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Vibrio ordalii is a major cause of vibriosis in wild and cultured marine salmonids and carries pMJ101, a 30-kb cryptic plasmid that replicates in the absence of DNA polymerase I without producing single-stranded intermediates. A recombinant derivative harboring the pMJ101 replication region proved to be compatible with pJM1, a plasmid containing the iron acquisition system required for the virulence of V. anguillarum 775, another important pathogen that causes vibriosis. Sequence analysis of a 1.56-kb fragment harboring the pMJ101 replication region revealed the presence of typical features found in DNA origins including an AT-rich region, 11 dam-methylation sites of which 5 are within the putative ori region, and five copies of the 9-bp consensus sequence for DnaA binding. Gel retardation assays demonstrated that the latter replication element indeed binds DnaA purified from Escherichia coli. A potential open reading frame encoding a hydrophilic protein with a predicted pI of 10.3 and an M(r) of 33,826 was found adjacent to the ori region. Although these properties are typical of DNA-binding proteins, no significant homology was found between this predicted protein, named RepM, and other previously characterized proteins. Reverse transcriptase-polymerase chain reaction analysis of total RNA demonstrated the presence of repM mRNA in V. ordalii. The major initiation site of this mRNA was located 187 nucleotides upstream of the GTG initiation codon as determined by nuclease S1 protection assays. This transcription initiation site is preceded by putative -10 and -35 promoter sequences that control the expression of the repM replication gene. These results demonstrate that the replication region of pMJ101 shares some structural and sequence similarities with other DNA replication regions, which include DnaA binding and methylation sites and an open reading frame encoding a distinct protein required for its replication., (Copyright 1999 Academic Press.)

Histamine production in bacteria-contaminated fish is the result of the presence of bacterial histidine decarboxylase activity, which converts histidine present in muscle proteins to histamine. The fish pathogen Vibrio anguillarum harbors a plasmid-encoded histidine decarboxylase gene (angH) that is essential for biosynthesis of the siderophore anguibactin. However, the role of angH in histamine biosynthesis by this pathogen has not been fully determined. Thus, the objectives of this study were to monitor the production and release of histamine by the wild-type as well as by a plasmidless strain and angH isogenic mutants generated by allelic exchange. Reverse transcription polymerase chain reaction showed that only the wild-type strain expressed angH, while no angH message was detected in the mutants and the plasmidless derivative. The iron uptake-deficient phenotype of one of the angH mutants confirmed the location of the mutation and the unique role of this gene in iron acquisition. Thin-layer chromatography, gas chromatography, and mass spectrometry showed that histamine was released by the strain harboring a wild-type angH gene when grown in excess histidine. This biogenic amine was not detected in the culture supernatants of the plasmidless derivative and the angH mutant when cultured under the same experimental conditions. These results indicate that angH is essential for histamine biosynthesis in V. anguillarum, a compound responsible for food poisoning and potentially involved in bacterial virulence. Thin-layer chromatography of wild-type culture supernatants and beta-galactosidase assays using the isogenic angH mutant demonstrated that the expression of this gene is independent of the histidine concentration of the medium under both iron-rich and iron-limiting conditions., (Copyright 1998 Academic Press.)

The 30-kb pMJ101 plasmid is found as a high-copy-number pool in all the pathogenic strains of Vibrio ordalii examined so far. The replication functions of pMJ101 were localized within a 2.4-kb EcoRV-HindIII restriction fragment by using different subclones in combination with Bal31 exonuclease deletions and Tn5 insertion mutants. Recombinant clones carrying this fragment were able to replicate in Escherichia coli cells deficient in either DNA Polymerase I (PolA-) or integration host factor functions. However, the viability of recombinant plasmids containing the pMJ101 origin of replication was dependent on the expression of the gene encoding the DnaA protein. Electrophoretic analysis of plasmid-encoded proteins in an in vitro transcription-translation coupled system revealed that the replication region of pMJ101 encodes a 36-kDa protein. The expression of this protein was correlated with the ability of different recombinant plasmids harboring this pMJ101 DNA region to replicate in the PolA- E. coli strain. Replication typing showed that pMJ101 is not related to any of the plasmid incompatibility groups contained in the bank of rep probes described by M. Couturier et al. (Microbiol. Rev. 52, 375-395, 1988).