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Applied and Environmental Microbiology, July 2002, p. 3537-3544, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3537-3544.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of the Enterococcus faecalis Tyrosine Decarboxylase Operon Involved in Tyramine Production

Nathalie Connil,1 Yoann Le Breton,2 Xavier Dousset,1 Yanick Auffray,2 Alain Rincé,2 and Hervé Prévost1*

Laboratoire de Microbiologie Alimentaire et Industrielle, ENITIAA, 44322 Nantes Cedex 03,1 Laboratoire de Microbiologie de l'Environnement, EA 956, USC INRA, IRBA, Université de Caen, 14032 Caen Cedex, France2

Received 27 November 2001/ Accepted 10 April 2002


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ABSTRACT
 
Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 µg ml-1 for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes.


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INTRODUCTION
 
Biogenic amines in food result mainly from microbial activity due to amino acid decarboxylation (16, 49). Histamine and tyramine have been the most studied biogenic amines due to the toxicological effects derived from their vasoactive and psychoactive properties. Histamine has been recognized as the causative agent of scromboid poisoning (histamine intoxication), whereas tyramine has been related to food-induced migraines and hypertensive crisis (39). Various tyramine concentrations have been found in many foods, including cheeses, drinks, and meat and fish products (30, 44, 45), and a dose of only 6 mg total tyramine intake may be dangerous for patients under antidepressive treatment who are receiving monoamine oxidase inhibitors (42). The formation of tyramine in foods depends on the concentration of free tyrosine and the presence of microorganisms having tyrosine decarboxylase activity. Many microorganisms could be implicated in tyramine production. For example, some bacteria belonging to the genera Enterococcus, Carnobacterium, and Lactobacillus have been found to be tyramine producers (5, 31, 35). However, while tyrosine decarboxylase enzymes have been well characterized in eukaryotes, for example, in parsley (Petroselinum crispum) and in Drosophila (24, 47), little is known about tyrosine decarboxylase in prokaryotes. Indeed, only a few reports have described physiological studies of the influence of some physicochemical factors, such as temperature, pH, NaCl, or tyrosine concentration, on tyramine production by Lactobacillus curvatus (46), Lactobacillus brevis (35), and Carnobacterium divergens (32). Tyrosine decarboxylase purification and characterization of the enzyme have been reported only for Enterococcus faecalis (previously called Streptococcus faecalis) (4) and for L. brevis IOEB 9809 and ATCC 367 (34, 36). These authors have shown that tyrosine decarboxylases in E. faecalis and L. brevis have an [{alpha}2] dimmer structure with two subunits of approximately 75 kDa for E. faecalis and 70 kDa for L. brevis. Furthermore, pyridoxal phosphate was necessary for activity, showing that these decarboxylases belong to the group of pyridoxal phosphate-dependent enzymes. Glutamate, lysine, and histidine decarboxylase genes have been described in various bacteria. However, to our knowledge, no data exist about the gene encoding tyrosine decarboxylase in prokaryotes.

The aim of this study was to isolate and characterize the gene encoding tyrosine decarboxylase in E. faecalis JH2-2. For this purpose, a library of E. faecalis JH2-2 insertional mutants was screened for mutations affecting tyramine production. Isolation and characterization of a tyrosine decarboxylase mutant allowed the identification and genetic analysis of the tyrosine decarboxylase determinants of E. faecalis JH2-2.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and culture conditions.
The present study was performed using the E. faecalis strain JH2-2 (57), which was obtained from the parental strain JH2 (22). E. faecalis was grown at 37°C in M17 medium supplemented with 0.5% glucose (GM17) (50). When necessary, the antibiotics erythromycin and chloramphenicol were used at 150 and 20 µg ml-1, respectively. Modified Maijala decarboxylation broth (29) containing 2 g of tyrosine per liter was used for screening tyrosine decarboxylase mutants in microtiter plates. Escherichia coli strain EC101 containing the repA gene for replication of pWV01-type plasmids (26) was grown in Luria-Bertani medium (40) with 100 µg of tetracycline or with 150 µg of erythromycin ml-1 to maintain the pORI19 plasmid and derivatives (26). Plasmid pG+host3 (previously named pVE6007) (28), encoding a thermosensitive RepA protein, was maintained in E. coli TG1 (Stratagene) at 30°C with 10 µg of chloramphenicol ml-1.

Isolation of insertional mutants of E. faecalis JH2-2.
The library of insertional mutants of E. faecalis JH2-2 used in this study was constructed with the strategy described by Law et al. (26) for Lactococcus lactis. Briefly, 200-bp to 1.5-kb chromosomal DNA fragments generated by partial digestion with endonuclease AluI were cloned into the pORI19 plasmid (pWV01 derived; Ori+ RepA-) using the RepA+ helper E. coli strain EC101 (26) to obtain a bank of approximately 37,200 recombinant plasmids. A mixture of these recombinant plasmids was then transferred into E. faecalis JH2-2, which had previously received the pWV01-derived Ori+ RepATS pG+host3 plasmid (28). Clones were grown at 30°C in GM17 medium containing erythromycin and chloramphenicol (the thermosensitive RepATS protein is active at 30°C and allowed replication of the pG+host3 and pORI19 recombinant plasmids). The cells were then transferred to GM17 containing erythromycin, and the incubation temperature was shifted to 42°C to inactivate the RepATS protein and consequently occasion the loss of pG+host3 and the integration of the pORI19 recombinant plasmid by homologous recombination.

