INTRODUCTION

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In all organisms, pyrimidine metabolism is required in order to supply the cell with building blocks for the synthesis of DNA, RNA, and certain coenzymes needed in central metabolic pathways. In Lactococcus lactis, this requirement can be fulfilled either by the use of nucleosides and nucleobases present in the growth medium (36-38) or by means of the pyrimidine biosynthetic pathway, which seems to be universal in all prototrophic organisms investigated so far and consists of six enzymatic steps leading to the formation of UMP.

Many metabolic genes in L. lactis such as genes involved in the biosynthesis of amino acids (4, 10, 18) and glycolysis (6, 7, 31) have been identified and sequenced. The amino acid biosynthetic genes were found to be members of large operons, organized like the ones identified in Bacillus subtilis. Likewise, the genes encoding the pyrimidine biosynthetic enzymes in different gram-positive bacteria like Bacillus caldolyticus (17), B. subtilis (40), Lactobacillus plantarum (12), and Enterococcus faecalis (30) have been identified as members of a single operon. In contrast, it was recently shown that the genes of the pyrimidine biosynthetic pathway in L. lactis are organized differently. A pyr operon, which consists of only three biosynthetic genes, has been found in L. lactis (2). Two of the genes are the well-known pyr genes pyrD and pyrF, which encode dihydroorotate dehydrogenase and OMP decarboxylase, respectively. The third gene, pyrK, was identified as a new pyr gene encoding a protein which was shown to be necessary for the dihydroorotate dehydrogenase activity encoded by the adjacent pyrDb gene (2). The lactococcal pyrKDbF operon is highly homologous to the corresponding part of the much larger pyr operons found in other gram-positive bacteria. An interesting exception occurs with Lactobacillus plantarum, in which the pyrK analogue is absent from the operon (12). Another surprising feature of the pyrimidine biosynthesis pathway in L. lactis is the presence of two different genes, pyrDa and pyrDb, both of which encode a dihydroorotate dehydrogenase. The pyrDb gene belongs to the same family as the genes encoding dihydroorotate dehydrogenases in other gram-positive bacteria, whereas the pyrDa gene is closely related to that of the dihydroorotate dehydrogenase of Saccharomyces cerevisiae (1). Only pyrDb was shown to be part of the identified pyr operon (2).

Carbamoylphosphate is formed from CO₂, ATP, and glutamine and is used in the biosynthesis of both pyrimidine and arginine (Fig. 1). It is synthesized by the heterodimeric enzyme carbamoylphosphate synthetase (CPSase). The small subunit of the enzyme functions as a glutamine amidotransferase, whereas the large subunit has other catalytic properties. In all procaryotes described so far, CPSase activity is encoded by two genes commonly called carA and carB, and there is no reason to believe that this is not true for L. lactis. Procaryotes are characterized by having either a single set of genes that is responsible for all carbamoylphosphate synthesized and that encodes a single CPSase or two different sets of genes that encode CPSase (9). The two sets of genes differ in their regulatory features; one set is regulated by the level of pyrimidines in the cell, whereas the other responds to changes in the concentration of arginine (9). The genes encoding the two subunits have been sequenced for many procaryotes and have been found almost exclusively to be transcribed as an operon in the order carA-carB. Exceptions have, however, been reported. In Pseudomonas aeruginosa and Neisseria spp., sequences between carA and carB have been found (26, 27).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Carbamoylphosphate pathways in L. lactis. Pathways of carbamoylphosphate in the formation of arginine and pyrimidines and its synthesis and degradation to ammonia and carbonate with formation of ATP are shown. CK, carbamate kinase; CPSase, carbamoylphosphate synthetase.

In this study we have cloned and determined the nucleotide sequence of the carB region of the L. lactis chromosome. Surprisingly, the gene is shown to be transcribed as a monocistronic unit. Moreover it is shown that L. lactis has only one carB gene, regulated by the pyrimidine level in the cell.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains and the plasmids used in this study are listed in Table 1. Plasmids pJS23 and pSJ24 were made in the following way. pKS2 was digested with HindIII and ligated into pSMA500 and pRC1, respectively. Competent L. lactis MG1363 cells were transformed with the Escherichia coli plasmids pSJ23 and pSJ24, which are unable to replicate in L. lactis but contain a selectable Em^r marker and cloned pieces of the lactococcal chromosome. Transformants were selected and purified on plates containing 1 µg of erythromycin per ml. Only transformants in which plasmids have recombined into the chromosome will result in Em^r colonies. Chromosomal DNA from strain MB35 was digested with SpeI, and the resulting linear molecules were circularized with T4 DNA ligase and transformed into E. coli DH5. A plasmid (pSJ50) conferring erythromycin resistance and containing a 3,200-bp SpeI-HindIII lactococcal DNA fragment was obtained.

Growth conditions and enzyme assay. Lactococcal cultures were grown either on M17 glucose broth (48) or on synthetic media that were based on MOPS (morpholinepropanesulfonic acid), contained seven vitamins and either 19 (SA) or 8 (BIV) amino acids (23), and were supplied with 1% glucose. E. coli cultures were grown on Luria-Bertani broth. L. lactis was cultured at 30°C in filled culture flasks without aeration. E. coli in batch cultures was grown at 37°C with vigorous shaking. For all plates, agar was added to 15 g/liter. When needed, the following compounds were added to the different media: arginine at 200 µg/ml, uracil at 20 µg/ml, erythromycin at 1 µg/ml for lactococci and 150 µg/ml for E. coli, and ampicillin at 100 µg/ml. For enzyme assays, the cells were grown in SA or BIV glucose medium and aliquots were harvested at different times during exponential growth between optical densities at 450 nm (OD₄₅₀s) of 0.2 and 0.8. The amount of -galactosidase in the cells was assayed as previously described (22), but the cell density was measured at 450 nm. Specific enzymatic activity was determined as follows: OD₄₂₀/(OD₄₅₀ × min × ml of culture).

Transformation. L. lactis was transformed by electroporation (21). E. coli cells were transformed as described previously (42).

DNA isolation, manipulations, and sequencing. Chromosomal lactococcal DNA was prepared as described by Johansen and Kibenich (24). The methods described by Sambrook et al. (42) were used for general DNA methods in vitro. DNA sequences were determined from plasmid DNA by the dideoxy-chain termination method (44) with a Thermo Sequenase-radiolabelled-terminator cycle sequencing kit (product no. US 79750; Amersham) in accordance with the protocol of the manufacturer.

Southern blot analysis. Southern blot analysis was performed with GeneScreen nylon membranes (New England Nuclear) and the digoxigenin system (Boehringer Mannheim) for colorimetric detection of hybridized products in accordance with the protocols of the manufacturers.

PCR amplification of DNA. L. lactis chromosomal DNA was amplified by PCR with 1 µg of DNA in a final volume of 100 µl containing deoxyribonucleoside triphosphates (0.25 mM each), oligonucleotides (10 µM), and 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer). Amplification was performed with 30 cycles of 95°C for 1 min and 55°C for 1 min, followed by 3 min at 72°C.

RNA extraction. L. lactis RNA was harvested from strain MG1363 grown exponentially in SA glucose medium to an OD₄₅₀ of approximately 0.8. Total RNA from a 20-ml culture was isolated according to the method of Arnau and coworkers (3).

Primer extension. A synthetic oligonucleotide, 5'-TTTCCTGTTCACAACCTTGC-3', complementary to the sense strand covering nucleotides 726 to 745 was radioactively labelled at its 5' end with [-³²P]ATP and T4 polynucleotide kinase and used for primer extensions on 20 µg of total RNA isolated from L. lactis MG1363 as previously described (16). The elongation was performed at 41°C with SuperScript II reverse transcriptase (Gibco BRL).

Nucleotide sequence accession number. The nucleotide sequence reported in this paper has been submitted to the EMBL data library and assigned the accession no. AJ000109.

	RESULTS

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Cloning and sequencing of the carB region of the chromosome. A part of the carB gene of L. lactis encoding the large subunit of CPSase (CPSase B) was obtained by chance. During the sequencing of clones obtained from a partial Sau3A library in pBR322, plasmid pKS2, which contains two Sau3A fragments from the L. lactis chromosome (3), turned out to include a 4-kb Sau3A fragment which was shown to harbor part of a putative open reading frame showing a high degree of similarity to those encoding the C-terminal parts of the large subunits of CPSases from different organisms. A 1,700-bp HindIII-Sau3A fragment was subcloned in the E. coli vector pRC1 (28), thus creating pSJ24. The fragment harbors an internal part of the carB open reading frame (Fig. 2). This plasmid was allowed to integrate into the chromosome of MG1363 by homologous recombination, resulting in strain MB35, which carries an insertion in carB (Fig. 2).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Genetic maps the carB regions of plasmids and strains used in this work. The physical map and the positions of selected restriction endonuclease sites are shown. The carB DNAs contained in the different plasmids are shown with lines. The dotted line in pKS2 indicates that the cloned lactococcal DNA extends to a Sau3A site at 6.1 kb. A P indicates the position of the carB promoter. The maps of the chromosomal DNA in the carB regions of the wild type (WT), MB35, MB36, MB37, and MB38 are shown. The broken lines represent the E. coli plasmid DNA, which is not drawn to scale. The erythromycin resistance genes of MB36, MB37, and MB38 are not shown. The carB terminator and the carB attenuator are shown by omega- and double-omega-like structures, respectively.

The carB gene is transcribed as a monocistronic message. Since the carB gene is preceded by the functionally unrelated gene gpo, which encodes glutathione peroxidase, it is tempting to believe that the carB gene is transcribed by a promoter present in the intercistronic region between gpo and carB. In order to assay promoter activity, a PCR fragment covering the entire intercistronic region and parts of the gpo and carB open reading frames was amplified and cloned into the promoter probe vector pAK80 (22), thus generating pSJ60 (Fig. 2). After transformation into MG1363, the specific -galactosidase activity was measured in exponentially growing cells and determined to be 0.3, thus demonstrating the presence of a promoter in the intercistronic region just upstream of carB, since the specific activity of cells harboring the pAK80 vector alone is less than 0.001 (Table 3).

View this table: [in this window] [in a new window]   TABLE 3.   -Galactosidase activities of strains carrying different carB::lacLM fusions

View larger version (40K): [in this window] [in a new window]   FIG. 3.   Primer extension mapping of the 5' end of the carB mRNA. Sequencing ladders generated with the oligonucleotide used for the primer extension were loaded next to the reaction mixture. The DNA sequence of the sense strand around the first nucleotide in the transcript (designated +1) is presented, and the 10 sequence is boxed.

View larger version (34K): [in this window] [in a new window]   FIG. 4.   Sequence of the gpo-carB intercistronic region. The numbers refer to the sequences submitted to the database. The amino acids of the glutathione peroxidase and CPSase large subunit derived from the DNA sequence are shown. The translational start site of carB is indicated with double underscoring. The 10 and 35 sequences of the carB promoter are indicated with underscoring, and the first nucleotide to be transcribed is marked +1. The right and left stems of loops I, II, and III (Fig. 5) are shown.

The physiological effect of a carB mutation. In order to elucidate the role of the carB gene product, the insertion mutant MB35 was subjected to phenotypic analysis. As previously mentioned, MB35 carries a truncated carB gene, lacking one third of its coding region for the C terminus (Fig. 2). Carbamoylphosphate is required for the biosynthesis of pyrimidines and arginine. To test whether the carB mutation would confer a pyrimidine requirement on the cell, the abilities of the strain to grow in the absence and presence of uracil were tested with the SA glucose medium, which includes arginine. An effect of uracil on growth was observed, since addition of uracil to the SA glucose medium resulted in a slight increase in the growth rate of MB35.

The expression of carB is regulated by pyrimidines. The leader of the carB mRNA has the potential to fold into two mutually exclusive structures: a putative terminator and a putative antiterminator (Fig. 5). Furthermore, sequence analysis showed that immediately after the transcriptional start site of the mRNA, the carB leader is equipped with a sequence that has extremely high levels of similarity to the three PyrR binding sites of B. subtilis, all of which are found at the same position with respect to that of the antiterminator (49). Exactly the same sequence is found in the pyrKDF operon of L. lactis (2). Moreover, pyrKDF mRNA can be folded into a structure similar to the one found in the carB leader (Fig. 5B). In order to analyze whether the expression of the carB gene is subject to regulation by pyrimidines, the -galactosidase content of L. lactis carrying a carB::lacLM fusion was monitored. The HindIII-Sau3A fragment from pKS2 harboring an internal part of the carB open reading frame and the PCR fragment covering the carB promoter used for construction of pSJ60 were subcloned in the E. coli vector pSMA500 (35) as 1,700- and 1,250-bp fragments, respectively (Fig. 2). The resulting plasmids were designated pSJ23 and pJS61. These plasmids were allowed to integrate into the chromosome of MG1363 by homologous recombination. The strain obtained by transformation with pSJ23 (MB36) has acquired a carB mutation identical to that found in MB35 in addition to the carB::lacLM fusion (Fig. 2), whereas MB37 which was obtained by transformation with pSJ61 is like the wild type with respect to carB despite the fact that this strain carries a carB::lacLM fusion on the chromosome. MB36 and MB37 were grown in BIV minimal medium supplied with arginine in the absence and presence of uracil, and their levels of -galactosidase synthesis were assayed. The results are presented in Table 3. The absence of uracil led to a 10-fold induction of expression of the carB gene in a carB mutant, whereas a wild-type background led to a 3-fold reduction in the induction of expression. It should be noted that since MB36 carries the same carB mutation as MB35, in the absence of uracil, strain MB36 must be starved for pyrimidines. In order to investigate whether the expression of the carB gene is regulated by arginine, the amounts of -galactosidase produced by strains growing in BIV minimal medium in the absence and presence of arginine were assayed. This experiment was conducted only with strain MB37, since MB36 is unable to grow in the absence of arginine. The expression of the carB gene was not repressed in the presence of exogenous arginine, whereas its expression was repressed threefold by exogenous uracil in the absence of arginine.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Sequences of the carB and pyrKDbF attenuators. (A) carB antiterminator structure IV. The putative binding site of the PyrR protein is indicated with a line marked pyrbox. The stems of the terminator are shown as IIIleft and IIIright. (B) carB and pyrKDF terminator structures. The putative PyrR binding site in domain I is indicated with a line marked pyrbox. The actual terminator structure is designated III.

	DISCUSSION

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L. lactis harbors only one carB allele. As previously stated, the carB strain requires arginine but not uracil for growth. Two different models may account for this observation: either only one carB allele is present, and the carbamoylphosphate requirement for pyrimidine biosynthesis is fulfilled by degradation of arginine, or the gene described in this paper is the arginine-specific carB allele used in the compartmentalized biosynthesis of arginine. It is highly unlikely that an arginine-specific CPSase is regulated by uracil. However, carB expression in L. lactis was found to be regulated by uracil. Therefore, the evidence points to the conclusion that L. lactis harbors only one carB allele.

The car genes are usually members of operons. The results presented in this work demonstrate that the carB gene from L. lactis is transcribed as a monocistronic message. This finding is in contrast to the observation made for most other organisms, namely, that the carB gene is part of an operon consisting of either the carA and carB genes alone or the carA and carB genes as members of a larger operon that includes other pyrimidine-biosynthetic genes. However, exceptions to this paradigm have been reported; in P. aeruginosa the existence of a 216-amino-acid-encoding open reading frame with unknown function between carA and carB has been demonstrated, although the three genes are part of the same operon transcribed from a promoter upstream of carA (26). Neisseria seems to be a true exception. Sequences between carA and carB that vary in size from 2.2 to 3.7 kb were found among different Neisseria species. Furthermore, putative transcription terminators in the intergenic DNA were identified by sequence analysis of one species. Whether these structures were of physiological relevance was not indicated by experimental data (27). Recently, the carA gene from L. lactis has been cloned in our lab, and both Southern blot analysis and PCR failed to demonstrate linkage between carA and carB in L. lactis (45).

Arginine is degraded in L. lactis. Originally, L. lactis subsp. lactis was distinguished from L. lactis subsp. cremoris by the ability of L. lactis subsp. lactis to degrade arginine by means of the arginine deiminase pathway (8). The first step in the pathway is the deamination of arginine to citrulline, which subsequently is phosphorolytically cleaved into ornithine and carbamoylphosphate. The latter energy-rich derivative can either be used for pyrimidine biosynthesis as described in this work or be degraded to carbon dioxide and ammonia with formation of ATP, thus generating one energy-rich bond per molecule of arginine. It has been shown that arginine uptake in lactococci is mediated by an energy-independent arginine-ornithine antiporter (11). Therefore, arginine can be used as an energy source. Strain MG1363 has been classified as L. lactis subsp. cremoris based on genetic evidence (19, 25, 41). Originally MG1363 was considered a strain of L. lactis subsp. lactis based on its physiological traits, including its capability to degrade arginine. The results presented here further confirm that L. lactis MG1363 degrades arginine through the arginine deiminase pathway.

Glutathione peroxidase encoded by the gpo gene protects against oxidizing elements. Glutathione is a tripeptide (L--glutamyl-L-cysteinylglycine) and is present in relatively large amounts in L. lactis (13). It is important as a scavenger of free radicals and in the control of the redox potential in the cell. In addition, glutathione is involved in transpeptidation and reduction of thiol groups in proteins and it acts as a cofactor in the reduction of ribonucleotides (39). In addition to completely reduced oxygen as found in water, partially reduced forms, such as singlet oxygen, superoxide anions, hydrogen peroxide, and hydroxyl radicals, are present in organisms growing in an aerobic environment. All these compounds are highly reactive, and they can oxidize proteins and damage DNA, and may oxidize membrane fatty acids, leading to peroxidation of the lipids. Aerobic organisms produce these compounds as metabolic by-products, but all oxygen-tolerant organisms, like L. lactis, are exposed to these powerful agents and must protect themselves against cell damage (14). In E. coli three different activities that protect against the reactive oxygen species noted above have been identified: superoxide dismutase, catalase, and peroxidase (20). In L. lactis, a gene encoding superoxide dismutase (sodA) has been identified (43). Based on the finding that an L. lactis mutant lacking superoxide dismutase is viable in an aerobic environment, Sanders and coworkers (43) concluded that an additional oxygen-protecting mechanism must be present in L. lactis. The glutathione peroxidase found in this work may fulfill this role.

The expression of the carB gene is regulated by pyrimidines. In this work we have been able to demonstrate that the carB gene is regulated by the presence of uracil in the growth medium. By analyzing the sequence of the carB leader, a structure including a pyrR binding site similar to the one found in the pyrKDF leader can be identified (2). The mechanism by which B. subtilis regulates its expression of the pyr operon by transcriptional attenuation through the PyrR regulatory protein has been studied in great detail (32-34). The structures that may be formed by the RNA transcribed from the carB and pyrKDF (2) operons in L. lactis are similar to the structures found in the RNA transcribed from the B. subtilis pyr operon (33). Figure 5B shows the structures believed to result in termination at the attenuator. Stem-loop structure I, including the PyrR binding site, is highly homologous to a similar structure found in the B. subtilis pyr operon designated the anti-antiterminator by Lu and coworkers (34). These findings strongly suggest the presence of a PyrR homologue in L. lactis.