Sequencing, characterization and transcriptional analysis of the histidine decarboxylase operon of Lactobacillus buchneri

M. Cruz Martín, María Fernández, Daniel M. Linares and Miguel A. Alvarez

Instituto de Productos Lácteos de Asturias, Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias, Spain

Correspondence
Miguel A. Alvarez
maag{at}ipla.csic.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The amplification of an internal fragment of the hdcA gene for histidine decarboxylase in Lactobacillus buchneri showed the gene to be located on the bacterial chromosome. Reverse PCR was then used to amplify both it and its adjacent genes. The histidine decarboxylase cluster was found to be composed of four genes: hdcC (expressed in Lactococcus lactis, the product of which is located in the membrane, suggesting it to be a histidine/histamine antiporter), hdcA (which encodes histidine decarboxylase), hdcB (of unknown function but co-transcribed as bicistronic mRNA together with hdcA) and hisS (the only copy of a gene encoding a histidyl-tRNA synthetase in Lb. buchneri). The expression of hisS depends on the histidine concentration of the growth medium, and it can be transcribed as monocistronic or hdcA–hdcB–hisS polycistronic mRNA.


Abbreviations: BA, biogenic amine; LAB, lactic acid bacteria

The GenBank/EMBL/DDBJ accession number for the sequences reported in this paper is AJ749838.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Lactic acid bacteria (LAB) play an essential role as biopreservatives in fermented foods, preventing spoilage by pathogenic micro-organisms through acidification, competition for essential nutrients, and/or the production of inhibitory compounds. They also contribute to the organoleptic characteristics of products. Some strains are even claimed to be beneficial to health because of their probiotic characteristics. The metabolic activity of some LAB strains can, however, produce undesirable products such as biogenic amines (BAs). Some BAs, such as histamine, have important biological functions in mammals, but if the quantities ingested are too great, or if the physiological detoxification process is inhibited (either by drugs or genetically), they can be toxic. Foods likely to contain high levels of BAs include fish and fish products, and fermented foods and beverages, such as cheese, certain meats, beer and wine (Silla Santos, 1996).

The most important BAs in foods and beverages are histamine, tyramine, tryptamine, putrescine, cadaverine, spermine, spermidine and {beta}-phenylethylamine. Of these, histamine is the most important with respect to food-borne intoxications; with the highest biological activity of all the BAs (Bodmer et al., 1999), this amine can cause hypertension, hypotension, headache, urticaria, nausea and vomiting.

Histamine is produced by enzymic decarboxylation of the histidine present in foods. There are two distinct classes of histidine decarboxylases: that of eukaryotic and Gram-negative bacteria, which requires pyridoxal phosphate as a cofactor, and that of Gram-positive bacteria, which uses a covalently bound pyruvoyl moiety as a prosthetic group. The second type of enzyme has so far been observed in Lactobacillus 30a (Chang & Snell, 1968), Clostridium perfringens (Recsei et al., 1983b), Micrococcus sp. (Prozorovski & Jörnvall, 1975; Alekseeva et al., 1976) and Oenococcus oeni (Coton et al., 1998). The best studied of these enzymes is the histidine decarboxylase of Lactobacillus 30a (EC 4.1.1.22; Riley & Snell, 1968, 1970; Hackert et al., 1981), which is now known to be a hexameric enzyme composed of six {beta} and six {alpha} chains. The active enzyme is derived from a hexameric proenzyme composed of six {pi} subunits. An unusual intramolecular reaction that involves cleavage of the {pi} chains at a single serine–serine peptide bond yields the {beta} and {alpha} chains of the active enzyme (Recsei et al., 1983a).

The gene encoding histidine decarboxylase (hdcA) has been identified in different Gram-positive micro-organisms, such as C. perfringens (van Poelje & Snell, 1990), O. oeni (Coton et al., 1998) and Lactobacillus 30a (Vanderslice et al., 1986). With the exception of C. perfringens, this gene is part of an operon which includes a second gene, hdcB. Although different functions such as regulation or transport have been suggested for the latter gene (Coton et al., 1998), its true function is still unknown.

Lactobacillus buchneri B301, a strain isolated from Gouda cheese, is able to decarboxylate histidine to produce histamine (Joosten & Northolt, 1989). The present study reports the amplification, sequencing, characterization and transcriptional analysis of the hdc cluster of Lb. buchneri B301. Besides hdcA and hdcB, a histidine/histamine antiporter gene (hdcC) and a histidyl-tRNA synthetase gene (hisS) were found. These are usually linked to genes encoding decarboxylases involved in BA synthesis (Van Bogelen et al., 1983; Connil et al., 2002; Lucas et al., 2003; Fernández et al., 2004); this is believed to be the first time they have been found in an hdc cluster. An in-depth study of the role and the regulation of the LAB genes involved in histidine decarboxylation will allow the design of rational strategies to avoid toxic histamine production in fermented foods.


   METHODS
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INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids, oligonucleotides and culture conditions.
Table 1 shows the bacterial strains, plasmids and oligonucleotides used in this study. Lactococcus lactis strains were grown at 30 °C in M17 (Oxoid) supplemented with 0·5 % glucose. The histamine producer Lb. buchneri B301 was grown in MRS (Oxoid), or LAPTg agar or broth (Raibaud et al., 1961), without aeration, at 37 °C. When indicated, LAPTg was supplemented with 2 g histidine l–1 (LAPTgHis). Standard media and growth conditions were used for Escherichia coli (Sambrook et al., 1989). When plasmid-containing clones were grown, the medium was supplemented with the appropriate antibiotics: 100 µg ampicillin ml–1 and 50 µg kanamycin ml–1 for E. coli, and 5 µg chloramphenicol ml–1 for Lc. lactis.


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Table 1. Bacterial strains, plasmids and primers

 
DNA manipulation procedures.
Lb. buchneri genomic DNA was prepared using 2x Kirby lytic mix, as described by Hopwood et al. (1985). Plasmid isolation was performed according to Scheirlink et al. (1989). Plasmid DNA was isolated from E. coli by the alkaline lysis method (Sambrook et al., 1989). Electrotransformation of E. coli was undertaken in a Bio-Rad pulser apparatus following the manufacturer's recommendations; Lc. lactis was electroporated as described by Kuipers et al. (1998). Restriction endonuclease digestions, alkaline phosphatase treatments, ligations and other manipulations were performed according to standard procedures (Sambrook et al., 1989). DNA hybridizations were performed using the non-radioactive DNA Labelling and Detection Kit (Roche Molecular Biochemicals) following the manufacturer's instructions.

Reverse PCR.
A reverse PCR strategy was used to obtain the complete hdc cluster sequence. Genomic DNA was digested independently with different enzymes (EcoRI, BglII and PstI), and then ligated in diluted conditions (to allow intramolecular ligation). Ligations were precipitated and resuspended in 10 µl TE buffer (10 mM Tris/HCl, 1 mM EDTA). A 0·5 µl volume of each ligation was used in the PCR amplification. The primers used are listed in Table 1. Amplifications were performed in a Mini Cycler (MJ Research) using Pwo polymerase and the Expand Long Template PCR System (Roche Molecular Biochemicals).

Nucleotide sequence analysis.
DNA was sequenced by the DNA Sequencing Service at the Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, using the BigDye Terminator cycle sequencing ready reaction FS kit and an ABI PRISM 3700 DNA sequencer (both from Applied Biosystems).

Sequence analysis was performed using the University of Wisconsin Genetics Computer Group software package (Devereux et al., 1984). The BLAST and BLASTP programs were used to determine the similarities of the deduced amino acid sequences with those present in databases (Altschul et al., 1997). Conserved regions within proteins were identified in the Conserved Domain Database (CDD) using the CD-Search software (Marchler-Bauer et al., 2003) developed by the National Center for Biotechnology Information (NCBI). Hydropathy analyses were performed using the TMpred method (Hofmann & Stoffel, 1993). The SOSUI program (Hirokawa et al., 1998) was used to determine the possible transmembrane segments. Phylogenetic trees were constructed from multiple-sequence alignments using the PILEUP program (Devereux et al., 1984). Multiple alignment was performed using BOXSHADE 3.21 from EMBnet.

Expression of hdcC in Lc. lactis NZ9000.
Initially, a PCR strategy was used to introduce six histidine codons downstream of the ATG in the nisA promoter. Primers pnis3, which has a 5' ‘add-on’ containing the six histidine codons (shown in bold in Table 1), and pnis1 (Table 1) were used to amplify an engineered nisA promoter from pNG8048E. The resulting amplicon was cloned into NcoI–BamHI-digested pUK21 (Vieira & Messing, 1991) to yield pEM149. A NcoI–BamHI fragment from this vector containing the PnisA–ATG(His)6NcoI was then inserted into the BglII–NcoI sites of plasmid pNG8048E to replace the PnisA promoter. The resulting vector was named pEM150. In parallel, the hdcC gene was amplified from Lb. buchneri DNA using the specific primers hdc29 (into which a NcoI site was introduced) and hdc30 (Table 1). Subsequently, the amplicon was digested with NcoI and SalI, and inserted into the equivalent sites of pGEM-5Zf(+) (Promega), resulting in plasmid pEM141. The NcoI–SacI fragment to pEM141 was then cloned into the same sites of the vector pEM150. This ligation was introduced by electroporation into Lc. lactis NZ9000, yielding the transformed derivative strain Lc. lactis EM156.

RNA isolation, Northern blotting and RT-PCR.
Total RNA was isolated from exponentially growing Lb. buchneri cultures by the Macaloid method described by Kuipers et al. (1993). For Northern analysis, RNA was separated on a 1 % formaldehyde agarose gel, and blotted and hybridized according to standard procedures (Sambrook et al., 1989). The probes used for hybridization were radiolabelled with [{alpha}-32P]dATP by nick translation. The size of the transcripts was determined relative to an RNA molecular mass marker I (Roche Molecular Biochemicals).

For RT-PCR, 10 µg total RNA was treated for 30 min at 37 °C with 20 U RNase-free DNaseI (Roche Molecular Biochemicals), followed by phenol/chloroform extraction and precipitation. Reactions were performed using the Titan One Tube RT-PCR Kit (Roche Molecular Biochemicals) with 0·4 µM of each primer and 1 µg RNA. After incubation at 50 °C for 30 min for the reverse transcription reaction, the amplifications were performed for 35 cycles (94 °C for 30 s, 55 °C for 30 s, and 68 °C for 3 min), and samples were analysed on a 1·5 % agarose gel in TAE [40 mM Tris/acetate (pH 8·0), 1 mM EDTA] buffer. The absence of contaminating DNA was controlled by non-reverse-transcribed PCR, which was performed under the same conditions without the reverse transcriptase.

Western blot analysis.
Lc. lactis EM156, containing the vector with a translational fusion of the nisA-promoter–His(x6)–hdcC-gene, was cultured to an OD600 of 0·6, and induced for 3 h with 3 ng nisin ml–1. Samples of 50 ml were collected from induced cultures. The bacterial sediments were washed twice with 25 mM Tris/HCl buffer (pH 8·0) containing 1 mM EDTA and 1 mM DTT, and finally suspended in 10 ml of the same buffer. All subsequent steps were performed at 0–4 °C. The cells were broken with a French pressure cell press (SLM-Aminco Instruments). Cell walls and unbroken cells were eliminated by centrifugation at 12 000 g for 1 h. The final supernatant was centrifuged at 75 000 g for 1 h to sediment the cell membranes. Samples of cell extract, membranes and cytoplasmic proteins were analysed using SDS-PAGE (12 % polyacrylamide gels) employing the Miniprotean II System (Bio-Rad). The gels were then transferred onto nitrocellulose membranes (Hybond ECL; Amersham Biosciences), stained with Ponceau S, washed with water, and blocked with TNT [20 mM Tris/HCl (pH 7·5), 500 mM NaCl, 0·05 % Tween 20] containing 5 % non-fat dry milk at room temperature for 2 h. The membranes were then incubated for 2 h with a 1 : 10 000 dilution of 6xHis mAb–HRP conjugate (BD Biosciences Clontech). After extensive membrane washing, twice with TNT, and once with TBS [10 mM Tris/HCl (pH 7·5), 150 mM NaCl], the binding of the primary antibody was visualized using the ECL plus Western Blotting Detection kit (Amersham Biosciences).


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Location of hdcA in the Lb. buchneri genome
To characterize the genes responsible for histamine synthesis in Lb. buchneri B301, a PCR reaction was performed using the oligonucleotides JV16HC and JV17HC. These primers were designed to detect hdcA genes from Gram-positive bacteria (Le Jeune et al., 1995). The sequence of the amplicon obtained was similar to those of hdcA genes found in databases; it may therefore encode HdcA. The 0·3 kb internal hdcA fragment was used as a probe to locate the gene in the Lb. buchneri genome. Hybridization fragments of 3·5 and 4·9 kb were detected when total DNA was digested with EcoRI and BglII, respectively, whereas two fragments of 1·6 and 4 kb were obtained after PstI digestion (data not shown). In addition, total and plasmid DNA samples extracted from three independent colonies of Lb. buchneri were digested with EcoRI, and transferred to a membrane. After hybridization, the expected 3·5 kb band was observed in the lanes corresponding to total DNA (Fig. 1B, lanes 2, 3 and 4), but no hybridization was observed in the lanes loaded with plasmid preparations (Fig. 1B, lanes 5, 6 and 7). Therefore, hdcA appears to be located on the chromosome of Lb. buchneri.



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Fig. 1. Southern blot analysis. (A) Total DNA (lanes 2, 3 and 4) and plasmid DNA (lanes 5, 6 and 7) extracted from three independent colonies, and digested with EcoRI, and (B) hybridized with an internal fragment of hdcA. Lane 1, {lambda} DNA digested with PstI.

 
Reverse PCR amplification of the hdc cluster from Lb. buchneri
Based on the Southern blot results, a reverse PCR strategy was designed to reveal the complete sequence of the hdcA gene and flanking regions. Lb. buchneri total DNA was digested with EcoRI, BglII and PstI. Each digestion was ligated as indicated in Methods, and used as a template for PCR reactions using the primers rev1, rev2, rev3, rev4, hdc6, hdc7, hdc8, hdc9, hdc10, hdc11, hdc12, hdc13, hdc14, hdc17 and hdc19 (Table 1). A 5775 bp DNA fragment was sequenced, and its restriction targets corresponded to the genetic organization found on the chromosome of Lb. buchneri by Southern analysis (see above).

Sequence analysis of the hdc cluster from Lb. buchneri
The sequence analysis showed the presence of four complete ORFs (Fig. 2A).



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Fig. 2. Transcriptional analysis of the hdc operon. (A) Organization of the hdc cluster of Lb. buchneri. Possible promoter and termination regions are indicated. RT-PCR primers are shown as 1 (hdc11), 2 (hdc50), 3 (rev2), 4 (hdc4), 5 (hdc51) and 6 (hdc52). Restriction endonuclease sites: BglII, B; EcoRI, ER; PstI, P. (B) Northern blot analysis of the hdc cluster. Total RNA was extracted from Lb. buchneri grown in LAPTg with histidine (B-I) or without histidine (B-II). The probes used were internal fragments of hdcA, lane 1; hdcB, lane 2; hisS, lane 3; and hdcC, lane 4. (C) RT-PCR amplification with four sets of primers designed to amplify intergenic regions: hdcC–hdcA, lane 1; hdcA–hdcB, lane 2; hdcB–hisS, lane 3; hdcC–hisS, lane 4. Negative controls were conducted without reverse transcriptase (lanes 1, 2, 3 and 4 of C-II).

 
hdcA.
The first gene analysed was hdcA. Two tandem ATG codons were found at the 5' end of the coding region, but only the second ATG was preceded by a reasonable Shine–Dalgarno consensus sequence (GGAGG). Possible promoter regions –35 and –10 were identified upstream of the start codon. The deduced protein had 315 residues, with a predicted mass of 36 141 Da, and a pI of 4·78. Analysis of the amino acid sequence showed that the protein could be included in the group of bacterial histidine decarboxylases that use a covalently bound pyruvoyl residue as a prosthetic group. All the important residues for proenzyme activation and catalysis were conserved: the two serine residues, located at positions 81 and 82 (involved in autocatalytic chain cleavage), the aspartate residue located at position 63 (hydrogen bonded to the imidazole side of the histamine), and a glutamate residue at position 197 (whose probable function is to donate the proton in the decarboxylation reaction). The phylogenetic tree for known HdcA sequences is shown in Fig. 3.



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Fig. 3. Relationships between the HdcA of different bacteria. The deduced primary sequence of the protein encoded by Lb. buchneri was compared to those of C. perfringens (J02880), Enterobacter aerogenes (M62745), Klebsiella planticola (M62746), Lactobacillus 30a (J02613), Lactobacillus sp. (P00862), Morganella morganii (J02577), O. oeni (U58865), Vibrio anguillarum (AY312585) and Lb. buchneri (AJ749838).

 
hdcB.
Downstream of hdcA, a second ORF, preceded by a possible ribosome-binding site (GGAG), was found in the same orientation (Fig. 2A). However, no consensus promoter regions were found. This ORF encoded a 174 aa polypeptide with a predicted mass of 19 536 Da, and a pI of 7·22. 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 (Le Jeune et al., 1995; Van Poelje & Snell, 1990). However, the protein appears to have no transmembrane motifs.

hisS.
Downstream of hdcB, another ORF was found in the same DNA strand. A putative promoter region and a potential ribosome-binding site (GGAG) upstream of the start codon were also identified. The deduced protein was 428 aa, with a molecular mass of 48 711 Da, and a pI of 4·88. It showed similarity to the class II histidyl-tRNA synthetases (HisRS), and to the ATP phosphoribosyltransferases, which act as regulators of histidine biosynthesis (HisZ; HisRS-like proteins) in several bacteria (Bond & Francklyn, 2000). However, higher similarities (between 60 and 70 %) were observed with members of the HisRS group than with the members of the HisZ family (45–59 %). As shown in Fig. 4, the encoded protein has all the conserved catalytic domains of the HisRS proteins, including those that contact with ATP (motifs 2 and 3), as well as A and B histidine-specific regions, which are considered necessary to be classed as a HisRS protein. In contrast, the only domain that is well conserved in HisZ proteins is Histidine B (Fig. 4). Accordingly, this ORF was termed hisS. It was then used as a Southern probe against Lb. buchneri total DNA. No homologous genes were detected (data not shown). Since these genes are very well conserved, this result suggests that hisS could be the only histidyl-tRNA synthetase gene present in the genome. In view of the fact that aminoacyl-tRNA synthetases play an indispensable catalytic role in protein biosynthesis, this function should be covered by hisS. On the other hand, the decarboxylation of amino acids has to be very well regulated, since they are essential to protein synthesis. It has been suggested that aminoacyl-tRNA synthetase genes in decarboxylation clusters might play a regulatory role as amino acid sensors (Fernández et al., 2004). Furthermore, it has been described that aminoacyl-tRNA synthetases participate in many other functions, such as the regulation of gene expression by attenuation mechanisms. The role of HisRS in attenuation control of the E. coli and Salmonella typhimurium his operons is well known (Yanofsky, 1981; Francklyn et al., 1998). It is possible that HisRS intervenes in protein synthesis in Lb. buchneri, and it may have a regulatory action on the expression of the hdc cluster.



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Fig. 4. Alignment of histidyl-tRNA synthetase (HisRS) and HisRS-like proteins (HisZ) from representative prokaryotic organisms. Identical residues are highlighted in black and similar residues in grey. The accession numbers of the sequences are as follows. HisS: Bacillus subtilis, NP_390634; E. coli O157:H/EDL933, NP_289067; S. typhimurium LT2, NP_417009; Yersinia pestis, NP_406383; Vibrio cholerae O1, NP_230409; Haemophilus influenzae Rd KW20, NP_438530; Pseudomonas aeruginosa PAO1, NP_252491; Streptococcus equisimilis, P30053; Streptococcus pneumoniae R2, NP_359522; Lc. lactis, NP_268124; Staphylococcus aureus, NP_372155; Listeria monocytogenes EGD-e, NP_465045; C. perfringens, NP_562850; and Lb. buchneri, AJ749838. HisZ: Pseudomonas fluorescens, ZP_00083254; Pseudomonas syringae, ZP_00125242; Microbulbifer degradans, ZP_00065006; Nitrosomonas europaea, NP_841331; Lc. lactis, Q021477; Lc. lactis IL1403, NC_002662; Clostridium thermocellum, ZP_00060493, Desulfitobacterium hafniense, ZP_00103827; Bacillus halodurans, NP_244451; Geobacter metallireducens, ZP_00082172; and Leuconostoc mesenteroides, ZP_00062659.

 
As in other aminoacyl-tRNA synthetases (Henkin, 1994), the region upstream of the start codon of hisS contains a putative promoter region (–35 TTGACT and –10 TATCAT), and a leader region with the sequence features of the tRNA-mediated anti-termination system described by Grundy & Henkin (1994). A folding model of this mRNA leader region with four stem–loop structures was constructed (Fig. 5), containing the typical T-box sequence, a histidine specifier CAC, and most of the other less-conserved boxes, including the terminator and the anti-terminator (with the anti-acceptor UGGA).



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Fig. 5. Structural model of the anti-termination sequence from the Lb. buchneri hisS leader region. The specifier codons (CAC), AGUA-I box, AG box, GAUG box (in stem–loop structure I), AGUA-II box (in stem–loop structure II), F-box (in stem–loop IIIB), anti-acceptor (UGGA in the anti-terminator) and T-box are in bold. (A) Terminator conformation, (B) anti-termination conformation, and (C) alternative conformation of stem II.

 
hdcC.
Upstream of hdcA, an additional ORF was identified in the same orientation; this was designated hdcC (Fig. 2A). The first ATG of hdcC was preceded by a typical Shine–Dalgarno sequence (AAGGA). A putative promoter region containing likely –10 and –35 sequences preceded the coding sequence. Downstream from the TAG stop codon, a stem–loop structure ({Delta}G=–21·7 kcal mol–1, 90·8 kJ mol–1), which might serve in transcription termination, was found (Fig. 2A). hdcC encodes a 488 aa protein of 52 kDa, with a pI of 4·88. Analyses of HdcC hydropathy using the TMpred method and SOSUI programs predicted the presence of 13–14 and 11 hydrophobic segments, respectively. Each of these segments is long enough to form a transmembrane helix (Fig. 6B). The similarities between HdcC and several amino acid/amine antiporter sequences from databases range between 67 and 49 % (Fig. 6A). It is noteworthy how antiporters with the same substrates are clustered together.