Excision and curing of integrated plasmids.
For sequencing experiments, excision of the plasmids integrated in mutants 16G10 and 16G12 was performed by transformation with pG+host3 and selection of the transformants at 30°C on GM17 plates with chloramphenicol and erythromycin. At this permissive temperature, the active RepATS protein allows the replication of both plasmids and thus favors the excision of the pORI19 recombinant plasmid. For curing of the integrated plasmid from mutant 16G10, transformants were selected at 30°C on GM17 plates with chloramphenicol, grown for 100 generations at 30°C on GM17 broth containing chloramphenicol, and then grown for 1 h at 30°C in GM17 without antibiotics and transferred at 42°C for 3 h before being plated in the same medium and incubated at 42°C. One of the 11% of the isolated clones was found to be sensitive to erythromycin. It was tested for its tyrosine decarboxylase activity using high-performance liquid chromatography (HPLC) quantification.

Extraction of chromosomal DNA.
Chromosomal DNA of E. faecalis was isolated from a 3-ml culture of strain JH2-2 as follows. Cells in stationary phase were harvested by centrifugation, resuspended in 0.5 ml of lysozyme (5 mg ml-1), and incubated for 5 min at 37°C. Then, 20 µl of proteinase K (2%) and 25 µl of a 10% sodium dodecyl sulfate (SDS) solution were added, and lysis was obtained after incubation at 60°C. DNA was then prepared as described by Sambrook et al. (40) and resuspended in 10 mM Tris-HCl-1 mM EDTA (pH 7.5) containing 500 µg of RNase ml-1.

Southern blot hybridization.
For Southern blot analysis, chromosomal DNA digested with restriction enzymes was electrophoresed on a 0.8% agarose gel and transfered to a Hybond-N+ membrane (Amersham) by capillary transfer as recommended by the membrane manufacturer. Membrane-bound nucleic acids were hybridized at a temperature of 55°C in 1 M sodium phosphate buffer (pH 7.0) containing 5% SDS with a 32P-labeled probe. After hybridization, the membranes were washed twice successively in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS (10 min) and twice in 0.5x SSC-0.1% SDS (10 min) at 55°C and exposed to a storage phosphor screen (Packard Instrument Company, Canberra, Australia) for 5 h.

Preparation of the double-stranded labeled probe was carried out by PCR using E. faecalis chromosomal DNA as a template; primers F4S1 and F3S2; Taq DNA polymerase; 2 µM (each) dCTP, dGTP, and dTTP; and 2 µCi of [{alpha}-32P]dATP.

DNA sequencing.
DNA sequencing was performed on PCR fragments purified with the QIAquick kit (Qiagen) and using the dideoxy chain termination method (41) with the ABI prism sequencing system (PE Biosystems). To eliminate the eventual effects of Taq DNA polymerase mistakes, a mix of three independent amplifications was sequenced.

Tyramine quantification by HPLC.
The growth and tyramine production of E. faecalis JH2-2 and the 16G10 mutant were studied in Maijala broth as follows. Maijala broth was inoculated at 107 CFU ml-1 with overnight precultures of E. faecalis JH2-2 and the 16G10 mutant in GM17. The cells were enumerated on GM17 agar for 24 h and simultaneously analyzed by HPLC for tyramine production. For this, cultures were centrifuged at 8,000 x g for 10 min, and the resulting supernatants were dansylated as previously described by Eerola and Hinkannen (13). Dansylated samples were analyzed on a C18 ODS2 Equisorb column (4.6 by 250 mm; particle size, 5 µm) mounted with a guard column and a Constametric apparatus (Ldc; Milton Roy). The elution gradient began with 45% 0.1 M ammonium acetate and 55% acetonitrile and ended at 10% 0.1 M ammonium acetate and 90% acetonitrile in 18 min at 40°C under a flow rate of 1 ml min-1. Dansylated tyramine was detected by UV absorption at 254 nm and was quantified using a calibration curve.

Analysis of mRNAs by Northern blotting and reverse transcription (RT)-PCR experiments.
Total RNA of E. faecalis JH2-2 was isolated from exponentially growing cells in Maijala broth by using the RNeasy Midi kit (Qiagen). Northern blots of exactly 10 µg of electrophoresed RNA were prepared by using Hybond-N+ membranes and standard procedures (40). The sizes of the transcripts were estimated by comparing band mobility with RNA ladder standards (0.56 to 9.4 kb) (Amersham International, Little Chalfont, United Kingdom). Membrane-bound nucleic acids were hybridized with single-stranded labeled probes as described for Southern blot hybridization. Preparation of the single-stranded labeled probes was as follows. First, a DNA fragment was amplified by PCR using chromosomal DNA of E. faecalis JH2-2 and primers F2S1 and F2S2 (Table 1) for ORF1, primers F5S1 and F5S2 for the tdc gene, and F8S1 and F8S2 for ORF3. Then, the probes were synthesized by elongation of the oligonucleotide F2S2, F5S2, or F8S2 using Taq DNA polymerase; 2 µM (each) dCTP, dGTP, and dTTP; 2 µCi of [{alpha}-32P]dATP; and 10 ng of the previously obtained PCR DNA fragments as a template. Thirty cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C were performed.


