^E regulates and is regulated by a small RNA in Escherichia coli

Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

^* To whom correspondence should be addressed. Email: susang{at}helix.nih.gov.

	Abstract

RybB is a small, Hfq-binding non-coding RNA originally identified in a screen of conserved intergenic regions in E. coli. Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB, negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multi-copy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the response regulator OmpR; the second contained mipA, a gene encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on ^E. The sequence upstream of the +1 of rybB contains a consensus ^E promoter. The activity of rybB-lacZ was increased either in cells lacking the RseA antisigma factor or when ^E was overproduced from a heterologous promoter. The activity of rybB-lacZ and the detection of RybB were totally abolished in an rpoE null strain. In vitro, ^E efficiently transcribes from this promoter. Both a rybB mutation and an hfq mutation significantly increase expression of both rybB-lacZ and rpoE-lacZ fusions, consistent with negative regulation of the ^E response by RybB and other small RNAs. Based on the plasmid screens, NsrR, a repressor sensitive to nitric oxide, was also found to negatively regulate ^E-dependent promoters in an RseA-independent fashion.