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Susan Gottesman

Publications and source records attributed to Susan Gottesman.

At least 19 recordsLinked to original sources
PubMed2007

The novel transcription factor SgrR coordinates the response to glucose-phosphate stress.

SgrR is the first characterized member of a family of bacterial transcription factors containing an N-terminal DNA binding domain and a C-terminal solute binding domain. Previously, we reported genetic evidence that SgrR activates the divergently transcribed gene sgrS, which encodes a small RNA required for recovery from glucose-phosphate stress. In this study, we examined the regulation of sgrR expression and found that SgrR negatively autoregulates its own transcription in the presence and absence of stress. An SgrR binding site in the sgrR-sgrS intergenic region is required in vivo for both SgrR-dependent activation of sgrS and autorepression of sgrR. Purified SgrR binds specifically to sgrS promoter DNA in vitro; a mutation in the site required for in vivo activation and autorepression abrogates in vitro SgrR binding. A plasmid library screen identified clones that alter expression of a P(sgrS)-lacZ fusion; some act by titrating endogenous SgrR. The yfdZ gene, encoding a putative aminotransferase, was identified in this screen; the yfdZ promoter contains an SgrR binding site, and transcriptional fusions indicate that yfdZ is activated by SgrR. Clones containing mlc, which encodes a glucose-specific repressor protein, also downregulate P(sgrS)-lacZ. The mlc clones do not appear to titrate the SgrR protein, indicating that Mlc affects sgrS expression by an alternative mechanism.

Base Sequence↗
PubMed2006

Modulating the outer membrane with small RNAs.

MicF, one of the first chromosomally encoded regulatory small RNAs (sRNAs) to be discovered, was found to modulate the expression of OmpF, an abundant outer membrane protein. Several recent papers have now shown that this is not an isolated case. At least five other sRNAs also regulate the synthesis of outer membrane porins, and additional sRNAs modulate the expression of other outer membrane proteins. Here we review what is known about these sRNAs and discuss the implications of this regulation.

Bacterial Outer Membrane Proteins↗
PubMed2006

The PhoP/PhoQ two-component system stabilizes the alternative sigma factor RpoS in Salmonella enterica.

The sigma factor RpoS regulates the expression of many stress response genes and is required for virulence in several bacterial species. We now report that RpoS accumulates when Salmonella enterica serovar Typhimurium is growing logarithmically in media with low Mg(2+) concentrations. This process requires the two-component regulatory system PhoP/PhoQ, which is specifically activated in low Mg(2+). We show that PhoP controls RpoS protein turnover by serving as a transcriptional activator of the iraP (yaiB) gene, which encodes a product that enhances RpoS stability by interacting with RssB, the protein that normally delivers RpoS to the ClpXP protease for degradation. Mutation of the phoP gene rendered Salmonella as sensitive to hydrogen peroxide as an rpoS mutant after growth in low Mg(2+). In Escherichia coli, low Mg(2+) leads to only modest RpoS stabilization, and iraP is not regulated by PhoP/PhoQ. These findings add the sigma factor RpoS to the regulatory proteins and two-component systems that are elevated in a PhoP/PhoQ-dependent fashion when Salmonella face low Mg(2+) environments. Our data also exemplify the critical differences in regulatory circuits that exist between the closely related enteric bacteria Salmonella and E. coli.

Bacterial Proteins↗
PubMed2006

Target prediction for small, noncoding RNAs in bacteria.

Many small, noncoding RNAs in bacteria act as post-transcriptional regulators by basepairing with target mRNAs. While the number of characterized small RNAs (sRNAs) has steadily increased, only a limited number of the corresponding mRNA targets have been identified. Here we present a program, TargetRNA, that predicts the targets of these bacterial RNA regulators. The program was evaluated by assessing whether previously known targets could be identified. The program was then used to predict targets for the Escherichia coli RNAs RyhB, OmrA, OmrB and OxyS, and the predictions were compared with changes in whole genome expression patterns observed upon expression of the sRNAs. Our results show that TargetRNA is a useful tool for finding mRNA targets of sRNAs, although its rate of success varies between sRNAs.

Base Pairing↗
PubMed2006

An antibody-based microarray assay for small RNA detection.

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.

Antibodies, Monoclonal↗
PubMed2006

Modulating RssB activity: IraP, a novel regulator of sigma(S) stability in Escherichia coli.