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  		TABLE 1. Primers used for PCR and sequencing experiments

For RTs, 10 µg of the RNA preparation was treated for 30 min at 37°C with 20 U of RNase-free DNase I (Roche Molecular Biochemicals, Indianapolis, Ind.) followed by a phenol-acid extraction (at 65°C). Reverse transcriptase reactions were performed at 55°C using the 3'/5' RACE kit (Roche Molecular Biochemicals) with 12.5 pmol of primer F2S2, F5S2, or F8S2 and 1 µg of RNA. The cDNAs were then purified with the QIAquick kit (Qiagen). RT-PCRs were performed using purified cDNA as a template, 20 pmol of primers, and Ready To Go PCR beads (Pharmacia Biotech). Amplifications were performed for 30 cycles consisting of 94°C for 30 s, 55°C for 30 s, and 72°C for 210 s, and the samples were analyzed on a 0.8% agarose gel in TAE (40 mM Tris-acetate [pH 8.0], 1 mM EDTA) buffer. The absence of contaminating genomic DNA was controlled by non-reverse-transcribed PCR performed under the same conditions, except that avian myeloblastosis virus reverse transcriptase was replaced by H2O.

General molecular methods.
Restriction endonucleases were obtained from Amersham International. PCRs were carried out with 5 µg of chromosomal DNA of E. faecalis JH2-2 and 20 pmol of primers using Ready To Go PCR beads (Pharmacia Biotech). E. coli and E. faecalis were transformed using a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, Calif.) as described by Dower et al. (11) and Holo and Nes (19), respectively. DNA and amino acid sequences were analyzed using the Mac Vector program (Kodak Scientific Imaging Systems), database searches were performed with the BLAST program (1), and amino acid sequence alignments were carried out with Clustal W (51). Other standard techniques were carried out as described by Sambrook et al. (40).

Nucleotide sequence accession number.
The DNA sequence of the tyrosine decarboxylase operon determined in this paper has been submitted to the EMBL, GenBank, and DDBJ nucleotide databases under accession number AF354231.


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RESULTS
 
Isolation of E. faecalis JH2-2 tyrosine decarboxylase mutants.
A library of 9,600 insertional mutants of E. faecalis JH2-2 constructed with the strategy described in Materials and Methods was screened for tyrosine decarboxylase mutants. Cultures of individual colonies were grown at 37°C in 96-well microtiter plates in GM17 broth containing erythromycin and replica plated in Maijala broth. Tyramine producer colonies led to a basification of Maijala broth (purple coloration), whereas wells containing mutants unable to produce tyramine remained green. After a 48-h incubation at 37°C, only two clones had the tyramine-negative phenotype. They were named 16G10 and 16G12. As a control, when Maijala broth was supplemented with 2 g of arginine per liter instead of tyrosine, basification of the media was still observed with the two mutants (data not shown).

Genomic characterization of the E. faecalis tyrosine decarboxylase mutants.
Integrated plasmids in mutants 16G10 and 16G12 were excised as described in Materials and Methods. DNA sequence analysis revealed that plasmids excised from both mutants contained identical 664-bp DNA inserts, so further characterizations were performed only with the 16G10 mutant. Comparison with the E. faecalis (V583) genomic sequence (available at http://www.tigr.org) revealed that this 664-bp segment corresponded to a central region of a 1,860-bp open reading frame (ORF), thereafter named tyrosine decarboxylase (tdc).

Oligonucleotides designed from the E. faecalis V583 genome (Table 1) were used to verify the integration locus of the 16G10 mutant and to sequence the corresponding gene and adjacent ORF from E. faecalis JH2-2. A Southern blot of total DNA extracted from the 16G10 mutant and from the wild-type JH2-2 strain and digested with EcoRI or KpnI plus SacI was hybridized with a 32P-labeled PCR DNA fragment corresponding to the 5' extremity of the tdc gene (Fig. 1). The results revealed bands whose sizes differed between the wild-type and the mutant strain and demonstrated that the integration occurred via homologous recombination between the genome and the insert of the recombinant plasmid. A 5,094-bp nucleotide sequence of the E. faecalis JH2-2 tdc gene and the adjacent ORF was determined from PCR products and deposited in the EMBL, GenBank, and DDBJ nucleotide databases.



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  		FIG. 1. Southern blot hybridization of EcoRI (lanes 1 and 2)- or KpnI plus SacI (lanes 3 and 4)-digested chromosomal DNA of E. faecalis JH2-2 (lanes 1 and 3) and an E. faecalis tdc mutant (lanes 2 and 4) with the F4S1-F3S2 DNA fragment as a probe. Migration of DNA fragments of the molecular size marker Smartladder (Eurogentec) is indicated on the left.

