1 Wageningen Centre for Food Sciences, Wageningen, The Netherlands
2 Flavour, Nutrition and Ingredients Department, NIZO Food Research, PO Box 20, 6710 BA Ede, The Netherlands
3 Institute of Molecular Genetics and Genetic Engineering, Belgrade, Yugoslavia
Correspondence
Michiel Kleerebezem
michiel.kleerebezem{at}nizo.nl
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ABSTRACT |
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Present address: Purac Biochem, Gorinchem, The Netherlands.
Present address: Instituto de Productos Lácteos de Asturias (CSIC) C/Infiesto s/n Villaviciosa, Asturias, Spain.
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INTRODUCTION |
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Genes of the cysteine regulon in E. coli and S. enterica serovar Typhimurium require a positive regulator protein CysB, a member of the LysR family of bacterial transcriptional regulators, as well as sulphur limitation and the presence of the inducer N-acetyl-L-serine, often provided as O-acetyl-L-serine (OAS) (Kredich, 1996; Ostrowski & Kredich, 1989
). OAS is a substrate for the cysteine synthase (CysK). The pathway involves transport and reduction of inorganic sulphate to sulphide in one branch and the synthesis of OAS from serine in another. The subsequent reaction of sulphide with OAS results in cysteine synthesis.
The genes involved in methionine biosynthesis in E. coli and S. enterica serovar Typhimurium are regulated by two regulator proteins, MetJ and MetR. The met regulon genes, except metH, are under negative transcriptional control of the MetJ repressor, with S-adenosylmethionine as a corepressor (Saint-Girons et al., 1988). In addition to MetJ-mediated negative control the metE, metA, metF, metH and glyA genes are under positive control of the MetR activator (Cowan et al., 1993
; Lorenz & Stauffer, 1996
; Mares et al., 1992
; Maxon et al., 1989
; Urbanowski & Stauffer, 1989
; Weissbach & Brot, 1991
).
Information on methionine and cysteine biosynthesis, and regulation of sulphur metabolism, in Gram-positive bacteria is limited. A number of Bacillus subtilis genes involved in cysteine and methionine biosynthesis contain a highly conserved sequence upstream of the coding region called the S-box (Grundy & Henkin, 1998). This motif contains a putative transcriptional terminator, suggesting that regulation is controlled via a transcription-termination mechanism. The S-box has a key role in the transcription control of the metIC operon (Auger et al., 2002
). In contrast, Mansilla et al. (2000)
showed that cysH operon transcription is independent of the S-box motif present upstream of this operon. CysL, a LysR-type transcriptional regulator, is the only regulator of sulphur genes identified in B. subtilis and acts as an activator of the cysJI operon (Guillouard et al., 2002
).
Previously, the metC gene and the encoded cystathionine -lyase, which has both
- and
-lyase activity, were characterized in L. lactis (Alting et al., 1995
; Fernández et al., 2000
). Cystathionine
-lyase is a tetrameric protein and its physiological function is the catalysis of a reaction in the methionine synthesis pathway, namely an
,
-elimination reaction from cystathionine to produce homocysteine, pyruvate and ammonia. Subsequently, the homocysteine is methylated to form methionine (Gottschalk, 1988
). The metC gene is present in an operon with the cysK gene, encoding a cysteine synthase, which catalyses the formation of cysteine from OAS and sulphide (Fernández et al., 2000
). Expression of the metCcysK operon is repressed by cysteine and, to a lesser extent, by methionine. Two genes, cmbR and cmbT, have been identified that are involved in regulation of metCcysK transcription (Fernández et al., 2000
).
