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Sample records for coli nusb-s10 transcription

  1. Transcription of foreign DNA in Escherichia coli

    PubMed Central

    Warren, René L.; Freeman, John D.; Levesque, Roger C.; Smailus, Duane E.; Flibotte, Stephane; Holt, Robert A.

    2008-01-01

    Propagation of heterologous DNA in E. coli host cells is central to molecular biology. DNA constructs are often engineered for expression of recombinant protein in E. coli, but the extent of incidental transcription arising from natural regulatory sequences in cloned DNA remains underexplored. Here, we have used programmable microarrays and RT-PCR to measure, comprehensively, the transcription of H. influenzae, P. aeruginosa, and human DNA propagating in E. coli as bacterial artificial chromosomes. We find evidence that at least half of all H. influenzae genes are transcribed in E. coli. Highly transcribed genes are principally involved in energy metabolism, and their proximal promoter regions are significantly enriched with E. coli σ70 (also known as RpoD) binding sites. H. influenzae genes acquired from an ancient bacteriophage Mu insertion are also highly transcribed. Compared with H. influenzae, a smaller proportion of P. aeruginosa genes are transcribed in E. coli, and in E. coli there is punctuated transcription of human DNA. The presence of foreign DNA in E. coli disturbs the host transcriptional profile, with expression of the E. coli phage shock protein operon and the flagellar gene cluster being particularly strongly up-regulated. While cross-species transcriptional activation is expected to be enabling for horizontal gene transfer in bacteria, incidental expression of toxic genes can be problematic for DNA cloning. Ongoing characterization of cross-expression will help inform the design of biosynthetic gene clusters and synthetic microbial genomes. PMID:18701636

  2. A comprehensive library of fluorescent transcriptional reporters for Escherichia coli.

    PubMed

    Zaslaver, Alon; Bren, Anat; Ronen, Michal; Itzkovitz, Shalev; Kikoin, Ilya; Shavit, Seagull; Liebermeister, Wolfram; Surette, Michael G; Alon, Uri

    2006-08-01

    E. coli is widely used for systems biology research; there exists a need, however, for tools that can be used to accurately and comprehensively measure expression dynamics in individual living cells. To address this we present a library of transcriptional fusions of gfp to each of about 2,000 different promoters in E. coli K12, covering the great majority of the promoters in the organism. Each promoter fusion is expressed from a low-copy plasmid. We demonstrate that this library can be used to obtain highly accurate dynamic measurements of promoter activity on a genomic scale, in a glucose-lactose diauxic shift experiment. The library allowed detection of about 80 previously uncharacterized transcription units in E. coli, including putative internal promoters within previously known operons, such as the lac operon. This library can serve as a tool for accurate, high-resolution analysis of transcription networks in living E. coli cells. PMID:16862137

  3. Gene transcription and chromosome replication in Escherichia coli.

    PubMed Central

    Zhou, P; Bogan, J A; Welch, K; Pickett, S R; Wang, H J; Zaritsky, A; Helmstetter, C E

    1997-01-01

    Transcript levels of several Escherichia coli genes involved in chromosome replication and cell division were measured in dnaC2(Ts) mutants synchronized for chromosome replication by temperature shifts. Levels of transcripts from four of the genes, dam, nrdA, mukB, and seqA, were reduced at a certain stage during chromosome replication. The magnitudes of the decreases were similar to those reported previously ftsQ and ftsZ (P. Zhou and C. E. Helmstetter, J. Bacteriol. 176:6100-6106, 1994) but considerably less than those seen with dnaA, gidA, and mioC (P. W. Theisen, J. E. Grimwade, A. C. Leonard, J. A. Bogan, and C. E. Helmstetter, Mol. Microbiol. 10:575-584, 1993). The decreases in transcripts appeared to correlate with the estimated time at which the genes replicated. This same conclusion was reached in studies with synchronous cultures obtained with the baby machine in those instances in which periodicities in transcript levels were clearly evident. The transcriptional levels for two genes, minE and tus, did not fluctuate significantly, whereas the transcripts for one gene, iciA, appeared to increase transiently. The results support the idea that cell cycle timing in E. coli is not governed by timed bursts of gene expression, since the overall findings summarized in this report are generally consistent with cell cycle-dependent transient inhibitions of transcription rather than stimulations. PMID:8981994

  4. The Transcription Unit Architecture of the Escherichia Coli Genome

    SciTech Connect

    Cho, Byung-Kwan; Zengler, Karsten; Qiu, Yu; Park, Young S.; Knight, Eric M.; Barrett, Christian; Gao, Yuan; Palsson, Bernhard O.

    2009-11-01

    Under EMSL User Proposal 25660, the authors reported that bacterial genomes are organized by structural and functional elements, including promoters, transcription start and termination sites, open reading frames, regulatory noncoding regions, untranslated regions and transcription units. Here, we iteratively integrate high-throughput, genome-wide measurements of RNA polymerase binding locations and mRNA transcript abundance, 5' sequences and translation into proteins to determine the organizational structure of the Escherichia coli K-12 MG1655 genome. Integration of the organizational elements provides an experimentally annotated transcription unit architecture, including alternative transcription start sites, 5' untranslated region, boundaries and open reading frames of each transcription unit. A total of 4,661 transcription units were identified, representing an increase of >530% over current knowledge. This comprehensive transcription unit architecture allows for the elucidation of condition-specific uses of alternative sigma factors at the genome scale. Furthermore, the transcription unit architecture provides a foundation on which to construct genome-scale transcriptional and translational regulatory networks.

  5. Transcription induces gyration of the DNA template in Escherichia coli.

    PubMed Central

    Figueroa, N; Bossi, L

    1988-01-01

    We show that transcription modulation of a plasmid sequence in exponentially growing Escherichia coli cells leads to a rapid change in the linking number of plasmid DNA. Activation of transcription is accompanied by an increase in the plasmid's level of negative supercoiling. The added superhelical turns, whose number is proportional to the strength of the promoter and to the length of the transcript, are promptly removed when transcription is turned off. The transcription-induced increase of template supercoiling can still be detected in the presence of an inhibitor of ATP-dependent DNA gyrase [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3]. Altogether, our results indicate that, in addition to being under a general control, DNA superhelicity can be modulated locally in response to the topological perturbations associated with DNA tracking processes. We discuss a model in which supercoiling changes are produced by differential swiveling activities on the opposite sides of a transcriptional flow during transcriptional modulation. Images PMID:2849103

  6. Transcriptional effects of CRP* expression in Escherichia coli

    PubMed Central

    Khankal, Reza; Chin, Jonathan W; Ghosh, Debashis; Cirino, Patrick C

    2009-01-01

    Background Escherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp*) enables co-utilization of glucose and other sugars in E. coli. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose. Results The glucose effect is significantly suppressed in strain PC05 relative to strain W3110. The expression levels of glucose-sensitive genes are generally not altered by glucose to the same extent in strain PCO5 as compared to W3110. Only 23 of the 80 genes showing significant differential expression in the presence of glucose for strain PC05 are present among the 418 genes believed to be directly regulated by CRP. Genes involved in central carbon metabolism (including several TCA cycle genes) and amino acid biosynthesis, as well as genes encoding nutrient transport systems are among those whose transcript levels are most significantly affected by CRP* expression. We present a detailed transcription analysis and relate these results to phenotypic differences between strains expressing wild-type CRP and CRP*. Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP. Meanwhile, a more drastic decrease in the NADPH/NADP+ ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase. Altered expression levels of transhydrogenase and TCA cycle genes

  7. Dissecting the stochastic transcription initiation process in live Escherichia coli

    PubMed Central

    Lloyd-Price, Jason; Startceva, Sofia; Kandavalli, Vinodh; Chandraseelan, Jerome G.; Goncalves, Nadia; Oliveira, Samuel M. D.; Häkkinen, Antti; Ribeiro, Andre S.

    2016-01-01

    We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics. PMID:27026687

  8. Dissecting the stochastic transcription initiation process in live Escherichia coli.

    PubMed

    Lloyd-Price, Jason; Startceva, Sofia; Kandavalli, Vinodh; Chandraseelan, Jerome G; Goncalves, Nadia; Oliveira, Samuel M D; Häkkinen, Antti; Ribeiro, Andre S

    2016-06-01

    We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics. PMID:27026687

  9. Control site location and transcriptional regulation in Escherichia coli.

    PubMed Central

    Collado-Vides, J; Magasanik, B; Gralla, J D

    1991-01-01

    The regulatory regions for 119 Escherichia coli promoters have been analyzed, and the locations of the regulatory sites have been cataloged. The following observations emerge. (i) More than 95% of promoters are coregulated with at least one other promoter. (ii) Virtually all sigma 70 promoters contain at least one regulatory site in a proximal position, touching at least position -65 with respect to the start point of transcription. There are not yet clear examples of upstream regulation in the absence of a proximal site. (iii) Operators within regulons appear in very variable proximal positions. By contrast, the proximal activation sites of regulons are much more fixed. (iv) There is a forbidden zone for activation elements downstream from approximately position -20 with respect to the start of transcription. By contrast, operators can occur throughout the proximal region. When activation elements appear in the forbidden zone, they repress. These latter examples usually involve autoregulation. (v) Approximately 40% of repressible promoters contain operator duplications. These occur either in certain regulons where duplication appears to be a requirement for repressor action or in promoters subject to complex regulation. (vi) Remote operator duplications occur in approximately 10% of repressible promoters. They generally appear when a multiple promoter region is coregulated by cyclic AMP receptor protein. (vii) Sigma 54 promoters do not require proximal or precisely positioned activator elements and are not generally subject to negative regulation. Rationales are presented for all of the above observations. PMID:1943993

  10. Combinatorial transcriptional control of the lactose operon of Escherichia coli.

    PubMed

    Kuhlman, Thomas; Zhang, Zhongge; Saier, Milton H; Hwa, Terence

    2007-04-01

    The goal of systems biology is to understand the behavior of the whole in terms of knowledge of the parts. This is hard to achieve in many cases due to the difficulty of characterizing the many constituents involved in a biological system and their complex web of interactions. The lac promoter of Escherichia coli offers the possibility of confronting "system-level" properties of transcriptional regulation with the known biochemistry of the molecular constituents and their mutual interactions. Such confrontations can reveal previously unknown constituents and interactions, as well as offer insight into how the components work together as a whole. Here we study the combinatorial control of the lac promoter by the regulators Lac repressor (LacR) and cAMP-receptor protein (CRP). A previous in vivo study [Setty Y, Mayo AE, Surette MG, Alon U (2003) Proc Natl Acad Sci USA 100:7702-7707] found gross disagreement between the observed promoter activities and the expected behavior based on the known molecular mechanisms. We repeated the study by identifying and removing several extraneous factors that significantly modulated the expression of the lac promoter. Through quantitative, systematic characterization of promoter activity for a number of key mutants and guided by the thermodynamic model of transcriptional regulation, we were able to account for the combinatorial control of the lac promoter quantitatively, in terms of a cooperative interaction between CRP and LacR-mediated DNA looping. Specifically, our analysis indicates that the sensitivity of the inducer response results from LacR-mediated DNA looping, which is significantly enhanced by CRP. PMID:17376875

  11. Transcription profile of Escherichia coli: genomic SELEX search for regulatory targets of transcription factors.

    PubMed

    Ishihama, Akira; Shimada, Tomohiro; Yamazaki, Yukiko

    2016-03-18

    Bacterial genomes are transcribed by DNA-dependent RNA polymerase (RNAP), which achieves gene selectivity through interaction with sigma factors that recognize promoters, and transcription factors (TFs) that control the activity and specificity of RNAP holoenzyme. To understand the molecular mechanisms of transcriptional regulation, the identification of regulatory targets is needed for all these factors. We then performed genomic SELEX screenings of targets under the control of each sigma factor and each TF. Here we describe the assembly of 156 SELEX patterns of a total of 116 TFs performed in the presence and absence of effector ligands. The results reveal several novel concepts: (i) each TF regulates more targets than hitherto recognized; (ii) each promoter is regulated by more TFs than hitherto recognized; and (iii) the binding sites of some TFs are located within operons and even inside open reading frames. The binding sites of a set of global regulators, including cAMP receptor protein, LeuO and Lrp, overlap with those of the silencer H-NS, suggesting that certain global regulators play an anti-silencing role. To facilitate sharing of these accumulated SELEX datasets with the research community, we compiled a database, 'Transcription Profile of Escherichia coli' (www.shigen.nig.ac.jp/ecoli/tec/). PMID:26843427

  12. Transcriptional Inhibitor of Virulence Factors in Enteropathogenic Escherichia coli

    PubMed Central

    Gauthier, Annick; Robertson, Marilyn L.; Lowden, Michael; Ibarra, J. Antonio; Puente, José Luis; Finlay, B. Brett

    2005-01-01

    The type III secretion system (TTSS) is a key virulence mechanism of many important gram-negative bacterial pathogens. The TTSS is conserved among different bacterial pathogens, and mutations and deletions to the system significantly decrease virulence, making the TTSS an important potential therapeutic target. We have developed a high-throughput assay to search for inhibitors of the TTSS. We screened a commercial library of 20,000 small molecules for their ability to inhibit type III secretion by enteropathogenic Escherichia coli (EPEC). After discarding compounds that had no effect on secretion, inhibited bacterial growth, and/or caused degradation of EPEC-secreted proteins, the search was focused on a class of compounds that, while not direct inhibitors of type III secretion, inhibit expression of TTSS-related genes and other genes involved in virulence. This class of compounds does not affect bacterial viability or motility, indicating that it is not significantly affecting the expression of essential genes and is specific to virulence-associated genes. Transcriptional fusion assays confirmed that virulence-associated promoters were more sensitive to inhibition by this class of compounds. Overall, we have identified a class of compounds that can be used as a tool to probe the mechanism(s) that regulates virulence gene expression in EPEC. PMID:16189086

  13. Meta-Analysis of Transcriptional Responses to Mastitis-Causing Escherichia coli

    PubMed Central

    Younis, Sidra; Javed, Qamar; Blumenberg, Miroslav

    2016-01-01

    Bovine mastitis is a widespread disease in dairy cows, and is often caused by bacterial mammary gland infection. Mastitis causes reduced milk production and leads to excessive use of antibiotics. We present meta-analysis of transcriptional profiles of bovine mastitis from 10 studies and 307 microarrays, allowing identification of much larger sets of affected genes than any individual study. Combining multiple studies provides insight into the molecular effects of Escherichia coli infection in vivo and uncovers differences between the consequences of E. coli vs. Staphylococcus aureus infection of primary mammary epithelial cells (PMECs). In udders, live E. coli elicits inflammatory and immune defenses through numerous cytokines and chemokines. Importantly, E. coli infection causes downregulation of genes encoding lipid biosynthesis enzymes that are involved in milk production. Additionally, host metabolism is generally suppressed. Finally, defensins and bacteria-recognition genes are upregulated, while the expression of the extracellular matrix protein transcripts is silenced. In PMECs, heat-inactivated E. coli elicits expression of ribosomal, cytoskeletal and angiogenic signaling genes, and causes suppression of the cell cycle and energy production genes. We hypothesize that heat-inactivated E. coli may have prophylactic effects against mastitis. Heat-inactivated S. aureus promotes stronger inflammatory and immune defenses than E. coli. Lipopolysaccharide by itself induces MHC antigen presentation components, an effect not seen in response to E. coli bacteria. These results provide the basis for strategies to prevent and treat mastitis and may lead to the reduction in the use of antibiotics. PMID:26933871

  14. Dynamic Transcriptional Response of Escherichia coli to Inclusion Body Formation

    PubMed Central

    Baig, Faraz; Fernando, Lawrence P.; Salazar, Mary Alice; Powell, Rhonda R.; Bruce, Terri F.; Harcum, Sarah W.

    2014-01-01

    Escherichia coli is used intensively for recombinant protein production, but one key challenge with recombinant E. coli is the tendency of recombinant proteins to misfold and aggregate into insoluble inclusion bodies (IBs). IBs contain high concentrations of inactive recombinant protein that require recovery steps to salvage a functional recombinant protein. Currently, no universally effective method exists to prevent IB formation in recombinant E. coli. In this study, DNA microarrays were used to compare the E. coli gene expression response dynamics to soluble and insoluble recombinant protein production. As expected and previously reported, the classical heat-shock genes had increased expression due to IB formation, including protein folding chaperones and proteases. Gene expression levels for protein synthesis-related and energy-synthesis pathways were also increased. Many transmembrane transporter and corresponding catabolic pathways genes had decreased expression for substrates not present in the culture medium. Additionally, putative genes represented over one-third of the genes identified to have significant expression changes due to IB formation, indicating many important cellular responses to IB formation still need to be characterized. Interestingly, cells grown in 3% ethanol had significantly reduced gene expression responses due to IB formation. Taken together, these results indicate that IB formation is complex, stimulates the heat-shock response, increases protein and energy synthesis needs, and streamlines transport and catabolic processes, while ethanol diminished all of these responses. PMID:24338599

  15. Mapping the Escherichia coli transcription elongation complex with exonuclease III

    PubMed Central

    Liu, Zhaokun; Artsimovitch, Irina

    2014-01-01

    Summary RNA polymerase interactions with the nucleic acids control every step of the transcription cycle. These contacts mediate RNA polymerase recruitment to promoters; induce pausing during RNA chain synthesis, and control transcription termination. These interactions are dissected using footprinting assays, in which a bound protein protects nucleic acids from the digestion by nucleases or modification by chemical probes. Exonuclease III is frequently employed to study protein-DNA interactions owing to relatively simple procedures and low background. Exonuclease III has been used to determine RNA polymerase position in transcription initiation and elongation complexes and to infer the translocation register of the enzyme. In this chapter, we describe probing the location and the conformation of transcription elongation complexes formed by walking of the RNA polymerase along an immobilized template. PMID:25665555

  16. Transcription termination controls prophage maintenance in Escherichia coli genomes

    PubMed Central

    Menouni, Rachid; Champ, Stéphanie; Espinosa, Leon; Boudvillain, Marc; Ansaldi, Mireille

    2013-01-01

    Prophages represent a large fraction of prokaryotic genomes and often provide new functions to their hosts, in particular virulence and fitness. How prokaryotic cells maintain such gene providers is central for understanding bacterial genome evolution by horizontal transfer. Prophage excision occurs through site-specific recombination mediated by a prophage-encoded integrase. In addition, a recombination directionality factor (or excisionase) directs the reaction toward excision and prevents the phage genome from being reintegrated. In this work, we describe the role of the transcription termination factor Rho in prophage maintenance through control of the synthesis of transcripts that mediate recombination directionality factor expression and, thus, excisive recombination. We show that Rho inhibition by bicyclomycin allows for the expression of prophage genes that lead to excisive recombination. Thus, besides its role in the silencing of horizontally acquired genes, Rho also maintains lysogeny of defective and functional prophages. PMID:23940369

  17. Transcriptional control of IS1 transposition in Escherichia coli.

    PubMed

    Biel, S W; Adelt, G; Berg, D E

    1984-04-01

    The movement of the bacterial insertion sequence IS1 often generates cointegrate structures in which donor and target replicons are connected by direct repeats of IS1. The experiments reported here were designed to understand how IS1 transposition is controlled. Our physical characterization of the structures of cointegrates between an F factor ( pOX38 ) and a set of pBR322::Tn9-related plasmids indicate that the relative mobilities of the two IS1 elements of Tn9 are inversely correlated with the strength of promoters upstream in the vector DNA. This implies that transcription across the ends of an IS1 element inhibits its transposition. Transcriptional inhibition may be due to interference with either the synthesis or the action of transposase. PMID:6325710

  18. Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli.

    PubMed

    Seo, Sang Woo; Kim, Donghyuk; Latif, Haythem; O'Brien, Edward J; Szubin, Richard; Palsson, Bernhard O

    2014-01-01

    The ferric uptake regulator (Fur) plays a critical role in the transcriptional regulation of iron metabolism. However, the full regulatory potential of Fur remains undefined. Here we comprehensively reconstruct the Fur transcriptional regulatory network in Escherichia coli K-12 MG1655 in response to iron availability using genome-wide measurements. Integrative data analysis reveals that a total of 81 genes in 42 transcription units are directly regulated by three different modes of Fur regulation, including apo- and holo-Fur activation and holo-Fur repression. We show that Fur connects iron transport and utilization enzymes with negative-feedback loop pairs for iron homeostasis. In addition, direct involvement of Fur in the regulation of DNA synthesis, energy metabolism and biofilm development is found. These results show how Fur exhibits a comprehensive regulatory role affecting many fundamental cellular processes linked to iron metabolism in order to coordinate the overall response of E. coli to iron availability. PMID:25222563

  19. YjjQ Represses Transcription of flhDC and Additional Loci in Escherichia coli

    PubMed Central

    Wiebe, Helene; Gürlebeck, Doreen; Groß, Jana; Dreck, Katrin; Pannen, Derk; Ewers, Christa; Wieler, Lothar H.

    2015-01-01

    ABSTRACT The presumptive transcriptional regulator YjjQ has been identified as being virulence associated in avian pathogenic Escherichia coli (APEC). In this work, we characterize YjjQ as transcriptional repressor of the flhDC operon, encoding the master regulator of flagellar synthesis, and of additional loci. The latter include gfc (capsule 4 synthesis), ompC (outer membrane porin C), yfiRNB (regulated c-di-GMP synthesis), and loci of poorly defined function (ybhL and ymiA-yciX). We identify the YjjQ DNA-binding sites at the flhDC and gfc promoters and characterize a DNA-binding sequence motif present at all promoters found to be repressed by YjjQ. At the flhDC promoter, the YjjQ DNA-binding site overlaps the RcsA-RcsB DNA-binding site. RcsA-RcsB likewise represses the flhDC promoter, but the repression by YjjQ and that by RcsA-RcsB are independent of each other. These data suggest that YjjQ is an additional regulator involved in the complex control of flhDC at the level of transcription initiation. Furthermore, we show that YjjQ represses motility of the E. coli K-12 laboratory strain and of uropathogenic E. coli (UPEC) strains CFT073 and 536. Regulation of flhDC, yfiRNB, and additional loci by YjjQ may be features relevant for pathogenicity. IMPORTANCE Escherichia coli is a commensal and pathogenic bacterium causing intra- and extraintestinal infections in humans and farm animals. The pathogenicity of E. coli strains is determined by their particular genome content, which includes essential and associated virulence factors that control the cellular physiology in the host environment. However, the gene pools of commensal and pathogenic E. coli are not clearly differentiated, and the function of virulence-associated loci needs to be characterized. In this study, we characterize the function of yjjQ, encoding a transcription regulator that was identified as being virulence associated in avian pathogenic E. coli (APEC). We characterize YjjQ as transcriptional

  20. Preliminary Characterization of the Transcriptional Response of the Porcine Intestinal Cell Line IPEC-J2 to Enterotoxigenic Escherichia coli, Escherichia coli, and E. coli Lipopolysaccharide

    PubMed Central

    Geens, Marisa M.; Niewold, Theo A.

    2010-01-01

    IPEC-J2, a promising in vitro model system, is not well characterized especially on the transcriptional level, in contrast to human counterparts. The aim of this study was to characterize the gene expression in IPEC-J2 cells when coincubated with enterotoxigenic Escherichia coli (ETEC), nonpathogenic E. coli, and E. coli endotoxin. Apical infection of polarized IPEC-J2 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TEER). Microarray analysis showed up-regulation of interleukins when IPEC-J2 were cocultured with E. coli strains this has so far never been measured in this cell line. Highest IL8 expression was found with the ETEC strain possessing the F4 fimbrium, suggesting IPEC-J2 cells to be F4 receptor positive, confirmed in a brush border membrane adhesion assay. It is concluded that the innate immune responses to pathogens and LPS makes the IPEC-J2 cell line a suitable model for research on intestinal host pathogen interaction. PMID:21318186

  1. Transcription of the Escherichia coli fliC gene is regulated by metal ions

    SciTech Connect

    Guzzo, A.; Diorio, C.; DuBow, M.S. )

    1991-08-01

    luxAB gene fusions in the Escherichia coli genome were used to screen for clones displaying transcriptional changes in the presence of aluminum. One clone was found that contained a luciferase gene fusion in which transcription was increased in the presence of aluminum and which was subsequently shown to be induced by copper, iron, and nickel. Cloning of the metal-regulated gene, hybridization to the ordered phage {lambda} bank of the E. coli chromosome, and sequencing of DNA adjacent to the luxAB fusion revealed that the insertion occurred within the fliC (hag) gene of E. coli. This gene encodes flagellin the filament subunit of the bacterial motility organ, and is under the control of several regulatory cascades. These results suggest that environmental metals may play a role in the regulation of the motility potential of E. coli and that this bioluminescent gene fusion clone (or derivatives thereof) may be used to prepare a biosensor for the rapid detection of metal contamination in water samples.

  2. Intracellular Concentrations of 65 Species of Transcription Factors with Known Regulatory Functions in Escherichia coli

    PubMed Central

    Kori, Ayako; Koshio, Etsuko; Yamada, Kayoko; Maeda, Hiroto; Shimada, Tomohiro; Makinoshima, Hideki; Iwata, Akira; Fujita, Nobuyuki

    2014-01-01

    The expression pattern of the Escherichia coli genome is controlled in part by regulating the utilization of a limited number of RNA polymerases among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in E. coli K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of E. coli under various stressful conditions in nature. PMID:24837290

  3. Environmental conditions and transcriptional regulation in Escherichia coli: a physiological integrative approach.

    PubMed

    Martínez-Antonio, Agustino; Salgado, Heladia; Gama-Castro, Socorro; Gutiérrez-Ríos, Rosa María; Jiménez-Jacinto, Verónica; Collado-Vides, Julio

    2003-12-30

    Bacteria develop a number of devices for sensing, responding, and adapting to different environmental conditions. Understanding within a genomic perspective how the transcriptional machinery of bacteria is modulated, as a response for changing conditions, is a major challenge for biologists. Knowledge of which genes are turned on or turned off under specific conditions is essential for our understanding of cell behavior. In this study we describe how the information pertaining to gene expression and associated growth conditions (even with very little knowledge of the associated regulatory mechanisms) is gathered from the literature and incorporated into RegulonDB, a database on transcriptional regulation and operon organization in E. coli. The link between growth conditions, signal transduction, and transcriptional regulation is modeled in the database in a simple format that highlights biological relevant information. As far as we know, there is no other database that explicitly clarifies the effect of environmental conditions on gene transcription. We discuss how this knowledge constitutes a benchmark that will impact future research aimed at integration of regulatory responses in the cell; for instance, analysis of microarrays, predicting culture behavior in biotechnological processes, and comprehension of dynamics of regulatory networks. This integrated knowledge will contribute to the future goal of modeling the behavior of E. coli as an entire cell. The RegulonDB database can be accessed on the web at the URL: http://www.cifn.unam.mx/Computational_Biology/regulondb/. PMID:14708114

  4. Functional characterization of alternate optimal solutions of Escherichia coli's transcriptional and translational machinery.

    PubMed

    Thiele, Ines; Fleming, Ronan M T; Bordbar, Aarash; Schellenberger, Jan; Palsson, Bernhard Ø

    2010-05-19

    The constraint-based reconstruction and analysis approach has recently been extended to describe Escherichia coli's transcriptional and translational machinery. Here, we introduce the concept of reaction coupling to represent the dependency between protein synthesis and utilization. These coupling constraints lead to a significant contraction of the feasible set of steady-state fluxes. The subset of alternate optimal solutions (AOS) consistent with maximal ribosome production was calculated. The majority of transcriptional and translational reactions were active for all of these AOS, showing that the network has a low degree of redundancy. Furthermore, all calculated AOS contained the qualitative expression of at least 92% of the known essential genes. Principal component analysis of AOS demonstrated that energy currencies (ATP, GTP, and phosphate) dominate the network's capability to produce ribosomes. Additionally, we identified regulatory control points of the network, which include the transcription reactions of sigma70 (RpoD) as well as that of a degradosome component (Rne) and of tRNA charging (ValS). These reactions contribute significant variance among AOS. These results show that constraint-based modeling can be applied to gain insight into the systemic properties of E. coli's transcriptional and translational machinery. PMID:20483314

  5. Escherichia coli pfs transcription: regulation and proposed roles in autoinducer-2 synthesis and purine excretion.

    PubMed

    Kim, Youngbae; Lew, Chih M; Gralla, Jay D

    2006-11-01

    Pfs expression is required for several metabolic pathways and limits the production of autoinducer-2, a molecule proposed to play a central role in interspecies quorum sensing. The present study reveals physiological conditions and promoter DNA elements that regulate Escherichia coli pfs transcription. Pfs transcription is shown to rely on both sigma 70 and sigma 38 (rpoS), and the latter is subject to induction that increases pfs expression. Transcription is maximal as the cells approach stationary phase, and this level can be increased by salt stress through induction of sigma 38-dependent expression. The pfs promoter is shown to contain both positive and negative elements, which can be used by both forms of RNA polymerase. The negative element is contained within the overlapping dgt promoter, which is involved in purine metabolism. Consideration of the physiological roles of sigma 38 and dgt leads to a model for how autoinducer production is controlled under changing physiological conditions. PMID:16950920

  6. Transcription profile of Escherichia coli: genomic SELEX search for regulatory targets of transcription factors

    PubMed Central

    Ishihama, Akira; Shimada, Tomohiro; Yamazaki, Yukiko

    2016-01-01

    Bacterial genomes are transcribed by DNA-dependent RNA polymerase (RNAP), which achieves gene selectivity through interaction with sigma factors that recognize promoters, and transcription factors (TFs) that control the activity and specificity of RNAP holoenzyme. To understand the molecular mechanisms of transcriptional regulation, the identification of regulatory targets is needed for all these factors. We then performed genomic SELEX screenings of targets under the control of each sigma factor and each TF. Here we describe the assembly of 156 SELEX patterns of a total of 116 TFs performed in the presence and absence of effector ligands. The results reveal several novel concepts: (i) each TF regulates more targets than hitherto recognized; (ii) each promoter is regulated by more TFs than hitherto recognized; and (iii) the binding sites of some TFs are located within operons and even inside open reading frames. The binding sites of a set of global regulators, including cAMP receptor protein, LeuO and Lrp, overlap with those of the silencer H-NS, suggesting that certain global regulators play an anti-silencing role. To facilitate sharing of these accumulated SELEX datasets with the research community, we compiled a database, ‘Transcription Profile of Escherichia coli’ (www.shigen.nig.ac.jp/ecoli/tec/). PMID:26843427

  7. Genes on a Wire: The Nucleoid-Associated Protein HU Insulates Transcription Units in Escherichia coli.

    PubMed

    Berger, Michael; Gerganova, Veneta; Berger, Petya; Rapiteanu, Radu; Lisicovas, Viktoras; Dobrindt, Ulrich

    2016-01-01

    The extent to which chromosomal gene position in prokaryotes affects local gene expression remains an open question. Several studies have shown that chromosomal re-positioning of bacterial transcription units does not alter their expression pattern, except for a general decrease in gene expression levels from chromosomal origin to terminus proximal positions, which is believed to result from gene dosage effects. Surprisingly, the question as to whether this chromosomal context independence is a cis encoded property of a bacterial transcription unit, or if position independence is a property conferred by factors acting in trans, has not been addressed so far. For this purpose, we established a genetic test system assessing the chromosomal positioning effects by means of identical promoter-fluorescent reporter gene fusions inserted equidistantly from OriC into both chromosomal replichores of Escherichia coli K-12. Our investigations of the reporter activities in mutant cells lacking the conserved nucleoid associated protein HU uncovered various drastic chromosomal positional effects on gene transcription. In addition we present evidence that these positional effects are caused by transcriptional activity nearby the insertion site of our reporter modules. We therefore suggest that the nucleoid-associated protein HU is functionally insulating transcription units, most likely by constraining transcription induced DNA supercoiling. PMID:27545593

  8. Genes on a Wire: The Nucleoid-Associated Protein HU Insulates Transcription Units in Escherichia coli

    PubMed Central

    Berger, Michael; Gerganova, Veneta; Berger, Petya; Rapiteanu, Radu; Lisicovas, Viktoras; Dobrindt, Ulrich

    2016-01-01

    The extent to which chromosomal gene position in prokaryotes affects local gene expression remains an open question. Several studies have shown that chromosomal re-positioning of bacterial transcription units does not alter their expression pattern, except for a general decrease in gene expression levels from chromosomal origin to terminus proximal positions, which is believed to result from gene dosage effects. Surprisingly, the question as to whether this chromosomal context independence is a cis encoded property of a bacterial transcription unit, or if position independence is a property conferred by factors acting in trans, has not been addressed so far. For this purpose, we established a genetic test system assessing the chromosomal positioning effects by means of identical promoter-fluorescent reporter gene fusions inserted equidistantly from OriC into both chromosomal replichores of Escherichia coli K-12. Our investigations of the reporter activities in mutant cells lacking the conserved nucleoid associated protein HU uncovered various drastic chromosomal positional effects on gene transcription. In addition we present evidence that these positional effects are caused by transcriptional activity nearby the insertion site of our reporter modules. We therefore suggest that the nucleoid-associated protein HU is functionally insulating transcription units, most likely by constraining transcription induced DNA supercoiling. PMID:27545593

  9. Model of transcriptional activation by MarA in escherichia coli

    SciTech Connect

    Wall, Michael E; Rosner, Judah L; Martin, Robert G

    2009-01-01

    The AraC family transcription factor MarA activates approximately 40 genes (the marA/soxS/rob regulon) of the Escherichia coli chromosome resulting in different levels of resistance to a wide array of antibiotics and to superoxides. Activation of marA/soxS/rob regulon promoters occurs in a well-defined order with respect to the level of MarA; however, the order of activation does not parallel the strength of MarA binding to promoter sequences. To understand this lack of correspondence, we developed a computational model of transcriptional activation in which a transcription factor either increases or decreases RNA polymerase binding, and either accelerates or retards post-binding events associated with transcription initiation. We used the model to analyze data characterizing MarA regulation of promoter activity. The model clearly explains the lack of correspondence between the order of activation and the MarA-DNA affinity and indicates that the order of activation can only be predicted using information about the strength of the full MarA-polymerase-DNA interaction. The analysis further suggests that MarA can activate without increasing polymerase binding and that activation can even involve a decrease in polymerase binding, which is opposite to the textbook model of activation by recruitment. These findings are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. We find that activation involving decreased polymerase binding yields lower latency in gene regulation and therefore might confer a competitive advantage to cells. Our model yields insights into requirements for predicting the order of activation of a regulon and enables us to suggest that activation might involve a decrease in polymerase binding which we expect to be an important theme of gene regulation in E. coli and beyond.

  10. dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product.

    PubMed Central

    Henrich, B; Becker, S; Schroeder, U; Plapp, R

    1993-01-01

    Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product. Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression. The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium opdA and the E. coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae. No potential export signals could be inferred from the amino acid sequence. Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E. coli and used to investigate some of its biochemical and biophysical properties. Images PMID:8226676

  11. The functional landscape bound to the transcription factors of Escherichia coli K-12.

    PubMed

    Pérez-Rueda, Ernesto; Tenorio-Salgado, Silvia; Huerta-Saquero, Alejandro; Balderas-Martínez, Yalbi I; Moreno-Hagelsieb, Gabriel

    2015-10-01

    Motivated by the experimental evidences accumulated in the last ten years and based on information deposited in RegulonDB, literature look up, and sequence analysis, we analyze the repertoire of 304 DNA-binding Transcription factors (TFs) in Escherichia coli K-12. These regulators were grouped in 78 evolutionary families and are regulating almost half of the total genes in this bacterium. In structural terms, 60% of TFs are composed by two-domains, 30% are monodomain, and 10% three- and four-structural domains. As previously noticed, the most abundant DNA-binding domain corresponds to the winged helix-turn-helix, with few alternative DNA-binding structures, resembling the hypothesis of successful protein structures with the emergence of new ones at low scales. In summary, we identified and described the characteristics associated to the DNA-binding TF in E. coli K-12. We also identified twelve functional modules based on a co-regulated gene matrix. Finally, diverse regulons were predicted based on direct associations between the TFs and potential regulated genes. This analysis should increase our knowledge about the gene regulation in the bacterium E. coli K-12, and provide more additional clues for comprehensive modelling of transcriptional regulatory networks in other bacteria. PMID:26094112

  12. Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding

    PubMed Central

    Zorzini, Valentina; Buts, Lieven; Schrank, Evelyne; Sterckx, Yann G.J.; Respondek, Michal; Engelberg-Kulka, Hanna; Loris, Remy; Zangger, Klaus; van Nuland, Nico A.J.

    2015-01-01

    Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses. The mazEF module encodes the MazF toxin and its cognate MazE antitoxin. The highly dynamic MazE possesses an N-terminal DNA binding domain through which it can negatively regulate its own promoter. Despite being one of the first TA systems studied, transcriptional regulation of Escherichia coli mazEF remains poorly understood. This paper presents the solution structure of C-terminal truncated E. coli MazE and a MazE-DNA model with a DNA palindrome sequence ∼10 bp upstream of the mazEF promoter. The work has led to a transcription regulator-DNA model, which has remained elusive thus far in the E. coli toxin–antitoxin family. Multiple complementary techniques including NMR, SAXS and ITC show that the long intrinsically disordered C-termini in MazE, required for MazF neutralization, does not affect the interactions between the antitoxin and its operator. Rather, the MazE C-terminus plays an important role in the MazF binding, which was found to increase the MazE affinity for the palindromic single site operator. PMID:25564525

  13. Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding.

    PubMed

    Zorzini, Valentina; Buts, Lieven; Schrank, Evelyne; Sterckx, Yann G J; Respondek, Michal; Engelberg-Kulka, Hanna; Loris, Remy; Zangger, Klaus; van Nuland, Nico A J

    2015-01-01

    Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses. The mazEF module encodes the MazF toxin and its cognate MazE antitoxin. The highly dynamic MazE possesses an N-terminal DNA binding domain through which it can negatively regulate its own promoter. Despite being one of the first TA systems studied, transcriptional regulation of Escherichia coli mazEF remains poorly understood. This paper presents the solution structure of C-terminal truncated E. coli MazE and a MazE-DNA model with a DNA palindrome sequence ∼ 10 bp upstream of the mazEF promoter. The work has led to a transcription regulator-DNA model, which has remained elusive thus far in the E. coli toxin-antitoxin family. Multiple complementary techniques including NMR, SAXS and ITC show that the long intrinsically disordered C-termini in MazE, required for MazF neutralization, does not affect the interactions between the antitoxin and its operator. Rather, the MazE C-terminus plays an important role in the MazF binding, which was found to increase the MazE affinity for the palindromic single site operator. PMID:25564525

  14. Model of Transcriptional Activation By MarA in Escherichia Coli

    NASA Astrophysics Data System (ADS)

    Wall, Michael E.; Markowitz, David A.; Rosner, Judah L.; Martin, Robert G.

    2010-01-01

    We have developed a mathematical model of transcriptional activation by MarA in Escherichia coli, and used the model to analyze measurements of MarA-dependent activity of the marRAB, sodA, and micF promoters in mar-rob- cells. The model rationalizes an unexpected poor correlation between the mid-point of in vivo promoter activity profiles and in vitro equilibrium constants for MarA binding to promoter sequences. Analysis of the promoter activity data using the model yielded the following predictions regarding activation mechanisms: (1) MarA activation of the marRAB, sodA, and micF promoters involves a net acceleration of the kinetics of transitions after RNA polymerase binding, up to and including promoter escape and message elongation; (2) RNA polymerase binds to these promoters with nearly unit occupancy in the absence of MarA, making recruitment of polymerase an insignificant factor in activation of these promoters; and (3) instead of recruitment, activation of the micF promoter might involve a repulsion of polymerase combined with a large acceleration of the kinetics of polymerase activity. These predictions are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. A lack of recruitment in transcriptional activation represents an exception to the textbook description of activation of bacterial sigma-70 promoters. However, use of accelerated polymerase kinetics instead of recruitment might confer a competitive advantage to E. coli by decreasing latency in gene regulation.

  15. Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli

    PubMed Central

    Seo, Sang Woo; Kim, Donghyuk; Latif, Haythem; O’Brien, Edward J.; Szubin, Richard; Palsson, Bernhard O.

    2014-01-01

    The ferric uptake regulator (Fur) plays a critical role in the transcriptional regulation of iron metabolism. However, the full regulatory potential of Fur remains undefined. Here we comprehensively reconstruct the Fur transcriptional regulatory network in Escherichia coli K-12 MG1655 in response to iron availability using genome-wide measurements (ChIP-exo and RNA-seq). Integrative data analysis reveals that a total of 81 genes in 42 transcription units are directly regulated by three different modes of Fur regulation, including apo- and holo-Fur activation and holo-Fur repression. We show that Fur connects iron transport and utilization enzymes with negative-feedback loop pairs for iron homeostasis. In addition, direct involvement of Fur in the regulation of DNA synthesis, energy metabolism, and biofilm development is found. These results show how Fur exhibits a comprehensive regulatory role affecting many fundamental cellular processes linked to iron metabolism in order to coordinate the overall response of E. coli to iron availability. PMID:25222563

  16. The spatial biology of transcription and translation in rapidly growing Escherichia coli

    PubMed Central

    Bakshi, Somenath; Choi, Heejun; Weisshaar, James C.

    2015-01-01

    Single-molecule fluorescence provides high resolution spatial distributions of ribosomes and RNA polymerase (RNAP) in live, rapidly growing Escherichia coli. Ribosomes are more strongly segregated from the nucleoids (chromosomal DNA) than previous widefield fluorescence studies suggested. While most transcription may be co-translational, the evidence indicates that most translation occurs on free mRNA copies that have diffused from the nucleoids to a ribosome-rich region. Analysis of time-resolved images of the nucleoid spatial distribution after treatment with the transcription-halting drug rifampicin and the translation-halting drug chloramphenicol shows that both drugs cause nucleoid contraction on the 0–3 min timescale. This is consistent with the transertion hypothesis. We suggest that the longer-term (20–30 min) nucleoid expansion after Rif treatment arises from conversion of 70S-polysomes to 30S and 50S subunits, which readily penetrate the nucleoids. Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits) explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone. A comprehensive model of the transcription-translation-transertion system incorporates this new information about the spatial organization of the E. coli cytoplasm. We propose that transertion, which radially expands the nucleoids, is essential for recycling of 30S and 50S subunits from ribosome-rich regions back into the nucleoids. There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain. Segregation of 70S-polysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription

  17. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription.

    PubMed

    Bell, A; Gaston, K; Williams, R; Chapman, K; Kolb, A; Buc, H; Minchin, S; Williams, J; Busby, S

    1990-12-25

    The effects of a number of mutations in the E. coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures. PMID:2259621

  18. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription.

    PubMed Central

    Bell, A; Gaston, K; Williams, R; Chapman, K; Kolb, A; Buc, H; Minchin, S; Williams, J; Busby, S

    1990-01-01

    The effects of a number of mutations in the E. coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures. Images PMID:2259621

  19. Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA.

    PubMed

    Weitzmann, C J; Cunningham, P R; Ofengand, J

    1990-06-25

    The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed. PMID:2194163

  20. DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro.

    PubMed Central

    Scarlato, V; Aricò, B; Rappuoli, R

    1993-01-01

    The bvg locus of Bordetella pertussis encodes an environmentally inducible operon essential for the expression of virulence genes. We show that in Escherichia coli, the PTOX promoter cloned in cis of the bvg locus is activated and environmentally regulated. Cotransformation of E. coli with the bvg locus cloned in a low-copy-number plasmid and with the PTOX promoter cloned in a high-copy-number plasmid can give rise to two different results. If the PTOX promoter is cloned in the pGem-3 vector, transcription is absent. If the PTOX promoter is cloned in the plasmid pKK232, containing the PTOX promoter between two ribosomal gene terminators of transcription, transcription occurs, although regulation of transcription is abolished. Under these conditions, the intracellular amount of RNA transcripts is increased by adding to the culture medium novobiocin, an inhibitor of bacterial gyrases. In vitro, the transcription of the PTOX promoter is activated on E. coli RNA polymerase supplemented with cell extracts from wild-type B. pertussis. Addition of DNA gyrase to the mixture dramatically reduces the amount of RNA synthesized. Our data show that the products of the bvg locus, BvgA and BvgS, are directly involved in the regulation of the PTOX promoter in E. coli and that DNA topology may play a role in the induction of transcription. Images PMID:8393006

  1. A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho.

    PubMed Central

    Geiselmann, J; Wang, Y; Seifried, S E; von Hippel, P H

    1993-01-01

    Transcription termination protein Rho of Escherichia coli interacts with newly synthesized RNA chains and brings about their release from elongation complexes paused at specific Rho-dependent termination sites. Rho is thought to accomplish this by binding to a specific Rho "loading site" on the nascent RNA and then translocating preferentially along the transcript in a 5'-->3' direction. On reaching the elongation complex, Rho releases the nascent RNA by a 5'-->3' RNA.DNA helicase activity. These translocation and helicase activities are driven by the RNA-dependent ATPase activity of Rho. In this paper we propose a mechanism for these processes that is based on the structure and properties of the Rho protein. Rho is a hexamer of identical subunits that are arranged as a trimer of asymmetric dimers with D3 symmetry. The binding of ATP and RNA to Rho also reflects this pattern; the Rho hexamer carries three strong and three weak binding sites for each of these entities. The asymmetric dimers of Rho correspond to functional dimers that can undergo conformational transitions driven by ATP hydrolysis. We propose that the quaternary structure of Rho coordinates the ATP-driven RNA binding and release processes to produce a biased random walk of the Rho hexamer along the RNA, followed by RNA.DNA helicase activity and transcript release. The proposed model may have implications for other hexameric DNA.DNA, RNA.DNA, and RNA.RNA helicases that function in replication and transcription. Images Fig. 2 PMID:7689228

  2. Transcriptional cross-activation between toxin-antitoxin systems of Escherichia coli

    PubMed Central

    2013-01-01

    Background Bacterial toxin-antitoxin (TA) systems are formed by potent regulatory or suicide factors (toxins) and their short-lived inhibitors (antitoxins). Antitoxins are DNA-binding proteins and auto-repress transcription of TA operons. Transcription of multiple TA operons is activated in temporarily non-growing persister cells that can resist killing by antibiotics. Consequently, the antitoxin levels of persisters must have been dropped and toxins are released of inhibition. Results Here, we describe transcriptional cross-activation between different TA systems of Escherichia coli. We find that the chromosomal relBEF operon is activated in response to production of the toxins MazF, MqsR, HicA, and HipA. Expression of the RelE toxin in turn induces transcription of several TA operons. We show that induction of mazEF during amino acid starvation depends on relBE and does not occur in a relBEF deletion mutant. Induction of TA operons has been previously shown to depend on Lon protease which is activated by polyphospate accumulation. We show that transcriptional cross-activation occurs also in strains deficient for Lon, ClpP, and HslV proteases and polyphosphate kinase. Furthermore, we find that toxins cleave the TA mRNA in vivo, which is followed by degradation of the antitoxin-encoding fragments and selective accumulation of the toxin-encoding regions. We show that these accumulating fragments can be translated to produce more toxin. Conclusion Transcriptional activation followed by cleavage of the mRNA and disproportionate production of the toxin constitutes a possible positive feedback loop, which can fire other TA systems and cause bistable growth heterogeneity. Cross-interacting TA systems have a potential to form a complex network of mutually activating regulators in bacteria. PMID:23432955

  3. Regulation of transcription of cell division genes in the Escherichia coli dcw cluster.

    PubMed

    Vicente, M; Gomez, M J; Ayala, J A

    1998-04-01

    The Escherichia coli dcw cluster contains cell division genes, such as the phylogenetically ubiquitous ftsZ, and genes involved in peptidoglycan synthesis. Transcription in the cluster proceeds in the same direction as the progress of the replication fork along the chromosome. Regulation is exerted at the transcriptional and post-transcriptional levels. The absence of transcriptional termination signals may, in principle, allow extension of the transcripts initiated at the up-stream promoter (mraZ1p) even to the furthest down-stream gene (envA). Complementation tests suggest that they extend into ftsW in the central part of the cluster. In addition, the cluster contains other promoters individually regulated by cis- and trans-acting signals. Dissociation of the expression of the ftsZ gene, located after ftsQ and A near the 3' end of the cluster, from its natural regulatory signals leads to an alteration in the physiology of cell division. The complexities observed in the regulation of gene expression in the cluster may then have an important biological role. Among them, LexA-binding SOS boxes have been found at the 5' end of the cluster, preceding promoters which direct the expression of ftsI (coding for PBP3, the penicillin-binding protein involved in septum formation). A gearbox promoter, ftsQ1p, forms part of the signals regulating the transcription of ftsQ, A and Z. It is an inversely growth-dependent mechanism driven by RNA polymerase containing sigma s, the factor involved in the expression of stationary phase-specific genes. Although the dcw cluster is conserved to a different extent in a variety of bacteria, the regulation of gene expression, the presence or absence of individual genes, and even the essentiality of some of them, show variations in the phylogenetic scale which may reflect adaptation to specific life cycles. PMID:9614967

  4. Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli.

    PubMed

    Zhou, Yan Ning; Lubkowska, Lucyna; Hui, Monica; Court, Carolyn; Chen, Shuo; Court, Donald L; Strathern, Jeffrey; Jin, Ding Jun; Kashlev, Mikhail

    2013-01-25

    Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the β subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity. PMID:23223236

  5. Isolation and Characterization of RNA Polymerase rpoB Mutations That Alter Transcription Slippage during Elongation in Escherichia coli*

    PubMed Central

    Zhou, Yan Ning; Lubkowska, Lucyna; Hui, Monica; Court, Carolyn; Chen, Shuo; Court, Donald L.; Strathern, Jeffrey; Jin, Ding Jun; Kashlev, Mikhail

    2013-01-01

    Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the β subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity. PMID:23223236

  6. Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction

    PubMed Central

    Tetteh, Antonia Y.; Sun, Katherine H.; Kittur, Farooqahmed S.; Ibeanu, Gordon C.

    2014-01-01

    Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se0), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process. PMID:24839442

  7. Genome-wide transcriptional response of an avian pathogenic Escherichia coli (APEC) pst mutant

    PubMed Central

    Crépin, Sébastien; Lamarche, Martin G; Garneau, Philippe; Séguin, Julie; Proulx, Julie; Dozois, Charles M; Harel, Josée

    2008-01-01

    Background Avian pathogenic E. coli (APEC) are associated with extraintestinal diseases in poultry. The pstSCAB-phoU operon belongs to the Pho regulon and encodes the phosphate specific transport (Pst) system. A functional Pst system is required for full virulence in APEC and other bacteria and contributes to resistance of APEC to serum, to cationic antimicrobial peptides and acid shock. The global mechanisms contributing to the attenuation and decreased resistance of the APEC pst mutant to environmental stresses have not been investigated at the transcriptional level. To determine the global effect of a pst mutation on gene expression, we compared the transcriptomes of APEC strain χ7122 and its isogenic pst mutant (K3) grown in phosphate-rich medium. Results Overall, 470 genes were differentially expressed by at least 1.5-fold. Interestingly, the pst mutant not only induced systems involved in phosphate acquisition and metabolism, despite phosphate availability, but also modulated stress response mechanisms. Indeed, transcriptional changes in genes associated with the general stress responses, including the oxidative stress response were among the major differences observed. Accordingly, the K3 strain was less resistant to reactive oxygen species (ROS) than the wild-type strain. In addition, the pst mutant demonstrated reduced expression of genes involved in lipopolysaccharide modifications and coding for cell surface components such as type 1 and F9 fimbriae. Phenotypic tests also established that the pst mutant was impaired in its capacity to produce type 1 fimbriae, as demonstrated by western blotting and agglutination of yeast cells, when compared to wild-type APEC strain χ7122. Conclusion Overall, our data elucidated the effects of a pst mutation on the transcriptional response, and further support the role of the Pho regulon as part of a complex network contributing to phosphate homeostasis, adaptive stress responses, and E. coli virulence. PMID:19038054

  8. Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism.

    PubMed

    Liu, Bin; Zuo, Yuhong; Steitz, Thomas A

    2016-04-12

    In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3'-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E coli transcription initiation complexes (TICs) containing the stress-responsive σ(S) factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σ(S)-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σ(S) factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the -10 element. In addition, σ(S)-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σ(S)-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation. PMID:27035955

  9. Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

    PubMed Central

    Liu, Bin; Zuo, Yuhong; Steitz, Thomas A.

    2016-01-01

    In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stress-responsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation. PMID:27035955

  10. DksA and ppGpp Directly Regulate Transcription of the Escherichia coli Flagellar Cascade

    PubMed Central

    Lemke, Justin J.; Durfee, Tim; Gourse, Richard L.

    2009-01-01

    The components of the Escherichia coli flagella apparatus are synthesized in a three-level transcriptional cascade activated by the master regulator FlhDC. The cascade coordinates the synthesis rates of a large number of gene products with each other and with nutritional conditions. Recent genome-wide studies have reported that flagellar transcription is altered in cells lacking the transcription regulators DksA or ppGpp, but some or all reported effects could be indirect, and some are contradictory. We report here that the activities of promoters at all three levels of the cascade are much higher in strains lacking dksA, resulting in overproduction of flagellin and hyperflagellated cells. In vitro, DksA/ppGpp inhibits the flhDC promoter and the σ70-dependent fliA promoter transcribing the gene for σ28. However, DksA and ppGpp do not affect the σ28-dependent fliA promoter or the σ28-dependent fliC promoter in vitro, suggesting that the dramatic effects on expression of those genes in vivo are mediated indirectly through direct effects of DksA/ppGpp on FlhDC and σ28 expression. We conclude that DksA/ppGpp inhibits expression of the flagellar cascade during stationary phase and following starvation, thereby coordinating flagella and ribosome assembly and preventing expenditure of scarce energy resources on synthesis of two of the cell’s largest macromolecular complexes. PMID:19889089

  11. Effects of post-transcriptional regulation on phenotypic noise in Escherichia coli

    PubMed Central

    Arbel-Goren, Rinat; Tal, Asaf; Friedlander, Tamar; Meshner, Shiri; Costantino, Nina; Court, Donald L.; Stavans, Joel

    2013-01-01

    Cell-to-cell variations in protein abundance, called noise, give rise to phenotypic variability between isogenic cells. Studies of noise have focused on stochasticity introduced at transcription, yet the effects of post-transcriptional regulatory processes on noise remain unknown. We study the effects of RyhB, a small-RNA of Escherichia coli produced on iron stress, on the phenotypic variability of two of its downregulated target proteins, using dual chromosomal fusions to fluorescent reporters and measurements in live individual cells. The total noise of each of the target proteins is remarkably constant over a wide range of RyhB production rates despite cells being in stress. In fact, coordinate downregulation of the two target proteins by RyhB reduces the correlation between their levels. Hence, an increase in phenotypic variability under stress is achieved by decoupling the expression of different target proteins in the same cell, rather than by an increase in the total noise of each. Extrinsic noise provides the dominant contribution to the total protein noise over the total range of RyhB production rates. Stochastic simulations reproduce qualitatively key features of our observations and show that a feed-forward loop formed by transcriptional extrinsic noise, an sRNA and its target genes exhibits strong noise filtration capabilities. PMID:23519613

  12. mioC transcription, initiation of replication, and the eclipse in Escherichia coli.

    PubMed Central

    Bogan, J A; Helmstetter, C E

    1996-01-01

    The potential role of mioC transcription as a negative regulator of initiation of chromosome replication in Escherichia coli was evaluated. When initiation was aligned by a shift of dnaC2(Ts) mutants to nonpermissive temperature (40 degrees C), mioC transcript levels measured at the 5' end or reading through oriC disappeared within one mass doubling. Upon return to permissive temperature (30 degrees C), the transcripts reappeared coordinately about 15 min after the first synchronized initiation and then declined sharply again 10 min later, just before the second initiation. Although these observations were consistent with the idea that mioC transcription might have to be terminated prior to initiation, it was found that the interval between initiations at permissive temperature, i.e., the eclipse period, was not influenced by the time required to shut down mioC transcription, since the eclipse was the same for chromosomes and minichromosomes which lacked mioC transcription. This finding did not, in itself, rule out the possibility that mioC transcription must be terminated prior to initiation of replication, since it might normally be shut off before initiation, and never be limiting, even during the eclipse. Therefore, experiments were performed to determine whether the continued presence of mioC transcription during the process of initiation altered the timing of initiation. It was found that minichromosomes possessing a deletion in the DnaA box upstream of the promoter transcribed mioC continuously and replicated with the same timing as those that either shut down expression prior to initiation or lacked expression entirely. It was further shown that mioC transcription was present throughout the induction of initiation by addition of chloramphenicol to a dnaA5(Ts) mutant growing at a semipermissive temperature. Thus, transcription through oriC emanating from the mioC gene promoter is normally inhibited prior to initiation of replication by the binding of Dna

  13. Identification of Candidate Adherent-Invasive E. coli Signature Transcripts by Genomic/Transcriptomic Analysis

    PubMed Central

    Zhang, Yuanhao; Rowehl, Leahana; Krumsiek, Julia M.; Orner, Erika P.; Shaikh, Nurmohammad; Tarr, Phillip I.; Sodergren, Erica; Weinstock, George M.; Boedeker, Edgar C.; Xiong, Xuejian; Parkinson, John; Frank, Daniel N.; Li, Ellen; Gathungu, Grace

    2015-01-01

    Adherent-invasive Escherichia coli (AIEC) strains are detected more frequently within mucosal lesions of patients with Crohn’s disease (CD). The AIEC phenotype consists of adherence and invasion of intestinal epithelial cells and survival within macrophages of these bacteria in vitro. Our aim was to identify candidate transcripts that distinguish AIEC from non-invasive E. coli (NIEC) strains and might be useful for rapid and accurate identification of AIEC by culture-independent technology. We performed comparative RNA-Sequence (RNASeq) analysis using AIEC strain LF82 and NIEC strain HS during exponential and stationary growth. Differential expression analysis of coding sequences (CDS) homologous to both strains demonstrated 224 and 241 genes with increased and decreased expression, respectively, in LF82 relative to HS. Transition metal transport and siderophore metabolism related pathway genes were up-regulated, while glycogen metabolic and oxidation-reduction related pathway genes were down-regulated, in LF82. Chemotaxis related transcripts were up-regulated in LF82 during the exponential phase, but flagellum-dependent motility pathway genes were down-regulated in LF82 during the stationary phase. CDS that mapped only to the LF82 genome accounted for 747 genes. We applied an in silico subtractive genomics approach to identify CDS specific to AIEC by incorporating the genomes of 10 other previously phenotyped NIEC. From this analysis, 166 CDS mapped to the LF82 genome and lacked homology to any of the 11 human NIEC strains. We compared these CDS across 13 AIEC, but none were homologous in each. Four LF82 gene loci belonging to clustered regularly interspaced short palindromic repeats region (CRISPR)—CRISPR-associated (Cas) genes were identified in 4 to 6 AIEC and absent from all non-pathogenic bacteria. As previously reported, AIEC strains were enriched for pdu operon genes. One CDS, encoding an excisionase, was shared by 9 AIEC strains. Reverse transcription

  14. ppGpp couples transcription to DNA repair in E. coli.

    PubMed

    Kamarthapu, Venu; Epshtein, Vitaly; Benjamin, Bradley; Proshkin, Sergey; Mironov, Alexander; Cashel, Michael; Nudler, Evgeny

    2016-05-20

    The small molecule alarmone (p)ppGpp mediates bacterial adaptation to nutrient deprivation by altering the initiation properties of RNA polymerase (RNAP). ppGpp is generated in Escherichia coli by two related enzymes, RelA and SpoT. We show that ppGpp is robustly, but transiently, induced in response to DNA damage and is required for efficient nucleotide excision DNA repair (NER). This explains why relA-spoT-deficient cells are sensitive to diverse genotoxic agents and ultraviolet radiation, whereas ppGpp induction renders them more resistant to such challenges. The mechanism of DNA protection by ppGpp involves promotion of UvrD-mediated RNAP backtracking. By rendering RNAP backtracking-prone, ppGpp couples transcription to DNA repair and prompts transitions between repair and recovery states. PMID:27199428

  15. Genome-Wide Transcriptional Profiling of the Escherichia coli Responses to Superoxide Stress and Sodium Salicylate

    PubMed Central

    Pomposiello, Pablo J.; Bennik, Marjon H. J.; Demple, Bruce

    2001-01-01

    Escherichia coli responds to oxidative stress by activating sets of coregulated genes that help the cell to maintain homeostasis. Identified previously by genetic and biochemical approaches, the soxRS system mediates the induction of 18 of these redox-inducible genes (including the soxS gene itself). An overlapping set of genes is activated by an assortment of structurally unrelated molecules with antibiotic activities; many genes in this response are controlled by the marRAB system. The activation of either the soxRS or the marRAB system results in enhanced resistance to both superoxide-generating agents and multiple antibiotics. In order to probe the extent of these regulatory networks, we have measured whole-genome transcriptional profiles of the E. coli response to the superoxide-generating agent paraquat (PQ), an inducer of the soxRS system, and to the weak acid salt sodium salicylate (NaSal), an inducer of the marRA system. A total of 112 genes was modulated in response to PQ, while 134 genes were modulated in response to NaSal. We have also obtained transcriptional profiles of the SoxS and MarA regulons in the absence of global stress, in order to establish the regulatory hierarchies within the global responses. Several previously unrelated genes were shown to be under SoxS or MarA control. The genetic responses to both environmental insults revealed several common themes, including the activation of genes coding for functions that replenish reducing potential; regulate iron transport and storage; and participate in sugar and amino acid transport, detoxification, protein modification, osmotic protection, and peptidoglycan synthesis. A large number of PQ- and NaSal-responsive genes have no known function, suggesting that many adaptive metabolic changes that ensue after stress remain uncharacterized. PMID:11395452

  16. Transcript degradation and noise of small RNA-controlled genes in a switch activated network in Escherichia coli.

    PubMed

    Arbel-Goren, Rinat; Tal, Asaf; Parasar, Bibudha; Dym, Alvah; Costantino, Nina; Muñoz-García, Javier; Court, Donald L; Stavans, Joel

    2016-08-19

    Post-transcriptional regulatory processes may change transcript levels and affect cell-to-cell variability or noise. We study small-RNA downregulation to elucidate its effects on noise in the iron homeostasis network of Escherichia coli In this network, the small-RNA RyhB undergoes stoichiometric degradation with the transcripts of target genes in response to iron stress. Using single-molecule fluorescence in situ hybridization, we measured transcript numbers of the RyhB-regulated genes sodB and fumA in individual cells as a function of iron deprivation. We observed a monotonic increase of noise with iron stress but no evidence of theoretically predicted, enhanced stoichiometric fluctuations in transcript numbers, nor of bistable behavior in transcript distributions. Direct detection of RyhB in individual cells shows that its noise is much smaller than that of these two targets, when RyhB production is significant. A generalized two-state model of bursty transcription that neglects RyhB fluctuations describes quantitatively the dependence of noise and transcript distributions on iron deprivation, enabling extraction of in vivo RyhB-mediated transcript degradation rates. The transcripts' threshold-linear behavior indicates that the effective in vivo interaction strength between RyhB and its two target transcripts is comparable. Strikingly, the bacterial cell response exhibits Fur-dependent, switch-like activation instead of a graded response to iron deprivation. PMID:27085802

  17. Transcription of ftsZ oscillates during the cell cycle of Escherichia coli.

    PubMed Central

    Garrido, T; Sánchez, M; Palacios, P; Aldea, M; Vicente, M

    1993-01-01

    The FtsZ protein is a key element controlling cell division in Escherichia coli. A powerful transcription titration assay was used to quantify the ftsZ mRNA present in synchronously dividing cells. The ftsZ mRNA levels oscillate during the cell cycle reaching a maximum at about the time DNA replication initiates. This cell cycle dependency is specifically due to the two proximal ftsZ promoters. A strain was constructed in which expression of ftsZ could be modulated by an exogenous inducer. In this strain cell size and cell division frequency were sensitive to the cellular FtsZ contents, demonstrating the rate-limiting role of this protein in cell division. Transcriptional activity of the ftsZ promoters was found to be independent of DnaA, indicating that DNA replication and cell division may be independently controlled at the time when new rounds of DNA replication are initiated. This suggests a parallelism between the prokaryotic cell cycle signals and the START point of eukaryotic cell cycles. Images PMID:8404863

  18. Effect of salt bridge on transcription activation of CRP-dependent lactose operon in Escherichia coli.

    PubMed

    Tutar, Yusuf; Harman, James G

    2006-09-15

    Expression of catabolite-sensitive operons in Escherichia coli is cAMP-dependent and mediated through the CRP:cAMP complex binding to specific sequences in DNA. Five specific ionic or polar interactions occur in cAMP binding pocket of CRP. E72 interacts with the cAMP 2' OH, R82 and S83 interact with the negatively charged phosphate moiety, and T127 and S128 interact with the adenine ring. There is evidence to suggest that E72 and R82 may mediate an essential CRP molecular switch mechanism. Therefore, stimulation of CRP transcription activation was examined by perturbing these residues. Further, CRP:cAMP complex was treated with a specific DNA sequence containing the lac CRP binding site along with RNA polymerase to mimic in vivo conditions. Biochemical and biophysical results revealed that regulation of transcription activation depends on alignment of CRP tertiary structure through inter-domain communication and it was concluded that positions 72 and 82 are essential in the activation of CRP by cAMP. PMID:16934214

  19. E. coli 6S RNA: a universal transcriptional regulator within the centre of growth adaptation.

    PubMed

    Geissen, René; Steuten, Benedikt; Polen, Tino; Wagner, Rolf

    2010-01-01

    Bacterial 6S RNA has been shown to bind with high affinity to σ(70)-containing RNA polymerase, suppressing σ(70)-dependent transcription during stationary phase, when 6S RNA concentrations are highest. We recently reported a genome-wide transcriptional comparison of wild-type and 6S RNA deficient E. coli strains. Contrary to the expected σ(70)- and stationary phase-specific regulatory effect of 6S RNA it turned out that mRNA levels derived from many alternative sigma factors, including σ(38) or σ(32), were affected during exponential and stationary growth. Among the most noticeably down-regulated genes at stationary growth are ribosomal proteins and factors involved in translation. In addition, a striking number of mRNA levels coding for enzymes involved in the purine metabolism, for transporters and stress regulators are altered both during log- and stationary phase. During the study we discovered a link between 6S RNA and the general stress alarmone ppGpp, which has a higher basal level in cells deficient in 6S RNA. This finding points to a functional interrelation of 6S RNA and the global network of stress and growth adaptation. PMID:20930516

  20. Control of transcription of gal repressor and isorepressor genes in Escherichia coli.

    PubMed

    Weickert, M J; Adhya, S

    1993-01-01

    Two regulatory proteins, Gal repressor and isorepressor, control the expression of the gal and mgl operons in Escherichia coli. The transcription start sites for galR and galS, the genes for the repressor and isorepressor, were determined by primer extension of in vivo transcripts. Study of the promoter-lacZ gene fusions introduced into the chromosome indicated that galS expression was elevated in cells in which the normal galS gene was interrupted, but not in cells in which the galR gene was deleted. When both genes were disrupted, galS expression was further elevated. Expression from the galS promoter was stimulated by the addition of D-fucose, repressed by glucose, and dependent on cyclic AMP receptor protein (CRP). Expression of a similar gene fusion of the galR promoter to lacZ was unregulated. Both galR and galS genes contain two potential operator sites (OE and OI) and a CRP-binding site. The arrangement of OE, OI, and the CRP-binding site in the galS gene is analogous to the arrangement in the gal and mgl promoters, but the arrangement in galR is atypical. The increased concentration of the isorepressor when inducer is present may facilitate early shutoff of the isorepressor-regulated genes of the gal regulon when inducer (substrate) concentration falls. PMID:8416900

  1. Identification of a Hypochlorite-specific Transcription Factor from Escherichia coli*

    PubMed Central

    Gebendorfer, Katharina M.; Drazic, Adrian; Le, Yan; Gundlach, Jasmin; Bepperling, Alexander; Kastenmüller, Andreas; Ganzinger, Kristina A.; Braun, Nathalie; Franzmann, Titus M.; Winter, Jeannette

    2012-01-01

    Hypochlorite is a powerful oxidant produced by neutrophils to kill invading microorganisms. Despite this important physiological role of HOCl in fighting bacterial infections, no hypochlorite-specific stress response has been identified yet. Here, we identified a hypochlorite-responsive transcription factor, YjiE, which is conserved in proteobacteria and eukaryotes. YjiE forms unusual dodecameric ring-like structures in vitro that undergo large DNA-induced conformational changes to form dimers and tetramers as shown by transmission electron microscopy and analytical ultracentrifugation. Such smaller oligomers are predominant in hypochlorite-stressed cells and are the active species as shown by fluorescence anisotropy and analytical ultracentrifugation. YjiE regulates a large number of genes upon hypochlorite stress. Among them are genes involved in cysteine, methionine biosynthesis, and sulfur metabolism (up-regulated) and genes involved in iron acquisition and homeostasis (down-regulated), thus supposedly replenishing oxidized metabolites and decreasing the hypochlorite-mediated amplification of intracellular reactive oxygen species. As a result, YjiE specifically confers hypochlorite resistance to E. coli cells. Thus, to our knowledge, YjiE is the first described hypochlorite-specific transcription factor. PMID:22223481

  2. Transcriptional analysis and adaptive evolution of Escherichia coli strains growing on acetate.

    PubMed

    Rajaraman, Eashwar; Agarwal, Ankit; Crigler, Jacob; Seipelt-Thiemann, Rebecca; Altman, Elliot; Eiteman, Mark A

    2016-09-01

    Eighteen strains of Escherichia coli were compared for maximum specific growth rate (μ MAX) on 85 mM acetate as the sole carbon source. The C strain ATCC8739 had the greatest growth rate (0.41 h(-1)) while SCS-1 had the slowest growth rate (0.15 h(-1)). Transcriptional analysis of three of the strains (ATCC8739, BL21, SMS-3-5) was conducted to elucidate why ATCC8739 had the greatest maximum growth rate. Seventy-one genes were upregulated 2-fold or greater in ATCC8739, while 128 genes were downregulated 2-fold or greater in ATCC8739 compared to BL21 and SMS-3-5. To generate a strain that could grow more quickly on acetate, ATCC8739 was cultured in a chemostat using a progressively increasing dilution rate. When the dilution rate reached 0.50 h(-1), three isolated colonies each grew faster than ATCC8739 on 85 mM acetate, with MEC136 growing the fastest with a growth rate of 0.51 h(-1), about 25 % greater than ATCC8739. Transcriptional analysis of MEC136 showed that eight genes were downregulated 2-fold or greater and one gene was upregulated 2-fold or greater compared to ATCC8739. Genomic sequencing revealed that MEC136 contained a single mutation, causing a serine to proline change in amino acid 266 of RpoA, the α subunit of the RNA polymerase core enzyme. The 260-270 amino acid region of RpoA has been shown to be a key region of the protein that affects the interaction of the α subunit of the RNA polymerase core enzyme with several global transcriptional activators, such as CRP and FNR. PMID:27448288

  3. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    PubMed

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  4. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

    PubMed Central

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J.; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  5. Control of the Escherichia coli Sialoregulon by Transcriptional Repressor NanR

    PubMed Central

    Kalivoda, Kathryn A.; Steenbergen, Susan M.

