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.

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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.

In order to determine the mechanism of histamine (Hm) formation in scombroid fish stored at low temperature, total viable and luminous bacterial counts (TVC and LBC), bacterial flora, Hm contents in several parts of mackerel stored in ice and at the temperature of ice , and Hm formation by the isolates were studied.In the first experiment (Exp. I), Hm was formed in the muscles during storage at the temperature of ice but not in ice. Hm content was far higher in the ventral muscles than in the dorsal muscles . P. phosphoreum grew in the abdominal walls in both storages (ca. 81% in LBC/TVC), and in the muscles during storage at the temperature of ice (ca. 2.5% in LBC/TVC). P. phosphoreum isolated formed a high level of Hm. In the second experiment (Exp. II), a small amount of Hm was formed in the muscles, especially the ventral muscles, and P. phosphoreum was not found in fish before and after storage. These results indicate that P. phosphoreum plays an exclusive role in histamine formation in mackerel stored at the temperature of ice.Both whole and halophilic bacterial floras were dominated by Vibrio in all the parts (skin, muscle, abdominal walls) of fish in Exp. I and by Pseudomonas or Moraxella in Exp. II. Photobacterium leiognathi was found in all the parts of fish before storage in Exp. I but not in Exp. II, and P. phosphoreum also grew only in fish after storage in Exp. I as above. Pseudomonas or Moraxella dominated also in Exp. I after growth of P. phosphoreum. These results suggest that Photobacterium grows in the flora dominated by Vibrio, especially halophilic floras, and hardly grows in the flora dominated by Pseudomonas or Moraxella.