The Listeria monocytogenes σB Regulon and Its Virulence-Associated Functions Are Inhibited by a Small Molecule

The stress-responsive alternative sigma factor σB is conserved across diverse Gram-positive bacterial genera. In Listeria monocytogenes, σB regulates transcription of >150 genes, including genes contributing to virulence and to bacterial survival under host-associated stress conditions, such as those encountered in the human gastrointestinal lumen. An inhibitor of L. monocytogenes σB activity was identified by screening ~57,000 natural and synthesized small molecules using a high-throughput cell-based assay. The compound fluoro-phenyl-styrene-sulfonamide (FPSS) (IC50 = 3.5 µM) downregulated the majority of genes previously identified as members of the σB regulon in L. monocytogenes 10403S, thus generating a transcriptional profile comparable to that of a 10403S ΔsigB strain. Specifically, of the 208 genes downregulated by FPSS, 75% had been identified previously as positively regulated by σB. Downregulated genes included key virulence and stress response genes, such as inlA, inlB, bsh, hfq, opuC, and bilE. From a functional perspective, FPSS also inhibited L. monocytogenes invasion of human intestinal epithelial cells and bile salt hydrolase activity. The ability of FPSS to inhibit σB activity in both L. monocytogenes and Bacillus subtilis indicates its utility as a specific inhibitor of σB across multiple Gram-positive genera.  The σB transcription factor regulates expression of genes responsible for bacterial survival under changing environmental conditions and for virulence; therefore, this alternative sigma factor is important for transmission of L. monocytogenes and other Gram-positive bacteria. Regulation of σB activity is complex and tightly controlled, reflecting the key role of this factor in bacterial metabolism. We present multiple lines of evidence indicating that fluoro-phenyl-styrene-sulfonamide (FPSS) specifically inhibits activity of σB across Gram-positive bacterial genera, i.e., in both Listeria monocytogenes and Bacillus subtilis. Therefore, FPSS is an important new tool that will enable novel approaches for exploring complex regulatory networks in L. monocytogenes and other Gram-positive pathogens and for investigating small-molecule applications for controlling pathogen transmission. The σB transcription factor regulates expression of genes responsible for bacterial survival under changing environmental conditions and for virulence; therefore, this alternative sigma factor is important for transmission of L. monocytogenes and other Gram-positive bacteria. Regulation of σB activity is complex and tightly controlled, reflecting the key role of this factor in bacterial metabolism. We present multiple lines of evidence indicating that fluoro-phenyl-styrene-sulfonamide (FPSS) specifically inhibits activity of σB across Gram-positive bacterial genera, i.e., in both Listeria monocytogenes and Bacillus subtilis. Therefore, FPSS is an important new tool that will enable novel approaches for exploring complex regulatory networks in L. monocytogenes and other Gram-positive pathogens and for investigating small-molecule applications for controlling pathogen transmission.

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title: Modulation of stress and virulence in Listeria monocytogenes.
abstract: Listeria monocytogenes can respond rapidly to changing environmental conditions, as illustrated by its ability to transition from a saprophyte to an orally transmitted facultative intracellular pathogen. Differential associations between various alternative sigma factors and a core RNA polymerase provide a transcriptional mechanism for regulating bacterial gene expression that is crucial for survival in rapidly changing conditions. Alternative sigma factors are key components of complex L. monocytogenes regulatory networks that include multiple transcriptional regulators of stress-response and virulence genes, regulation of genes encoding other regulators, and regulation of small RNAs. In this article, the contributions of various sigma factors to L. monocytogenes stress response and virulence are described.

title: The Listeria transcriptional landscape from saprophytism to virulence
abstract: The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5′ and 3′ untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

title: Listeria monocytogenes σB Regulates Stress Response and Virulence Functions
abstract: While the stress-responsive alternative sigma factor σB has been identified in different species of Bacillus, Listeria, and Staphylococcus, theσ B regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify σB-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidateσ B-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted σB-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and ΔsigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significantσ B-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting σB-dependent expression, 54 were preceded by a sequence resembling the σB promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the σB-dependent nature of a subset of eight selected promoter regions. Notably, theσ B-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, σB also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest thatσ B contributes to L. monocytogenes gene expression during infection.

title: Comparative Analysis of the σB-Dependent Stress Responses in Listeria monocytogenes and Listeria innocua Strains Exposed to Selected Stress Conditions
abstract: The alternative sigma factor σB contributes to transcription of stress response and virulence genes in diverse gram-positive bacterial species. The composition and functions of the Listeria monocytogenes and Listeria innocua σB regulons were hypothesized to differ due to virulence differences between these closely related species. Transcript levels in stationary-phase cells and in cells exposed to salt stress were characterized by microarray analyses for both species. In L. monocytogenes, 168 genes were positively regulated by σB; 145 of these genes were preceded by a putative σB consensus promoter. In L. innocua, 64 genes were positively regulated by σB. σB contributed to acid stress survival in log-phase cells for both species but to survival in stationary-phase cells only for L. monocytogenes. In summary, (i) the L. monocytogenes σB regulon includes >140 genes that are both directly and positively regulated by σB, including genes encoding proteins with importance in stress response, virulence, transcriptional regulation, carbohydrate metabolism, and transport; (ii) a number of L. monocytogenes genes encoding flagellar proteins show higher transcript levels in the ΔsigB mutant, and both L. monocytogenes and L. innocua ΔsigB null mutants have increased motility compared to the respective isogenic parent strains, suggesting that σB affects motility and chemotaxis; and (iii) although L. monocytogenes and L. innocua differ in σB-dependent acid stress resistance and have species-specific σB-dependent genes, the L. monocytogenes and L. innocua σB regulons show considerable conservation, with a common set of at least 49 genes that are σB dependent in both species.

title: Sigma B Contributes to Listeria monocytogenes Gastrointestinal Infection but Not to Systemic Spread in the Guinea Pig Infection Model
abstract: Contributions of the alternative sigma factor σB to Listeria monocytogenes infection were investigated using strains bearing null mutations in sigB, prfA, or inlA or in selected inlA or prfA promoter regions. The ΔP4inlA strain, which has a deletion in the σB-dependent P4inlA promoter, and the ΔsigB strain had significantly reduced invasion efficiencies relative to that of the wild-type strain in the Caco-2 human colorectal epithelial cell line, while the invasion efficiency of a strain bearing a deletion in the partially σB dependent P2prfA promoter region did not differ from that of the wild type. The virulence of the ΔsigB and ΔP4inlA strains was attenuated in intragastrically inoculated guinea pigs, with the ΔsigB strain showing greater attenuation, while the virulence capacity of the ΔP2prfA strain was similar to that of the wild-type strain, suggesting that attenuation of virulence due to the ΔsigB mutation does not result from loss of σB-dependent prfA transcription. Our results show that σB-dependent activation of inlA is important for cell invasion and gastrointestinal infection and suggest that σB-regulated genes in addition to inlA appear to contribute to gastrointestinal infection. Interestingly, the virulence of the ΔsigB strain was not attenuated in intravenously infected guinea pigs. We conclude that (i) L. monocytogenes σB plays a critical role in invasion of human host cells, (ii) σB-mediated contributions to invasion are, in part, due to direct effects on inlA transcription but not on prfA transcription, and (iii) σB plays a critical role during the gastrointestinal stage of listeriosis in the guinea pig but is not important for systemic spread of the organism.

title: In Vivo Transcriptional Profiling of Listeria monocytogenes and Mutagenesis Identify New Virulence Factors Involved in Infection
abstract: Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host–pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, ≈20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence.

title: Sigma B Contributes to PrfA-Mediated Virulence in Listeria monocytogenes
abstract: Transcription of the Listeria monocytogenes positive regulatory factor A protein (PrfA) is initiated from either of two promoters immediately upstream of prfA (prfAp1 and prfAp2) or from the upstream plcA promoter. We demonstrate that prfAp2 is a functional σB-dependent promoter and that a sigB deletion mutation affects the virulence phenotype of L. monocytogenes. Thus, the alternative sigma factor σB contributes to virulence in L. monocytogenes.

