Selective amplification of genes on the R plasmid, NR1, in Proteus mirabilis: an example of the induction of selective gene amplification.

Abstract

The dru-resistance plasmid, NRB, is a 37-,m circular DNA molecule that contains two components: the resistance transfer factor (29 ,unm) carrying the transfer genes and the genes for tetracycline resistance, and the r-determinant (8 Am) carrying the genes for resistance to several other antibiotics including chloramphenicol (Cm) In Proteus mirabifis, these two components are capable of independent replication, or they may replicate as a composite molecule. When cells of P. mirabilis containing NRB are cultured in medium containing Cm at 250 jag/ml a growth lag of 20-35 hr ensues. During this lag, Cm induces the selective amplification of the r-determinant, including the gene for resistance to Cm. The amplification results from the excision of the r-determinant from the R plasmid, the independent replication of the r-determinant to give polymeric as well as monomeric r-determinants, and the eventual reintegration of multiple tandem copies of the r-determinant with the resistance transfer factor to form a new R plasmid with multiple copies of the r-determinant. This mechanism represents a new level of control of gene expression in bacterial systems-namely, the induction of selective gene amplification.The transmissible drug-resistance plasmid NR1 is a polymorphic DNA molecule whose size and density in CsCl have been reported to vary depending on the host strain and culture conditions (refs. 1-4; for a review, see ref. 5). When isolated from strains of Proteus mirabilis, plasmids are found with CsCl buoyant densities of 1.712-1.718 g/ml (6). The 1.712 g/ml plasmid is a composite structure consisting of a 46 X 106 dalton resistance transfer factor (RTF-Tc; 1.710 g/ml) carrying both the tetracycline resistance and transfer genes, linked to a 14 X 106 dalton r-determinant (1.718 g/ml) carrying the genes for resistance to chloramphenicol (Cm), streptomycin, spectinomycin, and sulfonamide (4, 7). This plasmid is called the nonamplified plasmid (N plasmid). The RTF-Tc and r-determinant each contain at least one origin of replication (8). In contrast to the N plasmid, plasmids of higher density contain multiple, tandem copies of the r-determinant and are called amplified plasmids (A plasmids). These polymeric r-determinants can exist either in an autonomous state with a density of 1.718 g/ml or attached to an RTF-Tc to give an enlarged R plasmid (1.716-1.718 g/ml) (4, 7). Cells containing N plasmids and A plasmids are called "N cells" and "A cells," respectively. Clewell et al. (9) have recently described another plasmid that contains multiple copies of a tetracycline-resistance determinant in Streptococcus faecalis.Previous results have shown that, when a population of N

Contribution of Gene Amplification to Evolution of Increased Antibiotic Resistance inSalmonella typhimurium

“…It is of interest that plasmids carrying b-lactamase genes usually contain potential regions of homology for generating duplications (e.g., IS elements), and therefore the frequency of plasmid-borne preexisting bla gene duplications in a population is expected to be very high. This is supported by studies that have shown how the gene copy number of various resistance genes is increased in mutants with increased resistance (Hashimoto and Rownd 1975;Perlman and Stickgold 1977;Peterson and Rownd 1985;Nichols and Guay 1989;Hammond et al 2005;Nicoloff et al 2006Nicoloff et al , 2007. Furthermore, as demonstrated by our modeling, cells with some duplication or low amplification in place can very quickly expand the array to any copy number that is required to provide the necessary level of resistance.…”

Tandem multiplication of the IS26-flanked amplicon with theblaSHV-5gene within plasmid p1658/97

“…Interestingly, in the case of plasmid pXB2A with two tandem units, the negative results indicated that the initial duplication did not stimulate further amplification, even if the RecA is present. This observation was in opposition to that on the aforementioned plasmid NR1, in which duplication of the IS1-flanked region was essential for its further RecA-mediated amplification (Perlman & Sticgold, 1977;Peterson & Rownd, 1985).…”

Second EcoRI fragment of F capable of self-replication

“…Whereas such self-replicating fragments bear the only essential information for plasmid replication, it is possible that other genes outside these regions normally participate in replication of the large parent in some nonessential manner or lie quiescent and only become activated in the absence of the primary replication system or upon transfer to another host cell. A case in point may be the ability of the r-determinant region of plasmid R100.1 to replicate in Proteus mirabilis (22,23) and its failure to do so in Escherichia coli (15). In this paper we report the discovery of what appears to be a quiescent replicating system of the F plasmid in E. coli.…”