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Vibrio harveyi VIB 645, which is very pathogenic towards salmonids and produces extracellular product with a high titer of hemolytic activity towards fish erythrocytes, was found to contain two closely related hemolysin genes (designated vhhA and vhhB), whereas the majority of strains examined (11 of 13) carried only a single hemolysin gene. Both genes from VIB 645 were cloned and sequenced. The open reading frames (ORFs) of vhhA and vhhB shared a high level of identity (98.8%) and were predicted to encode identical polypeptides comprising 418 amino acid residues. The VHH protein shows homology to the lecithinase of V. mimicus and V. cholerae. Transformants of Escherichia coli containing the ORF of either vhhA or vhhB displayed weak hemolytic activity in rainbow trout blood agar. The hemolytic activity was very high when the ORF of vhhB was cloned in E. coli together with the native promoter. Surprisingly, the level of vhh-specific RNA transcript produced by VIB 645 was found to be very low. We conclude that the hemolytic phenotype of VIB 645 is not due to increased expression of one or both copies of the vhh gene.Vibrio harveyi is a gram-negative, luminous bacterium which is widely distributed in the marine environment (28,29). Recently, this organism has emerged as a serious pathogen of marine animals. For example, the organism has been reported as a primary pathogen of cultured penaeid shrimp, especially in South America and Asia (1,10,18,26,33,36). Additionally, V. harveyi has been associated with diseases in finfish (1,8,9,15,30) and pearl oysters (24). However, little is known about the pathogenicity mechanisms of V. harveyi. Liu et al. (19) considered that proteases, phospholipases, or hemolysins might be important for pathogenicity; cysteine protease has been reported as the major exotoxin to penaeid shrimp (16,17,20). Montero and Austin (21) suggested that the lipopolysaccharide might constitute the lethal toxin of V. harveyi E2 to penaeid shrimp. We have previously examined a large number of well-characterized V. harveyi cultures and found that the hemolysin activity in the extracellular product was involved in pathogenesis in salmonids. V. harveyi VIB 645, which was the most pathogenic isolate, produced extracellular product with the highest titer of hemolytic activity towards Atlantic salmon (1:256) and rainbow trout erythrocytes (1:32) (40).In general, bacterial hemolysins have been suggested to be important factors of pathogenic vibrios by causing hemorrhagic septicemia and diarrhea in the host. Many of these hemolysins are well characterized, and genes encoding them have been cloned from V. parahaemolyticus (22, 35), V. cholerae (3, 27), V. hollisae (38), V. mimicus (12), V. vulnificus (38), and V. anguillarum (7).In this study, we describe the cloning and characterization of the hemolysin gene of V. harveyi as a first step towards assessing the role of V. harveyi hemolysin in fish disease. MATERIALS AND METHODSBacterial strains and plasmids. Thirteen V. harveyi isolates from a diverse range of h...

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Maltose and maltotriose are the two most abundant fermentable sugars in brewer’s wort, and the rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. In spite of this, no information is currently available on the genetics of maltose and maltotriose uptake in brewing strains of yeast. In this work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and maltotriose transporter genes contained by them. To do this, we hybridized gene probes to chromosome blots. Studies performed with laboratory strains have shown that maltose utilization is conferred by any one of five unlinked but highly homologous MAL loci (MAL1 to MAL4 and MAL6). Gene 1 at each locus encodes a maltose transporter. All of the strains of brewer’s yeast examined except two were found to containMAL11 and MAL31 sequences, and only one of these strains lacked MAL41. MAL21 was not present in the five ale strains and 12 of the lager strains. MAL61 was not found in any of the yeast strains. In three of the lager strains, there was evidence that MAL transporter gene sequences occurred on chromosomes other than those known to carry MAL loci. Sequences corresponding to the AGT1 gene, which encodes a transporter of several α-glucosides, including maltose and maltotriose, were detected in all but one of the yeast strains. Homologues of AGT1 were identified in three of the lager strains, and two of these homologues were mapped, one to chromosome II and the other to chromosome XI. AGT1 appears to be a member of a family of closely related genes, which may have arisen in brewer’s yeast in response to selective pressure.

Inter-strain and inter-species inhibition mediated by a bacteriocin-like inhibitory substance (BLIS) from a pathogenic Vibrio harveyi strain VIB 571 was demonstrated against four isolates of the same species, and one culture each of a Vibrio sp., Vibrio fischeri, Vibrio gazogenes and Vibrio parahaemolyticus. The crude BLIS, which was obtained by ammonium-sulphate precipitation of the cell-free supernatant of a 72 h broth culture of strain VIB 571, was inactivated by lipase, proteinase K, pepsin, trypsin, pronase E, SDS and incubation at ¢60 6C for 10 min. The activity was stable between pH 2-11 for at least 5 h. Anion-exchange chromatography, gel filtration, SDS-PAGE and two-dimensional gel electrophoresis revealed the presence of a single major peak, comprising a protein with a pI of 5?4 and a molecular mass of~32 kDa. The N-terminal amino acid sequence of the protein comprised Asp-Glu-Tyr-Ile-Ser-X-Asn-Lys-X-Ser-Ser-Ala-Asp-Ile (with X representing cysteine or modified amino acid residues). A similarity search based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) generated peptide masses and the N-terminal sequence did not yield any significant matches.