Sequence analysis.
Observation of the nucleotide sequence immediately upstream of the E. faecalis JH2-2 tdc ORF gave evidence for the presence of a ribosome binding site sequence (GGAGG) located 9 bp upstream of the initiation codon (ATG). Translation of the entire ORF revealed that it encodes a 620-amino-acid protein of 70.0 kDa having homologies with decarboxylases (Fig. 2 ). The best homology scores were observed with the probable glutamate decarboxylase of Vibrio cholerae (24% identity; 40% similarity [17]). Furthermore, the tyrosine decarboxylase sequence of E. faecalis JH2-2 contained the consensus pattern observed for pyridoxal phosphate-dependent decarboxylases (also known as group II decarboxylases,S-[LIVMFYW]-X(5)-K-[LIVMFYWGH]-[LIVMFYWG]-X(3)-[LIVMFYW]-X-[CA]-X(2)-[LIVMFYWQ]-X(2)-[RK] (http://www.expasy.ch/cgi-bin/nicedoc.pl?PDOC00329), wherelysine (K) is the attachment site for the cofactor pyridoxal phosphate. However, in our case, the ultimate amino acid of the consensus pattern would not be arginine (R) or lysine but glutamine (Q) (Fig. 2). Furthermore, E. faecalis JH2-2 Tdc has 20% identity with the tyrosine decarboxylase II sequence from parsley (P. crispum), including the VHVDAAYmotif (Fig. 2) particularly conserved in pyridoxal phosphate-dependent decarboxylases (24). Upstream of the tdc gene, we can find an ORF (ORF1) corresponding to a 47.3-kDa protein having high homology with tyrosyl tRNA synthetases (54% identity and 72% similarity with the tyrosyl tRNA synthetase of Bacillus subtilis [18]). Tyrosyl tRNA synthetases are aminoacyl-tRNA synthetases of class I characterized by the HIGH and KMSKS amino acid motifs (14) involved in an ATP-dependent activation of tyrosine by forming an enzyme-bound tyrosyl-adenylate intermediate (56). The HIGH motif is perfectly conserved in E. faecalis tyrosyl tRNA synthetase, while the KMSKS motif is represented, as in E. coli and B. subtilis tyrosyl tRNA synthetases (3, 18), by the KFGKT sequence. The ORF located downstream of tdc (ORF3) encoded a 51.6-kDa protein with 12 hydrophobic fragments (data not shown). Homologies were found between the amino acid sequence deduced from ORF3 and proteins encoding amino acid antiporters (28% identity and 49% similarity with an amino acid antiporter of Chlamydia pneumoniae [38]).



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  		FIG. 2. Amino acid sequence comparison of E. faecalis Tdc (Tdc Ef) with the probable glutamate decarboxylases from V. cholerae (Gad Vc [17]) and Methanothermobacter thermautotrophicus (Gad Mt [43]); decarboxylases from Pyrococcus abyssi (Dec Pa; accession number NP 126894), Pyrococcus horikoshii (Dec Ph [25]), and Methanococcus jannaschii (Dec Mj [7]); and L-2,4-diaminobutyrate decarboxylases from Bacillus halodurans (Ddc Bh [48]) and from Haemophilus influenzae (Ddc Hi [15]). Amino acid residues that are shared by at least four proteins are shaded. An underlined number in a sequence indicates the number of amino acids omitted at that position to save space. The boxes represent the VHVDAAY motif and the pyridoxal phosphate attachment site.

Growth and tyramine quantification of E. faecalis JH2-2 and the 16G10 mutant.
The growth of E. faecalis JH2-2 and the 16G10 mutant in Maijala broth followed similar patterns (Fig. 3). Bacterial populations increased from 107 CFU ml-1 to 109 CFU ml-1 in 24 h. Whereas the tyramine production of E. faecalis JH2-2 rose to reach approximately 1,000 µg ml-1 in 24 h and the Maijala broth turned purple, no tyramine production was detected by HPLC for the 16G10 mutant, and the Maijala broth remained green (Fig. 3). To show that the tyramine production defect in mutant 16G10 was due to the plasmid insertion and not to an independent genomic mutation, the 16G10 mutant was cured from its pORI19-recombinant plasmid as described in Materials and Methods. This resulted in the restoration of tyramine production similar to that of the wild-type strain (data not shown).



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  		FIG. 3. Growth and tyramine production of E. faecalis JH2-2 and the 16G10 mutant in Maijala broth. Squares, JH2-2; circles, 16G10 mutant; solid symbols, log of UFC numbers; open symbols, tyramine concentration in supernatant.

Transcriptional analysis.
Northern blot hybridization of RNA extracted from exponentially growing cells in Maijala broth with the single-stranded F2S2-F2S1 probe (tyrosyl tRNA synthetase gene) revealed a 1.4-kb mRNA (Fig. 4A). The size of this mRNA corresponded to the transcription of the tyrosyl tRNA synthetase gene alone, and the 3' extremity of the transcript agreed with the presence of an inverted repeat 100 bp downstream of ORF1 ({Delta}G = -29.6 kcal/mol) which could act as a Rho-independent terminator. mRNAs corresponding to the tdc gene and to ORF3 were not visualized when the single-stranded probes F5S2-F5S1 or F8S2-F8S1 were used (data not shown), suggesting that theses genes are less transcribed or that their corresponding transcripts are less stable.



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  		FIG. 4. Transcriptional analysis. (A) Northern blot hybridization of RNA isolated from exponentially growing E. faecalis JH2-2 cells in Maijala broth. Hybridization was performed with the F2S1-F2S2 single-stranded labeled probe. The size of the transcript was estimated by comparing the band mobility with those of standards in an RNA ladder (0.56 to 9.4 kb) (Amersham International). (B) RT-PCR assays conducted on mRNA isolated from exponentially growing E. faecalis JH2-2 cells in Maijala broth. RTs were performed with oligonucleotides F2S2 for lanes 1 and 2, F5S2 for lanes 3 to 6, and F8S2 for lanes 7 to 10. To ensure the absence of genomic DNA, negative controls were performed without reverse transcriptase (even lanes). PCRs were performed with primers F2S1 and F2S2 (lanes 1 and 2), F3S1 and F5S2 (lanes 3 and 4), F5S1 and F5S2 (lanes 5 and 6), F3S1 and F8S2 (lanes 7 and 8), or F6S1 and F8S2 (lanes 9 and 10). Lanes M contain DNA fragments of the molecular weight marker Smartladder (Eurogentec).

