The HlyU Protein Is a Positive Regulator of rtxA1, a Gene Responsible for Cytotoxicity and Virulence in the Human Pathogen Vibrio vulnificus

ABSTRACT Vibrio vulnificus is an opportunistic human pathogen that preferentially infects compromised iron-overloaded patients, causing a fatal primary septicemia with very rapid progress, resulting in a high mortality rate. In this study we determined that the HlyU protein, a virulence factor in V. vulnificus CMCP6, up-regulates the expression of VV20479, a homologue of the Vibrio cholerae RTX (repeats in toxin) toxin gene that we named rtxA1. This gene is part of an operon together with two other open reading frames, VV20481 and VV20480, that encode two predicted proteins, a peptide chain release factor 1 and a hemolysin acyltransferase, respectively. A mutation in rtxA1 not only contributes to the loss of cytotoxic activity but also results in a decrease in virulence, whereas a deletion of VV20481 and VV20480 causes a slight decrease in virulence but with no effect in cytotoxicity. Activation of the expression of the rtxA1 operon by HlyU occurs at the transcription initiation level by binding of the HlyU protein to a region upstream of this operon.

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Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, Belonging to the “phiKMV-Like Viruses”

Applied and Environmental Microbiology ◽

10.1128/aem.00128-11 ◽

2011 ◽

Vol 77 (10) ◽

pp. 3443-3450 ◽

Cited By ~ 35

Author(s):

Evelien M. Adriaenssens ◽

Pieter-Jan Ceyssens ◽

Vincent Dunon ◽

Hans-Wolfgang Ackermann ◽

Johan Van Vaerenbergh ◽

...

Keyword(s):

Soil Samples ◽

Pantoea Agglomerans ◽

Open Reading Frames ◽

Human Pathogen ◽

Content Type ◽

Host Diversity ◽

Double Stranded Dna ◽

Opportunistic Human Pathogen ◽

Reading Frames

ABSTRACTPantoea agglomeransis a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight ofP. agglomeransare lytic phages, isolated from soil samples, belonging to thePodoviridaeand are the firstPantoeaphages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of theAutographivirinae, within the genus of the “phiKMV-like viruses.” Phylogenetic analysis of all the sequenced members of theAutographivirinaesupports the classification of phages LIMElight and LIMEzero as members of the “phiKMV-like viruses” and corroborates the subdivision into the different genera. These data expand the knowledge ofPantoeaphages and illustrate the wide host diversity of phages within the “phiKMV-like viruses.”

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Complex Control of a Genomic Island Governing Biofilm and Rugose Colony Development in Vibrio vulnificus

Journal of Bacteriology ◽

10.1128/jb.00190-18 ◽

2018 ◽

Vol 200 (16) ◽

Cited By ~ 5

Author(s):

Daniel M. Chodur ◽

Dean A. Rowe-Magnus

Keyword(s):

