In the last decade, the important role of small non-coding RNAs in regulation has been recognized and begun to be studied. Our laboratory, in collaboration with others, has undertaken two completed global searches for non-coding RNAs in Escherichia coli , contributing significantly to the more than 80 regulatory RNAs that are now identified. A large number of these RNAs bind tightly to the RNA chaperone Hfq. We and others have shown that every RNA that binds tightly to Hfq acts by pairing with target mRNAs, regulating stability and translation of the mRNA, either positively or negatively. Our lab has studied a number of these small RNAs in detail. We have found that expression of each small RNA is regulated by different stress conditions, and that the small RNA plays an important role in adapting to stress. One of the first studied small RNAs is RyhB. RyhB transcription is repressed by the Fur iron-dependent repressor, and the small RNA is therefore made in high quantities when intracellular iron is limiting. When it is made, it targets mRNAs that encode iron-binding proteins for degradation. Therefore, this small RNA, which is also found in Vibrio , Salmonella , Klebsiella , and Yersinia , reprograms iron use in the cells and may be an important component of virulence for some pathogens. Two other small RNAs, now called OmrA and OmrB, regulate a number of outer membrane proteins; these small RNAs are made at high osmolarity as part of the OmpR/EnvZ regulon, previously known for its regulation of major outer membrane porins. In addition, OmrA and OmrB also regulate their own transcriptional regulators, providing a negative feedback loop that may be a frequent component of these regulatory systems. RybB, another Hfq-binding RNA, is dependent on an alternative sigma factor, sigma(E), for transcription and down-regulates outer membrane proteins. Sigma(E) becomes active when misfolded outer membrane proteins accumulate in the periplasm of the cell. By down-regulating the outer membrane proteins, RybB negatively autoregulates sigma(E) activity. These RNAs are characteristic of a growing family of regulatory RNAs that regulate the cell surface, possibly important during infection. An additional outer membrane protein, OmpX, has now been shown to be regulated by CyaR, also an Hfq-binding RNA that is positively regulated by cyclic AMP (cAMP) and C-reactive protein (CRP). In addition to OmpX, believed to be important for cell adhesion, CyaR down-regulates the synthesis of other proteins, including LuxS, the synthase for a quorum-sensing molecule believed to work for a broad range of species. It seems possible that CyaR helps the cell down-regulate functions important in escape from biofilms under poor nutrient conditions (low glucose). Consistent with the idea that all major regulatory systems may have small RNA components, another Hfq-binding RNA is regulated by PhoP and PhoQ, a two-component system important for Salmonella virulence. PhoP and PhoQ activate synthesis of the RNA under low magnesium conditions; the small RNA inactivates the enzyme for cell surface modification, affecting the cell's sensitivity to antimicrobial peptides such as polymyxin. Previous studies had demonstrated the roles of two small RNAs, DsrA and RprA, in positively regulating translation of the stress sigma factor RpoS. More recently, we have shown that RprA also has a number of other mRNA targets, which are negatively regulated; these targets differ from those for DsrA. The new RprA targets expand the likely role of RprA and its regulators, RcsC, RcsD, and RcsB, in controlling biofilm formation by this bacteria. Studies on the mechanism of action of DsrA and RprA suggest that they increase both the stability and translation of rpoS mRNA, protecting it from degradation by RNAse E. All of these Hfq-binding sRNAs pair with target mRNAs, but the regions of pairing are often short, interrupted, and therefore difficult to identify. In addition, even when pairing can be predicted, regulation is not always seen. Examination of the details of pairing by the OmrA/B RNAs demonstrate that the 5 prime end of the RNA is required for pairing to all targets. It is possible that flexibility at the 5 prime end allows more efficient pairing. In other experiments, we have chosen genes with no known small RNA regulators, created translational fusions to them, and used genetic screens to identify sRNA translational regulators. In particular, we found that the dpiAB genes are