Unité de Microbiologie et Génétique, UMR 5122, Université Claude Bernard-Lyon 1, bât. Lwoff, 10 rue Dubois, F-69622 Villeurbanne Cedex, France1
Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blancs, CH-1000 Lausanne 26, Switzerland2
Author for correspondence: Dominique Aubel. Tel: +33 4 72 43 13 66. Fax: +33 4 72 43 26 86. e-mail: aubel{at}biomserv.univ-lyon1.fr
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ABSTRACT |
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Keywords: amino acid catabolism, ß-cystathionase, PatC, lactic acid bacteria, cheesemaking
Abbreviations: CBL, cystathionine ß-lyase; DMDS, dimethyldisulfide; DMTS; dimethyltrisulfide; PLP, pyridoxal 5'-phosphate
a The GenBank accession number for the sequence determined in this work is AF423071.
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INTRODUCTION |
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Cystathionine ß-lyase (CBL) (EC 4.4.1.8, also commonly referred to as ß-cystathionase) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme which catalyses mainly the cleavage of L-cystathionine to homocysteine, pyruvate and ammonia via an /ß-elimination reaction; that is, the enzyme splits the
CN (deaminating) and ßCS bonds (Clausen et al., 1997
). Subsequently, the homocysteine is methylated by the homocysteine methyltransferase to form methionine (Weimer et al., 1999
). However MetC, the purified CBL, from Lactococcus lactis subsp. cremoris B78 or MG1363 is also able to degrade cystathionine to cysteine,
-ketobutyrate and ammonia, or to degrade methionine (
CS linkage) in methanethiol via an
/
-elimination reaction (Alting et al., 1995
; Dobric et al., 2000
). In a following reaction, the methanethiol can be oxidized to DMDS or DMTS (Parliment et al., 1982
). However, no significant differences in the amount of DMDS formed were found between the MG1363 parental strain and the metC mutant, suggesting that other enzymes may play a more substantial role in the conversion of methionine (Fernandez et al., 2000
). In fact, the cystathionine
-lyase (Bruinenberg et al., 1997
), the aromatic aminotransferase AraT (Yvon et al., 1997
; Rijnen et al., 1999
), and the branched-chain aminotransferases AT-A and AT-B (Engels et al., 2000
) or ILvE (Atiles et al., 2000
) have also been reported to contribute to the conversion of methionine to flavour compounds.
Although the sulfur-containing amino acid catabolism of lactococci is well described, data for the genus Lactobacillus are as yet very limited. Dias & Weimer (1998) reported the presence of CBL and methionine aminotransferase activities in some strains of lactobacilli. Moreover, a cystathionine
-lyase was purified from Lactobacillus fermentum DT 41, a strain contained in the natural starter used for Parmesan cheese production (Smacchi & Gobetti, 1998
). This enzyme was found to be stable under the conditions of cheese ripening and has therefore been thought to contribute to the biosynthesis of volatile sulfur-containing compounds, although L-methionine and L-cysteine were not the optimal substrate for the purified enzyme. Recently, methionine aminotransferase activities have been detected in 30% of Lactobacillus casei and 23% of Lactobacillus plantarum strains (Amarita et al., 2001
).
To our knowledge there are no data available on the catabolism of sulfur-containing amino acids in Lactobacillus delbrueckii species. In the present work, 14 strains of Lb. delbrueckii subsp. bulgaricus or subsp. lactis were screened for CBL activity. The corresponding patC gene was cloned from Lb. delbrueckii subsp. bulgaricus NCDO 1489, sequenced, and its distribution and expression assessed among lactic acid bacteria.
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METHODS |
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RNA preparation and RT-PCR.
RNA was purified from 1 ml of a fresh bacterial culture grown in MRS medium and harvested at OD600 value of 1·0. Then, bacterial cells were treated with the Rneasy Mini kit (Qiagen) according to a modified procedure. Cells were lysed with TE buffer containing 5 mg lysozyme ml-1 (45 min at 37 °C). Genomic DNA was eliminated by the action of the Rnase-free Dnase I (Qiagen) during RNA purification on the Rneasy Mini column (40 min at room temperature). RNA preparations were analysed by agarose gel electrophoresis and RNA concentrations were determined by absorbance at 260 nm.
cDNAs from patC and prtB mRNA were generated by RT-PCR using the Qiagen OneStep RT-PCR kit according to the suppliers instructions. Two amounts of RNA (0·15 or 1·0 µg) were used for each RT-PCR assay. Primers specific to prtB or patC genes from Lb. delbrueckii subsp. bulgaricus NCDO 1489 were used for amplification (Fig. 1). Primers PRTBsens1 (5'-GACAACCCACTCACTGGACAAG-3') and PRTBrev1 (5'-CTCTTGATCGTAAGCCGGGC-3') , primers PRTBsens2 (5'-TTGACGAAGAAAGATCAGGCCG-3') and PATCrev1 (5'-AACGCCGCCAAGTTGAAGG-3') generated a 504 bp or a 3326 bp amplicon, respectively. cDNA of patC (674 bp) was generated with PATCsens1 (5'-GGGCAATTCCATCAAGTGGG-3') and PATCrev1 primers. Before any RT-PCR assays, all primers were controlled by direct PCR using a ready-to-use PCR mixture of Gibco-BRL (Invitrogen) and genomic DNAs of four studied strains as templates. In order to check DNA contamination of RNA preparations, direct PCR was also carried out with the same primers and Gibco-BRL mixture.