title: SigB-Dependent In Vitro Transcription of prfA and Some Newly Identified Genes of Listeria monocytogenes Whose Expression Is Affected by PrfA In Vivo
abstract: Recent studies have identified several new genes in Listeria monocytogenes which are positively or negatively affected by PrfA and grouped into three classes (E. Milohanic et al.,  2003). In vitro transcription performed with promoters of some class III genes showed strict SigB-dependent but PrfA-independent transcription initiation. Transcription starting at the prfA promoter PprfA2 was also optimal with SigB-loaded RNA polymerase, suggesting a direct link between SigB- and PrfA-dependent gene expression.

title: The Listeria monocytogenes prfAP2 promoter is regulated by sigma B in a growth phase dependent manner.
abstract: Listeria monocytogenes prfA, encoding positive regulatory factor A, is transcribed from three promoters (prfAP1, prfAP2, and PplcA). The prfAP2 promoter was previously proposed to be sigma B (sigma(B))-dependent. This hypothesis was tested by creating prfA promoter-gus transcriptional fusions in both L. monocytogenes wild-type (wt) and DeltasigB backgrounds and then measuring (i) beta-glucuronidase (GUS) activities; (ii) gus mRNA transcript levels; and (iii) the presence or absence of GUS in cells by immunofluorescence staining. prfAP2-directed expression increased as the wt L. monocytogenes strain entered stationary phase, whereas prfAP2-directed expression was greatly reduced in the DeltasigB strain, confirming both growth phase- and sigma(B)-dependent transcription of prfAP2. We conclude that prfAP2 is directly regulated by sigma(B).

title: Small-Molecule Inhibitor of Vibrio cholerae Virulence and Intestinal Colonization
abstract: Increasing antibiotic resistance requires the development of new approaches to combating infection. Virulence gene expression in vivo represents a target for antibiotic discovery that has not yet been explored. A high-throughput, phenotypic screen was used to identify a small molecule 4-[N-(1,8-naphthalimide)]-n-butyric acid, virstatin, that inhibits virulence regulation in Vibrio cholerae. By inhibiting the transcriptional regulator ToxT, virstatin prevents expression of two critical V. cholerae virulence factors, cholera toxin and the toxin coregulated pilus. Orogastric administration of virstatin protects infant mice from intestinal colonization by V. cholerae.

title: Analysis of the Role of OpuC, an Osmolyte Transport System, in Salt Tolerance and Virulence Potential of Listeria monocytogenes
abstract: The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the external environment prior to ingestion and subsequently within the animal host. Growth at high salt concentrations and low temperatures is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We utilized a novel system for generating chromosomal mutations (based on a lactococcal pWVO1-derived Ori+ RepA− vector, pORI19) to identify a listerial OpuC homologue. Mutating the operon in two strains of L. monocytogenes revealed significant strain variation in the observed activity of OpuC. Radiolabeled osmolyte uptake studies, together with growth experiments in defined media, linked OpuC to carnitine and glycine betaine uptake inListeria. We also investigated the role of OpuC in contributing to the growth and survival of Listeria in an animal (murine) model of infection. Altering OpuC resulted in a significant reduction in the ability of Listeria to colonize the upper small intestine and cause subsequent systemic infection following peroral inoculation.

title: Deep RNA sequencing of L. monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs
abstract: BackgroundIdentification of specific genes and gene expression patterns important for bacterial survival, transmission and pathogenesis is critically needed to enable development of more effective pathogen control strategies. The stationary phase stress response transcriptome, including many σB-dependent genes, was defined for the human bacterial pathogen Listeria monocytogenes using RNA sequencing (RNA-Seq) with the Illumina Genome Analyzer. Specifically, bacterial transcriptomes were compared between stationary phase cells of L. monocytogenes 10403S and an otherwise isogenic ΔsigB mutant, which does not express the alternative σ factor σB, a major regulator of genes contributing to stress response, including stresses encountered upon entry into stationary phase.ResultsOverall, 83% of all L. monocytogenes genes were transcribed in stationary phase cells; 42% of currently annotated L. monocytogenes genes showed medium to high transcript levels under these conditions. A total of 96 genes had significantly higher transcript levels in 10403S than in ΔsigB, indicating σB-dependent transcription of these genes. RNA-Seq analyses indicate that a total of 67 noncoding RNA molecules (ncRNAs) are transcribed in stationary phase L. monocytogenes, including 7 previously unrecognized putative ncRNAs. Application of a dynamically trained Hidden Markov Model, in combination with RNA-Seq data, identified 65 putative σB promoters upstream of 82 of the 96 σB-dependent genes and upstream of the one σB-dependent ncRNA. The RNA-Seq data also enabled annotation of putative operons as well as visualization of 5'- and 3'-UTR regions.ConclusionsThe results from these studies provide powerful evidence that RNA-Seq data combined with appropriate bioinformatics tools allow quantitative characterization of prokaryotic transcriptomes, thus providing exciting new strategies for exploring transcriptional regulatory networks in bacteria.See minireivew http://jbiol.com/content/8/12/107.

title: Invasiveness is a variable and heterogeneous phenotype in Listeria monocytogenes serotype strains.
abstract: The ability of Listeria monocytogenes to breach mucosal and endothelial barriers of the host during infection is a hallmark property mediated by the internalins (Inl) A and B. We examined the invasive property of several L. monocytogenes strains representing 13 serotypes. We found that invasiveness is a heterogeneous phenotype amongst L. monocytogenes serotype strains. Despite this, many of the poorly invasive and non-invasive strains of L. monocytogenes express internalins at levels comparable to those of invasive isolates. Introduction of the inlAB locus from EGD-e into several poorly invasive strains had no effect on their invasive properties. A strain from serotype 4b that exhibits highly invasive properties was further examined. Deletion of the inlAB locus abrogated invasion of this strain while reintroduction of the inlAB locus into this strain restored invasiveness. An analysis of regions flanking the inlAB locus revealed considerable differences in the strains studied. Our results suggest that efficacious entry of L. monocytogenes into eukaryotic cells is complex and requires additional factors apart from internalins. Data presented here also suggest that the inlAB locus was introduced into L. monocytogenes by horizontal gene transfer with subsequent deletion and rearrangements occurring during evolution of this species.

title: Identification of the Gene Encoding the Alternative Sigma Factor ςB from Listeria monocytogenesand Its Role in Osmotolerance
abstract: Listeria monocytogenes is well known for its robust physiology, which permits growth at low temperatures under conditions of high osmolarity and low pH. Although studies have provided insight into the mechanisms used by L. monocytogenes to allay the physiological consequences of these adverse environments, little is known about how these responses are coordinated. In the studies presented here, we have cloned the sigB gene and severalrsb genes from L. monocytogenes, encoding homologs of the alternative sigma factor ςB and the RsbUVWX proteins, which govern transcription of a general stress regulon in the related bacterium Bacillus subtilis. TheL. monocytogenes and B. subtilis sigB andrsb genes are similar in sequence and physical organization; however, we observed that the activity of ςB in L. monocytogenes was uniquely responsive to osmotic upshifting, temperature downshifting, and the presence of EDTA in the growth medium. The magnitude of the response was greatest after an osmotic upshift, suggesting a role for ςB in coordinating osmotic responses in L. monocytogenes. A null mutation in the sigB gene led to substantial defects in the ability of L. monocytogenesto use betaine and carnitine as osmoprotectants. Subsequent measurements of betaine transport confirmed that the absence of ςB reduced the ability of the cells to accumulate betaine. Thus, ςB coordinates responses to a variety of physical and chemical signals, and its function facilitates the growth of L. monocytogenes under conditions of high osmotic strength.

title: Intracellular Gene Expression Profile of Listeria monocytogenes
abstract: Listeria monocytogenes is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality. L. monocytogenes is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with L. monocytogenes strain EGD-e and examined the gene expression profile of L. monocytogenes inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that ∼17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic Listeria innocua CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by L. monocytogenes to escape the host cell responses.

title: The human anionic antimicrobial peptide dermcidin induces proteolytic defence mechanisms in staphylococci
abstract: Antimicrobial peptides (AMPs) represent a key component of innate host defence against bacterial pathogens. Bacterial resistance mechanisms usually depend on the characteristic positive charge of AMPs. However, several human cell types also produce anionic AMPs, mechanisms of resistance to which are poorly understood. Here we demonstrate that the skin commensal and leading nosocomial pathogen Staphylococcus epidermidis senses and efficiently inactivates the anionic AMP dermcidin. Dermcidin induced differential expression of global regulatory systems, leading to increased expression of proteases with the capacity to degrade dermcidin, particularly S. epidermidis SepA. A similar induction of extracellular proteolytic activity was found in Staphylococcus aureus, suggesting a common regulatory mechanism in staphylococci. Notably, human cationic AMPs also led to the activation of global regulators, but inactivation of dermcidin by SepA was much more effective than of the tested cationic peptides. The ability to react to the unusual, anionic dermcidin with effective countermeasures likely contributes to the extraordinary success of staphylococci as colonizers and infective agents on human epithelia. Our study indicates that staphylococci can react to human AMPs by specific mechanisms of resistance and establishes a crucial role for staphylococcal proteases in the interaction with human innate host defence.

