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Sequencing of the Tyrosine Decarboxylase

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Sequencing of the Tyrosine Decarboxylase 2521 Journal of Food Protection, Vol. 67, No. 11, 2004, Pages 2521±2529 Copyright Q, International Association for Food Protection Sequencing of the Tyrosine Decarboxylase Cluster of Lactococcus lactis IPLA 655 and the Development of a PCR Method for Detecting Tyrosine Decarboxylating Lactic Acid Bacteria MARIÂA FERNAÂ NDEZ, DANIEL M. LINARES, AND MIGUEL A. ALVAREZ* Instituto de Productos LaÂcteos de Asturias, Carretera de In®esto s/n, 33300 Villaviciosa, Asturias, Spain Downloaded from http://meridian.allenpress.com/jfp/article-pdf/67/11/2521/1674402/0362-028x-67_11_2521.pdf by guest on 01 October 2021 MS 04-109: Received 12 March 2004/Accepted 9 June 2004 ABSTRACT The enzymatic decarboxylation of tyrosine produces tyramine, the most abundant biogenic amine in dairy productsÐ especially in cheeses. The screening of lactic acid bacteria isolated from different artisanal cheeses and a number of microbial collections identi®ed 22 tyramine-producing strains belonging to different genera. The Lactococcus lactis strain IPLA 655 was selected, and the genes encoding a putative tyrosyl tRNA synthetase, a tyrosine decarboxylase (tdcA), and a tyrosine-tyramine antiporter, found together as a cluster, were sequenced. The disruption of tdcA yielded a strain unable to produce tyramine. Comparison of the L. lactis IPLA 655 tdcA gene with database tdcA sequences led to the design of two primers for use in a PCR method that identi®ed potential tyramine-producing strains. The proposed method can use puri®ed DNA, isolated colonies, milk, curd, and even cheese as a template. Molecular tools for the rapid detection of tyramine-producing bacteria at any time during the fermentation process could help prevent tyramine accumulation in fermented foods. The proposed technique could be of great use to the food industry. Lactic acid bacteria (LAB) are widely used in a variety tures. It is therefore useful to determine which strains pro- of food fermentation processes. These gram-positive bac- duce undesirable compounds so that these are not part of teria play an important role in the quality of the ®nal prod- starter cultures. ucts because they contribute to ¯avor formation, texture Histamine and tyramine are the most studied of the BA development, and biopreservation (the prevention of spoil- because they have toxicological effects that derive from age by undesirable and pathogenic microorganisms). Some their vasoactive and psychoactive properties (44). These strains even have probiotic properties. LAB metabolism compounds are formed from histidine and tyrosine, respec- strongly in¯uences the properties of fermented foods. Ami- tively, via an enzymatic decarboxylation reaction. The his- no acid catabolism is particularly important because it helps tidine decarboxylase genes (hdcA) of different gram-posi- produce desired ¯avor molecules (methylbutanal, isovaler- tive bacterial strainsÐLactobacillus sp. strain 30a (10, 53), ate, etc.), but it can also produce off-¯avor molecules (phe- Lactobacillus buchneri (26), Clostridium perfringens (26, nol, p-cresol, etc.) and even toxic compounds such as bio- 51), Micrococcus sp. (52), and Leuconostoc oenos (11)Ð genic amines (BA). BA are organic bases of low molecular have been characterized. The biochemical properties of his- mass that are formed and degraded during the normal me- tidine decarboxylase from Lactobacillus 30a have been tabolism of microorganisms, plants, and animals. They are studied extensively by van Poejie and Snell (51) and Schelp necessary for several physiological functions in humans. et al. (43). However, although tyrosine decarboxylase However, they are toxic if high concentrations are ingested (TDC) enzymes have been well characterized in eukary- or if the detoxi®cation process is inhibited (either geneti- otes, little is known about their prokaryotic counterparts. cally or by drugs) (2). Foods likely to contain high levels The puri®cation and characterization of this enzyme has of BA include ®shery products and fermented foods such been reported only for Enterococcus faecalis (4) and Lac- as cheese, wine, beer, and cured sausages (48). tobacillus brevis IOEB 9809 (33, 37). In addition, the E. BA form via the decarboxylation of their correspond- faecalis JH2-2 gene encoding the enzyme tyrosine decar- ing amino acids through the action of enzymes produced boxylase (tdcA) has been cloned (9), and the sequence for by microorganisms present in the food (48) (e.g., Entero- L. brevis IOEB 9809 is available (32, 33). In both strains, bacteriae, Pseudomonas spp., enterococci, and some LAB the tdcA gene is part of a cluster with a gene encoding a (21)). Nonetheless, production is more related to strain than putative tyrosine-tyramine antiporter (tyrP) and a tyrosyl species. Producer strains can appear as contaminants of fer- mented foods, but they can also form part of starter cul- tRNA synthetase gene (tyrS). Lactococcus lactis is the most widely used starter cul- * Author for correspondence. Tel: 134985893352; Fax: 134985892233; ture in the manufacture of cheese, other dairy products, and E-mail: [email protected]. fermented foods. Although some strains have been identi- 2522 FERNAÂ NDEZ ET AL. J. Food Prot., Vol. 67, No. 11 TABLE 1. Strains and plasmids used in this study DNA isolation and manipulation. Escherichia coli DH5a was used as an intermediate host. Cloning was performed accord- Strain/plasmid Relevant characteristica Source ing to standard procedures (42). The isolation of E. coli plasmid Strain DNA and the recombinant techniques used were those described E. coli by Sambrook et al. (42). Large-scale isolation of E. coli plasmids for nucleotide sequence analysis was performed with the Plasmid INVaF9 Invitrogen Midi Kit (Qiagen, Hilden, Germany) according to the manufac- DH5a (22) turer's instructions. Plasmid and chromosomal DNA of L. lactis L. lactis were isolated and transformed as described previously (15). IPLA 655 Isolated from artisanal chees- (13) Southern blot hybridization was performed at 658C, and blots es. IPLA collection. were washed with 0.13 SSC (i.e., 0.15 M NaCl 1 0.0015 M Plasmid sodium citrate) before autoradiography. The probes used for hy- bridization were radiolabeled with [a-32P]dATP by nick transla- pCR2.1 Ampr Invitrogen tion. pUC18Ery Eryr, 3.8-kb pUC19 carrying Downloaded from http://meridian.allenpress.com/jfp/article-pdf/67/11/2521/1674402/0362-028x-67_11_2521.pdf by guest on 01 October 2021 the ery gene of plL253 (51) Construction of plasmids. An internal fragment of the tdc 4 pM20 Amp , 4.7-kb derivative of gene (820 bp) from L. lactis IPLA 655 was obtained directly from pCR2.1 containing a 0.8-kb genomic DNA by PCR ampli®cation with the use of the degen- fragment of tdcA from erate oligonucleotides P2 (59-GAYATIATIGGIATIGGIYTIGAY L. lactis IPLA 655 This work CARG-39), and P1 (59-CCRTARTCIGGIATIGCRAARTCIGTRTG r pM24 Ery , 5.8-kb derivative of 39), where Y is C or T, R is A or G, and I is inosine (Table 2). pM20 containing the ery The design of these oligonucleotides was based on the sequence gene of pUC18Ery This work of tdcA from L. brevis IOEB 9809 (33). The PCR product ob- tained was cloned in pCR2.1 (Invitrogen, Carlsbad, Calif.), gen- a Ampr, ampicillin resistant; Eryr, erythromycin resistant. erating the plasmid pM20. The erythromycin resistance gene (ery) from pUC18Ery (50) was cloned as a BamHI-HindIII fragment into pM20, previously digested with the same enzymes, to yield ®ed as tyramine producers (20), the tdcA gene has not been pM24. This was used to construct a knock-out strain. characterized. Nucleotide sequence analysis. Nucleotide sequences were Although several qualitative and quantitative methods analyzed with an ABI Prism 373 A Strech automated sequencer have been developed to determine BA production, consum- at the DNA Sequencing Department (Centro de Investigaciones er demand for better and healthier foods has increased in- BioloÂgicas, Consejo Superior de Investigaciones Cienti®cas). Se- terest in the development of rapid and sensitive methods quences were assembled and analyzed with the CLONE program for detecting BA producers in food. Molecular methods (v. 5) and compared with those in the SwissProt library (February have been used for the detection of gram-positive hista- 2004 release) with the TFASTA program. The Clustal method (25) mine-producing strains (31) and, more recently, for gram- was used for the multiple alignment of sequences. negative strains (47). This paper reports the sequencing of the tdc cluster of Oligonucleotide design and PCR reaction conditions. Primers Tdc1 and Tdc2 (Table 2) were designed by comparing genes from L. lactis IPLA 655 (13), a strain isolated from the tdcA sequences of the gram-positive bacteria E. faecalis JH2- artisanal cheese. Besides tdcA, the cluster includes a puta- 2 (9), L. brevis IOEB 9809 (33), and L. lactis IPLA 655 with the tive tyrosine-tyramine antiporter gene and an aminoacyl Clustal program. DNA was ampli®ed in an iCycler thermal cycler tRNA synthetase gene. Disruption of the tdcA gene yielded (Bio-Rad, Hercules, Calif.). The PCR conditions involved an ini- a strain unable to produce tyramine. The comparison of the tial denaturation step (958C for 5 min), 35 ampli®cation cycles cluster sequence with those held in databases led to the (958C for 45 s, 508C for 1 min, and 728C for 1 min), and a ®nal design of speci®c oligonucleotides that ampli®ed an inter- extension step at 728C for 7 min. All ampli®cations were per- nal fragment of the tdcA gene of different LAB genera. To formed with the use of puRe Taq Ready-To-Go PCR beads fol- our knowledge, this is the ®rst PCR method to detect ty- lowing the manufacturer's instructions (Amersham-Biosciences, ramine-producing bacteria in foods. Buckinghamshire, UK) and 200 nM of oligonucleotides as prim- ers. MATERIAL AND METHODS When DNA was used as a template, 10 ng was diluted in 25 ml of MilliQ water and used directly in the reaction.
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