Food Chain ◽

Horizontal Transfer ◽

Vibrio Vulnificus ◽

Regulatory Control ◽

Colony Development ◽

Dependent Manner ◽

Human Pathogen ◽

Content Type ◽

Regulatory Pathways ◽

Opportunistic Human Pathogen

ABSTRACT Vibrio vulnificus is a potent opportunistic human pathogen that contaminates the human food chain by asymptomatically colonizing seafood. The expression of the 9-gene brp exopolysaccharide locus mediates surface adherence and is controlled by the secondary signaling molecule c-di-GMP and the regulator BrpT. Here, we show that c-di-GMP and BrpT also regulate the expression of an adjacent 5-gene cluster that includes the cabABC operon, brpT, and another VpsT-like transcriptional regulator gene, brpS. The expression of the 14 genes spanning the region increased with elevated intracellular c-di-GMP levels in a BrpT-dependent manner, save for brpS, which was positively regulated by c-di-GMP and repressed by BrpT. BrpS repressed brpA expression and was required for rugose colony development. The mutation of its consensus WFSA c-di-GMP binding motif blocked these activities, suggesting that BrpS function is dependent on binding c-di-GMP. BrpT specifically bound the cabA, brpT, and brpS promoters, and binding sites homologous to the Vibrio cholerae VpsT binding site were identified upstream of brpA and brpT. Transcription was initiated distal to brpA, and a conserved RfaH-recruiting ops element and a potential Rho utilization (rut) terminator site were identified within the 100-bp leader region, suggesting the integration of early termination and operon polarity suppression into the regulation of brp transcription. The GC content and codon usage of the 16-kb brp region was 5.5% lower relative to that of the flanking DNA, suggesting its recent assimilation via horizontal transfer. Thus, architecturally, the brp region can be considered an acquired biofilm and rugosity island that is subject to complex regulation. IMPORTANCE Biofilm and rugose colony formation are developmental programs that underpin the evolution of Vibrio vulnificus as a potent opportunistic human pathogen and successful environmental organism. A better understanding of the regulatory pathways governing theses phenotypes promotes the development and implementation of strategies to mitigate food chain contamination by this pathogen. c-di-GMP signaling is central to both pathways. We show that the molecule orchestrates the expression of 14 genes clustered in a 16-kb segment of the genome that governs biofilm and rugose colony development. This region exhibits the hallmarks of horizontal transfer, suggesting complex regulatory control of a recently assimilated genetic island governing the colonization response of V. vulnificus.

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Identification of Genetic Loci Required for Capsular Expression in Vibrio vulnificus

Infection and Immunity ◽

10.1128/iai.71.3.1091-1097.2003 ◽

2003 ◽

Vol 71 (3) ◽

pp. 1091-1097 ◽

Cited By ~ 41

Author(s):

Amy B. Smith ◽

Ronald J. Siebeling

Keyword(s):

Unknown Function ◽

Gene Locus ◽

Insertion Sequence ◽

Capsular Polysaccharide ◽

Vibrio Vulnificus ◽

Virulent Strain ◽

Gene Cassette ◽

Open Reading Frames ◽

Genetic Loci ◽

Reading Frames

ABSTRACT Transposon mutagenesis of an encapsulated, virulent strain of Vibrio vulnificus 1003(O) led to the identification of four genetic regions that are essential to capsular polysaccharide (CPS) expression and virulence. Of the four regions, three are believed to be part of a capsule gene locus comprised of biosynthesis, polymerization, and transport genes clustered on a single chromosomal fragment. Genes indicating a Wzy-dependent system of polymerization and transmembrane export are present, suggesting that the CPS of V. vulnificus is lipid linked. The fourth region, while it contains a gene essential for CPS expression, is characteristic of an integron-gene cassette region, similar to the super integron of V. cholerae. It is not believed to be part of a CPS gene locus and is located in a region of the chromosome separate from the putative CPS loci. It is comprised of open reading frames (ORFs) carrying genes of unknown function surrounded by direct repeats. This region also contains IS492, an insertion sequence located numerous times throughout a region of the genome, demonstrating a restriction fragment length polymorphism among an encapsulated and nonencapsulated morphotype of V. vulnificus. Collectively, 22 ORFs were recognized: 13 capsule synthesis genes, 4 insertion sequences, 1 truncated biosynthesis gene, and 4 genes of unknown function. This study has led to the identification of previously unrecognized genetic loci that may help to increase the understanding of capsular genetics and antigenic diversity among V. vulnificus strains.

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The groESL Chaperone Operon ofLactobacillus johnsonii

Applied and Environmental Microbiology ◽

10.1128/aem.65.7.3033-3041.1999 ◽

1999 ◽

Vol 65 (7) ◽

pp. 3033-3041 ◽

Cited By ~ 54

Author(s):

D. Carey Walker ◽

Hany S. Girgis ◽

Todd R. Klaenhammer

Keyword(s):