CmbR is a LysR-type regulator protein essential for expression of the metCcysK operon in L. lactis (Fernández et al., 2002). LysR-type transcriptional regulators (LTTRs) constitute a large family of prokaryotic regulator proteins that includes the well-characterized CysB and MetR regulators (Henikoff et al., 1988
). The CysB regulator is a coinducer-responsive transcription regulator, which positively regulates the transcription of the cys regulon and negatively regulates its own transcription and transcription of the hslJ gene. CysB binds to cognate promoters via a 15 bp dyad repeat with a common structure and position independently of the presence of a coinducer. The coinducer causes additional interactions of the regulator with sequences near the 35 RNA polymerase binding site that result in bending and transcription activation (Schell, 1993
; Jovanovic et al., 2003
).
By analogy with CysB, CmbR is expected to require a co-inducer (N-acetylserine) to allow binding to the activation site. However, the activator-binding sequences described for the CysB-responsive promoters in E. coli and S. enterica serovar Typhimurium (Hryniewicz & Kredich, 1995; Wu et al., 1995
) were not found in the lactococcal metC promoter (Fernández et al., 2002
). For a number of LTTRs such as AmpR (Lindquist et al., 1989
), IlvY (Wek & Hatfield, 1988
), NahR (Huang & Schell, 1991
), OccR (Cho & Winans, 1993
), TrpI (Chang & Crawford, 1991
) and MetR (Byerly et al., 1991
; Urbanowski & Stauffer, 1989
), dyad symmetry elements have been described in the binding sites, while both inverted and direct repeats have been found in NodD (Fisher & Long, 1993
; Goethals et al., 1992
; Wang & Stacey, 1991
) and CysB (Hryniewicz & Kredich, 1995
) binding sites.
In this paper we analyse the metC promoter in L. lactis, which contains three inverted repeat sequences and two direct repeats located upstream of the 35 region. We demonstrate the role of the second direct repeat sequence (DR2) in the regulation by CmbR.
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METHODS |
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Plasmid constructions.
To study the regulation of the metCcysK operon, deletion derivatives were made of pNZ9340 (Fernández et al., 2002) that contain the gusA gene under control of PmetC. A schematic representation of the deletion derivatives is shown in Fig. 1
. Smaller promoter regions were amplified by PCR using the primers PmetCF1 (5'-GCGCCGGGATCCCGAAAGATTTAGAAAATATTAAA-3'), PmetCF2 (5'-GCGCCGGGATCCGATGTCCTCGTTTTTTTAT-3'), PmetCF3 (5'-GCGCCGGGATCCATAAAAAAAGTTAATTCTGCT-3'), PmetCF4 (5'-GCGCCGGGATCCGTTAATTCTGCTATAAAAAAATCT-3') or PmetCF5 (5'-GCGCCGGGATCCCTTATAGCACTGGGCACAC-3') in combination with PmetCR (5'-GGCCGCGAATTCCAGCAAGTCCTGAACTAAATG-3'). All PCR amplifications were performed by using the Advantage Genomic Polymerase Mix (Clontech) in 25 successive cycles according to the instructions of the manufacturer. Resulting DNA fragments were digested with BamHI and EcoRI (sites introduced into the primers; underlined) and cloned into similarly digested pUC18 (Yanish-Perron et al., 1985
). The integrity of the inserts was confirmed by sequencing and, subsequently, the BamHIEcoRI fragments were subcloned upstream of the promoterless gusA gene in pNZ273 (Platteeuw et al., 1994
). The resulting PmetCgusA fusions were subcloned into the low-copy-number plasmid pIL252 (Simon & Chopin, 1988
) as EcoRIHindIII DNA fragments, generating the plasmids pNZ7211, pNZ7212, pNZ7213, pNZ7214 and pNZ7215, respectively.
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The cmbR gene was amplified using primers 5'-GCCGCGTCATGAATATTAAACAATTACGGTACG-3' and 5'-GCCGCGCTCGAGTTATCGCGAGAATTCATAGTTTTCAAAATAAACTTCA-3', and using plasmid pNZ9347 (Fernández et al., 2002) as a template. The PCR amplifications were performed using Pwo DNA Polymerase (Roche) in 25 successive cycles following the instructions of the manufacturer. The resulting DNA fragment was digested with RcaI and NruI (sites introduced in the primers; underlined) and cloned into similarly digested pNZ8113. The resulting plasmid, pNZ7200, was introduced into NZ9000cmbR, which was subsequently used for overexpression of CmbR-Histag protein under control of the NICE system (Kuipers et al., 1998
).
