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. B 13 as recipient. We have determined the complete nucleotide sequence of pB4. The plasmid is 79,370 by 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 Tn5719 containing genes characteristic of chromate resistance determinants, (2) the transposon Tn5393c carrying the widespread streptomycin resistance gene pair strA-strB, (3) the beta-lactam antibiotic resistance gene b/a(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 RNDMFP-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 zsp. 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.
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