scholarly journals A cryptic promoter in the LEE1 regulatory region of enterohaemorrhagic Escherichia coli: promoter specificity in AT-rich gene regulatory regions

2011 ◽  
Vol 436 (3) ◽  
pp. 681-686 ◽  
Author(s):  
Md. Shahidul Islam ◽  
Mark J. Pallen ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Regulatory Region ◽  
Cryptic Promoter ◽  
Regulatory Regions ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Enterocyte Effacement ◽  
Biochemical Analyses ◽  
Transcript Initiation ◽  
Coli Promoter

Transcription of the LEE1 operon in the locus of enterocyte effacement of enterohaemorrhagic Escherichia coli is due to the P1 promoter. Mutational and biochemical analyses reveal the existence of an overlapping promoter, designated P1A, which can drive transcript initiation 10 bp upstream of the P1 promoter transcript start point. Because of the overlap between P1 and P1A, P1A activity is unmasked only when the P1 promoter is inactivated by mutation. In the present paper, we report that mutation of the P1–10 element is less effective in unmasking P1A promoter activity than mutation of the P1–35 element. This suggests that the P1 promoter −35 element, which corresponds to the consensus, can sequester RNA polymerase even when P1 is inactive and thereby prevent RNA polymerase from serving the P1A promoter. We propose that such promoter elements may play a role in enforcing specificity in bacterial regulatory regions that contain alternative possible promoters.

Demonstration that the TyrR Protein and RNA Polymerase Complex Formed at the Divergent P3 Promoter Inhibits Binding of RNA Polymerase to the Major Promoter, P1, of the aroP Gene ofEscherichia coli

1998 ◽  
Vol 180 (20) ◽  
pp. 5466-5472 ◽  
Author(s):  
Peixiang Wang ◽  
Ji Yang ◽  
Akira Ishihama ◽  
A. J. Pittard
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Regulatory Region ◽  
Ofescherichia Coli ◽  
Mrna Synthesis ◽  
Polymerase Complex ◽  
Defective Mutant ◽  
Dna Fragment ◽  
Region Ii

ABSTRACT In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the Escherichia coli aroP gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206–4212, 1997). Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213–4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the aroP regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.

scholarly journals Location of the C-Terminal Domain of the RNA Polymerase   Subunit in Different Open Complexes at the Escherichia Coli Galactose Operon Regulatory Region

1996 ◽  
Vol 24 (12) ◽  
pp. 2243-2251 ◽  
Author(s):  
T. A. Belyaeva ◽  
J. A. Bown ◽  
N. Fujita ◽  
A. Ishihama ◽  
S. J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Regulatory Region ◽  
Terminal Domain

scholarly journals Control freaks—signals and cues governing the regulation of virulence in attaching and effacing pathogens

2018 ◽  
Vol 47 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Natasha C.A. Turner ◽  
James P.R. Connolly ◽  
Andrew J. Roe
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Regulation ◽  
Virulence Factors ◽  
Secretion System ◽  
Master Regulators ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Type 3 Secretion System ◽  
Enterocyte Effacement ◽  
Type 3 ◽  
Fine Tune

AbstractEnterohaemorrhagic Escherichia coli (EHEC) mediates disease using a type 3 secretion system (T3SS), which is encoded on the locus of enterocyte effacement (LEE) and is tightly controlled by master regulators. This system is further modulated by a number of signals that help to fine-tune virulence, including metabolic, environmental and chemical signals. Since the LEE and its master regulator, Ler, were established, there have been numerous scientific advancements in understanding the regulation and expression of virulence factors in EHEC. This review will discuss the recent advancements in this field since our previous review, with a focus on the transcriptional regulation of the LEE.

scholarly journals RNA polymerase supply and flux through the lac operon in Escherichia coli

2016 ◽  
Vol 371 (1707) ◽  
pp. 20160080 ◽  
Author(s):  
Bandar Sendy ◽  
David J. Lee ◽  
Stephen J. W. Busby ◽  
Jack A. Bryant
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Lac Operon ◽  
Lactose Operon ◽  
E Coli ◽  
Synthetic Promoters ◽  
Promoter Occupancy ◽  
Low Levels ◽  
Polymerase Binding ◽  
Transcript Initiation

Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli . By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase. This article is part of the themed issue ‘The new bacteriology’.

scholarly journals A Cyclic AMP Receptor Protein Mutant That Constitutively Activates an Escherichia coli Promoter Disrupted by an IS5 Insertion

1999 ◽  
Vol 181 (24) ◽  
pp. 7457-7463 ◽  
Author(s):  
Vladimir Podolny ◽  
E. C. C. Lin ◽  
Ann Hochschild
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Regulatory Region ◽  
Constitutive Expression ◽  
Single Amino Acid ◽  
Receptor Protein ◽  
Regulatory Sequences ◽  
Cyclic Amp Receptor Protein ◽  
Coli Promoter

ABSTRACT Previously an Escherichia coli mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated. This phenotype is the result of the constitutive expression of the fucO gene (in the fucAOoperon), which encodes one of the enzymes in the fucose metabolic pathway. The mutant was found to bear an IS5 insertion in the intergenic regulatory region between the divergently orientedfucAO and fucPIK operons. Though expression of the fucAO operon was constitutive, the fucPIKoperon became noninducible such that the mutant could no longer grow on fucose. A fucose-positive revertant which was found to contain a suppressor mutation in the crp gene was selected. Here we identify this crp mutation, which results in a single amino acid substitution (K52N) that has been proposed previously to uncover a cryptic activating region in the cyclic AMP receptor protein (CRP). We show that the mutant CRP constitutively activates transcription from both the IS5-disrupted and the wild-type fucPIKpromoters, and we identify the CRP-binding site that is required for this activity. Our results show that the fucPIK promoter, a complex promoter which ordinarily depends on both CRP and the fucose-specific regulator FucR for its activation, can be activated in the absence of FucR by a mutant CRP that uses three, rather than two, activating regions to contact RNA polymerase. For the IS5-disrupted promoter, which retains a single CRP-binding site, the additional activating region of the mutant CRP evidently compensates for the lack of upstream regulatory sequences.

scholarly journals Activation by NarL at the Escherichia coli ogt promoter

2020 ◽  
Vol 477 (15) ◽  
pp. 2807-2820
Author(s):  
Patcharawarin Ruanto ◽  
David L. Chismon ◽  
Joanne Hothersall ◽  
Rita E. Godfrey ◽  
David J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Response Regulator ◽  
Direct Interaction ◽  
Α Subunit ◽  
E Coli ◽  
Terminal Domain ◽  
Dna Alkyltransferase ◽  
Promoter Dna ◽  
Transcript Initiation

The Escherichia coli NarX/NarL two-component response-regulator system regulates gene expression in response to nitrate ions and the NarL protein is a global transcription factor, which activates transcript initiation at many target promoters. One such target, the E. coli ogt promoter, which controls the expression of an O6-alkylguanine-DNA-alkyltransferase, is dependent on NarL binding to two DNA targets centred at positions −44.5 and −77.5 upstream from the transcript start. Here, we describe ogt promoter derivatives that can be activated solely by NarL binding either at position −44.5 or position −77.5. We show that NarL can also activate the ogt promoter when located at position −67.5. We present data to argue that NarL-dependent activation of transcript initiation at the ogt promoter results from a direct interaction between NarL and a determinant in the C-terminal domain of the RNA polymerase α subunit. Footprinting experiments show that, at the −44.5 promoter, NarL and the C-terminal domain of the RNA polymerase α subunit bind to opposite faces of promoter DNA, suggesting an unusual mechanism of transcription activation. Our work suggests new organisations for activator-dependent transcription at promoters and future applications for biotechnology.

Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157 : H7 to HEp-2 cells

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2357-2571 ◽  
Author(s):  
Sunao Iyoda ◽  
Haruo Watanabe
Keyword(s):  
Escherichia Coli ◽  
Genomic Library ◽  
Regulatory Gene ◽  
Effector Proteins ◽  
Multicopy Plasmid ◽  
Positive Effects ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Dna Fragment ◽  
Enterocyte Effacement ◽  
Positive Effect

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) genomes contain a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes genes involved in the formation of attaching and effacing lesions on epithelial cells. To elucidate the regulatory mechanism of the LEE genes in EHEC, an EHEC O157 genomic library was screened for clones which modulated expression of the LEE genes. From more than 5000 clones, a DNA fragment was obtained containing a perC homologue as a positive regulator for the LEE genes. In EPEC, perC is known to be part of the per operon, along with perA and perB, located on the EPEC adherence factor plasmid, which is not found in EHEC. However, the complete genome sequence of EHEC O157 Sakai strain reveals that there are five perC-like sequences, but no perA and perB, on the chromosome. These five perC homologues were characterized, and it was found that three of the homologues (renamed perC homologue pchA, pchB and pchC) encoded 104 aa proteins, and when expressed on a multicopy plasmid enhanced the expression of LEE genes. In contrast, perC homologues encoding proteins of 89 and 90 aa, renamed pchD and pchE, respectively, had no significant effect. Deletion mutants of the pch genes were constructed, and the effect on the expression of LEE-encoded type III effector proteins, such as EspA, B and D, and adhesion phenotype to HEp-2 cells was examined. Deletion of pchA or pchB, but not pchC, decreased the expression of Esp proteins and adhesion to HEp-2 cells. Such effects were more apparent with mutants carrying double deletions of pchA/pchB or pchA/pchC, suggesting that pchA/B/C are all necessary for full expression of the LEE genes and adhesion to HEp-2 cells. Further study demonstrated that the positive effect of pchA/B/C was caused by enhanced transcription of the LEE-encoded regulatory gene, ler. Introduction of a multicopy plasmid carrying each pchA/B/C gene significantly induced microcolony formation by EHEC O157 on HEp-2 cells. These results suggest that the pchABC genes are necessary for full virulence of EHEC O157.

scholarly journals Coordinate Control of the Locus of Enterocyte Effacement and Enterohemolysin Genes by Multiple Common Virulence Regulators in Enterohemorrhagic Escherichia coli

2011 ◽  
Vol 79 (11) ◽  
pp. 4628-4637 ◽  
Author(s):  
Sunao Iyoda ◽  
Naoko Honda ◽  
Takehito Saitoh ◽  
Ken Shimuta ◽  
Jun Terajima ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Region ◽  
Regulatory System ◽  
Enterohemorrhagic Escherichia Coli ◽  
Mobility Shift ◽  
Content Type ◽  
Positive Regulators ◽  
Enterocyte Effacement ◽  
Gel Mobility Shift ◽  
K 12

ABSTRACTThe locus of enterocyte effacement (LEE) pathogenicity island is required for the intimate adhesion of enterohemorrhagicEscherichia coli(EHEC) to the intestinal epithelial cells. GrlR and GrlA are LEE-encoded negative and positive regulators, respectively. The interaction of these two regulators is important for controlling the transcription of LEE genes through Ler, a LEE-encoded central activator for the LEE. The GrlR-GrlA regulatory system controls not only LEE but also the expression of the flagellar and enterohemolysin (Ehx) genes in EHEC. Since Ehx levels were markedly induced in agrlRmutant but not in agrlR grlAdouble mutant and significantly increased by overexpression of GrlA in alermutant, GrlA is responsible for this regulation (T. Saitoh et al., J. Bacteriol.190:4822-4830, 2008). In this study, additional investigations of the regulation ofehxgene expression determined that Ler also acts as an activator for Ehx expression without requiring GrlA function. We recently reported that the LysR-type regulator LrhA positively controls LEE expression (N. Honda et al., Mol. Microbiol.74:1393-1411, 2009). The hemolytic activity of thelrhAmutant strain of EHEC was lower than that of the wild-type strain, and LrhA markedly inducedehxtranscription in anE. coliK-12 strain, suggesting that LrhA also activates the transcription ofehxwithout GrlA and Ler. Gel mobility shift assays demonstrated that Ler and LrhA directly bind to the regulatory region ofehxC. Together, these results indicate that transcription ofehxis positively regulated by Ler, GrlA, and LrhA, which all act as positive regulators for LEE expression.

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