Transcription of the Vibrio cholerae haemolysin gene, hlyA, and cloning of a positive regulatory locus, hlyU

Williams, Susan G.; Manning, Paul A.

doi:10.1111/j.1365-2958.1991.tb00825.x

Cited by 38 publications

(37 citation statements)

References 27 publications

(7 reference statements)

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“…Previous studies indicated that the hlyU gene is a conserved transcriptional regulator in many Vibrio species (11,12,22,23). We hypothesized that HlyU could be a putative regulator of the two hemolysin gene clusters in V. anguillarum.…”

Section: Resultsmentioning

confidence: 99%

“…While HlyU is a positive regulator of V. cholerae hlyA (22,23), there is no experimental evidence to demonstrate that HlyU binds to the hlyA promoter region. Therefore, it is still unclear if HlyU is a direct transcriptional activator binding to the hlyA promoter region or if it interferes with an unknown repressor of hlyA to cause derepression of hlyA by HlyU (17).…”

Section: Discussionmentioning

confidence: 99%

“…Since it had been reported that HlyU is a DNA binding protein (17) that positively regulates hlyA (homologue of vah1) in V. cholerae (23) and rtxA1 (homologue of rtxA) in V. vulnificus (11), we wanted to determine possible HlyU binding sites in FIG. 3.…”

Section: Resultsmentioning

confidence: 99%

“…It has been reported that HlyU is a positive regulator of hemolysin and toxin genes in Vibrio species. In Vibrio cholerae, the HlyU protein positively regulates expression of hemolysin gene hlyA, as well as the HlyA-coregulated gene hcp (22,23). Williams et al (22) reported that a mutation in hlyU attenuates V. cholerae O17 in the infant mouse cholera infection model.…”

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confidence: 99%

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HlyU Is a Positive Regulator of Hemolysin Expression in Vibrio anguillarum

Mou

Nelson

2011

J Bacteriol

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The two hemolysin gene clusters previously identified in Vibrio anguillarum, the vah1 cluster and the rtxACHBDE cluster, are responsible for the hemolytic and cytotoxic activities of V. anguillarum in fish. In this study, we used degenerate PCR to identify a positive hemolysin regulatory gene, hlyU, from the unsequenced V. anguillarum genome. The hlyU gene of V. anguillarum encodes a 92-amino-acid protein and is highly homologous to other bacterial HlyU proteins. An hlyU mutant was constructed, which exhibited an ϳ5-fold decrease in hemolytic activity on sheep blood agar with no statistically significant decrease in cytotoxicity of the wild-type strain. Complementation of the hlyU mutation restored both hemolytic activity and cytotoxic activity. Both semiquantitative reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR)were used to examine expression of the hemolysin genes under exponential and stationary-phase conditions in wild-type, hlyU mutant, and hlyU complemented strains. Compared to the wild-type strain, expression of rtx genes decreased in the hlyU mutant, while expression of vah1 and plp was not affected in the hlyU mutant. Complementation of the hlyU mutation restored expression of the rtx genes and increased vah1 and plp expression to levels higher than those in the wild type. The transcriptional start sites in both the vah1-plp and rtxH-rtxB genes' intergenic regions were determined using 5 random amplification of cDNA ends (5-RACE), and the binding sites for purified HlyU were discovered using DNA gel mobility shift experiments and DNase protection assays.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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HlyU Is a Positive Regulator of Hemolysin Expression in Vibrio anguillarum

