Comparative analysis and distribution of pP9014, a novel drug resistance IncP-1 plasmid from Photobacterium damselae subsp. piscicida.

Photobacterium damselae subsp. piscicida, a causative agent of pseudotuberculosis, often harbours resistance plasmids (R plasmids) that facilitate horizontal gene transfer of drug resistance genes. R plasmid pP9014 was isolated from P. damselae subsp. piscicida and its complete nucleotide sequence was determined using Next Generation Sequencing technology. A protein network analysis was conducted to determine the relatedness of protein coding sequences, and ClustalW was used for the full nucleotide sequences. The occurrence of pP9014-like plasmids compared with pP99-018-like plasmids in a specific region was determined using probes for their transfer regions. pP9014 is 55851bp long with an overall GC content of 44.4% encoding 61 open reading frames (ORFs) including antimicrobial resistance genes and two conjugative transfer regions (Tra and Trb). The backbone showed highest similarity to Marinobacter adhaerens pHP-42 and Methylophaga sp. JAM7. pP9014 is similar to several IncP plasmids but forms a different subgroup. pP9014 is a unique plasmid in P. damselae subsp. piscicida and was not commonly found in drug-resistant P. damselae subsp. piscicida isolated from different areas and years in Japan. Plasmids similar to the previously reported pP99-018 are more widely distributed. This rarity suggests that plasmids similar to pP99-018 are more compatible with γ-proteobacteria. pP9014 is the first reported IncP-1 plasmid from fish pathogens. Its similarity to other IncP plasmids isolated from soil and human pathogens suggests that plasmids of the IncP-1 incompatibility group are vectors for the transfer of drug resistance genes among diverse environments.

Cloning and sequencing of the histidine decarboxylase gene from Photobacterium phosphoreum and its functional expression in Escherichia coli.

The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. We reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10 degrees C. Moreover, histamine-forming ability was affected by two histidine decarboxylases (HDCs): constitutive and inducible enzymes. In this study, the gene encoding P. phosphoreum HDC was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA corresponding to the hdc gene revealed an open reading frame of 1,140 bp coding for a pyridoxal-5'-phosphate-dependent HDC of 380 amino acid residues with a predicted molecular mass of 42.6 kDa. The HDC amino acid sequences formed a phylogenetic clade with strong bootstrap support and revealed high sequence similarities among the P. phosphoreum isolate and species of the family Enterobacteriaceae and a separate phylogenetic branch with the lowest sequence similarity between the isolate and the taxonomically closer Listonella anguillarum. The T7 promoter was used to overexpress the hdc gene in E. coli cells. The recombinant clone, E. coli BL21(DE3), displayed significant levels of HDC activity. The recombinant hdc gene was suggested to code the inducible HDC; therefore, the optimum reaction conditions of the recombinant HDC were similar to those of the inducible HDC in the P. phosphoreum isolate. In addition, a putative catabolite-repressor protein binding site, amino acid permease gene, and histidine-tRNA synthetase gene were found in flanking regions of the hdc gene.

Activity of two histidine decarboxylases from Photobacterium phosphoreum at different temperatures, pHs, and NaCl concentrations.

The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. The authors reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10 degrees C. Moreover, histamine-forming ability was affected by two histidine decarboxylases: constitutive and inducible enzymes. This article reports the effect of various growth and reaction conditions, such as temperature, pH, and NaCl concentration, on the activity of two histidine decarboxylases that were isolated and separated by gel chromatography from cell-free extracts of P. phosphoreum. The histidine decarboxylase activity of the cell-free extracts was highest in 7 degrees C culture; in 5% NaCl, culture growth was inhibited, and growth was best in the culture grown at pH 6.0. Moreover, percent activity of the constitutive and inducible enzymes was highest for the inducible enzyme in cultures grown at 7 degrees C and pH 7.5 and in 5% NaCl. The temperature and pH dependences of histidine decarboxylase differed between the constitutive and inducible enzymes; that is, the activity of histidine decarboxylases was optimum at 30 degrees C and pH 6.5 for the inducible enzyme and 40 degrees C and pH 6.0 for the constitutive enzyme. The differences in the temperature and pH dependences between the two enzymes extended the activity range of histidine decarboxylase under reaction conditions. On the other hand, histidine decarboxylase activity was optimum in 0% NaCl for the two enzymes. Additionally, the effects of reaction temperature, pH, and NaCl concentration on the constitutive enzyme activity of the cell-free extracts were almost the same as those on the whole histidine decarboxylase activity of the cell-free extracts, suggesting that the constitutive enzyme activity reflected the whole histidine decarboxylase activity.

Cloning and nucleotide sequence analysis of the chloramphenicol resistance gene on conjugative R plasmids from the fish pathogen Photobacterium damselae subsp. piscicida.

Transferable resistance to various drugs was investigated in Photobacterium damselae subsp. piscicida from Japan. Drug resistances were transferred via plasmids of 100, 50, and 40 kb. Resistance to chloramphenicol (Cmr) was transferred on plasmids of all 3 sizes. The Cmr gene (cat) was cloned from the 50 kb plasmids pPDP8511 and pPDP9106 transferred from P. damselae subsp. piscicida strains isolated in different years and places in Japan. Subcloning localized the cat to within 1.5 kb HindIII-HincII (or PstI) fragments. Nucleotide sequences of the coding and flanking region of the cat were determined as 1607 bp (HindIII-HincII fragment) in pPDP8511 and 1568 bp (HindIII-PstI fragment) in pPDP9106, which corresponded with the sequence from nucleotides 40 to 1607 in pPDP8511. The nucleotide sequences identified an open reading frame (ORF) encoding 213 amino acid residues with a calculated molecular mass of about 24.8 kDa, a size consistent with the molecular mass of known cat gene products, and the ORF had maximum homology (99.5%) with a Type II CAT variant from Haemophilus influenzae.