title: GW domains of the Listeria monocytogenes invasion protein InlB are SH3‐like and mediate binding to host ligands
abstract: InlB, a surface‐localized protein of Listeria monocytogenes, induces phagocytosis in non‐phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q‐R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine‐rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline‐binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q‐R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB‐induced host membrane dynamics with bacterial proximity during invasion.

title: Identification of Listeria monocytogenes Genes Contributing to Intracellular Replication by Expression Profiling and Mutant Screening
abstract: A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.

title: Response regulator DegU of Listeria monocytogenes regulates the expression of flagella-specific genes.
abstract: An isogenic mutant of Listeria monocytogenes EGD with a deletion of the response regulator gene degU showed a lack of motility due to the absence of flagella. In the present study, we used two-dimensional gel electrophoresis, mass-spectrometry and microarray analyses to identify the listerial genes that depend on DegU for expression. We found that the two L. monocytogenes operons encoding flagella-specific genes and the monocistronically transcribed flaA gene are positively regulated by DegU at 24 degrees C, but are not expressed at 37 degrees C.

title: Characterization of DegU, a response regulator in Listeria monocytogenes, involved in regulation of motility and contributes to virulence.
abstract: The degU (lmo2515) gene encodes a putative response regulator in the food-borne pathogen Listeria monocytogenes. It has 63% amino acid identity to the DegU response regulator of Bacillus subtilis. We have characterized the degU gene product in L. monocytogenes EGD by generation of a deletion mutant. The DeltadegU mutant was found to be non-motile in motility plate assay and no flagellin was detected. The mutant was attenuated in challenge of mice. Northern blot analysis suggested that the degU gene product is a transcriptional activator of the flagellin gene, flaA, at 25 degrees C. However, the degU gene product had no influence on the transcription of prfA encoding the major virulence regulator, PrfA. The results indicate that the putative DegU response regulator is a pleiotropic regulator involved in expression of both motility at low temperature and in vivo virulence in mice.

title: Characterization of relA and codY mutants of Listeria monocytogenes: identification of the CodY regulon and its role in virulence
abstract: Listeria monocytogenes is a Gram‐positive intracellular parasite and the causative organism of human listeriosis. In this article we demonstrate that L. monocytogenes encodes a functional member of the CodY family of global regulatory proteins that is responsive to both GTP and branched chain amino acids. By transcript analyses we identified the CodY regulon in L. monocytogenes and demonstrated that it comprises genes involved in amino acid metabolism, nitrogen assimilation as well as genes involved in sugar uptake and incorporation, indicating a role for CodY in L. monocytogenes in both carbon and nitrogen assimilation. A ΔrelA mutation reduced expression of the CodY regulon in early stationary phase and introduction of a ΔcodY mutation into a ΔrelA strain restored virulence. These data indicate that the avirulence of the ΔrelA mutant can in part be explained by the continued repression of the CodY regulon. The phenotypes of ΔrelA and ΔcodY mutants were studied in J774.A1 and Caco‐2 cells and the ΔrelA mutation shown to effect intracellular growth. These results provide the first direct evidence that the activity of a CodY‐type protein influences pathogenesis and provides new information on the physiological adaptation of L. monocytogenes to post‐exponential phase growth and virulence.

title: Listeria monocytogenes regulates flagellar motility gene expression through MogR, a transcriptional repressor required for virulence.
abstract: Previous studies have shown that Listeria monocytogenes flagellar motility genes, including flaA, encoding flagellin, are transcriptionally down-regulated at 37 degrees C. For some L. monocytogenes strains, temperature-dependent motility gene expression is less stringent. By using flaA-lacZ transcriptional fusions, we identified regions upstream of the -35/-10 promoter elements that are necessary for temperature-dependent expression of flaA in L. monocytogenes strain EGDe. Whereas the sequence of the flaA promoter region was identical in L. monocytogenes strain 10403S, transcriptional activity was only partially down-regulated at 37 degrees C in 10403S. This finding suggested that a transacting regulatory protein with differential expression or activity in EGDe might be involved in temperature-dependent transcription of flaA. Indeed, a protein factor capable of specifically binding to the flaA promoter region was identified in cytoplasmic extracts of EGDe by using affinity purification and MS. Deletion of the factor-encoding gene (lmo0674) resulted in loss of temperature-dependent flaA expression and an increase in flaA promoter activity. Expression of other motility genes was also deregulated in the lmo0674 deletion. We have designated lmo0674 as mogR, indicating its role as a motility gene repressor. In tissue culture models, MogR repression of flaA during intracellular infection was independent of temperature and a deletion of mogR reduced the capacity for cell-to-cell spread. During in vivo infection, a deletion of mogR resulted in a 250-fold decrease in virulence. These studies indicate that regulation of flagellar motility gene expression and/or other genes controlled by MogR is required for full virulence of L. monocytogenes.

title: The MogR Transcriptional Repressor Regulates Nonhierarchal Expression of Flagellar Motility Genes and Virulence in Listeria monocytogenes
abstract: Flagella are surface structures critical for motility and virulence of many bacterial species. In Listeria monocytogenes, MogR tightly represses expression of flagellin (FlaA) during extracellular growth at 37 °C and during intracellular infection. MogR is also required for full virulence in a murine model of infection. Using in vitro and in vivo infection models, we determined that the severe virulence defect of MogR-negative bacteria is due to overexpression of FlaA. Specifically, overproduction of FlaA in MogR-negative bacteria caused pleiotropic defects in bacterial division (chaining phenotype), intracellular spread, and virulence in mice. DNA binding and microarray analyses revealed that MogR represses transcription of all known flagellar motility genes by binding directly to a minimum of two TTTT-N5-AAAA recognition sites positioned within promoter regions such that RNA polymerase binding is occluded. Analysis of MogR protein levels demonstrated that modulation of MogR repression activity confers the temperature-specificity to flagellar motility gene expression. Epistasis analysis revealed that MogR repression of transcription is antagonized in a temperature-dependent manner by the DegU response regulator and that DegU further regulates FlaA levels through a posttranscriptional mechanism. These studies provide the first known example to our knowledge of a transcriptional repressor functioning as a master regulator controlling nonhierarchal expression of flagellar motility genes.

title: A Small-Molecule Screen Identifies the Antipsychotic Drug Pimozide as an Inhibitor of Listeria monocytogenes Infection
abstract: We developed a screening procedure to identify small-molecule compounds that altered infection by Listeria monocytogenes to gain insights into bacterial/host cellular processes required for intracellular pathogenesis. A small-molecule library of 480 compounds with known biological functions was screened, and 21 compounds that altered the L. monocytogenes infection of murine bone marrow-derived macrophages (BMM) were identified. The identified compounds affected various cellular functions, such as actin polymerization, kinase/phosphatase activity, calcium signaling, and apoptosis. Pimozide, an FDA-approved drug used to treat severe Tourette's syndrome and schizophrenia, was further examined and shown to decrease the bacterial uptake and vacuole escape of L. monocytogenes in BMM. The inhibitory effect of pimozide on internalization was not specific for L. monocytogenes, as the phagocytosis of other bacterial species (Bacillus subtilis, Salmonella enterica serovar Typhimurium, and Escherichia coli K12) was significantly inhibited in the presence of pimozide. The invasion and cell-to-cell spread of L. monocytogenes during the infection of nonprofessional phagocytic cells also was decreased by pimozide treatment. Although pimozide has been reported to be an antagonist of mammalian cell calcium channels, the infection of BMM in a calcium-free medium did not relieve the inhibitory effects of pimozide on L. monocytogenes infection. Our results provide a generalizable screening approach for identifying small-molecule compounds that affect cellular pathways that are required for intracellular bacterial pathogenesis. We also have identified pimozide, a clinically approved antipsychotic drug, as a compound that may be suitable for further development as a therapeutic for intracellular bacterial infections.

