The pyrH gene of Lactococcus lactis subsp. cremoris encoding UMP kinase is transcribed as part of an operon including the frr1 gene encoding ribosomal recycling factor 1

Gene 241 (2000) 157–166 www.elsevier.com/locate/gene

The pyrH gene of Lactococcus lactis subsp. cremoris encoding UMP kinase is transcribed as part of an operon including the frr1 gene encoding ribosomal recycling factor 1 Steen L.L. Wadskov-Hansen, Jan Martinussen, Karin Hammer * Department of Microbiology, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark Received 23 July 1999; received in revised form 8 September 1999; accepted 8 October 1999 Received by D.L. Court

Abstract The pyrH gene of Lactococcus lactis subsp. cremoris MG1363, encoding UMP kinase, has been sequenced and cloned. It encodes a polypeptide of 239 amino acid residues (deduced molecular weight of 25 951), which was shown to complement a temperature sensitive pyrH mutation in Escherichia coli, thus establishing the ability of the encoded protein to synthesize UDP. The pyrH gene in L. lactis is flanked downstream by frr1 encoding ribosomal recycling factor 1 and upstream by an open reading frame, orfA, of unknown function. The three genes were shown to constitute an operon transcribed in the direction orfA-pyrHfrr1 from a promoter immediately in front of orfA. This operon belongs to an evolutionary highly conserved gene cluster, since the organization of pyrH on the chromosomal level in L. lactis shows a high resemblance to that found in Bacillus subtilis as well as in Escherichia coli and several other prokaryotes © 2000 Elsevier Science B.V. All rights reserved. Keywords: Conserved gene order; Pyrimidine metabolism; UMP kinase sequence

1. Introduction Specific engineering of organisms used for starter cultures in the dairy industry is a field of continuously growing importance. When the objective is to engineer strains with specific characteristics, research dealing with the central biochemical pathways of the organism of interest is important as a basic tool. The metabolic pathways of nucleotides, nucleosides and nucleobases are some of the most fundamental since all organisms need these compounds to synthesize DNA, RNA and several co-enzymes. This need for nucleotides can be fulfilled in two ways: (1) de novo synthesis of nucleotides

Abbreviations: aa, amino acids; bp, base pair(s); CMP, cytidine monophosphate; EMBL, European Molecular Biology Laboratory; GTP, guanosine triphosphate; kb, 1000 base pairs; MOPS, morpholinepropanesulfonic acid; MW, molecular weight; PCR, polymerase chain reaction; pI, isoelectric point; RBS, ribosome binding site; RTPCR, reverse transcriptase PCR; UDP, uridine diphosphate; UMP, uridine monophosphate; UTP, uridine triphosphate. * Corresponding author. Tel.: +45-45-25-24-96; fax: +45-45-88-26-60.. E-mail address: [email protected] ( K. Hammer)

or (2) exploiting nucleotides, nucleosides and nucleobases salvaged from the surroundings. Salvage of pyrimidines may vary among different organisms, but the pathways by which uracil, uridine, deoxyuridine, cytidine and deoxycytidine are metabolized in L. lactis are fairly similar to those of B. subtilis (Martinussen et al., 1994; Martinussen and Hammer 1995). The de-novo pathway of pyrimidine synthesis consists of six enzymatic reactions leading to UMP, which is subsequently converted into UTP and CTP. In contrast to all other organisms characterized so far, lactococci have two different pyrD genes encoding dihydroorotate dehydrogenase (Andersen et al., 1994). One of these is part of the dihydroorotate dehydrogenase B complex, together with the protein encoded by the pyrK gene. Furthermore, the pyrDb gene is organized as part of an operon, consisting also of pyrK and pyrF (Andersen et al., 1996). In all other Gram-positives characterized so far, all genes encoding pyrimidine biosynthetic enzymes needed to synthesize UMP are organized in one operon (Quinn et al., 1991; Ghim and Neuhard, 1994; Li et al., 1995, Elago¨z et al., 1996). Recently, the carB gene has been cloned from L. lactis (Martinussen and Hammer, 1998). Furthermore, the

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presence of the carA and pyrB genes on the L. lactis chromosome has been established (unpublished results), and the activity of the pyrC-encoded dihydroorotase and the pyrE-encoded orotate phosphoribosyltransferase has been measured in L. lactis (unpublished results). These observations verify that the universal pathway leading to synthesis of UMP is present in L. lactis, with the modification of the presence of two different pyrD encoded enzymes as mentioned above. Studies of the synthetic pathways after UMP were initiated with the recent description of a pyrG-encoded CTP synthetase, which is responsible for synthesis of CTP from UTP in L. lactis (unpublished results). The present communication reports a further investigation into the reactions leading from UMP to CTP since it establishes the presence of a pyrH encoded UMP kinase in L. lactis. Our studies included sequencing, cloning and genetical characterization of pyrH from Lactococcus lactis subsp. cremoris MG1363 and an attempt to construct a pyrH disruption mutant.

