Temperature- and H-NS-Dependent Regulation of a Plasmid-Encoded Virulence Operon Expressing Escherichia coli Hemolysin

ABSTRACT Proteins H-NS and Hha form a nucleoprotein complex that modulates expression of the thermoregulated hly operon of Escherichia coli. We have been able to identify two H-NS binding sites in the hly regulatory region. One of them partially overlaps the promoter region (site II), and the other is located about 2 kbp upstream (site I). In contrast, Hha protein did not show any preference for specific sequences. In vitro, temperature influences the affinity of H-NS for a DNA fragment containing both binding sites and H-NS-mediated repression of hly operon transcription. Deletion analysis of the hly regulatory region confirms the relevance of site I for thermoregulation of this operon. We present a model to explain the temperature-modulated repression of the hly operon, based on the experiments reported here and other, preexisting data.

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Bacterial-Chromatin Structural Proteins Regulate the Bimodal Expression of the Locus of Enterocyte Effacement (LEE) Pathogenicity Island in Enteropathogenic Escherichia coli

mBio ◽

10.1128/mbio.00773-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 10

Author(s):

Hervé Leh ◽

Ahmad Khodr ◽

Marie-Christine Bouger ◽

Bianca Sclavi ◽

Sylvie Rimsky ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Epigenetic Regulation ◽

Binding Sites ◽

Promoter Region ◽

Binding Proteins ◽

Bacterial Virulence ◽

Structural Modifications ◽

Chromatin Structural

ABSTRACT In enteropathogenic Escherichia coli (EPEC), the locus of enterocyte effacement (LEE) encodes a type 3 secretion system (T3SS) essential for pathogenesis. This pathogenicity island comprises five major operons (LEE1 to LEE5), with the LEE5 operon encoding T3SS effectors involved in the intimate adherence of bacteria to enterocytes. The first operon, LEE1, encodes Ler (LEE-encoded regulator), an H-NS (nucleoid structuring protein) paralog that alleviates the LEE H-NS silencing. We observed that the LEE5 and LEE1 promoters present a bimodal expression pattern, depending on environmental stimuli. One key regulator of bimodal LEE1 and LEE5 expression is ler expression, which fluctuates in response to different growth conditions. Under conditions in vitro considered to be equivalent to nonoptimal conditions for virulence, the opposing regulatory effects of H-NS and Ler can lead to the emergence of two bacterial subpopulations. H-NS and Ler share nucleation binding sites in the LEE5 promoter region, but H-NS binding results in local DNA structural modifications distinct from those generated through Ler binding, at least in vitro. Thus, we show how two nucleoid-binding proteins can contribute to the epigenetic regulation of bacterial virulence and lead to opposing bacterial fates. This finding implicates for the first time bacterial-chromatin structural proteins in the bimodal regulation of gene expression. IMPORTANCE Gene expression stochasticity is an emerging phenomenon in microbiology. In certain contexts, gene expression stochasticity can shape bacterial epigenetic regulation. In enteropathogenic Escherichia coli (EPEC), the interplay between H-NS (a nucleoid structuring protein) and Ler (an H-NS paralog) is required for bimodal LEE5 and LEE1 expression, leading to the emergence of two bacterial subpopulations (with low and high states of expression). The two proteins share mutual nucleation binding sites in the LEE5 promoter region. In vitro, the binding of H-NS to the LEE5 promoter results in local structural modifications of DNA distinct from those generated through Ler binding. Furthermore, ler expression is a key parameter modulating the variability of the proportions of bacterial subpopulations. Accordingly, modulating the production of Ler into a nonpathogenic E. coli strain reproduces the bimodal expression of LEE5. Finally, this study illustrates how two nucleoid-binding proteins can reshape the epigenetic regulation of bacterial virulence. IMPORTANCE Gene expression stochasticity is an emerging phenomenon in microbiology. In certain contexts, gene expression stochasticity can shape bacterial epigenetic regulation. In enteropathogenic Escherichia coli (EPEC), the interplay between H-NS (a nucleoid structuring protein) and Ler (an H-NS paralog) is required for bimodal LEE5 and LEE1 expression, leading to the emergence of two bacterial subpopulations (with low and high states of expression). The two proteins share mutual nucleation binding sites in the LEE5 promoter region. In vitro, the binding of H-NS to the LEE5 promoter results in local structural modifications of DNA distinct from those generated through Ler binding. Furthermore, ler expression is a key parameter modulating the variability of the proportions of bacterial subpopulations. Accordingly, modulating the production of Ler into a nonpathogenic E. coli strain reproduces the bimodal expression of LEE5. Finally, this study illustrates how two nucleoid-binding proteins can reshape the epigenetic regulation of bacterial virulence.

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Studies on the binding of the Escherichia coli MelR transcription activator protein to operator sequences at the MelAB promoter

Biochemical Journal ◽

10.1042/bj2870501 ◽

1992 ◽

Vol 287 (2) ◽

pp. 501-508 ◽

Cited By ~ 12

Author(s):

R Caswell ◽

C Webster ◽

S Busby

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Direct Contact ◽

Promoter Activity ◽

Mutational Analysis ◽

Dna Conformation ◽

Transcription Activator ◽

Transcription Start ◽

Upstream Site ◽

Dna Fragment

Escherichia coli MelR protein binds to two sites located upstream of the melAB transcription start site. Although both sites are required for optimal melibiose-dependent expression from the melAB promoter, some MelR-dependent expression is found if the upstream site is deleted or if the spacing between the two sites is altered. Gel retardation assays have been exploited to study MelR binding to a DNA fragment carrying just the upstream site. Methylation interference analysis was used to identify one guanine (at -104) which is important for MelR binding. Mutational analysis confirmed the importance of this base and revealed a second position (at -110) where mutations interfere with melAB promoter activity. Experiments using potassium permanganate as a probe suggested that the DNA sequence around -110 adopts a distorted conformation. We propose that the mutation at -104 alters MelR binding by interfering with a direct contact, whereas the mutation at -110 primarily affects DNA conformation. The binding of purified MelR protein to a melAB promoter fragment carrying both binding sites has also been studied: binding results in four retarded bands in gel assays. Methylation interference experiments have been exploited to identify the binding sites occupied in each complex. Although both binding sites share a common 18 bp sequence, MelR binding to the more upstream site is stronger. We could find no evidence for co-operative interactions between MelR and RNA polymerase and no major effects of melibiose. Some evidence for melibiose-dependent distortion in complexes between MelR and the melAB promoter is discussed.