title: The Alternative Sigma Factor σB of Bacillus cereus: Response to Stress and Role in Heat Adaptation
abstract: A gene cluster encoding the alternative sigma factor σB, three predicted regulators of σB (RsbV, RsbW, and RsbY), and one protein whose function is not known (Orf4) was identified in the genome sequence of the food pathogen Bacillus cereus ATCC 14579. Western blotting with polyclonal antibodies raised against σB revealed that there was 20.1-fold activation of σB after a heat shock from 30 to 42°C. Osmotic upshock and ethanol exposure also upregulated σB, albeit less than a heat shock. When the intracellular ATP concentration was decreased by exposure to carbonyl cyanide m-chlorophenylhydrazone (CCCP), only limited increases in σB levels were observed, revealing that stress due to ATP depletion is not an important factor in σB activation in B. cereus. Analysis of transcription of the sigB operon by Northern blotting and primer extension revealed the presence of a σB-dependent promoter upstream of the first open reading frame (rsbV) of the sigB operon, indicating that transcription of sigB is autoregulated. A second σB-dependent promoter was identified upstream of the last open reading frame (orf4) of the sigB operon. Production of virulence factors and the nonhemolytic enterotoxin Nhe in a sigB null mutant was the same as in the parent strain. However, σB was found to play a role in the protective heat shock response of B. cereus. The sigB null mutant was less protected against the lethal temperature of 50°C by a preadaptation to 42°C than the parent strain was, resulting in a more-than-100-fold-reduced survival of the mutant after 40 min at 50°C.

title: Vancomycin Activates σB in Vancomycin-Resistant Staphylococcus aureus Resulting in the Enhancement of Cytotoxicity
abstract: The alternative transcription factor σB is responsible for transcription in Staphylococcus aureus during the stress response. Many virulence-associated genes are directly or indirectly regulated by σB. We hypothesized that treatment with antibiotics may act as an environmental stressor that induces σB activity in antibiotic-resistant strains. Several antibiotics with distinct modes of action, including ampicillin (12 µg/ml), vancomycin (16 or 32 µg/ml), chloramphenicol (15 µg/ml), ciprofloxacin (0.25 µg/ml), and sulfamethoxazole/trimethoprim (SXT, 0.8 µg/ml), were investigated for their ability to activate this transcription factor. We were especially interested in the stress response in vancomycin-resistant S. aureus (VRSA) strains treated with vancomycin. The transcription levels of selected genes associated with virulence were also measured. Real-time quantitative reverse transcription PCR was employed to evaluate gene transcription levels. Contact hemolytic and cytotoxicity assays were used to evaluate cell damage following antibiotic treatment. Antibiotics that target the cell wall (vancomycin and ampicillin) and SXT induced σB activity in VRSA strains. Expression of σB-regulated virulence genes, including hla and fnbA, was associated with the vancomycin-induced σB activity in VRSA strains and the increase in cytotoxicity upon vancomycin treatment. These effects were not observed in the sigB-deficient strain but were observed in the complemented strain. We demonstrate that sub-minimum inhibitory concentration (sub-MIC) levels of antibiotics act as environmental stressors and activate the stress response sigma factor, σB. The improper use of antibiotics may alter the expression of virulence factors through the activation of σB in drug-resistant strains of S. aureus and lead to worse clinical outcomes.

title: Biofilm Formation by Staphylococcus epidermidis Depends on Functional RsbU, an Activator of thesigB Operon: Differential Activation Mechanisms Due to Ethanol and Salt Stress
abstract: Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of theicaADBC gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn917 insertions of two isogenic,icaADBC-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium S. epidermidis 1457. The insertion site was the same in both of the mutants and was located in the first gene, rsbU, of an operon highly homologous to thesigB operons of Staphylococcus aureus andBacillus subtilis. Supplementation of Trypticase soy broth with NaCl (TSBNaCl) or ethanol (TSBEtOH), both of which are known activators of sigB, led to increased biofilm formation and PIA synthesis by S. epidermidis 1457. Insertion of Tn917 into rsbU, a positive regulator of alternative sigma factor ςB, led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSBEtOH, the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSBNaCl, the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn917 insertion of mutant M15 to the independent and biofilm-producing strain S. epidermidis8400. In 11 clinical S. epidermidis strains, a restriction fragment length polymorphism of the sigB operon was detected which was independent of the presence of theicaADBC locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.

title: Microarray-Based Analysis of the Staphylococcus aureus σB Regulon
abstract: Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by σB activity. While σB was found to positively control 198 genes by a factor of ≥2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of σB. Gene products that were found to be influenced by σB are putatively involved in all manner of cellular processes, including cell envelope biosynthesis and turnover, intermediary metabolism, and signaling pathways. Most of the genes and/or operons identified as upregulated by σB were preceded by a nucleotide sequence that resembled the σB consensus promoter sequence of Bacillus subtilis. A conspicuous number of virulence-associated genes were identified as regulated by σB activity, with many adhesins upregulated and prominently represented in this group, while transcription of various exoproteins and toxins were repressed. The data presented here suggest that the σB of S. aureus controls a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of the agr locus. We propose that this alternative transcription factor may be of importance for the invading pathogen to fine-tune its virulence factor production in response to changing host environments.

title: General stress response of Bacillus subtilis and other bacteria.
abstract: One of the strongest and most noticeable responses of a Bacillus subtilis cell to a range of stress and starvation conditions is the dramatic induction of a large number of general stress proteins. The alternative sigma factor sigma B is responsible for the induction of the genes encoding these general stress proteins that occurs following heat, ethanol, salt or acid stress, or during energy depletion. sigma B was detected more than 20 years ago by Richard Losick and William Haldenwang as the first alternative sigma factor of bacteria, but interest in sigma B declined after it was realized that sigma B is not involved in sporulation. It later turned out that sigma B, whose activity itself is tightly controlled, is absolutely required for the induction of this regulon, not only in B. subtilis, but also in other Gram-positive bacteria. These findings may have been responsible for the recent revival of interest in sigma B. This chapter summarizes the current information on this sigma B response including the latest results on the signal transduction pathways, the structure of the regulon and its physiological role. More than 150 general stress proteins/genes belong to this sigma B regulon, which is believed to provide the non-growing cell with a non-specific, multiple and preventive stress resistance. sigma B-dependent stress proteins are involved in non-specific protection against oxidative stress and also protect cells against heat, acid, alkaline or osmotic stress. A cell in the transition from a growing to a non-growing state induced by energy depletion will be equipped with a comprehensive stress resistance machine to protect it against future stress. The protection against oxidative stress may be an essential part of this response. In addition, preloading of cells with sigma B-dependent stress proteins, induced by mild heat or salt stress, will protect cells against a severe, potentially lethal, future stress. Both the specific protection against an acute emerging stress, as well as the non-specific, prospective protection against future stress, are adaptive functions crucial for surviving stress and starvation in nature. We suggest that the sigma B response is one essential component of a survival strategy that ensures survival in a quiescent, vegetative state as an alternative to sporulation. The role of sigma B in related Gram-positive bacteria (including cyanobacteria) with special emphasis on pathogenic bacteria is discussed.

title: Listeria monocytogenes σB Contributes to Invasion of Human Intestinal Epithelial Cells
abstract: The role of σB in Listeria monocytogenes infection of human intestinal epithelial cells was investigated. Invasion defects associated with loss of σB paralleled those of a ΔinlA strain independently of the σB-dependent P2prfA promoter. Concomitantly, amounts of inlA transcript and InlA protein were significantly decreased in the ΔsigB strain.

title: Characterization of flagellin expression and its role in Listeria monocytogenes infection and immunity
abstract: Flagellin is the structural component of flagella produced by many pathogenic bacteria and is a potent proinflammatory molecule that mediates these effects through Toll‐like receptor (TLR) 5. In Listeria monocytogenes (LM), flagellin expression is regulated by temperature and has been described as being shut off at 37°C. In this study, we demonstrate that TLR5‐mediated cell activation and flagellin expression is maintained at 37°C in some laboratory‐adapted strains and in ≈ 20% of LM clinical isolates. To determine the role of flagellin in LM infection, a targeted mutation in the structural gene for flagellin (flaA) was generated in a parental LM strain that expressed flagellin under all conditions examined. In vitro studies demonstrated that this ΔflaA mutant was (i) non‐motile; (ii) not able to activate TLR5‐transfected HeLa cells; and (iii) induced tumour necrosis factor (TNF)‐α production in ≈ 50% fewer CD11b+ cells in splenocytes from normal mice compared with the parental strain. However, there was no significant alteration in virulence of the ΔflaA mutant after either intravenous or oral murine infection. Similarly, there was no difference in the generation of LM‐specific CD8 or CD4 T cells after intravenous or oral infection. These data indicate that flagellin is not essential for LM pathogenesis or for the induction of LM‐specific adaptive immune responses in normal mice.