Mou

Nelson

2011

J Bacteriol

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“…Total cellular RNA was prepared by the hot phenol method as described elsewhere (62). A total of 10 to 20 g of RNA and approximately 10 ng of oligonucleotide primer (5Ј labelled with [␥-32 P]dATP and T4 polynucleotide kinase) were ethanol precipitated, dried, and resuspended in 12 l of water.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Vibrio cholerae El Tor lipase operon lipAB and a protease gene downstream of the hly region

et al. 1997

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We have cloned and sequenced a region encoding a lipase operon and a putative, previously uncharacterized metalloprotease of Vibrio cholerae O1. These lie downstream of hlyA and hlyB, which encode the El Tor hemolysin and methyl-accepting chemotactic factor, respectively. Previous reports identified the hlyC gene downstream of hlyAB, encoding an 18.3-kDa protein. However, we now show that this open reading frame (ORF) encodes a 33-kDa protein, and since the amino acid sequence is highly homologous to the triacylglyceride-specific lipase of Pseudomonas spp., hlyC has been renamed lipA. LipA contains the highly conserved pentapeptide and catalytic triad amino acid regions of the catalytic sites of other lipases. The region downstream of lipA has been sequenced and has revealed ORFs lipB and prtV. The amino acid sequence of lipB is homologous to those of the accessory lipase proteins (lipase-specific foldase) required by Pseudomonas and various other bacterial species for the production of mature active lipase, and in agreement with this, we show that both lipA and lipB are required to restore a lipase-deficient lipA null mutant of V. cholerae. The intergenic stop codon for lipA overlaps the ribosome-binding site for lipB, and a stem-loop resembling a rho-independent terminator is present immediately downstream from lipB, suggesting that lipA and lipB form a lipase operon in V. cholerae. prtV lies downstream of lipAB but is transcribed in the opposite direction and is predicted to share the same putative transcriptional terminator with lipAB. The zinc-binding and catalytic domains conserved among many metalloproteases are present in PrtV, which is highly homologous to the immune inhibitor A (InA) metalloprotease of Bacillus thuringiensis. PrtV was visualized as approximately 102 kDa, which is consistent with the coding capacity of the gene. The genetic organization of this region suggests that it is possibly part of a pathogenicity island, encoding products capable of damaging host cells and/or involved in nutrient acquisition by V. cholerae. However, neither lipA nor prtV null mutants were attenuated in the infant mouse model, nor did they exhibit reduced colonization potential compared with wild type in competition experiments.The hemolysin (HlyA) produced by many El Tor and non-O1 strains of Vibrio cholerae has been shown to be a pore-forming toxin and has been implicated as a virulence determinant (4,25,28,29,63). The production of HlyA is controlled by levels of available iron, and thus, it has been suggested that HlyA may be involved in iron scavenging (54). HlyA expression is also upregulated by the DNA-binding protein HlyU, and studies of a hlyU null mutant suggest that HlyU may also regulate the expression of additional virulence determinants (63).The hlyA locus is closely linked to a number of other potential virulence determinants. A lecithinase-phospholipase gene, lecA, is transcribed divergently from hlyA (37), and downstream of hlyA lies hlyB, whose gene product is highly homologous to methyl-acceptin...

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The regulator HlyU, the repeat‐in‐toxin gene rtxA1, and their roles in the pathogenesis of Vibrio vulnificus infections

Liu

Crosa

2012

MicrobiologyOpen

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HlyU is a master regulator that plays an essential role in the virulence of the human pathogen Vibrio vulnificus. One of the most noteworthy characteristics of HlyU regulation in this organism is its positive control of the expression of the repeat-in-toxin (RtxA1) gene, one of the most important virulence factors accounting for the fulminating and damaging nature of V. vulnificus infections. In this work, we reviewed the latest studies of RtxA1 in this bacterium and highlight the mechanism of gene regulation of rtxA1 expression by HlyU under a broader gene regulatory network.

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Transcription of the Vibrio cholerae haemolysin gene, hlyA, and cloning of a positive regulatory locus, hlyU

Cited by 38 publications

References 27 publications

HlyU Is a Positive Regulator of Hemolysin Expression in Vibrio anguillarum

HlyU Is a Positive Regulator of Hemolysin Expression in Vibrio anguillarum

Characterization of the Vibrio cholerae El Tor lipase operon lipAB and a protease gene downstream of the hly region

The regulator HlyU, the repeat‐in‐toxin gene rtxA1, and their roles in the pathogenesis of Vibrio vulnificus infections

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