DNA sequencing.
Automatic double-stranded DNA sequence analysis was performed on both strands with an ALFred DNA sequencer (Amersham Biosciences). Sequencing reactions were accomplished by using the AutoRead sequencing kit, using Cy5-labelled universal and reverse primers according to the instructions of the manufacturer (Amersham Biosciences).
Enzyme assays.
For these assays the cells were grown in CDM with different methionine and cysteine concentrations to an OD600 of 0·7. For the preparation of extracts, cells were disrupted with zirconium beads in a Bio101 Fast Prep (two treatments of 30 s with intervals of 1 min on ice between treatments) and cellular debris was removed by centrifugation. The extracts were kept on ice, and enzyme assays were performed within 4 h. Activity of the -glucuronidase (GusA) enzyme was measured as described previously (Kuipers et al., 1995
); 40 µl of extract was added to 950 µl buffer (50 mM sodium phosphate, pH 7·0, 10 mM
-mercaptoethanol, 1 mM EDTA, 0·1 % Triton X-100) and 10 µl 100 mM p-nitrophenyl-
-D-glucuronic acid (Clontech). The mixture was incubated, and the increase in A405 was measured at 37 °C in a Cary 1E UVvisible spectrophotometer (Varian) with a thermostatically controlled compartment. Specific activity was calculated as increase in A405 min1 (mg protein)1. Protein concentrations were determined with a protein assay based on the method of Bradford (1976)
, using bovine serum albumin as a standard.
Preparation of extracts enriched in CmbR protein.
Cell-free extract (CFE) enriched in CmbR-Histag protein was isolated from NZ9000cmbR harbouring plasmid pNZ7200. As a negative control CFE was isolated from strain NZ9000cmbR without plasmid pNZ7200. Cells were grown to an OD600 of 0·5 and were subsequently induced with nisin (1 ng ml1). Following induction, growth was continued for 2 h, then cells were harvested and resuspended in 1 ml milliQ water. The cell-free extracts were prepared with 0·8 g zirconium beads (0·1 mm, Biospec Products) using a Bio101 FastPrep (two 30 s treatments, with cooling on ice between the runs). Samples were centrifuged for 1 min and the supernatants were used as CFEs. Samples were divided into small portions, frozen in liquid nitrogen, and stored at 70 °C until further use. Protein concentrations of the CFEs were determined as described above.
Gel mobility shift assay.
A DNA fragment containing the F1metC promoter region was generated by PCR using primers PmetCF1 and PmetCR. The PCR product obtained was 5'-labelled with [-32P]ATP using T4 polynucleotide kinase (Gibco-BRL), and purified by JETquick PCR Purification Spin Kit (Genomed). Binding reactions and gel mobility shift assay were performed as described previously (Lochowska et al., 2001
). Briefly, the reaction mixtures (40 µl) contained approximately 10 ng labelled DNA fragment and 1 µg poly(dIdC) (to reduce non-specific binding), in the CmbR buffer containing 40 mM Tris/HCl (pH 8·0), 10 mM MgCl2, 100 mM KCl, 1 mM dithiothreitol and 0·2 % Tween 20. Incubation (15 min at 37 °C) was initiated by adding a variable amount of CFE and OAS where indicated. OAS was always prepared fresh as a 10 mM stock solution. Samples were separated on 5 % acrylamide/bisacrylamide (30 : 0·8) vertical gels in 0·5 M Tris/borate/EDTA buffer (pH 8·3) for 1·5 h at 10 V cm1 and the radiolabelled bands were visualized by autoradiography.
Magnetic DNA affinity purification.
