Publications by authors named "Nadim Majdalani"

19 Publications

  • Page 1 of 1

Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

Sci Adv 2021 Mar 3;7(10). Epub 2021 Mar 3.

Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.
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http://dx.doi.org/10.1126/sciadv.abe0384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929499PMC
March 2021

IgaA negatively regulates the Rcs Phosphorelay via contact with the RcsD Phosphotransfer Protein.

PLoS Genet 2020 07 27;16(7):e1008610. Epub 2020 Jul 27.

National Cancer Institute, Bethesda, Maryland, United States of America.

Two-component systems and phosphorelays play central roles in the ability of bacteria to rapidly respond to changing environments. In E. coli and related enterobacteria, the complex Rcs phosphorelay is a critical player in the bacterial response to antimicrobial peptides, beta-lactam antibiotics, and other disruptions at the cell surface. The Rcs system is unusual in that an inner membrane protein, IgaA, is essential due to its negative regulation of the RcsC/RcsD/RcsB phosphorelay. While it is known that IgaA transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the phosphorelay has remained unknown. Here we performed in vivo interaction assays and genetic dissection of the critical proteins and found that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation. Interactions between IgaA and RcsD within their respective periplasmic domains of these two proteins anchor repression of signaling. However, the signaling response depends on a second interaction between cytoplasmic loop 1 of IgaA and a truncated Per-Arndt-Sim (PAS-like) domain in RcsD. A single point mutation in the PAS-like domain increased interactions between the two proteins and blocked induction of the phosphorelay. IgaA may regulate RcsC, the histidine kinase that initiates phosphotransfer through the phosphorelay, indirectly, via its contacts with RcsD. Unlike RcsD, and unlike many other histidine kinases, the periplasmic domain of RcsC is dispensable for the response to signals that induce the Rcs phosphorelay system. The multiple contacts between IgaA and RcsD constitute a poised sensing system, preventing potentially toxic over-activation of this phosphorelay while enabling it to rapidly and quantitatively respond to signals.
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http://dx.doi.org/10.1371/journal.pgen.1008610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418988PMC
July 2020

The Complex Rcs Regulatory Cascade.

Annu Rev Microbiol 2018 Sep 13;72:111-139. Epub 2018 Jun 13.

Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA; emails: , ,

RcsB, a response regulator of the FixJ/NarL family, is at the center of a complex network of regulatory inputs and outputs. Cell surface stress is sensed by an outer membrane lipoprotein, RcsF, which regulates interactions of the inner membrane protein IgaA, lifting negative regulation of a phosphorelay. In vivo evidence supports a pathway in which histidine kinase RcsC transfers phosphate to phosphotransfer protein RcsD, resulting in phosphorylation of RcsB. RcsB acts either alone or in combination with RcsA to positively regulate capsule synthesis and synthesis of small RNA (sRNA) RprA as well as other genes, and to negatively regulate motility. RcsB in combination with other FixJ/NarL auxiliary proteins regulates yet other functions, independent of RcsB phosphorylation. Proper expression of Rcs and its targets is critical for success of Escherichia coli commensal strains, for proper development of biofilm, and for virulence in some pathogens. New understanding of how the Rcs phosphorelay works provides insight into the flexibility of the two-component system paradigm.
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http://dx.doi.org/10.1146/annurev-micro-090817-062640DOI Listing
September 2018

Experimental Evolution of Escherichia coli K-12 at High pH and with RpoS Induction.

Appl Environ Microbiol 2018 08 17;84(15). Epub 2018 Jul 17.

Department of Biology, Kenyon College, Gambier, Ohio, USA

Experimental evolution of K-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS knockout of (encoding the positive regulator of the phosphate regulon). A :: knockout increased growth at high pH. mutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations in [poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions of , an inhibitor of TorR, which activates expression of (trimethylamine -oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay, deletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded by () (PhoPQ regulator), (nitrogen starvation sigma factor), , and , as well as envelope proteins, such as those encoded by and Overall, evolution at high pH selects for mutations in key transcriptional regulators, including and the stationary-phase sigma factor RpoS. in its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.
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http://dx.doi.org/10.1128/AEM.00520-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052260PMC
August 2018

Alternative pathways for Escherichia coli biofilm formation revealed by sRNA overproduction.

Mol Microbiol 2017 Jul 18;105(2):309-325. Epub 2017 May 18.

Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD, 20892, USA.

Small regulatory RNAs have major roles in many regulatory circuits in Escherichia coli and other bacteria, including the transition from planktonic to biofilm growth. We tested Hfq-dependent sRNAs in E. coli for their ability, when overproduced, to inhibit or stimulate biofilm formation, in two different growth media. We identify two mutually exclusive pathways for biofilm formation. In LB, PgaA, encoding an adhesion export protein, played a critical role; biofilm was independent of the general stress factor RpoS or CsgD, regulator of curli and other biofilm genes. The PgaA-dependent pathway was stimulated upon overproduction of DsrA, via negative regulation of H-NS, or of GadY, likely by titration of CsrA. In yeast extract casamino acids (YESCA) media, biofilm was dependent on RpoS and CsgD, but independent of PgaA; RpoS appears to indirectly negatively regulate the PgaA-dependent pathway in YESCA medium. Deletions of most sRNAs had very little effect on biofilm, although deletion of hfq, encoding an RNA chaperone, was defective in both LB and YESCA. Deletion of ArcZ, a small RNA activator of RpoS, decreased biofilm in YESCA; only a portion of this defect could be bypassed by overproduction of RpoS. Overall, sRNAs highlight different pathways to biofilm formation.
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http://dx.doi.org/10.1111/mmi.13702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5510166PMC
July 2017

Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens.

Structure 2016 06 5;24(6):965-976. Epub 2016 May 5.

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Incorporation of lipopolysaccharide (LPS) into the outer membrane of Gram-negative bacteria is essential for viability, and is accomplished by a two-protein complex called LptDE. We solved crystal structures of the core LptDE complexes from Yersinia pestis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and a full-length structure of the K. pneumoniae LptDE complex. Our structures adopt the same plug and 26-strand β-barrel architecture found recently for the Shigella flexneri and Salmonella typhimurium LptDE structures, illustrating a conserved fold across the family. A comparison of the only two full-length structures, SfLptDE and our KpLptDE, reveals a 21° rotation of the LptD N-terminal domain that may impart flexibility on the trans-envelope LptCAD scaffold. Utilizing mutagenesis coupled to an in vivo functional assay and molecular dynamics simulations, we demonstrate the critical role of Pro231 and Pro246 in the function of the LptD lateral gate that allows partitioning of LPS into the outer membrane.
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http://dx.doi.org/10.1016/j.str.2016.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899211PMC
June 2016

Stress sigma factor RpoS degradation and translation are sensitive to the state of central metabolism.

Proc Natl Acad Sci U S A 2015 Apr 6;112(16):5159-64. Epub 2015 Apr 6.

Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892

RpoS, the stationary phase/stress sigma factor of Escherichia coli, regulates a large cohort of genes important for the cell to deal with suboptimal conditions. Its level increases quickly in the cell in response to many stresses and returns to low levels when growth resumes. Increased RpoS results from increased translation and decreased RpoS degradation. Translation is positively regulated by small RNAs (sRNAs). Protein stability is positively regulated by anti-adaptors, which prevent the RssB adaptor-mediated degradation of RpoS by the ClpXP protease. Inactivation of aceE, a subunit of pyruvate dehydrogenase (PDH), was found to increase levels of RpoS by affecting both translation and protein degradation. The stabilization of RpoS in aceE mutants is dependent on increased transcription and translation of IraP and IraD, two known anti-adaptors. The aceE mutation also leads to a significant increase in rpoS translation. The sRNAs known to positively regulate RpoS are not responsible for the increased translation; sequences around the start codon are sufficient for the induction of translation. PDH synthesizes acetyl-CoA; acetate supplementation allows the cell to synthesize acetyl-CoA by an alternative, less favored pathway, in part dependent upon RpoS. Acetate addition suppressed the effects of the aceE mutant on induction of the anti-adaptors, RpoS stabilization, and rpoS translation. Thus, the bacterial cell responds to lowered levels of acetyl-CoA by inducing RpoS, allowing reprogramming of E. coli metabolism.
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http://dx.doi.org/10.1073/pnas.1504639112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413282PMC
April 2015

Production of recombinant protein by a novel oxygen-induced system in Escherichia coli.

Microb Cell Fact 2014 Apr 7;13(1):50. Epub 2014 Apr 7.

Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Background: The SoxRS regulon of E. coli is activated in response to elevated dissolved oxygen concentration likely to protect the bacteria from possible oxygen damage. The soxS expression can be increased up to 16 fold, making it a possible candidate for recombinant protein expression. Compared with the existing induction approaches, oxygen induction is advantageous because it does not involve addition or depletion of growth factors or nutrients, addition of chemical inducers or temperature changes that can affect growth and metabolism of the producing bacteria. It also does not affect the composition of the growth medium simplifying the recovery and purification processes.

Results: The soxS promoter was cloned into the commercial pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration. The efficiency and the regulatory properties of the soxS promoter were characterized by measuring the GFP expression when the culture dissolved oxygen concentration was increased from 30% to 300% air saturation. The expression level of recombinant GFP was proportional to the oxygen concentration, demonstrating that pAB49 is a controllable expression vector. A possible harmful effect of elevated oxygen concentration on the recombinant product was found to be negligible by determining the protein-carbonyl content and its specific fluorescence. By performing high density growth in modified LB medium, the cells were induced by increasing the oxygen concentration. After 3 hours at 300% air saturation, GFP fluorescence reached 109000 FU (494 mg of GFP/L), representing 3.4% of total protein, and the cell concentration reached 29.1 g/L (DW).

Conclusions: Induction of recombinant protein expression by increasing the dissolved oxygen concentration was found to be a simple and efficient alternative expression strategy that excludes the use of chemical, nutrient or thermal inducers that have a potential negative effect on cell growth or the product recovery.
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http://dx.doi.org/10.1186/1475-2859-13-50DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4234026PMC
April 2014

Reducing acetate excretion from E. coli K-12 by over-expressing the small RNA SgrS.

N Biotechnol 2013 Jan 16;30(2):269-73. Epub 2011 Nov 16.

Biotechnology Core Laboratory, NIDDK, NIH, Bethesda, MD, USA.

When exposed to the nonmetabolized glucose derivative alpha methyl glucoside (αMG), both Escherichia coli K-12 (JM109 and MG1655) and E. coli B (BL21) respond by reducing the concentration of the mRNA of the ptsG gene which is responsible for the biosynthesis of the glucose transporter EIICB(glu). This occurs through the over-expression of the noncoding small RNA SgrS, which interacts specifically with the mRNA of the ptsG gene and prevents its translation. However, when these bacteria are exposed to a glucose concentration of 40 g/L, over-expression of SgrS is observed only in E. coli B (BL21). Unlike E. coli K-12 (JM109 and MG1655), which are affected by high glucose concentration and produce higher levels of acetate, E. coli B (BL21) is not affected. Based on this information, it was assumed that over-expression of SgrS enables E. coli B (BL21) to reduce its acetate excretion by controlling the glucose transport. When SgrS was over-expressed in both E. coli K-12 strains from a multicopy plasmid, it was possible to reduce their acetate excretion levels to those seen in E. coli B. This observation opens a new approach towards controlling bacterial metabolism through the use of noncoding RNA.
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http://dx.doi.org/10.1016/j.nbt.2011.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322308PMC
January 2013

Adenovirus with hexon Tat-protein transduction domain modification exhibits increased therapeutic effect in experimental neuroblastoma and neuroendocrine tumors.

J Virol 2011 Dec 28;85(24):13114-23. Epub 2011 Sep 28.

Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185 Uppsala, Sweden.

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surfaces of tumor cells. Furthermore, infected cells overproduce adenovirus fiber proteins, which are released prior to cell lysis. The released fibers block CAR on noninfected neighboring cells, thereby preventing progeny virus entry. Our aim was to add a CAR-independent infection route to Ad5 to increase the infectivity of tumor cells with low CAR expression and prevent the fiber-masking problem. We constructed Ad5 viruses that encode the protein transduction domain (PTD) of the HIV-1 Tat protein (Tat-PTD) in hypervariable region 5 (HVR5) of the hexon protein. Tat-PTD functions as a cell-penetrating peptide, and Tat-PTD-modified Ad5 showed a dramatic increased transduction of CAR-negative cell lines compared to unmodified vector. Moreover, while tumor cell infectivity was severely reduced for Ad5 in the presence of fiber proteins, it was only marginally reduced for Tat-PTD-modified Ad5. Furthermore, because of the sequence alteration in the hexon HVR, coagulation factor X-mediated virus uptake was significantly reduced. Mice harboring human neuroblastoma and neuroendocrine tumors show suppressed tumor growths and prolonged survival when treated with Tat-PTD-modified oncolytic viruses. Our data suggest that modification of Ad5 with Tat-PTD in HVR5 expands its utility as an oncolytic agent.
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http://dx.doi.org/10.1128/JVI.05759-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3233137PMC
December 2011

The RpoS-mediated general stress response in Escherichia coli.

Annu Rev Microbiol 2011 ;65:189-213

Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA.

Under conditions of nutrient deprivation or stress, or as cells enter stationary phase, Escherichia coli and related bacteria increase the accumulation of RpoS, a specialized sigma factor. RpoS-dependent gene expression leads to general stress resistance of cells. During rapid growth, RpoS translation is inhibited and any RpoS protein that is synthesized is rapidly degraded. The complex transition from exponential growth to stationary phase has been partially dissected by analyzing the induction of RpoS after specific stress treatments. Different stress conditions lead to induction of specific sRNAs that stimulate RpoS translation or to induction of small-protein antiadaptors that stabilize the protein. Recent progress has led to a better, but still far from complete, understanding of how stresses lead to RpoS induction and what RpoS-dependent genes help the cell deal with the stress.
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http://dx.doi.org/10.1146/annurev-micro-090110-102946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356644PMC
January 2012

Mechanism of positive regulation by DsrA and RprA small noncoding RNAs: pairing increases translation and protects rpoS mRNA from degradation.

J Bacteriol 2010 Nov 27;192(21):5559-71. Epub 2010 Aug 27.

Laboratory of Molecular Biology, Bldg. 37, Room 5132, National Institutes of Health, Bethesda, MD 20892-4255, USA.

Small noncoding RNAs (sRNAs) regulate gene expression in Escherichia coli by base pairing with mRNAs and modulating translation and mRNA stability. The sRNAs DsrA and RprA stimulate the translation of the stress response transcription factor RpoS by base pairing with the 5' untranslated region of the rpoS mRNA. In the present study, we found that the rpoS mRNA was unstable in the absence of DsrA and RprA and that expression of these sRNAs increased both the accumulation and the half-life of the rpoS mRNA. Mutations in dsrA, rprA, or rpoS that disrupt the predicted pairing sequences and reduce translation of RpoS also destabilize the rpoS mRNA. We found that the rpoS mRNA accumulates in an RNase E mutant strain in the absence of sRNA expression and, therefore, is degraded by an RNase E-mediated mechanism. DsrA expression is required, however, for maximal translation even when rpoS mRNA is abundant. This suggests that DsrA protects rpoS mRNA from degradation by RNase E and that DsrA has a further activity in stimulating RpoS protein synthesis. rpoS mRNA is subject to degradation by an additional pathway, mediated by RNase III, which, in contrast to the RNase E-mediated pathway, occurs in the presence and absence of DsrA or RprA. rpoS mRNA and RpoS protein levels are increased in an RNase III mutant strain with or without the sRNAs, suggesting that the role of RNase III in this context is to reduce the translation of RpoS even when the sRNAs are acting to stimulate translation.
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http://dx.doi.org/10.1128/JB.00464-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953674PMC
November 2010

Positive regulation by small RNAs and the role of Hfq.

Proc Natl Acad Sci U S A 2010 May 10;107(21):9602-7. Epub 2010 May 10.

Johns Hopkins University, Program in Cell, Molecular, Developmental Biology and Biophysics, Baltimore, MD 21218, USA.

Bacterial small noncoding RNAs carry out both positive and negative regulation of gene expression by pairing with mRNAs; in Escherichia coli, this regulation often requires the RNA chaperone Hfq. Three small regulatory RNAs (sRNAs), DsrA, RprA, and ArcZ, positively regulate translation of the sigma factor RpoS, each pairing with the 5' leader to open up an inhibitory hairpin. In vitro, rpoS interaction with sRNAs depends upon an (AAN)(4) Hfq-binding site upstream of the pairing region. Here we show that both Hfq and this Hfq binding site are required for RprA or ArcZ to act in vivo and to form a stable complex with rpoS mRNA in vitro; both were partially dispensable for DsrA at 37 degrees C. ArcZ sRNA is processed from 121 nt to a stable 56 nt species that contains the pairing region; only the 56 nt ArcZ makes a strong Hfq-dependent complex with rpoS. For each of these sRNAs, the stability of the sRNA*mRNA complexes, rather than their rate of formation, best predicted in vivo activity. These studies demonstrate that binding of Hfq to the rpoS mRNA is critical for sRNA regulation under normal conditions, but if the stability of the sRNA*mRNA complex is sufficiently high, the requirement for Hfq can be bypassed.
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http://dx.doi.org/10.1073/pnas.1004435107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2906882PMC
May 2010

Genetic dissection of signaling through the Rcs phosphorelay.

Methods Enzymol 2007 ;423:349-62

Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD, USA.

The Rcs phosphorelay, consisting of a hybrid sensor kinase, a phosphotransferase, and a response regulator, regulates a large number of bacterial functions. These include capsule production, the target originally defined for these regulators, a small regulatory RNA, and a growing list of additional genes, many of unknown function. At the core of this phosphorelay is the response regulator RcsB that activates the expression of the target genes. In addition to RcsB, some but not all of these targets require a co-regulator. One such co-regulator is RcsA, which has not been described as working except with RcsB; RcsA is itself regulated at both the transcriptional and post-transcriptional levels. Signaling to the system is also complex, and numerous plasmids, mutations, and environmental conditions have been described as activating this system. Activation of the system on cell surfaces and the nature of some of the regulated functions suggest a role for this phosphorelay in biofilm formation. Here, we describe reporters and mutants that allow the genetic dissection of the system from two directions. In cases where a condition activates the system, for instance, causing an increase in capsule synthesis (a phenotype easily observed in colonies), specific tests can identify at what stage the signal feeds into the system. In cases where a target of the phosphorelay is identified, specific tests can define the genetic requirements for regulation of the target. Finally, in cases where overproduction of capsule interferes with other studies, mutants allow the study of cells in the absence of capsule formation.
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http://dx.doi.org/10.1016/S0076-6879(07)23016-2DOI Listing
January 2008

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

J Bacteriol 2005 Oct;187(19):6770-8

National Cancer Institute, 9000 Rockville Pike, Bldg. 37, Bethesda, MD 20892, USA.

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.
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http://dx.doi.org/10.1128/JB.187.19.6770-6778.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251585PMC
October 2005

The Rcs phosphorelay: a complex signal transduction system.

Annu Rev Microbiol 2005 ;59:379-405

Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA.

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.
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http://dx.doi.org/10.1146/annurev.micro.59.050405.101230DOI Listing
January 2006

Bacterial small RNA regulators.

Crit Rev Biochem Mol Biol 2005 Mar-Apr;40(2):93-113

Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892, USA.

Small regulatory RNAs can modify the activity of proteins and the stability and translation of mRNAs. They have now been found in a wide range of organisms, and can play previously unsuspected critical regulatory roles. The bacterial small RNAs include two major classes. The largest family(with at least 20 members in Escherichia coli K12) acts by base pairing with target mRNAs to modify mRNA translation or stability; this class of RNAs also uses an RNA chaperone protein, Hfq. DsrA is the best-studied example of this family of RNAs. It has been shown to positively regulate translation of the transcription factor RpoS by opening an inhibitory hairpin in the mRNA, and to negatively regulate translation of hns by pairing just beyond the translation initiation codon. The class of RNAs that modify activity of proteins is exemplified by CsrB and CsrC of E. coli, two RNAs that bind to and inhibit CsrA, a protein translational regulator. Homologs of CsrA and related regulatory RNAs have been implicated in the regulation of gluconeogenesis, biofilm formation,and virulence factor expression in plant and human pathogens.
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http://dx.doi.org/10.1080/10409230590918702DOI Listing
September 2005

Regulatory roles for small RNAs in bacteria.

Curr Opin Microbiol 2003 Apr;6(2):120-4

Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892, USA.

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.
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http://dx.doi.org/10.1016/s1369-5274(03)00027-4DOI Listing
April 2003

Regulation and mode of action of the second small RNA activator of RpoS translation, RprA.

Mol Microbiol 2002 Nov;46(3):813-26

Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA.

Translation of the stationary phase sigma factor RpoS is stimulated by at least two small RNAs, DsrA and RprA. DsrA disrupts an inhibitory secondary structure in the rpoS leader mRNA by pairing with the upstream RNA. Mutations in rprA and compensating mutations in the rpoS leader demonstrate that RprA interacts with the same region of the RpoS leader as DsrA. This is the first example of two different small RNAs regulating a common target. Regulation of these RNAs differs. DsrA synthesis is increased at low temperature. We find that RprA synthesis is regulated by the RcsC/RcsB phosphorelay system, previously found to regulate capsule synthesis and promoters of ftsZ and osmC. An rcsB null mutation abolishes the basal level, whereas mutations in rcsC that activate capsule synthesis also activate expression of the rprA promoter. An essential site with similarity to other RcsB-regulated promoters was defined in the rprA promoter. Activation of the RcsC/RcsB system leads to increased RpoS synthesis, in an RprA-dependent fashion. This work suggests a new signal for RpoS translation and extends the global regulation effected by the RcsC/RcsB system to coregulation of RpoS with capsule and FtsZ.
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http://dx.doi.org/10.1046/j.1365-2958.2002.03203.xDOI Listing
November 2002