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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 16 February 2010, Vol.107(7), pp.2854-9
    Description: Sequenced bacterial genomes provide a wealth of information but little understanding of transcriptional regulatory circuits largely because accurate prediction of promoters is difficult. We examined two important issues for accurate promoter prediction: (1) the ability to predict promoter strength and (2) the sequence properties that distinguish between active and weak/inactive promoters. We addressed promoter prediction using natural core promoters recognized by the well-studied alternative sigma factor, Escherichia coli sigma(E), as a representative of group 4 sigmas, the largest sigma group. To evaluate the contribution of sequence to promoter strength and function, we used modular position weight matrix models comprised of each promoter motif and a penalty score for suboptimal motif location. We find that a combination of select modules is moderately predictive of promoter strength and that imposing minimal motif scores distinguished active from weak/inactive promoters. The combined -35/-10 score is the most important predictor of activity. Our models also identified key sequence features associated with active promoters. A conserved "AAC" motif in the -35 region is likely to be a general predictor of function for promoters recognized by group 4 sigmas. These results provide valuable insights into sequences that govern promoter strength, distinguish active and inactive promoters for the first time, and are applicable to both in vivo and in vitro measures of promoter strength.
    Keywords: Models, Genetic ; Amino Acid Motifs -- Genetics ; Escherichia Coli -- Genetics ; Promoter Regions, Genetic -- Genetics ; Sigma Factor -- Genetics ; Transcription, Genetic -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 2011, Vol.108(31), pp.12875-12880
    Description: The Escherichia coli σE envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The σE stress response was characterized as a single-tier activation network comprised of ; p. 12875-12880.
    Keywords: Regulon ; Porins ; Messenger Rna ; Homeostasis ; Non-Coding Rna ; Stress Response ; Labor ; Viability ; Mica ; Gram-Negative Bacteria ; Escherichia Coli ; Gene Activation ; Transactivators
    ISSN: 0027-8424
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 02 August 2011, Vol.108(31), pp.12875-80
    Description: The Escherichia coli σ(E) envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The σ(E) stress response was characterized as a single-tier activation network comprised of ~100 genes, including the MicA and RybB noncoding sRNAs. These highly expressed sRNAs were thought to carry out the specialized function of halting de novo synthesis of several abundant porins when envelope homeostasis was perturbed. Using a systematic target profiling and validation approach we discovered that MicA and RybB are each global mRNA repressors of both distinct and shared targets, and that the two sRNAs constitute a posttranscriptional repression arm whose regulatory scope rivals that of the protein-based σ(E) activation arm. Intriguingly, porin mRNAs constitute only ~1/3 of all targets and new nonporin targets predict roles for MicA and RybB in crosstalk with other regulatory responses. This work also provides an example of evolutionarily unrelated sRNAs that are coinduced and bind the same targets, but at different sites. Our finding that expression of either MicA or RybB sRNA protects the cell from the loss of viability experienced when σ(E) activity is inadequate illustrates the importance of the posttranscriptional repression arm of the response. σ(E) is a paradigm of a single-tier stress response with a clear division of labor in which highly expressed noncoding RNAs (MicA, RybB) endow a transcriptional factor intrinsically restricted to gene activation (σ(E)) with the opposite repressor function.
    Keywords: Escherichia Coli Proteins -- Genetics ; RNA, Small Untranslated -- Genetics ; Regulon -- Genetics ; Sigma Factor -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    Language: English
    In: Applied and Environmental Microbiology, 2011, Vol.77(18), p.6622(8)
    Description: Quantitative reverse transcription (qRT)-PCR analysis was used to study the effect of poly(3-hydroxybutyrate) (PHB) accumulation, and that of phasins (PhaP) from Azotobacter sp. strain FA8, on the expression of stress-related genes in PHB-producing Escherichia coli. The results demonstrated the protective role of PhaP in PHB-synthesizing E. coli and linked the effects of the protein to the expression of stress-related genes, especially ibpA.
    Keywords: Bacterial Proteins – Research ; Ecological Stress – Research ; Escherichia Coli – Physiological Aspects ; Escherichia Coli – Genetic Aspects ; Gene Expression – Research ; Microbiological Synthesis – Research ; Protein Folding – Research
    ISSN: 0099-2240
    Source: Cengage Learning, Inc.
