INTRODUCTION

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Lactococcus lactis is a gram-positive bacterium related to members of the Streptococcus, Lactobacillus, and Bacillus genera (30). It obtains all energy by fermenting sugars to lactic acid and can be isolated from raw milk. While most strains of L. lactis are multiply auxotrophic for both amino acids and vitamins (30), the ability to synthesize both pyrimidine (15) and purine (21) nucleotides de novo is retained in all isolates of L. lactis tested. The de novo synthesis of purine nucleotides requires 10 enzymatic steps leading to IMP, which functions as a precursor for both AMP and GMP nucleotides (35). Purine nucleotides can also be formed by salvage reactions from exogenous purine nucleosides or bases (22). Whereas the de novo pathway appears to be conserved among most organisms, the salvage pathway can vary between organisms (22), and so far only one gene involved in purine salvage is known in L. lactis (21).

The organization of bacterial genes involved in purine metabolism and the regulation of the expression of these genes have been best studied in Escherichia coli, Salmonella typhimurium (19, 36), and Bacillus subtilis (19, 34, 35). In the gram-negative bacterium E. coli, the purine biosynthetic genes are scattered around the chromosome (19). However, the transcription of all of these genes is repressed by a single regulatory protein, the purR-encoded purine repressor (8, 13, 16, 23). Binding of the E. coli PurR repressor to its target DNA sequences (PurBox's) is stimulated by the corepressors guanine and hypoxanthine (17, 24). In the gram-positive bacterium B. subtilis, all de novo genes are organized in a single transcriptional unit, the purine operon (4). The transcription of the operon is controlled by two independent mechanisms. Initiation is controlled by a repressor which is encoded by the purR gene and which, at low 5-phosphoribosyl-1-pyrophosphate (PRPP) concentrations, binds specifically to a DNA sequence in the promoter region (5, 33). A rationale for the use of PRPP as an indicator of purine availability was put forward by Weng and coworkers (33). Upon uptake, adenine is converted to AMP, consuming PRPP in the process. The subsequent phosphorylation of AMP yields ADP, which is the primary inhibitor of PRPP synthetase. Thus, the combined inhibition of PRPP synthesis and increased PRPP consumption may explain why high extracellular adenine pool levels are correlated with low PRPP pool levels in B. subtilis (26). The purR genes from B. subtilis and E. coli are unrelated, and the B. subtilis enzyme shows a high degree of similarity with purine phosphoribosyltransferases (1, 12, 33), while the E. coli enzyme is a classical lacI-type repressor (28). The second mechanism controlling the expression of the pur operon in B. subtilis involves a terminator-antiterminator structure located between the promoter and the translation start site of the first gene. The formation of the antiterminator structure is believed to be prevented by the binding of an unidentified RNA binding protein in the presence of the purine base guanine (4) and hypoxanthine (1), thus resulting in premature termination of transcription.

We recently reported the nucleotide sequence and characterization of the purDEK operon from L. lactis CHCC285 (20). The transcription of the genes was shown to be down regulated more than 30-fold upon the addition of purines to a chemically defined medium. Deletion analysis of the region upstream of the purD reading frame made it possible to localize the promoter to a 133-bp fragment. By monitoring the promoter activity from different DNA fragments in a promoter fusion vector, we found that the 133-bp fragment also retained full purine regulation. A specific deletion mutant in which sequences from 78 bp upstream from the transcriptional start site was removed showed greatly reduced promoter activity. This result suggested that the affected region was a positive regulatory element (20).

Here we report the further characterization of the regulatory region located in front of the purD gene in L. lactis CHCC285. After cloning of the purC gene from the same strain, we were able to identify a common motif, designated a PurBox, which is present in two copies in purC and in one copy in purD. We present evidence that the PurBox is a positive regulatory site, most likely the binding site for a transcriptional activator.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. The strains used in the present study are listed in Table 1. Cultures of L. lactis were grown in either DN medium (3) or SA medium (11) supplemented with 1% glucose (GSA medium) and erythromycin at 2 µg/ml when required. Purine additions were made at the following final concentrations: guanosine (30 µg/ml), adenine (15 µg/ml), and hypoxanthine (15 µg/ml). SR plates (9) were used for plating transformants of L. lactis after electroporation.