    2013-01-01

    NanR, one of >8,500 GntR superfamily helix-turn-helix transcriptional regulators, controls expression of the genes required for catabolism of sialic acids in Escherichia coli. It is predicted to do the same in related bacteria harboring orthologs of nanR. The sialic acids are a family of over 40 naturally occurring nine-carbon keto-sugar acids found mainly in the animal lineage, which includes starfish to humans in the deuterostome lineage. Sialic acids function in development, immunity, protein localization and stability, and homeostasis. They also serve as microbial carbon and nitrogen sources and ligands for cell recognition during host colonization. The importance of microbial sialic acid metabolism for host-microbe interactions has made it a target for therapeutic development. Exploiting this target depends on understanding sialometabolic pathways in a wide range of evolutionarily distinct bacteria. Here, we show by transcriptome, genetic, and biochemical analyses that the most common sialic acid, N-acetylneuraminate, induces the nanATEK-yhcH, yjhATS (nanCMS), and yjhBC operons by directly inactivating NanR, converting the predominantly dimeric form of the repressor to an inactive monomer of approximately 30-kDa. Additionally, other results identify critical amino acid residues and nucleotides in the regulator and operator, respectively. The combined results better define how sialic acids, acting through NanR, affect the metabolic flux of an important group of host-derived metabolites. Thus, E. coli serves as a valuable model for understanding sialocatabolic pathways in bacteria. PMID:23935044

  6. H-NS controls pap and daa fimbrial transcription in Escherichia coli in response to multiple environmental cues.

    PubMed

    White-Ziegler, C A; Villapakkam, A; Ronaszeki, K; Young, S

    2000-11-01

    A comparative study was completed to determine the influence of various environmental stimuli on the transcription of three different fimbrial operons in Escherichia coli and to determine the role of the histone-like protein H-NS in this environmental regulation. The fimbrial operons studied included the pap operon, which encodes pyelonephritis-associated pili (P pili), the daa operon, which encodes F1845 fimbriae, and the fan operon, which encodes K99 fimbriae. Using lacZYA transcriptional fusions within each of the fimbrial operons, we tested temperature, osmolarity, carbon source, rich medium, oxygen levels, pH, amino acids, solid medium, and iron concentration for their effects on fimbrial gene expression. Low temperature, high osmolarity, glucose as a carbon source, and rich medium repressed transcription of all three operons. High iron did not alter transcription of any of the operons tested, whereas the remaining stimuli had effects on individual operons. For the pap and daa operons, introduction of the hns651 mutation relieved the repression, either fully or partially, due to low temperature, glucose as a carbon source, rich medium, and high osmolarity. Taken together, these data indicate that there are common environmental cues that regulate fimbrial transcription in E. coli and that H-NS is an important environmental regulator for fimbrial transcription in response to several stimuli. PMID:11053383

  7. Transcription-coupled Hypernegative Supercoiling of Plasmid DNA by T7 RNA Polymerase in Escherichia coli Topoisomerase I Deficient Strains

    PubMed Central

    Samul, Rebecca; Leng, Fenfei

    2007-01-01

    Summary Transcription by RNA polymerase can stimulate negative DNA supercoiling in Escherichia coli topA strains. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription in which positive DNA supercoils are generated in front of a translocating RNA polymerase and negative supercoils behind it. However, since a specific system is lacking to study the factors governing this biologically important process, the parameters regulating transcription-coupled DNA supercoiling (TCDS) in Escherichia coli still remain elusive. In this paper, we describe our efforts to study TCDS in Escherichia coli using a newly developed system. This system consists of a topA strain, VS111(DE3) or DM800(DE3), in which a λDE3 prophage containing a T7 RNA polymerase gene under control of the lacUV5 promoter has been integrated into the cell chromosome, along with a set of plasmids producing RNA transcripts of various lengths by T7 RNA polymerase. Using this system, we found that transcription by T7 RNA polymerase strikingly induced formation of hypernegatively supercoiled plasmid DNA. We also discovered, for the first time, that TCDS was dependent on the length of RNA transcripts in vivo, precisely predicted by the “twin-supercoiled-domain” model of transcription. Furthermore, our results demonstrated that hypernegative supercoiling of plasmid DNA by T7 RNA polymerase did not require anchoring of DNA to the bacterial cytoplasmic membrane. These results indicate that a transcribing RNA polymerase alone is sufficient to cause change of local DNA superhelicity, which can have a powerful impact on the conformation and function of critical DNA sequence elements, such as promoters and DNA replication origins. PMID:17980389

  8. Heat-Shock Response Transcriptional Program Enables High-Yield and High-Quality Recombinant Protein Production in Escherichia coli

    PubMed Central

    2014-01-01

    The biosynthesis of soluble, properly folded recombinant proteins in large quantities from Escherichia coli is desirable for academic research and industrial protein production. The basal E. coli protein homeostasis (proteostasis) network capacity is often insufficient to efficiently fold overexpressed proteins. Herein we demonstrate that a transcriptionally reprogrammed E. coli proteostasis network is generally superior for producing soluble, folded, and functional recombinant proteins. Reprogramming is accomplished by overexpressing a negative feedback deficient heat-shock response transcription factor before and during overexpression of the protein-of-interest. The advantage of transcriptional reprogramming versus simply overexpressing select proteostasis network components (e.g., chaperones and co-chaperones, which has been explored previously) is that a large number of proteostasis network components are upregulated at their evolved stoichiometry, thus maintaining the system capabilities of the proteostasis network that are currently incompletely understood. Transcriptional proteostasis network reprogramming mediated by stress-responsive signaling in the absence of stress should also be useful for protein production in other cells. PMID:25051296

  9. Region 4 of Rhizobium etli Primary Sigma Factor (SigA) Confers Transcriptional Laxity in Escherichia coli.

    PubMed

    Santillán, Orlando; Ramírez-Romero, Miguel A; Lozano, Luis; Checa, Alberto; Encarnación, Sergio M; Dávila, Guillermo

    2016-01-01

    Sigma factors are RNA polymerase subunits engaged in promoter recognition and DNA strand separation during transcription initiation in bacteria. Primary sigma factors are responsible for the expression of housekeeping genes and are essential for survival. RpoD, the primary sigma factor of Escherichia coli, a γ-proteobacteria, recognizes consensus promoter sequences highly similar to those of some α-proteobacteria species. Despite this resemblance, RpoD is unable to sustain transcription from most of the α-proteobacterial promoters tested so far. In contrast, we have found that SigA, the primary sigma factor of Rhizobium etli, an α-proteobacteria, is able to transcribe E. coli promoters, although it exhibits only 48% identity (98% coverage) to RpoD. We have called this the transcriptional laxity phenomenon. Here, we show that SigA partially complements the thermo-sensitive deficiency of RpoD285 from E. coli strain UQ285 and that the SigA region σ4 is responsible for this phenotype. Sixteen out of 74 residues (21.6%) within region σ4 are variable between RpoD and SigA. Mutating these residues significantly improves SigA ability to complement E. coli UQ285. Only six of these residues fall into positions already known to interact with promoter DNA and to comprise a helix-turn-helix motif. The remaining variable positions are located on previously unexplored sites inside region σ4, specifically into the first two α-helices of the region. Neither of the variable positions confined to these helices seem to interact directly with promoter sequence; instead, we adduce that these residues participate allosterically by contributing to correct region folding and/or positioning of the HTH motif. We propose that transcriptional laxity is a mechanism for ensuring transcription in spite of naturally occurring mutations from endogenous promoters and/or horizontally transferred DNA sequences, allowing survival and fast environmental adaptation of α-proteobacteria. PMID

  10. Region 4 of Rhizobium etli Primary Sigma Factor (SigA) Confers Transcriptional Laxity in Escherichia coli

    PubMed Central

    Santillán, Orlando; Ramírez-Romero, Miguel A.; Lozano, Luis; Checa, Alberto; Encarnación, Sergio M.; Dávila, Guillermo

    2016-01-01

    Sigma factors are RNA polymerase subunits engaged in promoter recognition and DNA strand separation during transcription initiation in bacteria. Primary sigma factors are responsible for the expression of housekeeping genes and are essential for survival. RpoD, the primary sigma factor of Escherichia coli, a γ-proteobacteria, recognizes consensus promoter sequences highly similar to those of some α-proteobacteria species. Despite this resemblance, RpoD is unable to sustain transcription from most of the α-proteobacterial promoters tested so far. In contrast, we have found that SigA, the primary sigma factor of Rhizobium etli, an α-proteobacteria, is able to transcribe E. coli promoters, although it exhibits only 48% identity (98% coverage) to RpoD. We have called this the transcriptional laxity phenomenon. Here, we show that SigA partially complements the thermo-sensitive deficiency of RpoD285 from E. coli strain UQ285 and that the SigA region σ4 is responsible for this phenotype. Sixteen out of 74 residues (21.6%) within region σ4 are variable between RpoD and SigA. Mutating these residues significantly improves SigA ability to complement E. coli UQ285. Only six of these residues fall into positions already known to interact with promoter DNA and to comprise a helix-turn-helix motif. The remaining variable positions are located on previously unexplored sites inside region σ4, specifically into the first two α-helices of the region. Neither of the variable positions confined to these helices seem to interact directly with promoter sequence; instead, we adduce that these residues participate allosterically by contributing to correct region folding and/or positioning of the HTH motif. We propose that transcriptional laxity is a mechanism for ensuring transcription in spite of naturally occurring mutations from endogenous promoters and/or horizontally transferred DNA sequences, allowing survival and fast environmental adaptation of α-proteobacteria. PMID

  11. PafR, a Novel Transcription Regulator, Is Important for Pathogenesis in Uropathogenic Escherichia coli

    PubMed Central

    Baum, Mordechai; Watad, Mobarak; Smith, Sara N.; Alteri, Christopher J.; Gordon, Noa; Rosenshine, Ilan; Mobley, Harry L.

    2014-01-01

    The metV genomic island in the chromosome of uropathogenic Escherichia coli (UPEC) encodes a putative transcription factor and a sugar permease of the phosphotransferase system (PTS), which are predicted to compose a Bgl-like sensory system. The presence of these two genes, hereby termed pafR and pafP, respectively, has been previously shown to correlate with isolates causing clinical syndromes. We show here that deletion of both genes impairs the ability of the resulting mutant to infect the CBA/J mouse model of ascending urinary tract infection compared to that of the parent strain, CFT073. Expressing the two genes in trans in the two-gene knockout mutant complemented full virulence. Deletion of either gene individually generated the same phenotype as the double knockout, indicating that both pafR and pafP are important to pathogenesis. We screened numerous environmental conditions but failed to detect expression from the promoter that precedes the paf genes in vitro, suggesting that they are in vivo induced (ivi). Although PafR is shown here to be capable of functioning as a transcriptional antiterminator, its targets in the UPEC genome are not known. Using microarray analysis, we have shown that expression of PafR from a heterologous promoter in CFT073 affects expression of genes related to bacterial virulence, biofilm formation, and metabolism. Expression of PafR also inhibits biofilm formation and motility. Taken together, our results suggest that the paf genes are implicated in pathogenesis and that PafR controls virulence genes, in particular biofilm formation genes. PMID:25069986

  12. Transcriptional Modulation of Enterotoxigenic Escherichia coli Virulence Genes in Response to Epithelial Cell Interactions

    PubMed Central

    Kansal, Rita; Rasko, David A.; Sahl, Jason W.; Munson, George P.; Roy, Koushik; Luo, Qingwei; Sheikh, Alaullah; Kuhne, Kurt J.

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens. PMID:23115039

  13. Application of neural networks and information theory to the identification of E. coli transcriptional promoters

    SciTech Connect

    Abremski, K. . Experimental Station); Sirotkin, K. ); Lapedes, A. )

    1991-01-01

    The Humane Genome Project has as its eventual goal the determination of the entire DNA sequence of man, which comprises approximately 3 billion base pairs. An important aspect of this project will be the analysis of the sequence to locate regions of biological importance. New computer methods will be needed to automate and facilitate this task. In this paper, we have investigated use of neural networks for the recognition of functional patterns in biological sequences. The prediction of Escherichia coli transcriptional promoters was chosen as a model system for these studies. Two approaches were employed. In the fist method, a mutual information analysis of promoter and nonpromoter sequences was carried out to demonstrate the informative base positions that help to distinguish promoter sequences from non-promoter sequences. These base positions were than used to train a Perceptron to predict new promoter sequences. In the second method, the experimental knowledge of promoters was used to indicate the important base positions in the sequence. These base positions were used to train a back propagation network with hidden units which represented regions of sequence conservation found in promoters. With both types of networks, prediction of new promoter sequences was greater than 96.9%. 12 refs., 1 fig., 4 tabs.

  14. Transcriptional modulation of enterotoxigenic Escherichia coli virulence genes in response to epithelial cell interactions.

    PubMed

    Kansal, Rita; Rasko, David A; Sahl, Jason W; Munson, George P; Roy, Koushik; Luo, Qingwei; Sheikh, Alaullah; Kuhne, Kurt J; Fleckenstein, James M

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens. PMID:23115039

  15. The highly conserved MraZ protein is a transcriptional regulator in Escherichia coli

    SciTech Connect

    Eraso, Jesus M.; Markillie, Lye Meng; Mitchell, Hugh D.; Taylor, Ronald C.; Orr, Galya; Margolin, William

    2014-05-05

    The mraZ and mraW genes are highly conserved in bacteria, both in sequence and location at the head of the division and cell wall (dcw) gene cluster. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin, and MraW is known to methylate ribosomal RNA, mraZ and mraW null mutants have no detectable growth phenotype in any species tested to date, hampering progress in understanding their physiological role. Here we show that overproduction of Escherichia coli MraZ perturbs cell division and the cell envelope, is more lethal at high levels or in minimal growth medium, and that MraW antagonizes these effects. MraZGFP localizes to the nucleoid, suggesting that it binds DNA. Indeed, purified MraZ directly binds a region upstream from its own promoter containing three direct repeats to regulate its own expression and that of downstream cell division and cell wall genes. MraZ-LacZ fusions are repressed by excess MraZ but not when DNA binding by MraZ is inhibited. RNAseq analysis indicates that MraZ is a global transcriptional regulator with numerous targets in addition to dcw genes. One of these targets, mioC, is directly bound by MraZ in a region with three direct repeats.

  16. The ribB FMN riboswitch from Escherichia coli operates at the transcriptional and translational level and regulates riboflavin biosynthesis.

    PubMed

    Pedrolli, Danielle; Langer, Simone; Hobl, Birgit; Schwarz, Julia; Hashimoto, Masayuki; Mack, Matthias

    2015-08-01

    FMN riboswitches are genetic elements that, in many bacteria, control genes responsible for biosynthesis and/or transport of riboflavin (vitamin B2 ). We report that the Escherichia coli ribB FMN riboswitch controls expression of the essential gene ribB coding for the riboflavin biosynthetic enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (RibB; EC 4.1.99.12). Our data show that the E. coli ribB FMN riboswitch is unusual because it operates at the transcriptional and also at the translational level. Expression of ribB is negatively affected by FMN and by the FMN analog roseoflavin mononucleotide, which is synthesized enzymatically from roseoflavin and ATP. Consequently, in addition to flavoenzymes, the E. coli ribB FMN riboswitch constitutes a target for the antibiotic roseoflavin produced by Streptomyces davawensis. PMID:25661987

  17. Sequence of the relB transcription unit from Escherichia coli and identification of the relB gene.

    PubMed Central

    Bech, F W; Jørgensen, S T; Diderichsen, B; Karlström, O H

    1985-01-01

    Escherichia coli relB mutants react to amino acid starvation by several abnormal responses, including accumulation of a translational inhibitor. We have isolated a relB-complementing plasmid from the Clarke and Carbon E. coli DNA library. From this plasmid we sequenced a 2140-bp segment which included the relB gene by the following two criteria: (i) it complements chromosomal relB mutations, (ii) the corresponding DNA segment cloned from chromosomal DNA of three relB mutants was defective in relB complementation. All three mutations fell within an open reading frame of 79 amino acids. A polypeptide of 9 kd compatible with this open reading frame was synthesized in maxicells and is in all probability the product of the relB gene. By nuclease S1 mapping we have determined the transcription start and stop of an 870 base transcript of the relB gene. Images Fig. 5. Fig. 8. PMID:2990907

  18. Decoding genome-wide GadEWX-transcriptional regulatory networks reveals multifaceted cellular responses to acid stress in Escherichia coli.

    PubMed

    Seo, Sang Woo; Kim, Donghyuk; O'Brien, Edward J; Szubin, Richard; Palsson, Bernhard O

    2015-01-01

    The regulators GadE, GadW and GadX (which we refer to as GadEWX) play a critical role in the transcriptional regulation of the glutamate-dependent acid resistance (GDAR) system in Escherichia coli K-12 MG1655. However, the genome-wide regulatory role of GadEWX is still unknown. Here we comprehensively reconstruct the genome-wide GadEWX transcriptional regulatory network and RpoS involvement in E. coli K-12 MG1655 under acidic stress. Integrative data analysis reveals that GadEWX regulons consist of 45 genes in 31 transcription units and 28 of these genes were associated with RpoS-binding sites. We demonstrate that GadEWX directly and coherently regulate several proton-generating/consuming enzymes with pairs of negative-feedback loops for pH homeostasis. In addition, GadEWX regulate genes with assorted functions, including molecular chaperones, acid resistance, stress response and other regulatory activities. These results show how GadEWX simultaneously coordinate many cellular processes to produce the overall response of E. coli to acid stress. PMID:26258987

  19. Decoding genome-wide GadEWX-transcriptional regulatory networks reveals multifaceted cellular responses to acid stress in Escherichia coli

    PubMed Central

    Seo, Sang Woo; Kim, Donghyuk; O'Brien, Edward J.; Szubin, Richard; Palsson, Bernhard O.

    2015-01-01

    The regulators GadE, GadW and GadX (which we refer to as GadEWX) play a critical role in the transcriptional regulation of the glutamate-dependent acid resistance (GDAR) system in Escherichia coli K-12 MG1655. However, the genome-wide regulatory role of GadEWX is still unknown. Here we comprehensively reconstruct the genome-wide GadEWX transcriptional regulatory network and RpoS involvement in E. coli K-12 MG1655 under acidic stress. Integrative data analysis reveals that GadEWX regulons consist of 45 genes in 31 transcription units and 28 of these genes were associated with RpoS-binding sites. We demonstrate that GadEWX directly and coherently regulate several proton-generating/consuming enzymes with pairs of negative-feedback loops for pH homeostasis. In addition, GadEWX regulate genes with assorted functions, including molecular chaperones, acid resistance, stress response and other regulatory activities. These results show how GadEWX simultaneously coordinate many cellular processes to produce the overall response of E. coli to acid stress. PMID:26258987

  20. Escherichia coli under Ionic Silver Stress: An Integrative Approach to Explore Transcriptional, Physiological and Biochemical Responses

    PubMed Central

    Saulou-Bérion, Claire; Gonzalez, Ignacio; Enjalbert, Brice; Audinot, Jean-Nicolas; Fourquaux, Isabelle; Jamme, Frédéric; Cocaign-Bousquet, Muriel; Mercier-Bonin, Muriel; Girbal, Laurence

    2015-01-01

    For a better understanding of the systemic effect of sub-lethal micromolar concentrations of ionic silver on Escherichia coli, we performed a multi-level characterization of cells under Ag+-mediated stress using an integrative biology approach combining physiological, biochemical and transcriptomic data. Physiological parameters, namely bacterial growth and survival after Ag+ exposure, were first quantified and related to the accumulation of intracellular silver, probed for the first time by nano secondary ion mass spectroscopy at sub-micrometer lateral resolution. Modifications in E. coli biochemical composition were evaluated under Ag+-mediated stress by in situ synchrotron Fourier-transform infrared microspectroscopy and a comprehensive transcriptome response was also determined. Using multivariate statistics, correlations between the physiological parameters, the extracellular concentration of AgNO3 and the intracellular silver content, gene expression profiles and micro-spectroscopic data were investigated. We identified Ag+-dependent regulation of gene expression required for growth (e.g. transporter genes, transcriptional regulators, ribosomal proteins), for ionic silver transport and detoxification (e.g. copA, cueO, mgtA, nhaR) and for coping with various types of stress (dnaK, pspA, metA,R, oxidoreductase genes). The silver-induced shortening of the acyl chain of fatty acids, mostly encountered in cell membrane, was highlighted by microspectroscopy and correlated with the down-regulated expression of genes involved in fatty acid transport (fadL) and synthesis/modification of lipid A (lpxA and arnA). The increase in the disordered secondary structure of proteins in the presence of Ag+ was assessed through the conformational shift shown for amides I and II, and further correlated with the up-regulated expression of peptidase (hfq) and chaperone (dnaJ), and regulation of transpeptidase expression (ycfS and ycbB). Interestingly, as these transpeptidases act on

  1. Nickel-Specific Response in the Transcriptional Regulator, 'Escherichia Coli'NikR

    SciTech Connect

    Leitch, S.; Bradley, M.J.; Rowe, J.L.; Chivers, P.T.; Maroney, M.J.; /Massachusetts U., Amherst /Washington U., St. Louis

    2007-07-10

    Studies of the transcriptional repression of the Ni-specific permease encoded by the Pnik operon by Escherichia coli NikR using a LacZ reporter assay establish that the NikR response is specific to nickel in vivo. Toward understanding this metal ion-specific response, X-ray absorption spectroscopy (XAS) analysis of various M-NikR complexes (M = Co(II), Ni(II), Cu(II), Cu(I), and Zn(II)) was used to show that each high-affinity binding site metal adopts a unique structure, with Ni(II) and Cu(II) being the only two metal ions to feature planar four-coordinate complexes. The results are consistent with an allosteric mechanism whereby the geometry and ligand selection of the metal present in the high-affinity site induce a unique conformation in NikR that subsequently influences DNA binding. The influence of the high-affinity metal on protein structure was examined using hydrogen/deuterium (H/D) exchange detected by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Each NikR complex gives rise to differing amounts of H/D exchange; Zn(II)- and Co(II)-NikR are most like apo-NikR, while the exchange time course is substantially different for Ni(II) and to a lesser extent for Cu(II). In addition to the high-affinity metal binding site, E. coli NikR has a low-affinity metal-binding site that affects DNA binding affinity. We have characterized this low-affinity site using XAS in heterobimetallic complexes of NikR. When Cu(II) occupies the high-affinity site and Ni(II) occupies the low-affinity site, the Ni K-edge XAS spectra show that the Ni site is composed of six N/O-donors. A similar low-affinity site structure is found for the NikR complex when Co(II) occupies the low-affinity site and Ni(II) occupies the high-affinity site, except that one of the Co(II) ligands is a chloride derived from the buffer.

  2. Nickel-Specific Response in the Transcriptional Regulator, Escherichia coli NikR

    SciTech Connect

    Leitch,S.; Bradley, M.; Rowe, J.; Chivers, P.; Maroney, M.

    2007-01-01

    Studies of the transcriptional repression of the Ni-specific permease encoded by the P{sub nik} operon by Escherichia coli NikR using a LacZ reporter assay establish that the NikR response is specific to nickel in vivo. Toward understanding this metal ion-specific response, X-ray absorption spectroscopy (XAS) analysis of various M-NikR complexes (M = Co(II), Ni(II), Cu(II), Cu(I), and Zn(II)) was used to show that each high-affinity binding site metal adopts a unique structure, with Ni(II) and Cu(II) being the only two metal ions to feature planar four-coordinate complexes. The results are consistent with an allosteric mechanism whereby the geometry and ligand selection of the metal present in the high-affinity site induce a unique conformation in NikR that subsequently influences DNA binding. The influence of the high-affinity metal on protein structure was examined using hydrogen/deuterium (H/D) exchange detected by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Each NikR complex gives rise to differing amounts of H/D exchange; Zn(II)- and Co(II)-NikR are most like apo-NikR, while the exchange time course is substantially different for Ni(II) and to a lesser extent for Cu(II). In addition to the high-affinity metal binding site, E. coli NikR has a low-affinity metal-binding site that affects DNA binding affinity. We have characterized this low-affinity site using XAS in heterobimetallic complexes of NikR. When Cu(II) occupies the high-affinity site and Ni(II) occupies the low-affinity site, the Ni K-edge XAS spectra show that the Ni site is composed of six N/O-donors. A similar low-affinity site structure is found for the NikR complex when Co(II) occupies the low-affinity site and Ni(II) occupies the high-affinity site, except that one of the Co(II) ligands is a chloride derived from the buffer.

  3. Shaping the landscape of the Escherichia coli chromosome: replication-transcription encounters in cells with an ectopic replication origin

    PubMed Central

    Ivanova, Darja; Taylor, Toni; Smith, Sarah L.; Dimude, Juachi U.; Upton, Amy L.; Mehrjouy, Mana M.; Skovgaard, Ole; Sherratt, David J.; Retkute, Renata; Rudolph, Christian J.

    2015-01-01

    Each cell division requires the unwinding of millions of DNA base pairs to allow chromosome duplication and gene transcription. As DNA replication and transcription share the same template, conflicts between both processes are unavoidable and head-on collisions are thought to be particularly problematic. Surprisingly, a recent study reported unperturbed cell cycle progression in Escherichia coli cells with an ectopic replication origin in which highly transcribed rrn operons were forced to be replicated opposite to normal. In this study we have re-generated a similar strain and found the doubling time to be twice that of normal cells. Replication profiles of this background revealed significant deviations in comparison to wild-type profiles, particularly in highly transcribed regions and the termination area. These deviations were alleviated by mutations that either inactivate the termination area or destabilise RNA polymerase complexes and allow their easier displacement by replication forks. Our data demonstrate that head-on replication-transcription conflicts are highly problematic. Indeed, analysis of the replication profile of the previously published E. coli construct revealed a chromosomal rearrangement that alleviates replication-transcription conflicts in an intriguingly simple way. Our data support the idea that avoiding head-on collisions has significantly contributed to shaping the distinct architecture of bacterial chromosomes. PMID:26160884

  4. Shaping the landscape of the Escherichia coli chromosome: replication-transcription encounters in cells with an ectopic replication origin.

    PubMed

    Ivanova, Darja; Taylor, Toni; Smith, Sarah L; Dimude, Juachi U; Upton, Amy L; Mehrjouy, Mana M; Skovgaard, Ole; Sherratt, David J; Retkute, Renata; Rudolph, Christian J

    2015-09-18

    Each cell division requires the unwinding of millions of DNA base pairs to allow chromosome duplication and gene transcription. As DNA replication and transcription share the same template, conflicts between both processes are unavoidable and head-on collisions are thought to be particularly problematic. Surprisingly, a recent study reported unperturbed cell cycle progression in Escherichia coli cells with an ectopic replication origin in which highly transcribed rrn operons were forced to be replicated opposite to normal. In this study we have re-generated a similar strain and found the doubling time to be twice that of normal cells. Replication profiles of this background revealed significant deviations in comparison to wild-type profiles, particularly in highly transcribed regions and the termination area. These deviations were alleviated by mutations that either inactivate the termination area or destabilise RNA polymerase complexes and allow their easier displacement by replication forks. Our data demonstrate that head-on replication-transcription conflicts are highly problematic. Indeed, analysis of the replication profile of the previously published E. coli construct revealed a chromosomal rearrangement that alleviates replication-transcription conflicts in an intriguingly simple way. Our data support the idea that avoiding head-on collisions has significantly contributed to shaping the distinct architecture of bacterial chromosomes. PMID:26160884

  5. Whole-Genome Transcriptional Analysis of Escherichia coli during Heat Inactivation Processes Related to Industrial Cooking

    PubMed Central

    Guernec, A.; Robichaud-Rincon, P.

    2013-01-01

    Escherichia coli K-12 was grown to the stationary phase, for maximum physiological resistance, in brain heart infusion (BHI) broth at 37°C. Cells were then heated at 58°C or 60°C to reach a process lethality value (Fo7010) of 2 or 3 or to a core temperature of 71°C (control industrial cooking temperature). Growth recovery and cell membrane integrity were evaluated immediately after heating, and a global transcription analysis was performed using gene expression microarrays. Only cells heated at 58°C with Fo = 2 were still able to grow on liquid or solid BHI broth after heat treatment. However, their transcriptome did not differ from that of bacteria heated at 58°C with Fo = 3 (P value for the false discovery rate [P-FDR] > 0.01), where no growth recovery was observed posttreatment. Genome-wide transcriptomic data obtained at 71°C were distinct from those of the other treatments without growth recovery. Quantification of heat shock gene expression by real-time PCR revealed that dnaK and groEL mRNA levels decreased significantly above 60°C to reach levels similar to those of control cells at 37°C (P < 0.0001). Furthermore, despite similar levels of cell inactivation measured by growth on BHI media after heating, 132 and 8 genes were differentially expressed at 71°C compared to 58°C and 60°C at Fo = 3, respectively (P-FDR < 0.01). Among them, genes such as aroA, citE, glyS, oppB, and asd, whose expression was upregulated at 71°C, may be worth investigating as good biomarkers for accurately determining the efficiency of heat treatments, especially when cells are too injured to be enumerated using growth media. PMID:23770902

  6. The Highly Conserved MraZ Protein Is a Transcriptional Regulator in Escherichia coli

    PubMed Central

    Eraso, Jesus M.; Markillie, Lye M.; Mitchell, Hugh D.; Taylor, Ronald C.; Orr, Galya

    2014-01-01

    The mraZ and mraW genes are highly conserved in bacteria, both in sequence and in their position at the head of the division and cell wall (dcw) gene cluster. Located directly upstream of the mraZ gene, the Pmra promoter drives the transcription of mraZ and mraW, as well as many essential cell division and cell wall genes, but no regulator of Pmra has been found to date. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin and MraW is known to methylate the 16S rRNA, mraZ and mraW null mutants have no detectable phenotypes. Here we show that overproduction of Escherichia coli MraZ inhibited cell division and was lethal in rich medium at high induction levels and in minimal medium at low induction levels. Co-overproduction of MraW suppressed MraZ toxicity, and loss of MraW enhanced MraZ toxicity, suggesting that MraZ and MraW have antagonistic functions. MraZ-green fluorescent protein localized to the nucleoid, suggesting that it binds DNA. Consistent with this idea, purified MraZ directly bound a region of DNA containing three direct repeats between Pmra and the mraZ gene. Excess MraZ reduced the expression of an mraZ-lacZ reporter, suggesting that MraZ acts as a repressor of Pmra, whereas a DNA-binding mutant form of MraZ failed to repress expression. Transcriptome sequencing (RNA-seq) analysis suggested that MraZ also regulates the expression of genes outside the dcw cluster. In support of this, purified MraZ could directly bind to a putative operator site upstream of mioC, one of the repressed genes identified by RNA-seq. PMID:24659771

  7. The trigger enzyme PepA (aminopeptidase A) of Escherichia coli, a transcriptional repressor that generates positive supercoiling.

    PubMed

    Nguyen Le Minh, Phu; Nadal, Marc; Charlier, Daniel

    2016-06-01

    Escherichia coli aminopeptidase A (PepA) is a trigger enzyme endowed with catalytic activity and DNA-binding properties prominent in transcriptional regulation and site-specific DNA recombination. The current work demonstrates that PepA is a repressor in its own right, capable of specifically inhibiting transcription initiation at promoter P1 of the carAB operon, encoding carbamoylphosphate synthase. Furthermore, in vitro topology studies performed with DNA minicircles demonstrate that PepA binding constrains a single positive supercoil in the carP1 control region. Such a topological event is understood to constitute an impediment to transcription initiation and may serve as a mechanism to regulate gene expression. PMID:27213286

  8. Examination of the Genome-Wide Transcriptional Response of Escherichia coli O157:H7 to Cinnamaldehyde Exposure

    PubMed Central

    Visvalingam, Jeyachchandran; Hernandez-Doria, Juan David

    2013-01-01

    Cinnamaldehyde is a natural antimicrobial that has been found to be effective against many food-borne pathogens, including Escherichia coli O157:H7. Although its antimicrobial effects have been well investigated, limited information is available on its effects at the molecular level. Sublethal treatment at 200 mg/liter cinnamaldehyde inhibited growth of E. coli O157:H7 at 37°C and for ≤2 h caused cell elongation, but from 2 to 4 h growth resumed and cells reverted to normal length. To understand this transient behavior, genome-wide transcriptional analysis of E. coli O157:H7 was performed at 2 and 4 h of exposure to cinnamaldehyde in conjunction with reverse-phase high-performance liquid chromatography (RP-HPLC) analysis for cinnamaldehyde and other cinnamic compounds. Drastically different gene expression profiles were obtained at 2 and 4 h. RP-HPLC analysis showed that cinnamaldehyde was structurally stable for at least 2 h. At 2 h of exposure, cinnamaldehyde induced expression of many oxidative stress-related genes and repressed expression of DNA, protein, O-antigen, and fimbrial synthetic genes. At 4 h, many cinnamaldehyde-induced repressive effects on E. coli O157:H7 gene expression were reversed, and cells became more motile and grew at a slightly higher rate. Data indicated that by 4 h, E. coli O157:H7 was able to convert cinnamaldehyde into the less toxic cinnamic alcohol using dehydrogenase/reductase enzymes (YqhD and DkgA). This is the first study to characterize the ability of E. coli O157:H7 to convert cinnamaldehyde into cinnamic alcohol which, in turn, showed that the antimicrobial activity of cinnamaldehyde is mainly attributable to its carbonyl aldehyde group. PMID:23183978

  9. Tailoring of global transcription sigma D factor by random mutagenesis to improve Escherichia coli tolerance towards low-pHs.

    PubMed

    Gao, Xi; Jiang, Ling; Zhu, Liying; Xu, Qing; Xu, Xian; Huang, He

    2016-04-20

    Bioconversion processes of organic acid or acid hydrolysis of raw material for microbial metabolism often suffer limitations as a result of microbial sensitivity in low-pH conditions. We adopted a three-step method called RAndom Insertional-deletional Strand Exchange mutagenesis (RAISE) to engineer the components of global regulator Sigma D factor (RpoD) of Escherichia coli to improve its acid tolerance. The best strain Mutant VII was identified from random mutagenesis libraries based on the growth performance, which exhibited much higher growth rate than the control (0.22h(-1) vs. 0.15h(-1)) at pH as low as 3.17. Combined transcriptome and phenome analysis of E. coli was carried out to better understand the global effects of RpoD on the regulatory networks. Our analysis showed that 95 (2.1%) of all E. coli genes were induced and 178 (4.0%) genes were repressed, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon metabolism, amino acid utilization, except for acid resistance. Also regulated were the master regulators (ArcA, EvgA, H-NS and RpoS) and gene/operon-specific transcription factors (GadX, GadW, AppY, YdeO, KdgR). These results demonstrated that RpoD acts as global regulator in the growth phase of E. coli and consequently improves acid tolerances. PMID:26971973

  10. Early transcriptional events in the udder and teat after intra-mammary Escherichia coli and Staphylococcus aureus challenge.

    PubMed

    Petzl, Wolfram; Günther, Juliane; Mühlbauer, Katharina; Seyfert, Hans-Martin; Schuberth, Hans-Joachim; Hussen, Jamal; Sauter-Louis, Carola; Hafner-Marx, Angela; Zerbe, Holm

    2016-05-01

    Intra-mammary bacterial infections can result in harmful clinical mastitis or subclinical mastitis with persistent infections. Research during the last decades closely examined the pathophysiology of inflamed udders. Initial events after pathogen perception but before the onset of mastitis have not been examined in vivo The objective of this study was to develop a mastitis model in cows by monitoring initial transcriptional pathogen-specific host response before clinical signs occur. We applied a short-term infection model to analyse transcripts encoding chemokines, cytokines and antimicrobial molecules in the teat cistern (TC) and lobulo-alveolar parenchyma (LP) up to 3 h after challenge with E and Staphylococcus aureus Both pathogens elicited an immune reaction by 1 h after challenge. Escherichia coli induced all analysed factors (CCL20, CXCL8, TNF, IL6, IL12B, IL10, LAP, S100A9); however, S. aureus failed to induce IL12B, IL10, LAP and S100A9 expression. The E. coli-induced up-regulation was 25-105 times greater than that after S. aureus challenge. Almost all the responses were restricted to the TC. The short-term mastitis model demonstrates that a divergent pathogen-specific response is generated during the first h. It confirms that the first transcripts are generated in the TC prior to a response in the LP. PMID:27012912

  11. Comparative Analysis of Regulatory Elements between Escherichia coli and Klebsiella pneumoniae by Genome-Wide Transcription Start Site Profiling

    PubMed Central

    Qiu, Yu; Nagarajan, Harish; Seo, Joo-Hyun; Cho, Byung-Kwan; Tsai, Shih-Feng; Palsson, Bernhard Ø.

    2012-01-01

    Genome-wide transcription start site (TSS) profiles of the enterobacteria Escherichia coli and Klebsiella pneumoniae were experimentally determined through modified 5′ RACE followed by deep sequencing of intact primary mRNA. This identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Experimentally determined TSSs were then used to define promoter regions and 5′ UTRs upstream of coding genes. Comparative analysis of these regulatory elements revealed the use of multiple TSSs, identical sequence motifs of promoter and Shine-Dalgarno sequence, reflecting conserved gene expression apparatuses between the two species. In both species, over 70% of primary transcripts were expressed from operons having orthologous genes during exponential growth. However, expressed orthologous genes in E. coli and K. pneumoniae showed a strikingly different organization of upstream regulatory regions with only 20% identical promoters with TSSs in both species. Over 40% of promoters had TSSs identified in only one species, despite conserved promoter sequences existing in the other species. 662 conserved promoters having TSSs in both species resulted in the same number of comparable 5′ UTR pairs, and that regulatory element was found to be the most variant region in sequence among promoter, 5′ UTR, and ORF. In K. pneumoniae, 48 sRNAs were predicted and 36 of them were expressed during exponential growth. Among them, 34 orthologous sRNAs between two species were analyzed in depth, and the analysis showed that many sRNAs of K. pneumoniae, including pleiotropic sRNAs such as rprA, arcZ, and sgrS, may work in the same way as in E. coli. These results reveal a new dimension of comparative genomics such that a comparison of two genomes needs to be comprehensive over all levels of genome organization. PMID:22912590

  12. In vitro transcription of the Bacillus subtilis phage phi 29 DNA by Bacillus subtilis and Escherichia coli RNA polymerases.

    PubMed Central

    Sogo, J M; Lozano, M; Salas, M

    1984-01-01

    The Escherichia coli RNA polymerase bound to phage phi 29 DNA has been visualized by electron microscopy. Thirteen specific binding sites have been observed at 1.7,2.6,5.5,10.4,13.7,25.2,25.7,26.3,33.5,59.5,69.2,91.7 and 99.6 DNA length units and they have been named A1,A1I,A1II,A1III,A1IV,A2,A2I, A3, A4,B1,B1I,C1 and C2, respectively. The binding sites A1,A2,A3,B1,C1 and C2 coincide with those found with Bacillus subtilis RNA polymerase. The transcription of phage phi 29 DNA with B. subtilis or E. coli RNA polymerases has been studied. With the B. subtilis RNA polymerase eight transcripts were found, starting at positions corresponding to the binding sites A1, A1III, A2,A3,B1I,B2,C1 and C2, respectively. With the E. coli RNA polymerase the same transcripts were found and a new one starting at position corresponding to the A4 binding site. The RNAs starting at binding sites A1,A1III,A2,B1I, B2,C1 and C2 are transcribed from right to left, as expected for early RNA. The RNAs which initiate at positions A3 and A4 are transcribed from left to right and probably correspond to late RNAs. Images PMID:6322128

  13. Bacteriophage λ N protein inhibits transcription slippage by Escherichia coli RNA polymerase.

    PubMed

    Parks, Adam R; Court, Carolyn; Lubkowska, Lucyna; Jin, Ding J; Kashlev, Mikhail; Court, Donald L

    2014-05-01

    Transcriptional slippage is a class of error in which ribonucleic acid (RNA) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (DNA) template. This phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. The bacteriophage λ N protein reduces transcriptional slippage within actively growing cells and in vitro. N appears to stabilize the RNA/DNA hybrid, particularly at the 5' end, preventing loss of register between transcript and template. This report provides the first evidence of a protein that directly influences transcriptional slippage, and provides a clue about the molecular mechanism of transcription termination and N-mediated antitermination. PMID:24711367

  14. Bacteriophage λ N protein inhibits transcription slippage by Escherichia coli RNA polymerase

    PubMed Central

    Parks, Adam R.; Court, Carolyn; Lubkowska, Lucyna; Jin, Ding J.; Kashlev, Mikhail; Court, Donald L.

    2014-01-01

    Transcriptional slippage is a class of error in which ribonucleic acid (RNA) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (DNA) template. This phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. The bacteriophage λ N protein reduces transcriptional slippage within actively growing cells and in vitro. N appears to stabilize the RNA/DNA hybrid, particularly at the 5′ end, preventing loss of register between transcript and template. This report provides the first evidence of a protein that directly influences transcriptional slippage, and provides a clue about the molecular mechanism of transcription termination and N-mediated antitermination. PMID:24711367

  15. The role of two surface exposed loops in transcription activation by the Escherichia coli CRP and FNR proteins.

    PubMed Central

    Williams, R; Bell, A; Sims, G; Busby, S

    1991-01-01

    We have investigated a number of mutations that alter the ability of the E. coli transcription factors CRP and FNR to activate transcription. In CRP, some mutations at position 159 (H159L, H159I and delta 159) prevent transcription activation at a number of naturally-occurring and semi-synthetic CRP-dependent promoters. We suggest that some feature of the surface-exposed turn around residue 159 is recognised by RNA polymerase during transcription activation at these promoters. Mutations at position 52 increase CRP activity and reverse the effects of H159L and delta 159, most likely by creating a new contact with RNA polymerase. However this new contact only gives increased expression when the CRP binding site is located 41 1/2 base pairs upstream of the transcription start site and fails to reverse the effects of H159L and delta 159 at promoters where the CRP site is located further upstream. To explain our results we propose that the two surface-exposed turns around residues 52 and 159 contain elements that are potential RNA polymerase docking sites: in the CRP dimer these two active patches are located on adjacent faces of different subunits. FNR, a related transcription activator, contains amino acid sequences homologous to the CRP sequence around position 52. Mutations in this zone (from residues 81-88 in FNR) reduce expression from an FNR-dependent promoter without stopping FNR binding to its target. This defines a patch on FNR, which is homologous to the CRP surface-exposed loop around position 52, which is involved in transcription activation, most likely by contacting RNA polymerase. PMID:1762901

  16. Transcription-Factor-Mediated DNA Looping Probed by High-Resolution, Single-Molecule Imaging in Live E. coli Cells

    PubMed Central

    Hensel, Zach; Xiao, Jie

    2013-01-01

    DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The “genetic switch” of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences OR and OL (separated by 2.3 kb), mediated by the λ repressor CI (accession number P03034), play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms. PMID:23853547

  17. Use of structural DNA properties for the prediction of transcription-factor binding sites in Escherichia coli

    PubMed Central

    Meysman, Pieter; Dang, Thanh Hai; Laukens, Kris; De Smet, Riet; Wu, Yan; Marchal, Kathleen; Engelen, Kristof

    2011-01-01

    Recognition of genomic binding sites by transcription factors can occur through base-specific recognition, or by recognition of variations within the structure of the DNA macromolecule. In this article, we investigate what information can be retrieved from local DNA structural properties that is relevant to transcription factor binding and that cannot be captured by the nucleotide sequence alone. More specifically, we explore the benefit of employing the structural characteristics of DNA to create binding-site models that encompass indirect recognition for the Escherichia coli model organism. We developed a novel methodology [Conditional Random fields of Smoothed Structural Data (CRoSSeD)], based on structural scales and conditional random fields to model and predict regulator binding sites. The value of relying on local structural-DNA properties is demonstrated by improved classifier performance on a large number of biological datasets, and by the detection of novel binding sites which could be validated by independent data sources, and which could not be identified using sequence data alone. We further show that the CRoSSeD-binding-site models can be related to the actual molecular mechanisms of the transcription factor DNA binding, and thus cannot only be used for prediction of novel sites, but might also give valuable insights into unknown binding mechanisms of transcription factors. PMID:21051340

  18. RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria

    PubMed Central

    Vogel, Jörg; Bartels, Verena; Tang, Thean Hock; Churakov, Gennady; Slagter-Jäger, Jacoba G.; Hüttenhofer, Alexander; Wagner, E. Gerhart H.

    2003-01-01

    Recent bioinformatics-aided searches have identified many new small RNAs (sRNAs) in the intergenic regions of the bacterium Escherichia coli. Here, a shot-gun cloning approach (RNomics) was used to generate cDNA libraries of small sized RNAs. Besides many of the known sRNAs, we found new species that were not predicted previously. The present work brings the number of sRNAs in E.coli to 62. Experimental transcription start site mapping showed that some sRNAs were encoded from independent genes, while others were processed from mRNA leaders or trailers, indicative of a parallel transcriptional output generating sRNAs co-expressed with mRNAs. Two of these RNAs (SroA and SroG) consist of known (THI and RFN) riboswitch elements. We also show that two recently identified sRNAs (RyeB and SraC/RyeA) interact, resulting in RNase III-dependent cleavage. To the best of our knowledge, this represents the first case of two non-coding RNAs interacting by a putative antisense mechanism. In addition, intracellular metabolic stabilities of sRNAs were determined, including ones from previous screens. The wide range of half-lives (<2 to >32 min) indicates that sRNAs cannot generally be assumed to be metabolically stable. The experimental characterization of sRNAs analyzed here suggests that the definition of an sRNA is more complex than previously assumed. PMID:14602901

  19. AutA and AutR, Two Novel Global Transcriptional Regulators, Facilitate Avian Pathogenic Escherichia coli Infection

    PubMed Central

    Zhuge, Xiangkai; Tang, Fang; Zhu, Hongfei; Mao, Xiang; Wang, Shaohui; Wu, Zongfu; Lu, Chengping; Dai, Jianjun; Fan, Hongjie

    2016-01-01

    Bacteria can change its lifestyle during inhabiting in host niches where they survive and replicate by rapidly altering gene expression pattern to accommodate the new environment. In this study, two novel regulators in avian pathogenic Escherichia coli (APEC) were identified and designated as AutA and AutR. RT-PCR and β-galactosidase assay results showed that AutA and AutR co-regulated the expression of adhesin UpaB in APEC strain DE205B. Electrophoretic mobility shift assay showed that AutA and AutR could directly bind the upaB promoter DNA. In vitro transcription assay indicated that AutA could activate the upaB transcription, while AutR inhibited the upaB transcription due to directly suppressing the activating effect of AutA on UpaB expression. Transcriptome analysis showed that AutA and AutR coherently affected the expression of hundreds of genes. Our study confirmed that AutA and AutR co-regulated the expression of DE205B K1 capsule and acid resistance systems in E. coli acid fitness island (AFI). Moreover, phenotypic heterogeneity in expression of K1 capsule and acid resistance systems in AFI during host–pathogen interaction was associated with the regulation of AutA and AutR. Collectively speaking, our studies presented that AutA and AutR are involved in APEC adaptive lifestyle change to facilitate its infection. PMID:27113849

  20. The post-transcriptional regulatory system CSR controls the balance of metabolic pools in upper glycolysis of Escherichia coli.

    PubMed

    Morin, Manon; Ropers, Delphine; Letisse, Fabien; Laguerre, Sandrine; Portais, Jean-Charles; Cocaign-Bousquet, Muriel; Enjalbert, Brice

    2016-05-01

    Metabolic control in Escherichia coli is a complex process involving multilevel regulatory systems but the involvement of post-transcriptional regulation is uncertain. The post-transcriptional factor CsrA is stated as being the only regulator essential for the use of glycolytic substrates. A dozen enzymes in the central carbon metabolism (CCM) have been reported as potentially controlled by CsrA, but its impact on the CCM functioning has not been demonstrated. Here, a multiscale analysis was performed in a wild-type strain and its isogenic mutant attenuated for CsrA (including growth parameters, gene expression levels, metabolite pools, abundance of enzymes and fluxes). Data integration and regulation analysis showed a coordinated control of the expression of glycolytic enzymes. This also revealed the imbalance of metabolite pools in the csrA mutant upper glycolysis, before the phosphofructokinase PfkA step. This imbalance is associated with a glucose-phosphate stress. Restoring PfkA activity in the csrA mutant strain suppressed this stress and increased the mutant growth rate on glucose. Thus, the carbon storage regulator system is essential for the effective functioning of the upper glycolysis mainly through its control of PfkA. This work demonstrates the pivotal role of post-transcriptional regulation to shape the carbon metabolism. PMID:26833659

  1. The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo

    PubMed Central

    Goldman, Seth R; Nair, Nikhil U; Wells, Christopher D; Nickels, Bryce E; Hochschild, Ann

    2015-01-01

    The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ70, can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ70 can bind in trans to TECs that emanate from either a σ70-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ70 to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ70-dependent promoters. DOI: http://dx.doi.org/10.7554/eLife.10514.001 PMID:26371553

  2. Interfering with Different Steps of Protein Synthesis Explored by Transcriptional Profiling of Escherichia coli K-12†

    PubMed Central

    Sabina, Jeffrey; Dover, Nir; Templeton, Lori J.; Smulski, Dana R.; Söll, Dieter; LaRossa, Robert A.

    2003-01-01

    Escherichia coli responses to four inhibitors that interfere with translation were monitored at the transcriptional level. A DNA microarray method provided a comprehensive view of changes in mRNA levels after exposure to these agents. Real-time reverse transcriptase PCRanalysis served to verify observations made with microarrays, and a chromosomal grpE::lux operon fusion was employed to specifically monitor the heat shock response. 4-Azaleucine, a competitive inhibitor of leucyl-tRNA synthetase, surprisingly triggered the heat shock response. Administration of mupirocin, an inhibitor of isoleucyl-tRNA synthetase activity, resulted in changes reminiscent of the stringent response. Treatment with kasugamycin and puromycin (targeting ribosomal subunit association as well as its peptidyl-transferase activity) caused accumulation of mRNAs from ribosomal protein operons. Abundant biosynthetic transcripts were often significantly diminished after treatment with any of these agents. Exposure of a relA strain to mupirocin resulted in accumulation of ribosomal protein operon transcripts. However, the relA strain's response to the other inhibitors was quite similar to that of the wild-type strain. PMID:14526028

  3. A topological model for transcription based on unwinding angle analysis of E. coli RNA polymerase binary, initiation and ternary complexes.

    PubMed

    Gamper, H B; Hearst, J E

    1982-05-01

    DNA unwinding induced by Escherichia coli RNA polymerase is measured for binary, initiation and ternary complexes formed from a unique promoter sequence on simian virus 40 DNA. At 37 degrees C the complexes all have an unwinding angle of 17 +/- 1 base pairs (580 degrees +/- 30 degrees). This unwinding is attributed to an enzyme-stabilized separation of the double helix at the promoter site, which is maintained throughout initiation and elongation. There is no heterogeneity in the unwinding angle of the ternary complex as it progresses down the helical template. The constant DNA unwinding during all phases of transcription leads us to propose the existence of unwindase and rewindase activities on the enzyme that allow it to travel down the helix like a nut on a DNA bolt. During elongation, the unwindase unwinds the DNA helix while the rewindase, lagging by 17 base pairs, displaces the RNA transcript and reseals the helix. Both activities induce a rotation in the DNA double helix relative to the polymerase. The RNA-DNA hybrid also rotates, maintaining both ends of that helix fixed relative to the catalytic and windase sites. Formation of an RNA-DNA hybrid which spans the distal end of the DNA unwound region is proposed as a possible mechanism for polymerase pausing and termination. This model requires that the polymerase direct the transcript past the noncoding DNA strand. Pausing occurs 16-20 nucleotides downstream from the centers of appropriately sized dyad symmetry elements. PMID:6286146

  4. Transcriptional organization and in vivo role of the Escherichia coli rsd gene, encoding the regulator of RNA polymerase sigma D.

    PubMed

    Jishage, M; Ishihama, A

    1999-06-01

    The regulator of sigma D (Rsd) was identified as an RNA polymerase sigma70-associated protein in stationary-phase Escherichia coli with the inhibitory activity of sigma70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953-4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, sigmaS-dependent P1 and sigma70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the sigma70- or sigmaS-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the rsd null mutant, the sigma70- and sigmaS-dependent gene expression was increased or decreased, respectively. On the other hand, the sigma70- or sigmaS-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the sigmaS-dependent transcription in the rsd mutant is overcome by increased production of the sigmaS subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase sigma subunit from sigma70 to sigmaS during the transition from exponential growth to the stationary phase. PMID:10368152

  5. Construction and characterization of plasmid and lambda phage vector systems for study of transcriptional control in Escherichia coli.

    PubMed

    Hirano, M; Shigesada, K; Imai, M

    1987-01-01

    We constructed a family of lambda phage and plasmid vectors which facilitate cloning and quantitative analysis of transcriptional regulator in both single and multiple copies. Their expression system was modified from the ara-trp-lac fusion operon of plasmid pMC81 [Casadaban and Cohen, J. Mol. Biol. 138 (1980) 179-207], which is designed to assay both promoters and terminators with a single vehicle. To eliminate transcriptional and translational polar effects liable to occur in the original fusion operon upon insertion of a foreign nucleotide sequence, intracistronic Rho-dependent terminators, that are present within the trpB gene and distal to the cloning site were deleted, and DNA spacers containing stop codons were introduced immediately before and after the cloning site. In analysis of the cloned trp regulatory region, the lambda phage system faithfully reproduced the tight regulation by tryptophan characteristic to the natural trp operon on the E. coli chromosome, whereas the plasmid counterpart exhibited a substantially relaxed response. Comparative studies on the relative strengths of various promoters and terminators have further demonstrated that the lambda phage vector system permits accurate assays of exceptionally strong promoters like Ptrp and lambda pL without disturbing the bacterial growth, while being sensitive enough for detecting low-level transcription under the control of weak promoters or potent terminators. Cloning with the lambda phage vector can be greatly facilitated by transferring the target regulatory site precloned with the plasmid onto the phage genome through in vivo recombination. PMID:2828183

  6. Transcription of the tRNA-tufB operon of Escherichia coli: activation, termination and antitermination.

    PubMed Central

    van Delft, J H; Mariñon, B; Schmidt, D S; Bosch, L

    1987-01-01

    Signals setting the level of transcription of the tRNA-tufB operon have been studied by deletion mapping. TufB transcription was measured in vivo with plasmid-borne tRNA-tufB:galk operon fusions. Removal of the sequences from -133 to -58 with respect to the transcription start point, results in a 90% decrease of tufB transcription. This demonstrates the presence of a region, upstream of the tRNA-tufB promoter, that enhances the expression of the operon. DNA fragments bearing this upstream activator region do not display an abnormal electrophoretic mobility, as has been observed for the rrnB P1 upstream activator. Deletions starting in the first tRNA gene and directing towards tufB reveal at least two sites that influence tufB transcription. One signals transcription termination in the intergenic region between thrT and tufB. The other may be involved in antitermination. Possible mechanisms underlying antitermination and termination are considered in the light of the nucleotide sequence. Images PMID:3317280

  7. Reconstructing genome-wide regulatory network of E. coli using transcriptome data and predicted transcription factor activities

    PubMed Central

    2011-01-01

    Background Gene regulatory networks play essential roles in living organisms to control growth, keep internal metabolism running and respond to external environmental changes. Understanding the connections and the activity levels of regulators is important for the research of gene regulatory networks. While relevance score based algorithms that reconstruct gene regulatory networks from transcriptome data can infer genome-wide gene regulatory networks, they are unfortunately prone to false positive results. Transcription factor activities (TFAs) quantitatively reflect the ability of the transcription factor to regulate target genes. However, classic relevance score based gene regulatory network reconstruction algorithms use models do not include the TFA layer, thus missing a key regulatory element. Results This work integrates TFA prediction algorithms with relevance score based network reconstruction algorithms to reconstruct gene regulatory networks with improved accuracy over classic relevance score based algorithms. This method is called Gene expression and Transcription factor activity based Relevance Network (GTRNetwork). Different combinations of TFA prediction algorithms and relevance score functions have been applied to find the most efficient combination. When the integrated GTRNetwork method was applied to E. coli data, the reconstructed genome-wide gene regulatory network predicted 381 new regulatory links. This reconstructed gene regulatory network including the predicted new regulatory links show promising biological significances. Many of the new links are verified by known TF binding site information, and many other links can be verified from the literature and databases such as EcoCyc. The reconstructed gene regulatory network is applied to a recent transcriptome analysis of E. coli during isobutanol stress. In addition to the 16 significantly changed TFAs detected in the original paper, another 7 significantly changed TFAs have been detected by

  8. Analysis of the human intestinal epithelial cell transcriptional response to Lactobacillus acidophilus, Lactobacillus salivarius, Bifidobacterium lactis and Escherichia coli.

    PubMed

    Putaala, H; Barrangou, R; Leyer, G J; Ouwehand, A C; Hansen, E Bech; Romero, D A; Rautonen, N

    2010-09-01

    The complex microbial population residing in the human gastrointestinal tract consists of commensal, potential pathogenic and beneficial species, which are probably perceived differently by the host and consequently could be expected to trigger specific transcriptional responses. Here, we provide a comparative analysis of the global in vitro transcriptional response of human intestinal epithelial cells to Lactobacillus acidophilus NCFM™, Lactobacillus salivarius Ls-33, Bifidobacterium animalis subsp. lactis 420, and enterohaemorrhagic Escherichia coli O157:H7 (EHEC). Interestingly, L. salivarius Ls-33 DCE-induced changes were overall more similar to those of B. lactis 420 than to L. acidophilus NCFM™, which is consistent with previously observed in vivo immunomodulation properties. In the gene ontology and pathway analyses both specific and unspecific changes were observed. Common to all was the regulation of apoptosis and adipogenesis, and lipid-metabolism related regulation by the probiotics. Specific changes such as regulation of cell-cell adhesion by B. lactis 420, superoxide metabolism by L. salivarius Ls-33, and regulation of MAPK pathway by L. acidophilus NCFM™ were noted. Furthermore, fundamental differences were observed between the pathogenic and probiotic treatments in the Toll-like receptor pathway, especially for adapter molecules with a lowered level of transcriptional activation of MyD88, TRIF, IRAK1 and TRAF6 by probiotics compared to EHEC. The results in this study provide insights into the relationship between probiotics and human intestinal epithelial cells, notably with regard to strain-specific responses, and highlight the differences between transcriptional responses to pathogenic and probiotic bacteria. PMID:21831765

  9. MalT, the regulatory protein of the Escherichia coli maltose system, is an ATP-dependent transcriptional activator.

    PubMed

    Richet, E; Raibaud, O

    1989-03-01

    We show that MalT, the transcriptional activator of the Escherichia coli maltose regulon, specifically binds ATP and dATP with a high affinity (Kd = 0.4 microM) and exhibits a weak ATPase activity. Using an abortive initiation assay, we further show that activation of open complex formation by MalT depends on the presence of ATP in addition to that of maltotriose, the inducer of the maltose system. Similar experiments in which ATP was replaced by ADP or AMP-PNP, a non-hydrolysable analogue of ATP, demonstrate that this reaction does not require ATP hydrolysis. As revealed by DNase I footprinting, both ATP and maltotriose are required for the binding of the MalT protein to the mal promoter DNA. PMID:2524384

  10. Self-association of the Escherichia coli transcription activator MalT in the presence of maltotriose and ATP.

    PubMed

    Schreiber, V; Richet, E

    1999-11-19

    MalT, the transcriptional activator of the Escherichia coli maltose regulon, binds the MalT-dependent promoters and activates transcription initiation only in the presence of maltotriose and ATP (or adenylyl imidodiphosphate (AMP-PNP)). Cooperative binding of MalT to the array of cognate sites present in the MalT-dependent promoters suggests that promoter binding involves MalT oligomerization. Gel filtration and sedimentation experiments were used to analyze the quaternary structure of MalT in solution in the absence or presence of maltotriose and/or AMP-PNP, ATP, or ADP. The protein is monomeric in the absence of ligands and in the presence of ADP. In the presence of maltotriose, AMP-PNP, or ATP only, the protein self-associates, but a large fraction of the protein remains monomeric. In the presence of both maltotriose and AMP-PNP (ATP or ADP), the protein is essentially oligomeric, with the difference being that the oligomerization is less favored in the presence of ADP + maltotriose than in the presence of AMP-PNP + maltotriose. We present evidence that the association pathway comprises the following steps: monomers --> dimers --> (MalT)(n) --> aggregates, where 3

  11. Global transcriptional regulation by H-NS and its biological influence on the virulence of Enterohemorrhagic Escherichia coli.

    PubMed

    Wan, Baoshan; Zhang, Qiufen; Tao, Jing; Zhou, Aiping; Yao, Yu-Feng; Ni, Jinjing

    2016-08-22

    As a global transcriptional regulator, H-NS, the histone-like nucleoid-associated DNA-binding and bridging protein, plays a wide range of biological roles in bacteria. In order to determine the role of H-NS in regulating gene transcription and further find out the biological significance of this protein in Enterohemorrhagic Escherichia coli (EHEC), we conducted transcriptome analysis of hns mutant by RNA sequencing. A total of 983 genes were identified to be regulated by H-NS in EHEC. 213 and 770 genes were down-regulated and up-regulated in the deletion mutant of hns, respectively. Interestingly, 34 of 97 genes on virulence plasmid pO157 were down-regulated by H-NS. Although the deletion mutant of hns showed a decreased survival rate in macrophage compared with the wild type strain, it exhibited the higher ability to colonize mice gut and became more virulent to BALB/c mice. The BALB/c mice infected with the deletion mutant of hns showed a lower survival rate, and a higher bacterial burden in the gut, compared with those infected with wild type strain, especially when the gut microbiota was not disturbed by antibiotic administration. These findings suggest that H-NS plays an important role in virulence of EHEC by interacting with host gut microbiota. PMID:27173635

  12. The MazF-regulon: a toolbox for the post-transcriptional stress response in Escherichia coli.

    PubMed

    Sauert, Martina; Wolfinger, Michael T; Vesper, Oliver; Müller, Christian; Byrgazov, Konstantin; Moll, Isabella

    2016-08-19

    Flexible adaptation to environmental stress is vital for bacteria. An energy-efficient post-transcriptional stress response mechanism in Escherichia coli is governed by the toxin MazF. After stress-induced activation the endoribonuclease MazF processes a distinct subset of transcripts as well as the 16S ribosomal RNA in the context of mature ribosomes. As these 'stress-ribosomes' are specific for the MazF-processed mRNAs, the translational program is changed. To identify this 'MazF-regulon' we employed Poly-seq (polysome fractionation coupled with RNA-seq analysis) and analyzed alterations introduced into the transcriptome and translatome after mazF overexpression. Unexpectedly, our results reveal that the corresponding protein products are involved in all cellular processes and do not particularly contribute to the general stress response. Moreover, our findings suggest that translational reprogramming serves as a fast-track reaction to harsh stress and highlight the so far underestimated significance of selective translation as a global regulatory mechanism in gene expression. Considering the reported implication of toxin-antitoxin (TA) systems in persistence, our results indicate that MazF acts as a prime effector during harsh stress that potentially introduces translational heterogeneity within a bacterial population thereby stimulating persister cell formation. PMID:26908653

  13. The Switch Regulating Transcription of the Escherichia coli Biotin Operon Does Not Require Extensive Protein-Protein Interactions

    PubMed Central

    Solbiati, José; Cronan, John E.

    2009-01-01

    Transcription of the Escherichia coli biotin (bio) operon is regulated by BirA, a protein that is not only the repressor that regulates bio operon expression by DNA binding but also the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein that is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (bio-AMP), the obligatory intermediate of the attachment reaction. The current model postulates that the unmodified acceptor protein binds the monomeric BirA:bio-AMP complex and thereby blocks assembly (dimerization) of the form of BirA that binds DNA. We report that expression of fusion proteins that carry synthetic biotin accepting peptide sequences was as effective as the natural acceptor protein in derepression of bio operon transcription. These peptide sequences have sequences that are remarkably dissimilar to that of the natural acceptor protein and thus our data argue that the regulatory switch does not require the extensive protein-protein interactions postulated in the current model. PMID:20142036

  14. Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase

    NASA Astrophysics Data System (ADS)

    Harada, Yoshie; Ohara, Osamu; Takatsuki, Akira; Itoh, Hiroyasu; Shimamoto, Nobuo; Kinosita, Kazuhiko

    2001-01-01

    Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5pNnm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

  15. An intersubunit contact stimulating transcription initiation by E. coli RNA polymerase: interaction of the α C-terminal domain and σ region 4

    PubMed Central

    Ross, Wilma; Schneider, David A.; Paul, Brian J.; Mertens, Aaron; Gourse, Richard L.

    2003-01-01

    The C-terminal domain of the Escherichia coli RNA polymerase (RNAP) alpha subunit (αCTD) stimulates transcription initiation by interacting with upstream (UP) element DNA and a variety of transcription activators. Here we identify specific substitutions in region 4.2 of sigma 70 (σ70) and in αCTD that decrease transcription initiation from promoters containing some, but not all, UP elements. This decrease in transcription derives from a decrease in the initial equilibrium constant for RNAP binding (KB). The open complexes formed by the mutant and wild-type RNAPs differ in DNAse I sensitivity at the junction of the αCTD and σ DNA binding sites, correlating with the differences in transcription. A model of the DNA–αCTD–σ region 4.2 ternary complex, constructed from the previously determined X-ray structures of the Thermus aquaticus σ region 4.2–DNA complex and the E. coli αCTD–DNA complex, indicates that the residues identified by mutation in σ region 4.2 and in αCTD are in very close proximity. Our results strongly suggest that αCTD, when bound to an UP element proximal subsite, contacts the RNAP σ70 subunit, increasing transcription. Previous data from the literature suggest that this same σ–αCTD interaction also plays a role in transcription factor-mediated activation. PMID:12756230

  16. Network analysis of the transcriptional pattern of young and old cells of Escherichia coli during lag phase

    PubMed Central

    2009-01-01

    Background The aging process of bacteria in stationary phase is halted if cells are subcultured and enter lag phase and it is then followed by cellular division. Network science has been applied to analyse the transcriptional response, during lag phase, of bacterial cells starved previously in stationary phase for 1 day (young cells) and 16 days (old cells). Results A genome scale network was constructed for E. coli K-12 by connecting genes with operons, transcription and sigma factors, metabolic pathways and cell functional categories. Most of the transcriptional changes were detected immediately upon entering lag phase and were maintained throughout this period. The lag period was longer for older cells and the analysis of the transcriptome revealed different intracellular activity in young and old cells. The number of genes differentially expressed was smaller in old cells (186) than in young cells (467). Relatively, few genes (62) were up- or down-regulated in both cultures. Transcription of genes related to osmotolerance, acid resistance, oxidative stress and adaptation to other stresses was down-regulated in both young and old cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were up-regulated in young cells while old cells up-regulated the Entner Doudoroff and gluconate pathways and down-regulated the pentose phosphate pathway. In both old and young cells, anaerobic respiration and fermentation pathways were down-regulated, but only young cells up-regulated aerobic respiration while there was no evidence of aerobic respiration in old cells. Numerous genes related to DNA maintenance and replication, translation, ribosomal biosynthesis and RNA processing as well as biosynthesis of the cell envelope and flagellum and several components of the chemotaxis signal transduction complex were up-regulated only in young cells. The genes for several transport proteins for iron compounds were up-regulated in both young and old cells. Numerous

  17. Internal promoter in the ilvGEDA transcription unit of Escherichia coli K-12.

    PubMed Central

    Calhoun, D H; Wallen, J W; Traub, L; Gray, J E; Kung, H F

    1985-01-01

    Segments of the ilvGEDA transcription unit have been cloned into the promoter tester plasmid pMC81. This vector contains cloning sites situated upstream of the lacZ gene coding for beta-galactosidase. Using this method we have quantitatively evaluated in vivo (i) the activity of previously described promoter, pG, preceding ilvG; (ii) the relative activity of pE promoter, previously postulated to be located between ilvG and ilvE; and (iii) the effect of the frameshift site present in the wild-type ilvG gene by comparison with mutant derivatives lacking this frameshift site. Isogenic derivatives of strain MC1000 were constructed by transduction with phage P1 grown on rho-120, delta(ilvGEDA), delta(ilvED), and ilvA538 hosts. The potential effects of these alleles that were previously postulated to affect ilvGEDA expression were assessed in vivo by monitoring beta-galactosidase production directed by ilv DNA fragments. Cloned ilv segments were also tested for activity in vitro with a DNA-directed coupled transcription and translation system. The production in vitro of ilv-directed ilv gene expression and beta-galactosidase expression with ara-ilv-lac fusions paralleled the in vivo activity. Images PMID:3917997

  18. LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli.

    PubMed

    Lehnen, D; Blumer, C; Polen, T; Wackwitz, B; Wendisch, V F; Unden, G

    2002-07-01

    The function of the LysR-type regulator LrhA of Escherichia coli was defined by comparing whole-genome mRNA profiles from wild-type E. coli and an isogenic lrhA mutant on a DNA microarray. In the lrhA mutant, a large number (48) of genes involved in flagellation, motility and chemotaxis showed relative mRNA abundances increased by factors between 3 and 80. When a representative set of five flagellar, motility and chemotaxis genes was tested in lacZ reporter gene fusions, similar factors for derepression were found in the lrhA mutant. In gel retardation experiments, the LrhA protein bound specifically to flhD and lrhA promoter DNA (apparent K(D) approximately 20 nM), whereas the promoters of fliC, fliA and trg were not bound by LrhA. The expression of flhDC (encoding FlhD(2)C(2)) was derepressed by a factor of 3.5 in the lrhA mutant. FlhD(2)C(2) is known as the master regulator for the expression of flagellar and chemotaxis genes. By DNase I footprinting, LrhA binding sites at the flhDC and lrhA promoters were identified. The lrhA gene was under positive autoregulation by LrhA as shown by gel retardation and lrhA expression studies. It is suggested that LrhA is a key regulator controlling the transcription of flagellar, motility and chemotaxis genes by regulating the synthesis and concentration of FlhD(2)C(2). PMID:12123461

  19. Molybdenum-sensitive transcriptional regulation of the chlD locus of Escherichia coli

    SciTech Connect

    Miller, J.B.; Scott, D.J.; Amy, N.K.

    1987-05-01

    The chlD gene in Escherichia coli is required for the incorporation and utilization of molybdenum when the cells are grown with low concentrations of molybdate. The authors constructed chlD-lac operon fusions and measured expressions of the fusion, Mo cofactor, and nitrate reductase activities under a variety of growth conditions. The chlD-lac fusion was highly expressed when cells were grown with less than 10 nm molybdate. Increasing concentrations of molybdate caused loss of activity, with less than 5% of the activity remaining at 500 nM molybdate; when tungstate replaced molybdate, it had an identical affect on chlD expression. Expression of chlD-lac was increased in cells grown with nitrate. Strains with chlD-lac plus an additional mutation in a chl or nar gene were constructed to test whether the regulation of chlD-lac required the concerted action of gene products involved with Mo cofactor or nitrate reductase synthesis. Mutations in narL prevented the increase in activity in response to nitrate; mutations in chlB, narC, or narI resulted in partial constitutive expression of the chlD-lac fusion: the fusion was regulated by molybdate, but it no longer required the presence of nitrate for maximal activity. Mutations in chlA, chlE, or chlG which affect Mo cofactor metabolism, did not affect the expression of chlD-lac.

  20. Global transcriptional response of Escherichia coli O157:H7 to growth transitions in glucose minimal medium

    PubMed Central

    Bergholz, Teresa M; Wick, Lukas M; Qi, Weihong; Riordan, James T; Ouellette, Lindsey M; Whittam, Thomas S

    2007-01-01

    Background: Global patterns of gene expression of Escherichia coli K-12 during growth transitions have been deeply investigated, however, comparable studies of E. coli O157:H7 have not been explored, particularly with respect to factors regulating virulence genes and genomic islands specific to this pathogen. To examine the impact of growth phase on the dynamics of the transcriptome, O157:H7 Sakai strain was cultured in MOPS minimal media (0.1% glucose), RNA harvested at 10 time points from early exponential to full stationary phase, and relative gene expression was measured by co-hybridization on high-density DNA microarrays. Expression levels of 14 genes, including those encoding Shiga toxins and other virulence factors associated with the locus of enterocyte effacement (LEE), were confirmed by Q-PCR. Results: Analysis of variance (R/MAANOVA, Fs test) identified 442 (36%) of 1239 O157-specific ORFs and 2110 (59%) of 3647 backbone ORFs that changed in expression significantly over time. QT cluster analysis placed 2468 of the 2552 significant ORFs into 12 groups; each group representing a distinct expression pattern. ORFs from the largest cluster (n = 1078) decreased in expression from late exponential to early stationary phase: most of these ORFs are involved in functions associated with steady state growth. Also represented in this cluster are ORFs of the TAI island, encoding tellurite resistance and urease activity, which decreased ~4-fold. Most ORFs of the LEE pathogenicity island also decreased ~2-fold by early stationary phase. The ORFs encoding proteins secreted via the LEE encoded type III secretion system, such as tccP and espJ, also decreased in expression from exponential to stationary phase. Three of the clusters (n = 154) comprised genes that are transiently upregulated at the transition into stationary phase and included genes involved in nutrient scavenging. Upregulated genes with an increase in mRNA levels from late exponential to early stationary

  1. NrdR Transcription Regulation: Global Proteome Analysis and Its Role in Escherichia coli Viability and Virulence

    PubMed Central

    Naveen, Vankadari; Hsiao, Chwan-Deng

    2016-01-01

    Bacterial ribonucleotide reductases (RNRs) play an important role in the synthesis of dNTPs and their expression is regulated by the transcription factors, NrdR and Fur. Recent transcriptomic studies using deletion mutants have indicated a role for NrdR in bacterial chemotaxis and in the maintenance of topoisomerase levels. However, NrdR deletion alone has no effect on bacterial growth or virulence in infected flies or in human blood cells. Furthermore, transcriptomic studies are limited to the deletion strain alone, and so are inadequate for drawing biological implications when the NrdR repressor is active or abundant. Therefore, further examination is warranted of changes in the cellular proteome in response to both NrdR overexpression, as well as deletion, to better understand its functional relevance as a bacterial transcription repressor. Here, we profile bacterial fate under conditions of overexpression and deletion of NrdR in E. coli. Biochemical assays show auxiliary zinc enhances the DNA binding activity of NrdR. We also demonstrate at the physiological level that increased nrdR expression causes a significant reduction in bacterial growth and fitness even at normal temperatures, and causes lethality at elevated temperatures. Corroborating these direct effects, global proteome analysis following NrdR overexpression showed a significant decrease in global protein expression. In parallel, studies on complementary expression of downregulated essential genes polA, eno and thiL showed partial rescue of the fitness defect caused by NrdR overexpression. Deletion of downregulated non-essential genes ygfK and trxA upon NrdR overexpression resulted in diminished bacterial growth and fitness suggesting an additional role for NrdR in regulating other genes. Moreover, in comparison with NrdR deletion, E. coli cells overexpressing NrdR showed significantly diminished adherence to human epithelial cells, reflecting decreased bacterial virulence. These results suggest

  2. Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli.

    PubMed

    Blumer, Caroline; Kleefeld, Alexandra; Lehnen, Daniela; Heintz, Margit; Dobrindt, Ulrich; Nagy, Gábor; Michaelis, Kai; Emödy, Levente; Polen, Tino; Rachel, Reinhard; Wendisch, Volker F; Unden, Gottfried

    2005-10-01

    Type 1 fimbriae of Escherichia coli facilitate attachment to the host mucosa and promote biofilm formation on abiotic surfaces. The transcriptional regulator LrhA, which is known as a repressor of flagellar, motility and chemotaxis genes, regulates biofilm formation and expression of type 1 fimbriae. Whole-genome expression profiling revealed that inactivation of lrhA results in an increased expression of structural components of type 1 fimbriae. In vitro, LrhA bound to the promoter regions of the two fim recombinases (FimB and FimE) that catalyse the inversion of the fimA promoter, and to the invertible element itself. Translational lacZ fusions with these genes and quantification of fimE transcript levels by real-time PCR showed that LrhA influences type 1 fimbrial phase variation, primarily via activation of FimE, which is required for the ON-to-OFF transition of the fim switch. Enhanced type 1 fimbrial expression as a result of lrhA disruption was confirmed by mannose-sensitive agglutination of yeast cells. Biofilm formation was stimulated by lrhA inactivation and completely suppressed upon LrhA overproduction. The effects of LrhA on biofilm formation were exerted via the changed levels of surface molecules, most probably both flagella and type 1 fimbriae. Together, the data show a role for LrhA as a repressor of type 1 fimbrial expression, and thus as a regulator of the initial stages of biofilm development and, presumably, bacterial adherence to epithelial host cells also. PMID:16207912

  3. Transcriptional analysis and regulation of the sfa determinant coding for S fimbriae of pathogenic Escherichia coli strains.

    PubMed

    Morschhäuser, J; Uhlin, B E; Hacker, J

    1993-04-01

    The sfa determinant codes for S fimbrial adhesins which constitute adherence factors of pathogenic Escherichia coli strains. We have recently shown that the sfa determinant is transcribed from three promoters, pA, pB, and pC. In comparison with the promoters pB and pC, promoter pA, which is located in front of the structural gene sfaA, showed very weak activity. Here we have determined the exact positions of the mRNA start points by primer extension studies. We have also shown that mRNAs of 500, 700 and 1400 bases can be detected using oligonucleotide probes specific for the genes sfaB, sfaC and sfaA. SfaB and SfaC are positive regulators influencing fimbriation and the production of the S-specific adhesin which is encoded by the gene sfaS located in the distal half of the determinant. In addition, it is demonstrated that SfaB and SfaC interfere with the regulatory effect of the histone-like protein H-NS, encoded by a locus termed drdX or osmZ. In a drdX+ strain the regulators are necessary for transcription of the sfa determinant. In contrast, sfa expression is activator-independent in a drdX- strain. In this latter genetic background, a substantial fraction of the sfa transcripts is initiated from promoter pA. On the basis of these data we discuss a model for the regulation of this adhesin-specific determinant. PMID:8097559

  4. Genome-Wide Transcriptional Response of Chemostat-Cultured Escherichia coli to Zinc

    PubMed Central

    Lee, Lucy J.; Barrett, Jason A.; Poole, Robert K.

    2005-01-01

    Zinc is an essential trace metal ion for growth, but an excess of Zn is toxic and microorganisms express diverse resistance mechanisms. To understand global bacterial responses to excess Zn, we conducted transcriptome profiling experiments comparing Escherichia coli MG1655 grown under control conditions and cells grown with a toxic, sublethal ZnSO4 concentration (0.2 mM). Cultures were grown in a defined medium lacking inorganic phosphate, permitting maximum Zn bioavailability, and in glycerol-limited chemostats at a constant growth rate and pH. Sixty-four genes were significantly up-regulated by Zn stress, including genes known to be involved in Zn tolerance, particularly zntA, zraP, and hydG. Microarray transcriptome profiling was confirmed by real-time PCR determinations of cusF (involved in Ag and Cu efflux), ais (an Al-inducible gene), asr (encoding an acid shock-inducible periplasmic protein), cpxP (a periplasmic chaperone gene), and basR. Five up-regulated genes, basR and basS [encoding a sensor-regulator implicated in Salmonella in Fe(III) sensing and antibiotic resistance], fliM (flagellar synthesis), and ycdM and yibD (both with unknown functions), are important for growth resistance to zinc, since mutants with mutations in these genes exhibited zinc sensitivity in liquid media and on metal gradient plates. Fifty-eight genes were significantly down-regulated by Zn stress; notably, several of these genes were involved in protection against acid stress. Since the mdt operon (encoding a multidrug resistance pump) was also up-regulated, these findings have important implications for understanding not only Zn homeostasis but also how bacterial antibiotic resistance is modulated by metal ions. PMID:15659689

  5. Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches

    PubMed Central

    Hoffmann, Stefan A.; Kruse, Sabrina M.; Arndt, Katja M.

    2016-01-01

    We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ70 dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a ‘forward’ gene interferes with the expression of a ‘reverse’ gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection. PMID:26932362

  6. The transcription elongation factor NusA is required for stress-induced mutagenesis in Escherichia coli.

    PubMed

    Cohen, Susan E; Walker, Graham C

    2010-01-12

    Stress-induced mutagenesis describes the accumulation of mutations that occur in nongrowing cells, in contrast to mutagenesis that occurs in actively dividing populations, and has been referred to as stationary-phase or adaptive mutagenesis. The most widely studied system for stress-induced mutagenesis involves monitoring the appearance of Lac(+) revertants of the strain FC40 under starvation conditions in Escherichia coli. The SOS-inducible translesion DNA polymerase DinB plays an important role in this phenomenon. Loss of DinB (DNA pol IV) function results in a severe reduction of Lac(+) revertants. We previously reported that NusA, an essential component of elongating RNA polymerases, interacts with DinB. Here we report our unexpected observation that wild-type NusA function is required for stress-induced mutagenesis. We present evidence that this effect is unlikely to be due to defects in transcription of lac genes but rather is due to an inability to adapt and mutate in response to environmental stress. Furthermore, we extended our analysis to the formation of stress-induced mutants in response to antibiotic treatment, observing the same striking abolition of mutagenesis under entirely different conditions. Our results are the first to implicate NusA as a crucial participant in the phenomenon of stress-induced mutagenesis. PMID:20036541

  7. Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches.

    PubMed

    Hoffmann, Stefan A; Kruse, Sabrina M; Arndt, Katja M

    2016-06-01

    We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ(70) dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a 'forward' gene interferes with the expression of a 'reverse' gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection. PMID:26932362

  8. Regulation of amino acid transport in Escherichia coli by transcription termination factor rho.

    PubMed Central

    Quay, S C; Oxender, D L

    1977-01-01

    Amino acid transport rates and amino acid binding proteins were examined in a strain containing the rho-120 mutation (formerly SuA), which has been shown to lower the rho-dependent, ribonucleic acid-activated adenosine triphosphatase activity to 9% of the rho activity in the isogenic wild-type strain. Tryptophan and proline transport, which occur by membrane-bound systems, were not altered. On the other hand, arginine, histidine, leucine, isoleucine, and valine transport were variably increased by a factor of 1.4 to 5.0. Kinetics of leucine transport showed that the LIV (leucine, isoleucine, and valine)-I (binding protein-associated) transport system is increased 8.5-fold, whereas the LIV-II (membrane-bound) system is increased 1.5-fold in the rho mutant under leucine-limited growth conditions. The leucine binding protein is increased fourfold under the same growth conditions. The difference in leucine transport in these strains was greatest during leucine-limited growth; growth on complex media repressed both strains to the same transport activity. We propose that rho-dependent transcriptional termination is important for leucine-specific repression of branched-chain amino acid transport, although rho-independent regulation, presumably by a corepressor-aporepressor-type mechanism, must also occur. PMID:324970

  9. Transcription frequency modulates the efficiency of an attenuator preceding the rpoBC RNA polymerase genes of Escherichia coli: possible autogenous control.

    PubMed Central

    Steward, K L; Linn, T

    1992-01-01

    Expression of the rpoBC genes encoding the beta and beta' RNA polymerase subunits of Escherichia coli is autogenously regulated. Although previous studies have demonstrated a post-transcriptional feedback mechanism, complex transcriptional controls of rpoBC expression may also contribute. We show that an attenuator (rpoBa) separating the ribosomal protein (rpl) genes from the rpoBC genes in the rplKAJLrpoBC gene cluster is modulated in its efficiency in response to changes in the frequency of transcription initiated by promoters located upstream. A series of rplJLrpoBalacZ transcriptional fusions was constructed on lambda vectors in which transcription into the rpoBa attenuator was varied by using a variety of promoters with different strengths. beta-galactosidase assays performed on monolysogens of the recombinant phage show that with transcription increasing over a 40-fold range, readthrough of rpoBa decreases from 61% to 19%. In contrast, two other well-characterized terminators show nearly constant efficiencies over a similar range of transcription frequencies. Using a set of phage P22 ant promoter variants with single-nucleotide changes in the promoter consensus sequences also demonstrates that the modulation of rpoBa function appears to be unrelated to the phenomenon of 'factor-independent antitermination' reported by others. The implications for autogenous control of RNA polymerase synthesis are discussed. PMID:1408790

  10. Physiological and Transcriptional Characterization of Escherichia Coli Strains Lacking Interconversion of Phosphoenolpyruvate and Pyruvate When Glucose and Acetate are Coutilized

    PubMed Central

    Sabido, Andrea; Sigala, Juan Carlos; Hernández-Chávez, Georgina; Flores, Noemí; Gosset, Guillermo; Bolívar, Francisco

    2013-01-01

    Phosphoenolpyruvate (PEP) is a precursor involved in the biosynthesis of aromatics and other valuable compounds in Escherichia coli. The PEP:carbohydrate phosphotransferase system (PTS) is the major glucose transport system and the largest PEP consumer. To increase intracellular PEP availability for aromatics production purposes, mutant strains of E. coli JM101 devoid of the ptsHIcrr operon (PB11 strain) have been previously generated. In this derivative, transport and growth rate on glucose decreased significantly. A laboratory evolved strain derived from PB11 that partially recovered its growth capacity on glucose was named PB12. In the present study, we blocked carbon skeletons interchange between PEP and pyruvate (PYR) in these ptsHIcrr− strains by deleting the pykA, pykF, and ppsA genes. The PB11 pykAF− ppsA− strain exhibited no growth on glucose or acetate alone, but it was viable when both substrates were consumed simultaneously. In contrast, the PB12 pykAF− ppsA− strain displayed a low growth rate on glucose or acetate alone, but in the mixture, growth was significantly improved. RT-qPCR expression analysis of PB11 pykAF− ppsA− growing with both carbon sources showed a downregulation of all central metabolic pathways compared with its parental PB11 strain. Under the same conditions, transcription of most of the genes in PB12 pykAF− ppsA− did not change, and few like aceBAK, sfcA, and poxB were overexpressed compared with PB12. We explored the aromatics production capabilities of both ptsHIcrr− pykAF− ppsA− strains and the engineered PB12 pykAF− ppsA− tyrR− pheAev2+/pJLBaroGfbrtktA enhanced the yield of aromatic compounds when coutilizing glucose and acetate compared with the control strain PB12 tyrR− pheAev2+/pJLBaroGfbrtktA. Biotechnol. Bioeng. 2014;111: 1150–1160. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:24375081

  11. Characterization of the [2Fe-2S] cluster of the Escherichia coli transcription factor IscR†

    PubMed Central

    Fleischhacker, Angela S.; Stubna, Audria; Hsueh, Kuang-Lung; Guo, Yisong; Teter, Sarah J.; Rose, Justin C.; Brunold, Thomas C.; Markley, John L.; Münck, Eckard; Kiley, Patricia J.

    2012-01-01

    IscR is a Fe-S cluster-containing transcription factor involved in a homeostatic mechanism that controls Fe-S cluster biogenesis in Escherichia coli. Although IscR has been proposed to act as a sensor of the cellular demands for Fe-S cluster biogenesis, the mechanism by which IscR performs this function is not known. In this study, we investigated the biochemical properties of the Fe-S cluster of IscR to gain insight into the proposed sensing activity. Mössbauer studies revealed that IscR contains predominantly a reduced [2Fe-2S]1+ cluster in vivo. However, upon anaerobic isolation of IscR some clusters became oxidized to the [2Fe-2S]2+ form. Cluster oxidation did not, however, alter the affinity of IscR for its binding site within the iscR promoter in vitro, indicating that cluster oxidation state is not important for regulation of DNA binding. Furthermore, characterization of anaerobically isolated IscR using resonance Raman, Mössbauer, and NMR spectroscopies leads to the proposal that the [2Fe-2S] cluster does not have full cysteinyl ligation. Mutagenesis studies indicate that, in addition to the three previously identified cysteine residues (Cys92, Cys98, and Cys104), the highly conserved residue His107 is essential for cluster ligation. Thus, these data suggest that IscR binds the cluster with an atypical ligation scheme of three cysteines and one histidine, a feature that may be relevant to the proposed function of IscR as a sensor of cellular Fe-S cluster status. PMID:22583201

  12. Transcriptional Regulation of the orf19 Gene and the tir-cesT-eae Operon of Enteropathogenic Escherichia coli

    PubMed Central

    Sánchez-SanMartín, Claudia; Bustamante, Víctor H.; Calva, Edmundo; Puente, José Luis

    2001-01-01

    To establish an intimate interaction with the host epithelial cell surface, enteropathogenic Escherichia coli (EPEC) produces Tir, a bacterial protein that upon translocation and insertion into the epithelial cell membrane constitutes the receptor for intimin. The tir gene is encoded by the locus for enterocyte effacement (LEE), where it is flanked upstream by orf19 and downstream by the cesT and eae genes. With the use of a series of cat transcriptional fusions and primer extension analysis, we confirmed that tir, cesT, and eae form the LEE5 operon, which is under the control of a promoter located upstream from tir, and found that the orf19 gene is transcribed as a monocistronic unit. We also demonstrated that the LEE-encoded regulator Ler was required for efficient activation of both the tir and the orf19 promoters and that a sequence motif located between positions −204 and −157 was needed for the Ler-dependent activation of the tir operon. Sequence elements located between positions −204 and −97 were determined to be required for the differential negative modulatory effects exerted by unknown regulatory factors under specific growth conditions. Upon deletion of the upstream sequences, the tir promoter was fully active even in the absence of Ler, indicating that tir expression is subject to a repression mechanism that is counteracted by this regulatory protein. However, its full activation was still repressed by growth in rich medium or at 25°C, suggesting that negative regulation also occurs at or downstream of the promoter. Expression of orf19, but not of the tir operon, became Ler independent in an hns mutant strain, suggesting that Ler overcomes the repression exerted by H-NS (histone-like nucleoid structuring protein) on this gene. PMID:11292802

  13. Characterization of the LysR-type transcriptional regulator YcjZ-like from Xylella fastidiosa overexpressed in Escherichia coli.

    PubMed

    Santiago, André S; Santos, Clelton A; Mendes, Juliano S; Toledo, Marcelo A S; Beloti, Lilian L; Souza, Alessandra A; Souza, Anete P

    2015-09-01

    The Xylella fastidiosa 9a5c strain is a xylem-limited phytopathogen that is the causal agent of citrus variegated chlorosis (CVC). This bacterium is able to form a biofilm and occlude the xylem vessels of susceptible plants, which leads to significant agricultural and economic losses. Biofilms are associated with bacterial pathogenicity because they are very resistant to antibiotics and other metal-based chemicals that are used in agriculture. The X. fastidiosa YcjZ-like (XfYcjZ-like) protein belongs to the LysR-type transcriptional regulator (LTTR) family and is involved in various cellular functions that range from quorum sensing to bacterial survival. In the present study, we report the cloning, expression and purification of XfYcjZ-like, which was overexpressed in Escherichia coli. The secondary folding of the recombinant and purified protein was assessed by circular dichroism, which revealed that XfYcjZ-like contains a typical α/β fold. An initial hydrodynamic characterization showed that XfYcjZ-like is a globular tetramer in solution. In addition, using a polyclonal antibody against XfYcjZ-like, we assessed the expression profile of this protein during the different developmental phases of X. fastidiosa in in vitro cultivated biofilm cells and demonstrated that XfYcjZ-like is upregulated in planktonic cells in response to a copper shock treatment. Finally, the ability of XfYcjZ-like to interact with its own predicted promoter was confirmed in vitro, which is a typical feature of LysR. Taken together, our findings indicated that the XfYcjZ-like protein is involved in both the organization of the architecture and the maturation of the bacterial biofilm and that it is responsive to oxidative stress. PMID:25979465

  14. Directional RNA-seq reveals highly complex condition-dependent transcriptomes in E. coli K12 through accurate full-length transcripts assembling

    PubMed Central

    2013-01-01

    Background Although prokaryotic gene transcription has been studied over decades, many aspects of the process remain poorly understood. Particularly, recent studies have revealed that transcriptomes in many prokaryotes are far more complex than previously thought. Genes in an operon are often alternatively and dynamically transcribed under different conditions, and a large portion of genes and intergenic regions have antisense RNA (asRNA) and non-coding RNA (ncRNA) transcripts, respectively. Ironically, similar studies have not been conducted in the model bacterium E coli K12, thus it is unknown whether or not the bacterium possesses similar complex transcriptomes. Furthermore, although RNA-seq becomes the major method for analyzing the complexity of prokaryotic transcriptome, it is still a challenging task to accurately assemble full length transcripts using short RNA-seq reads. Results To fill these gaps, we have profiled the transcriptomes of E. coli K12 under different culture conditions and growth phases using a highly specific directional RNA-seq technique that can capture various types of transcripts in the bacterial cells, combined with a highly accurate and robust algorithm and tool TruHMM (http://bioinfolab.uncc.edu/TruHmm_package/) for assembling full length transcripts. We found that 46.9 ~ 63.4% of expressed operons were utilized in their putative alternative forms, 72.23 ~ 89.54% genes had putative asRNA transcripts and 51.37 ~ 72.74% intergenic regions had putative ncRNA transcripts under different culture conditions and growth phases. Conclusions As has been demonstrated in many other prokaryotes, E. coli K12 also has a highly complex and dynamic transcriptomes under different culture conditions and growth phases. Such complex and dynamic transcriptomes might play important roles in the physiology of the bacterium. TruHMM is a highly accurate and robust algorithm for assembling full-length transcripts in prokaryotes using directional RNA

  15. Transcriptional Regulation of the Outer Membrane Porin Gene ompW Reveals its Physiological Role during the Transition from the Aerobic to the Anaerobic Lifestyle of Escherichia coli.

    PubMed

    Xiao, Minfeng; Lai, Yong; Sun, Jian; Chen, Guanhua; Yan, Aixin

    2016-01-01

    Understanding bacterial physiology relies on elucidating the regulatory mechanisms and cellular functions of those differentially expressed genes in response to environmental changes. A widespread Gram-negative bacterial outer membrane protein OmpW has been implicated in the adaptation to stresses in various species. It is recently found to be present in the regulon of the global anaerobic transcription factor FNR and ArcA in Escherichia coli. However, little is known about the physiological implications of this regulatory disposition. In this study, we demonstrate that transcription of ompW is indeed mediated by a series of global regulators involved in the anaerobiosis of E. coli. We show that FNR can both activate and repress the expression of ompW through its direct binding to two distinctive sites, -81.5 and -126.5 bp respectively, on ompW promoter. ArcA also participates in repression of ompW under anaerobic condition, but in an FNR dependent manner. Additionally, ompW is also subject to the regulation by CRP and NarL which senses the availability and types of carbon sources and respiration electron acceptors in the environment respectively, implying a role of OmpW in the carbon and energy metabolism of E. coli during its anaerobic adaptation. Molecular docking reveals that OmpW can bind fumarate, an alternative electron acceptor in anaerobic respiration, with sufficient affinity. Moreover, supplement of fumarate or succinate which belongs to the C4-dicarboxylates family of metabolite, to E. coli culture rescues OmpW-mediated colicin S4 killing. Taken together, we propose that OmpW is involved in anaerobic carbon and energy metabolism to mediate the transition from aerobic to anaerobic lifestyle in E. coli. PMID:27303386

  16. Transcriptional Regulation of the Outer Membrane Porin Gene ompW Reveals its Physiological Role during the Transition from the Aerobic to the Anaerobic Lifestyle of Escherichia coli

    PubMed Central

    Xiao, Minfeng; Lai, Yong; Sun, Jian; Chen, Guanhua; Yan, Aixin

    2016-01-01

    Understanding bacterial physiology relies on elucidating the regulatory mechanisms and cellular functions of those differentially expressed genes in response to environmental changes. A widespread Gram-negative bacterial outer membrane protein OmpW has been implicated in the adaptation to stresses in various species. It is recently found to be present in the regulon of the global anaerobic transcription factor FNR and ArcA in Escherichia coli. However, little is known about the physiological implications of this regulatory disposition. In this study, we demonstrate that transcription of ompW is indeed mediated by a series of global regulators involved in the anaerobiosis of E. coli. We show that FNR can both activate and repress the expression of ompW through its direct binding to two distinctive sites, -81.5 and -126.5 bp respectively, on ompW promoter. ArcA also participates in repression of ompW under anaerobic condition, but in an FNR dependent manner. Additionally, ompW is also subject to the regulation by CRP and NarL which senses the availability and types of carbon sources and respiration electron acceptors in the environment respectively, implying a role of OmpW in the carbon and energy metabolism of E. coli during its anaerobic adaptation. Molecular docking reveals that OmpW can bind fumarate, an alternative electron acceptor in anaerobic respiration, with sufficient affinity. Moreover, supplement of fumarate or succinate which belongs to the C4-dicarboxylates family of metabolite, to E. coli culture rescues OmpW-mediated colicin S4 killing. Taken together, we propose that OmpW is involved in anaerobic carbon and energy metabolism to mediate the transition from aerobic to anaerobic lifestyle in E. coli. PMID:27303386

  17. Identification and transcriptional analysis of the Escherichia coli htrE operon which is homologous to pap and related pilin operons.

    PubMed Central

    Raina, S; Missiakas, D; Baird, L; Kumar, S; Georgopoulos, C

    1993-01-01

    We have characterized a new Escherichia coli operon consisting of two genes, ecpD and htrE. The ecpD gene encodes a 27-kDa protein which is 40% identical at the amino acid level to the pilin chaperone PapD family of proteins. Immediately downstream of the ecpD gene is the htrE gene. The htrE gene encodes a polypeptide of 95 kDa which is processed to a 92-kDa mature species. The HtrE protein is 38% identical to the type II pilin porin protein PapC. The ecpD htrE operon is located at 3.3 min on the genetic map, corresponding to the region from kbp 153 to 157 of the E. coli physical map. The htrE gene was identified on the basis of a Tn5 insertion mutation which resulted in a temperature-sensitive growth phenotype above 43.5 degrees C. The transcription of this operon is induced with a temperature shift from 22 to 37 or 42 degrees C but not to higher temperatures, e.g., 50 degrees C. Consistent with this result, the temperature-induced transcription was shown to be independent of the rpoH gene product (sigma 32). The transcription of this operon was further shown to require functional integration host factor protein, since himA or himD mutant bacteria possessed lower levels of ecpD htrE transcripts. Among the three transcriptional start sites discovered, one, defined by the P2 promoter, was found to be under the positive regulation of the katF (rpoS) gene, which encodes a putative sigma factor required for the transcription of many growth phase-regulated genes. Images PMID:8102362

  18. Transcription of the Escherichia coli Fatty Acid Synthesis Operon fabHDG Is Directly Activated by FadR and Inhibited by ppGpp

    PubMed Central

    My, Laetitia; Rekoske, Brian; Lemke, Justin J.; Viala, Julie P.; Gourse, Richard L.

    2013-01-01

    In Escherichia coli, FadR and FabR are transcriptional regulators that control the expression of fatty acid degradation and unsaturated fatty acid synthesis genes, depending on the availability of fatty acids. In this report, we focus on the dual transcriptional regulator FadR. In the absence of fatty acids, FadR represses the transcription of fad genes required for fatty acid degradation. However, FadR is also an activator, stimulating transcription of the products of the fabA and fabB genes responsible for unsaturated fatty acid synthesis. In this study, we show that FadR directly activates another fatty acid synthesis promoter, PfabH, which transcribes the fabHDG operon, indicating that FadR is a global regulator of both fatty acid degradation and fatty acid synthesis. We also demonstrate that ppGpp and its cofactor DksA, known primarily for their role in regulation of the synthesis of the translational machinery, directly inhibit transcription from the fabH promoter. ppGpp also inhibits the fadR promoter, thereby reducing transcription activation of fabH by FadR indirectly. Our study shows that both ppGpp and FadR have direct roles in the control of fatty acid promoters, linking expression in response to both translation activity and fatty acid availability. PMID:23772072

  19. Nucleotide sequence and in vivo expression of the ilvY and ilvC genes in Escherichia coli K12. Transcription from divergent overlapping promoters.

    PubMed

    Wek, R C; Hatfield, G W

    1986-02-15

    The ilvC gene of Escherichia coli K12 encodes acetohydroxy acid isomeroreductase, the second enzyme in the parallel isoleucine-valine biosynthetic pathway. Previous data have shown that transcription of the ilvC gene is induced by the acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate, and that this substrate induction of ilvC expression is mediated by a positive activator encoded by the ilvY gene. We report here the isolation and complete nucleotide sequence of the ilvY and ilvC genes. The ilvY and ilvC genes encode polypeptides of Mr 33,200 and 54,000, respectively. In vitro transcription-translation of these gene templates results in the synthesis of gene products of these identical molecular weights. The ilvC gene is transcribed in the same direction as the genes of the adjacent ilvGMEDA operon. The ilvY gene is transcribed in a direction opposite to the ilvC and ilvGMEDA genes. The in vivo transcriptional initiation sites of the ilvY and ilvC genes have been determined by S1 nuclease protection experiments. These transcriptional initiation sites are 45 nucleotides apart, and transcription of the ilvY and ilvC genes is initiated via divergent overlapping promoters. The nucleotide sequence of the ilvY and ilvC promoters and 5'-coding regions of Salmonella typhimurium LT2 have been determined. A comparison of these sequences with E. coli K12 suggests regions important in the promotion, regulation, and translation of the ilvY and ilvC genes. A model is presented in which the ilvY-encoded activator binds to an operator site in the overlapping promoter region and reciprocally regulates the transcription of the ilvY and ilvC genes. The carboxyl-terminal amino acid sequence of threonine deaminase encoded by the ilvA gene of the ilv-GMEDA operon of E. coli K12 has been identified by homology with the previously deduced threonine deaminase amino acid sequence encoded by the ilv1 gene of Saccharomyces cerevisiae. Based on the deduced

  20. Repurposing Synechocystis PCC6803 UirS-UirR as a UV-Violet/Green Photoreversible Transcriptional Regulatory Tool in E. coli.

    PubMed

    Ramakrishnan, Prabha; Tabor, Jeffrey J

    2016-07-15

    We have previously engineered green/red and red/far red photoreversible E. coli phytochrome and cyanobacteriochrome (CBCR) two-component systems (TCSs) and utilized them to program tailor-made gene expression signals for gene circuit characterization. Here, we transport the UV-violet/green photoreversible CBCR TCS UirS-UirR from Synechocystis PCC6803 to E. coli. We demonstrate that the promoter of the small RNA csiR1, previously shown to be activated by inorganic carbon stress, is a UirS-UirR output. Additionally, in contrast to a recently proposed sequestration model, we show that the sensor histidine kinase UirS phosphorylates the response regulator UirR to activate PcsiR1 transcription in response to UV-violet light. Finally, we measure changes in UirS-UirR output minutes after a change in light input and exploit these rapid dynamics to program a challenging gene expression signal with high predictability. UirS-UirR is the first engineered transcriptional regulatory tool activated exclusively by UV-violet light, and the most blue shifted photoreversible transcriptional regulatory tool. PMID:27120220

  1. The Molybdate-Responsive Escherichia coli ModE Transcriptional Regulator Coordinates Periplasmic Nitrate Reductase (napFDAGHBC) Operon Expression with Nitrate and Molybdate Availability

    PubMed Central

    McNicholas, Paul M.; Gunsalus, Robert P.

    2002-01-01

    Expression of the Escherichia coli napFDAGHBC operon (also known as aeg46.5), which encodes the periplasmic molybdoenzyme for nitrate reduction, is increased in response to anaerobiosis and further stimulated by the addition of nitrate or to a lesser extent by nitrite to the cell culture medium. These changes are mediated by the transcription factors Fnr and NarP, respectively. Utilizing a napF-lacZ operon fusion, we demonstrate that napF gene expression is impaired in strain defective for the molybdate-responsive ModE transcription factor. This control abrogates nitrate- or nitrite-dependent induction during anaerobiosis. Gel shift and DNase I footprinting analyses establish that ModE binds to the napF promoter with an apparent Kd of about 35 nM at a position centered at −133.5 relative to the start of napF transcription. Although the ModE binding site sequence is similar to other E. coli ModE binding sites, the location is atypical, because it is not centered near the start of transcription. Introduction of point mutations in the ModE recognition site severely reduced or abolished ModE binding in vitro and conferred a modE phenotype (i.e., loss of molybdate-responsive gene expression) in vivo. In contrast, deletion of the upstream ModE region site rendered napF expression independent of modE. These findings indicate the involvement of an additional transcription factor to help coordinate nitrate- and molybdate-dependent napF expression by the Fnr, NarP, NarL, and ModE proteins. The upstream ModE regulatory site functions to override nitrate control of napF gene expression when the essential enzyme component, molybdate, is limiting in the cell environment. PMID:12029041

  2. Transcriptional Analysis of the sfa Determinant Revealing Multiple mRNA Processing Events in the Biogenesis of S Fimbriae in Pathogenic Escherichia coli

    PubMed Central

    Balsalobre, Carlos; Morschhäuser, Joachim; Jass, Jana; Hacker, Jörg; Uhlin, Bernt Eric

    2003-01-01

    Among the virulence factors present in pathogenic extraintestinal Escherichia coli strains, expression of fimbrial adhesins is necessary for attachment to the host tissues and subsequent colonization. Occurrence of the sfa determinant coding for the S fimbriae is widespread among the uropathogens and meningitis isolates. The sfa operon consists of nine genes. In the biogenesis of S fimbriae, the proteins encoded by the sfa genes are presumably required in a specific stoichiometry. In the present work we studied how differential expression of the sfa operon genes occurs. Our findings indicate that a number of endoribonucleolytic cleavages occur in the mRNA from the sfa operon, and we detected the presence of different distinct transcriptional products, including sfaBA, sfaA, sfaADE, and sfaGSH. The sfaGSH transcript represents the three distal genes of the sfa operon, which code for the minor subunits of the S fimbriae. Analysis of the proteins in S fimbriae suggested that expression of the sfaGSH transcript provides equimolar amounts of the minor subunits. Furthermore, we showed that in the generation of the major sfaA transcript, the processing included RNase E endoribonuceolytic cleavage of the precursor sfaBA transcript. We suggest that posttranscriptional mRNA processing events result in differential gene expression important to achieve the stoichiometry necessary for fimbrial adhesin biogenesis. PMID:12511509

  3. Novel two-component transmembrane transcription control: regulation of iron dicitrate transport in Escherichia coli K-12.

    PubMed Central

    Van Hove, B; Staudenmaier, H; Braun, V

    1990-01-01

    Citrate and iron have to enter only the periplasmic space in order to induce the citrate-dependent iron(III) transport system of Escherichia coli. The five transport genes fecABCDE form an operon and are transcribed from fecA to fecE. Two genes, termed fecI and fecR, that mediate induction by iron(III) dicitrate have been identified upstream of fecA. The fecI gene encodes a protein of 173 amino acids (molecular weight, 19,478); the fecR gene encodes a protein of 317 amino acids (molecular weight, 35,529). Chromosomal fecI::Mu d1 mutants were unable to grow with iron(III) dicitrate as the sole iron source and synthesized no FecA outer membrane receptor protein. Growth was restored by transformation with plasmids encoding fecI or fecI and fecR. FecA and beta-galactosidase syntheses under transcription control of the fecB gene (fecB::Mu d1) were constitutive in fecI transformants and were regulated by iron(III) dicitrate in fecI fecR transformants. The amino acid sequence of the FecI protein contains a region close to the carboxy-terminal end for which a helix-turn-helix motif is predicted, which is typical for DNA-binding regulatory proteins. The FecI protein was found in the membrane, and the FecR protein was found in the periplasmic fraction. It is proposed that the FecR protein is the sensor that recognizes iron(III) dicitrate in the periplasm. The FecI protein activates fec gene expression by binding to the fec operator region. In the absence of citrate, FecR inactivates FecI. The lack of sequence homologies to other transmembrane signaling proteins and the location of the two proteins suggest a new type of transmembrane control mechanism. Images PMID:2254251

  4. Novel two-component transmembrane transcription control: regulation of iron dicitrate transport in Escherichia coli K-12.

    PubMed

    Van Hove, B; Staudenmaier, H; Braun, V

    1990-12-01

    Citrate and iron have to enter only the periplasmic space in order to induce the citrate-dependent iron(III) transport system of Escherichia coli. The five transport genes fecABCDE form an operon and are transcribed from fecA to fecE. Two genes, termed fecI and fecR, that mediate induction by iron(III) dicitrate have been identified upstream of fecA. The fecI gene encodes a protein of 173 amino acids (molecular weight, 19,478); the fecR gene encodes a protein of 317 amino acids (molecular weight, 35,529). Chromosomal fecI::Mu d1 mutants were unable to grow with iron(III) dicitrate as the sole iron source and synthesized no FecA outer membrane receptor protein. Growth was restored by transformation with plasmids encoding fecI or fecI and fecR. FecA and beta-galactosidase syntheses under transcription control of the fecB gene (fecB::Mu d1) were constitutive in fecI transformants and were regulated by iron(III) dicitrate in fecI fecR transformants. The amino acid sequence of the FecI protein contains a region close to the carboxy-terminal end for which a helix-turn-helix motif is predicted, which is typical for DNA-binding regulatory proteins. The FecI protein was found in the membrane, and the FecR protein was found in the periplasmic fraction. It is proposed that the FecR protein is the sensor that recognizes iron(III) dicitrate in the periplasm. The FecI protein activates fec gene expression by binding to the fec operator region. In the absence of citrate, FecR inactivates FecI. The lack of sequence homologies to other transmembrane signaling proteins and the location of the two proteins suggest a new type of transmembrane control mechanism. PMID:2254251

  5. Global transcriptional analysis of Escherichia coli expressing IrrE, a regulator from Deinococcus radiodurans, in response to NaCl shock.

    PubMed

    Zhao, Peng; Zhou, Zhengfu; Zhang, Wei; Lin, Min; Chen, Ming; Wei, Gehong

    2015-04-01

    Improving the microbial tolerance to stresses is very important for bioprocesses. Our previous study showed that IrrE, a global regulator from the extremely radioresistant bacterium Deinococcus radiodurans, dramatically enhanced the multi-stress tolerance of Escherichia coli when expressed exogenously. However, the function of IrrE is still unclear. In this study, we used whole-genome microarray assays to profile the global gene expression of the IrrE-expressing E. coli strain MGE and the control strain MGT with or without salt shock. The analysis showed that IrrE expression led to many differentially expressed genes in E. coli, which were responsible for the transport and metabolism of trehalose and glycerol, nucleotide biosynthesis, carbon source utilization, amino acid utilization, and acid resistance, including many RpoS-dependent genes, e.g., the trehalose biosynthesis genes otsAB, the acid-resistance genes gadABC and uspB, the osmotic and oxidative stress response genes katE (response to DNA damage stimulus and stress) and osmBC (response to stress), and gadWX (which controls the transcription of pH-inducible genes). The intracellular content of trehalose and glycerol increased significantly in the IrrE-expressing strain after NaCl treatment for 0 and 60 min as determined by HPLC. These results indicated the possibility that IrrE regulates the global regulator RpoS. Interestingly, we found that although IrrE did not affect the level of the rpoS transcript, it enhanced the accumulation of the RpoS protein by increasing the expression of the antiadaptors, AppY, IraM and IraD, which inhibit RpoS degradation, suggesting that the accumulation of RpoS due to IrrE regulation is an important way to improve tolerance to salt and other stresses in E. coli. PMID:25703007

  6. Characterization of the Escherichia coli AF/R1 pilus operon: novel genes necessary for transcriptional regulation and for pilus-mediated adherence.

    PubMed

    Cantey, J R; Blake, R K; Williford, J R; Moseley, S L

    1999-05-01

    We isolated the genetic determinant of AF/R1 pilus production in attaching/effacing Escherichia coli RDEC-1 and identified seven genes required for pilus expression and function. DNA sequence analysis of the structural subunit gene afrA corrected an error in the published sequence and extended homology with the F18 pilus subunit of pig edema E. coli strains. AfrB and AfrC, encoded downstream from AfrA, were required for pilus expression. AfrB was related to the usher protein PefC of Salmonella typhimurium plasmid-encoded fimbriae, and AfrC was related to PefD, a chaperone protein. AfrD and AfrE, encoded downstream from AfrC, were not necessary for the expression of AF/R1 pili but were required for ileal adherence as assayed by ileal brush border aggregation. Thus, the adhesive subunit of the AF/R1 pilus is distinct from the structural subunit, as is the case for Pap pili and type 1 pili. AfrD was related to FedE of the F18 fimbrial operon of the E. coli strain that causes edema disease in pigs. AfrE was a novel protein. AfrR and AfrS are encoded upstream from AfrA, in the opposite orientation. AfrR is related to the AraC family of transcriptional regulators, and AfrR and AfrS interact to function in a novel mode of transcriptional activation of afrA. AF/R1 pili mediate the adherence to Peyer's patch M cells, ileal mucosa, and colonic mucosa in a rabbit model of diarrhea caused by enteropathogenic E. coli. Our observations will facilitate the further study of the phenomena of M-cell adherence. PMID:10225886

  7. Characterization of the Escherichia coli AF/R1 Pilus Operon: Novel Genes Necessary for Transcriptional Regulation and for Pilus-Mediated Adherence

    PubMed Central

    Cantey, J. Robert; Blake, R. K.; Williford, J. R.; Moseley, Steve L.

    1999-01-01

    We isolated the genetic determinant of AF/R1 pilus production in attaching/effacing Escherichia coli RDEC-1 and identified seven genes required for pilus expression and function. DNA sequence analysis of the structural subunit gene afrA corrected an error in the published sequence and extended homology with the F18 pilus subunit of pig edema E. coli strains. AfrB and AfrC, encoded downstream from AfrA, were required for pilus expression. AfrB was related to the usher protein PefC of Salmonella typhimurium plasmid-encoded fimbriae, and AfrC was related to PefD, a chaperone protein. AfrD and AfrE, encoded downstream from AfrC, were not necessary for the expression of AF/R1 pili but were required for ileal adherence as assayed by ileal brush border aggregation. Thus, the adhesive subunit of the AF/R1 pilus is distinct from the structural subunit, as is the case for Pap pili and type 1 pili. AfrD was related to FedE of the F18 fimbrial operon of the E. coli strain that causes edema disease in pigs. AfrE was a novel protein. AfrR and AfrS are encoded upstream from AfrA, in the opposite orientation. AfrR is related to the AraC family of transcriptional regulators, and AfrR and AfrS interact to function in a novel mode of transcriptional activation of afrA. AF/R1 pili mediate the adherence to Peyer’s patch M cells, ileal mucosa, and colonic mucosa in a rabbit model of diarrhea caused by enteropathogenic E. coli. Our observations will facilitate the further study of the phenomena of M-cell adherence. PMID:10225886

  8. RegulonDB version 7.0: transcriptional regulation of Escherichia coli K-12 integrated within genetic sensory response units (Gensor Units)

    PubMed Central

    Gama-Castro, Socorro; Salgado, Heladia; Peralta-Gil, Martin; Santos-Zavaleta, Alberto; Muñiz-Rascado, Luis; Solano-Lira, Hilda; Jimenez-Jacinto, Verónica; Weiss, Verena; García-Sotelo, Jair S.; López-Fuentes, Alejandra; Porrón-Sotelo, Liliana; Alquicira-Hernández, Shirley; Medina-Rivera, Alejandra; Martínez-Flores, Irma; Alquicira-Hernández, Kevin; Martínez-Adame, Ruth; Bonavides-Martínez, César; Miranda-Ríos, Juan; Huerta, Araceli M.; Mendoza-Vargas, Alfredo; Collado-Torres, Leonardo; Taboada, Blanca; Vega-Alvarado, Leticia; Olvera, Maricela; Olvera, Leticia; Grande, Ricardo; Morett, Enrique; Collado-Vides, Julio

    2011-01-01

    RegulonDB (http://regulondb.ccg.unam.mx/) is the primary reference database of the best-known regulatory network of any free-living organism, that of Escherichia coli K-12. The major conceptual change since 3 years ago is an expanded biological context so that transcriptional regulation is now part of a unit that initiates with the signal and continues with the signal transduction to the core of regulation, modifying expression of the affected target genes responsible for the response. We call these genetic sensory response units, or Gensor Units. We have initiated their high-level curation, with graphic maps and superreactions with links to other databases. Additional connectivity uses expandable submaps. RegulonDB has summaries for every transcription factor (TF) and TF-binding sites with internal symmetry. Several DNA-binding motifs and their sizes have been redefined and relocated. In addition to data from the literature, we have incorporated our own information on transcription start sites (TSSs) and transcriptional units (TUs), obtained by using high-throughput whole-genome sequencing technologies. A new portable drawing tool for genomic features is also now available, as well as new ways to download the data, including web services, files for several relational database manager systems and text files including BioPAX format. PMID:21051347

  9. Time-dependent Effects of Transcription- and Translation-halting Drugs on the Spatial Distributions of the E. coli Chromosome and Ribosomes

    PubMed Central

    Bakshi, Somenath; Choi, Heejun; Mondal, Jagannath; Weisshaar, James C.

    2014-01-01

    Summary Previously observed effects of rifampicin and chloramphenicol indicate that transcription and translation activity strongly affect the coarse spatial organization of the bacterial cytoplasm. Single-cell, time-resolved, quantitative imaging of chromosome and ribosome spatial distributions and ribosome diffusion in live E. coli provides insight into the underlying mechanisms. Monte Carlo simulations of model DNA-ribosome mixtures support a novel nucleoid-ribosome mixing hypothesis. In normal conditions, 70S-polysomes and the chromosomal DNA segregate, while 30S and 50S ribosomal subunits are able to penetrate the nucleoids. Growth conditions and drug treatments determine the partitioning of ribosomes into 70S-polysomes vs free 30S and 50S subunits. Entropic and excluded volume effects then dictate the resulting chromosome and ribosome spatial distributions. Direct observation of radial contraction of the nucleoids 0-5 min after treatment with either transcription- or translation-halting drugs supports the hypothesis that simultaneous transcription, translation, and insertion of proteins into the membrane (“transertion”) exerts an expanding force on the chromosomal DNA. Breaking of the DNA-RNA polymerase-mRNA-ribosome-membrane chain in either of two ways causes similar nucleoid contraction on a similar timescale. We suggest that chromosomal expansion due to transertion enables co-transcriptional translation throughout the nucleoids. PMID:25250841

  10. The Escherichia coli FIS protein is not required for the activation of tyrT transcription on entry into exponential growth.

    PubMed Central

    Lazarus, L R; Travers, A A

    1993-01-01

    The Escherichia coli DNA bending protein factor for inversion stimulation (FIS), is neither necessary nor responsible for the stimulation of transcription from the wild type promoter for the tyrT operon (encoding a species of tyrosine tRNA) that occurs upon resumption of exponential growth. This conclusion is unexpected given that the regulatory element required for optimal transcription of tyrT contains three binding sites for FIS protein. In addition, it is in apparent conflict with reports from other laboratories which have described FIS-dependent activation of the stable RNA promoters rrnB P1 and thrU(tufB) in vivo. However, tyrT transcription is stimulated in a FIS-dependent manner both in vivo and in vitro when promoter function is impaired by mutation of the promoter itself or by the addition of the polymerase effector guanosine 5'-diphosphate 3'-diphosphate. These conditions, which expose a requirement for activation of stable RNA synthesis by FIS, suggest that FIS serves an adaptive role permitting high levels of stable RNA transcription on nutritional shift-up when RNA polymerase levels are depleted. In principle such a mechanism could confer a significant selective advantage thus accounting for the conservation of FIS binding sites in the regulatory regions of stable RNA promoters. Images PMID:7685276

  11. Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32.

    PubMed Central

    Tomoyasu, T; Gamer, J; Bukau, B; Kanemori, M; Mori, H; Rutman, A J; Oppenheim, A B; Yura, T; Yamanaka, K; Niki, H

    1995-01-01

    Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E. coli heat-shock response. In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli. Images PMID:7781608

  12. Molecular structure of uvrC gene of Escherichia coli: identification of DNA sequences required for transcription of the uvrC gene.

    PubMed Central

    Sharma, S; Dowhan, W; Moses, R E

    1982-01-01

    We have carried out experiments to identify the regulatory regions of the uvrC gene of Escherichia coli. A uvrC+ plasmid, pUV7, containing the intact transcriptional unit for the uvrC gene, was used to subclone either the structural gene or combinations of the structural gene and 5'-flanking sequences. The plasmids so constructed were tested for ability to restore UV-resistant phenotype to uvrC- cells as an indication of expression of the uvrC gene. The chromosomal DNA in plasmid pUV7 was probed for strong binding with E. coli RNA polymerase in an attempt to identify a restriction fragment which bears the regulatory sequences for the uvrC transcriptional unit. The results indicate that DNA sequences at least 0.9 Kb upstream from the structural gene, but not the 5'-proximal sequences, regulate expression of the uvrC gene. Analysis of protein synthesis encoded by plasmid pUV7 and its derivatives suggest that there may be another gene that lies between the promoter and the uvrC gene and codes for a 27,000-Mr protein. The relation of this gene to uvrC function is not clear. Images PMID:6292835

  13. Genome-Scale Reconstruction of Escherichia coli's Transcriptional and Translational Machinery: A Knowledge Base, Its Mathematical Formulation, and Its Functional Characterization

    PubMed Central

    Thiele, Ines; Jamshidi, Neema; Fleming, Ronan M. T.; Palsson, Bernhard Ø.

    2009-01-01

    Metabolic network reconstructions represent valuable scaffolds for ‘-omics’ data integration and are used to computationally interrogate network properties. However, they do not explicitly account for the synthesis of macromolecules (i.e., proteins and RNA). Here, we present the first genome-scale, fine-grained reconstruction of Escherichia coli's transcriptional and translational machinery, which produces 423 functional gene products in a sequence-specific manner and accounts for all necessary chemical transformations. Legacy data from over 500 publications and three databases were reviewed, and many pathways were considered, including stable RNA maturation and modification, protein complex formation, and iron–sulfur cluster biogenesis. This reconstruction represents the most comprehensive knowledge base for these important cellular functions in E. coli and is unique in its scope. Furthermore, it was converted into a mathematical model and used to: (1) quantitatively integrate gene expression data as reaction constraints and (2) compute functional network states, which were compared to reported experimental data. For example, the model predicted accurately the ribosome production, without any parameterization. Also, in silico rRNA operon deletion suggested that a high RNA polymerase density on the remaining rRNA operons is needed to reproduce the reported experimental ribosome numbers. Moreover, functional protein modules were determined, and many were found to contain gene products from multiple subsystems, highlighting the functional interaction of these proteins. This genome-scale reconstruction of E. coli's transcriptional and translational machinery presents a milestone in systems biology because it will enable quantitative integration of ‘-omics’ datasets and thus the study of the mechanistic principles underlying the genotype–phenotype relationship. PMID:19282977

  14. Transcriptional analysis of the Escherichia coli ColV-Ia plasmid pS88 during growth in human serum and urine

    PubMed Central

    2012-01-01

    Background The sequenced O45:K1:H7 Escherichia coli meningitis strain S88 harbors a large virulence plasmid. To identify possible genetic determinants of pS88 virulence, we examined the transcriptomes of 88 plasmidic ORFs corresponding to known and putative virulence genes, and 35 ORFs of unknown function. Results Quantification of plasmidic transcripts was obtained by quantitative real-time reverse transcription of extracted RNA, normalized on three housekeeping genes. The transcriptome of E. coli strain S88 grown in human serum and urine ex vivo were compared to that obtained during growth in Luria Bertani broth, with and without iron depletion. We also analyzed the transcriptome of a pS88-like plasmid recovered from a neonate with urinary tract infection. The transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted LB broth. Genes encoding iron acquisition systems were strongly upregulated. ShiF and ORF 123, two ORFs encoding protein with hypothetical function and physically linked to aerobactin and salmochelin loci, respectively, were also highly expressed in iron-depleted conditions and may correspond to ancillary iron acquisition genes. Four ORFs were induced ex vivo, independently of the iron concentration. Other putative virulence genes such as iss, etsC, ompTp and hlyF were not upregulated in any of the conditions studied. Transcriptome analysis of the pS88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels. Conclusion We identify new pS88 genes potentially involved in the growth of E. coli meningitis strain S88 in human serum and urine. PMID:22720670

  15. Crystallization and preliminary crystallographic analysis of the transcriptional regulator RfaH from Escherichia coli and its complex with ops DNA

    SciTech Connect

    Vassylyeva, Marina N.; Svetlov, Vladimir; Klyuyev, Sergiy; Devedjiev, Yancho D.; Artsimovitch, Irina; Vassylyev, Dmitry G.

    2006-10-01

    The E. coli transcriptional regulator RfaH was cloned, expressed, purified and crystallized and the complex of RfaH with its target DNA oligonucleotide was cocrystallized. Complete diffraction data sets were collected for the apo protein and its nucleic acid complex at 2.4 and at 1.6 Å resolution, respectively. The bacterial transcriptional factor and virulence regulator RfaH binds to rapidly moving transcription elongation complexes through specific interactions with the exposed segment of the non-template DNA strand. To elucidate this unusual mechanism of recruitment, determination of the three-dimensional structure of RfaH and its complex with DNA was initiated. To this end, the Escherichia coli rfaH gene was cloned and expressed. The purified protein was crystallized by the sitting-drop vapor-diffusion technique. The space group was P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 45.46, c = 599.93 Å. A complex of RfaH and a nine-nucleotide oligodeoxyribonucleotide was crystallized by the same technique, but under different crystallization conditions, yielding crystals that belonged to space group P1 (unit-cell parameters a = 36.79, b = 44.01, c = 62.37 Å, α = 80.62, β = 75.37, γ = 75.41°). Complete diffraction data sets were collected for RfaH and its complex with DNA at 2.4 and 1.6 Å resolution, respectively. Crystals of selenomethionine-labeled proteins in both crystal forms were obtained by cross-microseeding using the native microcrystals. The structure determination of RfaH and its complex with DNA is in progress.

  16. Negative transcriptional regulation of a positive regulator: the expression of malT, encoding the transcriptional activator of the maltose regulon of Escherichia coli, is negatively controlled by Mlc.

    PubMed

    Decker, K; Plumbridge, J; Boos, W

    1998-01-01

    The maltose regulon consists of 10 genes encoding a multicomponent and binding protein-dependent ABC transporter for maltose and maltodextrins as well as enzymes necessary for the degradation of these sugars. MalT, the transcriptional activator of the system, is necessary for the transcription of all mal genes. MalK, the energy-transducing subunit of the transport system, acts phenotypically as repressor, particularly when overproduced. We isolated an insertion mutation that strongly reduced the repressing effect of overproduced MalK. The affected gene was sequenced and identified as mlc, a known gene encoding a protein of unknown function with homology to the Escherichia coli NagC protein. The loss of Mlc function led to a threefold increase in malT expression, and the presence of mlc on a multicopy plasmid reduced malT expression. By DNasel protection assay, we found that Mlc protected a DNA region comprising positions +1 to +23 of the malT transcriptional start point. Using a mlc-lacZ fusion in a mlc and mlc+ background, we found that Mlc represses its own expression. As Mlc also regulates another operon (manXYZ, see pages 369-379 of this issue), it may very well constitute a new global regulator of carbohydrate utilization. PMID:9484893

  17. Insights into transcription termination of Hfq-binding sRNAs of Escherichia coli and characterization of readthrough products

    PubMed Central

    Morita, Teppei; Ueda, Masaki; Kubo, Kento; Aiba, Hiroji

    2015-01-01

    The genes encoding Hfq-dependent sRNAs possess a typical Rho-independent transcription terminator. Here, we have studied the molecular events occurring at Rho-independent terminators of sRNA genes, focusing on two well-characterized Hfq-binding sRNAs, SgrS and RyhB. We constructed several hybrid genes in which the DNA sequence corresponding to a strong Rho-independent terminator was placed just downstream from the Rho-independent terminators of sRNA genes. By using this system, we demonstrate that transcripts frequently read through the Rho-independent terminators of sgrS and ryhB in normally growing cells. We show that Hfq does not affect the transcriptional readthrough event itself. We also find that the readthrough products no longer bind to Hfq in vivo. We have developed a competition assay based on a biotin–streptavidin system to analyze the interaction of Hfq and a particular RNA molecule in vitro. By using this method, we verify that the 3′-extended form of SgrS does not bind to Hfq in vitro. Finally, we demonstrate that transcription termination is significantly enhanced under stress conditions where transcription initiation of sRNA genes on the chromosome is induced. We conclude that the production of sRNAs is regulated not only at the step of transcription initiation but also at the step of transcription termination. The mechanism by which transcription termination is enhanced under stress conditions remains to be understood. PMID:26106215

  18. Insights into the architecture and stoichiometry of Escherichia coli PepA*DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy.

    PubMed

    Minh, Phu Nguyen Le; Devroede, Neel; Massant, Jan; Maes, Dominique; Charlier, Daniel

    2009-04-01

    Multifunctional Aminopeptidase A (PepA) from Escherichia coli is involved in the control of two distinct DNA transaction processes: transcriptional repression of the carAB operon, encoding carbamoyl phosphate synthase and site-specific resolution of ColE1-type plasmid multimers. Both processes require communication at a distance along a DNA molecule and PepA is the major structural component of the nucleoprotein complexes that underlie this communication. Atomic Force Microscopy was used to analyze the architecture of PepA.carAB and PepA.cer site complexes. Contour length measurements, bending angle analyses and volume determinations demonstrate that the carP1 operator is foreshortened by approximately 235 bp through wrapping around one PepA hexamer. The highly deformed part of the operator extends from slightly upstream of the -35 hexamer of the carP1 promoter to just downstream of the IHF-binding site, and comprises the binding sites for the PurR and RutR transcriptional regulators. This extreme remodeling of the carP1 control region provides a straightforward explanation for the strict requirement of PepA in the establishment of pyrimidine and purine-specific repression of carAB transcription. We further provide a direct physical proof that PepA is able to synapse two cer sites in direct repeat in a large interwrapped nucleoprotein complex, likely comprising two PepA hexamers. PMID:19136463

  19. Insights into the architecture and stoichiometry of Escherichia coli PepA•DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy

    PubMed Central

    Minh, Phu Nguyen Le; Devroede, Neel; Massant, Jan; Maes, Dominique; Charlier, Daniel

    2009-01-01

    Multifunctional Aminopeptidase A (PepA) from Escherichia coli is involved in the control of two distinct DNA transaction processes: transcriptional repression of the carAB operon, encoding carbamoyl phosphate synthase and site-specific resolution of ColE1-type plasmid multimers. Both processes require communication at a distance along a DNA molecule and PepA is the major structural component of the nucleoprotein complexes that underlie this communication. Atomic Force Microscopy was used to analyze the architecture of PepA·carAB and PepA·cer site complexes. Contour length measurements, bending angle analyses and volume determinations demonstrate that the carP1 operator is foreshortened by ∼235 bp through wrapping around one PepA hexamer. The highly deformed part of the operator extends from slightly upstream of the –35 hexamer of the carP1 promoter to just downstream of the IHF-binding site, and comprises the binding sites for the PurR and RutR transcriptional regulators. This extreme remodeling of the carP1 control region provides a straightforward explanation for the strict requirement of PepA in the establishment of pyrimidine and purine-specific repression of carAB transcription. We further provide a direct physical proof that PepA is able to synapse two cer sites in direct repeat in a large interwrapped nucleoprotein complex, likely comprising two PepA hexamers. PMID:19136463

  20. An Interplay among FIS, H-NS, and Guanosine Tetraphosphate Modulates Transcription of the Escherichia coli cspA Gene under Physiological Growth Conditions.

    PubMed

    Brandi, Anna; Giangrossi, Mara; Giuliodori, Anna M; Falconi, Maurizio

    2016-01-01

    CspA, the most characterized member of the csp gene family of Escherichia coli, is highly expressed not only in response to cold stress, but also during the early phase of growth at 37°C. Here, we investigate at molecular level the antagonistic role played by the nucleoid proteins FIS and H-NS in the regulation of cspA expression under non-stress conditions. By means of both probing experiments and immunological detection, we demonstrate in vitro the existence of binding sites for these proteins on the cspA regulatory region, in which FIS and H-NS bind simultaneously to form composite DNA-protein complexes. While the in vitro promoter activity of cspA is stimulated by FIS and repressed by H-NS, a compensatory effect is observed when both proteins are added in the transcription assay. Consistently with these findings, inactivation of fis and hns genes reversely affect the in vivo amount of cspA mRNA. In addition, by means of strains expressing a high level of the alarmone guanosine tetraphosphate ((p)ppGpp) and in vitro transcription assays, we show that the cspA promoter is sensitive to (p)ppGpp inhibition. The (p)ppGpp-mediated expression of fis and hns genes is also analyzed, thus clarifying some aspects of the regulatory loop governing cspA transcription. PMID:27252944

  1. An Interplay among FIS, H-NS, and Guanosine Tetraphosphate Modulates Transcription of the Escherichia coli cspA Gene under Physiological Growth Conditions

    PubMed Central

    Brandi, Anna; Giangrossi, Mara; Giuliodori, Anna M.; Falconi, Maurizio

    2016-01-01

    CspA, the most characterized member of the csp gene family of Escherichia coli, is highly expressed not only in response to cold stress, but also during the early phase of growth at 37°C. Here, we investigate at molecular level the antagonistic role played by the nucleoid proteins FIS and H-NS in the regulation of cspA expression under non-stress conditions. By means of both probing experiments and immunological detection, we demonstrate in vitro the existence of binding sites for these proteins on the cspA regulatory region, in which FIS and H-NS bind simultaneously to form composite DNA-protein complexes. While the in vitro promoter activity of cspA is stimulated by FIS and repressed by H-NS, a compensatory effect is observed when both proteins are added in the transcription assay. Consistently with these findings, inactivation of fis and hns genes reversely affect the in vivo amount of cspA mRNA. In addition, by means of strains expressing a high level of the alarmone guanosine tetraphosphate ((p)ppGpp) and in vitro transcription assays, we show that the cspA promoter is sensitive to (p)ppGpp inhibition. The (p)ppGpp-mediated expression of fis and hns genes is also analyzed, thus clarifying some aspects of the regulatory loop governing cspA transcription. PMID:27252944

  2. Genome-wide Reconstruction of OxyR and SoxRS Transcriptional Regulatory Networks under Oxidative Stress in Escherichia coli K-12 MG1655.

    PubMed

    Seo, Sang Woo; Kim, Donghyuk; Szubin, Richard; Palsson, Bernhard O

    2015-08-25

    Three transcription factors (TFs), OxyR, SoxR, and SoxS, play a critical role in transcriptional regulation of the defense system for oxidative stress in bacteria. However, their full genome-wide regulatory potential is unknown. Here, we perform a genome-scale reconstruction of the OxyR, SoxR, and SoxS regulons in Escherichia coli K-12 MG1655. Integrative data analysis reveals that a total of 68 genes in 51 transcription units (TUs) belong to these regulons. Among them, 48 genes showed more than 2-fold changes in expression level under single-TF-knockout conditions. This reconstruction expands the genome-wide roles of these factors to include direct activation of genes related to amino acid biosynthesis (methionine and aromatic amino acids), cell wall synthesis (lipid A biosynthesis and peptidoglycan growth), and divalent metal ion transport (Mn(2+), Zn(2+), and Mg(2+)). Investigating the co-regulation of these genes with other stress-response TFs reveals that they are independently regulated by stress-specific TFs. PMID:26279566

  3. Imprecise transcription termination within Escherichia coli greA leader gives rise to an array of short transcripts, GraL

    PubMed Central

    Potrykus, Katarzyna; Murphy, Helen; Chen, Xiongfong; Epstein, Jonathan A.; Cashel, Michael

    2010-01-01

    We report that greA expression is driven by two strong, overlapping P1 and P2 promoters. The P1 promoter is σ70-dependent and P2 is σE-dependent. Two-thirds of transcripts terminate within the leader region and the remaining third comprises greA mRNA. Termination efficiency seems to be unaffected by growth phase. Two collections of small 40–50 (initiating from P2) and 50–60 nt (from P1) RNA chains, termed GraL, are demonstrable in vivo and in vitro. We document that GraL arrays arise from an intrinsic terminator with an 11 bp stem followed by an AU7GCU2 sequence. Atypical chain termination occurs at multiple sites; the 3′-ends differ by 1 nt over a range of 10 nt. Transcripts observed are shown to be insensitive to Gre factors and physically released from RNAP–DNA complexes. The abundance of individual chains within each cluster displays a characteristic pattern, which can be differentially altered by oligonucleotide probes. Multiple termination sites are particularly sensitive to changes at the bottom of the stem. Evolutionarily conserved GraL stem structures and fitness assays suggest a biological function for the RNA clusters themselves. Although GraL overexpression induces ≥3-fold transcriptional changes of over 100 genes, a direct target remains elusive. PMID:20008510

  4. Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter.

    PubMed

    Donovan, R S; Robinson, C W; Glick, B R

    2000-06-01

    The expression of a monoclonal antibody Fab fragment in Escherichia coli strain RB791/pComb3, induced with either lactose or isopropyl-beta-D-thiogalactoside (IPTG), was compared to determine if lactose might provide an inexpensive alternative to induction with IPTG. Induction of Fab expression imposed a metabolic load on the recombinant cells, resulting in lower final cell yields compared to the non-induced controls. An IPTG concentration of 0.05 mM was sufficient to achieve maximal expression of soluble Fab protein when inducing in the early-, mid-, or late-log phases of batch cultures grown using either glucose or glycerol as a carbon source. The largest overall yield of Fab fragments when using 0.05 mM IPTG was achieved by increasing the final yield of cells through glycerol feeding following induction in late-log phase. Lactose was as effective as IPTG for inducing Fab expression in E. coli RB791/pComb3. The greatest overall level of Fab expression was found when cells grown on glycerol were induced with 2 g/L lactose in late-log phase. Since the cost of 0.05 mM of IPTG is significantly greater than the cost of 2 g/L lactose, lactose provides an inexpensive alternative to IPTG for inducing the expression of Fab fragments, and possibly other recombinant proteins, from the E. coli lac promoter. PMID:10913975

  5. Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator.

    PubMed Central

    Song, S; Park, C

    1997-01-01

    The metabolism of D-xylose in Escherichia coli K-12 is known to be mediated by the xylAB gene. However, the nearby xylFGHR genes were found by genome sequencing and predicted to be responsible for transport and regulation for xylose based on their sequence similarities to other functionally related genes. Here, we investigated transcriptional organization and functions of the xyl genes. An analysis with random transposon insertions revealed that the xyl genes are organized into two major transcriptional units, xylAB and xylFGHR, governed by the promoters PA and PF, respectively. However, there is an additional weak promoter, PR, which is specific for xylR. Sites of transcription initiation were determined by primer extension analysis. When studied with operon fusions to lacZ, the PA and PF promoters were activated by D-xylose and repressed by glucose. In contrast, the PR promoter was not regulated by these sugars. A mutation in xylR completely abolished expression from the PA and PF promoters, causing a defect in both growth and transport. Binding of XylR to the xyl promoter was enhanced by the presence of D-xylose, suggesting that transcription was positively regulated by XylR. In vivo footprinting analysis revealed that XylR binds to at least two DNA regions, IA and IF, each with a direct repeat. It is very likely that XylR interacts with IA and IF as a dimer. The presumed binding sites are located just upstream of the promoter consensus sequences (-35), while IA is additionally flanked by a cyclic AMP receptor protein-binding site on the other side. The proposed structure of xyl promoters is consistent with the regulation of xyl gene expression and with phenotypes of transposon insertions obtained in the promoter regions. PMID:9371449

  6. Transcription factor CecR (YbiH) regulates a set of genes affecting the sensitivity of Escherichia coli against cefoperazone and chloramphenicol.

    PubMed

    Yamanaka, Yuki; Shimada, Tomohiro; Yamamoto, Kaneyoshi; Ishihama, Akira

    2016-07-01

    Genomic SELEX (systematic evolution of ligands by exponential enrichment) screening was performed for identification of the binding site of YbiH, an as yet uncharacterized TetR-family transcription factor, on the Escherichia coli genome. YbiH was found to be a unique single-target regulator that binds in vitro within the intergenic spacer located between the divergently transcribed ybiH-ybhGFSR and rhlE operons. YbhG is an inner membrane protein and YbhFSR forms a membrane-associated ATP-binding cassette (ABC) transporter while RhlE is a ribosome-associated RNA helicase. Gel shift assay and DNase footprinting analyses indicated one clear binding site of YbiH, including a complete palindromic sequence of AATTAGTT-AACTAATT. An in vivo reporter assay indicated repression of the ybiH operon and activation of the rhlE operon by YbiH. After phenotype microarray screening, YbiH was indicated to confer resistance to chloramphenicol and cefazoline (a first-generation cephalosporin). A systematic survey of the participation of each of the predicted YbiH-regulated genes in the antibiotic sensitivity indicated involvement of the YbhFSR ABC-type transporter in the sensitivity to cefoperazone (a third-generation cephalosporin) and of the membrane protein YbhG in the control of sensitivity to chloramphenicol. Taken together with the growth test in the presence of these two antibiotics and in vitro transcription assay, it was concluded that the hitherto uncharacterized YbiH regulates transcription of both the bidirectional transcription units, the ybiH-ybhGFSR operon and the rhlE gene, which altogether are involved in the control of sensitivity to cefoperazone and chloramphenicol. We thus propose to rename YbiH as CecR (regulator of cefoperazone and chloramphenicol sensitivity). PMID:27112147

  7. Pho regulon promoter-mediated transcription of the key pathway gene aroGFbr improves the performance of an L-phenylalanine-producing Escherichia coli strain.

    PubMed

    Doroshenko, Vera G; Tsyrenzhapova, Irina S; Krylov, Alexander A; Kiseleva, Evgeniya M; Ermishev, Vladimir Yu; Kazakova, Svetlana M; Biryukova, Irina V; Mashko, Sergey V

    2010-12-01

    DAHP synthase (EC 4.1.2.15) is one of the key enzymes involved in aromatic amino acid biosynthesis in Escherichia coli. An approximately twofold decrease in DAHP synthase activity level was detected in the late growth phase of the L-phenylalanine (Phe)-producing E. coli strain, in which this enzyme encoded by aroG4 is resistant to feedback inhibition. An additional copy of aroG4 that is controlled by promoters of E. coli phoA or pstS genes was integrated into the chromosome of the Phe producer. The choice of promoter was based on the detected activation of the Pho regulon that occurs in response to the depletion of soluble inorganic orthophosphate (P(i)) in the medium, provided that the optical density of the Phe-producing culture did not exceed 70% of its maximum value. Pho-mediated aroG4 transcription increased both the accumulation of Phe and the level of DAHP synthase activity in the late stage of batch cultivation on glucose in P(i)-limited conditions. Disruption of rpoS led to the improved performance of a P(phoA)-aroG4 strain. The pstS promoter that is recognized by the σ(70)/σ(S)-associated core RNA polymerase resulted in the stable maintenance of DAHP synthase activity during long-drawn fed-batch cultivation of the RpoS(+) strain carrying the P(pstS)-aroG4. PMID:20730534

  8. CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli.

    PubMed

    Cress, Brady F; Toparlak, Ö Duhan; Guleria, Sanjay; Lebovich, Matthew; Stieglitz, Jessica T; Englaender, Jacob A; Jones, J Andrew; Linhardt, Robert J; Koffas, Mattheos A G

    2015-09-18

    Programmable control over an addressable global regulator would enable simultaneous repression of multiple genes and would have tremendous impact on the field of synthetic biology. It has recently been established that CRISPR/Cas systems can be engineered to repress gene transcription at nearly any desired location in a sequence-specific manner, but there remain only a handful of applications described to date. In this work, we report development of a vector possessing a CRISPathBrick feature, enabling rapid modular assembly of natural type II-A CRISPR arrays capable of simultaneously repressing multiple target genes in Escherichia coli. Iterative incorporation of spacers into this CRISPathBrick feature facilitates the combinatorial construction of arrays, from a small number of DNA parts, which can be utilized to generate a suite of complex phenotypes corresponding to an encoded genetic program. We show that CRISPathBrick can be used to tune expression of plasmid-based genes and repress chromosomal targets in probiotic, virulent, and commonly engineered E. coli strains. Furthermore, we describe development of pCRISPReporter, a fluorescent reporter plasmid utilized to quantify dCas9-mediated repression from endogenous promoters. Finally, we demonstrate that dCas9-mediated repression can be harnessed to assess the effect of downregulating both novel and computationally predicted metabolic engineering targets, improving the yield of a heterologous phytochemical through repression of endogenous genes. These tools provide a platform for rapid evaluation of multiplex metabolic engineering interventions. PMID:25822415

  9. Evaluation of the impact of quorum sensing transcriptional regulator SdiA on long-term persistence and fecal shedding of Escherichia coli O157:H7 in weaned calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quorum sensing transcriptional regulator SdiA has been shown to enhance the survival of Escherichia coli O157:H7 (O157) in the acidic compartment of bovine rumen in response to N-acyl-L-homoserine lactones (AHLs) produced by the rumen bacteria. Bacteria that survive the rumen environment subsequentl...

  10. Regulation of purE transcription in a purE::lac fusion strain of Escherichia coli.

    PubMed Central

    Levine, R A; Taylor, M W

    1982-01-01

    A purE::lac fusion strain was isolated by using a special Mu phage developed by M. Casadaban. In the presence of adenine (100 micrograms/ml), beta-galactosidase synthesis was repressed by greater than 90%. beta-Galactosidase activity could be detected 6 to 8 min after the removal of adenine and increased linearly for at least 20 min. purR- mutants were isolated and synthesized 1.7- to 1.8-fold-higher levels of beta-galactosidase compared with purR+ cells. Azaserine derepressed purE transcription approximately 1.7-fold by lowering purine nucleotide pools. Glutamine and pyrimidine supplementation or starvation had no effect on purE transcription. A comparison of the rate of de novo purine biosynthesis and purE transcription indicated that the in vivo rate of de novo purine biosynthesis was more sensitive to the inhibitory effects of adenine than was transcription at the purE locus. PMID:7037738

  11. Transcription Elongation Factor NusA Is a General Antagonist of Rho-dependent Termination in Escherichia coli.

    PubMed

    Qayyum, M Zuhaib; Dey, Debashish; Sen, Ranjan

    2016-04-01

    NusA is an essential protein that binds to RNA polymerase and also to the nascent RNA and influences transcription by inducing pausing and facilitating the process of transcription termination/antitermination. Its participation in Rho-dependent transcription termination has been perceived, but the molecular nature of this involvement is not known. We hypothesized that, because both Rho and NusA are RNA-binding proteins and have the potential to target the same RNA, the latter is likely to influence the global pattern of the Rho-dependent termination. Analyses of the nascent RNA binding properties and consequent effects on the Rho-dependent termination functions of specific NusA-RNA binding domain mutants revealed an existence of Rho-NusA direct competition for the overlappingnut(NusA-binding site) andrut(Rho-binding site) sites on the RNA. This leads to delayed entry of Rho at therutsite that inhibits the latter's RNA release process. High density tiling microarray profiles of these NusA mutants revealed that a significant number of genes, together with transcripts from intergenic regions, are up-regulated. Interestingly, the majority of these genes were also up-regulated when the Rho function was compromised. These results provide strong evidence for the existence of NusA-binding sites in different operons that are also the targets of Rho-dependent terminations. Our data strongly argue in favor of a direct competition between NusA and Rho for the access of specific sites on the nascent transcripts in different parts of the genome. We propose that this competition enables NusA to function as a global antagonist of the Rho function, which is unlike its role as a facilitator of hairpin-dependent termination. PMID:26872975

  12. Transcription activation at Escherichia coli promoters dependent on the cyclic AMP receptor protein: effects of binding sequences for the RNA polymerase alpha-subunit.

    PubMed Central

    Savery, N J; Rhodius, V A; Wing, H J; Busby, S J

    1995-01-01

    Transcription activation at two semi-synthetic Escherichia coli promoters, CC(-41.5) and CC(-72.5), is dependent on the cyclic AMP receptor protein (CRP) that binds to sites centred 41.5 and 72.5 bp upstream from the respective transcription startpoints. An UP-element that can bind the C-terminal domain of the RNA polymerase (RNAP) alpha-subunit was cloned upstream of the DNA site for CRP at CC(-41.5) and downstream of the DNA site for CRP at CC(-72.5). In both cases CRP-dependent promoter activity was increased by the UP-element, but CRP-independent activity was not increased. DNase I footprinting was exploited to investigate the juxtaposition of bound CRP and RNAP alpha-subunits. In both cases, CRP and RNAP alpha-subunits occupy their cognate binding sites in ternary CRP-RNAP promoter complexes. RNAP alpha-subunits can occupy the UP-element in the absence of CRP, but this is not sufficient for open complex formation. The positive effects of binding RNAP alpha-subunits upstream of the DNA site for CRP at -41.5 are suppressed if the UP-element is incorrectly positioned. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7619086

  13. Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: Potential implications for methylation-independent transcriptional repression

    SciTech Connect

    Horton, John R.; Zhang, Xing; Blumenthal, Robert M.; Cheng, Xiaodong

    2015-04-06

    DNA adenine methyltransferase (Dam) is widespread and conserved among the γ-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam and the promoter-proximal presence of GATC sequences. Here, we demonstrate that EcoDam interacts with a 5-base pair non-cognate sequence distinct from GATC. The crystal structure of a non-cognate complex allowed us to identify a DNA binding element, GTYTA/TARAC (where Y = C/T and R = A/G). This element immediately flanks GATC sites in some Dam-regulated promoters, including the Pap operon which specifies pyelonephritis-associated pili. In addition, Dam interacts with near-cognate GATC sequences (i.e. 3/4-site ATC and GAT). All together, these results imply that Dam, in addition to being responsible for GATC methylation, could also function as a methylation-independent transcriptional repressor.

  14. Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: Potential implications for methylation-independent transcriptional repression

    DOE PAGESBeta

    Horton, John R.; Zhang, Xing; Blumenthal, Robert M.; Cheng, Xiaodong

    2015-04-06

    DNA adenine methyltransferase (Dam) is widespread and conserved among the γ-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam and the promoter-proximal presence of GATC sequences. Here, we demonstrate that EcoDam interacts with a 5-base pair non-cognate sequence distinct from GATC. The crystal structure of a non-cognate complex allowed us to identify amore » DNA binding element, GTYTA/TARAC (where Y = C/T and R = A/G). This element immediately flanks GATC sites in some Dam-regulated promoters, including the Pap operon which specifies pyelonephritis-associated pili. In addition, Dam interacts with near-cognate GATC sequences (i.e. 3/4-site ATC and GAT). All together, these results imply that Dam, in addition to being responsible for GATC methylation, could also function as a methylation-independent transcriptional repressor.« less

  15. A factor that positively regulates cell division by activating transcription of the major cluster of essential cell division genes of Escherichia coli.

    PubMed Central

    Wang, X D; de Boer, P A; Rothfield, L I

    1991-01-01

    Cell division in Escherichia coli requires the products of the ftsQ, ftsA and ftsZ genes. It is not known how the cell regulates the cellular concentrations of these essential elements of the division system. We describe here a factor that activates cell division by specifically increasing transcription from one of the two promoters that lie immediately upstream of the ftsQAZ gene cluster. The trans-acting factor is the product of the sdiA gene, which was isolated on the basis of its ability to suppress the division inhibitory effect of the MinC/MinD division inhibitor. In addition, the sdiA gene product suppressed the action of other chromosomally encoded division inhibitors, induced minicell formation in wild type cells, and restored division activity to an ftsZ temperature-sensitive mutant grown under nonpermissive conditions. All of these properties were explained by the ability of the sdiA gene product specifically to increase transcription of the ftsQAZ gene cluster, resulting in an increase in cellular concentration of the FtsZ protein. The sdiA gene product is the first factor thus far identified that specifically regulates expression of this key group of cell division genes. Images PMID:1915297

  16. Orientation of functional activating regions in the Escherichia coli CRP protein during transcription activation at class II promoters.

    PubMed Central

    Williams, R M; Rhodius, V A; Bell, A I; Kolb, A; Busby, S J

    1996-01-01

    At class II CRP-dependent promoters the DNA site for CRP overlaps the DNA site for RNA polymerase, covering the -35 region. Transcription activation at class II CRP- dependent promoters requires a contact between an activating region in the upstream subunit of the bound CRP dimer and a contact site in the C-terminal domain of the alpha-subunit of RNA polymerase. Transcription activation is suppressed by amino acid substitutions in the activating region, but activation can be restored by second site substitutions at K52 or E96. These substitutions identify two separate regions on the surface of CRP that appear to be able to interact with RNA polymerase specifically at class II promoters. Using the method of 'oriented heterodimers' we show that these alternative activating regions are functional in the downstream subunit of the bound CRP dimer. PMID:8604346

  17. Non-Invasive Analysis of Recombinant mRNA Stability in Escherichia coli by a Combination of Transcriptional Inducer Wash-Out and qRT-PCR

    PubMed Central

    Kucharova, Veronika; Strand, Trine Aakvik; Almaas, Eivind; Naas, Adrian E.; Brautaset, Trygve; Valla, Svein

    2013-01-01

    mRNA stability is one among many parameters that can potentially affect the level of recombinant gene expression in bacteria. Blocking of the entire prokaryotic transcription machinery by addition of rifampicin is commonly used in protocols for analysis of mRNA stability. Here we show that such treatment can be effectively replaced by a simple, non-invasive method based on removal of the relevant transcriptional inducers and that the mRNA decay can then be followed by qRT-PCR. To establish the methodology we first used the m-toluate-inducible XylS/Pm expression cassette as a model system and analyzed several examples of DNA modifications causing gene expression stimulation in Escherichia coli. The new method allowed us to clearly discriminate whether an improvement in mRNA stability contributes to observed increases in transcript amounts for each individual case. To support the experimental data a simple mathematical fitting model was developed to calculate relative decay rates. We extended the relevance of the method by demonstrating its application also for an IPTG-inducible expression cassette (LacI/Ptac) and by analyzing features of the bacteriophage T7-based expression system. The results suggest that the methodology is useful in elucidating factors controlling mRNA stability as well as other specific features of inducible expression systems. Moreover, as expression systems based on diffusible inducers are almost universally available, the concept can be most likely used to measure mRNA decay for any gene in any cell type that is heavily used in molecular biology research. PMID:23840466

  18. Cloning and functional expression in E. coli of a polyphenol oxidase transcript from Coreopsis grandiflora involved in aurone formation☆

    PubMed Central

    Kaintz, Cornelia; Molitor, Christian; Thill, Jana; Kampatsikas, Ioannis; Michael, Claudia; Halbwirth, Heidi; Rompel, Annette

    2014-01-01

    Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4-deoxyaurone formation in Asteraceae was so far unknown. Three novel full-length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80 kb (cgAUS1) and 1.85 kb (cgAUS2a, 2b), encoding for proteins of 68–69 kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9 kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS–PAGE and shot-gun type nanoUHPLC–ESI-MS/MS. PMID:25109778

  19. CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress.

    PubMed

    Holyoake, Louise V; Hunt, Stuart; Sanguinetti, Guido; Cook, Gregory M; Howard, Mark J; Rowe, Michelle L; Poole, Robert K; Shepherd, Mark

    2016-03-15

    The glutathione/cysteine exporter CydDC maintains redox balance in Escherichia coli. A cydD mutant strain was used to probe the influence of CydDC upon reduced thiol export, gene expression, metabolic perturbations, intracellular pH homoeostasis and tolerance to nitric oxide (NO). Loss of CydDC was found to decrease extracytoplasmic thiol levels, whereas overexpression diminished the cytoplasmic thiol content. Transcriptomic analysis revealed a dramatic up-regulation of protein chaperones, protein degradation (via phenylpropionate/phenylacetate catabolism), β-oxidation of fatty acids and genes involved in nitrate/nitrite reduction. (1)H NMR metabolomics revealed elevated methionine and betaine and diminished acetate and NAD(+) in cydD cells, which was consistent with the transcriptomics-based metabolic model. The growth rate and ΔpH, however, were unaffected, although the cydD strain did exhibit sensitivity to the NO-releasing compound NOC-12. These observations are consistent with the hypothesis that the loss of CydDC-mediated reductant export promotes protein misfolding, adaptations to energy metabolism and sensitivity to NO. The addition of both glutathione and cysteine to the medium was found to complement the loss of bd-type cytochrome synthesis in a cydD strain (a key component of the pleiotropic cydDC phenotype), providing the first direct evidence that CydDC substrates are able to restore the correct assembly of this respiratory oxidase. These data provide an insight into the metabolic flexibility of E. coli, highlight the importance of bacterial redox homoeostasis during nitrosative stress, and report for the first time the ability of periplasmic low molecular weight thiols to restore haem incorporation into a cytochrome complex. PMID:26699904

  20. CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress

    PubMed Central

    Holyoake, Louise V.; Hunt, Stuart; Sanguinetti, Guido; Cook, Gregory M.; Howard, Mark J.; Rowe, Michelle L.; Poole, Robert K.; Shepherd, Mark

    2015-01-01

    The glutathione/cysteine exporter CydDC maintains redox balance in Escherichia coli. A cydD mutant strain was used to probe the influence of CydDC upon reduced thiol export, gene expression, metabolic perturbations, intracellular pH homoeostasis and tolerance to nitric oxide (NO). Loss of CydDC was found to decrease extracytoplasmic thiol levels, whereas overexpression diminished the cytoplasmic thiol content. Transcriptomic analysis revealed a dramatic up-regulation of protein chaperones, protein degradation (via phenylpropionate/phenylacetate catabolism), β-oxidation of fatty acids and genes involved in nitrate/nitrite reduction. 1H NMR metabolomics revealed elevated methionine and betaine and diminished acetate and NAD+ in cydD cells, which was consistent with the transcriptomics-based metabolic model. The growth rate and ΔpH, however, were unaffected, although the cydD strain did exhibit sensitivity to the NO-releasing compound NOC-12. These observations are consistent with the hypothesis that the loss of CydDC-mediated reductant export promotes protein misfolding, adaptations to energy metabolism and sensitivity to NO. The addition of both glutathione and cysteine to the medium was found to complement the loss of bd-type cytochrome synthesis in a cydD strain (a key component of the pleiotropic cydDC phenotype), providing the first direct evidence that CydDC substrates are able to restore the correct assembly of this respiratory oxidase. These data provide an insight into the metabolic flexibility of E. coli, highlight the importance of bacterial redox homoeostasis during nitrosative stress, and report for the first time the ability of periplasmic low molecular weight thiols to restore haem incorporation into a cytochrome complex. PMID:26699904

  1. RelB and RelE of Escherichia coli Form a Tight Complex That Represses Transcription via the Ribbon–Helix–Helix Motif in RelB

    PubMed Central

    Overgaard, Martin; Borch, Jonas; Gerdes, Kenn

    2009-01-01

    RelB, the ribbon–helix–helix (RHH) repressor encoded by the relBE toxin–antitoxin locus of Escherichia coli, interacts with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced by RelE; that is, RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate, and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional control of the relBE model operon. Using an in vivo screen for relB mutants, we identified multiple nucleotide changes that map to important amino acid positions within the DNA-binding domain formed by the N-terminal RHH motif of RelB. Analysis of DNA binding of a subset of these mutant RHH proteins by gel-shift assays, transcriptional fusion assays and a structure model of RelB–DNA revealed amino acid residues making crucial DNA–backbone contacts within the operator (relO) DNA. Mutational and footprinting analyses of relO showed that RelB dimers bind on the same face of the DNA helix and that the RHH motif recognizes four 6-bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between RelB and RelE confirmed that the proteins form a high-affinity complex with a 2:1 stoichiometry. Lon degraded RelB in vitro and degradation was inhibited by RelE, consistent with the proposal that RelE protects RelB from proteolysis by Lon in vivo. PMID:19747491

  2. A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing

    PubMed Central

    Romero, David A; Hasan, Ayad H; Lin, Yu-fei; Kime, Louise; Ruiz-Larrabeiti, Olatz; Urem, Mia; Bucca, Giselda; Mamanova, Lira; Laing, Emma E; van Wezel, Gilles P; Smith, Colin P; Kaberdin, Vladimir R; McDowall, Kenneth J

    2014-01-01

    Streptomyces coelicolor is a model for studying bacteria renowned as the foremost source of natural products used clinically. Post-genomic studies have revealed complex patterns of gene expression and links to growth, morphological development and individual genes. However, the underlying regulation remains largely obscure, but undoubtedly involves steps after transcription initiation. Here we identify sites involved in RNA processing and degradation as well as transcription within a nucleotide-resolution map of the transcriptional landscape. This was achieved by combining RNA-sequencing approaches suited to the analysis of GC-rich organisms. Escherichia coli was analysed in parallel to validate the methodology and allow comparison. Previously, sites of RNA processing and degradation had not been mapped on a transcriptome-wide scale for E. coli. Through examples, we show the value of our approach and data sets. This includes the identification of new layers of transcriptional complexity associated with several key regulators of secondary metabolism and morphological development in S. coelicolor and the identification of host-encoded leaderless mRNA and rRNA processing associated with the generation of specialized ribosomes in E. coli. New regulatory small RNAs were identified for both organisms. Overall the results illustrate the diversity in mechanisms used by different bacterial groups to facilitate and regulate gene expression. PMID:25266672

  3. The Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by E. coli RcnR‡

    PubMed Central

    Higgins, Khadine A.; Hu, Heidi Q.; Chivers, Peter T.; Maroney, Michael J.

    2013-01-01

    The RcnR metalloregulator represses the transcription of the Co(II) and Ni(II) exporter, RcnAB. Previous studies have shown that Co(II) and Ni(II) bind to RcnR in six-coordinate sites, resulting in de-repression. Here, the roles of His60, His64, and His67 in specific metal recognition are examined. His60 and His64 correspond to ligands that are important for Cu(I) binding in the homologous Cu(I)-responsive metalloregulator, CsoR. These residues are known to be functionally important in RcnR transcriptional regulation. XAS was used to examine the structure of bound cognate and non-cognate metal ions, and lacZ reporter assays were used to assess the transcription of rcnA in response to metal binding in the three His → Cys mutations, H60C, H64C and H67C. These studies confirm that both Ni(II) and Co(II) use His64 as a ligand. H64C-RcnR is also the only known mutation that retains a Co(II) response while eliminating the response to Ni(II) binding. XAS data indicate that His60 and His67 are potential Co(II) ligands. The effects of the mutations of His60, His64, and His67 residues on the structures of the non-cognate metal ions (Zn(II) and Cu(I)) reveals that these residues have distinctive roles in binding non-cognate metals. None of the His → Cys mutants in RcnR confer any response to Cu(I) binding, including H64C-RcnR, where the ligands involved in Cu(I) binding in CsoR are present. These data indicate that while the secondary, tertiary and quaternary structures of CsoR and RcnR are quite similar, small changes in primary sequence reveal that the specific mechanisms involved in metal recognition are quite different. PMID:23215580

  4. Structural modeling of the ExuR and UxuR transcription factors of E. coli: search for the ligands affecting their regulatory properties.

    PubMed

    Tutukina, Maria N; Potapova, Anna V; Vlasov, Peter K; Purtov, Yuri A; Ozoline, Olga N

    2016-10-01

    Gammaproteobacteria get energy for their growth from different carbon sources using either glycolysis or alternative metabolic pathways induced in stress conditions. These metabolic switches are coordinated by complex interplay of regulatory proteins sensing concentrations of available metabolites by mechanisms yet to be understood. Here, we use two transcriptional regulators, ExuR and UxuR, controlling d-galacturonate (d-gal) and d-glucuronate metabolism in Escherichia coli, as the targets for computational search of low-molecular compounds capable to bind their ligand-binding domains. Using a flexible molecular docking, we modeled the interactions of these proteins with substrates and intermediates of glycolysis, Ashwell and Entner-Doudoroff pathways. For UxuR, the two preferred sites of ligand binding were found: one is located within the C-terminal domain, while another occupies the interdomain space. For ExuR, the only one preferred site was detected in the interdomain area. Availability of this area to different ligands suggests that, similar to the Lac repressor, the DNA-binding properties of UxuR and ExuR may be changed by repositioning of their domains. Experimental assays confirmed the ability of ligands with highest affinities to bind the regulatory proteins and affect their interaction with DNA. d-gal that is carried into the cell by the ExuT transporter appeared to be the best ligand for repressor of the exuT transcription, ExuR. For UxuR, the highest affinity was found for d-fructuronate transported by GntP, which biosynthesis is repressed by UxuR. Providing a feedback loop to balance the concentrations of different nutrients, such ligand-mediated modulation can also coordinate switching between different metabolic pathways in bacteria. PMID:26549308

  5. Crystallization and preliminary X-ray diffraction studies of the transcriptional repressor PaaX, the main regulator of the phenylacetic acid degradation pathway in Escherichia coli W

    PubMed Central

    Rojas-Altuve, Alzoray; Carrasco-López, César; Hernández-Rocamora, Víctor M.; Sanz, Jesús M.; Hermoso, Juan A.

    2011-01-01

    PaaX is the main regulator of the phenylacetic acid aerobic degradation pathway in bacteria and acts as a transcriptional repressor in the absence of its inducer phenylacetyl-coenzyme A. The natural presence and the recent accumulation of a variety of highly toxic aromatic compounds owing to human pollution has created considerable interest in the study of degradation pathways in bacteria, the most important microorganisms capable of recycling these compounds, in order to design and apply novel bioremediation strategies. PaaX from Escherichia coli W was cloned, overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of 0.9 M Li2SO4 and 0.5 M sodium citrate pH 5.8. These crystals, which belonged to the monoclinic space group C2 with unit-cell parameters a = 167.88, b = 106.23, c = 85.87 Å, β = 108.33°, allowed the collection of an X-ray data set to 2.3 Å resolution. PMID:22102047

  6. The N terminus of the Escherichia coli transcription activator MalT is the domain of interaction with MalY.

    PubMed

    Schlegel, Anja; Danot, Olivier; Richet, Evelyne; Ferenci, Thomas; Boos, Winfried

    2002-06-01

    The maltose system of Escherichia coli consists of a number of genes encoding proteins involved in the uptake and metabolism of maltose and maltodextrins. The system is positively regulated by MalT, its transcriptional activator. MalT activity is controlled by two regulatory circuits: a positive one with maltotriose as effector and a negative one involving several proteins. MalK, the ATP-hydrolyzing subunit of the cognate ABC transporter, MalY, an enzyme with the activity of a cystathionase, and Aes, an acetyl esterase, phenotypically act as repressors of MalT activity. By in vivo titration assays, we have shown that the N-terminal 250 amino acids of MalT contain the interaction site for MalY but not for MalK. This was confirmed by gel filtration analysis, where MalY was shown to coelute with the N-terminal MalT structural domain. Mutants in MalT causing elevated mal gene expression in the absence of exogenous maltodextrins were tested in their response to the three repressors. The different MalT mutations exhibited a various degree of sensitivity towards these repressors, but none was resistant to all of them. Some of them became nearly completely resistant to Aes while still being sensitive to MalY. These mutations are located at positions 38, 220, 243, and 359, most likely defining the interaction patch with Aes on the three-dimensional structure of MalT. PMID:12003949

  7. Transcriptional and Physiological Characterizations of Escherichia coli MG1655 that have been grown under Low Shear Stress Environment for 1000 Generations

    NASA Astrophysics Data System (ADS)

    Karouia, Fathi; Tirumalai, Madhan R.; Nelman-Gonzalez, Mayra A.; Sams, Clarence F.; Ott, Mark C.; Pierson, Duane L.; Fofanov, Yuriy; Willson, Richard C.; Fox, George E.

    Human space travelers experience a unique environment that affects homeostasis and physio-logic adaptation. One of the important regulatory biology interactions affected by space flight is the alteration of the immune response. As such, the impairment of the immune system may lead to higher risk of bacterial and/or viral infection during human space flight missions. Mi-crobiological contaminants have been a source of concern over the years for NASA and there is evidence to suggest that microbes in space do not behave like they do on Earth. Previ-ous studies have examined the physiological response of bacteria when exposed to short-term microgravity either during spaceflight or in a Low Shear Modeled Microgravity (LSMMG) en-vironment. Exposure to these environments has been found to induce increased resistance to stresses and antibiotics, and in one case increase of virulence. As NASA increases the duration of space flight missions and is starting to envision human presence on the lunar surface and Mars, it becomes legitimate to question the long-term effects of microgravity on bacteria. The effect of long-term exposure to LSMMG on microbial gene expression and physiology in Escherichia coli (E. coli) is being examined using functional genomics, and molecular tech-niques. In previous E. coli short term studies, reproducible changes in transcription were seen but no direct responses to changes in the gravity vector were identified. Instead, absence of shear and a randomized gravity vector appeared to cause local extra-cellular environmental changes, which elicited cellular responses. In order to evaluate the long-term effects of micro-gravity on bacteria, E. coli was grown under simulated microgravity for 1000 generations and gene expression patterns and cellular physiology were analyzed in comparison with short-term exposure. The analysis revealed that the long-term response differed significantly from the short-term exposure and 357 genes were expressed

  8. Structural Basis of Low-Affinity Nickel Binding to the Nickel-Responsive Transcription Factor NikR from Escherichia coli

    SciTech Connect

    Phillips, C.; Schreiter, E; Stultz, C; Drennan, C

    2010-01-01

    Escherichia coli NikR regulates cellular nickel uptake by binding to the nik operon in the presence of nickel and blocking transcription of genes encoding the nickel uptake transporter. NikR has two binding affinities for the nik operon: a nanomolar dissociation constant with stoichiometric nickel and a picomolar dissociation constant with excess nickel [Bloom, S. L., and Zamble, D. B. (2004) Biochemistry 43, 10029-10038; Chivers, P. T., and Sauer, R. T. (2002) Chem. Biol. 9, 1141-1148]. While it is known that the stoichiometric nickel ions bind at the NikR tetrameric interface [Schreiter, E. R., et al. (2003) Nat. Struct. Biol. 10, 794-799; Schreiter, E. R., et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13676-13681], the binding sites for excess nickel ions have not been fully described. Here we have determined the crystal structure of NikR in the presence of excess nickel to 2.6 {angstrom} resolution and have obtained nickel anomalous data (1.4845 {angstrom}) in the presence of excess nickel for both NikR alone and NikR cocrystallized with a 30-nucleotide piece of double-stranded DNA containing the nik operon. These anomalous data show that excess nickel ions do not bind to a single location on NikR but instead reveal a total of 22 possible low-affinity nickel sites on the NikR tetramer. These sites, for which there are six different types, are all on the surface of NikR, and most are found in both the NikR alone and NikR-DNA structures. Using a combination of crystallographic data and molecular dynamics simulations, the nickel sites can be described as preferring octahedral geometry, utilizing one to three protein ligands (typically histidine) and at least two water molecules.

  9. New Insights into the Signaling Mechanism of the pH-responsive, Membrane-integrated Transcriptional Activator CadC of Escherichia coli*

    PubMed Central

    Haneburger, Ina; Eichinger, Andreas; Skerra, Arne; Jung, Kirsten

    2011-01-01

    The membrane-integrated transcriptional regulator CadC of Escherichia coli activates expression of the cadBA operon at low external pH with concomitantly available lysine, providing adaptation to mild acidic stress. CadC is a representative of the ToxR-like proteins that combine sensory, signal transduction, and DNA-binding activities within a single polypeptide. Although several ToxR-like regulators such as CadC, as well as the main regulator of Vibrio cholerae virulence, ToxR itself, which activate gene expression at acidic pH, have been intensively investigated, their molecular activation mechanism is still unclear. In this study, a structure-guided mutational analysis was performed to elucidate the mechanism by which CadC detects acidification of the external milieu. Thus, a cluster of negatively charged amino acids (Asp-198, Asp-200, Glu-461, Glu-468, and Asp-471) was found to be crucial for pH detection. These amino acids form a negatively charged patch on the surface of the periplasmic domain of CadC that stretches across its two subdomains. The results of different combinations of amino acid replacements within this patch indicated that the N-terminal subdomain integrates and transduces the signals coming from both subdomains to the transmembrane domain. Alterations in the phospholipid composition did not influence pH-dependent cadBA expression, and therefore, interplay of the acidic surface patch with the negatively charged headgroups is unlikely. Models are discussed according to which protonation of these acidic amino acid side chains reduces repulsive forces between the two subdomains and/or between two monomers within a CadC dimer and thereby enables receptor activation upon lowering of the environmental pH. PMID:21216950

  10. Escherichia coli (E. coli)

    MedlinePlus

    ... so you might hear about E. coli being found in drinking water, which are not themselves harmful, but indicate the ... at CDC Foodborne disease Travelers' Health: Safe Food & Water Healthy Swimming E. coli Infection & Farm ... Word file Microsoft Excel file Audio/Video file Apple ...

  11. Evidence for an arginine exporter encoded by yggA (argO) that is regulated by the LysR-type transcriptional regulator ArgP in Escherichia coli.

    PubMed

    Nandineni, Madhusudan R; Gowrishankar, J

    2004-06-01

    The anonymous open reading frame yggA of Escherichia coli was identified in this study as a gene that is under the transcriptional control of argP (previously called iciA), which encodes a LysR-type transcriptional regulator protein. Strains with null mutations in either yggA or argP were supersensitive to the arginine analog canavanine, and yggA-lac expression in vivo exhibited argP(+)-dependent induction by arginine. Lysine supplementation phenocopied the argP null mutation in that it virtually abolished yggA expression, even in the argP+ strain. The dipeptides arginylalanine and lysylalanine behaved much like arginine and lysine, respectively, to induce and to turn off yggA transcription. Dominant missense mutations in argP (argPd) that conferred canavanine resistance and rendered yggA-lac expression constitutive were obtained. The protein deduced to be encoded by yggA shares similarity with a basic amino acid exporter (LysE) of Corynebacterium glutamicum, and we obtained evidence for increased arginine efflux from E. coli strains with either the argPd mutation or multicopy yggA+. The null yggA mutation abolished the increased arginine efflux from the argPd strain. Our results suggest that yggA encodes an ArgP-regulated arginine exporter, and we have accordingly renamed it argO (for "arginine outward transport"). We propose that the physiological function of argO may be either to prevent the accumulation to toxic levels of canavanine (which is a plant-derived antimetabolite) or arginine or to maintain an appropriate balance between the intracellular lysine and arginine concentrations. PMID:15150242

  12. E. Coli

    MedlinePlus

    ... E. coli is short for the medical term Escherichia coli . The strange thing about these bacteria — and lots ... cause a very serious infection. Someone who has E. coli infection may have these symptoms: bad stomach cramps and ...

  13. Identifying Mechanisms by Which Escherichia coli O157:H7 Subverts Interferon-γ Mediated Signal Transducer and Activator of Transcription-1 Activation

    PubMed Central

    Ho, Nathan K.; Crandall, Ian; Sherman, Philip M.

    2012-01-01

    Enterohemorrhagic Escherichia coli serotype O157:H7 is a food borne enteric bacterial pathogen that causes significant morbidity and mortality in both developing and industrialized nations. E. coli O157:H7 infection of host epithelial cells inhibits the interferon gamma pro-inflammatory signaling pathway, which is important for host defense against microbial pathogens, through the inhibition of Stat-1 tyrosine phosphorylation. The aim of this study was to determine which bacterial factors are involved in the inhibition of Stat-1 tyrosine phosphorylation. Human epithelial cells were challenged with either live bacteria or bacterial-derived culture supernatants, stimulated with interferon-gamma, and epithelial cell protein extracts were then analyzed by immunoblotting. The results show that Stat-1 tyrosine phosphorylation was inhibited by E. coli O157:H7 secreted proteins. Using sequential anion exchange and size exclusion chromatography, YodA was identified, but not confirmed to mediate subversion of the Stat-1 signaling pathway using isogenic mutants. We conclude that E. coli O157:H7 subverts Stat-1 tyrosine phosphorylation in response to interferon-gamma through a still as yet unidentified secreted bacterial protein. PMID:22253910

  14. Identifying mechanisms by which Escherichia coli O157:H7 subverts interferon-γ mediated signal transducer and activator of transcription-1 activation.

    PubMed

    Ho, Nathan K; Crandall, Ian; Sherman, Philip M

    2012-01-01

    Enterohemorrhagic Escherichia coli serotype O157:H7 is a food borne enteric bacterial pathogen that causes significant morbidity and mortality in both developing and industrialized nations. E. coli O157:H7 infection of host epithelial cells inhibits the interferon gamma pro-inflammatory signaling pathway, which is important for host defense against microbial pathogens, through the inhibition of Stat-1 tyrosine phosphorylation. The aim of this study was to determine which bacterial factors are involved in the inhibition of Stat-1 tyrosine phosphorylation. Human epithelial cells were challenged with either live bacteria or bacterial-derived culture supernatants, stimulated with interferon-gamma, and epithelial cell protein extracts were then analyzed by immunoblotting. The results show that Stat-1 tyrosine phosphorylation was inhibited by E. coli O157:H7 secreted proteins. Using sequential anion exchange and size exclusion chromatography, YodA was identified, but not confirmed to mediate subversion of the Stat-1 signaling pathway using isogenic mutants. We conclude that E. coli O157:H7 subverts Stat-1 tyrosine phosphorylation in response to interferon-gamma through a still as yet unidentified secreted bacterial protein. PMID:22253910

  15. Hha controls Escherichia coli O157:H7 biofilm formation by differential regulation of global transcriptional regulators FlhDC and CsgD

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that produces a broad-spectrum of diarrheal illnesses in infected humans. Although molecular mechanisms enabling EHEC O157:H7 to produce characteristic adherence on epithelial cells are well characterized, regulatory mechanisms...

  16. Comparative transcriptional profiling reveals differential expression of pathways directly and indirectly influencing biofilm formation in Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Escherichia coli O157:H7 (O157) is a frequent cause of foodborne disease outbreaks. O157 encodes virulence factors for colonizing and survival in reservoir animals and the environment. For example, genetic factors promoting biofilm formation are linked to survival of O157 in and outsid...

  17. Distinct transcriptional profiles and phenotypes exhibited by Escherichia coli O157:H7 isolates related to the 2006 spinach-associated outbreak

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In 2006, an outbreak of Escherichia coli O157:H7 was linked to the consumption of ready-to-eat bagged spinach. The likely sources of pre-harvest spinach contamination were soil and water that became contaminated via cattle or feral pigs in the proximity of the spinach fields. In this study, we compa...

  18. Transcriptional cross-regulation between Gram-negative and gram-positive bacteria, demonstrated using ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum.

    PubMed

    Marbaniang, Carmelita N; Gowrishankar, J

    2012-10-01

    The protein-gene pairs ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum are orthologous, with the first member of each pair being a LysR-type transcriptional regulator and the second its target gene encoding a basic amino acid exporter. Whereas LysE is an exporter of arginine (Arg) and lysine (Lys) whose expression is induced by Arg, Lys, or histidine (His), ArgO exports Arg alone, and its expression is activated by Arg but not Lys or His. We have now reconstituted in E. coli the activation of lysE by LysG in the presence of its coeffectors and have shown that neither ArgP nor LysG can regulate expression of the noncognate orthologous target. Of several ArgP-dominant (ArgP(d)) variants that confer elevated Arg-independent argO expression, some (ArgP(d)-P274S, -S94L, and, to a lesser extent, -P108S) activated lysE expression in E. coli. However, the individual activating effects of LysG and ArgP(d) on lysE were mutually extinguished when both proteins were coexpressed in Arg- or His-supplemented cultures. In comparison with native ArgP, the active ArgP(d) variants exhibited higher affinity of binding to the lysE regulatory region and less DNA bending at both argO and lysE. We conclude that the transcription factor LysG from a Gram-positive bacterium, C. glutamicum, is able to engage appropriately with the RNA polymerase from a Gram-negative bacterium, E. coli, for activation of its cognate target lysE in vivo and that single-amino-acid-substitution variants of ArgP can also activate the distantly orthologous target lysE, but by a subtly different mechanism that renders them noninterchangeable with LysG. PMID:22904281

  19. Heat shock transcription factor δ³² is targeted for degradation via an ubiquitin-like protein ThiS in Escherichia coli.

    PubMed

    Xu, Xibing; Niu, Yulong; Liang, Ke; Wang, Jianmei; Li, Xufeng; Yang, Yi

    2015-04-01

    The posttranslational modification of proteins with ubiquitin and ubiquitin-like proteins (UBLs) plays an important role in eukaryote biology, through which substrate proteins are targeted for degradation by the proteasome. Prokaryotes have been thought to degrade proteins by an ubiquitin independent pathway. Here, we show that ThiS, an ubiquitin-like protein, is covalently attached to δ(32) and at least 27 other proteins, leading to their subsequent degradation by proteases, in a similar manner to the ubiquitin-proteasome system (UPS) in eukaryotes. Molecular biology and biochemical studies confirm that specific lysine sites in δ(32) can be modified by ThiS. The results presented here establish a new model for δ(32) degradation and show that Escherichia coli uses a small-protein modifier to control protein stability. PMID:25721662

  20. Expression, Functional Characterization and X-ray Analysis of HosA, A Member of MarR Family of Transcription Regulator from Uropathogenic Escherichia coli.

    PubMed

    Roy, Ajit; Reddi, Ravikumar; Sawhney, Bhavik; Ghosh, Debasish Kumar; Addlagatta, Anthony; Ranjan, Akash

    2016-08-01

    Regulators belonging to multiple antibiotic resistance regulator (MarR) family are widespread in prokaryotes and are involved in regulation of genes that are responsible for virulence and pathogenicity in most of the clinically important pathogens. Here we report the transcriptional, biophysical, and X-ray analyses of homologue of SlyA (HosA), a member of MarR family that is predominantly present in the pathogenic strains of Enterobacteriaceae family. The initiation of hosA transcription was observed to occur at two independent start sites and subsequent binding study has revealed that the purified HosA interacts with its upstream region suggesting a probable autoregulation. The secondary structure analysis through circular dichroism spectroscopy demonstrated that HosA is predominantly composed of the alpha helix with higher thermal stability. To further understand the three-dimensional structure, HosA was crystallized and the crystals were diffracted to maximum of 2.9 Ǻ on exposure to X-rays. Analysis of the X-ray crystallographic data suggested a primitive space group (P 6 ? 2 2), with unit cell parameters a = b = 64.19 Å and c = 244.25 Å. The solvent content and Matthews coefficient were 41 % and 2.11 Å(3) Da(-1), respectively, which indicated the existence of two molecules of HosA in the asymmetric unit of crystal. PMID:27325406

  1. Source-Related Effects of Wastewater on Transcription Factor (AhR, CAR and PXR)-Mediated Induction of Gene Expression in Cultured Rat Hepatocytes and Their Association with the Prevalence of Antimicrobial-Resistant Escherichia coli.

    PubMed

    Guruge, Keerthi S; Yamanaka, Noriko; Sonobe, Miyuki; Fujizono, Wataru; Yoshioka, Miyako; Akiba, Masato; Yamamoto, Takehisa; Joshua, Derrick I; Balakrishna, Keshava; Yamashita, Nobuyoshi; Kannan, Kurunthachalam; Tsutsui, Toshiyuki

    2015-01-01

    Extracts of wastewater collected from 4 sewage treatment plants (STPs) receiving effluents from different sources in South India were investigated for their levels of transcription factor-mediated gene induction in primary cultured rat hepatocytes. In addition, the relation between gene induction levels and the prevalence of antimicrobial-resistant Escherichia coli (E. coli) in wastewater was examined. STP-3, which treats only hospital wastewater, exhibited significantly greater induction potency of all 6 drug metabolizing cytochrome P450 (CYP) genes examined, CYP1A1, 1A2, 1B1, 2B15, 3A1, and 3A2, whereas the wastewater at STP-1, which exclusively receives domestic sewage, showed significantly diminished levels of induction of 3 CYP genes when compared to the levels of CYP induction at STP-2, which receives mixed wastewater. Samples collected during the monsoon season showed a significantly altered gene induction capacity compared to that of samples from the pre-monsoon period. The data suggest that the toxicity of wastewater in STPs was not significantly diminished during the treatment process. The chemical-gene interaction data predicted that a vast number of chemicals present in the wastewater would stimulate the genes studied in the rat hepatocytes. The multivariable logistic regression analysis demonstrated that the prevalence of isolates resistant to cefotaxime, imipenem and streptomycin was significantly correlated with the levels of induction of at least three CYP-isozymes in STP wastewater. In addition, the resistance of isolates in treatment plants was not altered by the treatment steps, whereas the sampling season did have an impact on the resistance to specific antimicrobials. The identification of receptor-mediated gene regulation capacities offers important data not limited to the (synergistic) physiological role of chemicals in biological systems but may provide new insight into the link between the effects of known/unknown drugs and prevalence of

  2. Source-Related Effects of Wastewater on Transcription Factor (AhR, CAR and PXR)-Mediated Induction of Gene Expression in Cultured Rat Hepatocytes and Their Association with the Prevalence of Antimicrobial-Resistant Escherichia coli

    PubMed Central

    Guruge, Keerthi S.; Yamanaka, Noriko; Sonobe, Miyuki; Fujizono, Wataru; Yoshioka, Miyako; Akiba, Masato; Yamamoto, Takehisa; Joshua, Derrick I.; Balakrishna, Keshava; Yamashita, Nobuyoshi; Kannan, Kurunthachalam; Tsutsui, Toshiyuki

    2015-01-01

    Extracts of wastewater collected from 4 sewage treatment plants (STPs) receiving effluents from different sources in South India were investigated for their levels of transcription factor-mediated gene induction in primary cultured rat hepatocytes. In addition, the relation between gene induction levels and the prevalence of antimicrobial-resistant Escherichia coli (E. coli) in wastewater was examined. STP-3, which treats only hospital wastewater, exhibited significantly greater induction potency of all 6 drug metabolizing cytochrome P450 (CYP) genes examined, CYP1A1, 1A2, 1B1, 2B15, 3A1, and 3A2, whereas the wastewater at STP-1, which exclusively receives domestic sewage, showed significantly diminished levels of induction of 3 CYP genes when compared to the levels of CYP induction at STP-2, which receives mixed wastewater. Samples collected during the monsoon season showed a significantly altered gene induction capacity compared to that of samples from the pre-monsoon period. The data suggest that the toxicity of wastewater in STPs was not significantly diminished during the treatment process. The chemical-gene interaction data predicted that a vast number of chemicals present in the wastewater would stimulate the genes studied in the rat hepatocytes. The multivariable logistic regression analysis demonstrated that the prevalence of isolates resistant to cefotaxime, imipenem and streptomycin was significantly correlated with the levels of induction of at least three CYP-isozymes in STP wastewater. In addition, the resistance of isolates in treatment plants was not altered by the treatment steps, whereas the sampling season did have an impact on the resistance to specific antimicrobials. The identification of receptor-mediated gene regulation capacities offers important data not limited to the (synergistic) physiological role of chemicals in biological systems but may provide new insight into the link between the effects of known/unknown drugs and prevalence of

  3. Galangin suppresses the proliferation of β-catenin response transcription-positive cancer cells by promoting adenomatous polyposis coli/Axin/glycogen synthase kinase-3β-independent β-catenin degradation.

    PubMed

    Gwak, Jungsug; Oh, Jingyo; Cho, Munju; Bae, Soo Kyung; Song, Im-Sook; Liu, Kwang-Hyeon; Jeong, Yongsu; Kim, Dong-Eun; Chung, Young-Hwa; Oh, Sangtaek

    2011-06-01

    Galangin is a naturally occurring bioflavonoid with anticancer activity against certain human cancers, yet little is known about its mechanism of action. Here, we used a chemical biology approach to reveal that galangin suppresses β-catenin response transcription (CRT), which is aberrantly up-regulated in colorectal and liver cancers, by promoting the degradation of intracellular β-catenin. Inhibition of glycogen synthase kinase-3β (GSK-3β) activity or mutation of the GSK-3β-targeted sequence from β-catenin was unable to abrogate the galangin-mediated degradation of β-catenin. In addition, galangin down-regulated the intracellular β-catenin levels in cancer cells with inactivating mutations of adenomatous polyposis coli (APC) or Axin, which are components of the β-catenin destruction complex. Galangin repressed the expression of β-catenin/T-cell factor-dependent genes, such as cyclin D1 and c-myc, and thus inhibited the proliferation of CRT-positive cancer cells. Structure-activity data indicated that the major structural requirements for galangin-mediated β-catenin degradation are hydroxyl groups at positions 3, 5, and 7. Our findings suggest that galangin exerts its anticancer activity by promoting APC/Axin/GSK-3β-independent proteasomal degradation of β-catenin. PMID:21406604

  4. Strategies for Protein Overproduction in Escherichia coli.

    ERIC Educational Resources Information Center

    Mott, John E.

    1984-01-01

    Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

  5. Evaluation of the impact of quorum sensing transcriptional regulator SdiA on long-term persistence and fecal shedding of Escherichia coli O157:H7 in weaned calves.

    PubMed

    Sharma, V K; Bearson, S M D

    2013-04-01

    Escherichia coli O157:H7 (O157) colonization of bovine intestine is mediated through the locus of enterocyte effacement (LEE)-encoded type III secretion system and secreted virulence proteins that promote colonization of the recto-anal junction (RAJ) of the large intestine of cattle. The quorum sensing transcriptional regulator SdiA, a homolog of LuxR, has been shown in vitro to repress LEE strongly when overexpressed from a multi-copy recombinant plasmid or when its activity is enhanced by the binding of N-acyl-L-homoserine lactones (AHLs), the quorum sensing signals that are detected by SdiA. Since LEE has been shown to be essential for colonization and persistence of O157 in bovine intestine, we examined whether a mutation in sdiA, which normally represses LEE in vitro, would also exert negative effect on colonization and long-term persistence of O157 in weaned calves. Ten-week old weaned calves (n = 4/group) were inoculated orally with 10(10) cfu of either the wild-type or sdiA mutant strain. Initial fecal shedding of the sdiA mutant and the wild-type strain were similar in magnitude and declined during the first 2 weeks post-inoculation. The sdiA mutant was detected in feces of only one of the four calves at low levels (≥10(2) cfu/g feces) from days 19 - 27 post-inoculation, whereas, the fecal shedding of the wild-type strain persisted at approximately 4-logs in all four calves from days 19 - 27. We also confirmed that SdiA represses ler, which encodes a positive transcriptional regulator of LEE, in response to AHLs, and reduces adherence of O157 to HEp-2 cells. In conclusion, this study demonstrates that although in vitro the sdiA gene represses LEE and LEE-mediated adherence to cultured cells, the presence of sdiA is necessary for colonization of bovine large intestine that in turn promotes persistent fecal shedding of O157 by these animals. PMID:23415735

  6. Structure of the Escherichia coli Antitoxin MqsA (YgiT/b3021) Bound to Its Gene Promoter Reveals Extensive Domain Rearrangements and the Specificity of Transcriptional Regulation

    SciTech Connect

    B Brown; T Wood; W Peti; R Page

    2011-12-31

    Bacterial cultures, especially biofilms, produce a small number of persister cells, a genetically identical subpopulation of wild type cells that are metabolically dormant, exhibit multidrug tolerance, and are highly enriched in bacterial toxins. The gene most highly up-regulated in Escherichia coli persisters is mqsR, a ribonuclease toxin that, along with mqsA, forms a novel toxin-antitoxin (TA) system. Like all known TA systems, both the MqsR-MqsA complex and MqsA alone regulate their own transcription. Despite the importance of TA systems in persistence and biofilms, very little is known about how TA modules, and antitoxins in particular, bind and recognize DNA at a molecular level. Here, we report the crystal structure of MqsA bound to a 26-bp fragment from the mqsRA promoter. We show that MqsA binds DNA predominantly via its C-terminal helix-turn-helix domain, with direct binding of recognition helix residues Asn{sup 97} and Arg{sup 010} to the DNA major groove. Unexpectedly, the structure also revealed that the MqsA N-terminal domain interacts with the DNA phosphate backbone. This results in a more than 105{sup o} rotation of the N-terminal domains between the free and complexed states, an unprecedented rearrangement for an antitoxin. The structure also shows that MqsA bends the DNA by more than 55{sup o} in order to achieve symmetrical binding. Finally, using a combination of biochemical and NMR studies, we show that the DNA sequence specificity of MqsA is mediated by direct readout.

  7. Na+-induced transcription of nhaA, which encodes an Na+/H+ antiporter in Escherichia coli, is positively regulated by nhaR and affected by hns.

    PubMed Central

    Dover, N; Higgins, C F; Carmel, O; Rimon, A; Pinner, E; Padan, E

    1996-01-01

    nhaA encodes an Na+/H+ antiporter in Escherichia coli which is essential for adaptation to high salinity and alkaline pH in the presence of Na+. We used Northern (RNA) analysis to measure directly the cellular levels of nhaA mRNA. NhaR belongs to the LysR family of regulatory proteins. Consistent with our previous data with an nhaA'-'lacZ fusion, NhaR was found to be a positive regulator and Na+ was found to be a specific inducer of nhaA transcription. In the nhaA'-'lacZ fusion, maximal induction was observed at alkaline pH. In contrast, in the nhaA+ strain both the level of nhaA expression and the induction ratio were lower at alkaline pH. This difference may be due to the activity of NhaA in the wild-type strain as NhaA efficiently excreted Na+ at alkaline pH and reduced the intracellular concentration of Na+, the signal for induction. We also showed that although the global regulator rpoS was not involved in nhaA regulation, the global regulator hns played a role. Thus, the expression of nhaA'-'lacZ was derepressed in strains bearing hns mutations and transformation with a low-copy-number plasmid carrying hns repressed expression and restored Na+ induction. The derepression in hns strains was nhaR independent. Most interestingly, multicopy nhaR, which in an hns+ background acted only as an Na+-dependent positive regulator, acted as a repressor in an hns strain in the absence of Na+ but was activated in the presence of the ion. Hence, an interplay between nhaR and hns in the regulation of nhaA was suggested. PMID:8932307

  8. E. Coli and Pregnancy

    MedlinePlus

    ... care provider. What is E. coli? E. coli (Escherichia coli) is a bacterium that lives in your colon ( ... 10):1411-1413. Jones B, et al. 2004. Escherichia coli: a growing problem in early onset neonatal sepsis. ...

  9. E. Coli Infection

    MedlinePlus

    ... is E. coli? E. coli is short for Escherichia coli -- bacteria (germs) that cause severe cramps and diarrhea. E. ... and especially in people who have another illness. E. coli infection is more common during the summer months and ...

  10. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  11. E. coli

    MedlinePlus

    ... sure that ground beef has reached a safe internal temperature of 160° F. Wash hands before preparing food, after diapering infants, and after contact with cows, sheep, or goats, their food or treats, or their living environment . General Information E. coli Infections (NIH MedlinePlus) Trusted ...

  12. Specificity and robustness in transcription control networks.

    PubMed

    Sengupta, Anirvan M; Djordjevic, Marko; Shraiman, Boris I

    2002-02-19

    Recognition by transcription factors of the regulatory DNA elements upstream of genes is the fundamental step in controlling gene expression. How does the necessity to provide stability with respect to mutation constrain the organization of transcription control networks? We examine the mutation load of a transcription factor interacting with a set of n regulatory response elements as a function of the factor/DNA binding specificity and conclude on theoretical grounds that the optimal specificity decreases with n. The predicted correlation between variability of binding sites (for a given transcription factor) and their number is supported by the genomic data for Escherichia coli. The analysis of E. coli genomic data was carried out using an algorithm suggested by the biophysical model of transcription factor/DNA binding. Complete results of the search for candidate transcription factor binding sites are available at http://www.physics.rockefeller.edu/~boris/public/search_ecoli. PMID:11854503

  13. Creating small transcription activating RNAs.

    PubMed

    Chappell, James; Takahashi, Melissa K; Lucks, Julius B

    2015-03-01

    We expanded the mechanistic capability of small RNAs by creating an entirely synthetic mode of regulation: small transcription activating RNAs (STARs). Using two strategies, we engineered synthetic STAR regulators to disrupt the formation of an intrinsic transcription terminator placed upstream of a gene in Escherichia coli. This resulted in a group of four highly orthogonal STARs that had up to 94-fold activation. By systematically modifying sequence features of this group, we derived design principles for STAR function, which we then used to forward engineer a STAR that targets a terminator found in the Escherichia coli genome. Finally, we showed that STARs could be combined in tandem to create previously unattainable RNA-only transcriptional logic gates. STARs provide a new mechanism of regulation that will expand our ability to use small RNAs to construct synthetic gene networks that precisely control gene expression. PMID:25643173

  14. Structural basis of transcription activation.

    PubMed

    Feng, Yu; Zhang, Yu; Ebright, Richard H

    2016-06-10

    Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme. PMID:27284196

  15. E. coli enteritis

    MedlinePlus

    Traveler's diarrhea - E. coli ; Food poisoning - E. coli ; E. coli diarrhea; Hamburger disease ... infected. The most common symptom is sudden, severe diarrhea that is often bloody. Other symptoms may include: ...

  16. Repression of the locus of the enterocyte effacement-encoded regulator of gene transcription of Escherichia coli O157:H7 by Lactobacillus reuteri culture supernatants is LuxS and strain dependent.

    PubMed

    Jelcić, Ivan; Hüfner, Eric; Schmidt, Herbert; Hertel, Christian

    2008-05-01

    Culture supernatants of Lactobacillus reuteri ATCC 55730 repressed ler expression in Escherichia coli O157:H7 cells, but neither the strain's isogenic luxS mutant nor the L. reuteri 100-23C wild-type strain and its luxS mutant elicited a comparable effect. Furthermore, the epinephrine-mediated induction of ler expression was repressed by secreted substance(s) of L. reuteri ATCC 55730. PMID:18378666

  17. Diarrheagenic Escherichia coli

    PubMed Central

    Nataro, James P.; Kaper, James B.

    1998-01-01

    Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler’s diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (enteropathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens. PMID:9457432

  18. E. Coli Infections

    MedlinePlus

    ... adults with weak immune systems. You can get E. coli infections by eating foods containing the bacteria. Symptoms of ... pool contaminated with human waste. Most cases of E. coli infection get better without treatment in 5 to 10 ...

  19. Effects of sigmaS and the transcriptional activator AppY on induction of the Escherichia coli hya and cbdAB-appA operons in response to carbon and phosphate starvation.

    PubMed Central

    Atlung, T; Knudsen, K; Heerfordt, L; Brøndsted, L

    1997-01-01

    The transcriptional regulation of two energy metabolism operons, hya and cbdAB-appA, has been investigated during carbon and phosphate starvation. The hya operon encodes hydrogenase 1, and the cbdAB-appA operon encodes cytochrome bd-II oxidase and acid phosphatase, pH 2.5. Both operons are targets for the transcriptional activator AppY. In exponential growth, expression of the hya and cbd operons was reduced in an rpoS mutant lacking the RNA polymerase sigmaS factor, and the induction of the two operons by entry into stationary phase in rich medium was strongly dependent on sigmaS. Both operons were induced by carbon starvation, but only induction of the hya operon was dependent on sigmaS, whereas that of the cbd promoter was dependent on AppY. The appY gene also showed sigmaS-dependent induction by carbon starvation. The cbd and hya operons were also found to exhibit a sigmaS-dependent transient twofold induction by osmotic upshift. Like the cbd operon, the hya operon was highly induced by phosphate starvation. For both operons the induction was strongly dependent on AppY. The induction ratio of the two operons was the same in rpoS+ and rpoS mutant strains, indicating that the phosphate starvation-induced increase in sigmaS concentration is not involved in the phosphate regulation of these operons. PMID:9079897

  20. Effects of sigmaS and the transcriptional activator AppY on induction of the Escherichia coli hya and cbdAB-appA operons in response to carbon and phosphate starvation.

    PubMed

    Atlung, T; Knudsen, K; Heerfordt, L; Brøndsted, L

    1997-04-01

    The transcriptional regulation of two energy metabolism operons, hya and cbdAB-appA, has been investigated during carbon and phosphate starvation. The hya operon encodes hydrogenase 1, and the cbdAB-appA operon encodes cytochrome bd-II oxidase and acid phosphatase, pH 2.5. Both operons are targets for the transcriptional activator AppY. In exponential growth, expression of the hya and cbd operons was reduced in an rpoS mutant lacking the RNA polymerase sigmaS factor, and the induction of the two operons by entry into stationary phase in rich medium was strongly dependent on sigmaS. Both operons were induced by carbon starvation, but only induction of the hya operon was dependent on sigmaS, whereas that of the cbd promoter was dependent on AppY. The appY gene also showed sigmaS-dependent induction by carbon starvation. The cbd and hya operons were also found to exhibit a sigmaS-dependent transient twofold induction by osmotic upshift. Like the cbd operon, the hya operon was highly induced by phosphate starvation. For both operons the induction was strongly dependent on AppY. The induction ratio of the two operons was the same in rpoS+ and rpoS mutant strains, indicating that the phosphate starvation-induced increase in sigmaS concentration is not involved in the phosphate regulation of these operons. PMID:9079897

  1. Dialogue between E. coli free radical pathways and the mitochondria of C. elegans

    PubMed Central

    Govindan, J. Amaranath; Jayamani, Elamparithi; Zhang, Xinrui; Mylonakis, Eleftherios; Ruvkun, Gary

    2015-01-01

    The microbial world presents a complex palette of opportunities and dangers to animals, which have developed surveillance and response strategies to hints of microbial intent. We show here that the mitochondrial homeostatic response pathway of the nematode Caenorhabditis elegans responds to Escherichia coli mutations that activate free radical detoxification pathways. Activation of C. elegans mitochondrial responses could be suppressed by additional mutations in E. coli, suggesting that C. elegans responds to products of E. coli to anticipate challenges to its mitochondrion. Out of 50 C. elegans gene inactivations known to mediate mitochondrial defense, we found that 7 genes were required for C. elegans response to a free radical producing E. coli mutant, including the bZip transcription factor atfs-1 (activating transcription factor associated with stress). An atfs-1 loss-of-function mutant was partially resistant to the effects of free radical-producing E. coli mutant, but a constitutively active atfs-1 mutant growing on wild-type E. coli inappropriately activated the pattern of mitochondrial responses normally induced by an E. coli free radical pathway mutant. Carbonylated proteins from free radical-producing E. coli mutant may directly activate the ATFS-1/bZIP transcription factor to induce mitochondrial stress response: feeding C. elegans with H2O2-treated E. coli induces the mitochondrial unfolded protein response, and inhibition of a gut peptide transporter partially suppressed C. elegans response to free radical damaged E. coli. PMID:26392561

  2. Problem-Solving Test: The Mechanism of Transcription Termination by the Rho Factor

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2012-01-01

    Transcription termination comes in two forms in "E. coli" cells. Rho-dependent termination requires the binding of a termination protein called Rho factor to the transcriptional machinery at the terminator region, whereas Rho-independent termination is achieved by conformational changes in the transcript itself. This article presents a test…

  3. Pathogenic Escherichia coli.

    PubMed

    Kaper, James B; Nataro, James P; Mobley, Harry L

    2004-02-01

    Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes. PMID:15040260

  4. Selective translation during stress in Escherichia coli

    PubMed Central

    Moll, Isabella; Engelberg-Kulka, Hanna

    2016-01-01

    The bacterial stress response, a strategy to cope with environmental changes, is generally known to operate on the transcriptional level. Here, we discuss a novel paradigm for stress adaptation at the post-transcriptional level, based on the recent discovery of a stress-induced modified form of the translation machinery in Escherichia coli that is generated by MazF, the toxin component of the toxin–antitoxin (TA) module mazEF. Under stress, the induced endoribonuclease MazF removes the 3′-terminal 43 nucleotides of the 16S rRNA of ribosomes and, concomitantly, the 5′-untranslated regions (UTRs) of specific transcripts. This elegant mechanism enables selective translation due to the complementary effect of MazF on ribosomes and mRNAs, and also represents the first example of functional ribosome heterogeneity based on rRNA alteration. PMID:22939840

  5. Pathogenic Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli, a member of the Enterobacteriaceae family, is a part of the normal flora of the intestinal tract of humans and a variety of animals. E. coli strains are classified on the basis of antigenic differences in two surface components (serotyping), the somatic antigen (O) of the lipopoly...

  6. PATHOGENIC ESCHERICHIA COLI

    EPA Science Inventory

    Escherichia coli is a bacterial species which inhabits the gastrointestinal tract of man and warm-blooded animals. Because of the ubiquity of this bacterium in the intestinal flora, it serves as an important indicator organism of fecal contamination. E. coli, aside from serving a...

  7. Synthetic in vitro transcriptional oscillators.

    PubMed

    Kim, Jongmin; Winfree, Erik

    2011-02-01

    The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system's robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells. PMID:21283141

  8. Complete Structural Model of Escherichia coli RNA Polymerase from a Hybrid Approach

    SciTech Connect

    Opalka, N.; Brown, J; Lane, W; Twist, K; Landick, R; Asturias, F; Darst, S

    2010-01-01

    The Escherichia coli transcription system is the best characterized from a biochemical and genetic point of view and has served as a model system. Nevertheless, a molecular understanding of the details of E. coli transcription and its regulation, and therefore its full exploitation as a model system, has been hampered by the absence of high-resolution structural information on E. coli RNA polymerase (RNAP). We use a combination of approaches, including high-resolution X-ray crystallography, ab initio structural prediction, homology modeling, and single-particle cryo-electron microscopy, to generate complete atomic models of E. coli core RNAP and an E. coli RNAP ternary elongation complex. The detailed and comprehensive structural descriptions can be used to help interpret previous biochemical and genetic data in a new light and provide a structural framework for designing experiments to understand the function of the E. coli lineage-specific insertions and their role in the E. coli transcription program. Transcription, or the synthesis of RNA from DNA, is one of the most important processes in the cell. The central enzyme of transcription is the DNA-dependent RNA polymerase (RNAP), a large, macromolecular assembly consisting of at least five subunits. Historically, much of our fundamental information on the process of transcription has come from genetic and biochemical studies of RNAP from the model bacterium Escherichia coli. More recently, major breakthroughs in our understanding of the mechanism of action of RNAP have come from high resolution crystal structures of various bacterial, archaebacterial, and eukaryotic enzymes. However, all of our high-resolution bacterial RNAP structures are of enzymes from the thermophiles Thermus aquaticus or T. thermophilus, organisms with poorly characterized transcription systems. It has thus far proven impossible to obtain a high-resolution structure of E. coli RNAP, which has made it difficult to relate the large collection

  9. E. Coli Infections

    MedlinePlus

    ... You can also get the infection by swallowing water in a swimming pool contaminated with human waste. Most cases of E. coli infection get better without treatment in 5 to 10 days. NIH: National Institute ...

  10. [Enterohemorrhagic E. coli (EHEC)].

    PubMed

    Goto, Tetsushi; Shirano, Michinori

    2012-08-01

    Escherichia coli (E. coli) is a bacterium that is commonly found in the gut of humans and warm-blooded animals. Most strains of E. coli are harmless. Some strains however, such as enterohemorrhagic E. coli (EHEC), can cause severe foodborne disease. It is transmitted to humans primarily through consumption of contaminated foods, such as raw meal. EHEC produces toxins, known as verotoxins. EHEC that induces bloody diarrhea leads to HUS in 10% of cases. The clinical manifestations of post-diarrheal HUS include acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia. The verocytotoxin can directly damage renal and endothelial cells. Thrombocytopenia occurs as platelets are consumed by clotting. Hemolytic anemia results from intravascular fibrin deposition, increased fragility of red blood cells, and fragmentation. PMID:22894069

  11. Recurrent Escherichia coli bacteremia.

    PubMed Central

    Maslow, J N; Mulligan, M E; Arbeit, R D

    1994-01-01

    Escherichia coli is the most common gram-negative organism associated with bacteremia. While recurrent E. coli urinary tract infections are well-described, recurrent E. coli bacteremia appears to be uncommon, with no episodes noted in multiple series of patients with gram-negative bacteremias. We report on 5 patients with recurrent bloodstream infections identified from a series of 163 patients with E. coli bacteremia. For each patient, the isolates from each episode were analyzed by pulsed-field gel electrophoresis (PFGE) and ribotyping and for the presence of E. coli virulence factors. For each of four patients, the index and recurrent episodes of bacteremia represented the same strain as defined by PFGE, and the strains were found to carry one or more virulence factors. The remaining patient, with two episodes of bloodstream infection separated by a 4-year interval, was infected with two isolates that did not carry any virulence factors and that were clonally related by ribotype analysis but differed by PFGE. All five patients had either a local host defense defect (three patients) or impaired systemic defenses (one patient) or both (one patient). Thus, recurrent E. coli bacteremia is likely to represent a multifactorial process that occurs in patients with impaired host defenses who are infected with virulent isolates. Images PMID:7910828

  12. A transcription map of a yeast centromere plasmid: unexpected transcripts and altered gene expression.

    PubMed Central

    Marczynski, G T; Jaehning, J A

    1985-01-01

    YCp19 is a yeast centromere plasmid capable of autonomous replication in both yeast and E. coli (J. Mol. Biol., 158: 157-179, 1982). It is stably maintained as a single copy in the yeast cell and is therefore a model yeast "minichromosome" and cloning vector. We have located the positions and measured the abundance of the in vivo yeast transcripts from YCp19. Transcripts from the selectable marker genes TRP1 and URA3 were present at increased levels relative to chromosomal copies of the genes. Unanticipated transcripts from the yeast CEN4 and E. coli pBR322 sequences were also found. Although much of the plasmid vector is actively transcribed in vivo, the regions around the most useful cloning sites (BamHI, EcoRI, SalI) are free of transcripts. We have analyzed transcription of BamHI inserts containing promoter variants of the HIS3 gene and determined that although initiation events are accurate, plasmid context may alter levels of gene expression. Images PMID:3909105

  13. Expression of the Serratia marcescens lipoproteins gene in Escherichia coli.

    PubMed Central

    Lee, N; Nakamura, K; Inouye, M

    1981-01-01

    The lipoprotein gene (lpp) of Serratia marcescens was cloned in a lambda phage vector (K. Nakamura and M. Inouye, Proc. Natl. Acad. Sci. U.S.A. 77: 1369-1373, 1980). This lpp gene was recloned in plasmid vectors pBR322 and pSC101. When a lipoprotein-deficient (lpp) mutant of Escherichia coli was transformed with pBR322 carrying the S. marcescens lpp gene, cells became nonleaky for ribonuclease, resistant to ethylenediaminetetraacetic acid, and sensitive to globomycin. The lipoprotein was found exclusively in the outer membrane fraction. These results indicate that the S. marcescens lipoprotein was normally secreted across the cytoplasmic membrane, modified, and assembled in the E. coli outer membrane. The amount of the free-form lipoprotein produced in this system was three times higher than that produced in lpp + C. coli cells, whereas there was no difference in the amount of the bound-form lipoprotein. On the other hand, lpp E. coli cells which harbored pSC101 carrying the S. marcescens lpp gene produced only one-third of the free-form lipoprotein produced in lpp E. coli cells which harbored pSC101 carrying the E. coli lpp gene. One of the major factors causing this difference in efficiency of gene expression between the lpp genes of S. marcescens and E. coli appears to be a deletion mutation at the transcription termination region found in the cloned S. marcescens lpp gene. The functional half-life of the S. marcescens lpp messenger ribonucleic acid in E. coli was found to be found half that of the E. coli lpp messenger ribonucleic acid. Images PMID:7016834

  14. Regulation of intestinal homeostasis and tumorigenesis by the transcriptional coregulator RIP140

    PubMed Central

    Cavaillès, Vincent; Lapierre, Marion

    2014-01-01

    Colon cancer frequently results from mutations that constitutively activate the Wnt signaling pathway, a major target being the tumor suppressor gene adenomatous polyposis coli (APC). We recently identified the transcription factor RIP140 as a new inducer of APC gene transcription that inhibits colon cancer cell growth and impedes the Wnt signaling pathway by reducing β-catenin activation. PMID:27308333

  15. Multichromatic control of gene expression in Escherichia coli

    PubMed Central

    Tabor, Jeffrey J.; Levskaya, Anselm; Voigt, Christopher A.

    2010-01-01

    Light is a powerful tool for manipulating living cells because it can be applied with high resolution across space and over time. We previously constructed a red-light sensitive E. coli transcription system based on a chimera between the red/far red switchable cyanobacterial phytochrome Cph1 and the E. coli EnvZ/OmpR two-component signaling pathways. Here we report the development of a green light inducible transcription system in E. coli based on a recently discovered green/red photoswitchable two-component system from cyanobacteria. We demonstrate that transcriptional output is proportional to the intensity of green light applied and that the green sensor is orthogonal to the red sensor at intensities of 532nm light less than 0.01W/m2. Expression of both sensors in a single cell allows two-color optical control of transcription in both batch culture and in patterns across a lawn of engineered cells. Because each sensor functions as a photoreversible switch, this system should allow the spatial and temporal control of the expression of multiple genes though different combinations of light wavelengths. This feature should aid precision single cell and population-level studies in systems and synthetic biology. PMID:21035461

  16. Comprehensive Mapping of the Escherichia coli Flagellar Regulatory Network

    PubMed Central

    Fitzgerald, Devon M.; Bonocora, Richard P.; Wade, Joseph T.

    2014-01-01

    Flagellar synthesis is a highly regulated process in all motile bacteria. In Escherichia coli and related species, the transcription factor FlhDC is the master regulator of a multi-tiered transcription network. FlhDC activates transcription of a number of genes, including some flagellar genes and the gene encoding the alternative Sigma factor FliA. Genes whose expression is required late in flagellar assembly are primarily transcribed by FliA, imparting temporal regulation of transcription and coupling expression to flagellar assembly. In this study, we use ChIP-seq and RNA-seq to comprehensively map the E. coli FlhDC and FliA regulons. We define a surprisingly restricted FlhDC regulon, including two novel regulated targets and two binding sites not associated with detectable regulation of surrounding genes. In contrast, we greatly expand the known FliA regulon. Surprisingly, 30 of the 52 FliA binding sites are located inside genes. Two of these intragenic promoters are associated with detectable noncoding RNAs, while the others either produce highly unstable RNAs or are inactive under these conditions. Together, our data redefine the E. coli flagellar regulatory network, and provide new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process. PMID:25275371

  17. Conserved rates and patterns of transcription errors across bacterial growth states and lifestyles.

    PubMed

    Traverse, Charles C; Ochman, Howard

    2016-03-22

    Errors that occur during transcription have received much less attention than the mutations that occur in DNA because transcription errors are not heritable and usually result in a very limited number of altered proteins. However, transcription error rates are typically several orders of magnitude higher than the mutation rate. Also, individual transcripts can be translated multiple times, so a single error can have substantial effects on the pool of proteins. Transcription errors can also contribute to cellular noise, thereby influencing cell survival under stressful conditions, such as starvation or antibiotic stress. Implementing a method that captures transcription errors genome-wide, we measured the rates and spectra of transcription errors in Escherichia coli and in endosymbionts for which mutation and/or substitution rates are greatly elevated over those of E. coli Under all tested conditions, across all species, and even for different categories of RNA sequences (mRNA and rRNAs), there were no significant differences in rates of transcription errors, which ranged from 2.3 × 10(-5) per nucleotide in mRNA of the endosymbiont Buchnera aphidicola to 5.2 × 10(-5) per nucleotide in rRNA of the endosymbiont Carsonella ruddii The similarity of transcription error rates in these bacterial endosymbionts to that in E. coli (4.63 × 10(-5) per nucleotide) is all the more surprising given that genomic erosion has resulted in the loss of transcription fidelity factors in both Buchnera and Carsonella. PMID:26884158

  18. Boosting transcription by transcription: enhancer-associated transcripts.

    PubMed

    Darrow, Emily M; Chadwick, Brian P

    2013-12-01

    Enhancers are traditionally viewed as DNA sequences located some distance from a promoter that act in cis and in an orientation-independent fashion to increase utilization of specific promoters and thereby regulate gene expression. Much progress has been made over the last decade toward understanding how these distant elements interact with target promoters, but how transcription is enhanced remains an object of active inquiry. Recent reports convey the prevalence and diversity of enhancer transcription and transcripts and support both as key factors with mechanistically distinct, but not mutually exclusive roles in enhancer function. Decoupling the causes and effects of transcription on the local chromatin landscape and understanding the role of enhancer transcripts in the context of long-range interactions are challenges that require additional attention. In this review, we focus on the possible functions of enhancer transcription by highlighting several recent enhancer RNA papers and, within the context of other enhancer studies, speculate on the role of enhancer transcription in regulating differential gene expression. PMID:24178450

  19. The different roles of tryptophan transfer RNA in regulating trp operon expression in E. coli versus B. subtilis.

    PubMed

    Yanofsky, Charles

    2004-08-01

    Escherichia coli and Bacillus subtilis use different mechanisms of sensing and responding to tryptophan and uncharged tRNA(Trp) as regulatory signals. In E. coli, tryptophan activates a repressor that binds to the trp promoter- operator, inhibiting transcription initiation. In B. subtilis, tryptophan activates an RNA-binding protein, TRAP, which binds to the trp operon leader RNA, causing transcription termination. In E. coli uncharged tRNA(Trp) accumulation stalls the ribosome attempting translation of tandem Trp codons in the leader-peptide coding region of the operon. This stalling permits the formation of an RNA antiterminator structure, preventing transcription termination. In B. subtilis uncharged tRNA(Trp) accumulation activates transcription and translation of the at operon. AT protein inhibits tryptophan-activated TRAP, thereby preventing TRAP-mediated transcription termination. These differences might reflect the unique organizational features of the respective trp operons and their ancestry. PMID:15262409

  20. Biophysical models of transcription in cells

    NASA Astrophysics Data System (ADS)

    Choubey, Sandeep

    Cells constantly face environmental challenges and deal with them by changing their gene expression patterns. They make decisions regarding which genes to express and which genes not to express based on intra-cellular and environmental cues. These decisions are often made by regulating the process of transcription. While the identities of the different molecules that take part in regulating transcription have been determined for a number of different genes, their dynamics inside the cell are still poorly understood. One key feature of these regulatory dynamics is that the numbers of the bio-molecules involved is typically small, resulting in large temporal fluctuations in transcriptional outputs (mRNA and protein). In this thesis I show that measurements of the cell-to-cell variability of the distribution of transcribing RNA polymerases along a gene provide a previously unexplored method for deciphering the mechanism of its transcription in vivo. First, I propose a simple kinetic model of transcription initiation and elongation from which I calculate transcribing RNA polymerase copy-number fluctuations. I test my theory against published data obtained for yeast genes and propose a novel mechanism of transcription. Rather than transcription being initiated through a single rate-limiting step, as was previously proposed, my single-cell analysis reveals the presence of at least two rate limiting steps. Second, I compute the distribution of inter-polymerase distance distribution along a gene and propose a method for analyzing inter-polymerase distance distributions acquired in experiments. By applying this method to images of polymerases transcribing ribosomal genes in E.coli I show that one model of regulation of these genes is consistent with inter-polymerase distance data while a number of other models are not. The analytical framework described in this thesis can be used to extract quantitative information about the dynamics of transcription from single

  1. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  2. Transcription in archaea

    NASA Technical Reports Server (NTRS)

    Kyrpides, N. C.; Ouzounis, C. A.; Woese, C. R. (Principal Investigator)

    1999-01-01

    Using the sequences of all the known transcription-associated proteins from Bacteria and Eucarya (a total of 4,147), we have identified their homologous counterparts in the four complete archaeal genomes. Through extensive sequence comparisons, we establish the presence of 280 predicted transcription factors or transcription-associated proteins in the four archaeal genomes, of which 168 have homologs only in Bacteria, 51 have homologs only in Eucarya, and the remaining 61 have homologs in both phylogenetic domains. Although bacterial and eukaryotic transcription have very few factors in common, each exclusively shares a significantly greater number with the Archaea, especially the Bacteria. This last fact contrasts with the obvious close relationship between the archaeal and eukaryotic transcription mechanisms per se, and in particular, basic transcription initiation. We interpret these results to mean that the archaeal transcription system has retained more ancestral characteristics than have the transcription mechanisms in either of the other two domains.

  3. Global Analysis of Extracytoplasmic Stress Signaling in Escherichia coli

    PubMed Central

    Bury-Moné, Stéphanie; Nomane, Yanoura; Reymond, Nancie; Barbet, Romain; Jacquet, Eric; Imbeaud, Sandrine; Jacq, Annick; Bouloc, Philippe

    2009-01-01

    The Bae, Cpx, Psp, Rcs, and σE pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signal-inducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response. PMID:19763168

  4. Chlamydial gene encoding a 70-kilodalton antigen in Escherichia coli: analysis of expression signals and identification of the gene product.

    PubMed Central

    Sardinia, L M; Engel, J N; Ganem, D

    1989-01-01

    In an attempt to identify chlamydial genes whose native promoters allow them to be expressed in Escherichia coli, we isolated and characterized a chlamydial gene identified by screening a library of chlamydial DNA with antichlamydial antibodies. This gene encodes a 70-kilodalton immunoreactive polypeptide in E. coli hosts. Sequence analysis of the 5' portion of the gene identified its product as the chlamydial homolog of the E. coli ribosomal protein S1. The site of transcription initiation of the mRNA in chlamydiae was determined, and its putative promoter regions were identified. These regions apparently do not function efficiently in E. coli; in vitro transcripts generated by using E. coli RNA polymerase did not start at the authentic chlamydial initiation site. Several in vitro transcripts both larger and smaller than the authentic transcript were seen; presumably, these transcripts result from adventitious promoterlike elements in adjacent chlamydial DNA and may be responsible for the expression of the gene in E. coli. Images PMID:2644193

  5. Metabolomic and transcriptomic stress response of Escherichia coli

    PubMed Central

    Jozefczuk, Szymon; Klie, Sebastian; Catchpole, Gareth; Szymanski, Jedrzej; Cuadros-Inostroza, Alvaro; Steinhauser, Dirk; Selbig, Joachim; Willmitzer, Lothar

    2010-01-01

    Environmental fluctuations lead to a rapid adjustment of the physiology of Escherichia coli, necessitating changes on every level of the underlying cellular and molecular network. Thus far, the majority of global analyses of E. coli stress responses have been limited to just one level, gene expression. Here, we incorporate the metabolite composition together with gene expression data to provide a more comprehensive insight on system level stress adjustments by describing detailed time-resolved E. coli response to five different perturbations (cold, heat, oxidative stress, lactose diauxie, and stationary phase). The metabolite response is more specific as compared with the general response observed on the transcript level and is reflected by much higher specificity during the early stress adaptation phase and when comparing the stationary phase response to other perturbations. Despite these differences, the response on both levels still follows the same dynamics and general strategy of energy conservation as reflected by rapid decrease of central carbon metabolism intermediates coinciding with downregulation of genes related to cell growth. Application of co-clustering and canonical correlation analysis on combined metabolite and transcript data identified a number of significant condition-dependent associations between metabolites and transcripts. The results confirm and extend existing models about co-regulation between gene expression and metabolites demonstrating the power of integrated systems oriented analysis. PMID:20461071

  6. Defects in polynucleotide phosphorylase impairs virulence in Escherichia coli O157:H7.

    PubMed

    Hu, Jia; Zhu, Mei-Jun

    2015-01-01

    Polynucleotide phosphorylase (PNPase) is reported to regulate virulence in Salmonella, Yersinia sp. and Campylobacter jejuni, yet its role in Escherichia coli O157:H7 has not been investigated. To gain insights into its roles in E. coli O157:H7 virulence, pnp deletion mutants were generated and the major virulence factors were compared to their parental wild type strains. Deletion of pnp in E. coli O157:H7 dramatically decreased stx2 mRNA expression and Stx2 protein production, and impaired lambdoid prophage activation in E. coli O157:H7. Quantitative PCR further confirmed that the Stx2 phage lytic growth was repressed by pnp deletion. Consistent with reduced Stx2 production and Stx2 phage activation, the transcriptional levels of genes involved in phage lysis and replication were down-regulated. In addition, disruption of pnp in E. coli O157:H7 decreased its adhesion to intestinal epithelial cells as well as cattle colonic explant tissues. On the other hand, PNPase inactivation in E. coli O157:H7 enhanced Tir protein content and the transcription of type three secretion system components, including genes encoding intimin, Tir, and EspB as well as locus of enterocyte and effacement positive regulator, Ler. Collectively, data indicate that PNPase has pleiotropic effects on the virulence of E. coli O157:H7. PMID:26347717

  7. Defects in polynucleotide phosphorylase impairs virulence in Escherichia coli O157:H7

    PubMed Central

    Hu, Jia; Zhu, Mei-Jun

    2015-01-01

    Polynucleotide phosphorylase (PNPase) is reported to regulate virulence in Salmonella, Yersinia sp. and Campylobacter jejuni, yet its role in Escherichia coli O157:H7 has not been investigated. To gain insights into its roles in E. coli O157:H7 virulence, pnp deletion mutants were generated and the major virulence factors were compared to their parental wild type strains. Deletion of pnp in E. coli O157:H7 dramatically decreased stx2 mRNA expression and Stx2 protein production, and impaired lambdoid prophage activation in E. coli O157:H7. Quantitative PCR further confirmed that the Stx2 phage lytic growth was repressed by pnp deletion. Consistent with reduced Stx2 production and Stx2 phage activation, the transcriptional levels of genes involved in phage lysis and replication were down-regulated. In addition, disruption of pnp in E. coli O157:H7 decreased its adhesion to intestinal epithelial cells as well as cattle colonic explant tissues. On the other hand, PNPase inactivation in E. coli O157:H7 enhanced Tir protein content and the transcription of type three secretion system components, including genes encoding intimin, Tir, and EspB as well as locus of enterocyte and effacement positive regulator, Ler. Collectively, data indicate that PNPase has pleiotropic effects on the virulence of E. coli O157:H7. PMID:26347717

  8. Transcription Regulation in Archaea.

    PubMed

    Gehring, Alexandra M; Walker, Julie E; Santangelo, Thomas J

    2016-07-15

    The known diversity of metabolic strategies and physiological adaptations of archaeal species to extreme environments is extraordinary. Accurate and responsive mechanisms to ensure that gene expression patterns match the needs of the cell necessitate regulatory strategies that control the activities and output of the archaeal transcription apparatus. Archaea are reliant on a single RNA polymerase for all transcription, and many of the known regulatory mechanisms employed for archaeal transcription mimic strategies also employed for eukaryotic and bacterial species. Novel mechanisms of transcription regulation have become apparent by increasingly sophisticated in vivo and in vitro investigations of archaeal species. This review emphasizes recent progress in understanding archaeal transcription regulatory mechanisms and highlights insights gained from studies of the influence of archaeal chromatin on transcription. PMID:27137495

  9. Transcription is regulated by NusA:NusG interaction

    PubMed Central

    Strauß, Martin; Vitiello, Christal; Schweimer, Kristian; Gottesman, Max; Rösch, Paul; Knauer, Stefan H.

    2016-01-01

    NusA and NusG are major regulators of bacterial transcription elongation, which act either in concert or antagonistically. Both bind to RNA polymerase (RNAP), regulating pausing as well as intrinsic and Rho-dependent termination. Here, we demonstrate by nuclear magnetic resonance spectroscopy that the Escherichia coli NusG amino-terminal domain forms a complex with the acidic repeat domain 2 (AR2) of NusA. The interaction surface of either transcription factor overlaps with the respective binding site for RNAP. We show that NusA-AR2 is able to remove NusG from RNAP. Our in vivo and in vitro results suggest that interaction between NusA and NusG could play various regulatory roles during transcription, including recruitment of NusG to RNAP, resynchronization of transcription:translation coupling, and modulation of termination efficiency. PMID:27174929

  10. Transcription is regulated by NusA:NusG interaction.

    PubMed

    Strauß, Martin; Vitiello, Christal; Schweimer, Kristian; Gottesman, Max; Rösch, Paul; Knauer, Stefan H

    2016-07-01

    NusA and NusG are major regulators of bacterial transcription elongation, which act either in concert or antagonistically. Both bind to RNA polymerase (RNAP), regulating pausing as well as intrinsic and Rho-dependent termination. Here, we demonstrate by nuclear magnetic resonance spectroscopy that the Escherichia coli NusG amino-terminal domain forms a complex with the acidic repeat domain 2 (AR2) of NusA. The interaction surface of either transcription factor overlaps with the respective binding site for RNAP. We show that NusA-AR2 is able to remove NusG from RNAP. Our in vivo and in vitro results suggest that interaction between NusA and NusG could play various regulatory roles during transcription, including recruitment of NusG to RNAP, resynchronization of transcription:translation coupling, and modulation of termination efficiency. PMID:27174929

  11. WRKY transcription factors

    PubMed Central

    Bakshi, Madhunita; Oelmüller, Ralf

    2014-01-01

    WRKY transcription factors are one of the largest families of transcriptional regulators found exclusively in plants. They have diverse biological functions in plant disease resistance, abiotic stress responses, nutrient deprivation, senescence, seed and trichome development, embryogenesis, as well as additional developmental and hormone-controlled processes. WRKYs can act as transcriptional activators or repressors, in various homo- and heterodimer combinations. Here we review recent progress on the function of WRKY transcription factors in Arabidopsis and other plant species such as rice, potato, and parsley, with a special focus on abiotic, developmental, and hormone-regulated processes. PMID:24492469

  12. Plant transcription factors.

    PubMed

    Meshi, T; Iwabuchi, M

    1995-12-01

    Transcriptional regulation of gene expression relies on the recognition of promoter elements by transcription factors. In the past several years, a considerable number of (putative) transcription factors have been identified in plants. Some genes coding for these factors were isolated by south-western screening with oligonucleotides as a probe or by homology-based screening, and others were initially isolated by genetic means and subsequently identified as the genes for transcription factors. These transcription factors often form families of structurally related proteins with similar DNA-binding specificities and in addition, they are sometimes involved in related phenomena. Some groups of factors homo- and/or heterodimerize to increase the length and variability of the target sequences. Transcriptional activators, in general, comprise a modular activation domain. The activities of the transcription factors are controlled by post-translational modification, like phosphorylation and glycosylation, as well as at the levels of nuclear transport, oligomerization, etc. In this review, we will summarize the current knowledge of plant transcription factors to help understand the mechanistic aspects of the transcriptional regulation of genes. PMID:8589926

  13. Transcriptional activators in yeast

    PubMed Central

    2006-01-01

    Eukaryotic transcription activation domains (ADs) are not well defined on the proteome scale. We systematicallly tested ∼6000 yeast proteins for transcriptional activity using a yeast one-hybrid system and identified 451 transcriptional activators. We then determined their transcription activation strength using fusions to the Gal4 DNA-binding domain and a His3 reporter gene which contained a promoter with a Gal4-binding site. Among the 132 strongest activators 32 are known transcription factors while another 35 have no known function. Although zinc fingers, helix–loop–helix domains and several other domains are highly overrepresented among the activators, only few contain characterized ADs. We also found some striking correlations: the stronger the activation activity, the more acidic, glutamine-rich, proline-rich or asparagine-rich the activators were. About 29% of the activators have been found previously to specifically interact with the transcription machinery, while 10% are known to be components of transcription regulatory complexes. Based on their transcriptional activity, localization and interaction patterns, at least six previously uncharacterized proteins are suggested to be bona fide transcriptional regulators (namely YFL049W, YJR070C, YDR520C, YGL066W/Sgf73, YKR064W and YCR082W/Ahc2). PMID:16464826

  14. DISTILLER: a data integration framework to reveal condition dependency of complex regulons in Escherichia coli

    PubMed Central

    Lemmens, Karen; De Bie, Tijl; Dhollander, Thomas; De Keersmaecker, Sigrid C; Thijs, Inge M; Schoofs, Geert; De Weerdt, Ami; De Moor, Bart; Vanderleyden, Jos; Collado-Vides, Julio; Engelen, Kristof; Marchal, Kathleen

    2009-01-01

    We present DISTILLER, a data integration framework for the inference of transcriptional module networks. Experimental validation of predicted targets for the well-studied fumarate nitrate reductase regulator showed the effectiveness of our approach in Escherichia coli. In addition, the condition dependency and modularity of the inferred transcriptional network was studied. Surprisingly, the level of regulatory complexity seemed lower than that which would be expected from RegulonDB, indicating that complex regulatory programs tend to decrease the degree of modularity. PMID:19265557

  15. Unusually Situated Binding Sites for Bacterial Transcription Factors Can Have Hidden Functionality

    PubMed Central

    Haycocks, James R. J.; Grainger, David C.

    2016-01-01

    A commonly accepted paradigm of molecular biology is that transcription factors control gene expression by binding sites at the 5' end of a gene. However, there is growing evidence that transcription factor targets can occur within genes or between convergent genes. In this work, we have investigated one such target for the cyclic AMP receptor protein (CRP) of enterotoxigenic Escherichia coli. We show that CRP binds between two convergent genes. When bound, CRP regulates transcription of a small open reading frame, which we term aatS, embedded within one of the adjacent genes. Our work demonstrates that non-canonical sites of transcription factor binding can have hidden functionality. PMID:27258043

  16. An arcane role of DNA in transcription activation.

    PubMed Central

    Ryu, S; Garges, S; Adhya, S

    1994-01-01

    The mechanism by which the cAMP receptor protein (CRP) activates transcription has been investigated using the lac promoter of Escherichia coli. For transcription activation, an interaction between DNA-bound CRP and RNA polymerase is not sufficient. CRP must bind to a site in the same DNA and close to the promoter. CRP action requires an intact spacer DNA to provide a rigid support in building a CRP-RNA polymerase protein bridge or to allow a conformational change in the DNA to be transmitted to the lac promoter using the protein bridge as a structural support. Images PMID:7811325

  17. The chemical structure of DNA sequence signals for RNA transcription

    NASA Technical Reports Server (NTRS)

    George, D. G.; Dayhoff, M. O.

    1982-01-01

    The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

  18. The extracellular RNA complement of Escherichia coli

    PubMed Central

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-01

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. PMID:25611733

  19. Divergent RNA transcription

    PubMed Central

    Naughton, Catherine; Corless, Samuel; Gilbert, Nick

    2013-01-01

    New approaches using biotinylated-psoralen as a probe for investigating DNA structure have revealed new insights into the relationship between DNA supercoiling, transcription and chromatin compaction. We explore a hypothesis that divergent RNA transcription generates negative supercoiling at promoters facilitating initiation complex formation and subsequent promoter clearance. PMID:23863199

  20. Emerging Enteropathogenic Escherichia coli Strains?

    PubMed Central

    Irino, Kinue; Girão, Dennys M.; Girão, Valéria B.C.; Guth, Beatriz E.C.; Vaz, Tânia M.I.; Moreira, Fabiana C.; Chinarelli, Silvia H.; Vieira, Mônica A.M.

    2004-01-01

    Escherichia coli strains of nonenteropathogenic serogroups carrying eae but lacking the enteropathogenic E. coli adherence factor plasmid and Shiga toxin DNA probe sequences were isolated from patients (children, adults, and AIDS patients) with and without diarrhea in Brazil. Although diverse in phenotype and genotype, some strains are potentially diarrheagenic. PMID:15504277

  1. A DNA structural atlas for Escherichia coli.

    PubMed

    Pedersen, A G; Jensen, L J; Brunak, S; Staerfeldt, H H; Ussery, D W

    2000-06-16

    We have performed a computational analysis of DNA structural features in 18 fully sequenced prokaryotic genomes using models for DNA curvature, DNA flexibility, and DNA stability. The structural values that are computed for the Escherichia coli chromosome are significantly different from (and generally more extreme than) that expected from the nucleotide composition. To aid this analysis, we have constructed tools that plot structural measures for all positions in a long DNA sequence (e.g. an entire chromosome) in the form of color-coded wheels (http://www.cbs.dtu. dk/services/GenomeAtlas/). We find that these "structural atlases" are useful for the discovery of interesting features that may then be investigated in more depth using statistical methods. From investigation of the E. coli structural atlas, we discovered a genome-wide trend, where an extended region encompassing the terminus displays a high of level curvature, a low level of flexibility, and a low degree of helix stability. The same situation is found in the distantly related Gram-positive bacterium Bacillus subtilis, suggesting that the phenomenon is biologically relevant. Based on a search for long DNA segments where all the independent structural measures agree, we have found a set of 20 regions with identical and very extreme structural properties. Due to their strong inherent curvature, we suggest that these may function as topological domain boundaries by efficiently organizing plectonemically supercoiled DNA. Interestingly, we find that in practically all the investigated eubacterial and archaeal genomes, there is a trend for promoter DNA being more curved, less flexible, and less stable than DNA in coding regions and in intergenic DNA without promoters. This trend is present regardless of the absolute levels of the structural parameters, and we suggest that this may be related to the requirement for helix unwinding during initiation of transcription, or perhaps to the previously observed

  2. Microarray analysis of norepinephrine grown E. coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To examine differences in the transcription profile of Escherichia coli O157:H7 during growth in the presence of catecholamines, the clinical-type isolate EDL933 (ATCC 43895) was grown using an in-vitro culture system made up of moderate initial inoculum and a serum-SAPI based medium. After 5 hrs of...

  3. Incomplete flagellar structures in Escherichia coli mutants.

    PubMed Central

    Suzuki, T; Komeda, Y

    1981-01-01

    Escherichia coli mutants with defects in 29 flagellar genes identified so far were examined by electron microscopy for possession of incomplete flagellar structures in membrane-associated fractions. The results are discussed in consideration of the known transcriptional interaction of flagellar genes. Hook-basal body structures were detected in flaD, flaS, flaT, flbC, and hag mutants. The flaE mutant had a polyhook-basal body structure. An intact basal body appeared in flaK mutants. Putative precursors of the basal body were detected in mutants with defects in flaM, flaU, flaV, and flaY. No structures homologous to the flagellar basal body or its parts were detected in mutants with defects in flaA, flaB, flaC, flaG, flaH, flaI, flaL, flaN, flaO, flaP, flaQ, flaR, flaW, flaX, flbA, flbB, and flbD. One flaZ mutant had an incomplete flagellar basal body structure and another formed no significant structure, suggesting that flaZ is responsible for both basal body assembly and the transcription of the hag gene. Images PMID:7007337

  4. ASTP Onboard Voice Transcription

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The transcription is presented of the Apollo-Soyuz Test Project voice communications as recorded on the command module data storage equipment. Data from this recorder are telemetered (dumped) to Space Tracking and Data Network sites for retransmission to the Johnson Space Center. The transcript is divided into three columns -- time, speaker, and text. The Greenwich mean time column consists of three two-digit numbers representing hours, minutes, and seconds (e.g., 22 34 14) for the Julian dates shown at the top of the page on which a new day begins. The speaker column indicates the source of a transmission; the text column contains the verbatim transcript of the communications.

  5. Transcriptional analysis and regulatory signals of the hom-thrB cluster of Brevibacterium lactofermentum.

    PubMed Central

    Mateos, L M; Pisabarro, A; Pátek, M; Malumbres, M; Guerrero, C; Eikmanns, B J; Sahm, H; Martín, J F

    1994-01-01

    Two genes, hom (encoding homoserine dehydrogenase) and thrB (encoding homoserine kinase), of the threonine biosynthetic pathway are clustered in the chromosome of Brevibacterium lactofermentum in the order 5' hom-thrB 3', separated by only 10 bp. The Brevibacterium thrB gene is expressed in Escherichia coli, in Brevibacterium lactofermentum, and in Corynebacterium glutamicum and complements auxotrophs of all three organisms deficient in homoserine kinase, whereas the Brevibacterium hom gene did not complement two different E. coli auxotrophs lacking homoserine dehydrogenase. However, complementation was obtained when the homoserine dehydrogenase was expressed as a fusion protein in E. coli. Northern (RNA) analysis showed that the hom-thrB cluster is transcribed, giving two different transcripts of 2.5 and 1.1 kb. The 2.5-kb transcript corresponds to the entire cluster hom-thrB (i.e., they form a bicistronic operon), and the short transcript (1.1 kb) originates from the thrB gene. The promoter in front of hom and the hom-internal promoter in front of thrB were subcloned in promoter-probe vectors of E. coli and corynebacteria. The thrB promoter is efficiently recognized both in E. coli and corynebacteria, whereas the hom promoter is functional in corynebacteria but not in E. coli. The transcription start points of both promoters have been identified by primer extension and S1 mapping analysis. The thrB promoter was located in an 87-bp fragment that overlaps with the end of the hom gene. A functional transcriptional terminator located downstream from the cluster was subcloned in terminator-probe vectors. Images PMID:7961509

  6. Common evolutionary origin of the ilvGMEDA attenuation locus and tRNA(1Leu) in Escherichia coli.

    PubMed

    Williamson, R M; Jackson, J H

    1987-06-01

    Published sequences of transcripts from ilvGMEDA leader regions of several enteric bacteria were compared with published sequences of the tRNAs from Escherichia coli. The analyses revealed homology between the ilvGMEDA leader peptide-coding region and tRNA(1Leu) in E. coli, Salmonella typhimurium, and Klebsiella aerogenes, whereas homology was not present in Serratia marcescens and Edwardsiella tarda. PMID:3294812

  7. Photoreactivation of Escherichia coli is impaired at high growth temperatures.

    PubMed

    Xu, Lei; Tian, Changqing; Lu, Xiaohua; Ling, Liefeng; Lv, Jun; Wu, Mingcai; Zhu, Guoping

    2015-06-01

    Photolyase repairs UV-induced lesions in DNA using light energy, which is the principle of photoreactivation. Active photolyase contains the two-electron-reduced flavin cofactor. We observed that photoreactivation of Escherichia coli was impaired at growth temperatures ⩾37°C, and growth in this temperature range also resulted in decreased photolyase protein levels in the cells. However, the levels of phr transcripts (encoding photolyase) were almost unchanged at the various growth temperatures. A lacZ-reporter under transcriptional control of the phr promoter showed no temperature-dependent expression. However, a translational reporter consisting of the photolyase N-terminal α/β domain-LacZ fusion protein exhibited lower β-galactosidase activity at high growth temperatures (37-42°C). These results indicated that the change in photolyase levels at different growth temperatures is post-transcriptional in nature. Limited proteolysis identified several susceptible cleavage sites in E. coli photolyase. In vitro differential scanning calorimetry and activity assays revealed that denaturation of active photolyase occurs at temperatures ⩾37°C, while apo-photolyase unfolds at temperatures ⩾25°C. Evidence from temperature-shift experiments also implies that active photolyase is protected from thermal unfolding and proteolysis in vivo, even at 42°C. These results suggest that thermal unfolding and proteolysis of newly synthesized apo-photolyase, but not active photolyase, is responsible for the impaired photoreactivation at high growth temperatures (37-42°C). PMID:25839748

  8. Thiophene metabolism by E. coli

    SciTech Connect

    Clark, D.P.

    1990-01-01

    The objective of this project is to investigate the mechanism of degradation of sulfur containing heterocyclic molecules by mutants of Escherichia coli K-12. We previously isolated multiple mutants of E. coli which were selected for improved oxidation of furan and thiophene derivatives. We have focused on the thdA mutation in our subsequent research as it appears to be of central importance in thiophene oxidation. We hope that analysis of the thd gene of E. coli will lead to improvement of our thiophene metabolizing bacterial strains.

  9. Thiophene metabolism by E. coli

    SciTech Connect

    Clark, D.P.

    1990-01-01

    The objective of this project is to investigate the mechanism of degradation of sulfur containing heterocyclic molecules such as those found in coal, by mutants of Escherichia coli K-12. We previously isolated multiple mutants of E. coli which were selected for improved oxidation of furan and thiophene derivatives. We have focused on the thdA mutation in our subsequent research as it appears to be of central importance in thiophene oxidation. We hope that analysis of the thd genes of E. coli will lead to improvement of our thiophene metabolizing bacterial strains. 1 tab.

  10. The impact of transcriptional tuning on in vitro integrated rRNA transcription and ribosome construction

    PubMed Central

    Fritz, Brian R.; Jewett, Michael C.

    2014-01-01

    In vitro ribosome construction could enable studies of ribosome assembly and function, provide a route toward constructing minimal cells for synthetic biology, and permit the construction of ribosome variants with new functions. Toward these long-term goals, we recently reported on an integrated, one-pot ribosomal RNA synthesis (rRNA), ribosome assembly, and translation technology (termed iSAT) for the construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. Here, we aimed to improve the activity of iSAT through transcriptional tuning. Specifically, we increased transcriptional efficiency through 3′ modifications to the rRNA gene sequences, optimized plasmid and polymerase concentrations, and demonstrated the use of a T7-promoted rRNA operon for stoichiometrically balanced rRNA synthesis and native rRNA processing. Our modifications produced a 45-fold improvement in iSAT protein synthesis activity, enabling synthesis of 429 ± 15 nmol/l green fluorescent protein in 6 h batch reactions. Further, we show that the translational activity of ribosomes purified from iSAT reactions is about 20% the activity of native ribosomes purified directly from E. coli cells. Looking forward, we believe iSAT will enable unique studies to unravel the systems biology of ribosome biogenesis and open the way to new methods for making and studying ribosomal variants. PMID:24792158

  11. The response of Escherichia coli to exposure to the biocide polyhexamethylene biguanide.

    PubMed

    Allen, Michael J; White, Graham F; Morby, Andrew P

    2006-04-01

    The global response of Escherichia coli to the broad-spectrum biocide polyhexamethylene biguanide (PHMB) was investigated using transcriptional profiling. The transcriptional analyses were validated by direct determination of the PHMB-tolerance phenotypes of derivatives of E. coli MG1655 carrying either insertionally inactivated genes and/or plasmids expressing the cognate open reading frames from a heterologous promoter in the corresponding chromosomally inactivated strains. The results showed that a wide range of genes was altered in transcriptional activity and that all of the corresponding knockout strains subsequently challenged with biocide were altered in tolerance. Of particular interest was the induction of the rhs genes and the implication of enzymes involved in the repair/binding of nucleic acids in the generation of tolerance, suggesting a novel dimension in the mechanism of action of PHMB based on its interaction with nucleic acids. PMID:16549663

  12. Endogenous protein S-Nitrosylation in E. coli: regulation by OxyR.

    PubMed

    Seth, Divya; Hausladen, Alfred; Wang, Ya-Juan; Stamler, Jonathan S

    2012-04-27

    Endogenous S-nitrosylation of proteins, a principal mechanism of cellular signaling in eukaryotes, has not been observed in microbes. We report that protein S-nitrosylation is an obligate concomitant of anaerobic respiration on nitrate in Escherichia coli. Endogenous S-nitrosylation during anaerobic respiration is controlled by the transcription factor OxyR, previously thought to operate only under aerobic conditions. Deletion of OxyR resulted in large increases in protein S-nitrosylation, and S-nitrosylation of OxyR induced transcription from a regulon that is distinct from the regulon induced by OxyR oxidation. Furthermore, products unique to the anaerobic regulon protected against S-nitrosothiols, and anaerobic growth of E. coli lacking OxyR was impaired on nitrate. Thus, OxyR serves as a master regulator of S-nitrosylation, and alternative posttranslational modifications of OxyR control distinct transcriptional responses. PMID:22539721

  13. Transformation and characterization of an arsenic gene operon from urease-positive thermophilic Campylobacter (UPTC) in Escherichia coli.

    PubMed

    Matsuda, M; Kuribayashi, T; Yamamoto, S; Millar, B C; Moore, J E

    2016-01-01

    An arsenate susceptibility test was performed with transformed and cultured Escherichia coli DH5α cells, which carried recombinant DNA of full-length arsenic (ars) operon, namely a putative membrane permease, ArsP; a transcriptional repressor, ArsR; an arsenate reductase, ArsC; and an arsenical-resistance membrane transporter, Acr3, from the Japanese urease-positive thermophilic Campylobacter lari (UPTC) CF89-12. The E. coli DH5α transformant showed reduced susceptibility to arsenate (~1536 μg/mL), compared to the control. Thus, these ars four-genes from the UPTC CF89-12 strain cells could confer a reduced susceptibility to arsenate in the transformed and E. coli DH5α cells. E. coli transformants with truncated ars operons, acr3 (acr3) and arsC-acr3 (∆arsC-acr3), of the ars operon, showed an MIC value of 384 μg/mL (~384 μg/mL), similar to the E. coli cells which carried the pGEM-T vector (control). Reverse transcription PCR confirmed in vivo transcription of recombinant full-length ars operon and deletion variants (∆acr3 and ∆arsC-acr3) in the transformed E. coli cells. PMID:26122364

  14. A novel bacteriophage-encoded RNA polymerase binding protein inhibits transcription initiation and abolishes transcription termination by host RNA polymerase.

    PubMed

    Nechaev, Sergei; Yuzenkova, Yulia; Niedziela-Majka, Anita; Heyduk, Tomasz; Severinov, Konstantin

    2002-06-28

    Xp10 is a lytic bacteriophage of Xanthomonas oryzae, a Gram-negative bacterium that causes rice blight. We purified an Xp10 protein, p7, that binds to and inhibits X. oryzae RNA polymerase (RNAP). P7 is a novel 73 amino acid-long protein; it does not bind to and hence does not affect transcription by Escherichia coli RNAP. Analysis of E. coli/X. oryzae RNAP hybrids locates the p7 binding site to the largest X. oryzae RNAP subunit, beta'. Binding of p7 to X. oryzae RNAP holoenzyme prevents large conformational change that places the sigma subunit region 4 into the correct position for interaction with the -35 promoter element. As a result, open promoter complex formation on the -10/-35 class promoters is inhibited. Inhibition of promoter complex formation on the extended -10 class promoters is less efficient. The p7 protein also abolishes factor-independent transcription termination by X. oryzae RNAP by preventing the release of nascent RNA at terminators. Further physiological and mechanistic studies of this novel transcription factor should provide additional insights into its biological role and the processes of promoter recognition and transcription termination. PMID:12079331

  15. Pneumatosis coli in a dog.

    PubMed

    Russell, Nicholas J; Tyrrell, Dayle; Irwin, Peter J; Beck, Catherine

    2008-01-01

    A 17-year-old, castrated male Maltese was presented with chronic polyphagia and a 2-week history of tenesmus, diarrhea, hematochezia, weight loss, and ribbon-like feces. Pneumatosis coli was diagnosed by abdominal radiography. Concurrent hyperadrenocorticism was suspected. The clinical signs of colorectal disease resolved within 2 days of initiating a lowresidue diet and oral metronidazole. Pneumatosis coli should be considered as a differential diagnosis for colorectal disease in dogs. PMID:18175797

  16. Soluble expression and stability enhancement of transcription factors using 30Kc19 cell-penetrating protein.

    PubMed

    Ryu, Jina; Park, Hee Ho; Park, Ju Hyun; Lee, Hong Jai; Rhee, Won Jong; Park, Tai Hyun

    2016-04-01

    Transcription factors have been studied as an important drug candidate. Ever since the successful generation of induced pluripotent stem cells (iPSCs), there has been tremendous interest in reprogramming transcription factors. Because of the safety risks involved in a virus-based approach, many researchers have been trying to deliver transcription factors using nonintegrating materials. Thus, delivery of transcription factors produced as recombinant proteins in E. coli was proposed as an alternative method. However, the low level of soluble expression and instability of such recombinant proteins are potential barriers. We engineered a Bombyx mori 30Kc19 protein as a fusion partner for transcription factors to overcome those problems. We have previously reported that 30Kc19 protein can be produced as a soluble form in E. coli and has a cell-penetrating property and a protein-stabilizing effect. Transcription factors fused with 30Kc19 (Oct4-30Kc19, Sox2-30Kc19, c-Myc-30Kc19, L-Myc-30Kc19, and Klf4-30Kc19) were produced as recombinant proteins. Interestingly, Oct4 and L-Myc were expressed as a soluble form by conjugating with 30Kc19 protein, whereas Oct4 alone and L-Myc alone aggregated. The 30Kc19 protein also enhanced the stability of transcription factors both in vitro and in cells. In addition, 30Kc19-conjugated transcription factors showed rapid delivery into cells and transcriptional activity significantly increased. Overall, 30Kc19 protein conjugation simultaneously enhanced soluble expression, stability, and transcriptional activity of transcription factors. We propose that the conjugation with 30Kc19 protein is a novel approach to solve the technical bottleneck of gene regulation using transcription factors. PMID:26668030

  17. Regulation of transcription by 6S RNAs

    PubMed Central

    Steuten, Benedikt; Hoch, Philipp G; Damm, Katrin; Schneider, Sabine; Köhler, Karen; Wagner, Rolf; Hartmann, Roland K

    2014-01-01

    Whereas, the majority of bacterial non-coding RNAs and functional RNA elements regulate post-transcriptional processes, either by interacting with other RNAs via base-pairing or through binding of small ligands (riboswitches), 6S RNAs affect transcription itself by binding to the housekeeping holoenzyme of RNA polymerase (RNAP). Remarkably, 6S RNAs serve as RNA templates for bacterial RNAP, giving rise to the de novo synthesis of short transcripts, termed pRNAs (product RNAs). Hence, 6S RNAs prompt the enzyme to act as an RNA-dependent RNA polymerase (RdRP). Synthesis of pRNAs exceeding a certain length limit (~13 nt) persistently rearrange the 6S RNA structure, which in turn, disrupts the 6S RNA:RNAP complex. This pRNA synthesis-mediated “reanimation” of sequestered RNAP molecules represents the conceivably fastest mechanism for resuming transcription in cells that enter a new exponential growth phase. The many different 6S RNAs found in a wide variety of bacteria do not share strong sequence homology but have in common a conserved rod-shaped structure with a large internal loop, termed the central bulge; this architecture mediates specific binding to the active site of RNAP. In this article, we summarize the overall state of knowledge as well as very recent findings on the structure, function, and physiological effects of 6S RNA examples from the two model organisms, Escherichia coli and Bacillus subtilis. Comparison of the presently known properties of 6S RNAs in the two organisms highlights common principles as well as diverse features. PMID:24786589

  18. Selection Shapes Transcriptional Logic and Regulatory Specialization in Genetic Networks

    PubMed Central

    Fogelmark, Karl; Peterson, Carsten; Troein, Carl

    2016-01-01

    Background Living organisms need to regulate their gene expression in response to environmental signals and internal cues. This is a computational task where genes act as logic gates that connect to form transcriptional networks, which are shaped at all scales by evolution. Large-scale mutations such as gene duplications and deletions add and remove network components, whereas smaller mutations alter the connections between them. Selection determines what mutations are accepted, but its importance for shaping the resulting networks has been debated. Methodology To investigate the effects of selection in the shaping of transcriptional networks, we derive transcriptional logic from a combinatorially powerful yet tractable model of the binding between DNA and transcription factors. By evolving the resulting networks based on their ability to function as either a simple decision system or a circadian clock, we obtain information on the regulation and logic rules encoded in functional transcriptional networks. Comparisons are made between networks evolved for different functions, as well as with structurally equivalent but non-functional (neutrally evolved) networks, and predictions are validated against the transcriptional network of E. coli. Principal Findings We find that the logic rules governing gene expression depend on the function performed by the network. Unlike the decision systems, the circadian clocks show strong cooperative binding and negative regulation, which achieves tight temporal control of gene expression. Furthermore, we find that transcription factors act preferentially as either activators or repressors, both when binding multiple sites for a single target gene and globally in the transcriptional networks. This separation into positive and negative regulators requires gene duplications, which highlights the interplay between mutation and selection in shaping the transcriptional networks. PMID:26927540

  19. Autogenous Regulation of Splicing of the Transcript of a Yeast Ribosomal Protein Gene

    NASA Astrophysics Data System (ADS)

    Dabeva, Mariana D.; Post-Beittenmiller, Martha A.; Warner, Jonathan R.

    1986-08-01

    The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

  20. Enterohemorrhagic Escherichia coli Adhesins.

    PubMed

    McWilliams, Brian D; Torres, Alfredo G

    2014-06-01

    Adhesins are a group of proteins in enterohemorrhagic Escherichia coli (EHEC) that are involved in the attachment or colonization of this pathogen to abiotic (plastic or steel) and biological surfaces, such as those found in bovine and human intestines. This review provides the most up-to-date information on these essential adhesion factors, summarizing important historical discoveries and analyzing the current and future state of this research. In doing so, the proteins intimin and Tir are discussed in depth, especially regarding their role in the development of attaching and effacing lesions and in EHEC virulence. Further, a series of fimbrial proteins (Lpf1, Lpf2, curli, ECP, F9, ELF, Sfp, HCP, and type 1 fimbria) are also described, emphasizing their various contributions to adherence and colonization of different surfaces and their potential use as genetic markers in detection and classification of different EHEC serotypes. This review also discusses the role of several autotransporter proteins (EhaA-D, EspP, Saa and Sab, and Cah), as well as other proteins associated with adherence, such as flagella, EibG, Iha, and OmpA. While these proteins have all been studied to varying degrees, all of the adhesins summarized in this article have been linked to different stages of the EHEC life cycle, making them good targets for the development of more effective diagnostics and therapeutics. PMID:26103974

  1. Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit.

    PubMed Central

    Hartmann, R K; Erdmann, V A

    1989-01-01

    A cloned 16S rRNA gene from the extreme thermophilic eubacterium Thermus thermophilus HB8 was used to characterize the in vivo expression of the 16S rRNA genes in this organism by nuclease S1 mapping. The gene represents an isolated transcription unit encoding solely 16S rRNA. Under exponential growth conditions, transcription was initiated at a single promoter, which represents the structural equivalent of Escherichia coli rrn P2 promoters. The promoter-leader region was very similar to the E. coli rrn P2 promoter-leader segment that is responsible for antitermination. The T. thermophilus leader region was approximately 85 nucleotides shorter than its E. coli P2 counterpart. Potential processing intermediates were correlated with a proposed secondary structure of T. thermophilus pre-16S rRNA. Images PMID:2722737

  2. Genetic and biochemical analysis of peptide transport in Escherichia coli

    SciTech Connect

    Andrews, J.C.

    1986-01-01

    E. coli peptide transport mutants have been isolated based on their resistance to toxic tripeptides. These genetic defects were found to map in two distinct chromosomal locations. The transport systems which require expression of the trp-linked opp genes and the oppE gene(s) for activity were shown to have different substrate preferences. Growth of E. coli in medium containing leucine results in increased entry of exogenously supplied tripeptides into the bacterial cell. This leucine-mediated elevation of peptide transport required expression of the trp-linked opp operon and was accompanied by increased sensitivity to toxic tripeptides, by an enhanced capacity to utilize nutritional peptides, and by an increase in both the velocity and apparent steady-state level of L-(U-/sup 14/C)alanyl-L-alanyl-L-alanine accumulation for E. coli grown in leucine-containing medium relative to these parameters of peptide transport measured with bacteria grown in media lacking leucine. Direct measurement of opp operon expression by pulse-labeling experiments demonstrated that growth of E. coli in the presence of leucine resulted in increased synthesis of the oppA-encoded periplasmic binding protein. The transcriptional regulation of the trp-linked opp operon of E. coli was investigated using lambda placMu51-generated lac operon fusions. Synthesis of ..beta..-galactosidase by strains harboring oppA-lac, oppB-lac, and oppD-lac fusions occurred at a basal level when the fusion-containing strains were grown in minimal medium.

  3. The N-degradome of Escherichia coli

    PubMed Central

    Humbard, Matthew A.; Surkov, Serhiy; De Donatis, Gian Marco; Jenkins, Lisa M.; Maurizi, Michael R.

    2013-01-01

    The N-end rule is a conserved mechanism found in Gram-negative bacteria and eukaryotes for marking proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In Escherichia coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. As ClpS recognizes N-terminal Phe, Trp, Tyr, and Leu, which are not found at the N terminus of proteins translated and processed by the canonical pathway, proteins must be post-translationally modified to expose an N-degron. One modification is catalyzed by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine; however, such proteins are also not generated by the canonical protein synthesis pathway. Thus, the mechanisms producing N-degrons in proteins and the frequency of their occurrence largely remain a mystery. To address these issues, we used a ClpS affinity column to isolate interacting proteins from E. coli cell lysates under non-denaturing conditions. We identified more than 100 proteins that differentially bound to a column charged with wild-type ClpS and eluted with a peptide bearing an N-degron. Thirty-two of 37 determined N-terminal peptides had N-degrons. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates. PMID:23960079

  4. Thiophenone Attenuates Enteropathogenic Escherichia coli O103:H2 Virulence by Interfering with AI-2 Signaling.

    PubMed

    Witsø, Ingun Lund; Valen Rukke, Håkon; Benneche, Tore; Aamdal Scheie, Anne

    2016-01-01

    Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli. PMID:27309855

  5. Thiophenone Attenuates Enteropathogenic Escherichia coli O103:H2 Virulence by Interfering with AI-2 Signaling

    PubMed Central

    Valen Rukke, Håkon; Benneche, Tore; Aamdal Scheie, Anne

    2016-01-01

    Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli. PMID:27309855

  6. The Transcription Factor Encyclopedia

    PubMed Central

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe. PMID:22458515

  7. The transcription factor encyclopedia.

    PubMed

    Yusuf, Dimas; Butland, Stefanie L; Swanson, Magdalena I; Bolotin, Eugene; Ticoll, Amy; Cheung, Warren A; Zhang, Xiao Yu Cindy; Dickman, Christopher T D; Fulton, Debra L; Lim, Jonathan S; Schnabl, Jake M; Ramos, Oscar H P; Vasseur-Cognet, Mireille; de Leeuw, Charles N; Simpson, Elizabeth M; Ryffel, Gerhart U; Lam, Eric W-F; Kist, Ralf; Wilson, Miranda S C; Marco-Ferreres, Raquel; Brosens, Jan J; Beccari, Leonardo L; Bovolenta, Paola; Benayoun, Bérénice A; Monteiro, Lara J; Schwenen, Helma D C; Grontved, Lars; Wederell, Elizabeth; Mandrup, Susanne; Veitia, Reiner A; Chakravarthy, Harini; Hoodless, Pamela A; Mancarelli, M Michela; Torbett, Bruce E; Banham, Alison H; Reddy, Sekhar P; Cullum, Rebecca L; Liedtke, Michaela; Tschan, Mario P; Vaz, Michelle; Rizzino, Angie; Zannini, Mariastella; Frietze, Seth; Farnham, Peggy J; Eijkelenboom, Astrid; Brown, Philip J; Laperrière, David; Leprince, Dominique; de Cristofaro, Tiziana; Prince, Kelly L; Putker, Marrit; del Peso, Luis; Camenisch, Gieri; Wenger, Roland H; Mikula, Michal; Rozendaal, Marieke; Mader, Sylvie; Ostrowski, Jerzy; Rhodes, Simon J; Van Rechem, Capucine; Boulay, Gaylor; Olechnowicz, Sam W Z; Breslin, Mary B; Lan, Michael S; Nanan, Kyster K; Wegner, Michael; Hou, Juan; Mullen, Rachel D; Colvin, Stephanie C; Noy, Peter John; Webb, Carol F; Witek, Matthew E; Ferrell, Scott; Daniel, Juliet M; Park, Jason; Waldman, Scott A; Peet, Daniel J; Taggart, Michael; Jayaraman, Padma-Sheela; Karrich, Julien J; Blom, Bianca; Vesuna, Farhad; O'Geen, Henriette; Sun, Yunfu; Gronostajski, Richard M; Woodcroft, Mark W; Hough, Margaret R; Chen, Edwin; Europe-Finner, G Nicholas; Karolczak-Bayatti, Magdalena; Bailey, Jarrod; Hankinson, Oliver; Raman, Venu; LeBrun, David P; Biswal, Shyam; Harvey, Christopher J; DeBruyne, Jason P; Hogenesch, John B; Hevner, Robert F; Héligon, Christophe; Luo, Xin M; Blank, Marissa Cathleen; Millen, Kathleen Joyce; Sharlin, David S; Forrest, Douglas; Dahlman-Wright, Karin; Zhao, Chunyan; Mishima, Yuriko; Sinha, Satrajit; Chakrabarti, Rumela; Portales-Casamar, Elodie; Sladek, Frances M; Bradley, Philip H; Wasserman, Wyeth W

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe. PMID:22458515

  8. Computational design of genomic transcriptional networks with adaptation to varying environments.

    PubMed

    Carrera, Javier; Elena, Santiago F; Jaramillo, Alfonso

    2012-09-18

    Transcriptional profiling has been widely used as a tool for unveiling the coregulations of genes in response to genetic and environmental perturbations. These coregulations have been used, in a few instances, to infer global transcriptional regulatory models. Here, using the large amount of transcriptomic information available for the bacterium Escherichia coli, we seek to understand the design principles determining the regulation of its transcriptome. Combining transcriptomic and signaling data, we develop an evolutionary computational procedure that allows obtaining alternative genomic transcriptional regulatory network (GTRN) that still maintains its adaptability to dynamic environments. We apply our methodology to an E. coli GTRN and show that it could be rewired to simpler transcriptional regulatory structures. These rewired GTRNs still maintain the global physiological response to fluctuating environments. Rewired GTRNs contain 73% fewer regulated operons. Genes with similar functions and coordinated patterns of expression across environments are clustered into longer regulated operons. These synthetic GTRNs are more sensitive and show a more robust response to challenging environments. This result illustrates that the natural configuration of E. coli GTRN does not necessarily result from selection for robustness to environmental perturbations, but that evolutionary contingencies may have been important as well. We also discuss the limitations of our methodology in the context of the demand theory. Our procedure will be useful as a novel way to analyze global transcription regulation networks and in synthetic biology for the de novo design of genomes. PMID:22927389

  9. Mapping Yeast Transcriptional Networks

    PubMed Central

    Hughes, Timothy R.; de Boer, Carl G.

    2013-01-01

    The term “transcriptional network” refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms. PMID:24018767

  10. Automatic Music Transcription

    NASA Astrophysics Data System (ADS)

    Klapuri, Anssi; Virtanen, Tuomas

    Written musical notation describes music in a symbolic form that is suitable for performing a piece using the available musical instruments. Traditionally, musical notation indicates the pitch, target instrument, timing, and duration of each sound to be played. The aim of music transcription either by humans or by a machine is to infer these musical parameters, given only the acoustic recording of a performance.

  11. Inhibition of transcription by oxidative DNA damage products

    SciTech Connect

    Byrd, S.; Reines, D.; Doetsch, P.W. )

    1991-03-11

    Thymine glycol is a major oxidative DNA base damage product that can be produced spontaneously in normal cells or by certain chemicals and ionizing radiation. This lesion as well as other oxidatively damaged bases are recognized and removed in eukaryotic cells by the DNA repair enzyme redoxyendonuclease which the authors have identified in a variety of cell types. Transcriptional regulation is a key element in the control of gene expression. Deficiencies in the various steps of transcription of an essential gene may have catastrophic effects for a cell. In terminally differentiated cells, the removal of RNA-polymerase blocking lesions could be viewed as a critical function for DNA repair systems in such cells. Very little information exists on the effects of oxidative base damage products on the process of transcription. The authors show here that thymine glycol containing DNA templates can inhibit transcriptional elongation when these lesions are chemically introduced into a DNA template. A DNA segment containing a region of the human H3.3 histone gene was utilized to determine the effects of oxidative DNA base damage on transcription by pure E. coli core RNA polymerase and rat liver RNA polymerase II. Both eukaryotic and prokaryotic RNA polymerases are blocked by the presence of thymine glycols appearing in certain clusters of thymines in the oxidatively damaged transcription template. To obtain quantitative efficiencies of transcriptional arrest, the authors are engineering a DNA template containing a single defined oxidatively damaged residue. The authors' results support the idea that an important function of DNA repair systems in terminally differentiated cells is to ensure the efficient transcription of genes necessary for normal cellular function.

  12. Statistical mechanical model of coupled transcription from multiple promoters due to transcription factor titration

    NASA Astrophysics Data System (ADS)

    Rydenfelt, Mattias; Cox, Robert Sidney, III; Garcia, Hernan; Phillips, Rob

    2014-01-01

    Transcription factors (TFs) with regulatory action at multiple promoter targets is the rule rather than the exception, with examples ranging from the cAMP receptor protein (CRP) in E. coli that regulates hundreds of different genes simultaneously to situations involving multiple copies of the same gene, such as plasmids, retrotransposons, or highly replicated viral DNA. When the number of TFs heavily exceeds the number of binding sites, TF binding to each promoter can be regarded as independent. However, when the number of TF molecules is comparable to the number of binding sites, TF titration will result in correlation (“promoter entanglement”) between transcription of different genes. We develop a statistical mechanical model which takes the TF titration effect into account and use it to predict both the level of gene expression for a general set of promoters and the resulting correlation in transcription rates of different genes. Our results show that the TF titration effect could be important for understanding gene expression in many regulatory settings.

  13. Characterization of a Novel Microcin That Kills Enterohemorrhagic Escherichia coli O157:H7 and O26

    PubMed Central

    Eberhart, Lauren J.; Deringer, James R.; Brayton, Kelly A.; Sawant, Ashish A.; Besser, Thomas E.

    2012-01-01

    A novel phenotype was recently identified in which specific strains of Escherichia coli inhibit competing E. coli strains via a mechanism that was designated “proximity-dependent inhibition” (PDI). PDI-expressing (PDI+) E. coli is known to inhibit susceptible (PDI−) E. coli strains, including several enterohemorrhagic (EHEC) and enterotoxigenic (ETEC) E. coli strains. In this study, every strain from a genetically diverse panel of E. coli O157:H7 (n = 25) and additional strains of E. coli serovar O26 were susceptible to the PDI phenotype. LIVE/DEAD staining was consistent with inhibition by killing of susceptible cells. Comparative genome analysis identified the genetic component of PDI, which is composed of a plasmid-borne (Incl1) operon encoding a putative microcin and associated genes for transport, immunity, and microcin activation. Transfer of the plasmid to a PDI− strain resulted in transfer of the phenotype, and deletion of the genes within the operon resulted in loss of the inhibition phenotype. Deletion of chromosomally encoded tolC also resulted in loss of the inhibitory phenotype, and this confirmed that the putative microcin is most likely secreted via a type I secretion pathway. Deletion of an unrelated plasmid gene did not affect the PDI phenotype. Quantitative reverse transcription (RT)-PCR demonstrated that microcin expression is correlated with logarithmic-phase growth. The ability to inhibit a diversity of E. coli strains indicates that this microcin may influence gut community composition and could be useful for control of important enteric pathogens. PMID:22773653

  14. Products of enteropathogenic Escherichia coli inhibit lymphocyte activation and lymphokine production.

    PubMed Central

    Klapproth, J M; Donnenberg, M S; Abraham, J M; Mobley, H L; James, S P

    1995-01-01

    The aim of this study was to determine whether products of enteric bacteria are able to regulate lymphocyte activation and cytokine production. Whole bacteria and bacterial lysates from different strains of Escherichia coli were tested for their ability to inhibit cytokine production by peripheral blood mononuclear cells as determined by reverse transcription-PCR, Northern (RNA) blotting of cellular RNA, or enzyme-linked immunosorbent assay for cytokine protein. Lysates from two pathogenic strains of E. coli, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli, inhibited mitogen-stimulated expression of interleukin-2 (IL-2), IL-4, IL-5, and gamma interferon. IL-1 beta, IL-6, IL-8, IL-10, IL-12, and Rantes mRNA expression was not affected. The inhibitory activity was dose dependent, protease and heat sensitive, nondialyzable, and not due to cellular toxicity. The inhibitory activity remained in EPEC strains having mutations in known virulence factors. Nonpathogenic E. coli HB101 transformed with a 22-kb cosmid clone derived from EPEC chromosomal DNA expressed the inhibitory activity. Thus, certain strains of pathogenic E. coli express a protein or proteins encoded by chromosomal genes that selectively inhibit lymphocyte activation and lymphokine production. Therefore, immunosuppressive factors produced by pathogenic bacteria could be important in modifying gastrointestinal immune responses in enteric bacterial infections or gastrointestinal autoimmune diseases. PMID:7768605

  15. Characterization of the YdeO Regulon in Escherichia coli

    PubMed Central

    Yamanaka, Yuki; Oshima, Taku; Ishihama, Akira; Yamamoto, Kaneyoshi

    2014-01-01

    Enterobacteria are able to survive under stressful conditions within animals, such as acidic conditions in the stomach, bile salts during transfer to the intestine and anaerobic conditions within the intestine. The glutamate-dependent (GAD) system plays a major role in acid resistance in Escherichia coli, and expression of the GAD system is controlled by the regulatory cascade consisting of EvgAS > YdeO > GadE. To understand the YdeO regulon in vivo, we used ChIP-chip to interrogate the E. coli genome for candidate YdeO binding sites. All of the seven operons identified by ChIP-chip as being potentially regulated by YdeO were confirmed as being under the direct control of YdeO using RT-qPCR, EMSA, DNaseI-footprinting and reporter assays. Within this YdeO regulon, we identified four stress-response transcription factors, DctR, NhaR, GadE, and GadW and enzymes for anaerobic respiration. Both GadE and GadW are involved in regulation of the GAD system and NhaR is an activator for the sodium/proton antiporter gene. In conjunction with co-transcribed Slp, DctR is involved in protection against metabolic endoproducts under acidic conditions. Taken all together, we suggest that YdeO is a key regulator of E. coli survival in both acidic and anaerobic conditions. PMID:25375160

  16. Production of 2-methyl-1-butanol in engineered Escherichia coli.

    PubMed

    Cann, Anthony F; Liao, James C

    2008-11-01

    Recent progress has been made in the production of higher alcohols by harnessing the power of natural amino acid biosynthetic pathways. Here, we describe the first strain of Escherichia coli developed to produce the higher alcohol and potential new biofuel 2-methyl-1-butanol (2MB). To accomplish this, we explored the biodiversity of enzymes catalyzing key parts of the isoleucine biosynthetic pathway, finding that AHAS II (ilvGM) from Salmonella typhimurium and threonine deaminase (ilvA) from Corynebacterium glutamicum improve 2MB production the most. Overexpression of the native threonine biosynthetic operon (thrABC) on plasmid without the native transcription regulation also improved 2MB production in E. coli. Finally, we knocked out competing pathways upstream of threonine production (DeltametA, Deltatdh) to increase its availability for further improvement of 2MB production. This work led to a strain of E. coli that produces 1.25 g/L 2MB in 24 h, a total alcohol content of 3 g/L, and with yields of up to 0.17 g 2MB/g glucose. PMID:18758769

  17. Analysis of Heme Biosynthetic Pathways in a Recombinant Escherichia coli.

    PubMed

    Pranawidjaja, Stephanie; Choi, Su-In; Lay, Bibiana W; Kim, Pil

    2015-06-01

    Bacterial heme was produced from a genetic-engineered Escherichia coli via the porphyrin pathway and it was useful as an iron resource for animal feed. The amount of the E. colisynthesized heme, however, was only few milligrams in a culture broth and it was not enough for industrial applications. To analyze heme biosynthetic pathways, an engineered E. coli artificially overexpressing ALA synthase (hemA from Rhodobacter sphaeroides) and pantothenate kinase (coaA gene from self geneome) was constructed as a bacterial heme-producing strain, and both the transcription levels of pathway genes and the intermediates concentrations were determined from batch and continuous cultures. Transcription levels of the pathway genes were not significantly changed among the tested conditions. Intracellular intermediate concentrations indicated that aminolevulinic acid (ALA) and coenzyme A (CoA) were enhanced by the hemA-coaA co-expression. Intracellular coproporphyrinogen I and protoporphyrin IX accumulation suggested that the bottleneck steps in the heme biosynthetic pathway could be the spontaneous conversion of HMB to coproporphyrinogen I and the limited conversion of protoporphyrin IX to heme, respectively. A strategy to increase the conversion of ALA to heme is discussed based on the results. PMID:25537720

  18. DNA Bending and Wrapping around RNA Polymerase: a “Revolutionary” Model Describing Transcriptional Mechanisms

    PubMed Central

    Coulombe, Benoit; Burton, Zachary F.

    1999-01-01

    A model is proposed in which bending and wrapping of DNA around RNA polymerase causes untwisting of the DNA helix at the RNA polymerase catalytic center to stimulate strand separation prior to initiation. During elongation, DNA bending through the RNA polymerase active site is proposed to lower the energetic barrier to the advance of the transcription bubble. Recent experiments with mammalian RNA polymerase II along with accumulating evidence from studies of Escherichia coli RNA polymerase indicate the importance of DNA bending and wrapping in transcriptional mechanisms. The DNA-wrapping model describes specific roles for general RNA polymerase II transcription factors (TATA-binding protein [TBP], TFIIB, TFIIF, TFIIE, and TFIIH), provides a plausible explanation for preinitiation complex isomerization, suggests mechanisms underlying the synergy between transcriptional activators, and suggests an unforseen role for TBP-associating factors in transcription. PMID:10357858

  19. Regulation of Transcription from Two ssrS Promoters in 6S RNA Biogenesis

    PubMed Central

    Lee, Ji Young; Park, Hongmarn; Bak, Geunu; Kim, Kwang-sun; Lee, Younghoon

    2013-01-01

    ssrS-encoded 6S RNA is an abundant noncoding RNA that binds σ70-RNA polymerase and regulates expression at a subset of promoters in Escherichia coli. It is transcribed from two tandem promoters, ssrS P1 and ssrS P2. Regulation of transcription from two ssrS promoters in 6S RNA biogenesis was examined. Both P1 and P2 were growth phase-dependently regulated. Depletion of 6S RNA had no effect on growth-phase-dependent transcription from either promoter, whereas overexpression of 6S RNA increased P1 transcription and decreased P2 transcription, suggesting that transcription from P1 and P2 is subject to feedback activation and feedback inhibition, respectively. This feedback regulation disappeared in Δfis strains, supporting involvement of Fis in this process. The differential feedback regulation may provide a means for maintaining appropriate cellular concentrations of 6S RNA. PMID:23864284

  20. Shiga Toxin Producing Escherichia coli.

    PubMed

    Bryan, Allen; Youngster, Ilan; McAdam, Alexander J

    2015-06-01

    Shiga toxin-producing Escherichia coli (STEC) is among the common causes of foodborne gastroenteritis. STEC is defined by the production of specific toxins, but within this pathotype there is a diverse group of organisms. This diversity has important consequences for understanding the pathogenesis of the organism, as well as for selecting the optimum strategy for diagnostic testing in the clinical laboratory. This review includes discussions of the mechanisms of pathogenesis, the range of manifestations of infection, and the several different methods of laboratory detection of Shiga toxin-producing E coli. PMID:26004641

  1. Experimental evolution of E. coli

    NASA Astrophysics Data System (ADS)

    Zhang, Mengshi

    The evolution from unicellular to multicellular behavior is an essential step in the history of life. Our aim is to investigate the emergence of collective behavior in the model organism Escherichia coli (E. coli) and its selection advantages, such as better utilization of public goods. Our preliminary results suggest that the evolution of collective behavior may be a natural response to stressed conditions. Mailing address: Room 306 Science Centre North Block, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong SAR. Phone: +852-3943-6354. Fax: +852-2603-5204. E-mail: mengshi0928@gmail.com.

  2. A modular strategy for engineering orthogonal chimeric RNA transcription regulators

    PubMed Central

    Takahashi, Melissa K.; Lucks, Julius B.

    2013-01-01

    Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli, we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry. PMID:23761434

  3. Identification and characterization of transcription networks in environmentally significant species

    SciTech Connect

    Lawrence, Charles E.; McCue, Lee Ann

    2005-11-30

    Understanding the regulation of gene expression, transcription regulation in particular, is one of the grand challenges of molecular biology. Transcription regulation is arguably the most important foundation of cellular function, since it exerts the most fundamental control of the abundance of virtually all of a cell's functional macromolecules. Nevertheless, this process, perhaps because of its difficulty, has been the subject of only a limited number of genomic level analyses. We have undertaken bioinformatics projects to address this issue by developing (1) a cross-species comparison method (i.e. phylogenetic footprinting) for the identification of transcription factor binding sites, (2) a Bayesian clustering method to identify regulons, (3) an improved scanning algorithm that uses a position weight matrix and several related species sequence data to locate transcription factor binding sites, and (4) a method to predict cognate binding sites for transcription factors of unknown specificity. These bioinformatics methods were developed using the model proteobacterium Escherichia coli, with further applications to the genomes of environmentally significant microbes (Rhodopseudomonas palustris, Shewanella oneidensis) in later years of the grant.

  4. Bacterial RNA polymerase can retain σ70 throughout transcription.

    PubMed

    Harden, Timothy T; Wells, Christopher D; Friedman, Larry J; Landick, Robert; Hochschild, Ann; Kondev, Jane; Gelles, Jeff

    2016-01-19

    Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ(70), can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ(70) retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ(70)-RNAP complex during initiation from the λ PR' promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ(70) and the kinetics of σ(70) release from actively transcribing complexes. σ(70) release from mature elongation complexes was slow (0.0038 s(-1)); a substantial subpopulation of elongation complexes retained σ(70) throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ(70) manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ(70) during transcription. PMID:26733675

  5. Serogroups of Escherichia coli from drinking water.

    PubMed

    Ramteke, P W; Tewari, Suman

    2007-07-01

    Fifty seven isolates of thermotolerant E. coli were recovered from 188 drinking water sources, 45 (78.9%) were typable of which 15 (26.3%) were pathogenic serotypes. Pathogenic serogroup obtained were 04 (Uropathogenic E. coli, UPEC), 025 (Enterotoxigenic E. coli, ETEC), 086 (Enteropathogenic E. coli, EPEC), 0103 (Shiga-toxin producing E. coli, STEC), 0157 (Shiga-toxin producing E. coli, STEC), 08 (Enterotoxigenic E. coli, ETEC) and 0113 (Shiga-toxin producing E. coli, STEC). All the pathogenic serotypes showed resistance to bacitracin and multiple heavy metal ions. Resistance to streptomycin and cotrimazole was detected in two strains whereas resistance to cephaloridine, polymixin-B and ampicillin was detected in one strain each. Transfer of resistances to drugs and metallic ions was observed in 9 out of 12 strains studied. Resistances to bacitracin were transferred in all nine strains. Among heavy metals resistance to As(3+) followed by Cr(6+) were transferred more frequently. PMID:17057960

  6. Investigating the Relatedness of Enteroinvasive Escherichia coli to Other E. coli and Shigella Isolates by Using Comparative Genomics.

    PubMed

    Hazen, Tracy H; Leonard, Susan R; Lampel, Keith A; Lacher, David W; Maurelli, Anthony T; Rasko, David A

    2016-08-01

    Enteroinvasive Escherichia coli (EIEC) is a unique pathovar that has a pathogenic mechanism nearly indistinguishable from that of Shigella species. In contrast to isolates of the four Shigella species, which are widespread and can be frequent causes of human illness, EIEC causes far fewer reported illnesses each year. In this study, we analyzed the genome sequences of 20 EIEC isolates, including 14 first described in this study. Phylogenomic analysis of the EIEC genomes demonstrated that 17 of the isolates are present in three distinct lineages that contained only EIEC genomes, compared to reference genomes from each of the E. coli pathovars and Shigella species. Comparative genomic analysis identified genes that were unique to each of the three identified EIEC lineages. While many of the EIEC lineage-specific genes have unknown functions, those with predicted functions included a colicin and putative proteins involved in transcriptional regulation or carbohydrate metabolism. In silico detection of the Shigella virulence plasmid (pINV), which is essential for the invasion of host cells, demonstrated that a form of pINV was present in nearly all EIEC genomes, but the Mxi-Spa-Ipa region of the plasmid that encodes the invasion-associated proteins was absent from several of the EIEC isolates. The comparative genomic findings in this study support the hypothesis that multiple EIEC lineages have evolved independently from multiple distinct lineages of E. coli via the acquisition of the Shigella virulence plasmid and, in some cases, the Shigella pathogenicity islands. PMID:27271741

  7. Vaccinia virus transcription.

    PubMed

    Broyles, Steven S

    2003-09-01

    Vaccinia virus replication takes place in the cytoplasm of the host cell. The nearly 200 kbp genome owes part of its complexity to encoding most of the proteins involved in genome and mRNA synthesis. The multisubunit vaccinia virus RNA polymerase requires a separate set of virus-encoded proteins for the transcription of the early, intermediate and late classes of genes. Cell fractionation studies have provided evidence for a role for host cell proteins in the initiation and termination of vaccinia virus intermediate and late gene transcription. Vaccinia virus resembles nuclear DNA viruses in the integration of viral and host proteins for viral mRNA synthesis, yet is markedly less reliant on host proteins than its nuclear counterparts. PMID:12917449

  8. "In Vitro" Synthesis and Activity of Reporter Proteins in an "Escherichia coli" S30 Extract System: An Undergraduate Experiment

    ERIC Educational Resources Information Center

    Higgins, Pamela J.

    2005-01-01

    This undergraduate laboratory experiment integrates multiple techniques ("in vitro" synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (beta-galactosidase and luciferase) within an "Escherichia coli" S30 transcription/translation extract. Comparison of the data suggests…

  9. Intrinsic disorder in transcription factors†

    PubMed Central

    Liu, Jiangang; Perumal, Narayanan B.; Oldfield, Christopher J.; Su, Eric W.; Uversky, Vladimir N.; Dunker, A. Keith

    2008-01-01

    Intrinsic disorder (ID) is highly abundant in eukaryotes, which reflect the greater need for disorder-associated signaling and transcriptional regulation in nucleated cells. Although several well-characterized examples of intrinsically disordered proteins in transcriptional regulation have been reported, no systematic analysis has been reported so far. To test for a general prevalence of intrinsic disorder in transcriptional regulation, we used the Predictor Of Natural Disorder Regions (PONDR) to analyze the abundance of intrinsic disorder in three transcription factor datasets and two control sets. This analysis revealed that from 94.13% to 82.63% of transcription factors posses extended regions of intrinsic disorder, relative to 54.51% and 18.64% of the proteins in two control datasets, which indicates the significant prevalence of intrinsic disorder in transcription factors. This propensity of transcription factors for intrinsic disorder was confirmed by cumulative distribution function analysis and charge-hydropathy plots. The amino acid composition analysis showed that all three transcription factor datasets were substantially depleted in order-promoting residues, and significantly enriched in disorder-promoting residues. Our analysis of the distribution of disorder within the transcription factor datasets revealed that: (a) The AT-hooks and basic regions of transcription factor DNA-binding domains are highly disordered; (b) The degree of disorder in transcription factor activation regions is much higher than that in DNA-binding domains; (c) The degree of disorder is significantly higher in eukaryotic transcription factors than in prokaryotic transcription factors; (d) The level of α-MoRFs (molecular recognition feature) prediction is much higher in transcription factors. Overall, our data reflected the fact that the eukaryotes with well-developed gene transcription machinery require transcription factor flexibility to be more efficient. PMID:16734424

  10. Molecular Ecology of Waterborne E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli is a common inhabitant of surface and ground waters in the developed and developing worlds. The majority of the E coli present in water are not particularly pathogenic to humans; however, there are E coli present in small proportion, that possess virulence genes that allow them to c...

  11. Relation of intracellular signal levels and promoter activities in the gal regulon of Escherichia coli.

    PubMed

    Krishna, Sandeep; Orosz, László; Sneppen, Kim; Adhya, Sankar; Semsey, Szabolcs

    2009-08-28

    Transcription of many genes is regulated by combinations of multiple signals. In Escherichia coli, combinatorial control is typical in the case of operons related to utilization of different sugars in the absence of glucose. To understand regulation of the transport and metabolic pathways in the galactose system, we measured activities of the six gal regulon promoters simultaneously, using an in vitro transcription system containing purified components. Input functions were computed on the basis of the experimental measurements. We observed four different shapes of input functions. From the results, we can conclude that the structure of the regulatory network is insufficient for the determination of signal integration. It is the actual structure of the promoter and regulatory region, the mechanism of transcription regulation, and the interplay between transcription factors that shape the input function to be suitable for adaptation. PMID:19559028

  12. EXTRAINTESTINAL PATHOGENIC ESCHERICHIA COLI (EXPEC)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Extraintestinal pathogenic Escherichia coli (ExPEC) possess virulence traits that allow them to invade, colonize, and induce disease in bodily sites outside of the gastrointestinal tract. Human diseases caused by ExPEC include urinary tract infections, neonatal meningitis, sepsis, pneumonia, surgic...

  13. In vivo footprinting analysis of Lrp binding to the ilvIH promoter region of Escherichia coli.

    PubMed Central

    Marasco, R; Varcamonti, M; La Cara, F; Ricca, E; De Felice, M; Sacco, M

    1994-01-01

    An in vivo footprinting analysis of the ilvIH regulatory region of Escherichia coli showed that the transcription activator Lrp binds to six sites, scattered over 250 bp upstream of the transcriptional start point. When Lrp-mediated activation was impaired by the presence of exogenous leucine, only one promoter-distal site (site 2) was partially protected by Lrp binding. Equilibrium dialysis experiments showed the formation of an Lrp-leucine complex in vitro. These results suggest that leucine negatively affects ilvIH transcription because its interaction with Lrp reduces the efficiency of binding of the regulatory protein to the promoter region. Images PMID:8071194

  14. Aromatic acid metabolites of Escherichia coli K-12 can induce the marRAB operon.

    PubMed

    Chubiz, Lon M; Rao, Christopher V

    2010-09-01

    MarR is a key regulator of the marRAB operon involved in antibiotic resistance and solvent stress tolerance in Escherichia coli. We show that two metabolic intermediates, 2,3-dihydroxybenzoate and anthranilate, involved in enterobactin and tryptophan biosynthesis, respectively, can activate marRAB transcription. We also found that a third intermediate involved in ubiquinone biosynthesis, 4-hydroxybenzoate, activates marRAB transcription in the absence of TolC. Of the three, however, only 2,3-dihydroxybenzoate directly binds MarR and affects its activity. PMID:20639340

  15. In vivo titration of mitomycin C action by four Escherichia coli genomic regions on multicopy plasmids.

    PubMed

    Wei, Y; Vollmer, A C; LaRossa, R A

    2001-04-01

    Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinct E. coli genes (mdfA, gyrl, rob, and sdiA) on high-copy-number vectors. mdfA encodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division. PMID:11244065

  16. In Vivo Titration of Mitomycin C Action by Four Escherichia coli Genomic Regions on Multicopy Plasmids

    PubMed Central

    Wei, Yan; Vollmer, Amy C.; LaRossa, Robert A.

    2001-01-01

    Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinct E. coli genes (mdfA, gyrl, rob, and sdiA) on high-copy-number vectors. mdfA encodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division. PMID:11244065

  17. Molecular Profiling: Catecholamine Modulation of Gene Expression in Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium.

    PubMed

    Bearson, Bradley L

    2016-01-01

    Investigations of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium have demonstrated that these bacterial pathogens can respond to the presence of catecholamines including norepinephrine and/or epinephrine in their environment by modulating gene expression and exhibiting various phenotypes. For example, one of the most intensively investigated phenotypes following exposure of E. coli and S. Typhimurium to norepinephrine is enhanced bacterial growth in a serum-based medium. Host-pathogen investigations have demonstrated that the mammalian host utilizes nutritional immunity to sequester iron and prevent extraintestinal growth by bacterial pathogens. However, Salmonella and certain E. coli strains have a genetic arsenal designed for subversion and subterfuge of the host. Norepinephrine enhances bacterial growth due, in part, to increased iron availability, and transcriptional profiling indicates differential expression of genes encoding iron acquisition and transport proteins. Bacterial motility of E. coli and S. Typhimurium is also enhanced in the presence of catecholamines and increased flagellar gene expression has been described. Furthermore, epinephrine and norepinephrine are chemoattractants for E. coli O157:H7. In S. Typhimurium, norepinephrine enhances horizontal gene transfer and increases expression of genes involved in plasmid transfer. Exposure of E. coli O157:H7 to norepinephrine increases expression of the genes encoding Shiga toxin and operons within the locus of enterocyte effacement (LEE). Alterations in the transcriptional response of enteric bacteria to catecholamine exposure in vivo are predicted to enhance bacterial colonization and pathogen virulence. This chapter will review the current literature on the transcriptional response of E. coli and S. Typhimurium to catecholamines. PMID:26589218

  18. Transcriptomic Analysis of Shiga-Toxigenic Bacteriophage Carriage Reveals a Profound Regulatory Effect on Acid Resistance in Escherichia coli

    PubMed Central

    Veses-Garcia, Marta; Liu, Xuan; Rigden, Daniel J.; Kenny, John G.; McCarthy, Alan J.

    2015-01-01

    Shiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on the Escherichia coli host. Here we use next-generation sequencing to compare the transcriptomes of E. coli strains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on the E. coli host. Known phage regulators were overexpressed in E. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of an E. coli O157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage and E. coli acid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology of E. coli. PMID:26386055

  19. Transcriptomic analysis of Shiga-toxigenic bacteriophage carriage reveals a profound regulatory effect on acid resistance in Escherichia coli.

    PubMed

    Veses-Garcia, Marta; Liu, Xuan; Rigden, Daniel J; Kenny, John G; McCarthy, Alan J; Allison, Heather E

    2015-12-01

    Shiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on the Escherichia coli host. Here we use next-generation sequencing to compare the transcriptomes of E. coli strains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on the E. coli host. Known phage regulators were overexpressed in E. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of an E. coli O157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage and E. coli acid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology of E. coli. PMID:26386055

  20. An evolutionarily conserved RNase-based mechanism for repression of transcriptional positive autoregulation

    PubMed Central

    Wurtmann, Elisabeth J.; Ratushny, Alexander V.; Pan, Min; Beer, Karlyn D.; Aitchison, John D.; Baliga, Nitin S.

    2014-01-01

    Summary It is known that environmental context influences the degree of regulation at the transcriptional and post-transcriptional levels. However, the principles governing the differential usage and interplay of regulation at these two levels are not clear. Here, we show that the integration of transcriptional and post-transcriptional regulatory mechanisms in a characteristic network motif drives efficient environment-dependent state transitions. Through phenotypic screening, systems analysis, and rigorous experimental validation, we discovered an RNase (VNG2099C) in Halobacterium salinarum that is transcriptionally co-regulated with genes of the aerobic physiologic state but acts on transcripts of the anaerobic state. Through modeling and experimentation we show that this arrangement generates an efficient state-transition switch, within which RNase-repression of a transcriptional positive autoregulation (RPAR) loop is critical for shutting down ATP-consuming active potassium uptake to reserve energy required for salinity adaptation under aerobic, high potassium, or dark conditions. Subsequently, we discovered that many Escherichia coli operons with energy-associated functions are also putatively controlled by RPAR indicating that this network motif may have evolved independently in phylogenetically distant organisms. Thus, our data suggest that interplay of transcriptional and post-transcriptional regulation in the RPAR motifis a generalized principle for efficient environment-dependent state transitions across prokaryotes. PMID:24612392

  1. Transcription initiation at the tet promoter and effect of mutations.

    PubMed Central

    Harley, C B; Lawrie, J; Betlach, M; Crea, R; Boyer, H W; Hedgpeth, J

    1988-01-01

    We have identified the startpoint for transcription in vitro of the tetracycline resistance gene (tet) of pBR322 and several deletion and insertion mutations which alter tet promoter structure. Tetracycline resistance in host bacteria correlates qualitatively with the efficiency of DNA fragments from these plasmids to promote tet transcription in vitro. Only in active promoters could we find by computer analysis promoter structures in which the -10 and -35 sequences and the relative spacing of the two regions agree with consensus sequence determinants. These data support the current model of the E. coli promoter sequence. Two promoter mutants gave heterogeneous 5' termini with additional A residues not encoded by the DNA sequence. Images PMID:3045754

  2. The complex choreography of transcription-coupled repair.

    PubMed

    Spivak, Graciela; Ganesan, Ann K

    2014-07-01

    A quarter of a century has elapsed since the discovery of transcription-coupled repair (TCR), and yet our fascination with this process has not diminished. Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. TCR, defined as a subpathway of NER, is dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, we will report on newly identified proteins, protein modifications, and protein complexes that participate in TCR in Escherichia coli and in human cells. We will discuss general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations. PMID:24751236

  3. Mechanisms of transcription-repair coupling and mutation frequency decline.

    PubMed Central

    Selby, C P; Sancar, A

    1994-01-01

    Mutation frequency decline is the rapid and irreversible decline in the suppressor mutation frequency of Escherichia coli cells if the cells are kept in nongrowth media immediately following the mutagenic treatment. The gene mfd, which is necessary for mutation frequency decline, encodes a protein of 130 kDa which couples transcription to excision repair by binding to RNA polymerase and to UvrA, which is the damage recognition subunit of the excision repair enzyme. Although current evidence suggests that transcription-repair coupling is the cause of the preferential repair of the transcribed strand of mRNA encoding genes as well as of suppressor tRNA genes, the decline occurs under stringent response conditions in which the tRNA genes are not efficiently transcribed. Thus, the mechanism of strand-specific repair is well understood, but some questions remain regarding the precise mechanism of mutation frequency decline. PMID:7968917

  4. An S6:S18 complex inhibits translation of E. coli rpsF

    PubMed Central

    Babina, Arianne M.; Soo, Mark W.; Fu, Yang; Meyer, Michelle M.

    2015-01-01

    More than half of the ribosomal protein operons in Escherichia coli are regulated by structures within the mRNA transcripts that interact with specific ribosomal proteins to inhibit further protein expression. This regulation is accomplished using a variety of mechanisms and the RNA structures responsible for regulation are often not conserved across bacterial phyla. A widely conserved mRNA structure preceding the ribosomal protein operon containing rpsF and rpsR (encoding S6 and S18) was recently identified through comparative genomics. Examples of this RNA from both E. coli and Bacillus subtilis were shown to interact in vitro with an S6:S18 complex. In this work, we demonstrate that in E. coli, this RNA structure regulates gene expression in response to the S6:S18 complex. β-galactosidase activity from a lacZ reporter translationally fused to the 5′ UTR and first nine codons of E. coli rpsF is reduced fourfold by overexpression of a genomic fragment encoding both S6 and S18 but not by overexpression of either protein individually. Mutations to the mRNA structure, as well as to the RNA-binding site of S18 and the S6–S18 interaction surfaces of S6 and S18, are sufficient to derepress β-galactosidase activity, indicating that the S6:S18 complex is the biologically active effector. Measurement of transcript levels shows that although reporter levels do not change upon protein overexpression, levels of the native transcript are reduced fourfold, suggesting that the mRNA regulator prevents translation and this effect is amplified on the native transcript by other mechanisms. PMID:26447183

  5. Infection strategies of enteric pathogenic Escherichia coli

    PubMed Central

    Clements, Abigail; Young, Joanna C.; Constantinou, Nicholas; Frankel, Gad

    2012-01-01

    Enteric Escherichia coli (E. coli) are both natural flora of humans and important pathogens causing significant morbidity and mortality worldwide. Traditionally enteric E. coli have been divided into 6 pathotypes, with further pathotypes often proposed. In this review we suggest expansion of the enteric E. coli into 8 pathotypes to include the emerging pathotypes of adherent invasive E. coli (AIEC) and Shiga-toxin producing enteroaggregative E. coli (STEAEC). The molecular mechanisms that allow enteric E. coli to colonize and cause disease in the human host are examined and for two of the pathotypes that express a type 3 secretion system (T3SS) we discuss the complex interplay between translocated effectors and manipulation of host cell signaling pathways that occurs during infection. PMID:22555463

  6. Clinical Implications of Enteroadherent Escherichia coli

    PubMed Central

    Arenas-Hernández, Margarita M.P.; Martínez-Laguna, Ygnacio; Torres, Alfredo G.

    2012-01-01

    Pathogenic Escherichia coli that colonize the small intestine primarily cause gastrointestinal illness in infants and travelers. The main categories of pathogenic E. coli that colonize the epithelial lining of the small intestine are enterotoxigenic E. coli enteropathogenic E. coli and enteroaggregative E. coli. These organisms accomplish their pathogenic process by a complex, coordinated multistage strategy, including non-intimate adherence mediated by various adhesins. These so called “enteroadherent E. coli ” categories subsequently produced toxins or effector proteins that are either secreted to the milieu or injected to the host cell. Finally, destruction of the intestinal microvilli results from the intimate adherence or the toxic effect exerted over the epithelia, resulting in water secretion and diarrhea. In this review, we summarize the current state of knowledge regarding these enteroadherent E. coli strains and the present clinical understanding of how these organisms colonize the human intestine and cause disease. PMID:22798032

  7. Clinical implications of enteroadherent Escherichia coli.

    PubMed

    Arenas-Hernández, Margarita M P; Martínez-Laguna, Ygnacio; Torres, Alfredo G

    2012-10-01

    Pathogenic Escherichia coli that colonize the small intestine primarily cause gastrointestinal illness in infants and travelers. The main categories of pathogenic E. coli that colonize the epithelial lining of the small intestine are enterotoxigenic E. coli, enteropathogenic E. coli, and enteroaggregative E. coli. These organisms accomplish their pathogenic process by a complex, coordinated multistage strategy, including nonintimate adherence mediated by various adhesins. These so called "enteroadherent E. coli" categories subsequently produce toxins or effector proteins that are either secreted to the milieu or injected to the host cell. Finally, destruction of the intestinal microvilli results from the intimate adherence or the toxic effect exerted over the epithelia, resulting in water secretion and diarrhea. In this review, we summarize the current state of knowledge regarding these enteroadherent E. coli strains and the present clinical understanding of how these organisms colonize the human intestine and cause disease. PMID:22798032

  8. Single Molecule Transcription Elongation

    PubMed Central

    Galburt, Eric A.; Grill, Stephan W.; Bustamante, Carlos

    2009-01-01

    Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study the dynamics of transcription elongation by RNA polymerase II from Saccharomyces cerevisiae. PMID:19426807

  9. Analysis of transcription of the exotoxin A gene of Pseudomonas aeruginosa.

    PubMed Central

    Grant, C C; Vasil, M L

    1986-01-01

    Analysis of RNA isolated from Pseudomonas aeruginosa PA103 and PAKS grown under Fe2+-limiting (0.08 microgram/ml) and Fe2+-sufficient (10 micrograms/ml) conditions demonstrated that exotoxin A (ETA) expression is regulated by Fe2+ at the level of transcription. S1 nuclease mapping revealed two 5' termini of the tox transcript, 89 base pairs (bp) (S1A) and 62 bp (S1B) 5' to the ETA initiation codon. There appeared to be no consensus promoter sequence for either tox transcript. An 8-bp direct repeat was found 5' to the start of transcript S1A. Transcript S1B mapped 8 bp upstream of a dodecamer sequence conserved between the ETA and phospholipase C genes of P. aeruginosa. Multicopy plasmids in which the expression of ETA is directed from the Escherichia coli trp promoter (ptrpETA-RSF1010) or the tox promoter (pCMtox) were constructed and mobilized into a Tox-P. aeruginosa strain, WR5. WR5 synthesized and secreted high levels of ETA when it was expressed from the E. coli trp promoter; however, the synthesis of ETA from its own promoter in this strain was very low. These and other data suggest that the expression of ETA is under a positive control mechanism. A fusion of the ETA promoter fragment to lacZ was constructed. Use of this fusion plasmid revealed that this DNA fragment directed the synthesis of beta-galactosidase in E. coli at very low levels and that the synthesis of beta-galactosidase from this fusion in E. coli was not regulated by Fe2+. Images PMID:2430945

  10. RNA-Based Detection Does not Accurately Enumerate Living Escherichia coli O157:H7 Cells on Plants

    PubMed Central

    Ju, Wenting; Moyne, Anne-Laure; Marco, Maria L.

    2016-01-01

    The capacity to distinguish between living and dead cells is an important, but often unrealized, attribute of rapid detection methods for foodborne pathogens. In this study, the numbers of enterohemorrhagic Escherichia coli O157:H7 after inoculation onto Romaine lettuce plants and on plastic (abiotic) surfaces were measured over time by culturing, and quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, and reverse transcriptase (RT)-qPCR targeting E. coli O157:H7 gapA, rfbE, eae, and lpfA genes and gene transcripts. On Romaine lettuce plants incubated at low relative humidity, E. coli O157:H7 cell numbers declined 107-fold within 96 h according to culture-based assessments. In contrast, there were no reductions in E. coli levels according to qPCR and only 100- and 1000-fold lower numbers per leaf by RT-qPCR and PMA-qPCR, respectively. Similar results were obtained upon exposure of E. coli O157:H7 to desiccation conditions on a sterile plastic surface. Subsequent investigation of mixtures of living and dead E. coli O157:H7 cells strongly indicated that PMA-qPCR detection was subject to false-positive enumerations of viable targets when in the presence of 100-fold higher numbers of dead cells. RT-qPCR measurements of killed E. coli O157:H7 as well as for RNaseA-treated E. coli RNA confirmed that transcripts from dead cells and highly degraded RNA were also amplified by RT-qPCR. These findings show that neither PMA-qPCR nor RT-qPCR provide accurate estimates of bacterial viability in environments where growth and survival is limited. PMID:26955370

  11. RNA-Based Detection Does not Accurately Enumerate Living Escherichia coli O157:H7 Cells on Plants.

    PubMed

    Ju, Wenting; Moyne, Anne-Laure; Marco, Maria L

    2016-01-01

    The capacity to distinguish between living and dead cells is an important, but often unrealized, attribute of rapid detection methods for foodborne pathogens. In this study, the numbers of enterohemorrhagic Escherichia coli O157:H7 after inoculation onto Romaine lettuce plants and on plastic (abiotic) surfaces were measured over time by culturing, and quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, and reverse transcriptase (RT)-qPCR targeting E. coli O157:H7 gapA, rfbE, eae, and lpfA genes and gene transcripts. On Romaine lettuce plants incubated at low relative humidity, E. coli O157:H7 cell numbers declined 10(7)-fold within 96 h according to culture-based assessments. In contrast, there were no reductions in E. coli levels according to qPCR and only 100- and 1000-fold lower numbers per leaf by RT-qPCR and PMA-qPCR, respectively. Similar results were obtained upon exposure of E. coli O157:H7 to desiccation conditions on a sterile plastic surface. Subsequent investigation of mixtures of living and dead E. coli O157:H7 cells strongly indicated that PMA-qPCR detection was subject to false-positive enumerations of viable targets when in the presence of 100-fold higher numbers of dead cells. RT-qPCR measurements of killed E. coli O157:H7 as well as for RNaseA-treated E. coli RNA confirmed that transcripts from dead cells and highly degraded RNA were also amplified by RT-qPCR. These findings show that neither PMA-qPCR nor RT-qPCR provide accurate estimates of bacterial viability in environments where growth and survival is limited. PMID:26955370

  12. Control of extracellular polysaccharide synthesis in Erwinia stewartii and Escherichia coli K-12: a common regulatory function.

    PubMed Central

    Torres-Cabassa, A; Gottesman, S; Frederick, R D; Dolph, P J; Coplin, D L

    1987-01-01

    A primary determinant of pathogenicity in Erwinia stewartii is the production of extracellular polysaccharide (EPS). A single mutation can abolish both EPS synthesis and pathogenicity; both properties are restored by a single cosmid clone. Subcloning and insertion analysis have defined a single positive regulatory function which shares a number of similarities with the rcsA function of Escherichia coli K-12, a positive regulator for capsular polysaccharide synthesis. In E. stewartii, the gene promotes the transcription of at least two operons (cps) involved in EPS synthesis; we have previously demonstrated a similar function for rcsA in E. coli. Both genes code for proteins of 25 to 27 kilodaltons; both proteins are unstable in E. coli. The E. stewartii RcsA protein was stabilized in E. coli lon mutants, as the RcsA product from E. coli is. The E. stewartii function complemented E. coli rcsA mutants, and the E. coli RcsA function increased cps expression and restored virulence in E. stewartii mutants. Therefore, these two gram-negative organisms share a similar component of their regulatory circuitry for the control of capsular polysaccharide synthesis. Images PMID:2820930

  13. Signal integration in the galactose network of Escherichia coli.

    PubMed

    Semsey, Szabolcs; Krishna, Sandeep; Sneppen, Kim; Adhya, Sankar

    2007-07-01

    The gal regulon of Escherichia coli contains genes involved in galactose transport and metabolism. Transcription of the gal regulon genes is regulated in different ways by two iso-regulatory proteins, Gal repressor (GalR) and Gal isorepressor (GalS), which recognize the same binding sites in the absence of d-galactose. DNA binding by both GalR and GalS is inhibited in the presence of d-galactose. Many of the gal regulon genes are activated in the presence of the adenosine cyclic-3',5'-monophosphate (cAMP)-cAMP receptor protein (CRP) complex. We studied transcriptional regulation of the gal regulon promoters simultaneously in a purified system and attempted to integrate the two small molecule signals, d-galactose and cAMP, that modulate the isoregulators and CRP respectively, at each promoter, using Boolean logic. Results show that similarly organized promoters can have different input functions. We also found that in some cases the activity of the promoter and the cognate gene can be described by different logic gates. We combined the transcriptional network of the galactose regulon, obtained from our experiments, with literature data to construct an integrated map of the galactose network. Structural analysis of the network shows that at the interface of the genetic and metabolic network, feedback loops are by far the most common motif. PMID:17630975

  14. Proteolytic regulation of stress response pathways in Escherichia coli.

    PubMed

    Micevski, Dimce; Dougan, David A

    2013-01-01

    Maintaining correct cellular function is a fundamental biological process for all forms of life. A critical aspect of this process is the maintenance of protein homeostasis (proteostasis) in the cell, which is largely performed by a group of proteins, referred to as the protein quality control (PQC) network. This network of proteins, comprised of chaperones and proteases, is critical for maintaining proteostasis not only during favourable growth conditions, but also in response to stress. Indeed proteases play a crucial role in the clearance of unwanted proteins that accumulate during stress, but more importantly, in the activation of various different stress response pathways. In bacteria, the cells response to stress is usually orchestrated by a specific transcription factor (sigma factor). In Escherichia coli there are seven different sigma factors, each of which responds to a particular stress, resulting in the rapid expression of a specific set of genes. The cellular concentration of each transcription factor is tightly controlled, at the level of transcription, translation and protein stability. Here we will focus on the proteolytic regulation of two sigma factors (σ(32) and σ(S)), which control the heat and general stress response pathways, respectively. This review will also briefly discuss the role proteolytic systems play in the clearance of unwanted proteins that accumulate during stress. PMID:23479439

  15. Examination of the internal promoter, PE, in the ilvGMEDA operon of E. coli K-12.

    PubMed Central

    Wek, R C; Hatfield, G W

    1986-01-01

    The ilvGMEDA operon of Escherichia coli K-12 contains an internal promoter, PE, in the distal portion of the ilvM gene immediately upstream from the ilvE gene. The location of this promoter was determined using S1 nuclease protection analyses of in vivo and in vitro transcripts. The transcriptional activity of the internal promoter was compared to the transcriptional activity of the operon-proximal promoter P1P2 using transcriptional fusion vectors and plasmid copy number determinations. These measurements showed that the P1P2 promoter is 52-fold stronger than the internal PE promoter. Estimates of the transcriptional role of the internal promoter on ilvE gene expression during growth conditions in excess and limiting branch chain amino acids is presented. Images PMID:2421252

  16. The Nun protein of bacteriophage HK022 inhibits translocation of Escherichia coli RNA polymerase without abolishing its catalytic activities

    PubMed Central

    Hung, Siu Chun; Gottesman, Max E.

    1997-01-01

    Bacteriophage HK022 Nun protein blocks transcription elongation by Escherichia coli RNA polymerase in vitro without dissociating the transcription complex. Nun is active on complexes located at any template site tested. Ultimately, only the 3′-OH terminal nucleotide of the nascent transcript in an arrested complex can turn over; it is removed by pyrophosphate and restored with NTPs. This suggests that Nun inhibits the translocation of RNA polymerase without abolishing its catalytic activities. Unlike spontaneously arrested complexes, Nun-arrested complexes cannot be reactivated by transcription factor GreB. The various complexes show distinct patterns of nucleotide incorporation and pyrophosphorolysis before or after treatment with Nun, suggesting that the configuration of RNAP, transcript, and template DNA is different in each complex. PMID:9334329

  17. Thiophene metabolism by E. coli

    SciTech Connect

    Clark, D.P.

    1991-01-01

    The objective of this project is to investigate the mechanism of degradation of sulfur-containing heterocyclic molecules by mutant strains of Escherichia coli K-12. We have previously isolated multiple mutants of E. coli which had gained the capacity to oxidize thiophene compounds and their furan analogs. We have focused on the thdA mutation in our subsequent research, as this appears to be in a regulatory gene central to the thiophene/furan oxidation system. The thdF gene appears to be more directly involved in the oxidation reactions, whereas thdC and thdD are apparently required for increased protection against the toxic effects of thiophene and furan compounds. 4 tabs.

  18. Transcriptional Network Growing Models Using Motif-Based Preferential Attachment

    PubMed Central

    Abdelzaher, Ahmed F.; Al-Musawi, Ahmad F.; Ghosh, Preetam; Mayo, Michael L.; Perkins, Edward J.

    2015-01-01

    Understanding relationships between architectural properties of gene-regulatory networks (GRNs) has been one of the major goals in systems biology and bioinformatics, as it can provide insights into, e.g., disease dynamics and drug development. Such GRNs are characterized by their scale-free degree distributions and existence of network motifs – i.e., small-node subgraphs that occur more abundantly in GRNs than expected from chance alone. Because these transcriptional modules represent “building blocks” of complex networks and exhibit a wide range of functional and dynamical properties, they may contribute to the remarkable robustness and dynamical stability associated with the whole of GRNs. Here, we developed network-construction models to better understand this relationship, which produce randomized GRNs by using transcriptional motifs as the fundamental growth unit in contrast to other methods that construct similar networks on a node-by-node basis. Because this model produces networks with a prescribed lower bound on the number of choice transcriptional motifs (e.g., downlinks, feed-forward loops), its fidelity to the motif distributions observed in model organisms represents an improvement over existing methods, which we validated by contrasting their resultant motif and degree distributions against existing network-growth models and data from the model organism of the bacterium Escherichia coli. These models may therefore serve as novel testbeds for further elucidating relationships between the topology of transcriptional motifs and network-wide dynamical properties. PMID:26528473

  19. Transcription Factors Exhibit Differential Conservation in Bacteria with Reduced Genomes.

    PubMed

    Galán-Vásquez, Edgardo; Sánchez-Osorio, Ismael; Martínez-Antonio, Agustino

    2016-01-01

    The description of transcriptional regulatory networks has been pivotal in the understanding of operating principles under which organisms respond and adapt to varying conditions. While the study of the topology and dynamics of these networks has been the subject of considerable work, the investigation of the evolution of their topology, as a result of the adaptation of organisms to different environmental conditions, has received little attention. In this work, we study the evolution of transcriptional regulatory networks in bacteria from a genome reduction perspective, which manifests itself as the loss of genes at different degrees. We used the transcriptional regulatory network of Escherichia coli as a reference to compare 113 smaller, phylogenetically-related γ-proteobacteria, including 19 genomes of symbionts. We found that the type of regulatory action exerted by transcription factors, as genomes get progressively smaller, correlates well with their degree of conservation, with dual regulators being more conserved than repressors and activators in conditions of extreme reduction. In addition, we found that the preponderant conservation of dual regulators might be due to their role as both global regulators and nucleoid-associated proteins. We summarize our results in a conceptual model of how each TF type is gradually lost as genomes become smaller and give a rationale for the order in which this phenomenon occurs. PMID:26766575

  20. The generation of promoter-mediated transcriptional noise in bacteria.

    PubMed

    Mitarai, Namiko; Dodd, Ian B; Crooks, Michael T; Sneppen, Kim

    2008-01-01

    Noise in the expression of a gene produces fluctuations in the concentration of the gene product. These fluctuations can interfere with optimal function or can be exploited to generate beneficial diversity between cells; gene expression noise is therefore expected to be subject to evolutionary pressure. Shifts between modes of high and low rates of transcription initiation at a promoter appear to contribute to this noise both in eukaryotes and prokaryotes. However, models invoked for eukaryotic promoter noise such as stable activation scaffolds or persistent nucleosome alterations seem unlikely to apply to prokaryotic promoters. We consider the relative importance of the steps required for transcription initiation. The 3-step transcription initiation model of McClure is extended into a mathematical model that can be used to predict consequences of additional promoter properties. We show in principle that the transcriptional bursting observed at an E. coli promoter by Golding et al. (2005) can be explained by stimulation of initiation by the negative supercoiling behind a transcribing RNA polymerase (RNAP) or by the formation of moribund or dead-end RNAP-promoter complexes. Both mechanisms are tunable by the alteration of promoter kinetics and therefore allow the optimization of promoter mediated noise. PMID:18617999

  1. Prediction of Transcriptional Terminators in Bacillus subtilis and Related Species

    PubMed Central

    2005-01-01

    In prokaryotes, genes belonging to the same operon are transcribed in a single mRNA molecule. Transcription starts as the RNA polymerase binds to the promoter and continues until it reaches a transcriptional terminator. Some terminators rely on the presence of the Rho protein, whereas others function independently of Rho. Such Rho-independent terminators consist of an inverted repeat followed by a stretch of thymine residues, allowing us to predict their presence directly from the DNA sequence. Unlike in Escherichia coli, the Rho protein is dispensable in Bacillus subtilis, suggesting a limited role for Rho-dependent termination in this organism and possibly in other Firmicutes. We analyzed 463 experimentally known terminating sequences in B. subtilis and found a decision rule to distinguish Rho-independent transcriptional terminators from non-terminating sequences. The decision rule allowed us to find the boundaries of operons in B. subtilis with a sensitivity and specificity of about 94%. Using the same decision rule, we found an average sensitivity of 94% for 57 bacteria belonging to the Firmicutes phylum, and a considerably lower sensitivity for other bacteria. Our analysis shows that Rho-independent termination is dominant for Firmicutes in general, and that the properties of the transcriptional terminators are conserved. Terminator prediction can be used to reliably predict the operon structure in these organisms, even in the absence of experimentally known operons. Genome-wide predictions of Rho-independent terminators for the 57 Firmicutes are available in the Supporting Information section. PMID:16110342

  2. Transcription Factors Exhibit Differential Conservation in Bacteria with Reduced Genomes

    PubMed Central

    Galán-Vásquez, Edgardo; Sánchez-Osorio, Ismael; Martínez-Antonio, Agustino

    2016-01-01

    The description of transcriptional regulatory networks has been pivotal in the understanding of operating principles under which organisms respond and adapt to varying conditions. While the study of the topology and dynamics of these networks has been the subject of considerable work, the investigation of the evolution of their topology, as a result of the adaptation of organisms to different environmental conditions, has received little attention. In this work, we study the evolution of transcriptional regulatory networks in bacteria from a genome reduction perspective, which manifests itself as the loss of genes at different degrees. We used the transcriptional regulatory network of Escherichia coli as a reference to compare 113 smaller, phylogenetically-related γ-proteobacteria, including 19 genomes of symbionts. We found that the type of regulatory action exerted by transcription factors, as genomes get progressively smaller, correlates well with their degree of conservation, with dual regulators being more conserved than repressors and activators in conditions of extreme reduction. In addition, we found that the preponderant conservation of dual regulators might be due to their role as both global regulators and nucleoid-associated proteins. We summarize our results in a conceptual model of how each TF type is gradually lost as genomes become smaller and give a rationale for the order in which this phenomenon occurs. PMID:26766575

  3. Optimality and thermodynamics determine the evolution of transcriptional regulatory networks†

    PubMed Central

    Yarmush, Martin L.

    2014-01-01

    Transcriptional motifs are small regulatory interaction patterns that regulate biological functions in highly-interacting cellular networks. Recently, attempts have been made to explain the significance of transcriptional motifs through dynamic function. However, fundamental questions remain unanswered. Why are certain transcriptional motifs with similar dynamic function abundant while others occur rarely? What are the criteria for topological generalization of these motifs into complex networks? Here, we present a novel paradigm that combines non-equilibrium thermodynamics with multiobjective-optimality for network analysis. We found that energetic cost, defined herein as specific dissipation energy, is minimal at the optimal environmental conditions and it correlates inversely with the abundance of the network motifs obtained experimentally for E. coli and S. cerevisiae. This yields evidence that dissipative energetics is the underlying criteria used during evolution for motif selection and that biological systems during transcription tend towards evolutionary selection of subgraphs which produces minimum specific heat dissipation under optimal conditions, thereby explaining the abundance/rare occurrence of some motifs. We show that although certain motifs had similar dynamical functionality, they had significantly different energetic cost, thus explaining the abundance/rare occurrence of these motifs. The presented insights may establish global thermodynamic analysis as a backbone in designing and understanding complex networks systems, such as metabolic and protein interaction networks. PMID:22076617

  4. Apolipoprotein A1 in channel catfish: Transcriptional analysis, antimicrobial activity, and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were to: 1) determine transcriptional profiles of apolipoprotein A1 (ApoA1) in collected channel catfish tissues after infection with A. hydrophila by bath immersion; 2) investigate whether recombinant channel catfish apolipoprotein A1 produced in E. coli expression syst...

  5. Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

    PubMed Central

    Polo, S; Ketner, G; Levis, R; Falgout, B

    1997-01-01

    The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts. PMID:9188607

  6. Effects of sound exposure on the growth and intracellular macromolecular synthesis of E. coli k-12.

    PubMed

    Gu, Shaobin; Zhang, Yongzhu; Wu, Ying

    2016-01-01

    Microbes, as one of the primary producers of the biosphere, play an important role in ecosystems. Exploring the mechanism of adaptation and resistance of microbial population to various environmental factors has come into focus in the fields of modern microbial ecology and molecular ecology. However, facing the increasingly serious problem of acoustic pollution, very few efforts have been put forth into studying the relation of single cell organisms and sound field exposure. Herein, we studied the biological effects of sound exposure on the growth of E. coli K-12 with different acoustic parameters. The effects of sound exposure on the intracellular macromolecular synthesis and cellular morphology of E. coli K-12 were also analyzed and discussed. Experimental results indicated that E. coli K-12 exposed to sound waves owned a higher biomass and a faster specific growth rate compared to the control group. Also, the average length of E. coli K-12 cells increased more than 27.26%. The maximum biomass and maximum specific growth rate of the stimulation group by 8000 Hz, 80dB sound wave was about 1.7 times and 2.5 times that of the control group, respectively. Moreover, it was observed that E. coli K-12 can respond rapidly to sound stress at both the transcriptional and posttranscriptional levels by promoting the synthesis of intracellular RNA and total protein. Some potential mechanisms may be involved in the responses of bacterial cells to sound stress. PMID:27077011

  7. Effects of sound exposure on the growth and intracellular macromolecular synthesis of E. coli k-12

    PubMed Central

    Zhang, Yongzhu; Wu, Ying

    2016-01-01

    Microbes, as one of the primary producers of the biosphere, play an important role in ecosystems. Exploring the mechanism of adaptation and resistance of microbial population to various environmental factors has come into focus in the fields of modern microbial ecology and molecular ecology. However, facing the increasingly serious problem of acoustic pollution, very few efforts have been put forth into studying the relation of single cell organisms and sound field exposure. Herein, we studied the biological effects of sound exposure on the growth of E. coli K-12 with different acoustic parameters. The effects of sound exposure on the intracellular macromolecular synthesis and cellular morphology of E. coli K-12 were also analyzed and discussed. Experimental results indicated that E. coli K-12 exposed to sound waves owned a higher biomass and a faster specific growth rate compared to the control group. Also, the average length of E. coli K-12 cells increased more than 27.26%. The maximum biomass and maximum specific growth rate of the stimulation group by 8000 Hz, 80dB sound wave was about 1.7 times and 2.5 times that of the control group, respectively. Moreover, it was observed that E. coli K-12 can respond rapidly to sound stress at both the transcriptional and posttranscriptional levels by promoting the synthesis of intracellular RNA and total protein. Some potential mechanisms may be involved in the responses of bacterial cells to sound stress. PMID:27077011

  8. Tellurite-exposed Escherichia coli exhibits increased intracellular {alpha}-ketoglutarate

    SciTech Connect

    Reinoso, Claudia A.; Auger, Christopher; Appanna, Vasu D.; Vasquez, Claudio C.

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Tellurite-exposed E. coli exhibits decreased {alpha}-KG dehydrogenase activity. Black-Right-Pointing-Pointer Cells lacking {alpha}-KGDH genes are more sensitive to ROS than isogenic, wt E. coli. Black-Right-Pointing-Pointer KG accumulation may serve to face tellurite-mediated oxidative damage in E. coli. -- Abstract: The tellurium oxyanion tellurite is toxic to most organisms because of its ability to generate oxidative stress. However, the detailed mechanism(s) how this toxicant interferes with cellular processes have yet to be fully understood. As part of our effort to decipher the molecular interactions of tellurite with living systems, we have evaluated the global metabolism of {alpha}-ketoglutarate a known antioxidant in Escherichia coli. Tellurite-exposed cells displayed reduced activity of the KG dehydrogenase complex (KGDHc), resulting in increased intracellular KG content. This complex's reduced activity seems to be due to decreased transcription in the stressed cells of sucA, a gene that encodes the E1 component of KGDHc. Furthermore, it was demonstrated that the increase in total reactive oxygen species and superoxide observed upon tellurite exposure was more evident in wild type cells than in E. coli with impaired KGDHc activity. These results indicate that KG may be playing a pivotal role in combating tellurite-mediated oxidative damage.

  9. O-GlcNAc modification of Sp3 and Sp4 transcription factors negatively regulates their transcriptional activities.

    PubMed

    Ha, Changhoon; Lim, Kihong

    2015-11-13

    The addition of O-linked N-acetylglucosamine (O-GlcNAc) on serine or threonine modifies a myriad of proteins and regulates their function, stability and localization. O-GlcNAc modification is common among chromosome-associated proteins, such as transcription factors, suggesting its extensive involvement in gene expression regulation. In this study, we demonstrate the O-GlcNAc status of the Sp family members of transcription factors and the functional impact on their transcriptional activities. We highlight the presence of O-GlcNAc residues in Sp3 and Sp4, but not Sp2, as demonstrated by their enrichment in GlcNAc positive protein fractions and by detection of O-GlcNAc residues on Sp3 and Sp4 co-expressed in Escherichia coli together with O-GlcNAc transferase (OGT) using an O-GlcNAc-specific antibody. Deletion mutants of Sp3 and Sp4 indicate that the majority of O-GlcNAc sites reside in their N-terminal transactivation domain. Overall, using reporter gene assays and co-immunoprecipitations, we demonstrate a functional inhibitory role of O-GlcNAc modifications in Sp3 and Sp4 transcription factors. Thereby, our study strengthens the current notion that O-GlcNAc modification is an important regulator of protein interactome. PMID:26431879

  10. Imaging Transcription in Living Cells

    PubMed Central

    Darzacq, Xavier; Yao, Jie; Larson, Daniel R.; Causse, Sebastien Z.; Bosanac, Lana; de Turris, Valeria; Ruda, Vera M.; Lionnet, Timothee; Zenklusen, Daniel; Guglielmi, Benjamin; Tjian, Robert; Singer, Robert H.

    2011-01-01

    The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding. PMID:19416065

  11. Mechanism of transcriptional repression at a bacterial promoter by analysis of single molecules.

    PubMed

    Sanchez, Alvaro; Osborne, Melisa L; Friedman, Larry J; Kondev, Jane; Gelles, Jeff

    2011-10-01

    The molecular basis for regulation of lactose metabolism in Escherichia coli is well studied. Nonetheless, the physical mechanism by which the Lac repressor protein prevents transcription of the lactose promoter remains unresolved. Using multi-wavelength single-molecule fluorescence microscopy, we visualized individual complexes of fluorescently tagged RNA polymerase holoenzyme bound to promoter DNA. Quantitative analysis of the single-molecule observations, including use of a novel statistical partitioning approach, reveals highly kinetically stable binding of polymerase to two different sites on the DNA, only one of which leads to transcription. Addition of Lac repressor directly demonstrates that bound repressor prevents the formation of transcriptionally productive open promoter complexes; discrepancies in earlier studies may be attributable to transcriptionally inactive polymerase binding. The single-molecule statistical partitioning approach is broadly applicable to elucidating mechanisms of regulatory systems including those that are kinetically rather than thermodynamically controlled. PMID:21829165

  12. Development of a LacZ-based transcriptional reporter system for use with Moraxella catarrhalis.

    PubMed

    Evans, Amanda S; Pybus, Christine; Hansen, Eric J

    2013-03-01

    The lack of a transcriptional reporter system for use in Moraxella catarrhalis has hindered studies of gene regulation in this pathogen. PCR and recombinant DNA methods were used to insert a multicloning site (MCS) and promoterless full-length Escherichia coli lacZ gene, flanked by transcriptional terminators both immediately upstream and downstream, into the M. catarrhalis recombinant plasmid pWW115. Insertion into the MCS in the newly constructed plasmid pASE222 of M. catarrhalis promoter regions controlled by either a repressor (i.e., NsrR) or activator (i.e., PhoB) yielded transcriptional fusion constructs that were appropriately responsive to signal inputs dependent on the host strain genotype, as measured quantitatively by means of a Miller β-galactosidase assay. The transcriptional reporter plasmid pASE222 should prove to be a useful tool for rapid screening of factors affecting gene expression in M. catarrhalis. PMID:23219721

  13. Understanding the Role of Housekeeping and Stress-Related Genes in Transcription-Regulatory Networks

    NASA Astrophysics Data System (ADS)

    Heath, Allison; Kavraki, Lydia; Balázsi, Gábor

    2008-03-01

    Despite the increasing number of completely sequenced genomes, much remains to be learned about how living cells process environmental information and respond to changes in their surroundings. Accumulating evidence indicates that eukaryotic and prokaryotic genes can be classified in two distinct categories that we will call class I and class II. Class I genes are housekeeping genes, often characterized by stable, noise resistant expression levels. In contrast, class II genes are stress-related genes and often have noisy, unstable expression levels. In this work we analyze the large scale transcription-regulatory networks (TRN) of E. coli and S. cerevisiae and preliminary data on H. sapien. We find that stable, housekeeping genes (class I) are preferentially utilized as transcriptional inputs while stress related, unstable genes (class II) are utilized as transcriptional integrators. This might be the result of convergent evolution that placed the appropriate genes in the appropriate locations within transcriptional networks according to some fundamental principles that govern cellular information processing.

  14. Transcriptional Regulation: a Genomic Overview

    PubMed Central

    Riechmann, José Luis

    2002-01-01

    The availability of the Arabidopsis thaliana genome sequence allows a comprehensive analysis of transcriptional regulation in plants using novel genomic approaches and methodologies. Such a genomic view of transcription first necessitates the compilation of lists of elements. Transcription factors are the most numerous of the different types of proteins involved in transcription in eukaryotes, and the Arabidopsis genome codes for more than 1,500 of them, or approximately 6% of its total number of genes. A genome-wide comparison of transcription factors across the three eukaryotic kingdoms reveals the evolutionary generation of diversity in the components of the regulatory machinery of transcription. However, as illustrated by Arabidopsis, transcription in plants follows similar basic principles and logic to those in animals and fungi. A global view and understanding of transcription at a cellular and organismal level requires the characterization of the Arabidopsis transcriptome and promoterome, as well as of the interactome, the localizome, and the phenome of the proteins involved in transcription. PMID:22303220

  15. Deregulated transcription factors in leukemia.

    PubMed

    Shima, Yutaka; Kitabayashi, Issay

    2011-08-01

    Specific chromosomal translocations and other mutations associated with acute myeloblastic leukemia (AML) often involve transcription factors and transcriptional coactivators. Such target genes include AML1, C/EBPα, RARα, MOZ, p300/CBP, and MLL, all of which are important in the regulation of hematopoiesis. The resultant fusion or mutant proteins deregulate the transcription of the affected genes and disrupt their essential role in hematopoiesis, causing differentiation block and abnormal proliferation and/or survival. This review focuses on such transcription factors and coactivators, and describes their roles in leukemogenesis and hematopoiesis. PMID:21823042

  16. The Stress Response of Escherichia coli under Microgravity.

    NASA Astrophysics Data System (ADS)

    Lynch, S.; Matin, A.

    At the onset of adverse environmental conditions, bacteria induce a controlled stress response to enable survival. Escherichia coli induces stress-specific reactions in response to a variety of environmental strains. A family of proteins termed sigma (s) factors is pivotal to the regulation of stress responses in bacteria. In particular Sigma S (ss) regulates several stress responses in E. coli and serves as an important global stress regulatory protein. Under optimal growth conditions, levels of ss are maintained at low cellular concentrations primarily via a proteolytic regulatory mechanism. At the onset of stress, ss levels increase due to increased stability of the molecule, facilitating transcriptional initiation and up regulation of specific stress related proteins. Concentrations of ss can therefore be indicative of cellular stress levels. Recent work by Kendrick et al demonstrated that Salmonella species grown under conditions of simulated microgravity display increased virulence - a stress-related phenotype. Using E. coli as a model system we aim to investigate the stress response elicited by the organism under conditions of simulated microgravity (SMG). SMG is generated in specially constructed rotary cell culture systems termed HARVs (High Aspect Ratio Vessels- Synthecon Inc.). By rotating at constant velocity around a vertical axis an environment is produced in which the gravitational vectors are randomized over the surface of the cell, resulting in an overall-time-averaged gravitational vector of 10-2 x g (4). E. coli cultures grown in HARVs under conditions of normal gravity (NG) and SMG repeatedly display slower growth kinetics under SMG. Western analysis of cells at exponential and stationary phase of growth from both cultures reveal similar levels of ss exist in exponential phase under both SMG and NG conditions. However, during stationary phase, levels of ss are at least 2-fold higher under conditions of SMG as compared to NG. Translational fusion

  17. A universal transcription pause sequence is an element of initiation factor σ70-dependent pausing.

    PubMed

    Bird, Jeremy G; Strobel, Eric J; Roberts, Jeffrey W

    2016-08-19

    The Escherichia coli σ70 initiation factor is required for a post-initiation, promoter-proximal pause essential for regulation of lambdoid phage late gene expression; potentially, σ70 acts at other sites during transcription elongation as well. The pause is induced by σ70 binding to a repeat of the promoter -10 sequence. After σ70 binding, further RNA synthesis occurs as DNA is drawn (or 'scrunched') into the enzyme complex, presumably exactly as occurs during initial synthesis from the promoter; this synthesis then pauses at a defined site several nucleotides downstream from the active center position when σ70 first engages the -10 sequence repeat. We show that the actual pause site in the stabilized, scrunched complex is the 'elemental pause sequence' recognized from its frequent occurrence in the E. coli genome. σ70 binding and the elemental pause sequence together, but neither alone, produce a substantial transcription pause. PMID:27098041

  18. Influence of Long Time Storage in Mineral Water on RNA Stability of Pseudomonas aeruginosa and Escherichia coli after Heat Inactivation

    PubMed Central

    Cenciarini, Claire; Courtois, Sophie; Raoult, Didier; La Scola, Bernard

    2008-01-01

    Background Research of RNA viability markers was previously studied for many bacterial species. Few and different targets of each species have been checked and motley results can be found in literature. No research has been done about Pseudomonas aeruginosa in this way. Methodology/Principal Findings Disappearance of 48 transcripts was analyzed by two-steps reverse transcription and real time polymerase chain reaction (RT-PCR) after heat-killing of Pseudomonas aeruginosa previously stored in mineral water or not. Differential results were obtained for each target. 16S rRNA, 23S rRNA, groEL, and rpmE were showed as the most persistent transcripts and rplP, rplV, rplE and rpsD were showed as the most labile transcripts after P. aeruginosa death. However, the labile targets appeared more persistent in bacteria previously stored in mineral water than freshly cultivated (non stored). These nine transcripts were also analyzed in Escherichia coli after heat-killing and different to opposite results were obtained, notably for groEL which was the most labile transcript of E. coli. Moreover, opposite results were obtained between mineral water stored and freshly cultivated E. coli. Conclusions and Significance This study highlights four potential viability markers for P. aeruginosa and four highly persistent transcripts. In a near future, these targets could be associated to develop an efficient viability kit. The present study also suggests that it would be difficult to determine universal RNA viability markers for environmental bacteria, since opposite results were obtained depending on the bacterial species and the physiological conditions. PMID:18941615

  19. Spatial organization of bacterial transcription and translation.

    PubMed

    Castellana, Michele; Hsin-Jung Li, Sophia; Wingreen, Ned S

    2016-08-16

    In bacteria such as Escherichia coli, DNA is compacted into a nucleoid near the cell center, whereas ribosomes-molecular complexes that translate mRNAs into proteins-are mainly localized to the poles. We study the impact of this spatial organization using a minimal reaction-diffusion model for the cellular transcriptional-translational machinery. Although genome-wide mRNA-nucleoid segregation still lacks experimental validation, our model predicts that [Formula: see text] of mRNAs are segregated to the poles. In addition, our analysis reveals a "circulation" of ribosomes driven by the flux of mRNAs, from synthesis in the nucleoid to degradation at the poles. We show that our results are robust with respect to multiple, biologically relevant factors, such as mRNA degradation by RNase enzymes, different phases of the cell division cycle and growth rates, and the existence of nonspecific, transient interactions between ribosomes and mRNAs. Finally, we confirm that the observed nucleoid size stems from a balance between the forces that the chromosome and mRNAs exert on each other. This suggests a potential global feedback circuit in which gene expression feeds back on itself via nucleoid compaction. PMID:27486246

  20. Uropathogenic Escherichia coli Epigenetically Manipulate Host Cell Death Pathways.

    PubMed

    Zhang, Zhengguo; Wang, Ming; Eisel, Florian; Tchatalbachev, Svetlin; Chakraborty, Trinad; Meinhardt, Andreas; Bhushan, Sudhanshu

    2016-04-01

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in human. It is well established that UPEC can subvert innate immune responses, but the role of UPEC in interfering with host cell death pathways is not known. Here, we show that UPEC abrogates activation of the host cell prosurvival protein kinase B signaling pathway, which results in the activation of mammalian forkhead box O (FOXO) transcription factors. Although FOXOs were localized in the nucleus and showed increased DNA-binding activity, no change in the expression levels of FOXO target genes were observed. UPEC can suppress BIM expression induced by LY249002, which results in attenuation of caspase 3 activation and blockage of apoptosis. Mechanistically, BIM expression appears to be epigenetically silenced by a decrease in histone 4 acetylation at the BIM promoter site. Taken together, these results suggest that UPEC can epigenetically silence BIM expression, a molecular switch that prevents apoptosis. PMID:26621912

  1. Phenotypic bistability in Escherichia coli's central carbon metabolism

    PubMed Central

    Kotte, Oliver; Volkmer, Benjamin; Radzikowski, Jakub L; Heinemann, Matthias

    2014-01-01

    Fluctuations in intracellular molecule abundance can lead to distinct, coexisting phenotypes in isogenic populations. Although metabolism continuously adapts to unpredictable environmental changes, and although bistability was found in certain substrate-uptake pathways, central carbon metabolism is thought to operate deterministically. Here, we combine experiment and theory to demonstrate that a clonal Escherichia coli population splits into two stochastically generated phenotypic subpopulations after glucose-gluconeogenic substrate shifts. Most cells refrain from growth, entering a dormant persister state that manifests as a lag phase in the population growth curve. The subpopulation-generating mechanism resides at the metabolic core, overarches the metabolic and transcriptional networks, and only allows the growth of cells initially achieving sufficiently high gluconeogenic flux. Thus, central metabolism does not ensure the gluconeogenic growth of individual cells, but uses a population-level adaptation resulting in responsive diversification upon nutrient changes. PMID:24987115

  2. Importance of understanding the main metabolic regulation in response to the specific pathway mutation for metabolic engineering of Escherichia coli

    PubMed Central

    Matsuoka, Yu; Shimizu, Kazuyuki

    2013-01-01

    Recent metabolic engineering practice was briefly reviewed in particular for the useful metabolite production such as natural products and biofuel productions. With the emphasis on systems biology approach, the metabolic regulation of the main metabolic pathways in E. coli was discussed from the points of view of enzyme level (allosteric and phosphorylation/ dephosphorylation) regulation, and gene level (transcriptional) regulation. Then the effects of the specific pathway gene knockout such as pts, pgi, zwf, gnd, pyk, ppc, pckA, lpdA, pfl gene knockout on the metabolism in E. coli were overviewed from the systems biology point of view with possible application for strain improvement point. PMID:24688678

  3. Regulation of ribonuclease E activity by the L4 ribosomal protein of Escherichia coli

    PubMed Central

    Singh, Dharam; Chang, Ssu-Jean; Lin, Pei-Hsun; Averina, Olga V.; Kaberdin, Vladimir R.; Lin-Chao, Sue

    2009-01-01

    Whereas ribosomal proteins (r-proteins) are known primarily as components of the translational machinery, certain of these r-proteins have been found to also have extraribosomal functions. Here we report the novel ability of an r-protein, L4, to regulate RNA degradation in Escherichia coli. We show by affinity purification, immunoprecipitation analysis, and E. coli two-hybrid screening that L4 interacts with a site outside of the catalytic domain of RNase E to regulate the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling plasmid DNA replication by stabilizing an antisense regulatory RNA normally attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli transcripts were shown by DNA microarray analysis, which revealed changes in the abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA, YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid metabolism and transport, transcription/translation, and DNA/RNA synthesis. Analysis of mRNA stability showed that the half lives of stress-responsive transcripts were increased by ectopic expression of L4, which normally increases along with other r-proteins in E. coli under stress conditions, and also by inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments. PMID:19144914

  4. Mechanosensitive mechanisms in transcriptional regulation

    PubMed Central

    Mammoto, Akiko; Mammoto, Tadanori; Ingber, Donald E.