title: Novel anti-infection agents: small-molecule inhibitors of bacterial transcription factors.
abstract: Structure-based drug design was utilized to identify potent small-molecule inhibitors of proteins within the AraC family of bacterial transcription factors, which control virulence in medically important microbes. These agents represent a novel approach to fight infectious disease and may be less likely to promote resistance development. These compounds lack intrinsic antibacterial activity in vitro and were able to limit a bacterial infection in a mouse model of urinary tract infection.

title: N-hydroxybenzimidazole inhibitors of the transcription factor LcrF in Yersinia: novel antivirulence agents.
abstract: LcrF, a multiple adaptational response (MAR) transcription factor, regulates virulence in Yersinia pestis and Yersinia pseudotuberculosis. In a search for small molecule inhibitors of LcrF, an acrylic amide series of N-hydroxybenzimidazoles was synthesized and the SAR (structure-activity relationship) was examined. Selected test compounds demonstrated inhibitory activity in a primary cell-free LcrF-DNA binding assay as well as in a secondary whole cell assay (type III secretion system dependent Y. pseudotuberculosis cytotoxicity assay). The inhibitors exhibited no measurable antibacterial activity in vitro, confirming that they do not target bacterial growth. These results demonstrate that N-hydroxybenzimidazole inhibitors, exemplified by 14, 22, and 36, are effective antivirulence agents and have the potential to prevent infections caused by Yersinia spp.

title: Development of a differential medium for bile salt hydrolase-active Lactobacillus spp
abstract: An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments.

title: LRET-based HTS of a small-compound library for inhibitors of bacterial RNA polymerase.
abstract: Resistance mechanisms against whole classes of antibiotics are currently developing faster than research generates new structurally different biologically active agents. The demand for new antimicrobial drugs has not been matched by the speed of discovery. The interface between sigma and core of bacterial RNA polymerase offers an attractive target for drug discovery, and we have previously described the development of a very robust high-throughput assay for this target based on luminescence resonance energy transfer. Here we describe a semiautomated screen of a commercially available library (Chembridge, San Diego, CA) that led to the identification of four small molecules, two of which have activity in preventing in vitro transcription and growth of Escherichia coli.

title: SoxS, an activator of superoxide stress genes in Escherichia coli. Purification and interaction with DNA.
abstract: A genetic response of Escherichia coli to nitric oxide or to superoxide-generating agents such as paraquat is controlled by the soxRS locus. The intracellular redox signals generated by these agents are sensed by the SoxR protein which, when activated, functions as a potent activator of soxS transcription. The resulting increased level of SoxS protein then activates approximately 10 genes that constitute the soxRS regulon. Although the SoxS protein is homologous to the COOH-terminal region of the AraC family of regulatory proteins, the mechanism by which SoxS protein activates the soxRS regulon promoters is unknown. We identified in extracts of cells expressing high levels of SoxS protein a DNA binding activity specific for fragments containing soxRS-regulated promoters. This binding activity was purified to physical homogeneity and proved to be the SoxS protein, as confirmed by NH2-terminal amino acid sequencing. The purified SoxS protein bound specifically to the promoters of the micF, zwf, nfo, and sodA genes. Multiple DNA-protein complexes were formed by SoxS in a concentration-dependent fashion with each of these promoters. This binding of SoxS protein also facilitated the subsequent binding of E. coli RNA polymerase to both the micF and the nfo promoters. The binding sites of SoxS in the zwf and micF promoters were identified by DNase I footprinting, which revealed an extended protected region immediately upstream of the respective -35 sites. These results indicate that the small SoxS protein (M(r) of only 12,900) is a direct transcriptional activator of the oxidative stress genes of the soxRS regulon, although the possible involvement of other proteins in transcription activation by SoxS has not been ruled out.

title: ToxR regulon of Vibrio cholerae and its expression in vibrios shed by cholera patients
abstract: Toxigenic Vibrio cholerae cause cholera, a severe diarrheal disease responsible for significant morbidity and mortality worldwide. Two determinants, cholera enterotoxin (CT) and toxin coregulated pilus (TCP) are critical factors responsible for this organism's virulence. The genes for these virulence determinants belong to a network of genes (the ToxR regulon) whose expression is modulated by transcriptional regulators encoded by the toxRS, tcpPH, and toxT genes. To define the ToxR regulon more fully, mutants defective in these regulatory genes were transcriptionally profiled by using V. cholerae genomic microarrays. This study identified 13 genes that were transcriptionally repressed by the toxT mutation (all involved in CT and TCP biogenesis), and 27 and 60 genes that were transcriptionally repressed by the tcpPH and toxRS mutations, respectively. During the course of this analysis, we validated the use of a genomic DNA-based reference sample as a means to standardize and normalize data obtained in different microarray experiments. This method allowed the accurate transcriptional profiling of V. cholerae cells present in stools from cholera patients and the comparison of these profiles to those of wild-type and mutant strains of V. cholerae grown under optimal conditions for CT and TCP expression. Our results suggest that vibrios present in cholera stools carry transcripts for these two virulence determinants, albeit at relatively low levels compared with optimal in vitro conditions. The transcriptional profile of vibrios present in cholera stools also suggests that the bacteria experienced conditions of anaerobiosis, iron limitation, and nutrient deprivation within the human gastrointestinal tract.

title: Common themes in microbial pathogenicity revisited.
abstract: Bacterial pathogens employ a number of genetic strategies to cause infection and, occasionally, disease in their hosts. Many of these virulence factors and their regulatory elements can be divided into a smaller number of groups based on the conservation of similar mechanisms. These common themes are found throughout bacterial virulence factors. For example, there are only a few general types of toxins, despite a large number of host targets. Similarly, there are only a few conserved ways to build the bacterial pilus and nonpilus adhesins used by pathogens to adhere to host substrates. Bacterial entry into host cells (invasion) is a complex mechanism. However, several common invasion themes exist in diverse microorganisms. Similarly, once inside a host cell, pathogens have a limited number of ways to ensure their survival, whether remaining within a host vacuole or by escaping into the cytoplasm. Avoidance of the host immune defenses is key to the success of a pathogen. Several common themes again are employed, including antigenic variation, camouflage by binding host molecules, and enzymatic degradation of host immune components. Most virulence factors are found on the bacterial surface or secreted into their immediate environment, yet virulence factors operate through a relatively small number of microbial secretion systems. The expression of bacterial pathogenicity is dependent upon complex regulatory circuits. However, pathogens use only a small number of biochemical families to express distinct functional factors at the appropriate time that causes infection. Finally, virulence factors maintained on mobile genetic elements and pathogenicity islands ensure that new strains of pathogens evolve constantly. Comprehension of these common themes in microbial pathogenicity is critical to the understanding and study of bacterial virulence mechanisms and to the development of new "anti-virulence" agents, which are so desperately needed to replace antibiotics.

title: A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid
abstract: We observed that glutamate greatly enhances the survival of Listeria monocytogenes in gastric fluid, a phenomenon that is directly linked to glutamate decarboxylase activity (GAD). Glutamate‐mediated acid tolerance has been associated in other intestinal genera with the GAD system, in which glutamate is internalized and converted to γ‐aminobutyrate (consuming an intracellular proton) that is subsequently exchanged for another extracellular glutamate via a membrane‐located antiporter. Molecular analysis of L. monocytogenes LO28 revealed the presence of two glutamate decarboxylase homologues, designated gadA and gadB, that are differentially expressed. The gadB gene is  in tandem with an upstream gene, gadC, which encodes a potential glutamate/γ‐aminobutyrate antiporter. Expression of this transcript is upregulated in response to mild acid stress (pH 5.5). In contrast, expression of the monocistronic gadA message was weaker and was not induced by mild acid treatment. Non‐polar deletion mutations resulted in a dramatic decrease in the level of GAD activity and a concomitant decrease in acid resistance in the order LO28 > ΔgadA > ΔgadB = ΔgadC > ΔgadAB for both stationary and logarithmic phase cultures. The exquisite sensitivity of the ΔgadAB mutant to ex vivo porcine and synthetic human gastric fluid demonstrates a clear role for the GAD system in facilitating survival of the organism in the stomach after ingestion and in other low‐pH environments. Furthermore, variations in levels of GAD activity between different strains of L. monocytogenes correlate significantly with levels of tolerance to gastric fluid. Sensitive strains, which include the sequenced L. monocytogenes EGD, exhibit reduced levels of GAD activity. It is clear from this study that expression of GAD by L. monocytogenes strains is an absolute requirement for survival in the stomach environment.

title: Influence of a Functional sigB Operon on the Global Regulators sar and agr inStaphylococcus aureus
abstract: The growth phase-dependent activity profile of the alternate transcription factor ςB and its effects on the expression of sar and agr were examined in three differentStaphylococcus aureus strains by Northern blot analyses and by the use of reporter gene fusion experiments. Significant ςB activity was detectable only in the clinical isolates MSSA1112 and Newman, carrying the wild-type rsbU allele, but not in the NCTC8325 derivative BB255, which is defective inrsbU. ςB activity peaked in the late exponential phase and diminished towards the stationary phase when bacteria were grown in Luria-Bertani medium. Transcriptional analysis and a sarP1-sarP2-sarP3(sarP1-P2-P3)-driven firefly luciferase (luc+) reporter gene fusion demonstrated a strong ςB activity- and growth phase-dependent increase in sar expression that was totally absent in either rsbU or ΔrsbUVWsigB mutants. In contrast, expression of theagr locus, as measured by RNAIII levels and by anhldp::luc+ fusion, was found to be higher in the absence of ςB activity, such as inrsbU or ΔrsbUVWsigB mutants, than in wild-type strains. Overexpression of ςB in BB255 derivatives resulted in a clear increase insarP1-P2-P3::luc+ expression as well as a strong decrease in hldp::luc+ expression. The data presented here suggest that ςBincreases sar expression while simultaneously reducing the RNAIII level in a growth phase-dependent manner.

title: The alternative sigma factor sigma B of Staphylococcus aureus modulates virulence in experimental central venous catheter-related infections.
abstract: The impact of the alternative sigma factor sigma B (SigB) on pathogenesis of Staphylococcus aureus is not conclusively clarified. In this study, a central venous catheter (CVC) related model of multiorgan infection was used to investigate the role of SigB for the pathogenesis of S. aureus infections and biofilm formation in vivo. Analysis of two SigB-positive wild-type strains and their isogenic mutants revealed uniformly that the wild-type was significantly more virulent than the SigB-deficient mutant. The observed difference in virulence was apparently not linked to the capability of the strains to form biofilms in vivo since wild-type and mutant strains were able to produce biofilm layers inside of the catheter. The data strongly indicate that the alternative sigma factor SigB plays a role in CVC-associated infections caused by S. aureus.

title: Sigma Factor B and RsbU Are Required for Virulence in Staphylococcus aureus-Induced Arthritis and Sepsis
abstract: The prototype Staphylococcus aureus strain 8325-4 produces high levels of hemolysins and proteases. Recently it has been shown that this property depends on a deficiency of sigma factor B (SigB) activity controlling the activation of regulatory genes such as agr and sarA. SigB deficiency is in turn due to a mutation in the rsbU gene, which is required for posttranslational activation of SigB. The rsbU defect of strain 8325-4 has recently been repaired, and we used this strain (SH1000), along with its isogenic sigB-negative mutant, to investigate the contributions of RsbU and SigB in a murine model of septic arthritis. Intravenous inoculation with the rsbU-repaired isogenic strain SH1000 resulted in significantly more severe arthritis, weight decrease, and mortality compared to those of the parental strain 8325-4 (rsbU-negative) or the isogenic sigB-negative mutant (MJH502). SH1000 also persisted more in kidneys and joints of infected mice. Our data strongly suggest that RsbU and SigB regulate important virulence factors, thereby contributing significantly to the outcome of staphylococcal infection.

title: Adoptive transfer of immunity to Listeria monocytogenes. The influence of in vitro stimulation on lymphocyte subset requirements.
abstract: BALB/c mice develop specific and relatively long lasting immunity after exposure to sublethal numbers of viable Listeria monocytogenes. This immunity can be passively transferred to naive recipients with maximal protection conferred by spleen cells obtained from donors 6 days after immunization. Immunity that can be directly transferred to syngeneic recipients is surprisingly short lived. Cell recipients lose immunity as early as 72 hr after transfer, and recipients express no detectable immunity after 1 wk. This short lived immunity requires both L3T4+ and Lyt-2+ T cell populations for full expression. Both the level of immunity transferred and the duration of the protective response expressed in recipients are dramatically increased if the spleen cell population is cultured in vitro with concanavalin A before cell transfer. Recipients of concanavalin A-activated cells express antigen-specific levels of immunity increased 100- to 1000-fold compared with syngeneic recipients of directly transferred immune spleen cells. In addition, this elevated level of adoptively transferred immunity remains constant for at least 8 wk. Transfer of this culture-enhanced immunity requires only an Lyt-2+ T cell population and is not influenced by cells of the L3T4+T cell subpopulation. Both direct as well as culture-enhanced transfer of immunity require major histocompatibility complex-compatible recipients. These findings suggest that two phenotypically distinct T cell subpopulations function in the development of the immune response to L. monocytogenes and that only one cell subpopulation is required for expression of immunity to this intracellular parasite.

title: Role of Listeria monocytogenes σB in Survival of Lethal Acidic Conditions and in the Acquired Acid Tolerance Response
abstract: The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, σB, which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and ΔsigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the ΔsigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both ΔsigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is σB independent. σB-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional σB reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms. σB does not appear to contribute to pHi homeostasis through regulation of net proton movement across the cell membrane or by regulation of pHi buffering by the GAD system under the conditions examined in this study. In summary, a functional σB protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.

title: sigmaB-dependent gene induction and expression in Listeria monocytogenes during osmotic and acid stress conditions simulating the intestinal environment.
abstract: Listeria monocytogenes must overcome a variety of stress conditions in the host digestive tract to cause foodborne infections. The alternative sigma factor sigma(B), encoded by sigB, is responsible for regulating transcription of several L. monocytogenes virulence and stress-response genes, including genes that contribute to establishment of gastrointestinal infections. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the virulence genes inlA and bsh, the stress-response genes opuCA and lmo0669 (encoding a carnitine transporter and an oxidoreductase, respectively) and the housekeeping gene rpoB. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting osmotic (0.3 M NaCl) or acid (pH 4.5) conditions typical for the human intestinal lumen. In exponential-phase cells, as well as under osmotic and acid stress, inlA, opuCA and bsh showed significantly lower absolute expression levels in a L. monocytogenes DeltasigB null mutant compared to wild-type. A statistical model that normalized target gene expression relative to rpoB showed that accumulation of inlA, opuCA and bsh transcripts was significantly increased in the wild-type strain within 5 min of acid and osmotic stress exposure; lmo0669 transcript accumulation increased significantly only after acid exposure. It was concluded that sigma(B) is essential for rapid induction of the tested stress-response and virulence genes under conditions typically encountered during gastrointestinal passage. As inlA, bsh and opuCA are critical for gastrointestinal infections in animal models, the data also suggest that sigma(B) contributes to the ability of L. monocytogenes to cause foodborne infections.

title: The 5′ untranslated region‐mediated enhancement of intracellular listeriolysin O production is required for Listeria monocytogenes pathogenicity
abstract: Listeriolysin O (LLO) and ActA are essential virulence determinants for Listeria monocytogenes pathogenesis. Transcription of actA and hly, encoding LLO, is regulated by PrfA and increases dramatically during intracellular infection. The 5′ untranslated regions (5′ UTRs) of actA and prfA have been shown to upregulate expression of their respective gene products. Here, we demonstrate that the hly 5′ UTR plays a critical role in regulating expression of LLO during intracellular infection. Deletion of the hly 5′ UTR, while retaining the hly ribosome binding site, had a moderate effect on LLO production during growth in broth culture, yet resulted in a marked decrease in LLO levels during intracellular infection. The diminished level of LLO resulted in a significant defect in bacterial cell‐to‐cell spread during intracellular infection and a 10‐fold reduction in virulence during in vivo infection of mice. Insertion of the hly 5′ UTR sequence between a heterologous promoter and reporter gene sequences indicated that the hly 5′ UTR functions independent of PrfA‐mediated transcription and can enhance expression of cis‐associated genes through a mechanism that appears to act at both a post‐transcriptional and translational level. The ability of the hly 5′ UTR to increase gene expression can be exploited to achieve PrfA‐independent complementation of virulence genes and high‐level expression of single copy heterologous genes in L. monocytogenes.

title: High-throughput Identification of Phage-derived Imaging Agents
abstract: The use of phage-displayed peptide libraries is a powerful method for selecting peptides with desired binding properties. However, the validation and prioritization of “hits” obtained from this screening approach remains challenging. Here, we describe the development and testing of a new analysis method to identify and display hits from phage-display experiments and high-throughput enzyme-linked immunosorbent assay screens. We test the method using a phage screen against activated macrophages to develop imaging agents with higher specificity for active disease processes. The new methodology should be useful in identifying phage hits and is extendable to other library screening methods such as small-molecule and nanoparticle libraries.

title: Relationship of stereochemical and skeletal diversity of small molecules to cellular measurement space.
abstract: Systematic and quantitative measurements of the roles of stereochemistry and skeleton-dependent conformational restriction were made using multidimensional screening. We first used diversity-oriented synthesis to synthesize the same number (122) of [10.4.0] bicyclic products (B) and their corresponding monocyclic precursors (M). We measured the ability of these compounds to modulate a broad swath of biology using 40 parallel cell-based assays. We analyzed the results using statistical methods that revealed illuminating relationships between stereochemistry, ring number, and assay outcomes. Conformational restriction by ring-closing metathesis increased the specificity of responses among active compounds and was the dominant factor in global activity patterns. Hierarchical clustering also revealed that stereochemistry was a second dominant factor; whereas the stereochemistry of macrocyclic appendages was a determinant for bicyclic compounds, the stereochemistry of the carbohydrates was a determinant for the monocyclic compounds of global activity patterns. These studies illustrate a quantitative method for measuring stereochemical and skeletal diversity of small molecules and their cellular consequences.

title: ChemBank: a small-molecule screening and cheminformatics resource database
abstract: ChemBank (http://chembank.broad.harvard.edu/) is a public, web-based informatics environment developed through a collaboration between the Chemical Biology Program and Platform at the Broad Institute of Harvard and MIT. This knowledge environment includes freely available data derived from small molecules and small-molecule screens and resources for studying these data. ChemBank is unique among small-molecule databases in its dedication to the storage of raw screening data, its rigorous definition of screening experiments in terms of statistical hypothesis testing, and its metadata-based organization of screening experiments into projects involving collections of related assays. ChemBank stores an increasingly varied set of measurements derived from cells and other biological assay systems treated with small molecules. Analysis tools are available and are continuously being developed that allow the relationships between small molecules, cell measurements, and cell states to be studied. Currently, ChemBank stores information on hundreds of thousands of small molecules and hundreds of biomedically relevant assays that have been performed at the Broad Institute by collaborators from the worldwide research community. The goal of ChemBank is to provide life scientists unfettered access to biomedically relevant data and tools heretofore available primarily in the private sector.

title: From Knowing to Controlling: A Path from Genomics to Drugs Using Small Molecule Probes
abstract: The National Cancer Institute Initiative in Chemical Genetics is designed to encourage the development of small molecular probes. The probes are useful for activating or inactivating protein functions, thereby providing resources that help discern the functions of gene products in normal and disease cells, as well as in tissues. This initiative includes “ChemBank,” a suite of informatics tools and databases aimed at promoting the development and use of chemical genetics by scientists worldwide. The information generated with such tools should provide a critical link from genomic discovery to drug development.

title: A method for the covalent capture and screening of diverse small molecules in a microarray format
abstract: This protocol describes a robust method for the covalent capture of small molecules with diverse reactive functional groups in microarray format, and outlines a procedure for probing small-molecule microarrays (SMMs) with proteins of interest. A vapor-catalyzed, isocyanate-mediated surface immobilization scheme is used to attach bioactive small molecules, natural products and small molecules derived from diversity-oriented synthesis pathways. Additionally, an optimized methodology for screening SMMs with purified proteins and cellular lysates is described. Finally, a suggested model for data analysis that is compatible with commercially available software is provided. These procedures enable a platform capability for discovering novel interactions with potential applications to immunoglobulin profiling, comparative analysis of cellular states and ligand discovery. With the appropriate materials and experimental setup, the printing of SMMs can be completed in 14 hours over 3 days. Screening and data analysis requires 2 days. A detailed timeline is provided.

title: A robust small-molecule microarray platform for screening cell lysates.
abstract: Herein we report the expanded functional group compatibility of small-molecule microarrays to include immobilization of primary alcohols, secondary alcohols, phenols, carboxylic acids, hydroxamic acids, thiols, and amines on a single slide surface. Small-molecule "diversity microarrays" containing nearly 10,000 known bioactive small molecules, natural products, and small molecules originating from several diversity-oriented syntheses were produced by using an isocyanate-mediated covalent capture strategy. Selected printed bioactive compounds were detected with antibodies against compounds of interest. The new surface of the diversity microarrays is highly compatible with approaches involving cellular lysates. This feature has enabled a robust, optimized screening methodology using cellular lysates, allowing the detection of specific interactions with a broad range of binding affinity by using epitope-tagged or chimeric fluorescent proteins without prior purification. We believe that this expanded research capability has considerable promise in biology and medicine.

title: A pipeline for ligand discovery using small-molecule microarrays.
abstract: Uncovering the functions of thousands of gene products, in various states of post-translational modification, is a key challenge in the post-genome era. To identify small-molecule probes for each protein function, high-throughput methods for ligand discovery are needed. In recent years, small-molecule microarrays (SMMs) have emerged as high-throughput and miniaturized screening tools for discovering protein-small-molecule interactions. Microarrays of small molecules from a variety of sources, including FDA-approved drugs, natural products and products of combinatorial chemistry and diversity-oriented synthesis, have been prepared and screened by several laboratories, leading to several newly discovered protein-ligand pairs.

title: Specific binding of the Listeria monocytogenes transcriptional regulator PrfA to target sequences requires additional factor(s) and is influenced by iron
abstract: The PrfA protein, which is a member of the Crp/Fnr family of prokaryotic transcription activators, regulates the virulence genes of Listeria monocytogenes. In this work, specific binding of PrfA to its target DNA was determined by electrophoretic mobility‐shift assays (EMSAs) using cell‐free extracts from the two L. monocytogenes strains EGD and NCTC 7973. PrfA‐specific binding differs between the two strains, even when the concentration of PrfA was adjusted to similar levels. Both strains exhibited increased PrfA‐specific binding after a shift into minimal essential medium (MEM) without showing a significant change in the amount of PrfA protein, relative to extracts from bacteria grown in brain–heart infusion (BHI). The purified PrfA protein from strain EGD produced in Escherichia coli did not exhibit specific binding to the target DNA but did so upon addition of PrfA‐free extracts from various Listeria species and Bacillus subtilis. The observed activation of PrfA seems to be caused by a PrfA‐activating factor (Paf), which is probably a protein since elevated temperature, but not RNase treatment, destroyed the activation potential of such PrfA‐free extracts. Moreover, fractionation of these extracts by sucrose gradient centrifugation yielded the Paf activity in a fraction sedimenting at 3.2 S. Specific binding of PrfA‐containing extracts from strain EGD to the hly and actA promoter sequences was strongly inhibited by iron, whereas that of extracts from strain NCTC 7973 was only slightly reduced. The iron effect seems to be mediated by Paf rather than by PrfA itself.

