scholarly journals Competition between NarL-dependent activation and Fis-dependent repression controls expression from the Escherichia coli yeaR and ogt promoters

2009 ◽  
Vol 420 (2) ◽  
pp. 249-257 ◽  
Author(s):  
Derrick J. P. Squire ◽  
Meng Xu ◽  
Jeffrey A. Cole ◽  
Stephen J. W. Busby ◽  
Douglas F. Browning
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Genetic Studies ◽  
Dnase I Footprinting ◽  
Single Target ◽  
Fnr Protein ◽  
Dna Alkyltransferase ◽  
Gene Regulatory ◽  
Nitrate And Nitrite ◽  
Target Promoters

The Escherichia coli NarL protein is a global gene regulatory factor that activates transcription at many target promoters in response to nitrate and nitrite ions. Although most NarL-dependent promoters are also co-dependent on a second transcription factor, FNR protein, two targets, the yeaR and ogt promoters, are activated by NarL alone with no involvement of FNR. Biochemical and genetic studies presented here show that activation of the yeaR promoter is dependent on the binding of NarL to a single target centred at position −43.5, whereas activation at the ogt promoter requires NarL binding to tandem DNA targets centred at position −45.5 and −78.5. NarL-dependent activation at both the yeaR and ogt promoters is decreased in rich medium and this depends on Fis, a nucleoid-associated protein. DNase I footprinting studies identified Fis-binding sites that overlap the yeaR promoter NarL site at position −43.5, and the ogt promoter NarL site at position −78.5, and suggest that Fis represses both promoters by displacing NarL. The ogt gene encodes an O6-alkylguanine DNA alkyltransferase and, hence, this is the first report of expression of a DNA repair function being controlled by nitrate ions.

scholarly journals Fnr-, NarP- and NarL-Dependent Regulation of Transcription Initiation from the Haemophilus influenzae Rd napF (Periplasmic Nitrate Reductase) Promoter in Escherichia coli K-12

2005 ◽  
Vol 187 (20) ◽  
pp. 6928-6935 ◽  
Author(s):  
Valley Stewart ◽  
Peggy J. Bledsoe
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Haemophilus Influenzae ◽  
Control Region ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Class I ◽  
E Coli ◽  
Fnr Protein

ABSTRACT Periplasmic nitrate reductase (napFDAGHBC operon product) functions in anaerobic respiration. Transcription initiation from the Escherichia coli napF operon control region is activated by the Fnr protein in response to anaerobiosis and by the NarQ-NarP two-component regulatory system in response to nitrate or nitrite. The binding sites for the Fnr and phospho-NarP proteins are centered at positions −64.5 and −44.5, respectively, with respect to the major transcription initiation point. The E. coli napF operon is a rare example of a class I Fnr-activated transcriptional control region, in which the Fnr protein binding site is located upstream of position −60. To broaden our understanding of napF operon transcriptional control, we studied the Haemophilus influenzae Rd napF operon control region, expressed as a napF-lacZ operon fusion in the surrogate host E. coli. Mutational analysis demonstrated that expression required binding sites for the Fnr and phospho-NarP proteins centered at positions −81.5 and −42.5, respectively. Transcription from the E. coli napF operon control region is activated by phospho-NarP but antagonized by the orthologous protein, phospho-NarL. By contrast, expression from the H. influenzae napF-lacZ operon fusion in E. coli was stimulated equally well by nitrate in both narP and narL null mutants, indicating that phospho-NarL and -NarP are equally effective regulators of this promoter. Overall, the H. influenzae napF operon control region provides a relatively simple model for studying synergistic transcription by the Fnr and phospho-NarP proteins acting from class I and class II locations, respectively.

scholarly journals CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from Enterotoxigenic Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 5060-5067 ◽  
Author(s):  
M. Carolina Pilonieta ◽  
Maria D. Bodero ◽  
George P. Munson
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Secretory Pathway ◽  
Dnase I ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Dnase I Footprinting ◽  
Virulence Regulator

ABSTRACT H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.

scholarly journals Identification of the DNA Binding Sites of PerA, the Transcriptional Activator of the bfp and per Operons in Enteropathogenic Escherichia coli

2003 ◽  
Vol 185 (9) ◽  
pp. 2835-2847 ◽  
Author(s):  
J. Antonio Ibarra ◽  
Miryam I. Villalba ◽  
José Luis Puente
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Promoter Sequence ◽  
Transcriptional Regulators ◽  
Mobility Shift ◽  
Dna Motifs ◽  
Dnase I Footprinting ◽  
Dna Binding Sites ◽  
Important Virulence Factor ◽  
Similar Affinity