RT-PCR experiments confirmed the transcription of the tyrosyl tRNA synthetase gene (Fig. 4B, lane 1) and provided evidence for the transcription of the tyrosine decarboxylase gene and the putative amino acid antiporter gene (Fig. 4B, lanes 3, 5, 7, and 9). When the cDNA from RT with primer F8S2 was amplified using primers F6S1 and F8S2, a large amount of amplimer was obtained (Fig. 4B, lane 9), showing that the tdc gene and the putative amino acid antiporter (ORF3) are cotranscribed. Moreover, observation of PCR products with primers F3S1 and F5S2 on cDNA from RT with F5S2 and with primers F3S1 and F8S2 on cDNA from RT with F8S2 (Fig. 4B, lanes 3 and 7, respectively), indicated that the tdc gene is also cotranscribed with the tyrosyl tRNA synthetase gene and that the three genes are cotranscribed. The existence of a cotranscription between the tyrosyl tRNA synthetase gene and genes encoding the tyrosine decarboxylase and the putative amino acid antiporter could be explained by an incomplete action of the putative Rho-independent terminator located between ORF1 and tdc.


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DISCUSSION
 
In order to identify the gene encoding tyrosine decarboxylase in E. faecalis, a library of E. faecalis JH2-2 insertional mutants was screened for mutations affecting tyramine production. Two mutants unable to produce tyramine were isolated, and their genetic characterization revealed a unique integration locus corresponding to the central region of a 1,860-bp ORF (tdc). Comparison of tyramine production during the growth of the wild-type E. faecalis JH2-2 strain and of one mutant (16G10) confirmed the absence of tyramine production for the mutant and showed that the wild-type E. faecalis JH2-2 strain is able to produce up to 1,000 µg of tyramine ml-1 after 24 h of culture in Maijala broth. The amount of tyramine produced by the wild-type strain E. faecalis JH2-2 is in agreement with the results obtained by Bover-Cid and Holzapfel (5), who found values from 601 to 4,986 µg ml-1 for different E. faecalis strains. In the wild-type strain, tyramine production seems to be correlated with growth and may protect the bacteria from acidification.

Genetic characterization of the integration locus revealed that the 1,860-bp ORF encoded a decarboxylase (tyrosine decarboxylase) which had homology with amino acid decarboxylases from prokaryotes and tyrosine decarboxylase from parsley (P. crispum) (24). Furthermore, the presence of a motif corresponding to the attachment site of the pyridoxal phosphate cofactor showed that the E. faecalis tyrosine decarboxylase belongs to the group of pyridoxal phosphate-dependent decarboxylases, which includes decarboxylases from eukaryotes and prokaryotes (21). To our knowledge, this study is the first description of a tyrosine decarboxylase gene in prokaryotes.

In Salmonella enterica serovar Typhimurium and E. coli, tyrosine is transported by a specific permease (a tyrP gene product) (55) and by the general aromatic amino acid transport system (an aroP gene product) (2, 6). Here, transcriptional analyses revealed that the tyrosine decarboxylase gene is cotranscribed with a gene encoding a potential amino acid antiporter, the latter probably being involved in tyrosine internalization. In E. coli, the lysine-cadaverine antiport cadB gene and the lysine decarboxylase cadA gene belong to the same operon, cadBA, and are cotranscribed under the control of the promoter Pcad (37). Recently, in Listeria monocytogenes and in E. coli, the glutamate decarboxylase gene, gadB, was also shown to be cotranscribed with gadC, a gene encoding a potential glutamate/{gamma}-aminobutyrate antiporter (the {gamma}-aminobutyrate is the product of glutamate decarboxylation) (8, 10). In Lactococcus lactis subspecies lactis, arginine is transported by an antiport system with ornithine (12), and in E. coli, the potE gene encodes a putrescine-ornithine antiporter (23). An antiport histidine-histamine has also been described in Lactobacillus buchneri (33). Similarly, in E. faecalis, the amino acid transporter encoded by the gene cotranscribed with tdc could be a tyrosine-tyramine antiporter.

The first tyrosine decarboxylase genetic sequence described here for a prokaryote organism (E. faecalis) could have an important impact. Indeed, it could be used to design primers that will allow fast and easy detection of tyramine producer strains in foods. Le Jeune et al. (27) compared the nucleotide sequences of histidine decarboxylase genes (hdcA) from Lactobacillus sp. strain 30A (52) and Clostridium perfringens (53) and compared the amino acid sequences of these histidine decarboxylases with those from L. buchneri (20) and Micrococcus (54). As alignment studies showed a high degree of relatedness among the hdcA gene products of gram-positive bacteria, these authors proposed PCR primers and genomic probes for identification of histidine-decarboxylating bacteria and tested them in numerous bacteria. Then, the primers were used to study efficiently the frequency and the distribution of histidine-decarboxylating bacteria in wines produced in southwest France (9). When more prokaryotic tyrosine decarboxylase genes are characterized, it should be possible to design primers in conserved regions for rapid detection of tyrosine-decarboxylating bacteria in foods. In this way, it may be possible in the future to prevent the toxicological effect of high tyramine consumption in food.


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ACKNOWLEDGMENTS
 
We are grateful to C. J. Leenhouts (Department of Genetics, University of Groningen, Groningen, The Netherlands) for providing us with the pORI19 plasmid and E. coli strain EC101 and E. Maguin (INRA, Jouy-en-Josas, France) for the plasmid pG+host3.