Oligonucleotide primers. The oligonucleotide primers used in the present study are listed in Table 2 and were obtained from T-A-G-Copenhagen ApS, Copenhagen, Denmark.

Cloning of the purC gene. Chromosomal DNA from L. lactis CHCC285 was digested with HindIII and inserted into the HindIII site of the L. lactis-E. coli shuttle vector pCI3340 (7). After transformation of E. coli DH5 with the ligation mixture, plasmid DNA was extracted from the resulting pool of transformants. Subsequently, the pur mutant DN207 (3) was transformed to chloramphenicol resistance with the pCI3340 library. Upon subsequent analysis of the transformants on solid DN medium in the absence and the presence of purines, purine prototrophic transformants were selected.

Nucleotide sequence determination. The nucleotide sequence was determined with either a Sequenase 2.0 sequencing kit (Stratagene) or a Thermosequenase kit (containing nucleotides labelled with ³³P-dideoxynucleoside triphosphates) (Amersham LifeScience).

Construction of purC-lacLM and purD-lacLM fusion plasmids in pAK80. The construction of the fusion plasmids pLN95, pLN96, and pLN97 has been described elsewhere (20). The DNA fragments inserted in plasmids pMK1013, pSH2, pSH4, pSH5, pSJ2, pSJ5, pSW1, pSW2, and pSW5 were all generated by PCR amplification of either CHCC285 chromosomal DNA or purified pLN95 plasmid DNA. The DNA template and oligonucleotide primers used in each construction are listed in Table 3. Each upstream primer has a HindIII site incorporated and each downstream primer has a PstI site incorporated for convenient insertion into the vector. pAK80 plasmid DNA was digested with the two restriction enzymes, and the linearized vector DNA was purified with a High Pure PCR purification kit from Boehringer. Each of the PCR-generated fragments was likewise digested with PstI and HindIII and purified. About 500 ng of digested pAK80 and 100 ng of digested PCR product were ligated in a total volume of 25 µl. Two microliters was used to transform MG1363 to erythromycin resistance, with selection on SR plates (9) containing erythromycin (2 µg/ml) and supplemented with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) (200 µg/ml) for the detection of promoter activity in the inserts. After purification of the transformants, the plasmid DNA was extracted (25). The nucleotide sequence of the inserts was determined after PCR amplification of the regions on the plasmid and the PAK80 and PAK80ERM primers (Table 2), specific for regions flanking the cloning sites (data not shown).

Growth conditions and determination of -galactosidase activity. The plasmid-containing strains were grown exponentially at 30°C in 50 ml of purine-free GSA medium supplemented with erythromycin (2 µg/ml) with slow magnetic stirring. For purine-excess conditions, guanosine (30 µg/ml), adenine (15 µg/ml), and hypoxanthine (15 µg/ml) were added. Growth was monitored by measuring the optical density at 450 nm (OD₄₅₀) of the culture. At an OD₄₅₀ of about 0.8, 10 ml of bacterial culture was harvested and washed in 0.9% NaCl. After resuspension in 500 µl of Z buffer (18), cells were disrupted by sonication two times for 1 min each time with an amplitude of 8 µm. The cell debris was removed by centrifugation at 20,000 × g for 10 min, and -galactosidase activity was determined from 10 and 50 µl of the crude extract in an assay volume of 600 µl. Specific activity is given as micromoles of ortho-nitrophenol (ONP) formed per minute per milligram of protein, with a molar extinction coefficient for ONP of 0.045 at 420 nm (18). Protein concentration was determined according to the method of Lowry et al. (14).

RNA extraction. For analysis of the purC transcriptional start site in L. lactis CHCC285, RNA was extracted from bacterial cultures after growth in purine-free GSA medium and GSA medium containing guanosine, adenine, and hypoxanthine. At an OD₄₅₀ of 0.6, 5 ml of culture was mixed with 5 ml of EPS solution (60% ethanol, 2% phenol, 0.9% NaCl) prechilled at 30°C to rapidly cool the cell culture (32). After centrifugation at 5,000 × g for 5 min at 4°C, the pellet was washed in EPS solution mixed 1:1 with 0.9% NaCl. The pellet was frozen at 80°C and lyophilized in a vacuum centrifuge without heating. The lyophilized pellet was ground with acid-washed glass beads (100 µm; Sigma) with the rounded tip of a melted pasteur pipette as a pestle, and RNA was extracted essentially as described by Vogel et al. (32). For detection of the start sites in fusion plasmids pLN95 and pSW1, plasmid-containing strains SH1 and SW1 were grown as described above except that erythromycin (2 µg/ml) was added to the cultures. RNA was extracted as described above.

Primer extension analysis. Primer extension analysis of purC transcripts from CHCC285 was done as previously described (20) with primer MKP58. A slightly modified procedure was used for the mapping of start sites from fusion plasmids in MG1363. About 10 pmol of oligonucleotide primer was end labeled at 37°C in 10 µl of kinase buffer (50 mM Tris-Cl [pH 7.6], 10 mM MgCl₂) containing 5 µl of -[³²P]ATP (>5,000 mCi/mmol, ~3 mM) and 1 U of polynucleotide kinase. After incubation for 30 min, the enzyme was inactivated at 95°C for 10 min. To 5 µl of this mixture was added 20 µg of RNA, 9 µl of 5× first-strand buffer (250 mM Tris-Cl [pH 8.3], 375 mM KCl, 15 mM MgCl₂, SuperScript reverse transcriptase [Gibco BRL Life Technologies]), and water to a final volume of 30 µl. Hybridization was performed with a thermocycler for 10 min at 94°C, followed by 5 min at each of the following temperatures: 75, 73, 71, 69, 67, 65, 63, 61, 59, 57, and 55°C. During incubation at 55°C, the following were added to the sample: 8.5 µl of preheated water, 4.5 µl of 100 mM dithiothreitol, 1 µl of 10 mM deoxynucleoside triphosphates (dNTPs) (10 mM each dATP, dGTP, dCTP, and dTTP), and 1 µl of SuperScript II reverse transcriptase (Moloney murine leukemia virus RNase H negative; Gibco BRL). Incubation was continued for 15 min. The nucleic acids were precipitated by the addition of 160 µl of TE (10 mM Tris-Cl, 1 mM EDTA) (pH 8) and 20 µl of 3 M sodium acetate (pH 4.8), followed by 500 µl of absolute ethanol. After centrifugation and drying of the pellet, the RNA-cDNA was dissolved in 6 µl of H₂O, and 4 µl of stop solution (from the Thermosequenase kit) was added. A 2.5-µl sample was applied to a sequencing gel beside a sequence ladder obtained with the same primer and a PCR-amplified fragment from MG1363 chromosomal DNA as a template.

Nucleotide sequence accession number. The nucleotide sequence for purC has been assigned GenBank accession no. AF054888.

	RESULTS AND DISCUSSION

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Cloning of the purC gene from L. lactis subsp. lactis CHCC285. Purine prototrophic transformants were selected on purine-deficient DN agar plates after transformation of the uncharacterized pur mutant DN207 (3) with recombinant plasmids from a library of CHCC285 DNA in plasmid pCI3340 (see Materials and Methods). Plasmid pLN63 was extracted from one such transformant. A preliminary determination of the nucleotide sequence (data not shown) of the 4.3-kb insert in plasmid pLN63 showed that the insert carried sequences homologous to the purC and purQ genes of B. subtilis (data not shown).