ABSTRACT The bundle-forming pilus (BFP) is an important virulence factor for enteropathogenic Escherichia coli (EPEC). Genes involved in its biogenesis and regulation are tightly regulated by PerA (BfpT), a member of the AraC/XylS family of transcriptional regulators. The aim of this work was to purify PerA and determine its association with bfpA and perA (bfpT) regulatory regions by electrophoretic mobility shift and DNase I footprinting assays. PerA was purified as a maltose-binding protein (MBP) fusion, which was capable of complementing bfpA expression and which was able to restore the localized adherence phenotype of an EPEC perA mutant strain. Upstream of bfpA and perA, MBP-PerA recognized with similar affinity asymmetric nucleotide sequences in which a 29-bp-long AT-rich consensus motif was identified. These DNA motifs share 66% identity and were previously shown, by deletion analysis, to be involved in the PerA-dependent expression of both genes. Interestingly, in perA, this motif spans the sequence between positions −75 and −47, approximately one helix turn upstream of the −35 promoter sequence, while in bfpA, it spans the sequence between positions −83 and −55, approximately two helix turns upstream from the promoter. An additional PerA binding site was identified at the 5′ end of the bfpA structural gene, which was not required for its activation. Experiments with LexA-PerA fusions suggested that PerA acts as a monomer to activate the transcription of both perA and bfpA, in contrast to what has been documented for other members of this family of transcriptional regulators.

scholarly journals Single-Target Regulators Constitute the Minority Group of Transcription Factors in Escherichia coli K-12

2021 ◽  
Vol 12 ◽  
Author(s):  
Tomohiro Shimada ◽  
Hiroshi Ogasawara ◽  
Ikki Kobayashi ◽  
Naoki Kobayashi ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
Transcription Factors ◽  
Binding Sites ◽  
Target Genes ◽  
Minority Group ◽  
E Coli ◽  
Single Target ◽  
K 12

The identification of regulatory targets of all transcription factors (TFs) is critical for understanding the entire network of genome regulation. A total of approximately 300 TFs exist in the model prokaryote Escherichia coli K-12, but the identification of whole sets of their direct targets is impossible with use of in vivo approaches. For this end, the most direct and quick approach is to identify the TF-binding sites in vitro on the genome. We then developed and utilized the gSELEX screening system in vitro for identification of more than 150 E. coli TF-binding sites along the E. coli genome. Based on the number of predicted regulatory targets, we classified E. coli K-12 TFs into four groups, altogether forming a hierarchy ranging from a single-target TF (ST-TF) to local TFs, global TFs, and nucleoid-associated TFs controlling as many as 1,000 targets. Using the collection of purified TFs and a library of genome DNA segments from a single and the same E. coli K-12, we identified here a total of 11 novel ST-TFs, CsqR, CusR, HprR, NorR, PepA, PutA, QseA, RspR, UvrY, ZraR, and YqhC. The regulation of single-target promoters was analyzed in details for the hitherto uncharacterized QseA and RspR. In most cases, the ST-TF gene and its regulatory target genes are adjacently located on the E. coli K-12 genome, implying their simultaneous transfer in the course of genome evolution. The newly identified 11 ST-TFs and the total of 13 hitherto identified altogether constitute the minority group of TFs in E. coli K-12.

scholarly journals Emergent properties in complex synthetic bacterial promoters

2017 ◽  
Author(s):  
Lummy Maria Oliveira Monteiro ◽  
Letícia Magalhães Arruda ◽  
Rafael Silva-Rocha
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Regulation Of Gene Expression ◽  
Emergent Properties ◽  
Global Regulators ◽  
Up Elements ◽  
The Individual ◽  
Target Promoters

SummaryRegulation of gene expression in bacteria results from the interplay between transcriptional factors (TFs) at target promoters, and how the arrangement of binding sites determines the regulatory logic of promoters is not well known. Here, we generated and fully characterized a library of synthetic complex promoters for the global regulators, CRP and IHF, inEscherichia coli, formed by a weak -35/-10 consensus sequence preceded by four combinatorial binding sites for these TFs. We found that whilecis-elements for CRP preferentially activate promoters when located immediately upstream of the promoter consensus, binding sites for IHF mainly function as “UP” elements and stimulate transcription in several different architectures in the absence of this protein. However, the combination of CRP- and IHF-binding sites resulted in emergent properties in these complex promoters, where the activity of combinatorial promoters cannot be predicted from the individual behavior of its components. Taken together, the results presented here add to the information on architecture-logic of complex promoters in bacteria.

scholarly journals Computation-Directed Identification of OxyR DNA Binding Sites in Escherichia coli

2001 ◽  
Vol 183 (15) ◽  
pp. 4571-4579 ◽  
Author(s):  
Ming Zheng ◽  
Xunde Wang ◽  
Bernard Doan ◽  
Karen A. Lewis ◽  
Thomas D. Schneider ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Binding Sites ◽  
Northern Blotting ◽  
Dnase I ◽  
Dependent Manner ◽  
E Coli ◽  
Dnase I Footprinting ◽  
Dna Binding Sites ◽  
Proximal Site

