Homology of TcpN, a putative regulatory protein of Vibrio cholerae, to the AraC family of transcriptional activators

Gene ◽  
1992 ◽  
Vol 116 (1) ◽  
pp. 93-97 ◽  
Author(s):  
Monica A. Ogierman ◽  
Paul A. Manning
Keyword(s):  
Vibrio Cholerae ◽  
Regulatory Protein ◽  
Transcriptional Activators ◽  
Arac Family

scholarly journals Characterization of Enhancer Binding by the Vibrio cholerae Flagellar Regulatory Protein FlrC

2005 ◽  
Vol 187 (9) ◽  
pp. 3158-3170 ◽  
Author(s):  
Nidia E. Correa ◽  
Karl E. Klose
Keyword(s):  
Binding Site ◽  
Vibrio Cholerae ◽  
Transcription Start Site ◽  
Regulatory Protein ◽  
Dnase I ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Dnase I Footprinting

ABSTRACT The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence. It has previously been demonstrated that the σ54-dependent activator FlrC is necessary for both flagellar synthesis and for enhanced intestinal colonization. In order to characterize FlrC binding, we analyzed two FlrC-dependent promoters, the highly transcribed flaA promoter and the weakly transcribed flgK promoter, utilizing transcriptional lacZ fusions, mobility shift assays, and DNase I footprinting. Promoter fusion studies showed that the smallest fragment with wild-type transcriptional activity for flaAp was from positions −54 to +137 with respect to the start site, and from −63 to +144 for flgKp. Gel mobility shift assays indicated that FlrC binds to a fragment containing the region from positions +24 to +95 in the flaAp, and DNase I footprinting identified a protected region between positions +24 and +85. Mobility shift and DNase I footprinting indicated weak binding of FlrC to a region downstream of the flgKp transcription start site. These results demonstrate a relatively novel σ54-dependent promoter architecture, with the activator FlrC binding downstream of the σ54-dependent transcription start sites. When the FlrC binding site(s) in the flaA promoter was moved a large distance (285 bp) upstream of the transcription start site of either flaAp or flgKp, high levels of FlrC-dependent transcription resulted, indicating that this binding region functions as an enhancer element. In contrast, the relatively weak FlrC binding site(s) in the flgK promoter failed to function as an enhancer element at either promoter, suggesting that FlrC binding strength contributes to enhancer activity. Our results suggest that the differences in FlrC binding to various flagellar promoters results in the differences in transcription levels that mirror the relative requirement for the flagellar components within the flagellum.

scholarly journals Identification of Multiple ς54-Dependent Transcriptional Activators inVibrio cholerae

1998 ◽  
Vol 180 (19) ◽  
pp. 5256-5259 ◽  
Author(s):  
Karl E. Klose ◽  
Veronica Novik ◽  
John J. Mekalanos
Keyword(s):  
Quorum Sensing ◽  
Vibrio Cholerae ◽  
Sigma Factor ◽  
Pathogenic Bacterium ◽  
Transcriptional Activators ◽  
High Homology ◽  
Chromosomal Dna ◽  
Degenerate Primers ◽  
Coding Sequences ◽  
Mutant Strains

ABSTRACT In the pathogenic bacterium Vibrio cholerae, the alternate sigma factor ς54 is required for expression of multiple sets of genes, including an unidentified gene(s) necessary for enhanced colonization within the host. To identify ς54-dependent transcriptional activators involved in colonization, PCR was performed with V. choleraechromosomal DNA and degenerate primers, revealing six novel and distinct coding sequences with homology to ς54-dependent activators. One sequence had high homology to the luxO gene of V. harveyi, which in that organism is involved in quorum sensing. Phenotypes of V. cholerae strains containing mutations in each of the six putative ς54-dependent activator genes identified one as a probable ntrChomologue. None of the mutant strains exhibited a defect in the ability to colonize infant mice, suggesting the presence of additional ς54-dependent activators not identified by this technique.