Total cellular extracts for enzyme assays.
Cells from 300 ml of an overnight culture of Lactobacillus strains grown in Difco MRS broth were harvested, washed with 100 mM Tris/HCl pH 8·2 and then resuspended in 3 ml of the same buffer. The cells were afterwards disrupted at 6 °C by three passages through an Aminco French pressure cell at 20000 p.s.i. (138 MPa). Intact cells and cell debris were removed by centrifugation at 12000 g for 30 min at 6 °C. The supernatant was collected and protein concentration was estimated by the method of Bradford (1976) using bovine serum albumin as the standard. The extracts were used for enzyme assays as described below and stored at -20 °C.
Detection of CBL activity by in situ staining.
CBL activity was monitored in PAGE gels with either L-cystine or L-cysteine as the substrate. Crude extracts (0·3 mg) supplemented with SDS-free sample buffer [5x buffer is composed of 150 mM Tris/HCl pH 6·8, 30% (v/v) glycerol, 0·005% (w/v) bromophenol blue] were loaded on the polyacrylamide gel. A discontinuous system consisting of stacking and separation gels was used to separate proteins. The lower separation gel consisted of 12% (w/v) polyacrylamide in 150 mM Tris/HCl pH 8·8 with 0·1% (w/v) ammonium persulfate and 0·1% (w/v) tetramethylenediamine (TEMED). The stacking gel was composed of 4·8% (w/v) polyacrylamide in 200 mM Tris/HCl pH 6·8 with polymerization reagents at the same concentration as above. The electrophoresis buffer contained 192 mM glycine, 25 mM Tris at pH 8·3. After electrophoresis, the polyacrylamide gel was incubated in 100 mM Tris/HCl buffer pH 8·2 prewarmed to 37 °C and supplemented with 10 mM L-cysteine or L-cystine, 0·5 mM Pb(NO3)2 and 0·4 mM PLP.
CBL assay and kinetic constant determination.
The assay used for determination of kinetic parameters was based on the formation of free thiol groups by spontaneous disulfide interchange with Ellmans reagent [5,5'-dithiobis-(2-nitrobenzoic acid)] or DTNB according to the method described by Uren (1987). Assay mixtures contained 0·2 mM DTNB, 0·1 mM PLP, various substrate concentrations (L-cystine or L-cystathionine dissolved in water and 0·04 M HCl, respectively) ranging from 0·5 to 4 mM and purified His-tagged PatCNCDO 1489 in a final volume of 1 ml Tris/HCl 100 mM pH 8. The increase in absorbance at 412 nm was measured in a Uvikon 930 spectrophotometer at 37 °C; it was recorded at 0·5 min intervals for 10 min. A molar absorption coefficient for the aryl mercaptide of 13200 l mol-1 cm-1. was used to calculate the enzyme activity. One enzyme unit represents the formation of 1 µmol aryl mercaptan min-1. The LineweaverBurk plot (1/v against 1/s) was employed to calculate Km and Vmax values. The values reported represent a mean of three independent experiments±SD.
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RESULTS AND DISCUSSION |
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A search for protein homology revealed significant similarity of the deduced amino acid sequence to the potential amino transferase (PatB) protein (37·3% identity) of Bacillus subtilis and the MalY protein (31·4% identity) of E. coli (Fig. 2). Whereas the biological function of PatB is still unknown (Trach & Hoch, 1993
), MalY has been described as a pleiotropic protein acting as a PLP-dependent CBL and as a repressor of the maltose regulon by interaction with MalT, the central transcription activator of the maltose regulon (Zdych et al., 1995
; Clausen et al., 2000
). Considering the significant identity of the deduced amino acid sequence with PatB from B. subtilis (Fig. 2
), the ORF located immediately downstream of prtB gene from Lb. delbrueckii subsp. bulgaricus NCDO 1489 was named patC. The DNA sequence of the Lb. delbrueckii subsp. bulgaricus NCDO 1489 patC gene has been assigned GenBank number AF423071.
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To test our hypothesis, plasmid pDL19 (harbouring the 1·4 kb PCR fragment encompassing patC from NCDO 1489 and its 217 bp upstream region) was constructed and introduced into E. coli strain CAG18475, which contains a mutation in the CBL-encoding gene (metC) essential in the methionine biosynthetic pathway (Singer et al., 1989 ). E. coli strain CAG18475 harbouring plasmid pDL19 was able to grow on minimal medium M63 supplemented with glucose or glycerol as the carbon source, whereas the same strain carrying the parental vector pJDC9 still required methionine to grow on the same medium. Thus, we clearly demonstrated that the overproduction of PatC, as with MalY (Zydch et al., 1995), complements the methionine requirement of a metC mutant and that PatC functions in vivo as a CBL. Moreover, we confirmed that the patC gene of strain NCDO 1489 is expressed under its own promoter since the fragment cloned in pDL19 is flanked by efficient transcriptional terminators which prevent any interference by vector promoters (Chen & Morrison, 1987
).
Although PatC and MalY share some features, they differ in their number of subunits and their capacity to repress the maltose regulon of E. coli. The native enzyme MalY consists of two identical subunits (Clausen et al., 2000 ), whereas PatC from Lb. delbrueckii subsp. bulgaricus NCDO 1489 is apparently a homotetramer (data not shown) as for CBL from numerous bacteria (Alting et al., 1995
; Belfaiza et al., 1986
). In contrast to MalY, PatC overproduction did not repress the expression of a malK''lacZ fusion (data not shown). So, PatC is not able to control the regulation of the maltose regulon (malT-dependent genes). This is not surprising, since most amino acid residues involved in the interaction with the transcriptional activator MalT are not conserved in PatC, as shown in Fig. 2
(Clausen et al., 2000
).
Purification of PatC and biochemical features
In order to produce large amounts of PatC, the patC gene from NCDO 1489 was amplified from genomic DNA using primers pmal4 and pmal5 (Fig. 1) and subsequently cloned in-frame with a His6-tag coding sequence (pDL22). Moreover, this hybrid patC gene was under control of the IPTG-inductible T5 coliphage promoter. The His-tagged PatC protein was purified under native conditions by affinity chromatography on nickel-nitrilotriacetic acid (Ni-NTA) resin and its purity was confirmed by SDS-PAGE. Indeed, a single band running at the expected size of 45 kDa was observed following staining with Coomassie blue as shown in Fig. 3(a
, lane 2).
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Previous results (Aubel et al., 2002 ) have shown that like most CBLs (Alting et al., 1995
; Smacchi & Gobetti, 1998
; Bruinenberg et al., 1997
; Gentry-Weeks et al., 1993
), PatC is a PLP-dependent enzyme that is sensitive to hydroxylamine. PatC is also sensitive to the thiol-reactive agent iodoacetic acid and, in this respect, is similar to the CBL of Bordetella avium (Gentry-Weeks et al., 1993
). Nevertheless, the two enzymes differ in their thermostability (Gentry-Weeks et al., 1993
; Aubel et al., 2002
) and substrate affinity. Indeed under the conditions used (see Methods), the PatC enzyme exhibited slightly higher affinity for L-cystine (Km 1·88±0·37 mM; Vmax 48·69±0·95 U mg-1) than for L-cystathionine (Km 5·13±0·42 mM; Vmax 13·17±1·01U mg-1). Both of these Km values were higher than those reported for the MetC enzyme of B. avium (Km 0·0700·077 mM for L-cystathionine and L-cystine, respectively; Gentry-Weeks et al., 1993
) and E. coli (Km 0·040·25 mM; Dwivedi et al., 1982
) or Salmonella (Km 0·220·80 mM; Guggenheim, 1971
). On the other hand, the affinity of PatC and MalY of E. coli (Km 1·70±0·10 mM; Zdych et al., 1995
) for L-cystine was quite similar.
Distribution of the patC gene among lactic acid bacteria
An ORF with 97·7 and 98·4% identity to patC was detected downstream of the prtB genes of Lb. delbrueckii subsp. lactis CNRZ 250 and ATCC 21815, respectively, which suggested that the locus encompassing both genes was common to most Lb. delbrueckii strains. In order to determine whether the patC gene was specific to Lb. delbrueckii species or widely distributed in lactic acid bacteria, Southern hybridizations were performed. The patC probe hybridized with a single DNA fragment in all eight Lb. delbrueckii subsp. bulgaricus and all five subsp. lactis strains studied, as shown in Fig. 4(a, b
). However, under the conditions used, no hybridizing band was detected in the genome of Lactobacillus helveticus (four strains), Lb. casei (one strain) or Lc. lactis (three strains) (data not shown). The absence of hybridation with the Lc. lactis genome is not surprising, because the identity between PatC and MetC from Lc. lactis (Fernandez et al., 2000
) is very low (18·3%). Our results suggest that the patC gene is only present in Lb. delbrueckii species.
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Received 20 December 2001;
revised 15 March 2002;
accepted 8 April 2002.