Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

Mahon, Vivienne; Smyth, Cyril J.; Smith, Stephen G.

doi:10.1099/mic.0.038521-0

Cited by 11 publications

(9 citation statements)

References 37 publications

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“…Similar to what has been reported for Rns, VirF Sh and ToxT [38], [39], [40], [41], the deletion of discrete and long NTD regions (data not shown) or alanine substitutions of single amino acids in this domain resulted in PerA mutants with impaired activation and DNA binding functions at both promoters. Similarly, NTD PerA mutants G37W, W68G and L94P were not able to activate a perA-lacZ transcriptional fusion [24], but the nature of this defect was not determined.…”

Section: Discussionsupporting

confidence: 85%

Further Characterization of Functional Domains of PerA, Role of Amino and Carboxy Terminal Domains in DNA Binding

et al. 2013

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PerA is a key regulator of virulence genes in enteropathogenic E. coli. PerA is a member of the AraC/XylS family of transcriptional regulators that directly regulates the expression of the bfp and per operons in response to different environmental cues. Here, we characterized mutants in both the amino (NTD) and carboxy (CTD) terminal domains of PerA that affect its ability to activate the expression of the bfp and per promoters. Mutants at residues predicted to be important for DNA binding within the CTD had a significant defect in their ability to bind to the regulatory regions of the bfp and per operons and, consequently, in transcriptional activation. Notably, mutants in specific NTD residues were also impaired to bind to DNA suggesting that this domain is involved in structuring the protein for correct DNA recognition. Mutations in residues E116 and D168, located in the vicinity of the putative linker region, significantly affected the activation of the perA promoter, without affecting PerA binding to the per or bfp regulatory sequences. Overall these results provide additional evidence of the importance of the N-terminal domain in PerA activity and suggest that the activation of these promoters involves differential interactions with the transcriptional machinery. This study further contributes to the characterization of the functional domains of PerA by identifying critical residues involved in DNA binding, differential promoter activation and, potentially, in the possible response to environmental cues.

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Section: Discussionsupporting

confidence: 85%

Further Characterization of Functional Domains of PerA, Role of Amino and Carboxy Terminal Domains in DNA Binding

et al. 2013

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“…Protein members of the AraC family have two structural domains: the C-terminal DNA binding domain and the N-terminal signaling domain, connected by a relatively unstructured linker (Mahon et al, 2010;Seedorff and Schleif, 2011). The DNA binding domain comprises a ca.…”

Section: Introductionmentioning

confidence: 99%

A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

Santiago

Yan

Tran

et al. 2016

Molecular Microbiology

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Summary AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays, and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholera, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.

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“…To date, the majority of the ETEC transcriptional studies have focused on the ETEC rns regulon (11), which has largely been associated with the regulation of CF expression (3,9,23), although binding motifs have been identified adjacent to other genes (44). No regulator has been identified that is associated with the expression of the enterotoxins.…”

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confidence: 99%

Analysis of Global Transcriptional Profiles of Enterotoxigenic Escherichia coli Isolate E24377A

Sahl

Rasko

2012

Infect Immun

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Enterotoxigenic Escherichia coli (ETEC) is an important pathogenic variant (pathovar) of E. coli in developing countries from a human health perspective, causing significant morbidity and mortality. Previous studies have examined specific regulatory networks in ETEC, although little is known about the global effects of inter-and intrakingdom signaling on the expression of virulence and colonization factors in ETEC. In this study, an E. coli/Shigella pan-genome microarray, combined with quantitative reverse transcriptase PCR (qRT-PCR) and RNA sequencing (RNA-seq), was used to quantify the expression of ETEC virulence and colonization factors. Biologically relevant chemical signals were combined with ETEC isolate E24377A during growth in either Luria broth (LB) or Dulbecco's modified Eagle medium (DMEM), and transcription was examined during different phases of the growth cycle; chemical signals examined included glucose, bile salts, and preconditioned media from E. coli/Shigella isolates. The results demonstrate that the presence of bile salts, which are found in the intestine and thought to be bactericidal, upregulates the expression of many ETEC virulence factors, including heat-stable (estA) and heat-labile (eltA) enterotoxin genes. In contrast, the ETEC colonization factors CS1 and CS3 were downregulated in the presence of bile, consistent with findings in studies of other enteric pathogens. RNA-seq analysis demonstrated that one of the most differentially expressed genes in the presence of bile is a unique plasmid-encoded AraC-like transcriptional regulator (peaR); other previously unknown genetic elements were found as well. These results provide transcriptional targets and putative mechanisms that should help improve understanding of the global regulatory networks and virulence expression in this important human pathogen. Despite the high incidence of death resulting from enterotoxigenic Escherichia coli (ETEC) infection, especially in children in developing countries (55), the mechanisms and regulatory networks of ETEC virulence are still relatively unexplored on a genomic scale. ETEC bacteria are characterized by the production of the plasmid-encoded heat-stable (ST) and/or heat-labile (LT) enterotoxin (11). ETEC release ST and/or LT into the host epithelium, which elevates intracellular cyclic AMP (cAMP) that then activates the cystic fibrosis transmembrane regulator (CFTR) chloride channel; this activation leads to the secretion of electrolytes and, subsequently, water as diarrhea (43). In addition to ST and LT, the enteroaggregative E. coli (EAEC) heat-stable enterotoxin 1 (EAST1) protein, first identified in enteroaggregative E. coli (45), has been found in many ETEC isolates with defined colonization factor profiles (58); however, the contribution of EAST1 to ETEC diarrheal disease is currently unknown (11).ETEC bacteria adhere to intestinal epithelium by fimbrial appendages known as colonization factors (CFs) (12). CFs are structurally and genetically diverse; to date, more than 25 CFs have been id...

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Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

Cited by 11 publications

References 37 publications

Further Characterization of Functional Domains of PerA, Role of Amino and Carboxy Terminal Domains in DNA Binding

Further Characterization of Functional Domains of PerA, Role of Amino and Carboxy Terminal Domains in DNA Binding

A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

Analysis of Global Transcriptional Profiles of Enterotoxigenic Escherichia coli Isolate E24377A

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