The sigma(S) subunit of Escherichia coli RNA polymerase regulates the expression of stationary phase and stress response genes. sigma(S) is highly unstable in exponentially growing cells, whereas its stability increases dramatically upon starvation or under certain stress conditions. The degradation of sigma(S) is controlled by the phosphorylatable adaptor protein RssB and the ClpXP protease. RssB specifically directs sigma(S) to ClpXP. An unanswered question is how RssB-mediated degradation of sigma(S) is blocked by conditions such as glucose or phosphate starvation. We report here the identification and characterization of a new regulator of sigma(S) stability, IraP (inhibitor of RssB activity during phosphate starvation), that stabilizes sigma(S) both in vivo and in vitro. Deletion of iraP interferes with sigma(S) stabilization during phosphate starvation, but not during carbon starvation, and has a partial effect in stationary phase and nitrogen starvation. IraP interferes with RssB-dependent degradation of sigma(S) through a direct protein-protein interaction with RssB. A point mutant of IraP was isolated and found to be defective both for inhibition of sigma(S) degradation and interaction with RssB. Our results reveal a novel mechanism of regulation of sigma(S) stability through the regulation of RssB activity and identify IraP as a member of a new class of regulators, the anti-adaptor proteins.

Bacterial Proteins↗
PubMed2006

Remodelling of the Escherichia coli outer membrane by two small regulatory RNAs.

Small non-coding RNAs that play important regulatory roles exist in numerous organisms. In Escherichia coli, about 60 small RNAs have been found and those that have been studied are involved in the response and adaptation to different stresses. RygA and RygB, two of these small RNAs, were identified on the basis of their conservation between different species and their ability to bind Hfq. They are adjacent on the chromosome and have sequence similarity at their 5' and 3' ends but distinct central regions, suggesting that they could regulate the expression of both common and distinct genes. A screen using a multicopy E. coli library led to identification of the response regulator OmpR and its associated sensor kinase EnvZ as positive regulators of rygA and rygB transcription. Therefore, RygA and RygB were renamed OmrA and OmrB respectively (for OmpR-regulated sRNAs A and B). When expressed at high levels, OmrA and OmrB RNAs negatively regulate the expression of several genes encoding multiple outer membrane proteins, including cirA, fecA, fepA and ompT. Taken together, these data suggest that OmrA and OmrB participate in the regulation of outer membrane composition in response to environmental conditions.

Bacterial Outer Membrane Proteins↗
PubMed2006

Modes of regulation of RpoS by H-NS.

Regulated degradation of RpoS requires RssB and ClpXP protease. Mutations in hns increase both RpoS synthesis and stability, causing a twofold increase in synthesis and almost complete stabilization of RpoS, independent of effects on synthesis and independent of phosphorylation of RssB. This suggests that H-NS regulates an RssB inhibitor or inhibitors.

Bacterial Proteins↗
PubMed2005

Micros for microbes: non-coding regulatory RNAs in bacteria.

Small non-coding RNAs with important regulatory roles are not confined to eukaryotes. Recent studies have led to the identification of numerous small regulatory RNAs in Escherichia coli and in other bacteria. As in eukaryotic cells, a major class of these small RNAs acts by base-pairing with target mRNAs, resulting in changes in the translation and stability of the mRNA. Roles for these non-coding pairing RNAs in bacteria have been demonstrated in several cases. Because these non-coding RNAs act post-transcriptionally, they impose a regulatory step that is independent of and epistatic to any transcriptional signals for their target mRNAs.

Bacterial Proteins↗
PubMed2005

Analysis of the Escherichia coli Alp phenotype: heat shock induction in ssrA mutants.

The major phenotypes of lon mutations, UV sensitivity and overproduction of capsule, are due to the stabilization of two substrates, SulA and RcsA. Inactivation of transfer mRNA (tmRNA) (encoded by ssrA), coupled with a multicopy kanamycin resistance determinant, suppressed both lon phenotypes and restored the rapid degradation of SulA. This novel protease activity was named Alp but was never identified further. We report here the identification, mapping, and characterization of a chromosomal mutation, faa (for function affecting Alp), that leads to full suppression of a Deltalon ssrA::cat host and thus bypasses the requirement for multicopy Kan(r); faa and ssrA mutants are additive in their ability to suppress lon mutants. The faa mutation was mapped to the C terminus of dnaJ(G232); dnaJ null mutants have similar effects. The identification of a lon suppressor in dnaJ suggested the possible involvement of heat shock. We find that ssrA mutants alone significantly induce the heat shock response. The suppression of UV sensitivity, both in the original Alp strain and in faa mutants, is reversed by mutations in clpY, encoding a subunit of the heat shock-induced ClpYQ protease that is known to degrade SulA. However, capsule synthesis is not restored by clpY mutants, probably because less RcsA accumulates in the Alp strain and because the RcsA that does accumulate is inactive. Both ssrA effects are partially relieved by ssrA derivatives encoding protease-resistant tags, implicating ribosome stalling as the primary defect. Thus, ssrA and faa each suppress two lon mutant phenotypes but by somewhat different mechanisms, with heat shock induction playing a major role.