title: Contributions of Listeria monocytogenes sigmaB and PrfA to expression of virulence and stress response genes during extra- and intracellular growth.
abstract: Listeria monocytogenes sigmaB and PrfA are pleiotropic regulators of stress response and virulence gene expression. Quantitative RT-PCR (qRT-PCR) was used to measure transcript levels of sigmaB- and PrfA-dependent genes in exponential-phase L. monocytogenes wild-type and DeltasigB strains as well as in bacteria exposed to environmental stresses (0.3 M NaCl or growth to stationary phase) or present in the vacuole or cytosol of human intestinal epithelial cells. Stationary-phase or NaCl-exposed L. monocytogenes showed sigmaB-dependent increases in opuCA (10- and 17-fold higher, respectively) and gadA transcript levels (77- and 14-fold higher, respectively) as compared to non-stressed, exponential-phase bacteria. While PrfA activity, as reflected by plcA transcript levels, was up to 95-fold higher in intracellular L. monocytogenes as compared to non-stressed bacteria, sigmaB activity was only slightly higher in intracellular than in non-stressed bacteria. Increased plcA transcript levels, which were similar in both host cell vacuole and cytosol, were associated with increases in both prfA expression and PrfA activity. qRT-PCR assays were designed to measure expression of prfA from each of its three promoter regions. Under all conditions, readthrough transcription from the upstream plcA promoter was very low. The relative contribution to total prfA transcription from the sigmaA-dependent P1prfA promoter ranged from approximately 17 % to 30 %, while the contribution of the P2prfA region, which appears to be transcribed by both sigmaA and sigmaB, ranged from approximately 70 % to 82 % of total prfA transcript levels. In summary (i) sigmaB is primarily activated during environmental stress and does not contribute to PrfA activation in intracellular L. monocytogenes and (ii) the partially sigmaB-dependent P2prfA promoter region contributes the majority of prfA transcripts in both intra- and extracellular bacteria.

title: SigmaB-dependent and sigmaB-independent mechanisms contribute to transcription of Listeria monocytogenes cold stress genes during cold shock and cold growth.
abstract: The role of the stress response regulator sigma(B) (encoded by sigB) in directing the expression of selected putative and confirmed cold response genes was evaluated using Listeria monocytogenes 10403S and an isogenic DeltasigB mutant, which were either cold shocked at 4 degrees C in brain heart infusion (BHI) broth for up to 30 min or grown at 4 degrees C in BHI for 12 days. Transcript levels of the housekeeping genes rpoB and gap, the sigma(B)-dependent genes opuCA and bsh, and the cold stress genes ltrC, oppA, and fri were measured using quantitative reverse transcriptase PCR. Transcriptional start sites for ltrC, oppA, and fri were determined using rapid amplification of cDNA ends PCR. Centrifugation was found to rapidly induce sigma(B)-dependent transcription, which necessitated the use of centrifugation-independent protocols to evaluate the contributions of sigma(B) to transcription during cold shock. Our data confirmed that transcription of the cold stress genes ltrC and fri is at least partially sigma(B) dependent and experimentally identified a sigma(B)-dependent ltrC promoter. In addition, our data indicate that (i) while sigma(B) activity is induced during 30 min of cold shock, this cold shock does not induce the transcription of sigma(B)-dependent or -independent cold shock genes; (ii) sigma(B) is not required for L. monocytogenes growth at 4 degrees C in BHI; and (iii) transcription of the putative cold stress genes opuCA, fri, and oppA is sigma(B) independent during growth at 4 degrees C, while both bsh and ltrC show growth phase and sigma(B)-dependent transcription during growth at 4 degrees C. We conclude that sigma(B)-dependent and sigma(B)-independent mechanisms contribute to the ability of L. monocytogenes to survive and grow at low temperatures.

title: Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles
abstract: Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.

title: Activation of Bacillus subtilis transcription factor sigma B by a regulatory pathway responsive to stationary-phase signals
abstract: Alternative transcription factor sigma B of Bacillus subtilis controls a stationary-phase regulon induced under growth conditions that do not favor sporulation. Little is known about the metabolic signals and protein factors regulating the activity of sigma B. The operon containing the sigma B structural gene has the gene order orfV-orfW-sigB-rsbX, and operon expression is autoregulated positively by sigma B and negatively by the rsbX product (rsbX = regulator of sigma B). To establish the roles of the orfV and orfW products, orfV and orfW null and missense mutations were constructed and tested for their effects on expression of the sigma B-dependent genes ctc and csbA. These mutations were tested in two contexts: in the first, the sigB operon was under control of its wild-type, sigma B-dependent promoter, and in the second, the sigB operon promoter was replaced by the inducible Pspac promoter. The principal findings are that (i) the orfV (now called rsbV) product is a positive regulator of sigma B-dependent gene expression; (ii) the orfW (now called rsbW) product is a negative regultor of such expression; (iii) sigma B is inactive during logarithmic growth unless the rsbW product is absent; (iv) the rsbX, rsbV, and rsbW products have a hierarchical order of action; and (v) both the rsbV and rsbW products appear to regulate sigma B activity posttranslationally. There are likely to be at least two routes by which information can enter the system to regulate sigma B: via the rsbX product, and via the rsbV and rsbW products.

title: Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor sigma B in response to environmental signals
abstract: sigma B of the gram-positive bacterium Bacillus subtilis is an alternative transcription factor activated by a variety of environmental stresses, including the stress imposed upon entry into the stationary growth phase. Previous reports have shown that this stationary-phase activation is enhanced when cells are grown in rich medium containing glucose and glutamine. The sigma B structural gene, sigB, lies in an operon with three other genes whose products have been shown to control sigma B activity in response to environmental stress. However, none of these is sufficient to explain the enhanced stationary-phase activation of sigma B in response to glucose. We show here that the four genes previously identified in the sigB operon constitute the downstream half of an eight-gene operon. The complete sigB operon is preceded by a sigma A-like promoter (PA) and has the order PA-orfR-orfS-orfT-orfU-PB-rsbV-rsbW-sig B-rsbX, where rsb stands for regulator of sigma-B and the previously identified sigma B-dependent promoter (PB) is an internal promoter preceding the downstream four-gene cluster. Although the genes downstream of PB were also transcribed by polymerase activity originating at PA, this transcription into the downstream cluster was not essential for normal induction of a sigma B-dependent ctc-lacZ fusion. However, deletion of all four upstream open reading frames was found to interfere with induction of the ctc-lacZ fusion in response to glucose. Additional deletion analysis and complementation studies showed that orfU was required for full glucose induction of sigma B-dependent genes. orfU encodes a trans-acting, positive factor with significant sequence identity to the RsbX negative regulator of sigma B. On the basis of these results, we rename orfU as rsbU to symbolize the regulatory role of its product.

title: Control of the Escherichia coli rrnB P1 promoter strength by ppGpp
abstract: Fusions of the rrnB P1 and P2 promoters, and of the tandem P1-P2 combination, to a wild-type lacZ gene were constructed on plasmids and recombined into the mal region of the bacterial chromosome, close to the normal location and in the normal orientation of rrnB. The upstream activator region (Fis-binding sites) was always present with the P1 promoter, and all constructs contained the box A antitermination site of rRNA genes. Using these constructs, beta-galactosidase specific activities were measured in Escherichia coli strains carrying either both ppGpp synthetases, PSI and PSII (relA+ spoT+), or only PSII (delta relA spoT+), or neither (delta relA delta spoT), using different media supporting growth rates between 0.6 and 2.8 doublings/h at 37 degrees C. The beta-galactosidase activities were used to estimate the relative strength of the rrnB P1 promoter in comparison to the isolated rrnB P2 promoter. Promoter strength (transcripts initiated per min per promoter per free RNA polymerase concentration) was distinguished from promoter activity (transcripts initiated per min per promoter). In ppGpp- synthesizing (wild-type) bacteria, the relative strength of the rrnB P1 promoter increased nearly 10-fold with increasing growth rate from 0.17 to 1.5, but in the ppGpp-less double mutants it decreased by 20% from 1.7 to 1.5. Thus, at low or zero levels of ppGpp, the P1 promoter was 1.5-1.7 times stronger than the isolated P2 promoter. These results indicate that the normal growth rate control of the rrnB P1 promoter strength requires ppGpp, and that the strength is reduced at basal levels of ppGpp found during exponential growth. No additional ppGpp- independent control of the rrnB P1 promoter strength was evident. From the beta-galactosidase data and previously determined values of rRNA gene activities, the activities of the isolated rrnB P1 and P2 promoters, and of the P2 promoter in the tandem combination, were estimated. With increasing growth rate, the activity of the isolated P2 promoter increased 6-fold from 6 to 33 initiations/min, while the activity of the isolated P1 promoter increased 24-fold from 2 to 54 initiations/min. The increasing activity of the isolated P2 promoter is assumed to reflect the increasing RNA polymerase concentration at constant promoter strength, whereas the steeper increase in P1 promoter activity reflects increases in both polymerase concentration and promoter strength. When in tandem with P1, the P2 promoter activity is inferred to decrease as the P1 promoter activity increases.