Nucleotide sequence of the purC promoter region and determination of the transcriptional start site. Figure 1A shows the nucleotide sequence of a region from about 250 bp before to 50 bp after the start codon in the purC gene present in plasmid pLN63. Translation of the purC gene is most likely initiated from a GTG start codon at position 255 which is preceded by a good ribosome binding site (GGAGG). This start site is consistent with the start site of purC from E. coli (31) (having the N-terminal sequence H₂N-MQKQAELYRGKAK-, where underlined amino acids are identical to those in the lactococcal enzyme). Upstream of the purC gene an open reading frame was detected from position 1 to position 110, where it encounters a stop codon (orfC; Fig. 1A). Following this reading frame is a putative terminator structure at positions 160 to 191, indicating that transcription from orfC does not proceed into the purC gene. Thus, the intercistronic space between orfC and purC is composed of 146 bp.

View larger version (46K): [in this window] [in a new window]   FIG. 1.   purC (A) and purD (B) promoter regions from L. lactis CHCC285. Transcriptional start sites (+1), putative 10 regions, putative operator sites (PurBox), start codons, and ribosome binding sites (Shine-Dalgarno [SD]) are shown in boldface, underlined, and marked above the nucleotide sequence, while the amino acids specified by the structural genes are shown below the sequence . End points of inserts in promoter fusion plasmid pAK80 (marked with the plasmid designations pLN95 to pLN97) are shown below the sequence as arrows, with the ending nucleotide aligned with the wavy line. Oligonucleotide primers (marked by an MKP designation) used for the generation of PCR products are also shown below the sequence, with the wavy lines aligned with the nucleotides present. The nucleotide sequence of the purD promoter region was modified from that given in reference 20.

View larger version (103K): [in this window] [in a new window]   FIG. 2.   Primer extension analysis of purC and purD transcriptional start sites. Primer extension experiments were performed by using 10 µg of RNA extracted from CHCC285 (purC) and primer MKP58 (A), SW1 (purC-lacLM) and primer PAK80 (B), and SH1 (purD-lacLM) and primer PAK80 (C). RNA was extracted from cells growing exponentially in GSA medium (lanes 2) or in the same medium supplemented with purines (lanes 1). Lanes G, A, T, and C, sequencing reactions with the same primers as in the primer extension experiments and with PCR-generated DNA as a template. Asterisks indicate the positions of the flanking nucleotides shown in the sequences adjacent to the lanes with the extension products. The picture was scanned at 300 dpi with a Scan Jet 4c/T (Hewlett-Packard Co.) and DeskScan II version 2.3 software. The TIF file was imported into Top Draw version 3.1 for the addition of text.

Identification of a putative regulatory region. When the purC promoter region was compared to the promoter region of the purDEK operon (20) from the same strain, we found that they share a number of features. Both promoters have A residues as the starting nucleotides, they both have reasonable 10 regions (TAGAAT for purC and TAAGAT for purD), and neither contains sequences with similarity to 35 regions (Fig. 1). Most striking, however, is the presence of an identical decanucleotide stretch (ACCGAACAAT) which can be found at exactly the same positions (positions 79 to 70) relative to the transcriptional start site (Fig. 1). The absence of well-defined 35 regions for the promoters and the conservation of the decanucleotide at about position 75 relative to the transcriptional start site suggested that the decanucleotide could serve as the binding site for a transcriptional activator (27). In a search for sequences resembling the decanucleotide, we found a number of regions in the purC and purD genes. These sequences are shown in Table 4. As shown, the similarity between the sequences extends past the decanucleotide, and a consensus sequence for the five sequences can be found (AWWWCCGAACWWT). Since it is likely that these regions represent binding sites for a purine-specific activator, we have termed the sequences PurBox's. Most interesting is a sequence (PurBox C1) positioned just upstream of the previously identified PurBox (PurBox C2) in purC. These PurBox sequences are so close that two activator proteins bound to each site would be likely to come in contact.

Deletion of PurBox sequences destroys purine regulation and lowers promoter activity. In order to analyze the importance of the PurBox sequences for purC and purD promoter activities, we constructed a set of promoter fusions to a reporter gene for each promoter region. The fusions were constructed by inserting DNA fragments in front of the promoterless lacLM operon in the transcriptional fusion vector pAK80. The resulting plasmids were introduced into L. lactis MG1363. After exponential growth of the transformants in GSA medium with or without purine additions, the level of plasmid-encoded -galactosidase activity was determined. Figure 3B shows the localization of the purD DNA fragments present in front of lacLM together with the resulting -galactosidase levels. The end points of the fragments, which were all generated by PCR amplification of DNA derived from strain CHCC285, are shown in Fig. 1B (see also Table 3 for details). When the levels of -galactosidase produced from the purD transcriptional fusion plasmids were analyzed (Fig. 1), the region upstream of the PurBox was found to play no role in purine regulation (compare pSH4 and pSJ2). The promoter 10 and +1 regions, however, were crucial for purD-lacLM transcription (compare pSH4 and pSH5). The importance of the PurBox is indicated by the constitutive low level of transcription which was produced from the promoter when the G7 residue in the PurBox was mutated to a C (compare pSJ5 and pSJ2).

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Expression of -galactosidase from purC-lacLM (A) and purD-lacLM (B) fusions. The locations of the DNA fragments inserted in each fusion plasmid are shown below the structures of the promoter regions. Black boxes correspond to structural genes, while open and gray boxes correspond to intact and mutated PurBox sequences, respectively. Specific -galactosidase activities are given as micromoles of ONP produced per minute per milligram of protein. The strains (MG1363 transformed with the indicated plasmids) were grown in GSA medium containing erythromycin (Pur) or in the same medium containing guanosine, adenine, and hypoxanthine (+Pur). MG1363 transformed with vector pAK80 showed a low and constant level of background activity (<2 U/mg of protein) under both growth conditions. Fold regulation is given as the values for Pur/+Pur.

Conclusion. From the analysis of the purine-mediated regulation of purC and purD transcription, we can conclude that efficient transcription requires a PurBox in the promoter region. We do not have enough data to conclude that the activating PurBox has to be located with the central G7 exactly at position 76 relative to the transcriptional start site, but the fact that both promoters share this feature leads us to expect that this is the case. It is interesting that the number of PurBox's in the purC promoter affects only the activity and not the regulation of the promoter. This finding may not, however, be too surprising when it is taken into account that the two PurBox's are spaced by 17 bp, which corresponds to 1.65 helical turns or an angular spacing of approximately 130°. So, if the RNA polymerase is able to make contact with an activator protein bound to PurBox C2, as we expect, it should be unable to make a similar contact with the corresponding region on an activator protein bound to PurBox C1, as this is turned 130° away. It is more likely that two activators are able to form protein-protein contacts while bound to the DNA and that the resulting dimer has an enhanced binding affinity due to the presence of two DNA binding sites, as has been seen for many repressors (2). If more PurBox's were present, it could be envisioned that the multimerization of activators would result in a helix-like protein structure along the DNA. This suggestion may seem a little premature in the present context, but in the accompanying report (12) we identified a regulatory gene, purR, which is required for pur gene expression, and we present genetic evidence that the PurBox's identified in the present study are binding sites for the PurR protein. Interestingly, the L. lactis PurR activator is very similar to the purR product from B. subtilis, which is a repressor of pur expression (33). Although the regulatory effects of the two PurR proteins are opposite, in the accompanying report (12) we present a unifying model which focuses on binding of the PurR proteins to PurBox's. Sequences resembling PurBox's are present in all PurR-regulated genes in both L. lactis and B. subtilis (12). The binding of the B. subtilis PurR repressor to its target DNA has been found to involve an extended DNA region of approximately 60 to 80 bp (29) which was proposed to be due to wrapping of the DNA around PurR (33). The data, however, could also be interpreted as the result of PurR multimerization along the DNA, as proposed above for the L. lactis PurR activator.