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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

Journal of Bacteriology ◽

10.1128/jb.180.20.5466-5472.1998 ◽

1998 ◽

Vol 180 (20) ◽

pp. 5466-5472 ◽

Cited By ~ 17

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.

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Induction of the Cellular E2F-1 Promoter by the Adenovirus E4-6/7 Protein

Journal of Virology ◽

10.1128/jvi.74.5.2084-2093.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2084-2093 ◽

Cited By ~ 23

Author(s):

Joel Schaley ◽

Robert J. O'Connor ◽

Laura J. Taylor ◽

Dafna Bar-Sagi ◽

Patrick Hearing

Keyword(s):

Dna Binding ◽

Binding Site ◽

Transient Expression ◽

Binding Sites ◽

Promoter Region ◽

Fluorescent Protein ◽

Protein Accumulation ◽

Promoter Regions ◽

Single Binding Site

ABSTRACT The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.

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The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

Biochemical Journal ◽

10.1042/bj2960851 ◽

1993 ◽

Vol 296 (3) ◽

pp. 851-857 ◽

Cited By ~ 23

Author(s):

T Belyaeva ◽

L Griffiths ◽

S Minchin ◽

J Cole ◽

S Busby

Keyword(s):

Escherichia Coli ◽

Point Mutations ◽

Growth Conditions ◽

Upstream Region ◽

E Coli ◽

Run Off ◽

A Site ◽

Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

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SlyA and H-NS Regulate Transcription of the Escherichia coli K5 Capsule Gene Cluster, and Expression of slyA in Escherichia coli Is Temperature-dependent, Positively Autoregulated, and Independent of H-NS

Journal of Biological Chemistry ◽

10.1074/jbc.m703465200 ◽

2007 ◽

Vol 282 (46) ◽

pp. 33326-33335 ◽

Cited By ~ 38

Author(s):

David Corbett ◽

Hayley J. Bennett ◽

Hamdia Askar ◽

Jeffrey Green ◽

Ian S. Roberts

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Regulation Of Transcription ◽

Temperature Dependent ◽

Mobility Shift ◽

E Coli ◽

Dnase I Footprinting ◽

Nucleoprotein Complex ◽

Electrophoretic Mobility Shift Assays

In this paper, we present the first evidence of a role for the transcriptional regulator SlyA in the regulation of transcription of the Escherichia coli K5 capsule gene cluster and demonstrate, using a combination of reporter gene fusions, DNase I footprinting, and electrophoretic mobility shift assays, the dependence of transcription on the functional interplay between H-NS and SlyA. Both SlyA and H-NS bind to multiple overlapping sites within the promoter in vitro, but their binding is not mutually exclusive, resulting in a remodeled nucleoprotein complex. In addition, we show that expression of the E. coli slyA gene is temperature-regulated, positively autoregulated, and independent of H-NS.

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

Journal of Bacteriology ◽

10.1128/jb.00131-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5060-5067 ◽

Cited By ~ 33

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.

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Bioinformatics Analysis of SNPs in IL-6 Gene Promoter of Jinghai Yellow Chickens

Genes ◽

10.3390/genes9090446 ◽

2018 ◽

Vol 9 (9) ◽

pp. 446 ◽

Cited By ~ 3

Author(s):

Shijie Xin ◽

Xiaohui Wang ◽

Guojun Dai ◽

Jingjing Zhang ◽

Tingting An ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Binding Sites ◽

Promoter Region ◽

Bioinformatics Analysis ◽

Cpg Islands ◽

Regulatory Region ◽

Transcription Factor Binding Sites ◽

Transcription Factor Binding ◽

Factor Binding

The proinflammatory cytokine, interleukin-6 (IL-6), plays a critical role in many chronic inflammatory diseases, particularly inflammatory bowel disease. To investigate the regulation of IL-6 gene expression at the molecular level, genomic DNA sequencing of Jinghai yellow chickens (Gallus gallus) was performed to detect single-nucleotide polymorphisms (SNPs) in the region −2200 base pairs (bp) upstream to 500 bp downstream of IL-6. Transcription factor binding sites and CpG islands in the IL-6 promoter region were predicted using bioinformatics software. Twenty-eight SNP sites were identified in IL-6. Four of these 28 SNPs, three [−357 (G > A), −447 (C > G), and −663 (A > G)] in the 5′ regulatory region and one in the 3′ non-coding region [3177 (C > T)] are not labelled in GenBank. Bioinformatics analysis revealed 11 SNPs within the promoter region that altered putative transcription factor binding sites. Furthermore, the C-939G mutation in the promoter region may change the number of CpG islands, and SNPs in the 5′ regulatory region may influence IL-6 gene expression by altering transcription factor binding or CpG methylation status. Genetic diversity analysis revealed that the newly discovered A-663G site significantly deviated from Hardy-Weinberg equilibrium. These results provide a basis for further exploration of the promoter function of the IL-6 gene and the relationships of these SNPs to intestinal inflammation resistance in chickens.

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