to Bacteroides spp, but it is not maintained in Bacteroides spp. If R751 carries the Bacteroides transposon Tn4351, it can be integrated into the Bacteroides chromosome. Previously we showed that R751, integrated in the chromosome of Bacteroides uniformis, cannot mobilize itself out of B. uniformis into E. coli or isogenic B. uniformis strains. In this report, we showed that if the Bacteroides conjugative tetracycline resistance element Tcr ERL was coresident with the R751 insertion in B. uniformis, derivatives of R751 were transferred to E. coli, where they were recovered as plasmids. The most common derivatives were R751::Tn4351 and R751::IS4351, but some strains transferred R751 derivatives, containing additional DNA segments ranging in size from 10 to 23 kilobases. These DNA inserts cross-hybridized with chromosomal DNA from B. uniformis which did not carry the Tcr ERL element. Therefore, the inserts appeared to be segments of the wild-type B. uniformis chromosome and were not associated with the Tcr ERL element. The transfer of integrated R751 from B. uniformis was independent of the RecA phenotype of the E. coli recipients and did not appear to be due to transfer of B. uniformis chromosomal DNA, followed by RecA-dependent recombination between homologous IS4351 sequences to form the resultant R751 plasmid derivatives. Consistent with this, no transfer of Tn4351 (associated with the cointegrated R751) from B. uniformis donors to isogenic B. uniformis recipients was detected (< 10(-8)). Our data support the hypothesis that R751 excises from the B. uniformis chromosome by recombination involving flanking Tn4351 or IS4351 sequences and forms nonreplicating circles. The mobilization of these circular forms out of B. uniformis to E.coli is then facilitated by the Tcr ERL element.     

Conjugal transfer of antibiotic resistance factors in Bacteroides fragilis: the btgA and btgB genes of plasmid pBFTM10 are required for its transfer from Bacteroides fragilis and for its mobilization by IncP beta plasmid R751 in Escherichia coli.

Transferable plasmids play an important role in the dissemination of clindamycin-erythromycin resistance in Bacteroides fragilis. We previously described the isolation and properties of pBFTM10, a 14.9-kb ClnR transfer factor from B. fragilis TMP10. We also reported the isolation of a transfer-deficient deletion derivative of pBFTM10 contained in the B. fragilis-Escherichia coli shuttle vector pGAT400. In the present study we used pGAT400 and a similar shuttle vector, pGAT550, to characterize and sequence a region of pBFTM10 required for its transfer from B. fragilis to B. fragilis or E. coli recipients and for its mobilization by the broad-host-range plasmid R751 from E. coli donors to E. coli recipients. Deletion of certain BglII restriction fragments from pBFTM10 resulted in partial or complete loss of transfer ability. Tn1000 insertions into this same region also resulted in altered transfer properties. We used the sites of Tn1000 insertions to determine the DNA sequence of the transfer region. Two potential open reading frames encoding proteins of 23.2 and 33.8 kDa, corresponding to two genes, btgA or btgB, were identified in the sequence. Tn1000 insertions within btgA or btgB or deletion of all or portions of btgA or btgB resulted in either a transfer deficiency or greatly reduced transfer from B. fragilis donors and alterations in mobilization by R751 in E. coli. A potential oriT sequence showing similarity in organization to the oriT regions of the IncP plasmids was also detected. Thus, pBFTM10 encodes and requires at least two proteins necessary for efficient transfer from B. fragilis. These same functions are expressed in E. coli and are required for mobilization by R751.