A biotin-labelled F1 PmetC fragment (F1b) was obtained by PCR by using the primers PmetCF1 and 5'-biotin-labelled PmetCR. One hundred microlitres of Magnasphere beads (Streptavidine Magne Sphere Paramagnetic Particles; Promega) were washed three times with 100 µl 0·5x SSC (1x SSC: 150 mM NaCl, 15 mM trisodium citrate) and three times in 100 µl TE buffer pH 8·0. Beads were resuspended in 100 µl TE buffer pH 8·0 containing the F1b fragment (4 µg), and incubated for 30 min at 25 °C with rotation until biotin-labelled DNA was bound to the beads. The beads were then washed three times with CmbR buffer to eliminate unbound DNA. Approximately 0·03 nmol of the F1b fragment was bound to the Magnasphere beads. A 1 ml sample of CFE enriched in CmbR-Histag protein (500 µg protein) in CmbR buffer was added and incubation was continued for 5 min at 30 °C. Unbound proteins were removed in five wash steps with 1 ml CmbR buffer. Proteins bound to the beads were eluted with 1 ml 1 M KCl. The solutions from the wash steps and elution were precipitated with TCA, and the precipitates were dissolved in 20 µl milliQ water. Residual TCA was neutralized by adding 4 µl 1 M Tris/HCl pH 8·0. Protein samples were run on 12·5 % SDS-PAGE and analysed by silver staining (Wray et al., 1981) or Western blotting. Western blotting was performed essentially as described by Towbin et al. (1979)
, using 1 : 5000 diluted anti-tetra-His antibody (Tetra-His Antibody, BSA-free; Qiagen) as a primary and 1 : 5000 diluted GAMPO [Goat-Anti-Mouse IgG (H+L) (HAS) Peroxidase Conjugate, Gibco-BRL] as a secondary antibody. Peroxidase activity was detected using 4-chloro-1-naphthol, according to the instructions of the manufacturer (Gibco-BRL).
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RESULTS AND DISCUSSION |
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Deletion analysis of the metCcysK promoter region
The region upstream of the transcription start of metC contains two direct (DR1 and DR2) and three inverted (IR1, IR2 and IR3) repeats (Fig. 1). These repeats were anticipated to play a role in the regulation of transcription of the metCcysK operon, which is mediated by the transcriptional activator CmbR (Fernández et al., 2002
). To determine which of the repeats is involved in the regulation of metCcysK transcription, we constructed fusions of metC promoter fragments of various lengths to the
-glucuronidase-encoding gusA gene in a low-copy plasmid (pIL252). All constructs contain the 35 and 10 sequences, the transcription start site, ribosome-binding site and the 5'-terminus of the metC gene (Fig. 1
). Plasmid pNZ9340 contains the entire metC promoter (fragment F0, Fig. 1
). Progressive 5'-truncations of the metC promoter fragment in pNZ9340 (Fig. 1
) were constructed removing, successively, DR1 (fragment F1, pNZ7211); DR1 and the 5'-half of IR1 (fragment F2, pNZ7212); DR1, IR1 and the 5'-half of IR2 (fragment F3, pNZ7213); DR1, IR1, IR2 and the 5'-half of DR2 (fragment F4, pNZ7214); and DR1, IR1, IR2, DR2 and the 5'-half of IR3 (fragment F5, pNZ7215).