( Israelsen et al., 1995) resulted in pSH62. Cloning of the same PCR fragment in the SmaI site of pGHost4 (Maguin et al., 1992) resulted in pSH621. Cloning of an approximately 400 bp PCR fragment covering the C-terminal amino acids of the pyrH open reading frame in the EcoRV site of pMOSBlue resulted in pSH61. The cloning of the approximately 400 bp fragment resulting from a BamHI/HincII digests of pSH61 in BamHI/SmaIdigested pGHost4 resulted in pSH611. Competent L. lactis MG1363 cells were transformed with plasmid pSH611 or pSH621 unable to replicate in L. lactis at temperatures above 30°C but containing a selectable EmR marker and cloned fragments of the lactococcal pyrH. Transformants were selected and purified on plates containing 1 mg/ml of erythromycin and 20 mg/ml of uracil at 28°C. Recombinants were attempted selected and purified on plates containing 1 mg/ml of erythromycin and 20 mg/ml of uracil at 37°C. Only plasmids recombining into the chromosome would result in EmR colonies. Competent E. coli KUR1244 were transformed with pSH601, and transformants were selected on plates containing 100 mg/ml of ampicilin.

2. Materials and methods 2.3. Transformation 2.1. Growth conditions Lactococcal cultures were grown either on M17 glucose broth ( Terzaghi and Sandine 1975) or on synthetic media (SA) based on MOPS and containing seven vitamins and 19 amino acids (Jensen and Hammer 1993) and supplemented with glucose to 1%. E. coli cultures were grown either on Luria–Bertani broth (LB) or synthetic medium (ABTG) (Clarck and Maaløe 1967). L. lactis was cultured at 30°C in filled culture flasks without aeration. E. coli was grown in batch cultures at 37°C with vigorous shaking. For all plates, agar was added to 15 g/l. When needed, the following was added to the different media: uracil 20 mg/ml, erythromycin at 1 mg/ml for lactococci and 150 mg/ml for E. coli, and ampicillin at 100 mg/ml 2.2. Bacterial strains and plasmids The bacterial strains and the plasmids used in this study are listed in Table 1. The plasmids pSH60, pSH61, pSH62, pSH601, pSH621 and pSH611 were made in the following way: an approximately 200 bp internal fragment of pyrH from L. lactis MG1363 obtained by PCR and cloned in the SmaI site of the commercial cloning vector pBluescript II KS was kindly provided by P.S. Andersen. This construct was named pSH60. The cloning of an approximately 1.1 kb PCR fragment covering pyrH from L. lactis in the EcoRV site of pMOSBlue resulted in pSH601. The cloning of a 400 bp PCR fragment covering the N-terminal amino acids of pyrH and upstream sequence in the SmaI site of pAK80

L. lactis was transformed by electroporation (Holo and Nes 1989). E. coli cells were transformed after CaCl treatment as described previously (Sambrook 2 et al., 1989). 2.4. DNA isolation, manipulations, and sequencing Chromosomal lactococcal DNA was prepared as described by Johansen and Kibenich (1992). The methods described by Sambrook et al. (1989) were used for general DNA methods in vitro. DNA sequences were determined from plasmid or PCR-product DNA by the dideoxy-chain termination method (Sanger et al., 1977) using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (Product number US 79750) from Amersham in accordance with the protocol of the manufacturer. 2.5. Southern blot analysis Southern blot analysis was performed with Hybond-N+ membranes (Amersham) and the DIG system (Boehringer Mannheim) for colorimetric detection of hybridized products in accordance with the protocols of the manufacturers. 2.6. PCR amplification of DNA L. lactis chromosomal DNA was amplified by PCR with 1 mg of DNA in a final volume of 100 ml containing deoxyribonucleoside triphosphates (0.25 mM each),

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S.L.L. Wadskov-Hansen et al. / Gene 241 (2000) 157–166 Table 1 Strains and plasmids Strain or plasmid

