Controlled expression of an rpoS antisense RNA can inhibit RpoS function in Escherichia coli.
We show that an inducible rpoS antisense RNA complementary to the rpoS message can inhibit expression of RpoS in both exponential and stationary phases and can attenuate expression of the rpoS regulon in Escherichia coli. Plasmids containing rpoS antisense DNA expressed under the control of the T7lac promoter and T7 RNA polymerase were constructed, and expression of the rpoS antisense RNA was optimized in the pET expression system. rpoS antisense RNA levels could be manipulated to effectively control the expression of RpoS and RpoS-dependent genes. RpoS expression was inhibited by the expression of rpoS antisense RNA in both exponential and stationary phases in E. coli. RpoS-dependent catalase HPII was also downregulated, as determined by catalase activity assays and with native polyacrylamide gels stained for catalase. Induced RpoS antisense expression also reduced the level of RpoS-dependent glycogen synthesis. These results demonstrate that controlled expression of antisense RNA can be used to attenuate expression of a regulator required for the expression of host adaptation functions and may offer a basis for designing effective antimicrobial agents.
Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada.
Guozhu
G
Chen
Cheryl L
CL
Patten
Herb E
HE
Schellhorn
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics*; D015152:Blotting, Northern; D015153:Blotting, Western; D002374:Catalase / Q000378:metabolism; D015342:DNA Probes / Q000138:chemical synthesis / Q000494:pharmacology; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D006003:Glycogen / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016372:RNA, Antisense / Q000096:biosynthesis* / Q000235:genetics*; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D018085:Regulon; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012808:Sigma Factor / Q000037:antagonists & inhibitors* / Q000235:genetics*; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D015342:DNA Probes; D004269:DNA, Bacterial; D016372:RNA, Antisense; D012329:RNA, Bacterial; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D006003:Glycogen; D002374:Catalase; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase