INTRODUCTION

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The de novo synthesis of pyrimidines is universal. The pathway consists of six enzymatic steps leading to the formation of UMP, which is further converted into UTP, CTP, dCTP, and dTTP. In order to coregulate expression, genes are often found to be organized in operons in procaryotes. The pyrimidine biosynthetic genes (pyr genes) have been found to constitute a single operon in a number of different gram-positive organisms including Bacillus subtilis (37), Bacillus caldolyticus (13), Enterococcus faecalis (23), and Lactobacillus plantarum (9). In addition a pyrimidine biosynthetic operon is found in Mycobacterium tuberculosis (6). The pyrimidine metabolism in Lactococcus lactis has been studied for a number of years. Surprisingly it was found that the pyr genes of L. lactis are scattered on the chromosome in small operons, the pyrKDbF operon (2), the carB gene (35), the pyrEC operon (5), and the pyrDa gene (1). This paper describes the genomic organization of the pyrimidine biosynthetic genes of L. lactis, since we demonstrate the presence of an operon including pyrB, encoding aspartate transcarbamoylase, and carA, encoding the small subunit of the carbamoyl-phosphate (CP) synthetase (CPSase). Moreover, the operon includes pyrR, the regulatory gene controlling expression of the pyr genes, and pyrP, encoding the uracil transporter.

Regulation of the expression of the pyr genes of L. lactis has not been subject to a detailed analysis. It has, however, been shown that the transcription of the carB gene is repressed by addition of uracil to the growth medium (35). Moreover, in front of both the carB gene (35) and the pyrKDbF operon (2) a putative attenuator similar to those found in B. subtilis (41) can be identified. These findings suggest that the expression of the pyr genes of L. lactis is regulated by the same attenuator mechanism as that suggested for B. subtilis (41). An RNA binding protein, PyrR, mediates the regulation of the expression of the pyr operon in B. subtilis by stabilizing the formation of a transcriptional terminator in the mRNA leader sequence. The binding of PyrR to the mRNA is dependent on the formation of a PyrR-UMP complex. In the absence of a PyrR-UMP complex, an antiterminator structure is preferentially formed (25). In the work presented here, the presence of a protein with a high degree of homology to the PyrR of B. subtilis and responsible for repressing expression of the pyrimidine biosynthetic enzymes by exogenous pyrimidines is documented.

The first step in the pyrimidine biosynthetic pathway is the formation of CP utilizing CO₂, ATP, and glutamine. CP is also a precursor for the biosynthesis of arginine. The formation of CP is catalyzed by CPSase. This enzyme consists of a small subunit and a large subunit. The small subunit of the enzyme functions as a glutamine amidotransferase, whereas the other catalytic properties are found in the large subunit. In all procaryotes described so far, two genes, commonly called carA and carB, encode the CPSase. Procaryotes may contain either a single CPSase encoded by a single set of genes responsible for all CP synthesized or, alternatively, two different sets of CPSase-encoding genes (8). The two sets differ in their regulatory features; the level of pyrimidines in the cell regulates the expression of one set of genes, whereas the other responds to changes in arginine concentration (8). The genes encoding the two subunits have been sequenced for many procaryotes and are almost exclusively transcribed as an operon in the order carA-carB. Previously we have been able to show that the carB gene in L. lactis is transcribed as a monocistronic message and that the carA gene is not in the immediate proximity of carB (35). This finding was confirmed by the publication of the gene map of another L. lactis strain, showing that the carA and carB genes are separated by approximately 240 kb (5). In this work we show that the carA gene is part of an operon including other genes involved in pyrimidine metabolism and regulation.

	MATERIALS AND METHODS

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Growth conditions. Lactococcal cultures were grown either on M17 glucose broth (39) or on synthetic media based on MOPS (morpholinepropanesulfonic acid) and containing seven vitamins and either 19 (SA) or 8 (BIV) amino acids (19) supplied with 1% glucose. Escherichia 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 or 200 µg/ml, uridine at 40 µg/ml, erythromycin at 1 µg/ml for lactococci and 150 µg/ml for E. coli, and ampicillin at 100 µg/ml. In order to assay for growth on pyrimidines and sensitivity to the toxic analogue 5-fluorouracil, cells were plated on minimal medium containing pyrimidine or the analogue at different concentrations. After incubation at 30°C, the colony sizes were estimated on an arbitrary scale from 0.1 to 1, where the size of the largest colony was set to 1.

Transformation, DNA isolation, manipulations, and sequencing. L. lactis was transformed by electroporation (17). E. coli cells were transformed as described previously (38). Chromosomal lactococcal DNA was prepared as described by Johansen and Kibenich (20). The methods described by Sambrook et al. (38) were used for general DNA methods in vitro. DNA sequences were determined from plasmid DNA by the dideoxy chain termination method using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (product no. US 79750) from 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 DIG 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 in one of the following two ways: (i) standard PCR, 30 cycles at 94°C for 1 min and 55°C for 1 min, followed by 3 min at 72°C; easy gene walking, 25 cycles at 94°C for 1 min, and 55°C for 1 min, followed by 3 min at 72°C (10 min for the last cycle).

pGhost9::ISS1 transposon mutagenesis and selection for pyrimidine auxotrophs. A pool of L. lactis strain MG1363 pGh9::ISS1 transposition mutants was obtained as previously described (21). After resuspension of the mutant library in SA medium supplemented with glucose and erythromycin but without pyrimidine addition, the cells were grown at 37°C for 30 min to stop the growth of auxotrophs. Then the culture was diluted 100-fold in the same medium, reaching an optical density at 450 nm (OD₄₅₀) of 0.8, and grown for one additional hour. Ampicillin counterselection for the auxotrophs was performed overnight at 37°C, after addition of ampicillin at 100 µg/ml to the diluted culture. After the washing and resuspension of the cells in 0.9% NaCl solution, aliquots of a 100-µl culture, both undiluted and 10-fold diluted, were plated on SA-glucose medium containing uracil. After incubation at 37°C overnight, colonies were screened for a pyrimidine requirement on SA-glucose medium with and without uracil. Pyrimidine-requiring strain MB400 was chosen for further analysis.

Plasmid rescue. Chromosomal DNA was extracted from MB400 and digested with SpeI. Following ligation and transformation of E. coli strain DH5 an erythromycin-resistant transformant was obtained. This strain was shown to harbor plasmid pJS50.

Construction of plasmids and strains. The strains and plasmids used in this study are shown in Table 1. The primers used for plasmid constructions are shown in Table 2 and the relevant genetic maps of strains and plasmids are presented in Fig. 1. Plasmid pAM111 was obtained in the following way. With primers PyrR-Nterm and PyrR_CTny and L. lactis MG1363 chromosomal DNA as the template a PCR fragment was obtained. Subsequently, the PCR fragment and pRC1 were digsted with XhoI and PstI, mixed, ligated, and transformed into DH5. Plasmid pAM122 was constructed in exactly the same way using primers PyrB-Nterm and PyrB-Cterm. By the same strategy, plasmid pAM117 was constructed using primers PyrR_4F and PyrB_14R and restriction enzymes HindIII and EcoRI. Strains MB411, MB417, and MB422 were constructed by transformations of MG1363 with nonreplicative plasmids pAM111, pAM117, and pAM122, respectively. Integrants were isolated on M17 plates supplied with glucose and 1 µg of erythromycin/ml and confirmed by PCR analysis of their chromosomal DNA.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Genetic maps of the pyrRPB-carA region, the plasmids, and the strains used in this work. The physical map and the positions of selected restriction endonuclease sites are shown in the top line. The scale indicates numbers of base pairs in the sequenced region. Lines below the map show the L. lactis DNA contained in the different plasmids. The dotted line in pJS50 indicates that the cloned lactococcal DNA extends to a distal SpeI site beyond the sequenced DNA. Arrow, position of the promoter. The maps of the chromosomal DNA in the pyrRPB-carA regions of the wild-type (WT) strain, MB411, MB417, and MB422 are shown. Hatched bars, E. coli plasmid DNA (not drawn to scale); omega-like and double-omega-like structures, terminator and attenuator of the operon, respectively.

RNA extraction. L. lactis RNA was harvested from strain MG1363 grown exponentially in SA-glucose medium to a cell density represented by an OD₄₅₀ of approximately 0.8. Total RNA from a 20-ml culture was isolated according to the method of Arnau and coworkers (4).

Primer extension. Synthetic oligonucleotide PyrR_3R, complementary to the sense strand, was radioactively labeled in the 5' end using [-³²P]ATP and T4 polynucleotide kinase and used for primer extensions on 20 µg of total RNA isolated from L. lactis strain MG1363 as previously described (11). The elongation was performed at 41°C using a SuperScript II reverse transcriptase (RT) (Gibco BRL).

RT-PCR. L. lactis RNA was used as the template in the Titan one-tube RT-PCR system from Boehringer Mannheim in accordance with the protocols of the manufacturer.

Enzyme assays. Exponentially growing cells were harvested at an OD₄₅₀ of 0.8, washed, and resuspended in 50 mM Tris-HC1 (pH 7.0)-1 mM EDTA-1 mM dithiothreitol, resulting in a 100-fold concentration. The cells were lysed using a French pressure cell press at 18,000 lb/in². Cell debris was removed by centrifugation, and the supernatant was used directly as the enzyme source in the assays. The protein concentration was determined as described by Lowry et al. (24). All assays were performed at 30°C using crude extracts with a final concentration of about 200 µg of protein/ml. Specific activities are expressed as milliunits per milligram of protein, and 1 mU is defined as 1nmol of product formed or substrate used per min.

(i) Aspartate transcarbamoylase (PyrB) activity. PyrB activity was determined at pH 7.0 in the following way. Potassium aspartate (50 mM), potassium phosphate (25 mM), and enzyme extract were mixed and equilibrated at 30°C. The assay was started by addition of CP (3 mM). Aliquots (150 µl) were extracted between 0 and 20 min, and the formation of carbamoylaspartate was measured using the colorimetric procedure described by Prescott and Jones (36), in which 10³ M carbamoylaspartate corresponds to an absorption of 18 at 560 nm.

(ii) Dihydroorotase (PyrC) activity. PyrC activity was determined essentially as described for the aspartate transcarbamoylase (PyrB) assay with the following alterations. Potassium aspartate and potassium phosphate were replaced by Tris-HCl (100 mM)-EDTA (2 mM), and the assay was started by adding dihydroorotate (2 mM) instead of CP.

(iii) Dihydroorotate dehydrogenase A (PyrDa) activity. PyrDa activity was determined by monitoring orotate formation in a spectrophotometer at 295 nm ( = 3.67 × 10³ M¹). The reaction mixture contained 0.1 M sodium phosphate (pH 7.0), 50 mM KCN, and 0.1 mM fumarate. After equilibration the assay was started by addition of dihydroorotate (0.1 mM).

(iv) Dihydroorotate dehydrogenase B (PyrDb) activity. PyrDb activity was assayed by the same procedure as that used for dihydroorotate dehydrogenase A, except that fumarate was replaced by 0.1 mM NAD⁺.

(v) Orotate phosphoribosyltransferase (PyrE) activity. PyrE activity was measured in a buffer at pH 7.5 containing Tris-HCl (20 mM), EDTA (2 mM), and orotate 300 µM. The reaction was monitored spectrophotometrically at 295 nm after addition of 5-phosphoribosyl-1-pyrophosphate (PRPP). A decrease in absorbancy of 3.67 is equivalent to an increase in OMP of 1 mM.

(vi) OMP decarboxylase (PyrF) activity. PyrF activity was determined in a buffer (pH 7.5) containing 20 mM Tris-HCl and 2 mM EDTA. After calibration at 30°C, the reaction was initiated by the addition of 50 µM OMP. The activity was monitored in a spectrophotometer at 285 nm. A reduction of the OMP concentration by 1 mM corresponds to a decrease in absorbancy of 1.65.

-Galactosidase. For enzyme assays the cells were grown in SA- or BIV-glucose medium and aliquots were harvested at different times during exponential growth between OD₄₅₀s of 0.2 and 0.8. The amount of -galactosidase in the cells was assayed as previously described (18), but the cell density was measured at 450 nm. The specific enzymatic activity was determined as OD₄₂₀/(OD₄₅₀ per minute per milliliter of culture).

Preparation and analysis of protein extracts from E. coli. E. coli cells were grown exponentially in Luria-Bertani broth exponentially in and, at an OD₄₅₀ of 0.7, IPTG (isopropyl--D-thiogalactopyranoside) was added, resulting in a final concentration of 1 mM. The cells were incubated at 37°C overnight. The 1-ml cell culture was harvested, washed, and resuspended in a 200-µl solution consisting of 0.5 M NaCl, 1 mM EDTA, and 50 mM Tris-HCl (pH 8.0). The proteins were extracted with 200 µl of phenol equilibrated with the buffer. The proteins were precipitated with 500 µl of ethanol and recovered by centrifugation. The protein pellets were resuspended in 100 µl of sodium dodecyl sulfate loading buffer and analyzed by 12.5% polyacrylamide gel electrophoresis as previously described (38).

Nucleotide sequence accession number. The nucleotide sequence reported in this paper has been submitted to the EMBL data library and assigned accession no. AJ132624.

	RESULTS

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Cloning and sequencing of the operon. From a library of approximately 6,000 pGh9::ISS1 transposon mutants four pyrimidine auxotrophic mutants were obtained after ampicilin counterselection in medium without pyrimidines added. Marker rescue experiments were conducted in order to obtain the DNA regions flanking the pGh9::ISS1 insertions. Marker rescues from two of the strains resulted in plasmids with chromosomal inserts. The nucleotide sequence of the Lactococcus chromosomal DNA was determined. The DNA in one clone was found to be identical to the previously cloned and sequenced carB gene from L. lactis (35). The other plasmid obtained by an SpeI digestion was termed pJS50 and was shown to contain a 5-kb chromosomal fragment, including part of an open reading frame showing extensive homology to those encoding aspartate transcarbamoylases from a number of different organisms. Consequently, the gene was named pyrB. The ISS1 element was inserted in the middle of pyrB. Despite numerous attempts using different restriction endonucleases, only clones harboring DNA encoding the C-terminal part of PyrB were obtained.

The open reading frames in the operon. On the 4.5-kb fragment, four open reading frames can be detected (Fig. 1 and Table 3). The Genemark program (29) predicts the identified open reading frames including ribosome binding sites as coding sequences. In order to assign a function to the open reading frames, Blast searches in protein databases were conducted. The first open reading frame showed homology to pyrimidine regulatory protein PyrR, first identified in B. subtilis (41). The second reading frame shows homology to that encoding uracil permease from a number of gram-positive organisms including B. subtilis (41). The third open reading frame was the already-identified PyrB gene, whereas the last one was homologous to carA, which encodes the small subunit of CPSase. The organization of the four open reading frames is shown in Fig. 1. In Table 3, the sizes, positions on the DNA, and translational initiation signals of the four open reading frames are presented. Moreover, the positions and sequences of the putative promoter and terminator are shown.

The four open reading frames are transcribed as an operon. In order to show whether the four open reading frames constitute an operon, two kinds of experiments were conducted. An RT-PCR experiment was used to define the size of the mRNA in vitro, whereas the phenotypes of strains constructed by gene disruption were used to define the transcriptional units in vivo.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   RT-PCR analysis of the transcription products of the pyrRPB-carA region. (A) Genetic map with the expected PCR products. Black box (T), putative terminator in the attenuator. Omega-like and double-omega-like structures are as defined for Fig. 1. (B) Agarose gel electrophoresis of the products obtained by the PCRs. Lanes R, results of the RT-PCR amplifications using RNA extracted from L. lactis; lanes D, standard PCRs with chromosomal DNA using the same primers. The following primers were used: I, PyrR_F4 and PyrR_4R; II, PyrRB_2F and PyrR_3R; III, PyrR_F5 and PyrR_3R; IV, PyrRB_3F and PyrRPintR; V, PyrP_F2 and PyrB_12R; VI, PyrB2 and CarA_R6. Marker (M), 100-bp DNA ladder; lane RNase, result of RT-PCR amplification with primers used in lanes VI after treatment of the RNA with RNase.

View larger version (56K): [in this window] [in a new window]   FIG. 3.   Primer extension mapping of the 5' end of the pyrRPB-carA 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 (+1) is presented, and the 10 sequence is boxed. The experiment was conducted on RNA extracted from cells growing exponentially in the presence (+) and absence () of uracil at 200 µg/ml.

PyrR is the pyr regulatory protein. As previously discussed, in front of all pyrimidine biosynthetic genes of L. lactis identified so far, except for pyrDa, a putative PyrR binding site identical to the PyrR boxes in B. subtilis has been found. Moreover, the pyr leaders of L. lactis can be folded in a manner similar to that found in B. subtilis forming alternative antiterminator or terminator structures, which suggests the presence of a transcriptional attenuator immediately in front of the structural genes. These findings strongly predict the presence of a PyrR homologue in L. lactis. Indeed, the PyrR open reading frame shows extensive homology to the PyrR open reading frame of B. subtilis. In order to identify the gene encoding the regulator of pyrimidine biosynthetic gene expression, the following experiment was conducted. Strain MB36 has a partial pyrimidine requirement due to a disruption of the carB gene by an integrative plasmid. Moreover, the truncated carB allele is fused to a promoterless lacLM gene encoding -galactosidase. It was previously shown that the expression of -galactosidase in this strain is repressed by the addition of uracil to the growth medium (35). Plasmid pG⁺host8::ISS1 is a transposon delivery vector conferring tetracycline resistance to the cell and is unable to replicate at 37°C (30). Strain MB36 was transformed with plasmid pG⁺host8::ISS1, and a transposon-induced mutant library comprising several thousand integrants was obtained. In a previous study, we were able to show that B. subtilis carrying a deletion of its pyrR allele is resistant to the toxic pyrimidine analog 5-fluorouracil at a concentration of 1 µg/ml (32). Therefore, strains were selected for growth in the presence of 5-fluorouracil and screened for high levels of -galactosidase by plating on SA-glucose medium supplied with 5-fluorouracil (1 µg/ml) and 160 µg of X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside)/ml. Two independent mutants with the expected phenotype were isolated, and their chromosomal DNA was extracted. Attempts to amplify the pyrR gene by PCR was unsuccessful, suggesting that the integration takes place in the pyrR gene (not shown). Furthermore, the presence of an ISS1 element in the pyrR gene was clearly shown in a Southern blot experiment (not shown). One of the strains was selected for further analysis and named MB401.

The pyrP gene encodes a uracil transporter. As previously stated PyrP shows extensive similarity to the uracil permease from B. subtilis, shown to be encoded by pyrP (41). To test whether pyrP from L. lactis encodes a uracil permease, strain MB417 (pyrP::pAM117) was tested on SA-glucose plates with increasing concentrations of uracil and uridine. This strain carries pyrP and has a pyrimidine requirement due to an interrupted transcription of the downstream genes, including pyrB. A wild-type strain and a strain carrying a mutated pyrF gene (PSA001) were included as controls. The results are presented in Fig. 4A, and it is clearly seen that a strain carrying a mutation in pyrP is unable to exploit uracil present at low concentrations, whereas no effect is seen at higher concentrations.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Growth dependence of an L. lactis pyrP mutant on different pyrimidine compounds. The growth was monitored by determining colony size after 40 h of incubation at 30°C on SA-glucose plates with the indicated additions. The size of the largest colonies was arbitrarily set to 1. (A) Growth of MB417 (pyrP pyrB) on uracil () and uridine (). The pyrF mutant PSA001 gave the same result with uracil and uridine as MB417 with uridine. Wild-type strain MG1363 was included as a control (). (B) Growth of MB417 on uridine (16.4 µM) and increasing concentrations of 5-fluorouracil (). As controls pyrF mutant PSA001 () and wild-type strain MG1363 () were included. All curves intercept the y axis at 1.0.

The pyrB allele of L. lactis MG1363 encodes a functional aspartate transcarbamoylase. In order to show the functional properties of the protein, a complementation experiment was conducted. The pyrB open reading frame was amplified from the chromosome of L. lactis MG1363 and cloned into E. coli vector pRC1, thus creating pAM122. Plasmid DNA was propagated in an E. coli wild-type strain and transformed into pyrimidine-requiring E. coli strain SØ990, which carries a defective pyrB allele. Transformed strains carrying either pAM122 or vector pRC1 were screened for their ability to grow in the absence of uracil, and only strains harboring pAM122 were able to grow, thus demonstrating that the pyrB allele of L. lactis cloned in this work complements the pyrB defect of SØ990.

The carA gene encodes the small subunit of CPSase. In a previous study it was concluded that, since no carA gene could be identified in the immediate proximity of the carB gene, it must be present somewhere else on the chromosome (35). Moreover it was shown that a carB mutation results in an arginine requirement, in accordance with CP being a precursor for both pyrimidine and arginine biosynthesis. In order to show that the carA gene actually encodes the small CPSase subunit, the ability of strain MB422 (carA) to grow in the absence of arginine was tested. Addition of arginine ensures the formation of CP for pyrimidine synthesis through the degradation of arginine by the arginine deiminase pathway (7). Together with MG1363 (wild type) and MB35 (carB), MB422 was plated on BIV-glucose plates without additional nutrients or BIV-glucose plates supplied with uracil, arginine, or uracil and arginine simultaneously. Strain MB422 had the same growth pattern as MB35, i.e., it requires arginine for growth, whereas uracil is unable to support growth of the strain. This finding suggests that carA is the sole gene encoding a functional small subunit in the CPSase of L. lactis.

	DISCUSSION

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Genetic organization of the pyrimidine biosynthetic genes. The pyrimidine biosynthetic genes in L. lactis are scattered on the chromosome in at least five different transcriptional units. Four of these, pyrKDbF (2), pyrDa (1), carB (35), and pyrRPB-carA (this work), have been described, whereas it is presently unknown whether pyrC and pyrE are cotranscribed or are members of two different transcriptional units. The carB gene encoding the large subunit of CPSase is transcribed as a monocistronic message and does not have the same message as carA, as is normally found (35). This, in conjunction with the observation that L. lactis harbors two different pyrD alleles (1), makes L. lactis a unique organism with respect to the organization of the pyr genes.

L. lactis harbors only one carA gene. The first step in the pyrimidine biosynthetic pathway is the formation of CP catalyzed by the CPSase complex comprising large and small subunits encoded by carB and carA, respectively. The small subunit is responsible for the binding of glutamine and the transfer of its amide nitrogen group to an ammonia binding site on the large subunit. The other activities in the formation of CP from ammonia, bicarbonate, and ATP are located on the large subunit (8). CP is not only used in the biosynthesis of pyrimidines but is also required for arginine biosynthesis. In a previous paper we presented evidence for the presence of only one CPSase activity in L. lactis, since a carB mutant required arginine for growth (35). In the work presented here, we were able to show that a carA mutant also acquired arginine, thus supporting the theory that only one CPSase is present in L. lactis.

The expression of the pyrRPB-carA operon is regulated by pyrimidines. The presented data show that the pyrRPB-carA operon is regulated by the presence of uracil in the growth medium. At the enzymatic level aspartate transcarbamoylase (PyrB) is reduced twofold by the addition of uracil, whereas the mRNA level is repressed threefold as judged by primer extension. Note that the primer binds within the pyrR open reading frame. By analyzing the sequence of the mRNA leader a structure including a PyrR binding site similar to the one found in the pyrKDbF (2) and carB (35) leaders can be identified. 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 (14, 15, 25-28). The structures that may be formed by the RNA in the carB and pyrKDbF leaders in L. lactis are similar to the structures found in the RNA transcribed from the B. subtilis pyr operon. Figure 5 shows the two mutually exclusive structures believed to result in antitermination (stem IV) or termination at stem III. Although the potential structures that may be formed by the pyrR leader are somewhat different from those found in the other pyr attenuators in L. lactis and B. subtilis, the functionality is preserved. A potential functional terminator (stem III) can be identified, and stem-loop structure I, including the PyrR binding site (Fig. 5), is highly homologous to a similar structure found in the B. subtilis pyr operon designated the antiantiterminator by Lu and coworkers (28).

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Sequence of the pyrRPB-carB attenuator. Boxes, sequences constituting the stems of the terminator. (A) pyrRPB-carA antiterminator including stem IV. (B) Structure of the terminator. Stem III indicates the putative terminator. PyrRbox (stem I), putative PyrR binding site.

The PyrR protein of L. lactis does not possess UPRTase activity. Unlike the finding for B. subtilis (32), the PyrR protein of L. lactis was shown not to encode UPRTase activity. The Blast search using the L. lactis PyrR amino acid sequence as the query revealed the presence of a class of proteins with a size of 170 to 193 amino acid residues with the highest similarity to PyrR from L. lactis. The different sequences are aligned in Fig. 6. The overall similarity among the different PyrR proteins is extended over the entire sequence, but especially the C-terminal part is highly conserved (Fig. 6). This part includes the binding site for PRPP. The most-pronounced deviation of the L. lactis PyrR sequence from those of the other PyrRs is the eight-amino-acid-residue deletion around amino acid residue 80 of the L. lactis PyrR sequence, corresponding to position 100 of the arbitrary scale in Fig. 6. The PyrR protein from E. faecalis has only a four-amino-acid-residue deletion at the same position (Fig. 6), and it has been shown that the PyrR protein from E. faecalis retains its catalytic activity (12). Moreover, the amino acid residues immediately before the 8-bp deletion (Fig. 6) are less conserved in the L. lactis pyrR. Assuming that the three-dimensional structure of PyrR from L. lactis is similar to that of PyrR from B. subtilis, this region is part of the "phosphoribosyltransferase flexible loop," important for enzyme activity (40). Moreover, flexible-loop motif LDITLYRDD, which is fully conserved in B. subtilis, B. caldolyticus, and E. faecalis, in which the enzymatic activity is known to be retained, is changed in L. lactis to LDTRPFRDD. Whether this change accounts for the loss of enzyme activity remains to be solved.

View larger version (51K): [in this window] [in a new window]   FIG. 6.   Clustal W alignment of amino acid sequences deduced from pyrR homologues of L. lactis (Ll), E. faecalis (Ef), L. plantarum (Lp), B. subtilis (Bs), B. caldolyticus (Bc), Deinococcus radiodurans (Dr), Haemophilus influenzae (Hi), Mycobacterium tuberculosis (Mt), Thermus aquaticus (Ta), Synechocystis sp. (Sy), Pseudomonas aeruginosa (Pa), and Pseudomonas putida (Pp). The sequences were retrieved from protein databases after a Blast search using the search engine at the National Center for Biotechnology Information. Solid triangles, amino acid residues conserved in all sequences; open triangles, positions where only conservative substitutions have occurred; asterisks, positions where the amino acid residues are conserved in more than 66% of sequences; single line, putative binding site for PRPP; double line, flexible-loop domain.