    2012-01-01

    Summary Transcriptional regulation contributes to the maintenance of pluripotency, self-renewal and differentiation in embryonic cells and in stem cells. Therefore, control of gene expression at the level of transcription is crucial for embryonic development, as well as for organogenesis, functional adaptation, and regeneration in adult tissues and organs. In the past, most work has focused on how transcriptional regulation results from the complex interplay between chemical cues, adhesion signals, transcription factors and their co-regulators during development. However, chemical signaling alone is not sufficient to explain how three-dimensional (3D) tissues and organs are constructed and maintained through the spatiotemporal control of transcriptional activities. Accumulated evidence indicates that mechanical cues, which include physical forces (e.g. tension, compression or shear stress), alterations in extracellular matrix (ECM) mechanics and changes in cell shape, are transmitted to the nucleus directly or indirectly to orchestrate transcriptional activities that are crucial for embryogenesis and organogenesis. In this Commentary, we review how the mechanical control of gene transcription contributes to the maintenance of pluripotency, determination of cell fate, pattern formation and organogenesis, as well as how it is involved in the control of cell and tissue function throughout embryogenesis and adult life. A deeper understanding of these mechanosensitive transcriptional control mechanisms should lead to new approaches to tissue engineering and regenerative medicine. PMID:22797927

  5. Escherichia coli proline tRNA: structure and recognition sites for prolyl-tRNA synthetase.

    PubMed

    Hasegawa, T; Yokogawa, T

    2000-01-01

    A major proline tRNA was purified from bulk Escherichia coli A19 tRNA by affinity chromatography with a biotinylated DNA probe. Its nucleotide sequence including modified nucleotides was determined by the post-labelling technique. In order to study the recognition sites of this proline tRNA for prolyl-tRNA synthetase, various mutant transcripts were prepared using an in vitro transcription system with T7 RNA polymerase. Based on the results of in vitro kinetic analyses of mutant transcripts, it was concluded that the second and third letters, G35 and G36, of the anticodon, G37 of the anticodon loop, the discriminator base A73, G72 of the acceptor stem, G49 and U17A that existed in the corner of an L-shaped structure are the recognition sites of proline tRNA for prolyl-tRNA synthetase. PMID:12903242

  6. Mitotic bookmarking by transcription factors

    PubMed Central

    2013-01-01

    Mitosis is accompanied by dramatic changes in chromatin organization and nuclear architecture. Transcription halts globally and most sequence-specific transcription factors and co-factors are ejected from mitotic chromatin. How then does the cell maintain its transcriptional identity throughout the cell division cycle? It has become clear that not all traces of active transcription and gene repression are erased within mitotic chromatin. Many histone modifications are stable or only partially diminished throughout mitosis. In addition, some sequence-specific DNA binding factors have emerged that remain bound to select sites within mitotic chromatin, raising the possibility that they function to transmit regulatory information through the transcriptionally silent mitotic phase, a concept that has been termed “mitotic bookmarking.” Here we review recent approaches to studying potential bookmarking factors with regards to their mitotic partitioning, and summarize emerging ideas concerning the in vivo functions of mitotically bound nuclear factors. PMID:23547918

  7. Why Is Carbonic Anhydrase Essential to Escherichia coli?

    PubMed Central

    Merlin, Christophe; Masters, Millicent; McAteer, Sean; Coulson, Andrew

    2003-01-01

    The can (previously yadF) gene of Escherichia coli encodes a β-class carbonic anhydrase (CA), an enzyme which interconverts CO2 and bicarbonate.Various essential metabolic processes require either CO2 or bicarbonate and, although carbon dioxide and bicarbonate spontaneously equilibrate in solution, the low concentration of CO2 in air and its rapid diffusion from the cell mean that insufficient bicarbonate is spontaneously made in vivo to meet metabolic and biosynthetic needs. We calculate that demand for bicarbonate is 103- to 104-fold greater than would be provided by uncatalyzed intracellular hydration and that enzymatic conversion of CO2 to bicarbonate is therefore necessary for growth. We find that can expression is ordinarily required for growth in air. It is dispensable if the atmospheric partial pressure of CO2 is high or during anaerobic growth in a closed vessel at low pH, where copious CO2 is generated endogenously. CynT, the single E. coli Can paralog, can, when induced with azide, replace Can; also, the γ-CA from Methanosarcina thermophila can at least partially replace it. Expression studies showed that can transcription does not appear to respond to carbon dioxide concentration or to be autoregulated. However, can expression is influenced by growth rate and the growth cycle; it is expressed best in slow-growing cultures and at higher culture densities. Expression can vary over a 10-fold range during the growth cycle and is also elevated during starvation or heat stress. PMID:14563877

  8. Promoters of Escherichia coli versus Promoter Islands: Function and Structure Comparison

    PubMed Central

    Panyukov, Valeriy V.; Ozoline, Olga N.

    2013-01-01

    Expression of bacterial genes takes place under the control of RNA polymerase with exchangeable σ-subunits and multiple transcription factors. A typical promoter region contains one or several overlapping promoters. In the latter case promoters have the same or different σ-specificity and are often subjected to different regulatory stimuli. Genes, transcribed from multiple promoters, have on average higher expression levels. However, recently in the genome of Escherichia coli we found 78 regions with an extremely large number of potential transcription start points (promoter islands, PIs). It was shown that all PIs interact with RNA polymerase in vivo and are able to form transcriptionally competent open complexes both in vitro and in vivo but their transcriptional activity measured by oligonucleotide microarrays was very low, if any. Here we confirmed transcriptional defectiveness of PIs by analyzing the 5′-end specific RNA-seq data, but showed their ability to produce short oligos (9–14 bases). This combination of functional properties indicated a deliberate suppression of transcriptional activity within PIs. According to our data this suppression may be due to a specific conformation of the DNA double helix, which provides an ideal platform for interaction with both RNA polymerase and the histone-like nucleoid protein H-NS. The genomic DNA of E.coli contains therefore several dozen sites optimized by evolution for staying in a heterochromatin-like state. Since almost all promoter islands are associated with horizontally acquired genes, we offer them as specific components of bacterial evolution involved in acquisition of foreign genetic material by turning off the expression of toxic or useless aliens or by providing optimal promoter for beneficial genes. The putative molecular mechanism underlying the appearance of promoter islands within recipient genomes is discussed. PMID:23717391

  9. RNA polymerase and the regulation of transcription

    SciTech Connect

    Reznikoff, W.S.; Gross, C.A.; Burgess, R.R.; Record, M.T.; Dahlberg, J.E.; Wickens, M.P.

    1987-01-01

    This book consists of eight sections, each containing several papers. The section titles are: RNA Polymerases; Transcription Initiation - Bacterial; Regulation of Bacterial Transcription Initiation; Stable RNA Synthesis in Eukaryotes: Chromatin Structure; Promoters; Enhancers; and the Global Control of Eukaryotic Transcription; Specific Eukaryotic Transcription Factors; Termination of Transcription; and Short Communications.

  10. Peptidoglycan Hydrolases of Escherichia coli

    PubMed Central

    van Heijenoort, Jean

    2011-01-01

    Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

  11. Mutations in the CRE pocket of bacterial RNA polymerase affect multiple steps of transcription

    PubMed Central

    Petushkov, Ivan; Pupov, Danil; Bass, Irina; Kulbachinskiy, Andrey

    2015-01-01

    During transcription, the catalytic core of RNA polymerase (RNAP) must interact with the DNA template with low-sequence specificity to ensure efficient enzyme translocation and RNA extension. Unexpectedly, recent structural studies of bacterial promoter complexes revealed specific interactions between the nontemplate DNA strand at the downstream edge of the transcription bubble (CRE, core recognition element) and a protein pocket formed by core RNAP (CRE pocket). We investigated the roles of these interactions in transcription by analyzing point amino acid substitutions and deletions in Escherichia coli RNAP. The mutations affected multiple steps of transcription, including promoter recognition, RNA elongation and termination. In particular, we showed that interactions of the CRE pocket with a nontemplate guanine immediately downstream of the active center stimulate RNA-hairpin-dependent transcription pausing but not other types of pausing. Thus, conformational changes of the elongation complex induced by nascent RNA can modulate CRE effects on transcription. The results highlight the roles of specific core RNAP–DNA interactions at different steps of RNA synthesis and suggest their importance for transcription regulation in various organisms. PMID:25990734

  12. Escherichia coli survival in waters: Temperature dependence

    EPA Science Inventory

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 mo...

  13. Diagnosisand Investigation of Diarrheagenic Escherichia coli.

    PubMed

    Nataro, J P; Martinez, J

    1998-01-01

    Although most Escherichia coli are harmless commensals of the human intestine, certain specific, highly-adapted E. coli strains are capable of causing urinary tract, systemic or enteric/diarrheagenic infection. Diarrheagenic E coli are divided into six distinct categories, or pathotypes, each with a distinct pathogenic scheme (Table 1). Combined, diarrheagenic E coli have emerged as perhaps the most important enteric pathogens of man. In the developing world, the E coli categories account for more cases of gastroenteiltis among infants than any other cause (1) In addition, E coli are also the most common cause of traveller's diarrhea, which afflicts more than one million travellers to the developing world annually (1). Enterohemorrhagic E coli (EHEC) are the cause of hemolytic uremic syndrome (HUS), which has become a major foodborne threat in many parts of the developed world (2). Table 1 Categories of Diarrheagenic E. coli Category Toxins Invasion Virulence plasmid Adhesin Clinical syndrome ETEC LT, ST - Many CFA/I, CFA/II, CFA/IV, others Watery diarrhea EPEC - + 60 MDa Bundle-forming pilus Watery diarrhea of infants EHEC SLT-1, SLT-2 - 60 MDa( a ) Intimin, Fimbriae( a ) Hemorrhagic colitis, HUS EAEC EAST1( a ) ? 65 MDa( a ) AAF/I, AAF/I Watery, persistent diarrhea EIEC EIET( a ) +++ 140 MDa Ipa's(?) Watery diarrhea, dysentery DAEC ? ? ? F1845( a ) Watery diarrhea ( a )Role in pathogenesis unproven. PMID:21390758

  14. E. coli survival in waters: temperature dependence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Knowing the survival rates of water-borne Escherichia coli is important for evaluating microbial contamination and in making appropriate management decisions. E. coli survival rates are dependent on temperature; this dependency is routinely expressed using an analog of the Q10 model. This suggestion...

  15. Third International E. coli genome meeting

    SciTech Connect

    1994-12-31

    Proceedings of the Third E. Coli Genome Meeting are provided. Presentations were divided into sessions entitled (1) Large Scale Sequencing, Sequence Analysis; (2) Databases; (3) Sequence Analysis; (4) Sequence Divergence in E. coli Strains; (5) Repeated Sequences and Regulatory Motifs; (6) Mutations, Rearrangements and Stress Responses; and (7) Origins of New Genes. The document provides a collection of abstracts of oral and poster presentations.

  16. Transcription of Trypanosoma brucei maxicircles

    SciTech Connect

    Michelotti, E.F.; Hajduk, S.L.

    1987-05-01

    Trypanosoma brucei is a protozoan parasite which developmentally regulates mitochondrial activity. In the mammal T. brucei produces ATP entirely by glycolysis while cytochrome mediated respiration resumes in the life-stage in the midgut of the insect vector. Using quantitative S1 nuclease protection assays two types of regulation of the steady state levels of the mitochondrial transcripts were found. Transcription of cytochrome b, cytochrome oxidase, and the rRNA genes is repressed in early bloodstream developmental stages, undergoes dramatic activation in later bloodstream stages, and finally a lesser activation in the insect developmental stage. Transcription of NADH dehydrogenase genes, however, is unregulated. Mitochondrial transcripts with a 5' triphosphate terminus, representing the site of transcription initiation, were capped using guanylyl transferase. The in vitro capped RNA hybridized to only one of eight mitochondrial restriction fragments on a Southern blot, however, hybridization of Southern blots with RNA from ..cap alpha..-/sup 32/P-UTP pulsed mitochondria labelled all restriction fragments equally. These results suggest that each DNA strand has a single promoter which directs the transcription of a full-length RNA which is subsequently processed. Different mitochondrial genes, despite being expressed on the same precursor RNA molecule, are independently regulated by both transcription initiation and RNA processing.

  17. AthaMap, integrating transcriptional and post-transcriptional data

    PubMed Central

    Bülow, Lorenz; Engelmann, Stefan; Schindler, Martin; Hehl, Reinhard

    2009-01-01

    The AthaMap database generates a map of predicted transcription factor binding sites (TFBS) for the whole Arabidopsis thaliana genome. AthaMap has now been extended to include data on post-transcriptional regulation. A total of 403 173 genomic positions of small RNAs have been mapped in the A. thaliana genome. These identify 5772 putative post-transcriptionally regulated target genes. AthaMap tools have been modified to improve the identification of common TFBS in co-regulated genes by subtracting post-transcriptionally regulated genes from such analyses. Furthermore, AthaMap was updated to the TAIR7 genome annotation, a graphic display of gene analysis results was implemented, and the TFBS data content was increased. AthaMap is freely available at http://www.athamap.de/. PMID:18842622

  18. AthaMap, integrating transcriptional and post-transcriptional data.

    PubMed

    Bülow, Lorenz; Engelmann, Stefan; Schindler, Martin; Hehl, Reinhard

    2009-01-01

    The AthaMap database generates a map of predicted transcription factor binding sites (TFBS) for the whole Arabidopsis thaliana genome. AthaMap has now been extended to include data on post-transcriptional regulation. A total of 403,173 genomic positions of small RNAs have been mapped in the A. thaliana genome. These identify 5772 putative post-transcriptionally regulated target genes. AthaMap tools have been modified to improve the identification of common TFBS in co-regulated genes by subtracting post-transcriptionally regulated genes from such analyses. Furthermore, AthaMap was updated to the TAIR7 genome annotation, a graphic display of gene analysis results was implemented, and the TFBS data content was increased. AthaMap is freely available at http://www.athamap.de/. PMID:18842622

  19. Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain.

    PubMed Central

    Provence, D L; Curtiss, R

    1994-01-01

    In this article, we report the isolation and characterization of a gene that may be important in the adherence of avian pathogenic Escherichia coli to the avian respiratory tract. The E. coli strain HB101, which is unable to agglutinate chicken erythrocytes, was transduced with cosmid libraries from the avian pathogenic E. coli strain chi 7122. Enrichment of transductants that could agglutinate chicken erythrocytes yielded 19 colonies. These isolates contained cosmids that encompassed four nonoverlapping regions of the E. coli chromosome. Only one group of cosmids, represented by pYA3104, would cause E. coli CC118 to agglutinate chicken erythrocytes. A 10-kb fragment of this cosmid was subcloned in pACYC184. Transposon mutagenesis of this fragment with Tn5seq1 indicated that a contiguous 4.4-kb region of cloned DNA was required for hemagglutination. In vitro transcription/translation assays indicated that this 4.4-kb region of DNA encoded one protein of approximately 140 kDa. The nucleotide sequence of this region was determined and found to encode one open reading frame of 4,134 nucleotides that would encode a protein of 1,377 amino acids with a deduced molecular weight of 148,226. This gene confers on E. coli K-12 a temperature-sensitive hemagglutination phenotype that is best expressed when cells are grown at 26 degrees C, and we have designated this gene tsh and the deduced gene product Tsh. Insertional mutagenesis of the chromosomal tsh gene in chi 7122 had no effect on hemagglutination titers. The deduced protein was found to contain significant homology to the Haemophilus influenzae and Neisseria gonorrhoeae immunoglobulin A1 proteases. These data indicate that (i) a single gene isolated from the avian pathogenic E. coli strain chi 7122 will confer on E. coli K-12 a hemagglutination-positive phenotype, (ii) chi 7122 contains at least two distinct mechanisms to allow hemagglutination to occur, and (iii) the hemagglutinin Tsh has homology with a class of

  20. Escherichia coli and Sudden Infant Death Syndrome

    PubMed Central

    Bettelheim, Karl A.; Goldwater, Paul N.

    2015-01-01

    This review examines the association of strains of Escherichia coli with sudden infant death syndrome (SIDS) and the possible role these bacteria play in this enigmatic condition. The review addresses evidence for E. coli in SIDS infants, potential sources of E. coli in the environment, colonization by commensal and pathogenic strains, the variety of currently accepted pathotypes, and how these pathotypes could compromise intestinal integrity and induce inflammation. Both intestinal and extraintestinal pathotypes are compared in relation to the apparent liability in which virulence traits can be gained or lost by strains of E. coli. The way in which E. coli infections fit with current views on infant sleeping position and other SIDS risk factors is highlighted. PMID:26191064

  1. Structures of the Porphyromonas gingivalis OxyR regulatory domain explain differences in expression of the OxyR regulon in Escherichia coli and P. gingivalis

    SciTech Connect

    Svintradze, David V.; Peterson, Darrell L.; Collazo-Santiago, Evys A.; Lewis, Janina P.; Wright, H. Tonie

    2013-10-01

    Differences in OxyR regulated expression of oxidative stress genes between Escherichia coli and Porphyromonas gingivalis are explained by very minor differences in structure and amino-acid sequence of the respective oxidized and reduced OxyR regulatory domains. These differences affect OxyR quaternary structures and are predicted from model building of full length OxyR–DNA complexes to confer distinct modes of DNA binding on this transcriptional regulator. OxyR transcriptionally regulates Escherichia coli oxidative stress response genes through a reversibly reducible cysteine disulfide biosensor of cellular redox status. Structural changes induced by redox changes in these cysteines are conformationally transmitted to the dimer subunit interfaces, which alters dimer and tetramer interactions with DNA. In contrast to E. coli OxyR regulatory-domain structures, crystal structures of Porphyromonas gingivalis OxyR regulatory domains show minimal differences in dimer configuration on changes in cysteine disulfide redox status. This locked configuration of the P. gingivalis OxyR regulatory-domain dimer closely resembles the oxidized (activating) form of the E. coli OxyR regulatory-domain dimer. It correlates with the observed constitutive activation of some oxidative stress genes in P. gingivalis and is attributable to a single amino-acid insertion in P. gingivalis OxyR relative to E. coli OxyR. Modelling of full-length P. gingivalis, E. coli and Neisseria meningitidis OxyR–DNA complexes predicts different modes of DNA binding for the reduced and oxidized forms of each.

  2. Survival and expression of acid resistance genes in Shiga toxin-producing Escherichia coli acid adapted in pineapple juice and exposed to synthetic gastric fluid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: The aim of this research was to examine relative transcriptional expression of acid resistance (AR) genes, rpoS, gadA and adiA, in O157:H7 and non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes after adaptation to pineapple juice (PJ) and subsequently to determine survival with e...

  3. Autoregulation of topoisomerase I expression by supercoiling sensitive transcription

    PubMed Central

    Ahmed, Wareed; Menon, Shruti; D. N. B. Karthik, Pullela V.; Nagaraja, Valakunja

    2016-01-01

    The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Escherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobacterium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of sub-optimal spacing between the −35 and −10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the −35 and −10 elements to facilitate the optimal transcription of topoI. PMID:26496944

  4. A dual switch controls bacterial enhancer-dependent transcription

    PubMed Central

    Wiesler, Simone C.; Burrows, Patricia C.; Buck, Martin

    2012-01-01

    Bacterial RNA polymerases (RNAPs) are targets for antibiotics. Myxopyronin binds to the RNAP switch regions to block structural rearrangements needed for formation of open promoter complexes. Bacterial RNAPs containing the major variant σ54 factor are activated by enhancer-binding proteins (bEBPs) and transcribe genes whose products are needed in pathogenicity and stress responses. We show that (i) enhancer-dependent RNAPs help Escherichia coli to survive in the presence of myxopyronin, (ii) enhancer-dependent RNAPs partially resist inhibition by myxopyronin and (iii) ATP hydrolysis catalysed by bEBPs is obligatory for functional interaction of the RNAP switch regions with the transcription start site. We demonstrate that enhancer-dependent promoters contain two barriers to full DNA opening, allowing tight regulation of transcription initiation. bEBPs engage in a dual switch to (i) allow propagation of nucleated DNA melting from an upstream DNA fork junction and (ii) complete the formation of the transcription bubble and downstream DNA fork junction at the RNA synthesis start site, resulting in switch region-dependent RNAP clamp closure and open promoter complex formation. PMID:22965125

  5. Structure of the Escherichia coli RNA polymerase α subunit C-terminal domain

    SciTech Connect

    Lara-González, Samuel; Birktoft, Jens J.; Lawson, Catherine L.

    2010-07-01

    The crystal structure of the dimethyllysine derivative of the E. coli RNA polymerase α subunit C-terminal domain is reported at 2.0 Å resolution. The α subunit C-terminal domain (αCTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli αCTD (α subunit residues 245–329) determined to 2.0 Å resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P2{sub 1} and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, R{sub free} = 0.236) has improved geometry compared with prior lower resolution determinations of the αCTD structure [Jeon et al. (1995 ▶), Science, 270, 1495–1497; Benoff et al. (2002 ▶), Science, 297, 1562–1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of αCTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction.

  6. RegR virulence regulon of rabbit-specific enteropathogenic Escherichia coli strain E22.

    PubMed

    Srikhanta, Yogitha N; Hocking, Dianna M; Praszkier, Judyta; Wakefield, Matthew J; Robins-Browne, Roy M; Yang, Ji; Tauschek, Marija

    2013-04-01

    AraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli, enteroaggregative E. coli, and Citrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, of C. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target, sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression of sefA by binding to a region upstream of the sefA promoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22. PMID:23340312

  7. Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy

    PubMed Central

    Nemani, Krishnamurthy V.; Ennis, Riley C.; Griswold, Karl E.; Gimi, Barjor

    2015-01-01

    Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional control of thermo-regulatory λpL-cI857 promoter cassette which provides a thermal switch to trigger enzyme synthesis. Enhanced cytosine deaminase expression was observed in cultures incubated at 42 °C as compared to 30 °C, and enzyme expression was further substantiated by spectrophotometric assays indicating enhanced conversion of 5-fluorocytosine to 5-fluorouracil. The engineered cells were subsequently co-encapsulated with magnetic iron oxide nanoparticles in immunoprotective alginate microcapsules, and cytosine deaminase expression was triggered remotely by alternating magnetic field-induced hyperthermia. The combination of 5-fluorocytosine with AMF-activated microcapsules demonstrated tumor cell cytotoxicity comparable to direct treatment with 5-fluorouracil chemotherapy. Such enzyme-prodrug therapy, based on engineered and immunoisolated E. coli, may ultimately yield an improved therapeutic index relative to monotherapy, as AMF mediated hyperthermia might be expected to pre-sensitize tumors to chemotherapy under appropriate conditions. PMID:25820125

  8. Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7210 expressed in Escherichia coli

    SciTech Connect

    Gurevitz, M.; Somerville, C.R.; McIntosh, L.

    1985-10-01

    The authors have placed the genes encoding ribulosebisphosphate carboxylase/oxygenase from the Anabaena 7120 operon under transcriptional control of the lac promoter carried on the Escherichia coli plasmid pUC19. The genes encoding both the large and small subunit polypeptides (rbcL and rbcS) are transcribed and translated so that approx. = 0.6% of the soluble protein in E. coli extracts is a fully functional holoenzyme with a sedimentation coefficient of approximately 18S, which contains stoichiometric amounts of the two subunits. However, expression of the large subunit polypeptide vastly exceeds that of the small subunit because the majority of transcripts terminate in the intergenic region between the rbcL and rbcS genes. As a result, excess large subunit is synthesized and accumulates in E. coli as an insoluble and catalytically inactive form. Because small subunit is found only in the high molecular weight soluble form of ribulosebisphosphate carboxylase/oxygenase, the authors propose that the small subunit promotes assembly of the hexadecameric form of the enzyme via heterodimers of large and small subunits.

  9. Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy.

    PubMed

    Nemani, Krishnamurthy V; Ennis, Riley C; Griswold, Karl E; Gimi, Barjor

    2015-06-10

    Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional control of thermo-regulatory λpL-cI857 promoter cassette which provides a thermal switch to trigger enzyme synthesis. Enhanced cytosine deaminase expression was observed in cultures incubated at 42°C as compared to 30°C, and enzyme expression was further substantiated by spectrophotometric assays indicating enhanced conversion of 5-fluorocytosine to 5-fluorouracil. The engineered cells were subsequently co-encapsulated with magnetic iron oxide nanoparticles in immunoprotective alginate microcapsules, and cytosine deaminase expression was triggered remotely by alternating magnetic field-induced hyperthermia. The combination of 5-fluorocytosine with AMF-activated microcapsules demonstrated tumor cell cytotoxicity comparable to direct treatment with 5-fluorouracil chemotherapy. Such enzyme-prodrug therapy, based on engineered and immunoisolated E. coli, may ultimately yield an improved therapeutic index relative to monotherapy, as AMF mediated hyperthermia might be expected to pre-sensitize tumors to chemotherapy under appropriate conditions. PMID:25820125

  10. Conserved Units of Co-Expression in Bacterial Genomes: An Evolutionary Insight into Transcriptional Regulation

    PubMed Central

    Junier, Ivan; Rivoire, Olivier

    2016-01-01

    Genome-wide measurements of transcriptional activity in bacteria indicate that the transcription of successive genes is strongly correlated beyond the scale of operons. Here, we analyze hundreds of bacterial genomes to identify supra-operonic segments of genes that are proximal in a large number of genomes. We show that these synteny segments correspond to genomic units of strong transcriptional co-expression. Structurally, the segments contain operons with specific relative orientations (co-directional or divergent) and nucleoid-associated proteins are found to bind at their boundaries. Functionally, operons inside a same segment are highly co-expressed even in the apparent absence of regulatory factors at their promoter regions. Remote operons along DNA can also be co-expressed if their corresponding segments share a transcriptional or sigma factor, without requiring these factors to bind directly to the promoters of the operons. As evidence that these results apply across the bacterial kingdom, we demonstrate them both in the Gram-negative bacterium Escherichia coli and in the Gram-positive bacterium Bacillus subtilis. The underlying process that we propose involves only RNA-polymerases and DNA: it implies that the transcription of an operon mechanically enhances the transcription of adjacent operons. In support of a primary role of this regulation by facilitated co-transcription, we show that the transcription en bloc of successive operons as a result of transcriptional read-through is strongly and specifically enhanced in synteny segments. Finally, our analysis indicates that facilitated co-transcription may be evolutionary primitive and may apply beyond bacteria. PMID:27195891

  11. relA enhances the adherence of enteropathogenic Escherichia coli.

    PubMed

    Spira, Beny; Ferreira, Gerson Moura; de Almeida, Luiz Gustavo

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) is a known causative agent of diarrhea in children. In the process of colonization of the small intestine, EPEC synthesizes two types of adhesins, the bundle-forming pilus (BFP) and intimin. The BFP pilus is an adhesin associated with the initial stages of adherence of EPEC to epithelial cells, while the outer membrane protein intimin carries out the intimate adherence that takes place at the third stage of infection. BFP is encoded by the bfp operon located in plasmid EAF, present only in typical EPEC isolates, while eae, the gene that encodes intimin is situated in the LEE, a chromosomal pathogenicity island. Transcription of bfp and eae is regulated by the products of the perABC operon, also present in plasmid EAF. Here we show that deletion of relA, that encodes a guanosine penta and tetraphosphate synthetase impairs EPEC adherence to epithelial cells in vitro. In the absence of relA, the transcription of the regulatory operon perABC is reduced, resulting in lower levels of BFP and intimin. Bacterial adherence, BFP and intimin synthesis and perABC expression are restored upon complementation with the wild-type relA allele. PMID:24643076

  12. Genotype to Phenotype Mapping of the E. coli lac Promoter

    NASA Astrophysics Data System (ADS)

    Otwinowski, Jakub; Nemenman, Ilya

    2014-03-01

    Genotype-to-phenotype maps and the related fitness landscapes that include epistatic interactions are difficult to measure because of their high dimensional structure. Here we construct such a map using the recently collected corpora of high-throughput sequence data from the 75 base pairs long mutagenized E. coli lac promoter region, where each sequence is associated with induced transcriptional activity measured by a fluorescent reporter. We find that the additive (non-epistatic) contributions of individual mutations account for about two-thirds of the explainable phenotype variance, while pairwise epistasis explains about 7% of the variance for the full mutagenized sequence and about 15% for the subsequence associated with protein binding sites. Surprisingly, there is no evidence for third order epistatic contributions, and our inferred fitness landscape is essentially single peaked, with a small amount of antagonistic epistasis. We identify transcription factor (CRP) and RNA polymerase binding sites in the promotor region and their interactions. We conclude with a cautionary note that inferred properties of fitness landscapes may be severely influenced by biases in the sequence data. Funded in part by HFSP and James S. McDonnell Foundation.

  13. Multiscale Spatial Organization of RNA Polymerase in Escherichia coli

    PubMed Central

    Endesfelder, Ulrike; Finan, Kieran; Holden, Seamus J.; Cook, Peter R.; Kapanidis, Achillefs N.; Heilemann, Mike

    2013-01-01

    Nucleic acid synthesis is spatially organized in many organisms. In bacteria, however, the spatial distribution of transcription remains obscure, owing largely to the diffraction limit of conventional light microscopy (200–300 nm). Here, we use photoactivated localization microscopy to localize individual molecules of RNA polymerase (RNAP) in Escherichia coli with a spatial resolution of ∼40 nm. In cells growing rapidly in nutrient-rich media, we find that RNAP is organized in 2–8 bands. The band number scaled directly with cell size (and so with the chromosome number), and bands often contained clusters of >70 tightly packed RNAPs (possibly engaged on one long ribosomal RNA operon of 6000 bp) and clusters of such clusters (perhaps reflecting a structure like the eukaryotic nucleolus where many different ribosomal RNA operons are transcribed). In nutrient-poor media, RNAPs were located in only 1–2 bands; within these bands, a disproportionate number of RNAPs were found in clusters containing ∼20–50 RNAPs. Apart from their importance for bacterial transcription, our studies pave the way for molecular-level analysis of several cellular processes at the nanometer scale. PMID:23823236

  14. Zooming in on Transcription Preinitiation.

    PubMed

    Gupta, Kapil; Sari-Ak, Duygu; Haffke, Matthias; Trowitzsch, Simon; Berger, Imre

    2016-06-19

    Class II gene transcription commences with the assembly of the Preinitiation Complex (PIC) from a plethora of proteins and protein assemblies in the nucleus, including the General Transcription Factors (GTFs), RNA polymerase II (RNA pol II), co-activators, co-repressors, and more. TFIID, a megadalton-sized multiprotein complex comprising 20 subunits, is among the first GTFs to bind the core promoter. TFIID assists in nucleating PIC formation, completed by binding of further factors in a highly regulated stepwise fashion. Recent results indicate that TFIID itself is built from distinct preformed submodules, which reside in the nucleus but also in the cytosol of cells. Here, we highlight recent insights in transcription factor assembly and the regulation of transcription preinitiation. PMID:27067110

  15. The transcriptional foundation of pluripotency.

    PubMed

    Chambers, Ian; Tomlinson, Simon R

    2009-07-01

    A fundamental goal in biology is to understand the molecular basis of cell identity. Pluripotent embryonic stem (ES) cell identity is governed by a set of transcription factors centred on the triumvirate of Oct4, Sox2 and Nanog. These proteins often bind to closely localised genomic sites. Recent studies have identified additional transcriptional modulators that bind to chromatin near sites occupied by Oct4, Sox2 and Nanog. This suggests that the combinatorial control of gene transcription might be fundamental to the ES cell state. Here we discuss how these observations advance our understanding of the transcription factor network that controls pluripotent identity and highlight unresolved issues that arise from these studies. PMID:19542351

  16. Transcriptional Regulation of Hepatic Lipogenesis

    PubMed Central

    Wang, Yuhui; Viscarra, Jose; Kim, Sun-Joong; Sul, Hei Sook

    2016-01-01

    Fatty acid and fat synthesis in liver is a highly regulated metabolic pathway critical for energy distribution. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcription level. Transcription factors, such as USF, SREBP-1c, LXR and ChREBP play critical roles in this process. Recently, insights have been gained into how various signaling pathways regulate these transcription factors. After feeding, high blood glucose and insulin induce lipogenic genes through several pathways, including DNA-PK, aPKC and Akt-mTOR. Various transcription factors and coregulators undergo specific modifications, such as phosphorylation, acetylation, or ubiquitination, which affect their function, stability, or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance. PMID:26490400

  17. RNA-guided transcriptional regulation

    DOEpatents

    Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

    2016-02-23

    Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

  18. Expression and Genetic Activation of Cyclic Di-GMP-Specific Phosphodiesterases in Escherichia coli

    PubMed Central

    Reinders, Alberto; Hee, Chee-Seng; Ozaki, Shogo; Mazur, Adam; Boehm, Alex; Schirmer, Tilman

    2015-01-01

    ABSTRACT Intracellular levels of the bacterial second messenger cyclic di-GMP (c-di-GMP) are controlled by antagonistic activities of diguanylate cyclases and phosphodiesterases. The phosphodiesterase PdeH was identified as a key regulator of motility in Escherichia coli, while deletions of any of the other 12 genes encoding potential phosphodiesterases did not interfere with motility. To analyze the roles of E. coli phosphodiesterases, we demonstrated that most of these proteins are expressed under laboratory conditions. We next isolated suppressor mutations in six phosphodiesterase genes, which reinstate motility in the absence of PdeH by reducing cellular levels of c-di-GMP. Expression of all mutant alleles also led to a reduction of biofilm formation. Thus, all of these proteins are bona fide phosphodiesterases that are capable of interfering with different c-di-GMP-responsive output systems by affecting the global c-di-GMP pool. This argues that E. coli possesses several phosphodiesterases that are inactive under laboratory conditions because they lack appropriate input signals. Finally, one of these phosphodiesterases, PdeL, was studied in more detail. We demonstrated that this protein acts as a transcription factor to control its own expression. Motile suppressor alleles led to a strong increase of PdeL activity and elevated pdeL transcription, suggesting that enzymatic activity and transcriptional control are coupled. In agreement with this, we showed that overall cellular levels of c-di-GMP control pdeL transcription and that this control depends on PdeL itself. We thus propose that PdeL acts both as an enzyme and as a c-di-GMP sensor to couple transcriptional activity to the c-di-GMP status of the cell. IMPORTANCE Most bacteria possess multiple diguanylate cyclases and phosphodiesterases. Genetic studies have proposed that these enzymes show signaling specificity by contributing to distinct cellular processes without much cross talk. Thus, spatial

  19. CovR alleviates transcriptional silencing by a nucleoid-associated histone-like protein in Streptococcus mutans.

    PubMed

    Biswas, Indranil; Mohapatra, Saswat Sourav

    2012-04-01

    In Streptococcus mutans, the global response regulator CovR plays an important role in biofilm formation, stress tolerance response, and caries production. We have previously demonstrated that CovR activates a large gene cluster, which is a part of a genomic island, TnSmu2. In this article, we have further characterized CovR at the molecular level to understand the gene activation mechanism. Toward this end, we mapped the transcription start site of the operon that lies upstream of the SMU.1348 gene (P(SMU.1348)), the first gene of the cluster. We constructed a transcriptional reporter fusion and showed that CovR induces expression from P(SMU.1348). We also demonstrated that purified CovR protects the sequence surrounding the -10 region of P(SMU.1348). In an in vitro transcription assay, we showed that histone-like protein (HLP), a homologue of Escherichia coli HU protein, represses transcription from P(SMU.1348). In vivo overexpression of HLP in trans also represses transcription from P(SMU.1348). Addition of CovR to the HLP-repressed P(SMU.1348) resulted in increased transcription from the promoter, suggesting a role for CovR in countering HLP silencing. Moreover, addition of SMU.1349, a transcriptional activator of the operon, to the in vitro assay further stimulated the transcription. Based on our in vivo and in vitro results, we propose a model for transcriptional activation of the operon. PMID:22343292

  20. RHON1 Mediates a Rho-Like Activity for Transcription Termination in Plastids of Arabidopsis thaliana[C][W

    PubMed Central

    Chi, Wei; He, Baoye; Manavski, Nikolay; Mao, Juan; Ji, Daili; Lu, Congming; Rochaix, Jean David; Meurer, Jörg; Zhang, Lixin

    2014-01-01

    Although transcription termination is essential to generate functional RNAs, its underlying molecular mechanisms are still poorly understood in plastids of vascular plants. Here, we show that the RNA binding protein RHON1 participates in transcriptional termination of rbcL (encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) in Arabidopsis thaliana. Inactivation of RHON1 leads to enhanced rbcL read-through transcription and to aberrant accD (encoding β-subunit of the acetyl-CoA carboxylase) transcriptional initiation, which may result from inefficient transcription termination of rbcL. RHON1 can bind to the mRNA as well as to single-stranded DNA of rbcL, displays an RNA-dependent ATPase activity, and terminates transcription of rbcL in vitro. These results suggest that RHON1 terminates rbcL transcription using an ATP-driven mechanism similar to that of Rho of Escherichia coli. This RHON1-dependent transcription termination occurs in Arabidopsis but not in rice (Oryza sativa) and appears to reflect a fundamental difference between plastomes of dicotyledonous and monocotyledonous plants. Our results point to the importance and significance of plastid transcription termination and provide insights into its machinery in an evolutionary context. PMID:25480370

  1. CovR Alleviates Transcriptional Silencing by a Nucleoid-Associated Histone-Like Protein in Streptococcus mutans

    PubMed Central

    Mohapatra, Saswat Sourav

    2012-01-01

    In Streptococcus mutans, the global response regulator CovR plays an important role in biofilm formation, stress tolerance response, and caries production. We have previously demonstrated that CovR activates a large gene cluster, which is a part of a genomic island, TnSmu2. In this article, we have further characterized CovR at the molecular level to understand the gene activation mechanism. Toward this end, we mapped the transcription start site of the operon that lies upstream of the SMU.1348 gene (PSMU.1348), the first gene of the cluster. We constructed a transcriptional reporter fusion and showed that CovR induces expression from PSMU.1348. We also demonstrated that purified CovR protects the sequence surrounding the −10 region of PSMU.1348. In an in vitro transcription assay, we showed that histone-like protein (HLP), a homologue of Escherichia coli HU protein, represses transcription from PSMU.1348. In vivo overexpression of HLP in trans also represses transcription from PSMU.1348. Addition of CovR to the HLP-repressed PSMU.1348 resulted in increased transcription from the promoter, suggesting a role for CovR in countering HLP silencing. Moreover, addition of SMU.1349, a transcriptional activator of the operon, to the in vitro assay further stimulated the transcription. Based on our in vivo and in vitro results, we propose a model for transcriptional activation of the operon. PMID:22343292

  2. Forcing FAK into Transcriptional Activity.

    PubMed

    Lietha, Daniel

    2016-08-01

    Focal adhesion kinase (FAK) has known signaling roles in cytoplasmic adhesion structures, but was recently shown to act as a transcriptional regulator in the nucleus. In this issue of Structure, Cardoso et al. (2016) report that mechanical forces translocate FAK to the nucleus of cardiomyocytes, and provide structural insights into how FAK interacts with the MEF2 transcription factor to control cardiac hypertrophy. PMID:27486913

  3. Insights into the complex levels of regulation imposed on Escherichia coli DNA polymerase V.

    PubMed

    Goodman, Myron F; McDonald, John P; Jaszczur, Malgorzata M; Woodgate, Roger

    2016-08-01

    It is now close to 40 years since the isolation of non-mutable umu/uvm strains of Escherichia coli and the realization that damage induced mutagenesis in E.coli is not a passive process. Early models of mutagenesis envisioned the Umu proteins as accessory factors to the cell's replicase that not only reduced its normally high fidelity, but also allowed the enzyme to traverse otherwise replication-blocking lesions in the genome. However, these models underwent a radical revision approximately 15 years ago, with the discovery that the Umu proteins actually encode for a DNA polymerase, E.coli pol V. The polymerase lacks 3'→5' exonucleolytic proofreading activity and is inherently error-prone when replicating both undamaged and damage DNA. So as to limit any "gratuitous" mutagenesis, the activity of pol V is strictly regulated in the cell at multiple levels. This review will summarize our current understanding of the myriad levels of regulation imposed on pol V including transcriptional control, posttranslational modification, targeted proteolysis, activation of the catalytic activity of pol V through protein-protein interactions and the very recently described intracellular spatial regulation of pol V. Remarkably, despite the multiple levels at which pol V is regulated, the enzyme is nevertheless able to contribute to the genetic diversity and evolutionary fitness of E.coli. PMID:27236212

  4. A novel nucleoid protein of Escherichia coli induced under anaerobiotic growth conditions.

    PubMed

    Teramoto, Jun; Yoshimura, Shige H; Takeyasu, Kunio; Ishihama, Akira

    2010-06-01

    A systematic search was performed for DNA-binding sequences of YgiP, an uncharacterized transcription factor of Escherichia coli, by using the Genomic SELEX. A total of 688 YgiP-binding loci were identified after genome-wide profiling of SELEX fragments with a high-density microarray (SELEX-chip). Gel shift and DNase-I footprinting assays indicated that YgiP binds to multiple sites along DNA probes with a consensus GTTNATT sequence. Atomic force microscope observation indicated that at low concentrations, YgiP associates at various sites on DNA probes, but at high concentrations, YgiP covers the entire DNA surface supposedly through protein-protein contact. The intracellular concentration of YgiP is very low in growing E. coli cells under aerobic conditions, but increases more than 100-fold to the level as high as the major nucleoid proteins under anaerobic conditions. An E. coli mutant lacking ygiP showed retarded growth under anaerobic conditions. High abundance and large number of binding sites together indicate that YgiP is a nucleoid-associated protein with both architectural and regulatory roles as the nucleoid proteins Fis and IHF. We then propose that YgiP is a novel nucleoid protein of E. coli under anaerobiosis and propose to rename it Dan (DNA-binding protein under anaerobic conditions). PMID:20156994

  5. Characterization of cellulose production in Escherichia coli Nissle 1917 and its biological consequences.

    PubMed

    Monteiro, Cláudia; Saxena, Inder; Wang, Xiaoda; Kader, Abdul; Bokranz, Werner; Simm, Roger; Nobles, David; Chromek, Milan; Brauner, Annelie; Brown, R Malcolm; Römling, Ute

    2009-05-01

    Bacterial species of the Enterobacteriaceae family produce cellulose and curli fimbriae as extracellular matrix components, and their synthesis is positively regulated by the transcriptional activator CsgD. In this group of bacteria, cellulose biosynthesis is commonly regulated by CsgD via the GGDEF domain protein AdrA, a diguanylate cyclase that produces cyclic-diguanylic acid (c-di-GMP), an allosteric activator of cellulose synthase. In the probiotic Escherichia coli strain Nissle 1917 and its recent clonal isolates, CsgD activates the production of curli fimbriae at 28 degrees C, but neither CsgD nor AdrA is required for the c-di-GMP-dependent biosynthesis of cellulose at 28 degrees C and 37 degrees C. In these strains, the GGDEF domain protein YedQ, a diguanylate cyclase that activates cellulose biosynthesis in certain E. coli strains, is not required for cellulose biosynthesis and it has in fact evolved into a novel protein. Cellulose production in Nissle 1917 is required for adhesion of bacteria to the gastrointestinal epithelial cell line HT-29, to the mouse epithelium in vivo, and for enhanced cytokine production. The role of cellulose in this strain is in contrast to the role of cellulose in the commensal strain E. coli TOB1. Consequently, the role of cellulose in bacterial-host interaction is dependent on the E. coli strain background. PMID:19175667

  6. Cloning and expression of the phospho-beta-galactosidase gene of Staphylococcus aureus in Escherichia coli.

    PubMed Central

    Breidt, F; Stewart, G C

    1986-01-01

    The phospho-beta-galactosidase gene of Staphylococcus aureus was cloned in Escherichia coli. This was done by first isolating a staphylococcal transposon Tn551-induced mutant which rendered phospho-beta-galactosidase synthesis partially constitutive because of an insertion nearby this lac structural gene. This allowed selection in E. coli of chimeric plasmids which expressed the erythromycin resistance determinant of Tn551. A 26-kilobase (kb) BamHI insert in plasmid pBR322 was isolated which encoded phospho-beta-galactosidase, as determined by phospho-beta-galactosidase activity measurements. Maxicell experiments showed the presence of 56-, 13.5-, and 31-kilodalton proteins encoded by the staphylococcal DNA. The presence of the 56-kilodalton protein correlated with phospho-beta-galactosidase activity and corresponded in molecular weight to the reported value for the purified enzyme. The nature of the other proteins is unknown. Phospho-beta-galactosidase was apparently expressed in E. coli by a promoter contained within a 2.1-kb EcoRI chromosomal DNA fragment. This fragment, when inserted into a chloramphenicol acetyl transferase promoter detection plasmid, was transcriptionally active in both E. coli and Bacillus subtilis but was much more active in the latter host. Images PMID:3011732

  7. Genome engineering Escherichia coli for L-DOPA overproduction from glucose

    PubMed Central

    Wei, Tao; Cheng, Bi-Yan; Liu, Jian-Zhong

    2016-01-01

    Genome engineering has become a powerful tool for creating useful strains in research and industry. In this study, we applied singleplex and multiplex genome engineering approaches to construct an E. coli strain for the production of L-DOPA from glucose. We first used the singleplex genome engineering approach to create an L-DOPA-producing strain, E. coli DOPA-1, by deleting transcriptional regulators (tyrosine repressor tyrR and carbon storage regulator A csrA), altering glucose transport from the phosphotransferase system (PTS) to ATP-dependent uptake and the phosphorylation system overexpressing galactose permease gene (galP) and glucokinase gene (glk), knocking out glucose-6-phosphate dehydrogenase gene (zwf) and prephenate dehydratase and its leader peptide genes (pheLA) and integrating the fusion protein chimera of the downstream pathway of chorismate. Then, multiplex automated genome engineering (MAGE) based on 23 targets was used to further improve L-DOPA production. The resulting strain, E. coli DOPA-30N, produced 8.67 g/L of L-DOPA in 60 h in a 5 L fed-batch fermentation. This titer is the highest achieved in metabolically engineered E. coli having PHAH activity from glucose. PMID:27417146

  8. UV disinfection induces a VBNC state in Escherichia coli and Pseudomonas aeruginosa.

    PubMed

    Zhang, Shenghua; Ye, Chengsong; Lin, Huirong; Lv, Lu; Yu, Xin

    2015-02-01

    The occurrence of a viable but nonculturable (VBNC) state in bacteria may dramatically underestimate the health risks associated with drinking water. Therefore, the potential for UV treatment to induce a VBNC state in Escherichia coli and Pseudomonas aeruginosa was investigated. UV disinfection effectively reduced the culturability of E. coli and P. aeruginosa, with the destruction of nucleic acids demonstrated using gadA long gene fragment qPCR amplification. Following UV radiation, copy numbers for the high transcriptional levels of the 16S rRNA gene varied insignificantly in both strains, confirming results from plate counting assays indicating that VBNC states were induced in both strains. Furthermore, the virulence genes gadA and oprL remained highly expressed, suggesting that the VBNC bacteria still displayed pathogenicity. Propidium monoazide qPCR indicated that cell membranes remained intact even at a UV dose of 300 mJ/cm(2). The RT-qPCR results after UV and chlorine treatments in E. coli were significantly different (8.41 and 5.59 log units, respectively), further confirming the induction of VBNC bacteria induced by UV radiation. Finally, resuscitation was achieved, with E. coli showing greater resuscitation ability than P. aeruginosa. These results systematically revealed the potential health risks of UV disinfection and strongly suggest a combined disinfection strategy. PMID:25584685

  9. Investigating direct interaction between Escherichia coli topoisomerase I and RecA.

    PubMed

    Banda, Srikanth; Tiwari, Purushottam Babu; Darici, Yesim; Tse-Dinh, Yuk-Ching

    2016-07-01

    Protein-protein interactions are of special importance in cellular processes, including replication, transcription, recombination, and repair. Escherichia coli topoisomerase I (EcTOP1) is primarily involved in the relaxation of negative DNA supercoiling. E. coli RecA, the key protein for homologous recombination and SOS DNA-damage response, has been shown to stimulate the relaxation activity of EcTOP1. The evidence for their direct protein-protein interaction has not been previously established. We report here the direct physical interaction between E. coli RecA and topoisomerase I. We demonstrated the RecA-topoisomerase I interaction via pull-down assays, and surface plasmon resonance measurements. Molecular docking supports the observation that the interaction involves the topoisomerase I N-terminal domains that form the active site. Our results from pull-down assays showed that ATP, although not required, enhances the RecA-EcTOP1 interaction. We propose that E. coli RecA physically interacts with topoisomerase I to modulate the chromosomal DNA supercoiling. PMID:27001450

  10. Cloning, expression, purification and characterization of the stress kinase YeaG from Escherichia coli.

    PubMed

    Tagourti, Jihen; Landoulsi, Ahmed; Richarme, Gilbert

    2008-05-01

    We cloned, overexpressed and purified the Escherichia coli yeaG gene product, whose amino acid sequence displays homology to prokaryotic serine protein kinases. The gene coding for YeaG was generated by amplifying the yeaG gene from E. coli by polymerase chain reaction. It was inserted into the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21(DE3) E. coli strain transformed with the YeaG-expression vector pET-21a-yeaG accumulates large amounts of a soluble protein with a molecular mass of 76kDa in SDS-PAGE, which matches the expected YeaG molecular weight of 74.5kDa. YeaG, although soluble, has a marked tendency to aggregate in the absence of detergents, so that it was purified in the presence of 0.1% Triton X-100, by ion exchange chromatography and hydroxyapatite chromatography. The purified protein is monomeric and displays autokinase and casein kinase activities which are optimal in the presence of 10mM Mn(2+). The purification of the active protein kinase YeaG described in this study should allow us to characterize its biochemical target(s) in E. coli extracts. PMID:18276156

  11. Bacterial invasion augments epithelial cytokine responses to Escherichia coli through a lipopolysaccharide-dependent mechanism.

    PubMed

    Schilling, J D; Mulvey, M A; Vincent, C D; Lorenz, R G; Hultgren, S J

    2001-01-15

    One mechanism of initiating innate host defenses against uropathogenic Escherichia coli (UPEC) is the production of cytokines by bladder epithelial cells; however, the means by which these cells recognize bacterial pathogens is poorly understood. Type 1 pili, expressed by the majority of UPEC, have been shown to have a critical role in inducing the expression of IL-6 in bladder epithelial cells after exposure to E. coli. In this study, we demonstrate that type 1 pili are not sufficient to activate IL-6 production by bladder epithelial cells. Instead, it was shown that bacterial invasion mediated by type 1 pili augments bladder epithelial responses to E. coli via an LPS-dependent mechanism, leading to the production of IL-6. RNA transcripts for the LPSR Toll-like receptor 4 (TLR4) was detected in cultured bladder epithelial cells. The in vivo role of TLR4 was assessed using C3H/HeJ mice, which express a dominant negative form of TLR4. After infection with UPEC, C3H/HeJ mice have large foci of intracellular bacteria that persist within the bladder epithelium in the absence of any notable inflammatory response. These results indicate that LPS is required for bacterial invasion to enhance host responses to E. coli within the bladder. PMID:11145696

  12. Most RNAs regulating ribosomal protein biosynthesis in Escherichia coli are narrowly distributed to Gammaproteobacteria

    PubMed Central

    Fu, Yang; Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M.

    2013-01-01

    In Escherichia coli, 12 distinct RNA structures within the transcripts encoding ribosomal proteins interact with specific ribosomal proteins to allow autogenous regulation of expression from large multi-gene operons, thus coordinating ribosomal protein biosynthesis across multiple operons. However, these RNA structures are typically not represented in the RNA Families Database or annotated in genomic sequences databases, and their phylogenetic distribution is largely unknown. To investigate the extent to which these RNA structures are conserved across eubacterial phyla, we created multiple sequence alignments representing 10 of these messenger RNA (mRNA) structures in E. coli. We find that while three RNA structures are widely distributed across many phyla of bacteria, seven of the RNAs are narrowly distributed to a few orders of Gammaproteobacteria. To experimentally validate our computational predictions, we biochemically confirmed dual L1-binding sites identified in many Firmicute species. This work reveals that RNA-based regulation of ribosomal protein biosynthesis is used in nearly all eubacterial phyla, but the specific RNA structures that regulate ribosomal protein biosynthesis in E. coli are narrowly distributed. These results highlight the limits of our knowledge regarding ribosomal protein biosynthesis regulation outside of E. coli, and the potential for alternative RNA structures responsible for regulating ribosomal proteins in other eubacteria. PMID:23396277

  13. Comparative Genomics Provides Insight into the Diversity of the Attaching and Effacing Escherichia coli Virulence Plasmids

    PubMed Central

    Hazen, Tracy H.; Kaper, James B.; Nataro, James P.

    2015-01-01

    Attaching and effacing Escherichia coli (AEEC) strains are a genomically diverse group of diarrheagenic E. coli strains that are characterized by the presence of the locus of enterocyte effacement (LEE) genomic island, which encodes a type III secretion system that is essential to virulence. AEEC strains can be further classified as either enterohemorrhagic E. coli (EHEC), typical enteropathogenic E. coli (EPEC), or atypical EPEC, depending on the presence or absence of the Shiga toxin genes or bundle-forming pilus (BFP) genes. Recent AEEC genomic studies have focused on the diversity of the core genome, and less is known regarding the genetic diversity and relatedness of AEEC plasmids. Comparative genomic analyses in this study demonstrated genetic similarity among AEEC plasmid genes involved in plasmid replication conjugative transfer and maintenance, while the remainder of the plasmids had sequence variability. Investigation of the EPEC adherence factor (EAF) plasmids, which carry the BFP genes, demonstrated significant plasmid diversity even among isolates within the same phylogenomic lineage, suggesting that these EAF-like plasmids have undergone genetic modifications or have been lost and acquired multiple times. Global transcriptional analyses of the EPEC prototype isolate E2348/69 and two EAF plasmid mutants of this isolate demonstrated that the plasmid genes influence the expression of a number of chromosomal genes in addition to the LEE. This suggests that the genetic diversity of the EAF plasmids could contribute to differences in the global virulence regulons of EPEC isolates. PMID:26238712

  14. Genome image programs: visualization and interpretation of Escherichia coli microarray experiments.

    PubMed Central

    Zimmer, Daniel P; Paliy, Oleg; Thomas, Brian; Gyaneshwar, Prasad; Kustu, Sydney

    2004-01-01

    We have developed programs to facilitate analysis of microarray data in Escherichia coli. They fall into two categories: manipulation of microarray images and identification of known biological relationships among lists of genes. A program in the first category arranges spots from glass-slide DNA microarrays according to their position in the E. coli genome and displays them compactly in genome order. The resulting genome image is presented in a web browser with an image map that allows the user to identify genes in the reordered image. Another program in the first category aligns genome images from two or more experiments. These images assist in visualizing regions of the genome with common transcriptional control. Such regions include multigene operons and clusters of operons, which are easily identified as strings of adjacent, similarly colored spots. The images are also useful for assessing the overall quality of experiments. The second category of programs includes a database and a number of tools for displaying biological information about many E. coli genes simultaneously rather than one gene at a time, which facilitates identifying relationships among them. These programs have accelerated and enhanced our interpretation of results from E. coli DNA microarray experiments. Examples are given. PMID:15342544

  15. Genome engineering Escherichia coli for L-DOPA overproduction from glucose.

    PubMed

    Wei, Tao; Cheng, Bi-Yan; Liu, Jian-Zhong

    2016-01-01

    Genome engineering has become a powerful tool for creating useful strains in research and industry. In this study, we applied singleplex and multiplex genome engineering approaches to construct an E. coli strain for the production of L-DOPA from glucose. We first used the singleplex genome engineering approach to create an L-DOPA-producing strain, E. coli DOPA-1, by deleting transcriptional regulators (tyrosine repressor tyrR and carbon storage regulator A csrA), altering glucose transport from the phosphotransferase system (PTS) to ATP-dependent uptake and the phosphorylation system overexpressing galactose permease gene (galP) and glucokinase gene (glk), knocking out glucose-6-phosphate dehydrogenase gene (zwf) and prephenate dehydratase and its leader peptide genes (pheLA) and integrating the fusion protein chimera of the downstream pathway of chorismate. Then, multiplex automated genome engineering (MAGE) based on 23 targets was used to further improve L-DOPA production. The resulting strain, E. coli DOPA-30N, produced 8.67 g/L of L-DOPA in 60 h in a 5 L fed-batch fermentation. This titer is the highest achieved in metabolically engineered E. coli having PHAH activity from glucose. PMID:27417146

  16. Propagation of restriction fragments from the mitochondrial DNA of Saccharomyces cerevisiae in E. coli by means of plasmid vectors.

    PubMed Central

    Berg, P E; Lewin, A; Christianson, T; Rabinowitz, M

    1979-01-01

    Some of the EcoRI fragments of yeast (Saccharomyces cerevisiae) mitochondrial DNA were cloned into E. coli using plasmid pMB9. The five smallest fragments in molecular weight appeared to be preferentially retained by E coli; partial fragments derived from larger mitochondrial DNA fragments were also found. One of the fragments, R7 (2.4 kb), may contain the OII gene. Cloned R7 DNA was stable under a variety of growth conditions, but showed some changes in molecular weight after transfer to different E. coli strains. Fragment R7 is transcribed in minicells, producing RNA that hybridizes specifically to mitochondrial DNA. Both DNA strands are transcribed, in contrast to the asymmetric transcription found in mitochondria. No new polypeptides were observed in minicells containing cloned fragment 7. Images PMID:379817

  17. Cloning, nucleotide sequence, and transcriptional analysis of the Pediococcus acidilactici L-(+)-lactate dehydrogenase gene.

    PubMed Central

    Garmyn, D; Ferain, T; Bernard, N; Hols, P; Delcour, J

    1995-01-01

    Recombinant plasmids containing the Pediococcus acidilactici L-(+)-lactate dehydrogenase gene (ldhL) were isolated by complementing for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase-pyruvate formate lyase double mutant. The nucleotide sequence of the ldhL gene predicted a protein of 323 amino acids showing significant similarity with other bacterial L-(+)-lactate dehydrogenases and especially with that of Lactobacillus plantarum. The ldhL transcription start points in P. acidilactici were defined by primer extension, and the promoter sequence was identified as TCAAT-(17 bp)-TATAAT. This sequence is closely related to the consensus sequence of vegetative promoters from gram-positive bacteria as well as from E. coli. Northern analysis of P. acidilactici RNA showed a 1.1-kb ldhL transcript whose abundance is growth rate regulated. These data, together with the presence of a putative rho-independent transcriptional terminator, suggest that ldhL is expressed as a monocistronic transcript in P. acidilactici. PMID:7887607

  18. Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases

    PubMed Central

    Raindlová, Veronika; Janoušková, Martina; Slavíčková, Michaela; Perlíková, Pavla; Boháčová, Soňa; Milisavljevič, Nemanja; Šanderová, Hana; Benda, Martin; Barvík, Ivan; Krásný, Libor; Hocek, Michal

    2016-01-01

    DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2′-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli. Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. coli enzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription. PMID:27001521

  19. Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch

    PubMed Central

    Perdrizet, George A.; Artsimovitch, Irina; Furman, Ran; Sosnick, Tobin R.; Pan, Tao

    2012-01-01

    Riboswitches are cis-acting elements that regulate gene expression by affecting transcriptional termination or translational initiation in response to binding of a metabolite. A typical riboswitch is made of an upstream aptamer domain and a downstream expression platform. Both domains participate in the folding and structural rearrangement in the absence or presence of its cognate metabolite. RNA polymerase pausing is a fundamental property of transcription that can influence RNA folding. Here we show that pausing plays an important role in the folding and conformational rearrangement of the Escherichia coli btuB riboswitch during transcription by the E. coli RNA polymerase. This riboswitch consists of an approximately 200 nucleotide, coenzyme B12 binding aptamer domain and an approximately 40 nucleotide expression platform that controls the ribosome access for translational initiation. We found that transcriptional pauses at strategic locations facilitate folding and structural rearrangement of the full-length riboswitch, but have minimal effect on the folding of the isolated aptamer domain. Pausing at these regulatory sites blocks the formation of alternate structures and plays a chaperoning role that couples folding of the aptamer domain and the expression platform. Pausing at strategic locations may be a general mechanism for coordinated folding and conformational rearrangements of riboswitch structures that underlie their response to environmental cues. PMID:22331895

  20. Nonchemotactic Mutants of Escherichia coli

    PubMed Central

    Armstrong, John B.; Adler, Julius; Dahl, Margaret M.

    1967-01-01

    We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged. Images PMID:5335897

  1. The cost of expression of Escherichia coli lac operon proteins is in the process, not in the products.

    PubMed

    Stoebel, Daniel M; Dean, Antony M; Dykhuizen, Daniel E

    2008-03-01

    Transcriptional regulatory networks allow bacteria to express proteins only when they are needed. Adaptive hypotheses explaining the evolution of regulatory networks assume that unneeded expression is costly and therefore decreases fitness, but the proximate cause of this cost is not clear. We show that the cost in fitness to Escherichia coli strains constitutively expressing the lactose operon when lactose is absent is associated with the process of making the lac gene products, i.e., associated with the acts of transcription and/or translation. These results reject the hypotheses that regulation exists to prevent the waste of amino acids in useless protein or the detrimental activity of unnecessary proteins. While the cost of the process of protein expression occurs in all of the environments that we tested, the expression of the lactose permease could be costly or beneficial, depending on the environment. Our results identify the basis of a single selective pressure likely acting across the entire E. coli transcriptome. PMID:18245823

  2. The rpoE gene of Escherichia coli, which encodes sigma E, is essential for bacterial growth at high temperature.

    PubMed Central

    Hiratsu, K; Amemura, M; Nashimoto, H; Shinagawa, H; Makino, K

    1995-01-01

    In vitro transcription analysis has shown that only RNA polymerase containing an alternative sigma subunit, sigma E, activates transcription from one of the rpoH promoters and the htrA promoter. The location of the rpoE gene encoding sigma E on the Escherichia coli chromosome has recently been established, but no rpoE mutant has yet become available for phenotypic testing. We cloned the rpoE gene from the lambda-ordered clones of the E. coli genome and confirmed that the reconstituted RNA polymerase containing the gene product (E sigma E) can transcribe htrA in vitro. We constructed an rpoE-defective strain by gene disruption using the cloned rpoE gene. We demonstrate that expression of htrA is completely dependent on the rpoE gene in vivo and that the rpoE gene is essential for bacterial growth at high temperature. PMID:7751307

  3. Gas signatures from Escherichia coli and Escherichia coli-inoculated human whole blood

    PubMed Central

    2013-01-01

    Background The gaseous headspace above naïve Escherichia Coli (E. coli) cultures and whole human blood inoculated with E. coli were collected and analyzed for the presence of trace gases that may have the potential to be used as novel, non-invasive markers of infectious disease. Methods The naïve E. coli culture, LB broth, and human whole blood or E. coli inoculated whole blood were incubated in hermetically sealable glass bioreactors at 37°C for 24 hrs. LB broth and whole human blood were used as controls for background volatile organic compounds (VOCs). The headspace gases were collected after incubation and analyzed using a gas chromatographic system with multiple column/detector combinations. Results Six VOCs were observed to be produced by E. coli-infected whole blood while there existed nearly zero to relatively negligible amounts of these gases in the whole blood alone, LB broth, or E. coli-inoculated LB broth. These VOCs included dimethyl sulfide (DMS), carbon disulfide (CS2), ethanol, acetaldehyde, methyl butanoate, and an unidentified gas S. In contrast, there were several VOCs significantly elevated in the headspace above the E. coli in LB broth, but not present in the E. coli/blood mixture. These VOCs included dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), methyl propanoate, 1-propanol, methylcyclohexane, and unidentified gases R2 and Q. Conclusions This study demonstrates 1) that cultivated E. coli in LB broth produce distinct gas profiles, 2) for the first time, the ability to modify E. coli-specific gas profiles by the addition of whole human blood, and 3) that E. coli-human whole blood interactions present different gas emission profiles that have the potential to be used as non-invasive volatile biomarkers of E. coli infection. PMID:23842518

  4. Structure of Escherichia coli tryptophanase.

    PubMed

    Ku, Shao Yang; Yip, Patrick; Howell, P Lynne

    2006-07-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the alpha-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the alpha-proton of the substrate for beta-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal. PMID:16790938

  5. Structure of Escherichia Coli Tryptophanase

    SciTech Connect

    Ku,S.; Yip, P.; Howell, P.