Heat Shock ◽

Transcription Initiation ◽

Shuttle Vector ◽

Stress Protein ◽

Open Reading Frames ◽

Northern Analysis ◽

Temperature Sensitive ◽

Insertion Element ◽

Translation Start ◽

Reading Frames

ABSTRACT The Lactobacillus johnsonii VPI 11088groESL operon was localized on the chromosome near the insertion element IS1223. The operon was initially cloned as a series of three overlapping PCR fragments, which were sequenced and used to design primers to amplify the entire operon. The amplified fragment was used as a probe to recover the chromosomal copy of thegroESL operon from a partial library of L. johnsonii VPI 11088 (NCK88) DNA, cloned in the shuttle vector pTRKH2. The 2,253-bp groESL fragment contained three putative open reading frames, two of which encoded the ubiquitous GroES and GroEL chaperone proteins. Analysis of the groESLpromoter region revealed three transcription initiation sites, as well as three sets of inverted repeats (IR) positioned between the transcription and translation start sites. Two of the three IR sets bore significant homology to the CIRCE elements, implicated in negative regulation of the heat shock response in many bacteria. Northern analysis and primer extension revealed that multiple temperature-sensitive promoters preceded the groESLchaperone operon, suggesting that stress protein production in L. johnsonii is strongly regulated. Maximum groESLtranscription activity was observed following a shift to 55°C, and a 15 to 30-min exposure of log-phase cells to this temperature increased the recovery of freeze-thawed L. johnsonii VPI 11088. These results suggest that a brief, preconditioning heat shock can be used to trigger increased chaperone production and provide significant cross-protection from the stresses imposed during the production of frozen culture concentrates.

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Cyclo-(L-Phe-L-Pro), a Quorum-Sensing Signal ofVibrio vulnificus, Induces Expression of Hydroperoxidase through a ToxR-LeuO-HU-RpoS Signaling Pathway To Confer Resistance against Oxidative Stress

Infection and Immunity ◽

10.1128/iai.00932-17 ◽

2018 ◽

Vol 86 (9) ◽

Cited By ~ 3

Author(s):

In Hwang Kim ◽

So-Yeon Kim ◽

Na-Young Park ◽

Yancheng Wen ◽

Keun-Woo Lee ◽

...

Keyword(s):

Oxidative Stress ◽

Signaling Pathway ◽

Virulence Factor ◽

Vibrio Vulnificus ◽

Transcriptional Regulator ◽

Oxygen Species ◽

Human Pathogen ◽

Content Type ◽

Opportunistic Human Pathogen ◽

Bacterial Genes

ABSTRACTVibrio vulnificus, an opportunistic human pathogen, produces cyclo-(l-Phe-l-Pro) (cFP), which serves as a signaling molecule controlling the ToxR-dependent expression of innate bacterial genes, and also as a virulence factor eliciting pathogenic effects on human cells by enhancing intracellular reactive oxygen species levels. We found that cFP facilitated the protection ofV. vulnificusagainst hydrogen peroxide. At a concentration of 1 mM, cFP enhanced the level of the transcriptional regulator RpoS, which in turn induced expression ofkatG, encoding hydroperoxidase I, an enzyme that detoxifies H2O2to overcome oxidative stress. We found that cFP upregulated the transcription of the histone-like proteins vHUα and vHUβ through the cFP-dependent regulator LeuO. LeuO binds directly to upstream regions ofvhuAandvhuBto enhance transcription. vHUα and vHUβ then enhance the level of RpoS posttranscriptionally by stabilizing the mRNA. This cFP-mediated ToxR-LeuO-vHUαβ-RpoS pathway also upregulates genes known to be members of the RpoS regulon, suggesting that cFP acts as a cue for the signaling pathway responsible for both the RpoS and the LeuO regulons. Taken together, this study shows that cFP plays an important role as a virulence factor, as well as a signal for the protection of the cognate pathogen.

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Chromatin Remodeling around Nucleosome-Free Regions Leads to Repression of Noncoding RNA Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.00602-10 ◽

2010 ◽

Vol 30 (21) ◽

pp. 5110-5122 ◽

Cited By ~ 57

Author(s):

Adam N. Yadon ◽

Daniel Van de Mark ◽

Ryan Basom ◽

Jeffrey Delrow ◽

Iestyn Whitehouse ◽

...

