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^E Regulates and Is Regulated by a Small RNA in Escherichia coli ^,

Karl M. Thompson,^1, Virgil A. Rhodius,² and Susan Gottesman^1*

Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892,¹ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California 94143²

Received 4 January 2007/ Accepted 26 March 2007

RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia 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. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA, 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 in cells lacking the RseA anti-sigma factor and 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 increased 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.

Published ahead of print on 6 April 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: Dept. of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298-0678.