ABSTRACT A computational search was carried out to identify additional targets for the Escherichia coli OxyR transcription factor. This approach predicted OxyR binding sites upstream ofdsbG, encoding a periplasmic disulfide bond chaperone-isomerase; upstream of fhuF, encoding a protein required for iron uptake; and within yfdI. DNase I footprinting assays confirmed that oxidized OxyR bound to the predicted site centered 54 bp upstream of the dsbG gene and 238 bp upstream of a known OxyR binding site in the promoter region of the divergently transcribed ahpC gene. Although the new binding site was near dsbG, Northern blotting and primer extension assays showed that OxyR binding to thedsbG-proximal site led to the induction of a secondahpCF transcript, while OxyR binding to theahpCF-proximal site leads to the induction of bothdsbG and ahpC transcripts. Oxidized OxyR binding to the predicted site centered 40 bp upstream of thefhuF gene was confirmed by DNase I footprinting, but these assays further revealed a second higher-affinity site in thefhuF promoter. Interestingly, the two OxyR sites in thefhuF promoter overlapped with two regions bound by the Fur repressor. Expression analysis revealed that fhuFwas repressed by hydrogen peroxide in an OxyR-dependent manner. Finally, DNase I footprinting experiments showed OxyR binding to the site predicted to be within the coding sequence of yfdI. These results demonstrate the versatile modes of regulation by OxyR and illustrate the need to learn more about the ensembles of binding sites and transcripts in the E. coli genome.

scholarly journals Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in Escherichia coli K-12

2007 ◽  
Vol 189 (21) ◽  
pp. 7539-7548 ◽  
Author(s):  
Hsia-Yin Lin ◽  
Peggy J. Bledsoe ◽  
Valley Stewart
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Transcription Initiation ◽  
Shewanella Oneidensis ◽  
Regulatory System ◽  
Anaerobic Growth ◽  
Transcription Activator ◽  
Fnr Protein ◽  
Nitrate And Nitrite ◽  
K 12

ABSTRACT The facultative aerobe Escherichia coli K-12 can use respiratory nitrate ammonification to generate energy during anaerobic growth. The toxic compound nitric oxide is a by-product of this metabolism. Previous transcript microarray studies identified the yeaR-yoaG operon, encoding proteins of unknown function, among genes whose transcription is induced in response to nitrate, nitrite, or nitric oxide. Nitrate and nitrite regulate anaerobic respiratory gene expression through the NarX-NarL and NarQ-NarP two-component systems. All known Nar-activated genes also require the oxygen-responsive Fnr transcription activator. However, previous studies indicated that yeaR-yoaG operon transcription does not require Fnr activation. Here, we report results from mutational analyses demonstrating that yeaR-yoaG operon transcription is activated by phospho-NarL protein independent of the Fnr protein. The phospho-NarL protein binding site is centered at position −43.5 with respect to the transcription initiation site. Expression from the Shewanella oneidensis MR-1 nnrS gene promoter, cloned into E. coli, similarly was activated by phospho-NarL protein independent of the Fnr protein. Recently, yeaR-yoaG operon transcription was shown to be regulated by the nitric oxide-responsive NsrR repressor (N. Filenko et al., J. Bacteriol. 189:4410-4417, 2007). Our mutational analyses reveal the individual contributions of the Nar and NsrR regulators to overall yeaR-yoaG operon expression and document the NsrR operator centered at position −32. Thus, control of yeaR-yoaG operon transcription provides an example of overlapping regulation by nitrate and nitrite, acting through the Nar regulatory system, and nitric oxide, acting through the NsrR repressor.

scholarly journals Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration for ε-caprolactone production

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jinghui Xiong ◽  
Hefeng Chen ◽  
Ran Liu ◽  
Hao Yu ◽  
Min Zhuo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
Whole Cell ◽  
Cyclohexanone Monooxygenase ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Whole Cell Biocatalyst ◽  
Substrate Tolerance

Abstractε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.

scholarly journals Shared associations identify causal relationships between gene expression and immune cell phenotypes

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Christiane Gasperi ◽  
Sung Chun ◽  
Shamil R. Sunyaev ◽  
Chris Cotsapas
Keyword(s):  
Complex Traits ◽  
Direct Evidence ◽  
Immune Cell ◽  
Genetic Locus ◽  
Causal Variant ◽  
Causal Relationships ◽  
Genetic Studies ◽  
Two Factors ◽  
Gene Regulatory ◽  
Cell Phenotypes

AbstractGenetic mapping studies have identified thousands of associations between common variants and hundreds of human traits. Translating these associations into mechanisms is complicated by two factors: they fall into gene regulatory regions; and they are rarely mapped to one causal variant. One way around these limitations is to find groups of traits that share associations, using this genetic link to infer a biological connection. Here, we assess how many trait associations in the same locus are due to the same genetic variant, and thus shared; and if these shared associations are due to causal relationships between traits. We find that only a subset of traits share associations, with many due to causal relationships rather than pleiotropy. We therefore suggest that simply observing overlapping associations at a genetic locus is insufficient to infer causality; direct evidence of shared associations is required to support mechanistic hypotheses in genetic studies of complex traits.

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