Keyword(s):

Chromatin Remodeling ◽

Transcription Initiation ◽

Noncoding Rna ◽

Open Reading Frames ◽

Yeast Genome ◽

Transcription Start Sites ◽

Transcriptional Regulatory ◽

First Time ◽

Reading Frames

ABSTRACT Nucleosome-free regions (NFRs) at the 5′ and 3′ ends of genes are general sites of transcription initiation for mRNA and noncoding RNA (ncRNA). The presence of NFRs within transcriptional regulatory regions and the conserved location of transcription start sites at NFRs strongly suggest that the regulation of NFRs profoundly affects transcription initiation. To date, multiple factors are known to facilitate transcription initiation by positively regulating the formation and/or size of NFRs in vivo. However, mechanisms to repress transcription by negatively regulating the size of NFRs have not been identified. We identified four distinct classes of NFRs located at the 5′ and 3′ ends of genes, within open reading frames (ORFs), and far from ORFs. The ATP-dependent chromatin-remodeling enzyme Isw2 was found enriched at all classes of NFRs. Analysis of RNA levels also demonstrated Isw2 is required to repress ncRNA transcription from many of these NFRs. Thus, by the systematic annotation of NFRs across the yeast genome and analysis of ncRNA transcription, we established, for the first time, a mechanism by which NFR size is negatively regulated to repress ncRNA transcription from NFRs. Finally, we provide evidence suggesting that one biological consequence of repression of ncRNA, by Isw2 or by the exosome, is prevention of transcriptional interference of mRNA.

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Bacterial adherence and biofilm behavior of Vibrio vulnificus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147727 ◽

1993 ◽

Vol 51 ◽

pp. 378-379

Author(s):

B. D. Tall ◽

R. T. Gray ◽

D. B. Shah

Keyword(s):

Electron Microscopy ◽

Biofilm Formation ◽

Vibrio Vulnificus ◽

Ruthenium Red ◽

Human Pathogen ◽

Normal Microflora ◽

Microscopic Studies ◽

Plate Counts ◽

Opportunistic Human Pathogen ◽

Phase Variants

Vibrio vulnificus, an opportunistic human pathogen, is found as member of the normal microflora of shellfish and other seafoods, many of which are eaten raw. Though usually not harmful, V. vulnificus is responsible for causing fulminating septicemia in immunocompromised individuals. In previous light microscopic studies, we showed data suggesting that isogenic unencapsulated phase variants were more adherent to HeLa cells than were counterpart encapsulated phase variants. In this study, we extended our observations by comparing phase variant capsular morphology stained with Alcian blue (AB) and Ruthenium red (RR), and investigated the dynamics of biofilm formation by these organisms to glass coverslips (CS) using quantitative plate counts and scanning electron microscopy (SEM).To characterize the morphology of capsules expressed by these organisms, we stained cells grown on trypticase soy agar containing 1 % NaCl (TSA/NaCl) with AB and then prepared them for electron microscopy (EM) according to the method described by Hendley et al.

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Phage ϕC2 Mediates Transduction of Tn6215, Encoding Erythromycin Resistance, between Clostridium difficile Strains

mBio ◽

10.1128/mbio.00840-13 ◽

2013 ◽

Vol 4 (6) ◽

Cited By ~ 48

Author(s):

Shan Goh ◽

Haitham Hussain ◽

Barbara J. Chang ◽

Warren Emmett ◽

Thomas V. Riley ◽

...

Keyword(s):

Clostridium Difficile ◽

Genetic Manipulation ◽

Recipient Strain ◽

Open Reading Frames ◽

Human Pathogen ◽

Erythromycin Resistance ◽

Content Type ◽

Filter Mating ◽

First Time ◽

Reading Frames

ABSTRACTIn this work, we show thatClostridium difficilephage ϕC2 transduceserm(B), which confers erythromycin resistance, from a donor to a recipient strain at a frequency of 10−6per PFU. The transductants were lysogenic for ϕC2 and contained theerm(B) gene in a novel transposon, Tn6215. This element is 13,008 bp in length and contains 17 putative open reading frames (ORFs). It could also be transferred at a lower frequency by filter mating.IMPORTANCEClostridium difficileis a major human pathogen that causes diarrhea that can be persistent and difficult to resolve using antibiotics.C. difficileis potentially zoonotic and has been detected in animals, food, and environmental samples.C. difficilegenomes contain large portions of horizontally acquired genetic elements. The conjugative elements have been reasonably well studied, but transduction has not yet been demonstrated. Here, we show for the first time transduction as a mechanism for the transfer of a novel genetic element inC. difficile. Transduction may also be a useful tool for the genetic manipulation ofC. difficile.

