Dataset Information

Characterization of heme uptake cluster genes in the fish pathogen Vibrio anguillarum.

ABSTRACT: Vibrio anguillarum can utilize hemin and hemoglobin as sole iron sources. In previous work we identified HuvA, the V. anguillarum outer membrane heme receptor by complementation of a heme utilization mutant with a cosmid clone (pML1) isolated from a genomic library of V. anguillarum. In the present study, we describe a gene cluster contained in cosmid pML1, coding for nine potential heme uptake and utilization proteins: HuvA, the heme receptor; HuvZ and HuvX; TonB, ExbB, and ExbD; HuvB, the putative periplasmic binding protein; HuvC, the putative inner membrane permease; and HuvD, the putative ABC transporter ATPase. A V. anguillarum strain with an in-frame chromosomal deletion of the nine-gene cluster was impaired for growth with heme or hemoglobin as the sole iron source. Single-gene in-frame deletions were constructed, demonstrating that each of the huvAZBCD genes are essential for utilization of heme as an iron source in V. anguillarum, whereas huvX is not. When expressed in Escherichia coli hemA (strain EB53), a plasmid carrying the gene for the heme receptor, HuvA, was sufficient to allow the use of heme as the porphyrin source. For utilization of heme as an iron source in E. coli ent (strain 101ESD), the tonB exbBD and huvBCD genes were required in addition to huvA. The V. anguillarum heme uptake cluster shows some differences in gene arrangement when compared to homologous clusters described for other Vibrio species.

SUBMITTER: Mourino S

PROVIDER: S-EPMC515166 | biostudies-literature | 2004 Sep

REPOSITORIES: biostudies-literature

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Characterization of heme uptake cluster genes in the fish pathogen Vibrio anguillarum.

Mouriño Susana S Osorio Carlos R CR Lemos Manuel L ML

Journal of bacteriology 20040901 18

Vibrio anguillarum can utilize hemin and hemoglobin as sole iron sources. In previous work we identified HuvA, the V. anguillarum outer membrane heme receptor by complementation of a heme utilization mutant with a cosmid clone (pML1) isolated from a genomic library of V. anguillarum. In the present study, we describe a gene cluster contained in cosmid pML1, coding for nine potential heme uptake and utilization proteins: HuvA, the heme receptor; HuvZ and HuvX; TonB, ExbB, and ExbD; HuvB, the puta ...[more]

PMID: 15342586

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Project description:Vibrio anguillarum is a causative agent of vibriosis in fish. Hemolytic activity has been suggested as a virulence factor by contributing to hemorrhagic septicemia and diarrhea. In order to identify and characterize the hemolysin genes and examine the role of hemolytic activity in virulence, a mini-Tn10Kan mutagenesis clone bank of V. anguillarum was screened. While no hemolysin-negative strains were observed, several mutants with two- to threefold-increased hemolytic activity were found. The region containing the insertion mutation was cloned, sequenced, and found to contain the V. anguillarum hemolysin (vah1) and two other open reading frames, coding for a putative lactonizing lipase (llpA) and a putative phospholipase (plp). The mini-Tn10Kan was inserted into plp. Site-directed mutagenesis of each gene revealed that mutations in vah1 and llpA did not affect hemolytic activity, but insertions into plp caused a two- to threefold increase in hemolysis. Double mutations in plp and either vah1 or llpA resulted in wild-type hemolytic activity. Complementation of plp restored hemolytic activity to wild-type levels. Spectrophotometric determination of hemolysin specific activity revealed that activity on a per cell basis peaked during the first 2 h of growth in LB20. Real-time quantitative reverse transcriptase PCR used to quantitate transcription of the hemolysin genes plp and vah1 in V. anguillarum wild-type strains M93Sm and NB10 revealed that transcription of plp and vah1 peaked at 2 h of growth in LB20. Additionally, expression of vah1 measured in the plp mutant strain, JL01, during the first 2 h of growth was >8 times higher than that in M93Sm. Mutations in plp and llpA did not affect virulence of V. anguillarum. The mutation in vah1 attenuated V. anguillarum virulence in fish. These data show that several genes are responsible for hemolytic activity in V. anguillarum. At least three genes (plp, llpA, and vah1) are responsible for one hemolytic activity. The data also suggest that plp acts as a negative regulator of vah1 and llpA.

Dataset Information

Characterization of heme uptake cluster genes in the fish pathogen Vibrio anguillarum.

Publications

Characterization of heme uptake cluster genes in the fish pathogen Vibrio anguillarum.

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