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FOOTNOTES
 
* Corresponding author. Mailing address: LMAI, ENITIAA, rue de la Géraudière, BP82225, 44322 Nantes Cedex 03, France. Phone: 33 2 51 78 55 24. Fax: 33 2 51 78 55 20. E-mail: prevost{at}enitiaa-nantes.fr. Back


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REFERENCES
 
    1
  1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.[CrossRef][Medline]
    2. 2
  2. Ames, G. F. 1964. Uptake of amino acids by Salmonella typhimurium. Arch. Biochem. Biophys. 104:1-18.[CrossRef][Medline]
    3. 3
  3. Barker, D. G., C. J. Bruton, and G. Winter. 1982. The tyrosyl-tRNA synthetase from Escherichia coli. Complete nucleotide sequence of the structural gene. FEBS Lett. 150:419-423.[CrossRef][Medline]
    4. 4
  4. Borresen, T., N. K. Klausen, L. M. Larsen, and H. Sorensen. 1989. Purification and characterisation of tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase. Biochim. Biophys. Acta 993:108-115.[Medline]
    5. 5
  5. Bover-Cid, S., and W. H. Holzapfel. 1999. Improved screening procedure for biogenic amine production by lactic acid bacteria. Int. J. Food Microbiol. 53:33-41.[CrossRef][Medline]
    6. 6
  6. Brown, K. D. 1970. Formation of aromatic amino acid pools in Escherichia coli K12. J. Bacteriol. 104:177-188.[Abstract/Free Full Text]
    7. 7
  7. Bult, C. J., O. White, G. J. Olsen, L. Zhou, R. D. Fleischmann, G. G. Sutton, J. A. Blake, L. M. FitzGerald, R. A. Clayton, J. D. Gocayne, A. R. Kerlavage, B. A. Dougherty, J.-F. Tomb, M. D. Adams, C. I. Reich, R. Overbeek, E. F. Kirkness, K. G. Weinstock, J. M. Merrick, A. Glodek, J. D. Scott, N. S. Geoghagen, J. F. Weidman, J. L. Fuhrmann, D. T. Nguyen, T. Utterback, J. M. Kelley, J. D. Peterson, P. W. Sadow, M. C. Hanna, M. D. Cotton, M. A. Hurst, K. M. Roberts, B. B. Kaine, M. Borodovsky, H. P. Klenk, C. M. Fraser, H. O. Smith, C. R. Woese, and J. C. Venter. 1996. Complete genome sequence of the methanogenic archaeon Methanococcus jannaschii. Science 273:1058-1073.[Abstract]
    8. 8
  8. Castanie-Cornet, M. P., and J. W. Foster. 2001. Escherichia coli acid resistance: cAMP receptor protein and a 20 bp cis-acting sequence control pH and stationary phase expression of the gadA and gadBC glutamate decarboxylase genes. Microbiology 147:709-715.[Abstract/Free Full Text]
    9. 9
  9. Coton, E., G. Rollan, A. Bertrand, and A. Lonvaud-Funel. 1998. Histamine-producing lactic acid bacteria in wine: early detection, frequency, and distribution. Am. J. Enol. Vitic. 49:199-204.[Abstract/Free Full Text]
    10. 10
  10. Cotter, P. D., C. G. Gahan, and C. Hill. 2001. A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid. Mol. Microbiol. 40:465-475.[CrossRef][Medline]
    11. 11
  11. Dower, J. W., J. F. Miller, and C. W. Ragsdale. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16:6127-6145.[Abstract/Free Full Text]
    12. 12
  12. Driessen, A. J., D. Molenaar, and W. N. Konings. 1989. Kinetic mechanism and specificity of the arginine-ornithine antiporter of Lactococcus lactis. J. Biol. Chem. 264:10361-10370.[Abstract/Free Full Text]
    13. 13
  13. Eerola, S., and R. Hinkannen. 1993. Liquid chromatographic determination of biogenic amines in dry sausages. J. AOAC Int. 76:575-578.[Medline]
    14. 14
  14. Eriani, G., M. Delarue, O. Poch, J. Gangloff, and D. Moras. 1990. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 347:203-206.[CrossRef][Medline]
    15. 15
  15. Fleischmann, R. D., M. D. Adams, O. White, R. A. Clayton, E. F. Kirkness, A. R. Kerlavage, C. J. Bult, J.-F. Tomb, B. A. Dougherty, J. M. Merrick, K. McKenney, G. G. Sutton, W. FitzHugh, C. A. Fields, J. D. Gocayne, J. D. Scott, R. Shirley, L. I. Liu, A. Glodek, J. M. Kelley, J. F. Weidman, C. A. Phillips, T. Spriggs, E. Hedblom, M. D.Cotton, T. Utterback, M. C. Hanna, D. T. Nguyen, D. M. Saudek, R. C. Brandon, L. D. Fine, J. L. Fritchman, J. L. Fuhrmann, N. S. Geoghagen, C. L. Gnehm, L. A. McDonald, K. V. Small, C. M. Fraser, H. O. Smith, and J. C. Venter. 1995. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496-512.[Abstract/Free Full Text]
    16. 16
  16. Halasz, A., A. Barath, L. Simon-Sarkadi, and W. H. Holzapfel. 1994. Biogenic amines and their production by microorganisms in food. Trends Food Sci. Technol. 5:42-49.
    17. 17