scholarly journals Construction and Use of an ipb DNA Module To Generate Pseudomonas Strains with Constitutive Trichloroethene and Isopropylbenzene Oxidation Activity

1998 ◽  
Vol 64 (7) ◽  
pp. 2454-2462 ◽  
Author(s):  
Frank Berendes ◽  
Nicolas Sabarth ◽  
Beate Averhoff ◽  
Gerhard Gottschalk
Keyword(s):  
Dna Sequences ◽  
Promoter Region ◽  
Sequence Similarity ◽  
Regulatory Protein ◽  
Continuous Cultivation ◽  
Oxidation Activity ◽  
Translational Initiation ◽  
Degradation Rates ◽  
Tce Degradation ◽  
Arac Family

ABSTRACT Pseudomonas sp. strain JR1 exhibits trichloroethene (TCE) oxidation activity with isopropylbenzene (IPB) as the inducer substrate. We previously reported the genes encoding the first three enzymes of the IPB-degradative pathway (ipbA1,ipbA2, ipbA3, ipbA4,ipbB, and ipbC) and identified the initial IPB dioxygenase (IpbA1A2A3A4) as responsible for TCE cooxidation (U. Pflugmacher, B. Averhoff, and G. Gottschalk, Appl. Environ. Microbiol. 62:3967–3977, 1996). Primer extension analyses revealed multiple transcriptional start points located upstream of the translational initiation codon of ipbA1. The transcription from these start sites was found to be IPB dependent. Thirty-one base pairs upstream of the first transcriptional start point tandemly repeated DNA sequences overlapping the −35 region of a putative ς70 promoter were found. These repeats exhibit significant sequence similarity to the operator-promoter region of thexyl meta operon in Pseudomonas putida, which is required for the binding of XylS, a regulatory protein of the XylS (also called AraC) family. These similarities suggest that the transcription of the IPB dioxygenase genes is modulated by a regulatory protein of the XylS/AraC family. The construction of an ipbDNA module devoid of this ipb operator-promoter region and the stable insertion of this DNA module into the genomes of differentPseudomonas strains resulted in pseudomonads with constitutive IPB and TCE oxidation activities. Constitutive TCE oxidation of two such Pseudomonas hybrid strains, JR1A::ipb and CBS-3::ipb, was found to be stable for more than 120 generations in antibiotic-free medium. Evaluation of constitutive TCE degradation rates revealed that continuous cultivation of strain JR1A::ipbresulted in a significant increase in rates of TCE degradation.

scholarly journals A Novel Regulatory Protein Involved in Motility of Vibrio cholerae

2009 ◽  
Vol 191 (22) ◽  
pp. 7027-7038 ◽  
Author(s):  
Manuel Moisi ◽  
Christian Jenul ◽  
Susan M. Butler ◽  
Aaron New ◽  
Sarah Tutz ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Gene Transcription ◽  
Deletion Mutant ◽  
Expression Patterns ◽  
Regulatory Protein ◽  
Regulatory Elements ◽  
Flagellar Gene ◽  
Class Iii ◽  
Reading Frame ◽  
Infant Mouse

ABSTRACT The facultative pathogen Vibrio cholerae is the causative agent of the human intestinal disease cholera. Both motility and chemotaxis of V. cholerae have been shown to contribute to the virulence and spread of cholera. The flagellar gene operons are organized into a hierarchy composed of four classes (I to IV) based on their temporal expression patterns. Some regulatory elements involved in flagellar gene expression have been elucidated, but regulation is complex and flagellar biogenesis in V. cholerae is not completely understood. In this study, we determined that the virulence defect of a V. cholerae cheW1 deletion mutant was due to polar effects on the downstream open reading frame VC2058 (flrD). Expression of flrD in trans restored the virulence defect of the cheW1 deletion mutant, and deletion of flrD resulted in a V. cholerae strain attenuated for virulence, as determined by using the infant mouse intestinal colonization model. The flrD mutant strain exhibited decreased transcription of class III and IV flagellar genes and reduced motility. Transcription of the flrD promoter, which lies within the coding sequence of cheW1, is independent of the flagellar transcriptional activators FlrA and RpoN, which activate class II genes, indicating that flrD does not fit into any of the four flagellar gene classes. Genetic epistasis studies revealed that the two-component system FlrBC, which is required for class III and IV flagellar gene transcription, acts downstream of flrD. We hypothesize that the inner membrane protein FlrD interacts with the cytoplasmic FlrBC complex to activate class III and IV gene transcription.

scholarly journals Bicarbonate Induces Vibrio cholerae Virulence Gene Expression by Enhancing ToxT Activity

2009 ◽  
Vol 77 (9) ◽  
pp. 4111-4120 ◽  
Author(s):  
Basel H. Abuaita ◽  
Jeffrey H. Withey
Keyword(s):  
Gene Expression ◽  
Carbonic Anhydrase ◽  
Vibrio Cholerae ◽  
Regulatory Protein ◽  
Virulence Gene ◽  
Chemical Stimulus ◽  
High Concentration ◽  
Virulence Gene Expression ◽  
Regulatory Cascade ◽  
El Tor

ABSTRACT Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera, a severe diarrheal illness. The two biotypes of V. cholerae O1 capable of causing cholera, classical and El Tor, require different in vitro growth conditions for induction of virulence gene expression. Growth under the inducing conditions or infection of a host initiates a complex regulatory cascade that results in production of ToxT, a regulatory protein that directly activates transcription of the genes encoding cholera toxin (CT), toxin-coregulated pilus (TCP), and other virulence genes. Previous studies have shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype. However, the mechanism for bicarbonate-mediated CT induction has not been defined. In this study, we demonstrate that bicarbonate stimulates virulence gene expression by enhancing ToxT activity. Both the classical and El Tor biotypes produce inactive ToxT protein when they are cultured statically in the absence of bicarbonate. Addition of bicarbonate to the culture medium does not affect ToxT production but causes a significant increase in CT and TCP expression in both biotypes. Ethoxyzolamide, a potent carbonic anhydrase inhibitor, inhibits bicarbonate-mediated virulence induction, suggesting that conversion of CO2 into bicarbonate by carbonic anhydrase plays a role in virulence induction. Thus, bicarbonate is the first positive effector for ToxT activity to be identified. Given that bicarbonate is present at high concentration in the upper small intestine where V. cholerae colonizes, bicarbonate is likely an important chemical stimulus that V. cholerae senses and that induces virulence during the natural course of infection.

scholarly journals Vibrio cholerae H-NS Silences Virulence Gene Expression at Multiple Steps in the ToxR Regulatory Cascade

2000 ◽  
Vol 182 (15) ◽  
pp. 4295-4303 ◽  
Author(s):  
Melinda B. Nye ◽  
James D. Pfau ◽  
Karen Skorupski ◽  
Ronald K. Taylor
Keyword(s):  
Gene Expression ◽  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Transcriptional Start Site ◽  
Regulatory Protein ◽  
Virulence Gene ◽  
Virulence Gene Expression ◽  
Activator Proteins ◽  
Regulatory Cascade ◽  
Genes Encoding

ABSTRACT H-NS is an abundant nucleoid-associated protein involved in the maintenance of chromosomal architecture in bacteria. H-NS also has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. In this study we demonstrate a role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon ofVibrio cholerae. Deletion of hns resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin-coregulated pilus, and the ToxT virulence gene regulatory protein. For the cholera toxin- and ToxT-encoding genes, elevated expression in an hns mutant was found to occur in the absence of the cognate activator proteins, suggesting that H-NS functions directly at these promoters to decrease gene expression. Deletion analysis of the region upstream of toxT suggests that an extensive region located far upstream of the transcriptional start site is required for complete H-NS-mediated repression of gene expression. These data indicate that H-NS negatively influences multiple levels of gene expression within the V. choleraevirulence cascade and raise the possibility that the transcriptional activator proteins in the ToxR regulon function to counteract the repressive effects of H-NS at the various promoters as well as to recruit RNA polymerase.

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