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  • 5
    Language: English
    In: Applied and environmental microbiology, September 2011, Vol.77(18), pp.6622-9
    Description: Phasins (PhaP) are proteins normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated by many bacteria as a reserve molecule. These proteins enhance growth and polymer production in natural and recombinant PHB producers. It has been shown that the production of PHB causes stress in recombinant Escherichia coli, revealed by an increase in the concentrations of several heat stress proteins. In this work, quantitative reverse transcription (qRT)-PCR analysis was used to study the effect of PHB accumulation, and that of PhaP from Azotobacter sp. strain FA8, on the expression of stress-related genes in PHB-producing E. coli. While PHB accumulation was found to increase the transcription of dnaK and ibpA, the expression of these genes and of groES, groEL, rpoH, dps, and yfiD was reduced, when PhaP was coexpressed, to levels even lower than those detected in the non-PHB-accumulating control. These results demonstrated the protective role of PhaP in PHB-synthesizing E. coli and linked the effects of the protein to the expression of stress-related genes, especially ibpA. The effect of PhaP was also analyzed in non-PHB-synthesizing strains, showing that expression of this heterologous protein has an unexpected protective effect in E. coli, under both normal and stress conditions, resulting in increased growth and higher resistance to both heat shock and superoxide stress by paraquat. In addition, PhaP expression was shown to reduce RpoH protein levels during heat shock, probably by reducing or titrating the levels of misfolded proteins.
    Keywords: Stress, Physiological ; Bacterial Proteins -- Metabolism ; DNA-Binding Proteins -- Metabolism ; Escherichia Coli -- Physiology ; Hydroxybutyrates -- Metabolism ; Polyesters -- Metabolism
    ISSN: 00992240
    E-ISSN: 1098-5336
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  • 6
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 03 September 2002, Vol.99(18), pp.11772-7
    Description: We present an algorithm that extracts the binding sites (represented by position-specific weight matrices) for many different transcription factors from the regulatory regions of a genome, without the need for delineating groups of coregulated genes. The algorithm uses the fact that many DNA-binding proteins in bacteria bind to a bipartite motif with two short segments more conserved than the intervening region. It identifies all statistically significant patterns of the form W(1)N(x)W(2), where W(1) and W(2) are two short oligonucleotides separated by x arbitrary bases, and groups them into clusters of similar patterns. These clusters are then used to derive quantitative recognition profiles of putative regulatory proteins. For a given cluster, the algorithm finds the matching sequences plus the flanking regions in the genome and performs a multiple sequence alignment to derive position-specific weight matrices. We have analyzed the Escherichia coli genome with this algorithm and found approximately 1,500 significant patterns, which give rise to approximately 160 distinct position-specific weight matrices. A fraction of these matrices match the binding sites of one-third of the approximately 60 characterized transcription factors with high statistical significance. Many of the remaining matrices are likely to describe binding sites and regulons of uncharacterized transcription factors. The significance of these matrices was evaluated by their specificity, the location of the predicted sites, and the biological functions of the corresponding regulons, allowing us to suggest putative regulatory functions. The algorithm is efficient for analyzing newly sequenced bacterial genomes for which little is known about transcriptional regulation.
    Keywords: Genome, Bacterial ; Bacterial Proteins -- Metabolism
    ISSN: 0027-8424
    E-ISSN: 10916490
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  • 7
    Language: English
    In: Journal of Bacteriology, June, 2007, Vol.189(11-12), p.4243(14)
    Description: RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli. Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB, negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA, encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on [[sigma].sup.E]. The sequence upstream of the + 1 of rybB contains a consensus [[sigma].sup.E] promoter. The activity of rybB-lacZ was increased in cells lacking the RseA anti-sigma factor and when [[sigma].sup.E] was overproduced from a heterologous promoter. The activity of rybB-lacZ and the detection of RybB were totally abolished in an rpoE-null strain. In vitro, [[sigma].sup.E] efficiently transcribes from this promoter. Both a rybB mutation and an hfq mutation significantly increased expression of both rybB-lacZ and rpoE-lacZ fusions, consistent with negative regulation of the [[sigma].sup.E] response by RybB and other small RNAs. Based on the plasmid screens, NsrR, a repressor sensitive to nitric oxide, was also found to negatively regulate [[sigma].sup.E]-dependent promoters in an RseA-independent fashion.
    Keywords: Escherichia Coli -- Genetic Aspects ; Escherichia Coli -- Research ; Genetic Regulation -- Research ; Plasmids -- Research ; Cell Surface Antigens -- Research
    ISSN: 0021-9193
    E-ISSN: 10985530
    Source: Cengage Learning, Inc.