  17. Heidelberg, J. F., J. A. Eisen, W. C. Nelson, R. A. Clayton, M. L. Gwinn, R. J. Dodson, D. H. Haft, E. K. Hickey, J. D. Peterson, L. A. Umayam, S. R. Gill, K. E. Nelson, T. D. Read, H. Tettelin, D. Richardson, M. D. Ermolaeva, J. Vamathevan, S. Bass, H. Qin, I. Dragoi, P. Sellers, L. McDonald, T. Utterback, R. D. Fleishmann, W. C. Nierman, O. White, S. L. Salzberg, H. O. Smith, R. R. Colwell, J. J. Mekalanos, J. C. Venter, and C. M. Fraser. 2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406:477-483.[CrossRef][Medline]
    18. 18
  18. Henkin, T. M., B. L. Glass, and F. J. Grundy. 1992. Analysis of the Bacillus subtilis tyrS gene: conservation of a regulatory sequence in multiple tRNA synthetase genes. J. Bacteriol. 174:1299-1306.[Abstract/Free Full Text]
    19. 19
  19. Holo, H., and I. F. Nes. 1995. Transformation of Lactococcus by electroporation. Methods Mol. Biol. 47:195-199.[Medline]
    20. 20
  20. Huynh, Q. K., and E. E. Snell. 1985. Pyruvoyl-dependent histidine decarboxylase. Preparation and amino acid sequences of the ß chains of histidine decarboxylase from Clostridium perfringens and Lactobacillus buchneri. J. Biol. Chem. 260:2798-2803.[Abstract/Free Full Text]
    21. 21
  21. Jackson, F. R. 1990. Prokaryotic and Eukaryotic pyridoxal dependent decarboxylases are homologous. J. Mol. Evol. 31:325-329.[CrossRef][Medline]
    22. 22
  22. Jacob, A. E., and S. J. Hobbs. 1974. Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J. Bacteriol. 117:360-372.[Abstract/Free Full Text]
    23. 23
  23. Kashiwagi, K., S. Miyamoto, F. Suzuki, H. Kobayashi, and K. Igarashi. 1992. Excretion of putrescine by the putrescine-ornithine antiporter encoded by the potE gene of Escherichia coli. Proc. Natl. Acad. Sci. USA 89:4529-4533.[Abstract/Free Full Text]
    24. 24
  24. Kawalleck, P., H. Keller, K. Hahlbrock, D. Scheel, and I. E. Somssich. 1993. A pathogen-responsive gene of parsley encodes tyrosine decarboxylase. J. Biol. Chem. 268:2189-2194.[Abstract/Free Full Text]
    25. 25
  25. Kawarabayasi, Y., M. Sawada, H. Horikawa, Y. Haikawa, Y. Hino, S. Yamamoto, M. Sekine, S. Baba, H. Kosugi, A. Hosoyama, Y. Nagai, M. Sakai, K. Ogura, R. Otsuka, H. Nakazawa, M. Takamiya, Y. Ohfuku, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, K. Aoki, Y. Nakamura, F. T. Robb, K. Horikoshi, Y. Masuchi, H. Shizuya, and H. Kikuchi. 1998. Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. DNA Res. 5:55-76.[Abstract]
    26. 26
  26. Law, J., G. Buist, A. Haandrikman, J. Kok, G. Venema, and K. Leenhouts. 1995. A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J. Bacteriol. 177:7011-7018.[Abstract/Free Full Text]
    27. 27
  27. Le Jeune, C., A. Lonvaud-Funel, B. ten Brink, H. Hofstra, and J. M. B. M. van der Vossen. 1995. Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test. J. Appl. Bacteriol. 78:316-326.[Medline]
    28. 28
  28. Maguin, E., T. Duwat, T. Hege, D. Ehrlich, and A. Gruss. 1992. New thermosensitive plasmid for gram-positive bacteria. J. Bacteriol. 174:5633-5638.[Abstract/Free Full Text]
    29. 29
  29. Maijala, R. L. 1993. Formation of histamine and tyramine by some lactic acid bacteria in MRS-broth and modified decarboxylation agar. Lett. Appl. Microbiol. 17:40-43.
    30. 30
  30. Mariné-Font, A., M. C. Vidal-Carou, M. Izquierdo-Pulido, M. T. Veciana-Nogués, and T. Hernandez-Jover. 1995. Les amines biogènes dans les aliments: leur signification, leur analyse. Ann. Fals. Exp. Chim. 88:119-140.
    31. 31
  31. Masson, F., R. Talon, and M. C. Montel. 1996. Histamine and tyramine production by bacteria from meat production. Int. J. Food Microbiol. 32:199-207.[CrossRef][Medline]
    32. 32
  32. Masson, F., A. Lebert, R. Talon, and M. C. Montel. 1997. Effects of physico-chemical factors influencing tyramine production by Carnobacterium divergens. J. Appl. Microbiol. 83:36-42.[CrossRef][Medline]
    33. 33
  33. Molenaar, D., J. S. Bosscher, B. ten Brink, A. J. M. Driessen, and W. N. Konings. 1993. Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri. J. Bacteriol. 175:2864-2870.[Abstract/Free Full Text]
    34. 34
  34. Moreno-Arribas, V., and A. Lonvaud-Funel. 1999. Tyrosine decarboxylase activity of Lactobacillus brevis IOEB 9809 isolated from wine and L. brevis ATCC 367. FEMS Microbiol. Lett. 180:55-60.[CrossRef][Medline]
    35. 35
  35. Moreno-Arribas, V., S. Torlois, A. Joyeux, A. Bertrand, and A. Lonvaud-Funel. 2000. Isolation, properties and behaviour of tyramine-producing lactic acid bacteria from wine. J. Appl. Microbiol. 88:584-593.[CrossRef][Medline]
    36. 36
  36. Moreno-Arribas, V., and A. Lonvaud-Funel. 2001. Purification and characterization of tyrosine decarboxylase of Lactobacillus brevis IOEB 9809 isolated from wine. FEMS Microbiol. Lett. 195:103-107.[CrossRef][Medline]
    37. 37