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Characterization of the Multiple-Copy Host-Selective Toxin Gene, ToxB, in Pathogenic and Nonpathogenic Isolates of Pyrenophora tritici-repentis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.5.467 ◽

2004 ◽

Vol 17 (5) ◽

pp. 467-474 ◽

Cited By ~ 69

Author(s):

J. Patrick Martinez ◽

Nicholas W. Oesch ◽

Lynda M. Ciuffetti

Keyword(s):

Sequence Data ◽

Pathogenic Fungi ◽

Single Copy ◽

Culture Conditions ◽

Open Reading Frames ◽

Toxin Gene ◽

Pyrenophora Tritici Repentis ◽

Race 4 ◽

Reading Frames

ToxB, a gene that encodes a 6.6-kDa host-selective toxin (HST), is present in several races of the wheat pathogen Pyrenophora tritici-repentis. To learn more about the multiple ToxB open reading frames (ORFs), six of the estimated nine copies from a race 5 isolate were cloned and analyzed. All six copies of ToxB have identical 261-bp ORFs and thus encode the same form of Ptr ToxB. Sequence analysis of regions flanking the cloned ToxB loci revealed that the majority of loci are associated with portions of retrotransposons and a transposon-like sequence. Data indicate that ToxB loci reside on two chromosomes, 3.5 and 2.7 Mb, with the majority of copies located on the 2.7 Mb chromosome. A related gene, referred to as toxb, from a nonpathogenic race 4 isolate was also cloned and characterized. This is interesting because, until now, HST genes have only been found in toxin-producing, pathogenic isolates of plant pathogenic fungi. The toxb gene from nonpathogenic isolates is 86% similar to ToxB, and data suggest that toxb is a single-copy gene. No toxb transcript was detected under culture conditions that favor the expression of ToxB; therefore, these genes differ in their transcriptional regulation.

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Regulation of Hexuronate Utilization inBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.181.2.426-433.1999 ◽

1999 ◽

Vol 181 (2) ◽

pp. 426-433 ◽

Cited By ~ 34

Author(s):

Kathleen R. Mekjian ◽

Edward M. Bryan ◽

Bernard W. Beall ◽

Charles P. Moran

Keyword(s):

Protein A ◽

Repeat Sequence ◽

Open Reading Frames ◽

Erwinia Chrysanthemi ◽

Inverted Repeat Sequence ◽

Metabolic Genes ◽

Catabolite Control Protein A ◽

Regulated Expression ◽

Control Protein ◽

Reading Frames

ABSTRACT We have identified a locus essential for galacturonate utilization in Bacillus subtilis. Genes homologous to Escherichia coli and Erwinia chrysanthemi glucuronate and galacturonate metabolic genes were found in a cluster consisting of 10 open reading frames (ORFs) in the B. subtilis chromosome. A mutant of B. subtilis containing a replacement of the second and third ORFs was unable to grow with galacturonate as its primary carbon source. Galacturonate induced expression from a ςA-dependent promoter, exuP1, located upstream from the first ORF. The eighth ORF in this cluster (theexu locus) encodes a LacI and GalR homolog that negatively regulated expression from exuP1. A 26-bp inverted repeat sequence centered 15 bp downstream from the exuP1 start point of transcription acted in cis to negatively regulate expression from exuP1 under noninducing conditions. Expression from the exuP1 promoter was repressed by high levels of glucose, which is probably mediated by CcpA (catabolite control protein A). A ςE-dependent promoter,exuP2, was localized between the second and third ORFs and was active during sporulation.

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