Chromosomes, Bacterial↗
PubMed2005

Role of RcsF in signaling to the Rcs phosphorelay pathway in Escherichia coli.

The rcs phosphorelay pathway components were originally identified as regulators of capsule synthesis. In addition to the transmembrane sensor kinase RcsC, the RcsA coregulator, and the response regulator RcsB, two new components have been characterized, RcsD and RcsF. RcsD, the product of the yojN gene, now renamed rcsD, acts as a phosphorelay between RcsC and RcsB. Transcription of genes for capsule synthesis (cps) requires both RcsA and RcsB; transcription of other promoters, including that for the small RNA RprA, requires only RcsB. RcsF was described as an alternative sensor kinase for RcsB. We have examined the role of RcsF in the activation of both the rprA and cps promoters. We find that a number of signals that lead to activation of the phosphorelay require both RcsF and RcsC; epistasis experiments place RcsF upstream of RcsC. The RcsF sequence is characteristic of lipoproteins, consistent with a role in sensing cell surface perturbation and transmitting this signal to RcsC. Activation of RcsF does not require increased transcription of the gene, suggesting that modification of the RcsF protein may act as an activating signal. Signals from RcsC require RcsD to activate RcsB. Sequencing of an rcsC allele, rcsC137, that leads to high-level constitutive expression of both cps and rprA suggests that the response regulator domain of RcsC plays a role in negatively regulating the kinase activity of RcsC. The phosphorelay and the variation in the activation mechanism (dependent upon or independent of RcsA) provide multiple steps for modulating the output from this system.

Escherichia coli↗
PubMed2005

Effect of RyhB small RNA on global iron use in Escherichia coli.

RyhB is a noncoding RNA regulated by the Fur repressor. It has previously been shown to cause the rapid degradation of a number of mRNAs that encode proteins that utilize iron. Here we examine the effect of ectopic RyhB production on global gene expression by microarray analysis. Many of the previously identified targets were found, as well as other mRNAs encoding iron-binding proteins, bringing the total number of regulated operons to at least 18, encoding 56 genes. The two major operons involved in Fe-S cluster assembly showed different behavior; the isc operon appears to be a direct target of RyhB action, while the suf operon does not. This is consistent with previous findings suggesting that the suf genes but not the isc genes are important for Fe-S cluster synthesis under iron-limiting conditions, presumably for essential iron-binding proteins. In addition, we observed repression of Fur-regulated genes upon RyhB expression, interpreted as due to intracellular iron sparing resulting from reduced synthesis of iron-binding proteins. Our results demonstrate the broad effects of a single noncoding RNA on iron homeostasis.

Bacterial Proteins↗
PubMed2005

The Rcs phosphorelay: a complex signal transduction system.

RcsC, RcsB, and RcsA were first identified as a sensor kinase, a response regulator, and an auxiliary regulatory protein, respectively, regulating the genes of capsular polysaccharide synthesis. Recent advances have demonstrated that these proteins are part of a complex phosphorelay, in which phosphate travels from the histidine kinase domain in RcsC to a response regulator domain in the same protein; from there to a phosphotransfer protein, RcsD; and from there to RcsB. In addition to capsule synthesis, which requires the unstable regulatory protein RcsA, RcsB also stimulates transcription of a small RNA, RprA; the cell division gene ftsZ; and genes encoding membrane and periplasmic proteins, including the osmotically inducible genes osmB and osmC. The Rcs system appears to play an important role in the later stages of biofilm development; induction of Rcs signaling by surfaces is consistent with this role.

Bacterial Capsules↗
PubMed2004

Small RNAs shed some light.

Small regulatory RNAs can act by pairing with their target messages, targeting themselves and the mRNA for degradation; Lenz et al. (this issue of Cell) now report that multiple small RNAs are essential regulators of the quorum-sensing systems of Vibrio species, including the regulation of virulence in V. cholerae.

