Summary In bacteria, many small regulatory RNAs (sRNAs) are induced in response to specific environmental signals or stresses and act by base pairing with mRNA targets to affect protein translation or mRNA stability. In Escherichia coli, the gene for the sRNA IS061/IsrA, here renamed McaS, was predicted to reside in an intergenic region between abgR, encoding a transcription regulator and ydaL, encoding a small MutS-related protein. We show that McaS is a ~95 nt transcript whose expression increases over growth, peaking in early-to-mid stationary phase, or when glucose is limiting. McaS uses three discrete single-stranded regions to regulate mRNA targets involved in various aspects of biofilm formation. McaS represses csgD, the transcription regulator of curli biogenesis and activates flhD, the master transcription regulator of flagella synthesis leading to increased motility, a process not previously reported to be regulated by sRNAs. McaS also regulates pgaA, a porin required for the export of the polysaccharide poly β-1,6 N-acetyl-D-glucosamine. Consequently, high levels of McaS result in increased biofilm formation while a strain lacking mcaS shows reduced biofilm formation. Based on our observations, we propose that, in response to limited nutrient availability, increasing levels of McaS modulate steps in the progression to a sessile lifestyle.

A large group of bacterial small regulatory RNAs (sRNAs) use the Hfq chaperone to mediate pairing with and regulation of mRNAs. Recent findings help to clarify how Hfq acts and highlight the role of the endonuclease RNase E and its associated proteins (the degradosome) in negative regulation by these sRNAs. sRNAs frequently uncouple transcription and translation by blocking ribosome access to the mRNA, allowing other proteins access to the mRNA. As more examples of sRNA-mediated regulation are studied, more variations on how Hfq, RNase E, and other proteins collaborate to bring about sRNAbased regulation are being found. Post-transcriptional Regulation by Small Noncoding RNAs in BacteriaThe idea that RNAs could function as regulators of gene expression has been around since the earliest studies of gene regulation. In their seminal paper entitled "Genetic Regulatory Mechanisms in the Synthesis of Proteins", Jacob and Monod originally hypothesized, "The specific 'repressor' (RNA?), acting with a given operator, is synthesized by a regulator gene" (1). Although the repressor in the case of the lac operon turned out to be the Lac repressor protein, the later discovery of small RNA (sRNA) 3 regulators confirmed their original hypothesis. Currently, examples of this form of gene regulation are widespread among organisms. Here, we will focus on pairing sRNAs in bacteria and, specifically, those that are often termed transencoding sRNAs. These RNAs are expressed from the DNA in trans, i.e. the sRNA genes are far from the genes encoding their mRNA target(s) and have limited complementarity with their target mRNAs. These bacterial sRNAs typically range in length from ϳ50 to 300 nucleotides. Many of these sRNAs are highly expressed when cells are undergoing some type of stress (for instance, oxidative stress, sugar phosphate accumulation, or nutrient starvation). The sRNAs base pair with their mRNA targets, leading to a variety of outcomes. Base pairing can lead to stabilization and/or translational activation of an mRNA target. Usually, activation occurs by base pairing within the 5Ј-UTR, changing folding of the 5Ј-UTR to allow entry of the ribosome and translation to occur (reviewed in Refs. 2 and 3). Another mode of action by sRNAs ultimately leads to translational repression and/or degradation of an mRNA target. In the majority of characterized cases, an sRNA base pairs at or around the ribosome-binding site (RBS) of an mRNA target. This leads to inhibition of translational initiation and, in most cases, the subsequent destabilization of the target. Negative regulation can also occur in other ways, as discussed below. Degradation of the mRNA target reinforces the translational repression and makes it irreversible.In many bacteria, an RNA chaperone, Hfq, is required for efficient base pairing between an sRNA and its target mRNA (reviewed in Ref. 4). In this minireview, we will focus on recent advances in understanding sRNA-mediated negative gene regulation in Escherichia coli and Salmonella enterica. More specifically, ...

Small Hfq-dependent non-coding regulatory RNAs (sRNAs) that alter mRNA stability and expression by pairing with target mRNAs have increasingly been shown to be important in influencing the behaviour of bacteria. In Escherichia coli, flhD and flhC, which encode the master regulator of flagellar synthesis, are co-transcribed from a promoter that is regulated by multiple transcription factors that respond to different environmental cues. Here, we show that the 5' untranslated region (5' UTR) of the flhDC mRNA also serves as a hub to integrate additional environmental cues into the decision to make flagella. Four sRNAs, ArcZ, OmrA, OmrB and OxyS, negatively regulated and one sRNA, McaS, positively regulated motility and flhDC expression by base-pairing with the 5' UTR of this mRNA. Another sRNA, MicA, positively regulated motility independent of regulation of flhDC. Furthermore, we demonstrate that the regulation of motility by the ArcB/A two component system is in part due to its regulation of ArcZ. flhDC is the first mRNA that has been shown to be both positively and negatively regulated by direct pairing to sRNAs. Moreover, both positive regulation by McaS and negative regulation by ArcZ require the same binding site in the flhDC mRNA.

Small noncoding regulatory RNAs (sRNAs) play a key role in regulating the expression of many genes in Escherichia coli and other bacteria. Many of the sRNAs identified in E. coli bind to mRNAs in an Hfq-dependent manner and stimulate or inhibit translation of the mRNAs. Several sRNAs are regulated by well-studied global regulators. Here, we report characterization of the CyaR (RyeE) sRNA, which was previously identified in a global search for sRNAs in E. coli. We demonstrated that CyaR is positively regulated by the global regulator Crp under conditions in which cyclic AMP levels are high. We showed by using microarray analysis and Northern blotting that several genes are negatively regulated by CyaR, including ompX, encoding a major outer membrane protein; luxS, encoding the autoinducer-2 synthase; nadE, encoding an essential NAD synthetase; and yqaE, encoding a predicted membrane protein with an unknown function. Using translational lacZ fusions to yqaE, ompX, nadE, and luxS, we demonstrated that the negative regulation of these genes by CyaR occurs at the posttranscriptional level and is direct. Different portions of a highly conserved 3 region of CyaR are predicted to pair with sequences near the ribosome binding site of each of these targets; mutations in this sequence affected regulation, and compensatory mutations in the target mRNA restored regulation, confirming that there is direct regulation by the sRNA. These results provide insight into the mechanisms by which Crp negatively regulates genes such as luxS and ompX and provide a link between catabolite repression, quorum sensing, and nitrogen assimilation in E. coli.