L. lactis NZ9000 derivatives harbouring one of the metC promoter constructs were grown in CDM without cysteine and with 0·1x methionine (0·1xM), or with 10x methionine and 10x cysteine (10xM/10xC). Quantitative GusA analysis showed that the promoter fragments F0, F1, F2 and F3 could drive the high-level expression of -glucuronidase in growth media with low methionine and without cysteine. In contrast,
-glucuronidase activity levels were barely detectable in cells harbouring the F4 and F5 promoter fusion constructs under the same conditions (Fig. 2
). However, the apparent discrepancy between the
-glucuronidase activities in cells harbouring F0, F1, F2 and F3 promoter fusion constructs could be explained by different lengths of the DNA fragments. It is well known that upstream sequences can mediate the promoter activity (Watson et al., 1988
). For example, the nucleotide sequence in the deleted region could represent a target site for topoisomerases that change local DNA supercoiling. Alternatively, it is possible that differences in the upstream region might affect the bending of DNA in the DR2, resulting in different affinity of CmbR for its target sequence, considering the fact that LTTRs do not recognize consensus sequences, but DNA topography. Moreover, the bending of DNA could cause the difference in RNA polymerase promoter-binding affinity. Since the addition of methionine and cysteine (10xM/10xC) resulted in an almost complete loss of
-glucuronidase activity, the low concentration of methionine and lack of cysteine in the growth medium were essential for activation of the metC promoter fragments F0, F1, F2 and F3.
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Interaction of the CmbR protein with PmetC
Initially, interaction of CmbR with PmetC was evaluated by magnetic DNA affinity purification using a biotin-labelled 407 bp F1b PCR fragment, which contains the metC promoter region from positions 135 to +258 relative to the transcription start site. CFE of L. lactis NZ9000cmbR harbouring pNZ7200 in CmbR buffer was added to the beads. The magnasphere beads were washed with CmbR buffer to remove unbound proteins (lanes 15 in Fig. 3), after which bound proteins were eluted using a high-salt solution (EL in Fig. 3
). Analysis of the protein fractions obtained by SDS-PAGE showed that the last wash step contained only minor amounts of protein, while the elution fraction contained a prominent protein band of approximately 36 kDa. This molecular mass is in good agreement with the predicted value for CmbR-Histag (35 691 Da) (Fig. 3a
). Moreover, Western blot analysis using anti-tetra-His antibodies strongly suggested that the protein bound to the F1b fragment was CmbR-Histag (Fig. 3b
).
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Many LTTRs bind to their target sequences without the presence of coinducer. The coinducer causes a conformational change of the LTTR, altering the bending of the DNA and activating transcription (Schell, 1993). This has been demonstrated for promoters that are positively regulated by MetR, with homocysteine as a co-inducer (Urbanowski & Stauffer, 1989
; Martin et al., 1986
; Wek & Hatfield, 1988
), and for promoters that are positively regulated by CysB (cysK, cysP), with N-acetylserine as a co-inducer (Hryniewicz & Kredich, 1995
; Monroe et al., 1990
). In the binding experiments performed with CysB and its regulated promoters, slow and fast complexes were detected (Monroe et al., 1990
). A model was proposed in which a single molecule of CysB bends the cysK promoter by binding simultaneously to two cognate sites, in the absence of acetyl-L-serine. This DNAprotein interaction is unfavourable for transcriptional activation of the promoter. As the bending affects electrophoretic mobility, this complex migrates slowly during gel electrophoresis. Acetyl-L-serine is thought to induce a conformational change of CysB, allowing the protein to interact preferentially with one of the cognate sites. Bending is eliminated and fast complex is formed. In this study we did not observe slow and fast complexes of CmbR bound to PmetC in the presence and absence of OAS. CmbR binds to PmetC in the presence of OAS, while in the absence of OAS only a small amount of DNA fragment was bound. These results suggest that the presence of OAS is essential for the binding of CmbR to the metC promoter, and that in contrast to CysB, it is more likely that OAS does not induce the conformational change of already existing complex but facilitates the binding of CmbR to its binding site.
In conclusion, we have demonstrated that PmetC is regulated at the transcriptional level by interaction of the activator protein CmbR with PmetC, and shown that the nucleotide sequence positioned from 80 to 67 is involved in this interaction. The presence of OAS is essential for proper binding of CmbR to the promoter region. This mechanism of gene expression regulation by CmbR from L. lactis is different from that of CysB and MetR from E. coli, in which their respective coinducers cause a conformational change of the regulator that is already bound to the DNA.
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ACKNOWLEDGEMENTS |
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Received 16 June 2004;
revised 30 September 2004;
accepted 26 October 2004.
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