    2006-01-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the {alpha}-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the {alpha}-proton of the substrate for {beta}-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  6. The Role of α-CTD in the Genome-Wide Transcriptional Regulation of the Bacillus subtilis Cells

    PubMed Central

    Murayama, Satohiko; Ishikawa, Shu; Chumsakul, Onuma; Ogasawara, Naotake; Oshima, Taku

    2015-01-01

    The amino acid sequence of the RNA polymerase (RNAP) α-subunit is well conserved throughout the Eubacteria. Its C-terminal domain (α-CTD) is important for the transcriptional regulation of specific promoters in both Escherichia coli and Bacillus subtilis, through interactions with transcription factors and/or a DNA element called the “UP element”. However, there is only limited information regarding the α-CTD regulated genes in B. subtilis and the importance of this subunit in the transcriptional regulation of B. subtilis. Here, we established strains and the growth conditions in which the α-subunit of RNAP was replaced with a C-terminally truncated version. Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis. In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis. Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria. PMID:26154296

  7. Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase Expression Revisited▿ †

    PubMed Central

    Carzaniga, Thomas; Briani, Federica; Zangrossi, Sandro; Merlino, Giuseppe; Marchi, Paolo; Dehò, Gianni

    2009-01-01

    The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5′-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5′ end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5′ double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5′ end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5′ fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript. PMID:19136586

  8. Network regulation of the Escherichia coli maltose system.

    PubMed

    Schlegel, Anja; Böhm, Alex; Lee, Sung-Jae; Peist, Ralf; Decker, Katja; Boos, Winfried

    2002-05-01

    The genes of the Escherichia coli maltose regulon are controlled by MalT, the specific transcriptional activator which, together with the inducer maltotriose and ATP, is essential for mal gene transcription. Network regulation in this system affects the function of MalT and occurs on two levels. The first concerns the expression of malT. It has long been known that malT is under catabolite repression and thus under the control of the cAMP/CAP complex. We found that, in addition, the global regulator Mlc is a repressor for malT transcription. The repressor activity of Mlc is controlled by the transport status of the glucose-specific enzyme EIICB of the PTS that causes sequestration (and inactivation as a repressor) of Mlc when glucose is transported. The second level of MalT regulation affects its activity. MalT is activated by maltotriose which is not only formed when the cells are growing on any maltodextrin but also, in low amounts, endogenously when the cells grow on non-maltodextrin carbon sources. Thus, cellular metabolism, for instance degradation of galactose or trehalose, can cause mal gene induction. It was found that unphosphorylated internal glucose takes part in endogenous maltodextrin biosynthesis and is therefore a key element in endogenous mal gene expression. In addition to the maltotriose-dependent activation, MalT can interact with three different enzymes that lead to its inactivation as a transcriptional activator. The first is MaIK, the energy transducing ABC subunit of the maltodextrin transport system. Transport controls the interaction of MalK and MalT thus affecting gene expression. The second enzyme is MalY, a pyridoxal phosphate containing enzyme exhibiting cystathionase activity. The crystal structure of MalY was established and mutations in MalY that reduce mal gene repression map in a hydrophobic MalT interaction patch on the surface of the enzyme. The last enzyme is a soluble esterase of as yet unknown function. When overproduced, this

  9. Escherichia coli O157:H7 gene expression in the presence of catecholamine norepinephrine.

    PubMed

    Dowd, Scot E

    2007-08-01

    Various forms of host stresses (e.g. physiological, psychological) are thought to influence susceptibility to pathogenic microorganisms. Catecholamines such as norepinephrine are released into the GI environment during acute stress and may influence the infective process of bacterial pathogens associated with the GI tract. To examine the effects of norepinephrine on expression of virulence factors in Escherichia coli O157:H7, the clinical-type isolate EDL933 (ATCC 43895) was grown in serum-Standard American Petroleum Institute media in the presence or absence of norepinephrine. After 5 h of exposure to norepinephrine, treatment and control cultures (not exposed to norepinephrine) were harvested, their RNA isolated, and gene expression evaluated. There was a dramatic increase in the expression of virulence factor transcripts including stx1, stx2, and eae. Also induced were transcripts involved in iron metabolism. Conversely, there was comparative repression of iron acquisition and phage shock protein-related transcripts in the presence of norepinephrine. Novel observations from these data suggested that exposure to norepinephrine induced glutamate decarboxylase acid resistance as well as an SOS response in E. coli O157:H7. The results corroborate many of the previous findings detailed in the literature and provide new observations that could expand the scope of microbial endocrinology. PMID:17573936

  10. Nitrogen stress response and stringent response are coupled in Escherichia coli

    PubMed Central

    Brown, Daniel R.; Barton, Geraint; Pan, Zhensheng; Buck, Martin; Wigneshweraraj, Sivaramesh

    2014-01-01

    Assimilation of nitrogen is an essential process in bacteria. The nitrogen regulation stress response is an adaptive mechanism used by nitrogen-starved Escherichia coli to scavenge for alternative nitrogen sources and requires the global transcriptional regulator NtrC. In addition, nitrogen-starved E. coli cells synthesize a signal molecule, guanosine tetraphosphate (ppGpp), which serves as an effector molecule of many processes including transcription to initiate global physiological changes, collectively termed the stringent response. The regulatory mechanisms leading to elevated ppGpp levels during nutritional stresses remain elusive. Here, we show that transcription of relA, a key gene responsible for the synthesis of ppGpp, is activated by NtrC during nitrogen starvation. The results reveal that NtrC couples these two major bacterial stress responses to manage conditions of nitrogen limitation, and provide novel mechanistic insights into how a specific nutritional stress leads to elevating ppGpp levels in bacteria. PMID:24947454

  11. Direct linking of metabolism and gene expression in the proline utilization A protein from Escherichia coli

    PubMed Central

    Zhou, Yuzhen; Zhu, Weidong; Bellur, Padmanetra S.; Rewinkel, Dustin; Becker, Donald F.

    2009-01-01

    Summary The control of gene expression by enzymes provides a direct pathway for cells to respond to fluctuations in metabolites and nutrients. One example is the proline utilization A (PutA) protein from Escherichia coli. PutA is a membrane-associated enzyme that catalyzes the oxidation of L-proline to glutamate using a flavin containing proline dehydrogenase domain and a NAD+ dependent Δ1-pyrroline-5-carboxylate dehydrogenase domain. In some Gram-negative bacteria such as E. coli, PutA is also endowed with a ribbon-helix-helix DNA-binding domain and acts as a transcriptional repressor of the proline utilization genes. PutA switches between transcriptional repressor and enzymatic functions in response to proline availability. Molecular insights into the redox based mechanism of PutA functional switching from recent studies are reviewed. In addition, new results from cell-based transcription assays are presented which correlate PutA membrane localization with put gene expression levels. General membrane localization of PutA, however, is not sufficient to activate the put genes. PMID:18324349

  12. Proteomic Analyses Reveal the Mechanism of Dunaliella salina Ds-26-16 Gene Enhancing Salt Tolerance in Escherichia coli

    PubMed Central

    Wang, Yanlong; Hu, Bin; Du, Shipeng; Gao, Shan; Chen, Xiwen; Chen, Defu

    2016-01-01

    We previously screened the novel gene Ds-26-16 from a 4 M salt-stressed Dunaliella salina cDNA library and discovered that this gene conferred salt tolerance to broad-spectrum organisms, including E. coli (Escherichia coli), Haematococcus pluvialis and tobacco. To determine the mechanism of this gene conferring salt tolerance, we studied the proteome of E. coli overexpressing the full-length cDNA of Ds-26-16 using the iTRAQ (isobaric tags for relative and absolute quantification) approach. A total of 1,610 proteins were identified, which comprised 39.4% of the whole proteome. Of the 559 differential proteins, 259 were up-regulated and 300 were down-regulated. GO (gene ontology) and KEGG (Kyoto encyclopedia of genes and genomes) enrichment analyses identified 202 major proteins, including those involved in amino acid and organic acid metabolism, energy metabolism, carbon metabolism, ROS (reactive oxygen species) scavenging, membrane proteins and ABC (ATP binding cassette) transporters, and peptidoglycan synthesis, as well as 5 up-regulated transcription factors. Our iTRAQ data suggest that Ds-26-16 up-regulates the transcription factors in E. coli to enhance salt resistance through osmotic balance, energy metabolism, and oxidative stress protection. Changes in the proteome were also observed in E. coli overexpressing the ORF (open reading frame) of Ds-26-16. Furthermore, pH, nitric oxide and glycerol content analyses indicated that Ds-26-16 overexpression increases nitric oxide content but has no effect on glycerol content, thus confirming that enhanced nitric oxide synthesis via lower intercellular pH was one of the mechanisms by which Ds-26-16 confers salt tolerance to E. coli. PMID:27135411

  13. Proteomic Analyses Reveal the Mechanism of Dunaliella salina Ds-26-16 Gene Enhancing Salt Tolerance in Escherichia coli.

    PubMed

    Wang, Yanlong; Hu, Bin; Du, Shipeng; Gao, Shan; Chen, Xiwen; Chen, Defu

    2016-01-01

    We previously screened the novel gene Ds-26-16 from a 4 M salt-stressed Dunaliella salina cDNA library and discovered that this gene conferred salt tolerance to broad-spectrum organisms, including E. coli (Escherichia coli), Haematococcus pluvialis and tobacco. To determine the mechanism of this gene conferring salt tolerance, we studied the proteome of E. coli overexpressing the full-length cDNA of Ds-26-16 using the iTRAQ (isobaric tags for relative and absolute quantification) approach. A total of 1,610 proteins were identified, which comprised 39.4% of the whole proteome. Of the 559 differential proteins, 259 were up-regulated and 300 were down-regulated. GO (gene ontology) and KEGG (Kyoto encyclopedia of genes and genomes) enrichment analyses identified 202 major proteins, including those involved in amino acid and organic acid metabolism, energy metabolism, carbon metabolism, ROS (reactive oxygen species) scavenging, membrane proteins and ABC (ATP binding cassette) transporters, and peptidoglycan synthesis, as well as 5 up-regulated transcription factors. Our iTRAQ data suggest that Ds-26-16 up-regulates the transcription factors in E. coli to enhance salt resistance through osmotic balance, energy metabolism, and oxidative stress protection. Changes in the proteome were also observed in E. coli overexpressing the ORF (open reading frame) of Ds-26-16. Furthermore, pH, nitric oxide and glycerol content analyses indicated that Ds-26-16 overexpression increases nitric oxide content but has no effect on glycerol content, thus confirming that enhanced nitric oxide synthesis via lower intercellular pH was one of the mechanisms by which Ds-26-16 confers salt tolerance to E. coli. PMID:27135411

  14. Interaction of T4 AsiA with its target sites in the RNA polymerase sigma70 subunit leads to distinct and opposite effects on transcription.

    PubMed

    Minakhin, Leonid; Niedziela-Majka, Anita; Kuznedelov, Konstantin; Adelman, Karen; Urbauer, Jeffrey L; Heyduk, Tomasz; Severinov, Konstantin

    2003-02-21

    Bacteriophage T4 AsiA is a homodimeric protein that orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the sigma(70) subunit of Escherichia coli RNA polymerase holoenzyme (Esigma(70)) and preventing promoter complex formation on most E.coli and early T4 promoters. In addition, Esigma(70)AsiA, but not Esigma(70), is a substrate of transcription activation by T4-encoded DNA-binding protein MotA, a co-activator of transcription from middle viral promoters. The molecular determinants of sigma(70)-AsiA interaction necessary for transcription inhibition reside in the sigma(70) conserved region 4.2, which recognizes the -35 promoter consensus element. The molecular determinants of sigma(70)-AsiA interaction necessary for MotA-dependent transcription activation have not been identified. Here, we show that in the absence of sigma(70) region 4.2, AsiA interacts with sigma(70) conserved region 4.1 and activates transcription in a MotA-independent manner. Further, we show that the AsiA dimer must dissociate to interact with either region 4.2 or region 4.1 of sigma(70). We propose that MotA may co-activate transcription by restricting AsiA binding to sigma(70) region 4.1. PMID:12581632

  15. Influence of transcriptional and translational control sequences on the expression of foreign genes in Caulobacter crescentus.

    PubMed Central

    Yap, W H; Thanabalu, T; Porter, A G

    1994-01-01

    The influence of expression control sequences (ECSs; promoters and ribosome-binding sites [RBSs]), transcriptional terminators, and gene orientation on the expression of the Escherichia coli lacZ gene in the gram-negative microorganisms Caulobacter crescentus and E. coli was investigated. A series of broad-host-range expression vectors, based on the RK2 plasmid derivative pRK248, were constructed. The ECSs included the tac promoter, the promoter for the surface layer protein of C. crescentus, and promoters from a number of gram-positive bacteria together with their associated RBSs. In addition, synthetic ECSs were constructed by using different combinations of promoters and RBSs. lacZ expression was found to be dependent on the nature of the promoter and RBS and, to a lesser extent, on the presence of a transcriptional terminator and the orientation of the promoter-lacZ construct in pRK248. The relative efficiencies of the various ECSs in driving lacZ expression differed markedly in C. crescentus and E. coli. In C. crescentus, the ECS ptac1 (tac promoter and consensus RBS for C. crescentus mRNAs) appeared to be the most efficient, producing 12-fold-higher activity than did pSL (promoter for the surface layer protein of C. crescentus and its putative RBS). pSL was not transcribed in E. coli, whereas various promoters from gram-positive microorganisms were transcribed in both C. crescentus and E. coli. A number of ECSs were also used to drive mosquitocidal toxin gene expression in C. crescentus, and a correlation between toxin expression and lacZ expression was observed. PMID:8169208

  16. TATA-binding protein and transcription factor IIB induce transcript slipping during early transcription by RNA polymerase II.

    PubMed

    Gilman, Benjamin; Drullinger, Linda F; Kugel, Jennifer F; Goodrich, James A

    2009-04-01

    To better understand the mechanism of steps in early transcription by RNA polymerase II (pol II), we investigated the molecular determinants of transcript slipping within complexes assembled on promoters containing a pre-melted transcription bubble from -9 to +3. Transcript slippage occurs when an RNA transcript contains a repetitive sequence that allows the transcript to slip back and pair with the template strand of the DNA at a new register before transcription continues. We established the contributions of individual transcription factors, DNA elements, and RNA length to slipping on a heteroduplex template using a highly purified human pol II transcription system. We found that transcripts slip at a very defined point in the transcription reaction, after pol II completes phosphodiester bond synthesis at register +5. This point is set by the position of the polymerase active site on the DNA template, as opposed to the length of the transcript, as well as by a repetitive CUCU sequence that must occur from +2 to +5. Interestingly, slipping at this juncture is induced by TATA-binding protein and transcription factor IIB and requires a TATA box but not a transcription factor IIB recognition sequence. We propose a model in which transcribing complexes, upon completing phosphodiester bond synthesis at register +5, enter one of two branches in which they either complete productive synthesis of the transcript or undergo multiple rounds of transcript slipping. PMID:19193635

  17. Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate.

    PubMed

    Sokolova, Ekaterina; Spruijt, Evan; Hansen, Maike M K; Dubuc, Emilien; Groen, Joost; Chokkalingam, Venkatachalam; Piruska, Aigars; Heus, Hans A; Huck, Wilhelm T S

    2013-07-16

    Liquid-liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation. PMID:23818642

  18. Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate

    PubMed Central

    Sokolova, Ekaterina; Spruijt, Evan; Hansen, Maike M. K.; Dubuc, Emilien; Groen, Joost; Chokkalingam, Venkatachalam; Piruska, Aigars; Heus, Hans A.; Huck, Wilhelm T. S.

    2013-01-01

    Liquid–liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation. PMID:23818642

  19. A transcription activator-like effector induction system mediated by proteolysis

    PubMed Central

    Copeland, Matthew F.; Politz, Mark C.; Johnson, Charles B.; Markley, Andrew L.; Pfleger, Brian F.

    2016-01-01

    Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications due to their customizable DNA binding specificity. In this work we expand the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded following the induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator agnostic. PMID:26854666

  20. Escherichia coli K1 internalization via caveolae requires caveolin-1 and protein kinase Calpha interaction in human brain microvascular endothelial cells.

    PubMed

    Sukumaran, Sunil K; Quon, Michael J; Prasadarao, Nemani V

    2002-12-27

    The morbidity and mortality associated with Escherichia coli K1 meningitis during the neonatal period have remained significant over the last decade and are once again on the rise. Transcytosis of brain microvascular endothelial cells (BMEC) by E. coli within an endosome to avoid lysosomal fusion is crucial for dissemination into the central nervous system. Central to E. coli internalization of BMEC is the expression of OmpA (outer membrane protein A), which interacts with its receptor for the actin reorganization that leads to invasion. However, nothing is known about the nature of the signaling events for the formation of endosomes containing E. coli K1. We show here that E. coli K1 infection of human BMEC (HBMEC) results in activation of caveolin-1 for bacterial uptake via caveolae. The interaction of caveolin-1 with phosphorylated protein kinase Calpha (PKCalpha) at the E. coli attachment site is critical for the invasion of HBMEC. Optical sectioning of confocal images of infected HBMEC indicates continuing association of caveolin-1 with E. coli during transcytosis. Overexpression of a dominant-negative form of caveolin-1 containing mutations in the scaffolding domain blocked the interaction of phospho-PKCalpha with caveolin-1 and the E. coli invasion of HBMEC, but not actin cytoskeleton rearrangement or the phosphorylation of PKCalpha. The interaction of caveolin-1 with phospho-PKCalpha was completely abrogated in HBMEC overexpressing dominant-negative forms of either focal adhesion kinase or PKCalpha. Treatment of HBMEC with a cell-permeable peptide that represents the scaffolding domain, which was coupled to an antennapedia motif of a Drosophila transcription factor significantly blocked the interaction of caveolin-1 with phospho-PKCalpha and E. coli invasion. These results show that E. coli K1 internalizes HBMEC via caveolae and that the scaffolding domain of caveolin-1 plays a significant role in the formation of endosomes. PMID:12386163

  1. Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry

    PubMed Central

    Jha, Ramesh K.; Kern, Theresa L.; Fox, David T.; M. Strauss, Charlie E.

    2014-01-01

    We created a single cell sorting system to screen for enzyme activity in Escherichia coli producing 3,4 dihydroxy benzoate (34DHB). To do so, we engineered a transcription factor regulon controlling the expression of green fluorescent protein (GFP) for induction by 34DHB. An autoregulated transcription factor, pcaU, was borrowed from Acinetobacter sp ADP1 to E. coli and its promoter region adapted for activity in E. Coli. The engineered pcaU regulon was inducible at >5 μM exogenous 34DHB, making it a sensitive biosensor for this industrially significant nylon precursor. Addition of a second plasmid provided IPTG inducible expression of dehydroshikimate dehydratase enzyme (AsbF), which converts endogenous dehydroshikimate to 34DHB. This system produced GFP fluorescence in an IPTG dose-dependent manner, and was easily detected in single cell on flow cytometer despite a moderate catalytic efficiency of AsbF. Using fluorescence-activated cell sorting (FACS), individual cells carrying the active AsbF could be isolated even when diluted into a decoy population of cells carrying a mutant (inactivated) AsbF variant at one part in a million. The same biosensor was also effective for further optimization of itself. FACS on E. coli carrying randomized loci in the promoter showed several variants with enhanced response to 34DHB. PMID:24861620

  2. Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

    PubMed

    Ruano-Gallego, David; Álvarez, Beatriz; Fernández, Luis Ángel

    2015-09-18

    Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these "molecular syringes" for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells. PMID:26017572

  3. Tweedle cuticular protein BmCPT1 is involved in innate immunity by participating in recognition of Escherichia coli.

    PubMed

    Liang, Jiubo; Wang, Ting; Xiang, Zhonghuai; He, Ningjia

    2015-03-01

    Bombyx mori, a lepidopteran insect, is one of the earliest models for pattern recognition of Gram-negative bacteria, which may induce the IMD pathway for production of antibacterial peptides. So far, several recognition proteins have been reported in B. mori. However, the connection between pattern recognition of Gram negative bacteria and activation of BmRelish1, a transcription factor controlled by the IMD pathway remains largely unknown. In the present study, we identify BmCPT1, a cuticle protein bearing a Tweedle domain. Its gene expression is co-regulated by NF-kappaB and juvenile hormone signals. BmCPT1 is induced by Escherichia coli in fat bodies and hemocytes, but is constitutively expressed in the epidermis. In vitro binding assays indicate that BmCPT1 protein recognizes and binds to E. coli peptidoglycan. Post-transcriptionally modified BmCPT1 in the hemolymph binds to E. coli cells through interactions with peptidoglycan recognition protein-5 (BmPGRP5) and lipopolysaccharide binding protein (BmLBP). Transgenic overexpression of BmCPT1 causes the upregulated expression of BmRelish1 and clear induction of two gloverin genes. Therefore, BmCPT1 may work along with BmPGRP-S5 and BmLBP to recognize E. coli in the hemolymph and indirectly activate BmRelish1 to induce antimicrobial peptide synthesis. PMID:25449127

  4. A self-inducible heterologous protein expression system in Escherichia coli

    PubMed Central

    Briand, L.; Marcion, G.; Kriznik, A.; Heydel, J. M.; Artur, Y.; Garrido, C.; Seigneuric, R.; Neiers, F.

    2016-01-01

    Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter’s transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale. PMID:27611846

  5. The Crystal Structure of the Escherichia coli RNase E Apoprotein and a Mechanism for RNA Degradation

    PubMed Central

    Koslover, Daniel J.; Callaghan, Anastasia J.; Marcaida, Maria J.; Garman, Elspeth F.; Martick, Monika; Scott, William G.; Luisi, Ben F.

    2008-01-01

    Summary RNase E is an essential bacterial endoribonuclease involved in the turnover of messenger RNA and the maturation of structured RNA precursors in Escherichia coli. Here, we present the crystal structure of the E. coli RNase E catalytic domain in the apo-state at 3.3 Å. This structure indicates that, upon catalytic activation, RNase E undergoes a marked conformational change characterized by the coupled movement of two RNA-binding domains to organize the active site. The structural data suggest a mechanism of RNA recognition and cleavage that explains the enzyme's preference for substrates possessing a 5′-monophosphate and accounts for the protective effect of a triphosphate cap for most transcripts. Internal flexibility within the quaternary structure is also observed, a finding that has implications for recognition of structured RNA substrates and for the mechanism of internal entry for a subset of substrates that are cleaved without 5′-end requirements. PMID:18682225

  6. The crystal structure of the Escherichia coli RNase E apoprotein and a mechanism for RNA degradation.

    PubMed

    Koslover, Daniel J; Callaghan, Anastasia J; Marcaida, Maria J; Garman, Elspeth F; Martick, Monika; Scott, William G; Luisi, Ben F

    2008-08-01

    RNase E is an essential bacterial endoribonuclease involved in the turnover of messenger RNA and the maturation of structured RNA precursors in Escherichia coli. Here, we present the crystal structure of the E. coli RNase E catalytic domain in the apo-state at 3.3 A. This structure indicates that, upon catalytic activation, RNase E undergoes a marked conformational change characterized by the coupled movement of two RNA-binding domains to organize the active site. The structural data suggest a mechanism of RNA recognition and cleavage that explains the enzyme's preference for substrates possessing a 5'-monophosphate and accounts for the protective effect of a triphosphate cap for most transcripts. Internal flexibility within the quaternary structure is also observed, a finding that has implications for recognition of structured RNA substrates and for the mechanism of internal entry for a subset of substrates that are cleaved without 5'-end requirements. PMID:18682225

  7. A self-inducible heterologous protein expression system in Escherichia coli.

    PubMed

    Briand, L; Marcion, G; Kriznik, A; Heydel, J M; Artur, Y; Garrido, C; Seigneuric, R; Neiers, F

    2016-01-01

    Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter's transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale. PMID:27611846

  8. The E.coli fis promoter is subject to stringent control and autoregulation.

    PubMed Central

    Ninnemann, O; Koch, C; Kahmann, R

    1992-01-01

    The DNA binding protein FIS is involved in processes like site specific DNA inversion, lambda excision and stimulation of stable RNA synthesis in Escherichia coli. The amount of FIS protein is subject to dramatic changes during growth. We demonstrate that fis is part of an operon with one ORF of unknown function preceding the fis gene. Regulation of fis synthesis occurs at the transcriptional level. Within 15 min after nutritional upshift a large burst of fis mRNA is produced which levels off when cells begin to grow. By mutational analysis using promoter-lacZ fusions we demonstrate that the fis promoter is autoregulated by FIS. Growth phase regulation of the fis promoter depends on the presence of a GC motif downstream of the -10 region. We show that the fis promoter is subject to stringent control and discuss this unusual feature with respect to the known and putative functions FIS serves in E. coli. Images PMID:1547773

  9. The structure of full-length LysR-type transcriptional regulators. Modeling of the full-length OxyR transcription factor dimer

    PubMed Central

    Zaim, Jolanta; Kierzek, Andrzej M.

    2003-01-01

    The LysR-type transcriptional regulators (LTTRs) comprise the largest family of prokaryotic transcription factors. These proteins are composed of an N-terminal DNA binding domain (DBD) and a C-terminal cofactor binding domain. To date, no structure of the DBD has been solved. According to the SUPERFAMILY and MODBASE databases, a reliable homology model of LTTR DBDs may be built using the structure of the Escherichia coli ModE transcription factor, containing a winged helix– turn–helix (HTH) motif, as a template. The remote, but statistically significant, sequence similarity between ModE and LTTR DBDs and an alignment generated using SUPERFAMILY and MODBASE methods was independently confirmed by alignment of sequence profiles representing ModE and LTTR family DBDs. Using the crystal structure of the E.coli OxyR C-terminal domain and the DBD alignments we constructed a structural model of the full-length dimer of this LTTR family member and used it to investigate the mode of protein–DNA interaction. We also applied the model to interpret, in a structural context, the results of numerous biochemical studies of mutated LTTRs. A comparison of the LTTR DBD model with the structures of other HTH proteins also provides insights into the interaction of LTTRs with the C-terminal domain of the RNA polymerase α subunit. PMID:12595552

  10. Survival of pathogenic Escherichia coli on basil, lettuce, and spinach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The contamination of lettuce, spinach and basil with pathogenic E. coli has caused numerous illnesses over the past decade. E. coli O157:H7, E. coli O104:H4 and avian pathogenic E. coli (APECstx- and APECstx+) were inoculated on basil plants and in promix soiless substrate using drip and overhead ir...

  11. Advances in genoserotyping and subtyping of Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E. coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera a...

  12. Global regulation by horizontally transferred regulators establishes the pathogenicity of Escherichia coli.

    PubMed

    Abe, Hiroyuki; Miyahara, Akira; Oshima, Taku; Tashiro, Kosuke; Ogura, Yoshitoshi; Kuhara, Satoru; Ogasawara, Naotake; Hayashi, Tetsuya; Tobe, Toru

    2008-02-29

    Enterohemorrhagic Escherichia coli is an emerging pathogen that causes diarrhea and hemolytic uremic syndrome. Much of the genomic information that affects virulence is acquired by horizontal transfer. Genes necessary for attaching and effacing lesions are located in the locus for enterocyte effacement (LEE) pathogenicity island. LEE gene transcription is positively regulated by Ler, which is also encoded by the LEE, and by Pch regulators, which are encoded at other loci. Here we identified genes whose transcription profiles were similar to those of the LEE genes, by comparing the effects of altering ler and pch transcript levels. We assigned these genes into two classes, according to their transcription profiles. By determining the binding profiles for Ler and Pch, we showed that both were involved in regulating one class of genes, but only Pch was involved in regulating the other class. Binding sites were found in the coding region as well as the promoter region of regulated genes, which include genes common to K12 strains as well as 0157-specific genes, suggesting that both act as a global regulator. These results indicate that Ler and Pch orchestrate the transcription of virulence genes, which are captured by horizontal transfer and scattered throughout the chromosome. PMID:18222925

  13. High Levels of Transcription Stimulate Transversions at GC Base Pairs in Yeast

    PubMed Central

    Alexander, Matthew P.; Begins, Kaitlyn J.; Crall, William C.; Holmes, Margaret P.; Lippert, Malcolm J.

    2016-01-01

    High-levels of transcription through a gene stimulate spontaneous mutation rate, a phenomenon termed transcription-associated mutation (TAM). While transcriptional effects on specific mutation classes have been identified using forward mutation and frameshift-reversion assays, little is yet known about transcription-associated base substitutions in yeast. To address this issue, we developed a new base substitution reversion assay (the lys2-TAG allele). We report a 22-fold increase in overall reversion rate in the high- relative to the low-transcription strain (from 2.1- to 47- × 10−9). While all detectable base substitution types increased in the high-transcription strain, G→T and G→C transversions increased disproportionately by 58- and 52-fold, respectively. To assess a potential role of DNA damage in the TAM events, we measured mutation rates and spectra in individual strains defective in the repair of specific DNA lesions or null for the error-prone translesion DNA polymerase zeta (Pol zeta). Results exclude a role of 8-oxoGuanine, general oxidative damage, or apurinic/apyrimidinic sites in the generation of TAM G→T and G→C transversions. In contrast, the TAM transversions at GC base pairs depend on Pol zeta for occurrence implicating DNA damage, other than oxidative lesions or AP sites, in the TAM mechanism. Results further indicate that transcription-dependent G→T transversions in yeast differ mechanistically from equivalent events in E. coli reported by others. Given their occurrences in repair-proficient cells, transcription-associated G→T and G→C events represent a novel type of transcription-associated mutagenesis in normal cells with potentially important implications for evolution and genetic disease. PMID:23055242

  14. Engineering an allosteric transcription factor to respond to new ligands

    PubMed Central

    Taylor, Noah D; Garruss, Alexander S; Moretti, Rocco; Chan, Sum; Arbing, Mark A; Cascio, Duilio; Rogers, Jameson K; Isaacs, Farren J; Kosuri, Sriram; Baker, David; Fields, Stanley; Church, George M; Raman, Srivatsan

    2016-01-01

    Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol or sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits. PMID:26689263

  15. Engineering an allosteric transcription factor to respond to new ligands.

    PubMed

    Taylor, Noah D; Garruss, Alexander S; Moretti, Rocco; Chan, Sum; Arbing, Mark A; Cascio, Duilio; Rogers, Jameson K; Isaacs, Farren J; Kosuri, Sriram; Baker, David; Fields, Stanley; Church, George M; Raman, Srivatsan

    2016-02-01

    Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits. PMID:26689263

  16. Comparative genomic reconstruction of transcriptional networks controlling central metabolism in the Shewanella genus

    PubMed Central

    2011-01-01

    Background Genome-scale prediction of gene regulation and reconstruction of transcriptional regulatory networks in bacteria is one of the critical tasks of modern genomics. The Shewanella genus is comprised of metabolically versatile gamma-proteobacteria, whose lifestyles and natural environments are substantially different from Escherichia coli and other model bacterial species. The comparative genomics approaches and computational identification of regulatory sites are useful for the in silico reconstruction of transcriptional regulatory networks in bacteria. Results To explore conservation and variations in the Shewanella transcriptional networks we analyzed the repertoire of transcription factors and performed genomics-based reconstruction and comparative analysis of regulons in 16 Shewanella genomes. The inferred regulatory network includes 82 transcription factors and their DNA binding sites, 8 riboswitches and 6 translational attenuators. Forty five regulons were newly inferred from the genome context analysis, whereas others were propagated from previously characterized regulons in the Enterobacteria and Pseudomonas spp.. Multiple variations in regulatory strategies between the Shewanella spp. and E. coli include regulon contraction and expansion (as in the case of PdhR, HexR, FadR), numerous cases of recruiting non-orthologous regulators to control equivalent pathways (e.g. PsrA for fatty acid degradation) and, conversely, orthologous regulators to control distinct pathways (e.g. TyrR, ArgR, Crp). Conclusions We tentatively defined the first reference collection of ~100 transcriptional regulons in 16 Shewanella genomes. The resulting regulatory network contains ~600 regulated genes per genome that are mostly involved in metabolism of carbohydrates, amino acids, fatty acids, vitamins, metals, and stress responses. Several reconstructed regulons including NagR for N-acetylglucosamine catabolism were experimentally validated in S. oneidensis MR-1. Analysis of

  17. High-Density Transcriptional Initiation Signals Underline Genomic Islands in Bacteria

    PubMed Central

    Huang, Qianli; Cheng, Xuanjin; Cheung, Man Kit; Kiselev, Sergey S.; Ozoline, Olga N.; Kwan, Hoi Shan

    2012-01-01

    Genomic islands (GIs), frequently associated with the pathogenicity of bacteria and having a substantial influence on bacterial evolution, are groups of “alien” elements which probably undergo special temporal–spatial regulation in the host genome. Are there particular hallmark transcriptional signals for these “exotic” regions? We here explore the potential transcriptional signals that underline the GIs beyond the conventional views on basic sequence composition, such as codon usage and GC property bias. It showed that there is a significant enrichment of the transcription start positions (TSPs) in the GI regions compared to the whole genome of Salmonella enterica and Escherichia coli. There was up to a four-fold increase for the 70% GIs, implying high-density TSPs profile can potentially differentiate the GI regions. Based on this feature, we developed a new sliding window method GIST, Genomic-island Identification by Signals of Transcription, to identify these regions. Subsequently, we compared the known GI-associated features of the GIs detected by GIST and by the existing method Islandviewer to those of the whole genome. Our method demonstrates high sensitivity in detecting GIs harboring genes with biased GI-like function, preferred subcellular localization, skewed GC property, shorter gene length and biased “non-optimal” codon usage. The special transcriptional signals discovered here may contribute to the coordinate expression regulation of foreign genes. Finally, by using GIST, we detected many interesting GIs in the 2011 German E. coli O104:H4 outbreak strain TY-2482, including the microcin H47 system and gene cluster ycgXEFZ-ymgABC that activates the production of biofilm matrix. The aforesaid findings highlight the power of GIST to predict GIs with distinct intrinsic features to the genome. The heterogeneity of cumulative TSPs profiles may not only be a better identity for “alien” regions, but also provide hints to the special

  18. Identification of mutator genes and mutational pathways in Escherichia coli using a multicopy cloning approach.

    PubMed

    Yang, Hanjing; Wolff, Erika; Kim, Mandy; Diep, Amy; Miller, Jeffrey H

    2004-07-01

    We searched for genes that create mutator phenotypes when put on to a multicopy plasmid in Escherichia coli. In many cases, this will result in overexpression of the gene in question. We constructed a random shotgun library with E. coli genomic fragments between 3 and 5 kbp in length on a multicopy plasmid vector that was transformed into E. coli to screen for frameshift mutators. We identified a total of 115 independent genomic fragments that covered 17 regions on the E. coli chromosome. Further studies identified 12 genes not previously known as causing mutator phenotypes when overproduced. A striking finding is that overproduction of the multidrug resistance transcription regulator, EmrR, results in a large increase in frameshift and base substitution mutagenesis. This suggests a link between multidrug resistance and mutagenesis. Other identified genes include those encoding DNA helicases (UvrD, RecG, RecQ), truncated forms of the DNA mismatch repair protein (MutS) and a primosomal component (DnaT), a negative modulator of initiation of replication/GATC-binding protein (SeqA), a stationary phase regulator AppY, a transcriptional regulator PaaX and three putative open reading frames, ycgW, yfjY and yjiD, encoding hypothetical proteins. In addition, we found three genes encoding proteins that were previously known to cause mutator effects under overexpression conditions: error-prone polymerase IV (DinB), DNA methylase (Dam) and sigma S factor (RpoS). This genomic strategy offers an approach to identify novel mutator effects resulting from the multicopy cloning (MCC) of specific genes and therefore complementing the conventional gene inactivation approach to finding mutators. PMID:15225322

  19. Transcript analysis of nrrF, a Fur repressed sRNA of Neisseria gonorrhoeae

    PubMed Central

    Ducey, Thomas F.; Jackson, Lydgia; Orvis, Joshua; Dyer, David W.

    2016-01-01

    Like most microorganisms, Neisseria gonorrhoeae alters gene expression in response to iron availability. The ferric uptake regulator Fur has been shown to be involved in controlling this response, but the extent of this involvement remains unknown. It is known that in addition to working directly to repress gene expression, Fur may also work indirectly by controlling additional regulatory elements. Using in silico analysis, we identified a putative small RNA (sRNA) homolog of the meningococcal nrrF locus, and demonstrate that this sRNA is iron-repressible, suggesting that this is the gonococcal analog of the rhyB locus in Escherichia coli. Quantitative real-time RT-PCR analysis indicates that this transcript may also be temporally regulated. Transcript analysis identified the 5′ start of the transcript, using a single reaction, fluorescent-based, primer extension assay. This protocol allows for the rapid identification of transcriptional start sites of RNA transcripts, and could be used for high-throughput transcript mapping. PMID:19162160

  20. lac repressor blocks in vivo transcription of lac control region DNA.

    PubMed Central

    Sellitti, M A; Pavco, P A; Steege, D A

    1987-01-01

    Transcription of the Escherichia coli lac repressor gene (lacI) in vivo produces monocistronic mRNAs with discrete 3' ends in the lac control region, although the DNA sequence of this region does not specify a strong termination signal of the traditional form. Direct analysis of lac transcripts was used to show that the DNA sequence alone does not provide the signal to end the repressor mRNA and to establish that of the proteins with specific binding sites on control region DNA only the lac repressor has a striking effect on the continuity of lacI gene transcription. RNAs with 3' ends in the control region sequence are major mRNA species produced from a repressor-bound template, reflecting as much as a 50-fold increase over their levels in the repressor's absence. Repressor binding to the operator thus has a dual function. In addition to blocking initiation of transcription from the lacZ promoter, repressor serves as a termination factor by setting the length of its own transcript and separating lacI and lacZYA into two distinct transcription units. Images PMID:3554233

  1. Reconstruction of transcriptional network from microarray data using combined mutual information and network-assisted regression.

    PubMed

    Wang, X-D; Qi, Y-X; Jiang, Z-L

    2011-03-01

    Many methods had been developed on inferring transcriptional network from gene expression. However, it is still necessary to design new method that discloses more detailed and exact network information. Using network-assisted regression, the authors combined the averaged three-way mutual information (AMI3) and non-linear ordinary differential equation (ODE) model to infer the transcriptional network, and to obtain both the topological structure and the regulatory dynamics. Synthetic and experimental data were used to evaluate the performance of the above approach. In comparison with the previous methods based on mutual information, AMI3 obtained higher precision with the same sensitivity. To describe the regulatory dynamics between transcription factors and target genes, network-assisted regression and regression without network, respectively, were applied in the steady-state and time series microarray data. The results revealed that comparing with regression without network, network-assisted regression increased the precision, but decreased the fitting goodness. Then, the authors reconstructed the transcriptional network of Escherichia coli and simulated the regulatory dynamics of genes. Furthermore, the authors' approach identified potential transcription factors regulating yeast cell cycle. In conclusion, network-assisted regression, combined AMI3 and ODE model, was a more precisely to infer the topological structure and the regulatory dynamics of transcriptional network from microarray data. [Includes supplementary material]. PMID:21405197

  2. Genomic mapping of cAMP receptor protein (CRPMt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor

    PubMed Central

    Kahramanoglou, Christina; Cortes, Teresa; Matange, Nishad; Hunt, Debbie M.; Visweswariah, Sandhya S.; Young, Douglas B.; Buxton, Roger S.

    2014-01-01

    Chromatin immunoprecipitation identified 191 binding sites of Mycobacterium tuberculosis cAMP receptor protein (CRPMt) at endogenous expression levels using a specific α-CRPMt antibody. Under these native conditions an equal distribution between intragenic and intergenic locations was observed. CRPMt binding overlapped a palindromic consensus sequence. Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRPMt during exponential growth, and in response to nutrient starvation. Differential expression of genes with a CRPMt-binding site represented only a minor portion of this transcriptional reprogramming with ∼19% of those representing transcriptional regulators potentially controlled by CRPMt. The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation. CRPMt can function as a classical transcription factor in M. tuberculosis, though this occurs at only a subset of CRPMt-binding sites. PMID:24957601

  3. Phylogenetic and Transcription Analysis of Chrysanthemum WRKY Transcription Factors

    PubMed Central

    Song, Aiping; Li, Peiling; Jiang, Jiafu; Chen, Sumei; Li, Huiyun; Zeng, Jun; Shao, Yafeng; Zhu, Lu; Zhang, Zhaohe; Chen, Fadi

    2014-01-01

    WRKY transcription factors are known to function in a number of plant processes. Here we have characterized 15 WRKY family genes of the important ornamental species chrysanthemum (Chrysanthemum morifolium). A total of 15 distinct sequences were isolated; initially internal fragments were amplified based on transcriptomic sequence, and then the full length cDNAs were obtained using RACE (rapid amplification of cDNA ends) PCR. The transcription of these 15 genes in response to a variety of phytohormone treatments and both biotic and abiotic stresses was characterized. Some of the genes behaved as would be predicted based on their homology with Arabidopsis thaliana WRKY genes, but others showed divergent behavior. PMID:25196345

  4. In depth analysis of genes and pathways of the mammary gland involved in the pathogenesis of bovine Escherichia coli-mastitis

    PubMed Central

    2011-01-01

    Background Bovine mastitis is one of the most costly and prevalent diseases affecting dairy cows worldwide. In order to develop new strategies to prevent Escherichia coli-induced mastitis, a detailed understanding of the molecular mechanisms underlying the host immune response to an E. coli infection is necessary. To this end, we performed a global gene-expression analysis of mammary gland tissue collected from dairy cows that had been exposed to a controlled E. coli infection. Biopsy samples of healthy and infected utter tissue were collected at T = 24 h post-infection (p.i.) and at T = 192 h p.i. to represent the acute phase response (APR) and chronic stage, respectively. Differentially expressed (DE) genes for each stage were analyzed and the DE genes detected at T = 24 h were also compared to data collected from two previous E. coli mastitis studies that were carried out on post mortem tissue. Results Nine-hundred-eighty-two transcripts were found to be differentially expressed in infected tissue at T = 24 (P < 0.05). Up-regulated transcripts (699) were largely associated with immune response functions, while the down-regulated transcripts (229) were principally involved in fat metabolism. At T = 192 h, all of the up-regulated transcripts were associated with tissue healing processes. Comparison of T = 24 h DE genes detected in the three E. coli mastitis studies revealed 248 were common and mainly involved immune response functions. KEGG pathway analysis indicated that these genes were involved in 12 pathways related to the pro-inflammatory response and APR, but also identified significant representation of two unexpected pathways: natural killer cell-mediated cytotoxicity pathway (KEGG04650) and the Rig-I-like receptor signalling pathway (KEGG04622). Conclusions In E. coli-induced mastitis, infected mammary gland tissue was found to significantly up-regulate expression of genes related to the immune response and down-regulate genes related to fat metabolism

  5. Biofuels from E. Coli: Engineering E. coli as an Electrofuels Chassis for Isooctane Production

    SciTech Connect

    2010-07-16

    Electrofuels Project: Ginkgo Bioworks is bypassing photosynthesis and engineering E. coli to directly use carbon dioxide (CO2) to produce biofuels. E. coli doesn’t naturally metabolize CO2, but Ginkgo Bioworks is manipulating and incorporating the genes responsible for CO2 metabolism into the microorganism. By genetically modifying E. coli, Ginkgo Bioworks will enhance its rate of CO2 consumption and liquid fuel production. Ginkgo Bioworks is delivering CO2 to E. coli as formic acid, a simple industrial chemical that provides energy and CO2 to the bacterial system.

  6. Transcriptional gene silencing in humans.

    PubMed

    Weinberg, Marc S; Morris, Kevin V

    2016-08-19

    It has been over a decade since the first observation that small non-coding RNAs can functionally modulate epigenetic states in human cells to achieve functional transcriptional gene silencing (TGS). TGS is mechanistically distinct from the RNA interference (RNAi) gene-silencing pathway. TGS can result in long-term stable epigenetic modifications to gene expression that can be passed on to daughter cells during cell division, whereas RNAi does not. Early studies of TGS have been largely overlooked, overshadowed by subsequent discoveries of small RNA-directed post-TGS and RNAi. A reappraisal of early work has been brought about by recent findings in human cells where endogenous long non-coding RNAs function to regulate the epigenome. There are distinct and common overlaps between the proteins involved in small and long non-coding RNA transcriptional regulatory mechanisms, suggesting that the early studies using small non-coding RNAs to modulate transcription were making use of a previously unrecognized endogenous mechanism of RNA-directed gene regulation. Here we review how non-coding RNA plays a role in regulation of transcription and epigenetic gene silencing in human cells by revisiting these earlier studies and the mechanistic insights gained to date. We also provide a list of mammalian genes that have been shown to be transcriptionally regulated by non-coding RNAs. Lastly, we explore how TGS may serve as the basis for development of future therapeutic agents. PMID:27060137

  7. First international E. coli genome meeting

    SciTech Connect

    Not Available

    1992-01-01

    This volume is a collection of abstracts of oral presentations and poster sessions of studies reported at the First International E. Coli Genome Meeting, held September 10-14, 1992 at the University of Wisconsin.

  8. First international E. coli genome meeting

    SciTech Connect

    Not Available

    1992-12-31

    This volume is a collection of abstracts of oral presentations and poster sessions of studies reported at the First International E. Coli Genome Meeting, held September 10-14, 1992 at the University of Wisconsin.

  9. Escherichia coli bacteriuria and contraceptive method.

    PubMed

    Hooton, T M; Hillier, S; Johnson, C; Roberts, P L; Stamm, W E

    1991-01-01

    We evaluated the effects of contraceptive method on the occurrence of bacteriuria and vaginal colonization with Escherichia coli in 104 women who were evaluated prior to having sexual intercourse, the morning after intercourse, and 24 hours later. After intercourse, the prevalence of E coli bacteriuria increased slightly in oral contraceptive users but dramatically in both foam and condom users and diaphragm-spermicide users. Twenty-four hours later, the prevalence of bacteriuria remained significantly elevated only in the latter two groups. Similarly, vaginal colonization with E coli was more dramatic and persistent in users of diaphragm-spermicide and foam and condoms. Vaginal colonization with Candida species, enterococci, and staphylococci also increased significantly in diaphragm-spermicide users after intercourse. We conclude that use of the diaphragm with spermicidal jelly or use of a spermicidal foam with a condom markedly alters normal vaginal flora and strongly predisposes users to the development of vaginal colonization and bacteriuria with E coli. PMID:1859519

  10. Adhesion behaviors of Escherichia coli on hydroxyapatite.

    PubMed

    Kamitakahara, Masanobu; Takahashi, Shohei; Yokoi, Taishi; Inoue, Chihiro; Ioku, Koji

    2016-04-01

    Optimum design of support materials for microorganisms is required for the construction of bioreactors. However, the effects of support materials on microorganisms are still unclear. In this study, we investigated the adhesion behavior of Escherichia coli (E. coli) on hydroxyapatite (HA), polyurethane (PU), poly(vinyl chloride) (PVC), and carbon (Carbon) to obtain basic knowledge for the design of support materials. The total metabolic activity and number of E. coli adhering on the samples followed the order of HA ≈ Carbon>PVC>PU. On the other hand, the water contact angle of the pellet surfaces followed the order of HAcoli. The results implied that HA has a potential as a support material for microorganisms used in bioreactors. PMID:26838837

  11. Escherichia coli in retail processed food.

    PubMed Central

    Pinegar, J. A.; Cooke, E. M.

    1985-01-01

    Four thousand two hundred and forty six samples of retail processed food were examined for the presence of Escherichia coli. Overall 12% of samples contained this organism, cakes and confectionery being more frequently contaminated (28%) than meat and meat based products (9%). Contamination was more frequent in the summer months than in the colder weather and 27% of the contaminated foods contained greater than 10(3) E. coli/g. E. coli from meat and meat based products were more commonly resistant to one or more antibiotics (14%) than were confectionery strains (1%). The significance of these findings in relation to the E. coli population of the human bowel is discussed. PMID:3894508

  12. Escherichia coli in retail processed food.

    PubMed

    Pinegar, J A; Cooke, E M

    1985-08-01

    Four thousand two hundred and forty six samples of retail processed food were examined for the presence of Escherichia coli. Overall 12% of samples contained this organism, cakes and confectionery being more frequently contaminated (28%) than meat and meat based products (9%). Contamination was more frequent in the summer months than in the colder weather and 27% of the contaminated foods contained greater than 10(3) E. coli/g. E. coli from meat and meat based products were more commonly resistant to one or more antibiotics (14%) than were confectionery strains (1%). The significance of these findings in relation to the E. coli population of the human bowel is discussed. PMID:3894508

  13. Vaginal Lactobacillus isolates inhibit uropathogenic Escherichia coli.

    PubMed

    Atassi, Fabrice; Brassart, Dominique; Grob, Philipp; Graf, Federico; Servin, Alain L

    2006-04-01

    The purpose of this study was to investigate the antibacterial activities of Lactobacillus jensenii KS119.1 and KS121.1, and Lactobacillus gasserii KS120.1 and KS124.3 strains isolated from the vaginal microflora of healthy women, against uropathogenic, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strains IH11128 and 7372 involved in recurrent cystitis. We observed that some of the Lactobacillus isolates inhibited the growth and decreased the viability of E. coli IH11128 and 7372. In addition, we observed that adhering Lactobacillus strains inhibited adhesion of E. coli IH11128 onto HeLa cells, and inhibited internalization of E. coli IH11128 within HeLa cells. PMID:16553843

  14. FTIR nanobiosensors for Escherichia coli detection

    PubMed Central

    Greppi, Gianfranco; Marongiu, Maria Laura; Roggero, Pier Paolo; Ravindranath, Sandeep P; Mauer, Lisa J; Schibeci, Nicoletta; Perria, Francesco; Piccinini, Massimo; Innocenzi, Plinio; Irudayaraj, Joseph

    2012-01-01

    Summary Infections due to enterohaemorrhagic E. coli (Escherichia coli) have a low incidence but can have severe and sometimes fatal health consequences, and thus represent some of the most serious diseases due to the contamination of water and food. New, fast and simple devices that monitor these pathogens are necessary to improve the safety of our food supply chain. In this work we report on mesoporous titania thin-film substrates as sensors to detect E. coli O157:H7. Titania films treated with APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde) were functionalized with specific antibodies and the absorption properties monitored. The film-based biosensors showed a detection limit for E. coli of 1 × 102 CFU/mL, constituting a simple and selective method for the effective screening of water samples. PMID:23019542

  15. Transcription Profiling of the Stringent Response in Escherichia coli▿ †

    PubMed Central

    Durfee, Tim; Hansen, Anne-Marie; Zhi, Huijun; Blattner, Frederick R.; Jin, Ding Jun

    2008-01-01

    The bacterial stringent response serves as a paradigm for understanding global regulatory processes. It can be triggered by nutrient downshifts or starvation and is characterized by a rapid RelA-dependent increase in the alarmone (p)ppGpp. One hallmark of the response is the switch from maximum-growth-promoting to biosynthesis-related gene expression. However, the global transcription patterns accompanying the stringent response in Escherichia coli have not been analyzed comprehensively. Here, we present a time series of gene expression profiles for two serine hydroxymate-treated cultures: (i) MG1655, a wild-type E. coli K-12 strain, and (ii) an isogenic relAΔ251 derivative defective in the stringent response. The stringent response in MG1655 develops in a hierarchical manner, ultimately involving almost 500 differentially expressed genes, while the relAΔ251 mutant response is both delayed and limited in scope. We show that in addition to the down-regulation of stable RNA-encoding genes, flagellar and chemotaxis gene expression is also under stringent control. Reduced transcription of these systems, as well as metabolic and transporter-encoding genes, constitutes much of the down-regulated expression pattern. Conversely, a significantly larger number of genes are up-regulated. Under the conditions used, induction of amino acid biosynthetic genes is limited to the leader sequences of attenuator-regulated operons. Instead, up-regulated genes with known functions, including both regulators (e.g., rpoE, rpoH, and rpoS) and effectors, are largely involved in stress responses. However, one-half of the up-regulated genes have unknown functions. How these results are correlated with the various effects of (p)ppGpp (in particular, RNA polymerase redistribution) is discussed. PMID:18039766

  16. Entire sequence of the colonization factor coli surface antigen 6-encoding plasmid pCss165 from an enterotoxigenic Escherichia coli clinical isolate.

    PubMed

    Wajima, Takeaki; Sabui, Subrata; Kano, Shigeyuki; Ramamurthy, Thandavarayan; Chatterjee, Nabendu Sekhar; Hamabata, Takashi

    2013-11-01

    Coli surface antigen 6 (CS6) is one of the most prevalent colonization factors among enterotoxigenic Escherichia coli (ETEC) isolated in developing countries. Although it is known that CS6 is encoded by a plasmid, there are no reports on the sequence analysis of the CS6-encoding plasmid or genes exhibiting similar behavior to CS6. Here, we report the isolation of the CS6-encoding plasmid, pCss165Kan, from 4266 ΔcssB::kanamycin (Km) and its complete nucleotide sequence. This plasmid consisted of 165,311bp and 222 predicted coding sequences. Remarkably, there were many insertion sequence (IS) elements, which comprised 24.4% of the entire sequence. Virulence-associated genes such as heat-stable enterotoxin, homologues of ATP-binding cassette transporter in enteroaggregative E. coli (EAEC), and ETEC autotransporter A were also present, although the ETEC autotransporter A gene was disrupted by the integration of IS629. We found that 2 transcriptional regulators belonging to the AraC family were not involved in CS6 expression. Interestingly, pCss165 had conjugative transfer genes, as well as 3 toxin-antitoxin systems that potentially exclude other plasmid-free host bacteria. These genes might be involved in the prevalence of CS6 among ETEC isolates. PMID:23933356

  17. Biodegradation of Aromatic Compounds by Escherichia coli

    PubMed Central

    Díaz, Eduardo; Ferrández, Abel; Prieto, María A.; García, José L.

    2001-01-01

    Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications. PMID:11729263

  18. Fosfomycin Resistance in Escherichia coli, Pennsylvania, USA

    PubMed Central

    Alrowais, Hind; McElheny, Christi L.; Spychala, Caressa N.; Sastry, Sangeeta; Guo, Qinglan; Butt, Adeel A.

    2015-01-01

    Fosfomycin resistance in Escherichia coli is rare in the United States. An extended-spectrum β-lactamase–producing E. coli clinical strain identified in Pennsylvania, USA, showed high-level fosfomycin resistance caused by the fosA3 gene. The IncFII plasmid carrying this gene had a structure similar to those found in China, where fosfomycin resistance is commonly described. PMID:26488485

  19. Circadian Control of Global Transcription

    PubMed Central

    Li, Shujing; Zhang, Luoying

    2015-01-01

    Circadian rhythms exist in most if not all organisms on the Earth and manifest in various aspects of physiology and behavior. These rhythmic processes are believed to be driven by endogenous molecular clocks that regulate rhythmic expression of clock-controlled genes (CCGs). CCGs consist of a significant portion of the genome and are involved in diverse biological pathways. The transcription of CCGs is tuned by rhythmic actions of transcription factors and circadian alterations in chromatin. Here, we review the circadian control of CCG transcription in five model organisms that are widely used, including cyanobacterium, fungus, plant, fruit fly, and mouse. Comparing the similarity and differences in the five organisms could help us better understand the function of the circadian clock, as well as its output mechanisms adapted to meet the demands of diverse environmental conditions. PMID:26682214

  20. Dnmt1/Transcription Factor Interactions

    PubMed Central

    Hervouet, Eric; Vallette, François M.; Cartron, Pierre-François

    2010-01-01

    DNA methylation inheritance is the process of copying, via the DNA methyltransferase 1 (Dnmt1), the pre-existing methylation patterns onto the new DNA strand during DNA replication. Experiments of chromatin immunoprecipitation, measurement of maintenance methyltransferase activity, proximity ligation in situ assays (P-LISA, Duolink/Olink), and transcription factor arrays demonstrate that Dnmt1 interacts with transcription factors to promote site-specific DNA methylation inheritance, while the Dnmt1-PCNA-UHRF1 complex promotes the DNA methylation inheritance without site preference. We also show that the Dnmt1-PCNA-UHRF1 and Dnmt1/transcription factor complexes methylate DNA by acting as a single player or in cooperation. Thus, our data establish that the copying of the pre-existing methylation pattern is governed by the orchestration of the untargeted and the targeted mechanisms of DNA methylation inheritance, which are themselves dictated by the partners of Dnmt1. PMID:21779454

  1. Transcriptional Landscape of Cardiomyocyte Maturation

    PubMed Central

    Uosaki, Hideki; Cahan, Patrick; Lee, Dong I.; Wang, Songnan; Miyamoto, Matthew; Fernandez, Laviel; Kass, David A.; Kwon, Chulan

    2015-01-01

    SUMMARY Decades of progress in developmental cardiology has advanced our understanding of the early aspects of heart development, including cardiomyocyte (CM) differentiation. However, control of CM maturation which is subsequently required to generate adult myocytes, remains elusive. Here, we analyzed over 200 microarray datasets from early embryonic to adult hearts and identified a large number of genes whose expression shifts gradually and continuously during maturation. We generated an atlas of integrated gene expression, biological pathways, transcriptional regulators, and gene regulatory networks (GRNs), which show discrete sets of key transcriptional regulators and pathways activated or suppressed during CM maturation. We developed a GRN-based program named MatStatCM that indexes CM maturation status. MatStatCM reveals that pluripotent stem cell-derived CMs mature early in culture, but are arrested at the late embryonic stage with aberrant regulation of key transcription factors. Our study provides a foundation for understanding CM maturation. PMID:26586429

  2. Natural plasmid transformation in Escherichia coli.

    PubMed

    Tsen, Suh-Der; Fang, Suh-Sen; Chen, Mei-Jye; Chien, Jun-Yi; Lee, Chih-Chun; Tsen, Darwin Han-Lin

    2002-01-01

    Although Escherichia coli does not have a natural transformation process, strains of E. coli can incorporate extracellular plasmids into cytoplasm 'naturally' at low frequencies. A standard method was developed in which stationary phase cells were concentrated, mixed with plasmids, and then plated on agar plates with nutrients which allowed cells to grow. Transformed cells could then be selected by harvesting cells and plating again on selective agar plates. Competence developed in the lag phase, but disappeared during exponential growth. As more plasmids were added to the cell suspension, the number of transformants increased, eventually reaching a plateau. Supercoiled monomeric or linear concatemeric DNA could transform cells, while linear monomeric DNA could not. Plasmid transformation was not related to conjugation and was recA-independent. Most of the E. coli strains surveyed had this process. All tested plasmids, except pACYC184, could transform E. coli. Insertion of a DNA fragment containing the ampicillin resistance gene into pACYC184 made the plasmid transformable. By inserting random 20-base-pair oligonucleotides into pACYC184 and selecting for transformable plasmids, a most frequent sequence was identified. This sequence resembled the bacterial interspersed medium repetitive sequence of E. coli, suggesting the existence of a recognition sequence. We conclude that plasmid natural transformation exists in E. coli. PMID:12065899

  3. Systems Metabolic Engineering of Escherichia coli.

    PubMed

    Choi, Kyeong Rok; Shin, Jae Ho; Cho, Jae Sung; Yang, Dongsoo; Lee, Sang Yup

    2016-05-01

    Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass. PMID:27223822

  4. Mechanism of Sperm Immobilization by Escherichia coli

    PubMed Central

    Prabha, Vijay; Sandhu, Ravneet; Kaur, Siftjit; Kaur, Kiranjeet; Sarwal, Abha; Mavuduru, Ravimohan S.; Singh, Shravan Kumar

    2010-01-01

    Aim. To explore the influence of Escherichia coli on the motility of human spermatozoa and its possible mechanism. Methods. Highly motile preparations of spermatozoa from normozoospermic patients were coincubated with Escherichia coli for 4 hours. At 1, 2 and 4 hours of incubation, sperm motility was determined. The factor responsible for sperm immobilization without agglutination was isolated and purified from filtrates. Results. This report confirms the immobilization of spermatozoa by E. coli and demonstrates sperm immobilization factor (SIF) excreted by E. coli. Further this factor was purified by ammonium sulfate precipitation, gel permeation chromatography, and ion-exchange chromatography. Purified SIF (56 kDa) caused instant immobilization without agglutination of human spermatozoa at 800 μg/mL and death at 2.1 mg/mL. Spermatozoa incubated with SIF revealed multiple and profound alterations involving all superficial structures of spermatozoa as observed by scanning electron microscopy. Conclusion. In conclusion, these results have shown immobilization of spermatozoa by E. coli and demonstrate a factor (SIF) produced and secreted by E. coli which causes variable structural damage as probable morphological correlates of immobilization. PMID:20379358

  5. Chemotaxis of Escherichia coli to norepinephrine (NE) requires conversion of NE to 3,4-dihydroxymandelic acid.

    PubMed

    Pasupuleti, Sasikiran; Sule, Nitesh; Cohn, William B; MacKenzie, Duncan S; Jayaraman, Arul; Manson, Michael D

    2014-12-01

    Norepinephrine (NE), the primary neurotransmitter of the sympathetic nervous system, has been reported to be a chemoattractant for enterohemorrhagic Escherichia coli (EHEC). Here we show that nonpathogenic E. coli K-12 grown in the presence of 2 μM NE is also attracted to NE. Growth with NE induces transcription of genes encoding the tyramine oxidase, TynA, and the aromatic aldehyde dehydrogenase, FeaB, whose respective activities can, in principle, convert NE to 3,4-dihydroxymandelic acid (DHMA). Our results indicate that the apparent attractant response to NE is in fact chemotaxis to DHMA, which was found to be a strong attractant for E. coli. Only strains of E. coli K-12 that produce TynA and FeaB exhibited an attractant response to NE. We demonstrate that DHMA is sensed by the serine chemoreceptor Tsr and that the chemotaxis response requires an intact serine-binding site. The threshold concentration for detection is ≤5 nM DHMA, and the response is inhibited at DHMA concentrations above 50 μM. Cells producing a heterodimeric Tsr receptor containing only one functional serine-binding site still respond like the wild type to low concentrations of DHMA, but their response persists at higher concentrations. We propose that chemotaxis to DHMA generated from NE by bacteria that have already colonized the intestinal epithelium may recruit E. coli and other enteric bacteria that possess a Tsr-like receptor to preferred sites of infection. PMID:25182492

  6. Genetic Features of MCR-1-Producing Colistin-Resistant Escherichia coli Isolates in South Africa.

    PubMed

    Poirel, Laurent; Kieffer, Nicolas; Brink, Adrian; Coetze, Jennifer; Jayol, Aurélie; Nordmann, Patrice

    2016-07-01

    A series of colistin-resistant Escherichia coli clinical isolates was recovered from hospitalized and community patients in South Africa. Seven clonally unrelated isolates harbored the mcr-1 gene located on different plasmid backbones. Two distinct plasmids were fully sequenced, and identical 2,600-bp-long DNA sequences defining a mcr-1 cassette were identified. Promoter sequences responsible for the expression of mcr-1, deduced from the precise identification of the +1 transcription start site for mcr-1, were characterized. PMID:27161623

  7. Transcriptional activation in yeast cells lacking transcription factor IIA.

    PubMed Central

    Chou, S; Chatterjee, S; Lee, M; Struhl, K

    1999-01-01

    The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA. PMID:10581267

  8. [Selective detection of viable pathogenic bacteria in water using reverse transcription quantitative PCR].

    PubMed

    Lin, Yi-Wen; Li, Dan; Wu, Shu-Xu; He, Miao; Yang, Tian

    2012-11-01

    A reverse transcription q quantitative PCR (RT-qPCR) assay method was established, which can quantify the copy numbers of RNA in pathogenic bacteria of E. coli and Enterococcus faecium. The results showed that cDNA was generated with the RT-PCR reagents, target gene was quantified with the qPCR, the copy numbers of RNA were stable at about 1 copies x CFU(-1) for E. coli and 7.98 x 10(2) copies x CFU(-1) for Enterococcus faecium respectively during the stationary grow phase for the both indicator bacteria [E. coli (6-18 h) and Enterococcus faecium (10-38 h)]. The established RT-qPCR method can quantify the numbers of viable bacteria through detecting bacterial RNA targets. Through detecting the heat-treated E. coli and Enterococcus faecium by three methods (culture method, qPCR, RT-qPCR), we found that the qPCR and RT-qPCR can distinguish 1.43 lg copy non-viable E. coli and 2.5 lg copy non-viable Enterococcus faecium. These results indicated that the established methods could effectively distinguish viable bacteria from non-viable bacteria. Finally we used this method to evaluate the real effluents of the secondary sedimentation of wastewater treatment plant (WWTP), the results showed that the correlation coefficients (R2) between RT-qPCR and culture method were 0.930 (E. coli) and 0.948 (Enterococcus faecium), and this established RT-PCR method can rapidly detect viable pathogenic bacteria in genuine waters. PMID:23323443

  9. Making Sense of Transcription Networks

    PubMed Central

    Sorrells, Trevor R; Johnson, Alexander D

    2015-01-01

    When transcription regulatory networks are compared among distantly related eukaryotes, a number of striking similarities are observed: a larger-than-expected number of genes, extensive overlapping connections, and an apparently high degree of functional redundancy. It is often assumed that the complexity of these networks represents optimized solutions, precisely sculpted by natural selection; their common features are often asserted to be adaptive. Here, we discuss support for an alternative hypothesis: the common structural features of transcription networks arise from evolutionary trajectories of “least resistance,” that is, the relative ease by which certain types of network structures are formed during their evolution. PMID:25957680

  10. Transcription rates in DNA brushes.

    PubMed

    Yamamoto, Tetsuya; Safran, S A

    2015-04-21

    We theoretically predict the rate of transcription (TX) in DNA brushes by introducing the concept of TX dipoles that takes into account the unidirectional motion of enzymes (RNAP) along DNA during transcription as correlated pairs of sources and sinks in the relevant diffusion equation. Our theory predicts that the TX rates dramatically change upon the inversion of the orientation of the TX dipoles relative to the substrate because TX dipoles modulate the concentrations of RNAP in the solution. Comparing our theory with experiments suggests that, in some cases, DNA chain segments are relatively uniformly distributed in the brush, in contrast to the parabolic profile expected for flexible polymer brushes. PMID:25736601

  11. Succinate production in Escherichia coli

    PubMed Central

    Thakker, Chandresh; Martínez, Irene; San, Ka-Yiu; Bennett, George N.

    2012-01-01

    Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for production of the four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon that is channeled to the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose as well as other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, as well as the requirement for efficient recovery of succinate and the reliability of the performance under scale-up are important in the overall process. The costs of the overall biorefinery compatible process will determine the economical commercialization of succinate and its impact in larger chemical markets. PMID:21932253

  12. E. coli on the move

    NASA Astrophysics Data System (ADS)

    Calne, S.

    2012-04-01

    Lynn Grove High School in Great Yarmouth, UK has been awarded a Royal Society partnership grant. Lynn Grove pupils aged between 11 and 16 years will carry out an investigation collaborating with scientists at the John Innes Centre in Norwich, UK to investigate the distribution of E.coli and other coliform bacteria within a school. The information will be used as an evidence base to educate pupils about the transmission of microbes and about methods of control. Through this work pupils will gain an appreciation of the diversity of microbial biochemistry and the chemistry behind chromogenic detection methodologies for specific bacterial enzymes. Inferences from the use of diagnostic selective media will be confirmed by carrying out DNA isolation and PCR to identify the genes responsible for the biochemical reactions. PCR will also be used to identify species of coliforms by reference to genomic sequence databases. These techniques will allow pupils to look into an unseen world in a way which has direct relevance to their everyday lives. Furthermore this partnership study will demonstrate to pupils that solving scientific questions requires the integration of a variety of scientific disciplines. The project will run from January 2012 until June 2012. We will present our preliminary results from the investigation and outline our future plans.

  13. Enterohemorrhagic E. coli (EHEC) pathogenesis

    PubMed Central

    Nguyen, Y; Sperandio, Vanessa

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) w