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Fig. 6. (A) Dendrogram showing relationships among different amino acid/biogenic amine antiporters. The accession numbers of the sequences are as follows. ArcD: C. perfringens, NP 561082; Lactobacillus sakei, O53092; V. cholerae, NP_230087; E. coli, NP_753892; S. aureus, NP_373157. CadB: S. typhimurium LT2, NP_461493; Vibrio vulnificus, AF324470; E. coli, NP_313141; and V. cholerae, NP_229936. PotE: H. influenzae RD KW20, NP_438748; Salmonella typhi, NP_455261; and V. cholerae O1, NP_233444. TdcP: Lb. brevis, AF446085; Ent. faecalis V583, AE016948; and Lc. lactis IPLA655, AJ630043. Putative histidine/histamine antiporter: Lb. buchneri, AJ749838; and C. perfringens, NC_003366. (B) Hydrophobicity plot of the HdcC protein. The hydropathy profile was calculated using TMpred methods. (C) Western blot analysis: Ponceau S staining (C-I) and immunodetection (C-II) after SDS-PAGE of fractionated extracts of Lc. lactis EM156. Lanes: 1, total extract; 2, cytoplasmic fraction; 3, membrane fraction.

 
The expression of hdcC in Lc. lactis indicates that HdcC is a membrane protein
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. As indicated in Methods, the gene was manipulated to incorporate an N-terminal His6 tag in the encoded protein. After nisin induction, cells were broken, and fractionated into membrane and cytoplasmic fractions by centrifugation. Fig. 6(C) shows a Western blot with a monoclonal antibody against His-tag. A protein band of the expected size was clearly recognized by the antibodies in both the total extracts and the membrane fraction (Fig. 6C-II, lanes 1 and 3). However, no protein band was observed in the cytoplasmic fraction (Fig. 6C-II, lane 2), although it showed a greater concentration of proteins when stained with Ponceau S (Fig. 6C-I, lane 2). The results allow the conclusion that the protein encoded by hdcC is located in the membrane. This result, together with the evidence that Lb. buchneri has an electrogenic histidine/histamine antiport (Molenaar et al., 1993), and sequence data, suggests that hdcC could be responsible for histamine/histidine exchange in Lb. buchneri. To our knowledge, this is the first time such a gene has been described.

Transcriptional analysis of the hdc cluster
RNA isolated from Lb. buchneri grown in LAPTg and LAPTgHis was used for Northern blot analysis. Internal fragments of hdcA, hdcB, hdcC and hisS were generated by PCR, labelled, and used as DNA probes. Using hisS as a probe, two different bands were detected in the cultures grown in LAPTg media without histidine (Fig. 2B-II, lane 3). The size of the small band corresponded to a monocistronic transcript (expected size, 1·5 kb), while the other could correspond to a polycistronic mRNA including hdcA, hdcB and hisS (expected size, 3·3 kb). In LAPTgHis, no signal was detected, indicating that hisS transcription depends on the histidine concentration in the medium. When using hdcA or hdcB as probes, a single band of the same size was obtained in media containing histidine. The size of the band suggests that hdcA and hdcB are transcribed as bicistronic mRNA (expected size 1·6 kb) (Fig. 2B-I, lanes 1 and 2). In media without histidine (Fig. 2B-II, lanes 1 and 2), an additional band of the same size as the larger one detected with the hisS probe was seen, reinforcing the idea of an hdcA–hisB–hdcS polycistronic mRNA. However, hdcC was transcribed as single monocistronic mRNA (expected size, 1·5 kb), and no differences were observed in the expression of the gene when the strain was grown with (Fig. 2B-I, lane 4) or without histidine (Fig. 2B-II, lane 4).

To confirm these results, total RNA of Lb. buchneri grown in the absence of histidine was used in RT-PCR experiments with four sets of primers designed to amplify regions spanning gene junctions. RT-PCR using primers rev2 and hdc4 (Fig. 2A, primers 3 and 4) confirmed the co-transcription of hdcA and hdcB genes (Fig. 2C-I, lane 2). When primers hdc51 and hdc52 (Fig. 2A, primers 5 and 6) were used, the expected amplimer was obtained (Fig. 2C-I, lane 3), showing that hdcB and hisS can also be co-transcribed. However, PCR products were not observed either with primers hdc11 and hdc50 (Fig. 2A, primers 1 and 2), or with primers hdc11 and hdc52 (Fig. 2A, primers 1 and 6; Fig. 2C-I, lanes 1 and 4, respectively), confirming that the hdcC gene is transcribed as a single monocistronic mRNA.

