title: HlyU is a positive regulator of hemolysin expression in Vibrio anguillarum.
abstract: 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 ∼5-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.
authors.affiliation: Department of Cell and Molecular Biology, 120 Flagg Rd., University of Rhode Island, Kingston, RI 02881, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Xiangyu
authors.initials: X
authors.lastname: Mou
authors.forename: David R
authors.initials: DR
authors.lastname: Nelson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis*; D002460:Cell Line; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004912:Erythrocytes / Q000187:drug effects; D005399:Fishes; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006460:Hemolysin Proteins / Q000096:biosynthesis*; D006461:Hemolysis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site; D014733:Vibrio / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; D014157:Transcription Factors; C073845:hemolysin, Vibrio