regulated by a previously uncharacterized Hfq-binding RNA, RybC. Because DpiAB are involved in helping cells survive in the presence of low levels of antibiotic, it is possible the small RNA is involved in this response. The approach used to study dpiAB has been developed into a very flexible set of strains to allow the rapid analysis of many targets, already being used for many lab projects. Our work, combined with work from other labs on this family of regulators, suggests that a large number of genes in bacteria will be subject to this post-transcriptional regulation. Tiling arrays allow a detailed examination of the RNA transcripts in the cell. In collaboration with Dr. G. Storz, we are using an E. coli tiling array to examine whether any additional small RNAs are present in E. coli and to define possible mRNA target RNAs. RpoS is subject to control at the level of protein turnover as well. RpoS is rapidly degraded during active growth, in a process that requires the energy-dependent ClpXP protease and the adaptor protein RssB, a phosphorylatable protein that presents RpoS to the protease. RpoS becomes stable after various stress or starvation treatments; the mode of stabilization has been a mystery. Recent studies in our lab and others demonstrated significant regulation of degradation in the absence of phosphorylation. A genetic screen for regulators of RpoS degradation led to discovery of a small, previously uncharacterized protein, YaiB, now renamed IraP. Mutants of iraP have somewhat decreased stability of RpoS under normal growth conditions and totally abolish the stabilization of RpoS after phosphate starvation. IraP blocks RpoS turnover in a purified in vitro system, and directly interacts with RssB. In E. coli , phosphate starvation is sensed by an increase in the levels of the small molecule ppGpp, and the iraP promoter is positively regulated by ppGpp. In Salmonella , expression of this gene is also induced in response to starvation for magnesium. This anti-adaptor is only the first of these proteins. We have now identified two other small proteins that also act to stabilize RpoS in a purified in vitro system, YcgW (renamed IraM), and YjiD (renamed IraD). IraM is made in response to magnesium starvation, dependent on the PhoP and PhoQ regulators; IraD is important after DNA damage. We also have identified AppY, a transcriptional regulator, as involved in RpoS stabilization; when it is overproduced, RpoS is stable, independent of IraP, IraM, or IraD. We believe it acts by regulating synthesis of a novel anti-adaptor and a mutational search for this has been developed. Thus, the anti-adaptors define a new level of regulatory control, interacting with the RssB adaptor protein and [summary truncated at 7800 characters]

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De Lay, Nicholas; Gottesman, Susan (2009) The Crp-activated small noncoding regulatory RNA CyaR (RyeE) links nutritional status to group behavior. J Bacteriol 191:461-76
Bougdour, Alexandre; Cunning, Christofer; Baptiste, Patrick Jean et al. (2008) Multiple pathways for regulation of sigmaS (RpoS) stability in Escherichia coli via the action of multiple anti-adaptors. Mol Microbiol 68:298-313
Ranquet, Caroline; Gottesman, Susan (2007) Translational regulation of the Escherichia coli stress factor RpoS: a role for SsrA and Lon. J Bacteriol 189:4872-9
Bougdour, Alexandre; Gottesman, Susan (2007) ppGpp regulation of RpoS degradation via anti-adaptor protein IraP. Proc Natl Acad Sci U S A 104:12896-901
Majdalani, Nadim; Gottesman, Susan (2007) Genetic dissection of signaling through the Rcs phosphorelay. Methods Enzymol 423:349-62
Thompson, Karl M; Rhodius, Virgil A; Gottesman, Susan (2007) SigmaE regulates and is regulated by a small RNA in Escherichia coli. J Bacteriol 189:4243-56
Vanderpool, Carin K; Gottesman, Susan (2007) The novel transcription factor SgrR coordinates the response to glucose-phosphate stress. J Bacteriol 189:2238-48
Bougdour, Alexandre; Wickner, Sue; Gottesman, Susan (2006) Modulating RssB activity: IraP, a novel regulator of sigma(S) stability in Escherichia coli. Genes Dev 20:884-97
Zhou, YanNing; Gottesman, Susan (2006) Modes of regulation of RpoS by H-NS. J Bacteriol 188:7022-5
Tu, Xuanlin; Latifi, Tammy; Bougdour, Alexandre et al. (2006) The PhoP/PhoQ two-component system stabilizes the alternative sigma factor RpoS in Salmonella enterica. Proc Natl Acad Sci U S A 103:13503-8

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