Small deletion variants of the replication protein, pi, and their potential for over-replication-based antimicrobial activity.
The emergence of multiply antibiotic-resistant microorganisms in the environment has become a serious public health threat. To address this, our lab has devised a methodology in which antimicrobial agents are transferred into unwanted cells using the process of bacterial conjugation. In the work described here, we pursued proteins that cause plasmid over-replication as potential antimicrobial agents. Our focus was on the pir-encoded pi protein of plasmid R6K that possesses both positive and negative functions in controlling gamma origin-based replication. We observed that three of four pir mutations examined, including two in-frame deletions, severely impaired negative plasmid-replication control. The resulting over-replication phenotype was particularly strong when a pir mutant was placed in cis to gamma origin. In conjugative mating experiments with several representatives of the family Enterobacteriaceae, the plasmids expressed postconjugational antimicrobial activity. The potential utility of a conjugation-based antimicrobial approach is discussed. Additionally, we describe the replication inhibitory function of a novel and useful Rep protein variant, pi*M36A;M38A, which binds iteron DNA exclusively as dimers.
Peng
Yanyu
Y
Department of Bacteriology, University of Wisconsin, Madison, USA.
Rakowski
Sheryl A
SA
Filutowicz
Marcin
M
D003227:Conjugation, Genetic; D004265:DNA Helicases / Q000235:genetics*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics*; D004755:Enterobacteriaceae / Q000235:genetics / Q000254:growth & development*; D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D011815:R Factors / Q000235:genetics*; D018741:Replication Origin; D015534:Trans-Activators / Q000235:genetics*
D004268:DNA-Binding Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases