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Susan Gottesman

Publications and source records attributed to Susan Gottesman.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed2004

The small RNA regulators of Escherichia coli: roles and mechanisms*.

Small noncoding RNAs have been found in all organisms, primarily as regulators of translation and message stability. The most exhaustive searches have taken place in E. coli, resulting in identification of more than 50 small RNAs, or 1%-2% of the number of protein-coding genes. One large class of these small RNAs uses the RNA chaperone Hfq; members of this class act by pairing to target messenger RNAs. Among the members of this class are DsrA and RprA, which positively regulate rpoS translation, OxyS, which negatively regulates rpoS translation and fhlA translation, RyhB, which reapportions iron use in the cell by downregulating translation of many genes that encode Fe-containing proteins, and Spot 42, which changes the polarity of translation in the gal operon. The promoters of these small RNAs are tightly regulated, frequently as part of well-understood regulons. Lessons learned from the study of small RNAs in E. coli can be applied to finding these important regulators in other organisms.

Bacterial Outer Membrane Proteins↗
PubMed2003

Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli.

RyhB is a small antisense regulatory RNA that is repressed by the Fur repressor and negatively regulates at least six mRNAs encoding Fe-binding or Fe-storage proteins in Escherichia coli. When Fe is limiting, RyhB levels rise, and target mRNAs are rapidly degraded. RyhB is very stable when measured after treatment of cells with the transcription inhibitor rifampicin, but is unstable when overall mRNA transcription continues. We propose that RyhB turnover is coupled to and dependent on pairing with the target mRNAs. Degradation of both mRNA targets and RyhB is dependent on RNase E and is slowed in degradosome mutants. RyhB requires the RNA chaperone Hfq. In the absence of Hfq, RyhB is unstable, even when general transcription is inhibited; degradation is dependent upon RNase E. Hfq and RNase E bind similar sites on the RNA; pairing may allow loss of Hfq and access by RNase E. Two other Hfq-dependent small RNAs, DsrA and OxyS, are also stable when overall transcription is off, and unstable when it is not, suggesting that they, too, are degraded when their target mRNAs are available for pairing. Thus, this large class of regulatory RNAs share an unexpected intrinsic mechanism for shutting off their action.

Arabinose↗
PubMed2003

Regulatory roles for small RNAs in bacteria.

Small RNAs can act to regulate both the synthesis of proteins, by affecting mRNA transcription, translation and stability, and the activity of specific proteins by binding to them. As a result of recent genome-wide screens, around 50 small RNAs have now been identified in Escherichia coli. These include many that require the RNA-binding protein Hfq for their activity; most of these RNAs act by pairing with their target mRNAs. Small RNAs can both positively and negatively regulate translation, can simultaneously regulate multiple mRNA targets, and can change the pattern of polarity within an operon.

Bacterial Proteins↗
PubMed2003

Global analysis of small RNA and mRNA targets of Hfq.

Hfq, a bacterial member of the Sm family of RNA-binding proteins, is required for the action of many small regulatory RNAs that act by basepairing with target mRNAs. Hfq binds this family of small RNAs efficiently. We have used co-immunoprecipitation with Hfq and direct detection of the bound RNAs on genomic microarrays to identify members of this small RNA family. This approach was extremely sensitive; even Hfq-binding small RNAs expressed at low levels were readily detected. At least 15 of 46 known small RNAs in E. coli interact with Hfq. In addition, high signals in other intergenic regions suggested up to 20 previously unidentified small RNAs bind Hfq; five were confirmed by Northern analysis. Strong signals within genes and operons also were detected, some of which correspond to known Hfq targets. Within the argX-hisR-leuT-proM operon, Hfq appears to compete with RNase E and modulate RNA processing and degradation. Thus Hfq immunoprecipitation followed by microarray analysis is a highly effective method for detecting a major class of small RNAs as well as identifying new Hfq functions.

Escherichia coli↗
PubMed2003

Proteolysis in bacterial regulatory circuits.

Proteolysis by cytoplasmic, energy-dependent proteases plays a critical role in many regulatory circuits, keeping basal levels of regulatory proteins low and rapidly removing proteins when they are no longer needed. In bacteria, four families of energy-dependent proteases carry out degradation. In all of them, substrates are first recognized and bound by ATPase domains and then unfolded and translocated to a sequestered proteolytic chamber. Substrate selection depends not on ubiquitin but on intrinsic recognition signals within the proteins and, in some cases, on adaptor or effector proteins that participate in delivering the substrate to the protease. For some, the activity of these adaptors can be regulated, which results in regulated proteolysis. Recognition motifs for proteolysis are frequently found at the N and C termini of substrates. Proteolytic switches appear to be critical for cell cycle development in Caulobacter crescentus, for proper sporulation in Bacillus subtilis, and for the transition in and out of stationary phase in Escherichia coli. In eukaryotes, the same proteases are found in organelles, where they also play important roles.

Adaptor Proteins, Vesicular Transport↗