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Dna sequence of genes involved in histamine production

Histamine is the biogenic amine the most frequently involved in food intoxications. It is produced in fermented beverages and foods by strains of lactic acid bacteria (LAB) that belong to diverse species. All these strains share a similar enzyme, the histidine decarboxylase (HDC) catalysing the conversion of histidine to histamine. Genes coding for the HDC of some LAB were identified during the past two decades.

However, it was not known if they were associated with other genes. We have sequenced a 5732 bp DNA region surrounding the HDC gene of the wine LAB Lactobacillus hilgardii IOEB 0006. Four complete open reading frames were detected in this sequence. They code for the HDC, a protein of unknown function, a putative histidyl-tRNA synthetase and a membrane transporter. A biochemical characterisation of the transporter was performed in the Laboratory of Molecular Microbiology of the University of Groningen. It was demonstrated that this enzyme is a histidine/histamine exchanger.

The combination of the HDC and the histidine/histamine exchanger forms a typical amino acid decarboxylation system involved in metabolic energy production and/or acid stress resistance. The role of the two other proteins is unknown but it is supposed that they play a role in the histamine-producing pathway. Further works have shown that the four genes of this pathway are located on a large plasmid of L. hilgardii. This plasmid was unstable since it could be lost by LAB grown in specific culture media.

To determine if this histamine-producing pathway was conserved in other LAB of wine and fermented foods, we have analysed five other LAB strains producing histamine and belonging to different species: Oenococcus oeni IOEB 9204 from wine, Lactobacillus 30a from horse digestive tract, Lactobacillus buchneri DSM 5987 from cheese, Lactobacillus sakei LTH 2076 from sauerkraut and Tetragenococcus muriaticus LMG 18498 from squid liver sauce.

A similar four-gene cluster was detected in all the strains except in L. 30a in which the gene coding for the histidyl-tRNA synthetase was apparently missing. As found in L. hilgardii, the genes were located on a plasmid in L. sakei, T. muriaticus and O. oeni. In contrast, they were detected on the chromosome in L. buchneri and L. 30a. These differences of genetic locations may be important for the stability and the dissemination of the histamine-producing pathway.

Sequencing of the histamine decarboxylase cluster of Lactobacillus buchneri B301.
To characterize the genes responsible for histamine synthesis in L. buchneri B301, a PCR reaction was performed using the oligonucleotides JV16HC and JV17HC (le Jeune et al., 1995). The sequence of the obtained amplicon was similar to those of hdcA genes found in databases; it may therefore encode HdcA. This 0.3 kb internal hdcA fragment was located in the L. buchneri chromosome. A reverse PCR strategy was designed to reveal the complete sequence of hdcA gene and the flanking regions. A 5,775 bp DNA fragment was sequenced.

The sequence analysis showed the presence of four complete ORFs: hdcA. Analysis of the amino acid sequence showed the protein can be included in the group of bacterial histidine decarboxylases that use a covalently bound pyruvoyl residue as a prosthetic group. hdcB. Downstream of hdcA, a second ORF was found in the same orientation. Comparisons of the deduced amino acid sequence with those present in databases revealed similarities of 89 %, 81 % and 69 % with HdcB of Tetragenococcus muriaticus, O. oeni, and Lactobacillus 30A, respectively. Some authors have associated this protein with the regulation of the operon or transport.

However, the protein appears to have no transmembrane motifs. hisS. Downstream of hdcB, another ORF was found in the same DNA strand. The deduced protein showed similarity to the class II histidyl-tRNA synthetases. As in other aminoacyl-tRNA synthetases, the region upstream of the start codon of hisS contains a putative promoter region and a leader region with the sequence features of the tRNA-mediated anti-termination system. hdcC. Upstream of hdcA an additional ORF was identified in the same orientation; this was designated hdcC. Analyses of HdcC hydropathy predicted 11 hydrophobic segments long enough to form a transmembrane helix.

To determine whether the product of the hdcC gene was a membrane protein, as suggested by sequence analysis, it was expressed in Lc. lactis NZ9000 under the control of the nisin promoter. The results allow the conclusion that the protein encoded by hdcC is located in the membrane. This result, together with the sequence data, suggests that hdcC could be responsible for histamine/histidine exchange in L. buchneri. To our knowledge, this is the first time such a gene has been described.

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Faculty of Enology of Bordeaux 2 University
351, Cours de la Libération
33405 Talence
France
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