  37. Neely, M. N., and E. R. Olson. 1996. Kinetics of expression of the Escherichia coli cad operon as a function of pH and lysine. J. Bacteriol. 178:5522-5528.[Abstract/Free Full Text]
    38. 38
  38. Read, T. D., R. C. Brunham, C. Shen, S. R. Gill, J. F. Heidelberg, O. White, E. K. Hickey, J. Peterson, T. Utterback, K. Berry, S. Bass, K. Linher, J. Weidman, H. Khouri, B. Craven, C. Bowman, R. Dodson, M. Gwinn, W. Nelson, R. DeBoy, J. Kolonay, G. McClarty, S. L. Salzberg, J. Eisen, and C. M. Fraser. 2000. Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucleic Acids Res. 28:1397-1406.[Abstract/Free Full Text]
    39. 39
  39. Rice, S. L., R. R. Eitenmiller, and P. E. Koehler. 1976. Biologically active amines in food: a review. J. Milk Food Technol. 39:353-358.
    40. 40
  40. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    41. 41
  41. Sanger, K., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.[Abstract/Free Full Text]
    42. 42
  42. Silla Santos, M. H. 1996. Biogenic amines: their importance in foods. Int. J. Food Microbiol. 29:213-231.[CrossRef][Medline]
    43. 43
  43. Smith, D. R., L. A. Doucette-Stamm, C. Deloughery, H.-M. Lee, J. Dubois, T. Aldredge, R. Bashirzadeh, D. Blakely, R. Cook, K. Gilbert, D. Harrison, L. Hoang, P. Keagle, W. Lumm, B. Pothier, D. Qiu, R. Spadafora, R. Vicare, Y. Wang, J. Wierzbowski, R. Gibson, N. Jiwani, A. Caruso, D. Bush, H. Safer, D. Patwell, S. Prabhakar, S. McDougall, G. Shimer, A. Goyal, S. Pietrovski, G. M. Church, C. J. Daniels, J.-I. Mao, P. Rice, J. Nolling, and J. N. Reeve. 1997. Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics. J. Bacteriol. 179:7135-7155.[Abstract/Free Full Text]
    44. 44
  44. Smith, T. A. 1981. Amines in food. Food Chem. 6:169-200.
    45. 45
  45. Stratton, J. E., R. W. Hutkins, and S. L. Taylor. 1991. Biogenic amines in cheese and other fermented foods: a review. J. Food Prot. 54:460-470.
    46. 46
  46. Straub, B. W., P. S. Tichaczek, M. Kicherer, and W. P. Hammes. 1994. Formation of tyramine by Lactobacillus curvatus LTH 972. Z. Lebensm. Unters. Forsch. 199:9-12.[CrossRef][Medline]
    47. 47
  47. Sukhanova, M. J., L. V. Shumnaya, L. G. Grenback, N. E. Grutenko, T. M. Khlebodarova, and I. Y. Rauschenbach. 1997. Tyrosine decarboxylase and dopa decarboxylase in Drosophila virilis under normal conditions and heat stress: genetic and physiological aspects. Biochem. Genet. 35:91-103.[CrossRef][Medline]
    48. 48
  48. Takami, H., K. Nakasone, Y. Takaki, G. Maeno, Y. Sasaki, N. Masui, F. Fuji, C. Hirama, Y. Nakamura, N. Ogasawara, S. Kuhara, and K. Horikoshi. 2000. Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res. 28:4317-4331.[Abstract/Free Full Text]
    49. 49
  49. ten Brink, B., C. Damink, H. J. Joosten, and J. Huis in't Veld. 1990. Occurrence and formation of biologically active amines in foods. Int. J. Food Microbiol. 11:73-84.[CrossRef][Medline]
    50. 50
  50. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Environ. Microbiol. 29:807-813.[Abstract/Free Full Text]
    51. 51
  51. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
    52. 52
  52. Vanderslice, P., W. C. Copeland, and J. D. Robertus. 1986. Cloning and nucleotide sequence of wild type and a mutant histidine decarboxylase from Lactobacillus 30A. J. Biol. Chem. 261:15186-15191.[Abstract/Free Full Text]
    53. 53
  53. Van Poelje, P. D., and E. E. Snell. 1990. Cloning, sequencing, expression, and site-directed mutagenesis of the gene from Clostridium perfringens encoding pyruvoyl-dependent histidine decarboxylase. Biochemistry 29:132-139.[CrossRef][Medline]
    54. 54
  54. Van Poelje, P. D., and E. E. Snell. 1990. Pyruvoyl-dependent enzymes. Annu. Rev. Biochem. 59:29-59.[Medline]
    55. 55
  55. Wookey, P. J., J. Pittard, S. M. Forrest, and B. E. Davidson. 1984. Cloning of the tyrP gene and further characterization of the tyrosine specific transport system in Escherichia coli K12. J. Bacteriol. 160:169-174.[Abstract/Free Full Text]
    56. 56
  56. Xin, Y., W. Li, D. S. Dwyer, and E. A. First. 2000. Correlating amino acid conservation with function in tyrosyl-tRNA synthetase. J. Mol. Biol. 303:287-298.[CrossRef][Medline]
    57. 57
  57. Yagi, Y., and D. B. Clewell. 1980. Recombination-deficient mutant of Streptococcus faecalis. J. Bacteriol. 143:966-970.[Abstract/Free Full Text]

Applied and Environmental Microbiology, July 2002, p. 3537-3544, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3537-3544.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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