Gene Expression Regulation, Bacterial↗
PubMed2004

Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis.

In many bacteria, iron homeostasis is controlled primarily by the ferric uptake regulator (Fur), a transcriptional repressor. However, some genes, including those involved in iron storage, are positively regulated by Fur. A Fur-repressed regulatory small RNA (sRNA), RyhB, has been identified in Escherichia coli, and it has been demonstrated that negative regulation of genes by this sRNA is responsible for the positive regulation of some genes by Fur. No RyhB sequence homologs were found in Pseudomonas aeruginosa, despite the identification of genes positively regulated by its Fur homolog. A bioinformatics approach identified two tandem sRNAs in P. aeruginosa that were candidates for functional homologs of RyhB. These sRNAs (PrrF1 and PrrF2) are >95% identical to each other, and a functional Fur box precedes each. Their expression is induced under iron limitation. Deletion of both sRNAs is required to affect the iron-dependent regulation of an array of genes, including those involved in resistance to oxidative stress, iron storage, and intermediary metabolism. As in E. coli, induction of the PrrF sRNAs leads to the rapid loss of mRNAs for sodB (superoxide dismutase), sdh (succinate dehydrogenase), and a gene encoding a bacterioferritin. Thus, the PrrF sRNAs are the functional homologs of RyhB sRNA. At least one gene, bfrB, is positively regulated by Fur and Fe(2+), even in the absence of the PrrF sRNAs. This work suggests that the role of sRNAs in bacterial iron homeostasis may be broad, and approaches similar to those described here may identify these sRNAs in other organisms.

Bacterial Proteins↗
PubMed2004

Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM-yoeB toxin-antitoxin system.

In Escherichia coli, the Lon ATP-dependent protease is responsible for degradation of several regulatory proteins and for the elimination of abnormal proteins. Previous studies have shown that the overproduction of Lon is lethal. Here, we showed that Lon overproduction specifically inhibits translation through at least two different pathways. We have identified one of the pathways as being the chromosomal yefM-yoeB toxin-antitoxin system. The existence of a second pathway is demonstrated by the observation that the deletion of the yefM-yoeB system did not completely suppress lethality and translation inhibition. We also showed that the YoeB toxin induces cleavage of translated mRNAs and that Lon overproduction specifically activates YoeB-dependent mRNAs cleavage. Indeed, none of the other identified chromosomal toxin-antitoxin systems (relBE, mazEF, chpB and dinJ-yafQ) was involved in Lon-dependent lethality, translation inhibition and mRNA cleavage even though the RelB and MazE antitoxins are known to be Lon substrates. Based on our results and other studies, translation inhibition appears to be the key element that triggers chromosomal toxin-antitoxin systems. We propose that under Lon overproduction conditions, translation inhibition is mediated by Lon degradation of a component of the YoeB-independent pathway, in turn activating the YoeB toxin by preventing synthesis of its unstable YefM antidote.

ATP-Dependent Proteases↗
PubMed2004

Involvement of a novel transcriptional activator and small RNA in post-transcriptional regulation of the glucose phosphoenolpyruvate phosphotransferase system.

RyaA is a small non-coding RNA in Escherichia coli that was identified by its ability to bind tightly to the RNA chaperone Hfq. This study reports the role of RyaA in mediating the cellular response to glucose-specific phosphoenolypyruvate phosphotransferase system (PTS)-dependent phosphosugar stress. Aiba and co-workers have shown that a block in the metabolism of glucose 6-phosphate causes transient growth inhibition and post-transcriptional regulation of ptsG, encoding the glucose-specific PTS transporter. We found that RyaA synthesis was induced by a non-metabolizable glucose phosphate analogue and was necessary for relief of the toxicity of glucose phosphate stress. Expression of RyaA was sufficient to cause a rapid loss of ptsG mRNA, probably reflecting degradation of the message mediated by RyaA:ptsG pairing. The ryaA gene was renamed sgrS, for sugar transport-related sRNA. Expression of sgrS is regulated by a novel transcriptional activator, SgrR (formerly YabN), which has a putative DNA-binding domain and a solute-binding domain similar to those found in certain transport proteins. Our results suggest that under conditions of glucose phosphate accumulation, SgrR activates SgrS synthesis, causing degradation of ptsG mRNA. Decreased ptsG mRNA results in decreased production of glucose transport machinery, thus limiting further accumulation of glucose phosphate.

Base Sequence↗