The transcriptional analysis results are consistent with the DNA sequence study. A putative promoter and a stem–loop structure region, which could serve as a transcriptional termination structure, were found in hdcC. Possible promoter regions were identified upstream of the start codon of hdcA, but no obvious terminator sequences where found between this gene and hdcB. Consensus promoter regions were not found in this region either. The region upstream of the start codon of hisS contains a putative promoter, and a leader region with the sequence features of a tRNA-mediated anti-termination system (Fig. 5) that seems to work as previously described for similar structures (Grundy & Henkin, 1994; Delorme et al., 1999). In the absence of histidine, the tRNA destabilizes the terminator, allowing transcription from the promoter. It would also explain the polycistronic hdcA–hdcB–hisS mRNA found in the absence of histidine in the media. In the presence of histidine, the histidyl-tRNA would not interact with the structure, and the terminator would stop transcription from its own promoter, and from upstream promoters. The polycistronic mRNA including hdcA, hdcB and hisS detected in the absence of histidine had a weaker signal than the bicistronic hdcA–hdcB or the monocistronic hisS mRNAs. This larger transcript may be more unstable, or the different stem–loops in the anti-termination structure might interfere with transcription from promoters located upstream.

Comparative analysis of the hdc cluster organization
The hdc gene cluster organization was compared to other clusters (Fig. 7). In the Gram-negative Photobacterium phosphoreum (AY223843), the hdc genes mapped in a similar way to that observed in Lb. buchneri. The analysis of the complete genome sequence of C. perfringens (Shimizu et al., 2002) shows that the histidine decarboxylase gene (dchS) is upstream of CPE0389, an ORF annotated as a putative arginine/ornithine antiporter (ArcD) in databases (NC_003366). According to the dendrogram of Fig. 6(A), this ORF should be a histidine/histamine antiporter; therefore, its organization would be different. With respect to other amino acid decarboxylation clusters, only the genetic organization of the lysine decarboxylase cluster from E. coli (Meng & Bennett, 1992) is similar to that of the Lb. buchneri hdc cluster. In the tyrosine decarboxylase cluster of Lactobacillus brevis (Lucas et al., 2003), Enterococcus faecalis (Connil et al., 2002) and Lc. lactis IPLA 655 (Fernández et al., 2004), the gene that encodes the tyrosine/tyramine antiporter is located downstream of the decarboxylase gene, which as just as it is with the putrescine/ornithine cluster in E. coli (Kashiwagi et al., 1992). It can therefore be concluded that in all clusters, the permease and decarboxylase genes are present. Aminoacyl-tRNA synthetase was located in some clusters only (Neely et al., 1994; Lucas et al., 2003; Connil et al., 2002; Fernández et al., 2004).



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Fig. 7. Comparison of the genetic organization of the amino acid decarboxylase region of different bacteria. C. perfringens, NC 003366; E. coli K-12, NC 000913; Photobacterium phosphoreum, AY223843; Ent. faecalis, AF354231; Lactobacillus 30a, J02613; Lb. brevis, AF446085; O. oeni, U58865; Lc. lactis IPLA 655, AJ630043; and Lb. buchneri, AJ749838. DC, decarboxylase; P, permease; aaRS, aminoacyl-tRNA synthetase; R, regulatory protein.

 


   ACKNOWLEDGEMENTS
 
We thank J. E. Suárez for his encouragement during the initial phases of this work, M. R. Rodicio for helpful discussions, and Ann-Katrin Becker for technical assistance. We are grateful to NIZO food research for providing Lb. buchneri B301 and Lc. lactis NZ9000. D. M. Linares was the recipient of a fellowship from the Spanish Ministry of Science and Technology. This research was supported by project QLK1-CT-2002-02388 (European Union).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 2 July 2004; revised 9 December 2004; accepted 24 December 2004.



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