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  • 8
    Language: English
    In: Methods in Enzymology, 2011, Vol.497, pp.75-113
    Description: In recent years, the capability of synthetic biology to design large genetic circuits has dramatically increased due to rapid advances in DNA synthesis technology and development of tools for large-scale assembly of DNA fragments. Large genetic circuits require more components (parts), especially regulators such as transcription factors, sigma factors, and viral RNA polymerases to provide increased regulatory capability, and also devices such as sensors, receivers, and signaling molecules. All these parts may have a potential impact upon the host that needs to be considered when designing and fabricating circuits. DNA microarrays are a well-established technique for global monitoring of gene expression and therefore are an ideal tool for systematically assessing the impact of expressing parts of genetic circuits in host cells. Knowledge of part impact on the host enables the user to design circuits from libraries of parts taking into account their potential impact and also to possibly modify the host to better tolerate stresses induced by the engineered circuit. In this chapter, we present the complete methodology of performing microarrays from choice of array platform, experimental design, preparing samples for array hybridization, and associated data analysis including preprocessing, normalization, clustering, identifying significantly differentially expressed genes, and interpreting the data based on known biology. With these methodologies, we also include lists of bioinformatic resources and tools for performing data analysis. The aim of this chapter is to provide the reader with the information necessary to be able to systematically catalog the impact of genetic parts on the host and also to optimize the operation of fully engineered genetic circuits.
    Keywords: Anatomy & Physiology;
    ISBN: 978-0-12-385075-1
    ISSN: 0076-6879
    ISSN: 15577988
    E-ISSN: 15577988
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  • 9
    Language: English
    In: Nucleic acids research, April 2012, Vol.40(7), pp.2907-24
    Description: Predicting the location and strength of promoters from genomic sequence requires accurate sequenced-based promoter models. We present the first model of a full-length bacterial promoter, encompassing both upstream sequences (UP-elements) and core promoter modules, based on a set of 60 promoters dependent on σ(E), an alternative ECF-type σ factor. UP-element contribution, best described by the length and frequency of A- and T-tracts, in combination with a PWM-based core promoter model, accurately predicted promoter strength both in vivo and in vitro. This model also distinguished active from weak/inactive promoters. Systematic examination of promoter strength as a function of RNA polymerase (RNAP) concentration revealed that UP-element contribution varied with RNAP availability and that the σ(E) regulon is comprised of two promoter types, one of which is active only at high concentrations of RNAP. Distinct promoter types may be a general mechanism for increasing the regulatory capacity of the ECF group of alternative σ's. Our findings provide important insights into the sequence requirements for the strength and function of full-length promoters and establish guidelines for promoter prediction and for forward engineering promoters of specific strengths.
    Keywords: Promoter Regions, Genetic ; Escherichia Coli -- Genetics ; Escherichia Coli Proteins -- Metabolism ; Sigma Factor -- Metabolism
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 10
    In: The Journal of Bacteriology, 2009, Vol. 191(23), p.7279
    Description: The [[sigma].sup.E]-directed envelope stress response maintains outer membrane homeostasis and is an important virulence determinant upon host infection in Escherichia coli and related bacteria, ore is activated by at least two distinct mechanisms: accumulation of outer membrane porin precursors and an increase in the alarmone ppGpp upon transition to stationary phase. Expression of the [[sigma].sup.E] regulon is driven from a suite of approximately 60 [[sigma].sup.E]-dependent promoters. Using green fluorescent protein fusions to each of these promoters, we dissected promoter contributions to the output of the regulon under a variety of in vivo conditions. We found that the [[sigma].sup.E] promoters exhibit a large dynamic range, with a few strong and many weak promoters. Interestingly, the strongest promoters control either transcriptional regulators or functions related to porin homeostasis, the very functions conserved among E. coli and its close relatives. We found that (i) the strength of most promoters is significantly affected by the presence of the upstream (-35 to -65) region of the promoter, which encompasses the UP element, a binding site for the C-terminal domain of the [alpha]-subunit of RNA polymerase; (ii) ppGpp generally activates [[sigma].sup.E] promoters, and (iii) [[sigma].sup.E] promoters are responsive to changing ore holoenzyme levels under physiological conditions, reinforcing the idea that the orE regulon is extremely dynamic, enabling cellular adaptation to a constantly changing environment. doi: 10.1128/JB.01047-09
    Keywords: Water Quality;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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