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Cloning and Verification of the Lactococcus lactis pyrG Gene and Characterization of the Gene Product, CTP Synthase

2001; Elsevier BV; Volume: 276; Issue: 41 Linguagem: Inglês

10.1074/jbc.m100531200

1083-351X

Steen L. L. Wadskov-Hansen, Martin Willemoës, Jan Martinussen, Karin Hammer, Jan Neuhard, Sine Larsen,

Folate and B Vitamins Research

The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactisstrains harboring disrupted pyrG alleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrG product is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrG mutant could be constructed in the presence of a functionalcdd gene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mm for dUTP makes it unlikely that this reaction plays a significant physiological role. As for other CTP synthases, the oligomeric structure of the lactococcal enzyme was found to be a tetramer, but unlike most of the other previously characterized enzymes, the tetramer was very stable even at dilute enzyme concentrations. The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactisstrains harboring disrupted pyrG alleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrG product is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrG mutant could be constructed in the presence of a functionalcdd gene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mm for dUTP makes it unlikely that this reaction plays a significant physiological role. As for other CTP synthases, the oligomeric structure of the lactococcal enzyme was found to be a tetramer, but unlike most of the other previously characterized enzymes, the tetramer was very stable even at dilute enzyme concentrations. polymerase chain reaction dithiothreitol polyacrylamide gel electrophoresis 4-morpholinepropanesulfonic acid Any growing organism needs nucleotides for the synthesis of DNA, RNA, and several co-enzymes. This demand can be met in two ways, either by de novo synthesis of nucleotides or by exploiting nucleotides, nucleosides, and nucleobases taken up from the surroundings through the salvage pathways. The de novosynthesis of pyrimidines seems to be universal. The pathway consists of six enzymatic reactions leading to UMP, which is subsequently converted into UTP and CTP (see Fig. 1). Most prokaryotes for which the de novo synthesis of pyrimidines has been studied possess only one allele of each gene responsible for the six enzymatic reactions. In lactococci there are, however, two different pyrD genes encoding dihydroorotate dehydrogenase (1Andersen P.S. Jansen P.J. Hammer K. J. Bacteriol. 1994; 176: 3975-3982Crossref PubMed Google Scholar). Furthermore, the protein encoded by the pyrDb gene needs the activity of the protein encoded by the pyrK gene to be active and is organized as part of an operon, consisting also of pyrK andpyrF (2Andersen P.S. Martinussen J. Hammer K. J. Bacteriol. 1996; 178: 5005-5012Crossref PubMed Google Scholar). The Lactococcus lactis carB gene encoding the catalytic subunit of carbamyl phosphate synthase has been cloned and was shown to be monocistronically transcribed (3Martinussen J. Hammer K. J. Bacteriol. 1998; 180: 4380-4386Crossref PubMed Google Scholar). ThepyrB gene encoding aspartate transcarbamylase and thecarA gene encoding the glutaminase subunit of carbamyl phosphate synthase are members of a four cistronic operon including the genes encoding the PyrR (pyrR) regulator and the high affinity uracil transporter (pyrP) (4Martinussen J. Schallert J. Andersen B. Hammer K. J. Bacteriol. 2001; 183: 2785-2794Crossref PubMed Scopus (50) Google Scholar). Furthermore, the presence of the genes pyrC encoding dihydroorotase andpyrE encoding orotate phosphoribosyltransferase has been shown, 1Jan Martinussen, unpublished results.1Jan Martinussen, unpublished results. thus establishing that the universal pathway leading to synthesis of UMP is also present in L. lactis. In most other Gram-positive bacteria the pyrimidine biosynthetic genes are organized in one large operon (5Elagöz A. Abdi A. Hubert J.C. Kammerer B. Gene. 1996; 182: 37-43Crossref PubMed Scopus (34) Google Scholar, 6Ghim S.Y. Neuhard J. J. Bacteriol. 1994; 176: 3698-3707Crossref PubMed Google Scholar, 7Li X. Weinstock G.M. Murray B.E. J. Bacteriol. 1995; 177: 6866-6873Crossref PubMed Google Scholar, 8Quinn C.L. Stephenson B.T. Switzer R.L. J. Biol. Chem. 1991; 266: 9113-9127Abstract Full Text PDF PubMed Google Scholar). Knowledge of the salvage pathways is important when studying the phenotype of auxotrophic pyr mutants. In contrast to thede novo synthesis of pyrimidines, the salvage pathways may vary between different organisms. However, the pathways by which uracil, uridine, deoxyuridine, cytidine, and deoxycytidine are metabolized in L. lactis seem to be quite similar to those found in Bacillus subtilis and Escherichia coli. Compared with E. coli, the only exceptions are that lactococci are unable to utilize cytosine and that L. lactisonly contain one pyrimidine nucleoside phosphorylase encoded bypdp (9Martinussen J. Andersen P.S. Hammer K. J. Bacteriol. 1994; 176: 1514-1516Crossref PubMed Google Scholar, 10Martinussen J. Hammer K. Microbiology. 1995; 141: 1883-1890Crossref PubMed Scopus (31) Google Scholar). As part of the elucidation of the salvage pathways in L. lactis, mutants blocked in various pyrimidine salvage genes have been isolated using different 5-fluoropyrimidine analogues (10Martinussen J. Hammer K. Microbiology. 1995; 141: 1883-1890Crossref PubMed Scopus (31) Google Scholar). These include mutations in genes encoding uracil phosphoribosyltransferase (upp), uridine/cytidine kinase (udk), pyrimidine nucleoside phosphorylase (pdp), cytidine/deoxycytidine deaminase (cdd), and thymidine kinase (tdk). Furthermore, the upp gene of L. lactis has been cloned and characterized (11Martinussen J. Hammer K. J. Bacteriol. 1994; 176: 6457-6463Crossref PubMed Google Scholar). The pyrimidine salvage pathways, as verified in the present in L. lactis, are shown in Fig. 1. Studies of the pathways converting UMP to other pyrimidine derivatives in lactococci were initiated with the recent description of apyrH-encoded UMP kinase as responsible for synthesis of UDP from UMP (12Wadskov-Hansen S.L. Martinussen J. Hammer K. Gene. 2000; 241: 157-166Crossref PubMed Scopus (15) Google Scholar). These pathways are further elucidated with the present communication, which establishes the presence of apyrG-encoded CTP synthase (EC 6.3.4.2.) as responsible for synthesis of CTP from UTP in L. lactis. In the presence of Mg2+ the enzyme catalyzes the reaction ATP + UTP + glutamine → ADP + Pi + CTP + glutamate. The glutamine-dependent amination of UTP to CTP is activated allosterically by GTP. Our studies included cloning and sequencing ofpyrG from L. lactis and the construction of different pyrG disruption and deletion mutants as well as enzymatic characterization of the gene product. The methods described by Sambrook et al. (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989Google Scholar) were used for the extraction and manipulation of plasmid DNA and general DNA in vitromethods. Chromosomal lactococcal DNA was prepared as described by Johansen and Kibenich (14Johansen E. Kibenich A. J. Dairy Sci. 1992; 75: 1186-1191Abstract Full Text PDF Scopus (58) Google Scholar). DNA sequences were determined by the dideoxy chain termination method using the Thermo Sequenase radiolabeled terminator cycle sequencing kit in accordance with the protocol of the manufacturer (Amersham Pharmacia Biotech) or using an ABI PRISM 310 DNA Sequencer as recommended by the supplier (PerkinElmer Life Sciences). PCR2amplification of DNA was performed using standard methods. E. coli cells were transformed after CaCl2 treatment as described (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989Google Scholar), and L. lactis was transformed by electroporation (15Holo H. Nes I.F. Methods Mol. Biol. 1995; 47: 195-199PubMed Google Scholar). The plasmids pSH105 and pSH106 (Fig.2) were made in the following way. A 1.4-kilobase internal fragment of pyrG from L. lactis MG1363 (16Gasson M.J. J. Bacteriol. 1983; 154: 1-9Crossref PubMed Google Scholar) was obtained by PCR using the degenerate primers pyrG1a, 5′-CCCAAGCTTAYATHAAYGTNGAYCC-3′, and pyrG2b, 5′-CGGGATCCRAAYTCNGGRTGRAAYTG-3′, which were designed based on a primary sequence alignment of the CTP synthases from E. coliand B. subtilis. Subsequently the fragment was cloned in theEcoRV site of the commercial PCR cloning vector pMOSBlue T-vector (Amersham Pharmacia Biotech). From this construct the 1.4-kilobase fragment was excised with BamHI and ligated into BamHI-digested pRC1, and the plasmid was named pSH105. pRC1 (17Le Bourgeois P. Lautier M. Mata M. Ritzenthaler P. Gene. 1992; 111: 109-114Crossref PubMed Scopus (46) Google Scholar) cannot replicate in L. lactis but contains an erm gene conferring erythromycin resistance in both E. coli and L. lactis. Plasmid pSH106 was constructed from pSH105 by deleting a 0.4-kilobase SacI fragment. Hence, pSH106 also cannot replicate in L. lactisbut is carrying the erythromycin resistance gene. The deoxy oligonucleotides PYRG LL5A, 5′-GGAATTCGAGGAGATTTAGATGTCAAC-3′, that overlap the 5′ end and PYRG LL3A, 5′-AAAACTGCAGTTATTTACTATTTTCAACAGC-3′, that overlap the 3′ end of L. lactis pyrG were used to amplify the gene by PCR using chromosomal DNA from MG1363 as a template. The sequences encoding the pyrG ribosome binding site, the start codon, and translation stop signal are indicated by italicized letters. The unique EcoRI and PstI restrictions sites of the PCR product inserted by the oligonucleotides are indicated in the sequence by underlining and were used for cloning of the pyrG fragment after the PA1 promotor (18Lutz R. Bujard H. Nucleic Acids Res. 1997; 25: 1203-1210Crossref PubMed Scopus (1221) Google Scholar) of the vector pUHE23–2. The resultant plasmid pMW602 was verified by sequencing and transformed into SØ5393, a Leu+ derivative of the E. coli pyrG strain JF622 (19Friesen J.D. Parker J. Watson R.J. Fill N.P. Pedersen S. Pedersen F.S. J. Bacteriol. 1976; 127: 917-922Crossref PubMed Google Scholar), which also carries an F′ with lacIQ and Tn5 in lacZ. The resulting plasmid-carrying strain SØ5399 was used for overexpression of the L. lactis pyrG. Lactococcal cultures were grown either on M17 glucose broth (20Terzaghi B.E. Sandine W.E. Appl. Environ. Microbiol. 1975; 29: 807-813Crossref Google Scholar) or on synthetic SA medium based on MOPS and containing 7 vitamins and 19 amino acids (21Jensen P.R. Hammer K. Appl. Environ. Microbiol. 1993; 59: 4363-4366Crossref PubMed Google Scholar), in both cases supplied with glucose to 1% (w/v).E. coli cultures were grown either on LB medium (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989Google Scholar) or synthetic AB medium (22Clark D.J. Maaloe O. J. Mol. Biol. 1967; 23: 99-112Crossref Scopus (706) Google Scholar). 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−1. When needed, the following was added to the different media: cytidine at 20 or 50 μg ml−1, uracil at 20 μg ml−1, erythromycin at 1 μg ml−1 for lactococci, and 150 μg ml−1for E. coli, and ampicillin at 100 μg ml−1. PyrG mutants were constructed in L .lactis subsp.cremoris MG1363 (12Wadskov-Hansen S.L. Martinussen J. Hammer K. Gene. 2000; 241: 157-166Crossref PubMed Scopus (15) Google Scholar) and its cdd derivative MB109 (9Martinussen J. Andersen P.S. Hammer K. J. Bacteriol. 1994; 176: 1514-1516Crossref PubMed Google Scholar) by transforming the strains with the plasmid pSH106, which contains a disrupted pyrG gene. Since the plasmid cannot replicate inL. lactis, selection for erythromycin results in transformants that harbor the nonreplicating plasmid integrated into the chromosome by homologous recombination in the pyrGregion. The selections were performed using SA medium and cytidine 50 μg ml−1 and erythromycin at 1 μg ml−1. LKH 278 was derived in this way from MG1363, whereas LKH 280 was derived from MB109. Cytidine deaminase activity in crude extracts fromL. lactis was assayed at 30 °C as previously described (9Martinussen J. Andersen P.S. Hammer K. J. Bacteriol. 1994; 176: 1514-1516Crossref PubMed Google Scholar) using the spectroscopic assay developed by Beck et al. (24Beck C.F. Ingraham J.L. Neuhard J. Thomassen E. J. Bacteriol. 1972; 110: 219-228Crossref PubMed Google Scholar). The pyrG gene fromL. lactis MG1363 was sequenced using the Easy Gene Walking method (25Harrison R.W. Miller J.C. D'Souza M.J. Kampo G. Biotechniques. 1997; 22: 650-653Crossref PubMed Scopus (21) Google Scholar). For PCR with Chromosomal DNA from MG1363 two sets of oligonucleotides pyrG6a, 5′-GAAAAATGGTTCACGCCG-3′, pyrG7a, 5′-TGCCAACGATGTGACCG-3′, and pyrG8a, 5′-GGCAAAAAATTCTTCGTTGC-3′, and pyrG6b, 5′-GAAATATAAGCATCTGGC-3′, pyrG7b 5′-TTGGCACTTCACTGCGC-3′, and pyrG8b, 5′-TCAGTTGTTGTTGCTGC-3′, designed to anneal to each end of the internal pyrG fragment of pSH105, were used together with partly degenerate oligonucleotides containing anEcoRI (5′-NNNNNNNNNNGAATTC-3′), HindIII (5′-NNNNNNNNNNAAGCTT-3′), or Sau3AI (5′-NNNNNNNNNNGATC-3′) restriction site. Fragments covering both ends of the DNA regions flanking pyrG were obtained in combination with all three degenerate oligonucleotides. Direct sequencing of the PCR fragments was done without prior cloning to eliminate the risk of errors caused by mutations in individual PCR fragments. Based on the sequences obtained, a PCR fragment covering the entire pyrG open reading frame and adjacent sequence was made using the oligonucleotides SLLH6, 5′-ACTTTGACAAAAAAGG-3′, and SLLH7, 5′-TACAAAAGATTTTGGGC-3′, and chromosomal DNA from MG1353 as a template. The direct sequencing of this PCR fragment on both strands revealed minor errors in the sequence determined by Easy Gene Walking (less than 0.5%). By combining the sequence data, the total sequence of pyrG and flanking regions was obtained. E. coli strain SØ5399 was grown in 1 liter of LB medium with the addition of ampicillin to an OD436 of 0.8. Then isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm, and cultivation was continued for 2.5 h. The cells were harvested by centrifugation at 8000 rpm for 5 min in a Sorvall rotor SLA3000. The cells were washed in 50 mm potassium phosphate, pH 7.5, divided in three portions of ∼1 g of wet cell paste and stored at −-20 °C. The purification was performed at 4 °C unless otherwise noted and is described for the amount of protein obtained from about 1 g of cell paste. Cells were thawed on ice, resuspended in 15 ml of extraction buffer (50 mm potassium phosphate, pH 7.5 and 2 mm DTT), and opened using a Sonics Vibra-Cell ultrasonic processor. Cell debris was sedimented by centrifugation in a Sorvall SS34 rotor at 14,000 rpm for 15 min. The supernatant was made 1% (w/v) with streptomycin sulfate, and centrifugation was repeated as above. A fractionated ammonium sulfate precipitation was performed by first adding 6.3 g of ammonium sulfate (∼ 35% saturated) to the supernatant while gently stirring the precipitate and centrifugation was repeated as above. To the supernatant, 4.2 g of ammonium sulfate (∼60% saturated) was added, and centrifugation was repeated as above. The precipitate (about 10–15 mg of CTP synthase) was dissolved in 10 ml of extraction buffer and loaded on to a column (2.6 × 28 cm) of phenyl-Sepharose CL-4B (Amerham Pharmacia Biotech) equilibrated with the same buffer and placed at room temperature. With a flow rate of 3 ml min−1, the column was washed with 300 ml of extraction buffer followed by an isocratic elution of the protein with a solution containing 30% ethylene glycol, 0.5 mm potassium phosphate, pH 7.5, and 2 mm DTT. The fractions containing CTP synthase were pooled, and 200 mm potassium phosphate, pH 7.5, with 20 mm DTT was added to a final concentration of 20 mm potassium phosphate. The protein was precipitated by adding ammonium sulfate to 60% saturation and collected by centrifugation as above. The precipitate was dissolved in 5 ml of extraction buffer and loaded onto a column (2.6 × 7.5) of phenyl-Sepharose equilibrated with the same buffer. The protein started to elute after one column volume of buffer (40 ml), and the next 80 ml of eluent were collected. The CTP synthase-containing fractions were precipitated by 60% ammonium sulfate as above and redissolved in 2 ml of buffer (50 mm Hepes, pH 8.0, and 2 mm DTT). The protein was dialyzed against 2 × 1 liter of the same buffer for 24 h. Finally, the enzyme was loaded on a HiTrap desalting column (Amersham Pharmacia Biotech) and eluted with the same buffer as above with a flow rate of 1 ml min−1 in portions of 1 ml, and the protein fractions were collected. The highly reproducible purification procedure presented above involving two runs of hydrophobic chromatography appears to take advantage of a hysteretic effect on the retaining capacity of phenyl-Sepharose for L. lactis CTP synthase that depends on the protein concentration of the loaded sample. CTP synthase was prepared to near homogeneity as judged from SDS-PAGE (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar). The overall purification fold was not determined but could be estimated from SDS-PAGE to be about 2–3-fold due to the very high overproduction of enzyme. The protein concentration was determined by the bicinchoninic acid procedure (27Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18590) Google Scholar) with reagents provided by Pierce and with bovine serum albumin as a standard. The specific activity of the L. lactis CTP synthase ranged from 2.5 to 3 μmol min−1mg−1 when determined as described below. The enzyme could be stored frozen at -20 °C for several months without loss of activity. All chemicals were from Sigma. Enzyme activity was determined at 30 °C by following the increase in absorbance at 291 nm using a PerkinElmer Life Sciences Lambda 17 UV-visible spectrophotometer (28Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Abstract Full Text PDF PubMed Google Scholar), and activities were calculated using the molar extinction coefficients Δε = 1338 m−1cm−1 or Δε = 1664 m−1cm−1 (29Pappas A. Park T.S. Carman G.M. Biochemistry. 1999; 38: 16671-16677Crossref PubMed Scopus (14) Google Scholar) for the conversion of UTP to CTP or dUTP to dCTP, respectively. The standard assay was performed in 150 μl of 50 mm Hepes, pH 8.0, 2 mm DTT, 27 mm MgCl2, 1 mm ATP, 1 mm UTP, 0.1 mm GTP, and 10 mmglutamine. When nucleotide concentrations were varied, the MgCl2 concentration was always maintained at an excess of 25 mm. CTP synthase was added to concentrations between 3 and 25 μg ml−1. All initial velocities were determined in duplicate at two different enzyme concentrations. Calculation of kinetic constants was performed by fitting the initial velocities to one of the five equations below using the computer program UltraFit (BioSoft, version 3.01). The reported standard errors are those calculated by the computer program. Equation 1 is for a two-substrate sequential mechanism where each substrate shows cooperative binding that is independent of the binding of the other. Equations 2 and 3apply to hyperbolic and sigmoid saturation kinetics, respectively, Equation 4 applies to hyperbolic activation, and Equation 5 is for cooperative inhibition.υ=VappAnABnB/(Si0.5AnAS0.5BnB+S0.5AnABnB+S0.5BnBAnA+AnABnB)Equation 1 υ=VappA/(KM+A)Equation 2 υ=VappAn/(S0.5n+An)Equation 3 υ=V1+V2A/(KA+A)Equation 4 υ=VappIC50n/(IC50n+In)Equation 5 where v is the initial velocity,Vapp is the apparent maximal velocity,Si0.5 and S0.5 are the concentrations of substrates or activators, A or B, denoted when appropriate, at apparent half-maximal velocity at infinite small or saturating concentrations of the other substrate or activator, respectively, Km and KA are the apparent Michaelis-Menten constant for substrate or activator A, respectively, n is the Hill coefficient, denoted when appropriate, V1 and V2are the velocities in the absence and in the presence of activator, respectively, IC50 is the concentration of inhibitor I at half-maximal inhibition. Unless otherwise noted, all reported kinetic velocities are in μmol of CTP min−1 mg−1. The calculation of the free Mg2+ concentration was performed using a stability constant of 73,000m−1 for the Mg2+ and nucleoside triphosphate complexes (30O'Sullivan W.J. Smithers G.W. Methods Enzymol. 1979; 63: 294-336Crossref PubMed Scopus (188) Google Scholar). For gel-filtration experiments, a prepacked Bio-Prep S.E.1000/17 column from Bio-Rad was equilibrated at room temperature with buffer (50 mm Hepes, pH 8.0, and 2 mm DTT). Fifty-microliter samples of (i) a calibration standard (Bio-Rad) containing thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B-12 (1.35 kDa), (ii) a mix of thyroglobuline, catalase (232 kDa), and ovalbumin (Amersham Pharmacia Biotech), or (iii) CTP synthase (13–65 μg) was loaded onto the column with a flow rate of 0.5 ml min−1. Protein elution from the column was monitored at 280 nm. Furthermore, the position of elution of the individual proteins in the calibration standards was also evaluated by SDS-PAGE analysis of the collected fractions. Chemical cross-linking experiments were performed in two ways. (i) CTP synthase (0.75 mg ml−1) was incubated at room temperature in 100 μl of 50 mm Hepes, pH 8.0, 2 mm DTT with either 0.1% or 1% of glutaraldehyde for 30 min. Apart from the difference in glutaraldehyde concentration four incubation conditions were used that contained enzyme alone, enzyme in the presence of 10 mm MgCl2, enzyme in the presence of 10 mm MgCl2 and 2 mm ATP, or enzyme in the presence of 10 mm MgCl2 and 2 mm UTP. Samples were then removed and prepared for analysis by SDS-PAGE (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar). (ii) CTP synthase (25 or 250 μg ml−1) was incubated in the absence of MgCl2 and nucleotides with 0.1% or 1% glutaraldehyde as above, and the reactions were terminated by the addition of glycine to a final concentration of 200 mm. Subsequently, all samples were concentrated on Millipore ULTRAFREE-MC 30.000 NMWL filter units, and about 10 μg of protein from each sample was subjected to SDS-PAGE analysis. The molecular weight of the protein bands was calculated using a high range molecular weight marker from Bio-Rad. Dynamic light-scattering was performed on a DynaPro MSXTC apparatus thermostated to 30 °C, and samples were prepared in 50 mm Hepes, pH 8.0, 2 mm DTT. Light-scattering was determined twice on 15-μl samples of CTP synthase at concentrations ranging from 5 to 0.05 mg ml−1. Each protein dilution was passed through an ULTRAFREE-MC 0.22-μm filter unit. One measurement consisted of 20 acquisitions of 10 s. Analysis of the light-scattering data was done with the software Dynamics V6 supplied with the DynaPro instrument using standard settings for a general aqueous buffer solution of protein (phosphate-buffered saline) with a refractive index at 589 nm and 20 °C of 1.33, a viscosity coefficient of 1.019 at 20 °C, a Cauchy coefficient of 3119 nm2, a solvent intensity of 0, and a temperature model for an aqueous solution. From the peaks derived from CTP synthase, the calculated hydrodynamic radii and polydispersities are reported and compared with those obtained with catalase whose tetramer has a similar molecular mass as the CTP synthase tetramer. The sequence of L. lactis PyrG was obtained as described under “Experimental Procedures.” Southern blot analysis verified that the PCR fragment cloned into pSH105 (Fig. 2) originated from MG1363 (data not shown). Analysis of the DNA sequence using the GeneMark program (31Lukashin A.V. Borodovsky M. Nucleic Acids Res. 1998; 26: 1107-1115Crossref PubMed Scopus (1209) Google Scholar) indicated a consensus Shine-Dalgarno motif (GAGGAG) spaced six nucleotides upstream of the deduced translational initiation site of thepyrG reading frame (nucleotide 898–2505) encoding a 537-amino acid polypeptide with a Mr of 59,455. When the deduced amino acid sequence of the L. lactis CTP synthase was submitted to a BLAST search, it revealed identities between 36 and 77% with that of other CTP synthases. Of particular interest is maybe the E. coli sequence, which showed 47% identity with that of L. lactis CTP synthase. The lactococcal pyrG is flanked by two open reading frames. The upstream reading frame orf81 (nucleotides 1–248) encodes 81 C-terminal amino acids of a polypeptide that shows homology to several aspartate aminotransferases. The downstream open reading frame (nucleotide 2717–3052) encoding polypeptide of 116 amino acids, named orf116, is preceded with a spacing of 5 base pairs by a good ribosome binding site motif (AGGAAA). The orf116 does not show homology to any known open reading frames in the data bases. The DNA sequence was submitted to the EMBL data library and was assigned the accession number AJ010153. IfpyrG is the only functional gene encoding CTP synthase inL. lactis, a gene disruption would result in a cytidine requirement, i.e. the cell would be restricted to acquire CTP through uptake and subsequent phosphorylation of cytidine. InE. coli and Salmonella typhimurium, exogenously added cytidine is rapidly deaminated to uridine by cytidine deaminase encoded by cdd, and hence, a cdd inactivation is needed to isolate a pyrG mutant (32Beck C.F. Ingraham J.L. Mol. Gen. Genet. 1971; 111: 303-316Crossref PubMed Scopus (15) Google Scholar, 33Weng M. Makaroff C.A. Zalkin H. J. Biol. Chem. 1986; 261: 5568-5574Abstract Full Text PDF PubMed Google Scholar). Consequently, thepyrG mutation in L. lactis was established in MB109 (10Martinussen J. Hammer K. Microbiology. 1995; 141: 1883-1890Crossref PubMed Scopus (31) Google Scholar), a cdd derivative of MG1363. Plasmid pSH106 was transformed into MB109 and, by selecting for erythromycin resistance, transformants were obtained in which this nonreplicating plasmid had integrated into the chromosome. 12 transformants were tested for the ability to grow without cytidine, and all 12 were found to have a cytidine requirement, which, as expected, could not be satisfied by the addition of uracil. These results strongly indicate that the clonedpyrG gene encodes the only physiological relevant CTP synthase in L. lactis. Chromosomal DNA from one of thepyrG mutants was extracted, and the pyrG mutation due to the integration of the plasmid was verified by PCR. The strain was kept as LKH280 (Fig. 2). Next we tested whether a pyrG mutant could be established in L. lactis without an additional mutation in cdd. The wild type strain MG1363 was transformed with pSH106, and erythromycin-resistant colonies were readily obtained. Again all transformants were shown to have a cytidine requirement, thus showing that the pyrG mutation could be established inL. lactis even in the presence of a wild type cddgene. The mutated pyrG region was verified by PCR on chromosomal DNA extracted from the strain. Based on the observations made in E. coli (33Weng M. Makaroff C.A. Zalkin H. J. Biol. Chem. 1986; 261: 5568-5574Abstract Full Text PDF PubMed Google Scholar, 34Beck C.F. Ingraham J.L. Neuhard J. Mol. Gen. Genet. 1972; 115: 208-215Crossref PubMed Scopus (19) Google Scholar), we speculated that such mutants would require a high external cytidine concentration to avoid cytidine depletion due to the deamination to uridine. Hence, mutants were isolated using a cytidine concentration of 50 μg ml−1but were later proved able to grow even at a cytidine concentration of 20 μg ml−1. One of these mutants was kept as LKH278 (Fig. 2). In E. coli it has only been possible to isolate a mutant defective in pyrG if the strain also contained acdd mutation. This difference could be explained if the activity of cytidine deaminase varies between the two bacterial genera. Without added cytidine, the levels of cytidine deaminase are of the same order of magnitude in the two genera (9Martinussen J. Andersen P.S. Hammer K. J. Bacteriol. 1994; 176: 1514-1516Crossref PubMed Google Scholar). However, the presence of cytidine in the medium induces the expression of cdd 30-fold in E. coli (36Hammer-Jespersen K. Munch-Petersen A. Mol. Gen. Genet. 1973; 126: 177-186Crossref PubMed Scopus (21) Google Scholar) and ensures a very rapid degradation of cytidine in this organism. In fact, cytidine is the effector for the CytR repressor in E. coli (37Hammer-Jespersen K. Munch-Petersen A. Metabolism of Nucleotides, Nucle