Genotype description and/or construction

Reference or origin

Strains L. lactis subsp. cremoris MG1363 E. coli XL1Blue

Plasmid-free strain

Gasson (1983)

endA1 hsdR17(r -m+ )supE44 thi-1 recA1 gyrA96 relA1 k12 k12 lac[F ∞ proA+B+ lacI qZDM15 :Tn10(Tcr)] pyrH88 thi-1 leuB6 proA2 lacY1 galK2 mtl-1 xyl-5 ara-14 supE44

Laboratory strain Smallshaw and Kelln (1992)

bla; direct selection E. coli vector ermAM; L. lactis integration vector bla; direct selection E. coli vector erm, lacLM; L. lactis promotor probe vector bla internal pyrH fragment bla C-terminal pyrH fragment erm upstream pyrH fragment bla pyrH+ ermAM C-terminal pyrH fragment ermAM upstream pyrH fragment

Amersham Maguin et al. (1992) Stratagene Israelsen et al. (1995) This study This study This study This study This study This study

KUR1244 Plasmids pMOSBlue pGHost4 pBluescript pAK80 pSH60 pSH61 pSH62 pSH601 pSH611 pSH621

oligonucleotides (10 mM ) and 2.5 u of AmpliTaq DNA polymerase (Perkin Elmer). Amplification was performed in one of the following ways: (1) for standard PCR, 30 cycles at 94°C for 1 min, 55°C for 1 min, followed by 3 min at 72°C; (2) for easy gene walking, 25 cycles at 94°C for 1 min, 55°C for 1 min, followed by 3 min at 72°C (10 min for last cycle). All synthetic oligonucleotides used in this study are listed in Table 2.

All synthetic oligonucleotides used in this study are listed in Table 2.

2.7. RNA extraction

3. Results and discussion

L. lactis RNA was harvested from strain MG1363 grown exponentially in SA glucose medium to a cell density of approximately OD =0.8. Total RNA from 450 200 ml of culture was isolated using the fast prep system (BIO101) using the protocols of the manufacturers.

3.1. Determination of a pyrH sequence from L. lactis

2.8. Primer extension Three synthetic oligonucleotides, pyrHext1 complementary to the sense strand covering nucleotide 815– 785, pyrHext2 complementary to the sense strand covering nucleotide 242–207 and pyrHext3 complementary to the sense strand covering nucleotide 306–268, was radioactively labeled in the 5∞-end using [c-33P]ATP and T4 polynucleotide kinase and used for primer extensions on 10 mg total RNA isolated from L. lactis strain MG1363. The elongation was performed at 48°C, using a SuperScript II reverse transcriptase (GibcoBRL) 2.9. RT-PCR L. lactis RNA was used as template in the Titan@ One Tube RT-PCR System from Boehringer Mannheim in accordance with the protocols of the manufacturers.

2.10. Nucleotide sequence Accession No. The nucleotide sequence reported in this paper has been submitted to the EMBL Data Library and assigned the Accession No. AJ011960.

A PCR-fragment from L. lactis subsp. cremoris MG1363 obtained by chance showed an extended sequence similarity to UMP kinases from other prokaryotes. A Southern blot analysis verified that the PCR fragment cloned originated from MG1363 (data not shown). Thus, we attempted to obtain the remaining sequence of the gene from L. lactis MG1363, using the Easy Genome Walking method (Harrison et al., 1997). This method is based on nested PCR. Two different sets of nested oligonucleotides, pyrH5, pyrH6 and pyrH1 or pyrH3, pyrH2 and pyrH4, were used together with partly degenerate oligonucleotides containing either an EcoRI, HindIII or Sau3AI restriction site in the 3∞ end (refer to Table 2 for oligonucleotides). Fragments covering the flanking DNA in both ends were obtained. These PCR fragments were sequenced without prior cloning, which eliminated the risk of errors due to mutations in individual PCR fragments. However, in contrast to the original procedure (Harrison et al., 1997), this was done with an internal oligonucleotide and not that used in the third round of amplification since the sequence obtained in this way was found to be far superior to

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Table 2 Oligonucleotides Name

Oligonucleotide sequencea

Coordinatesb

pyrH1 pyrH2 pyrH3 pyrH4 pyrH5 pyrH6 pyrH7 pyrH9 pyrH10 pyrH11 pyrHext1 pyrHext2 pyrHext3 SLLH5 SLLH11 SLLH12 SLLH14 SLLH15 SLLH16 SLLH22 EcoRI HindIII Sau3AI

5∞-TTGCGGTGGTGGTAATGTTTGG-3∞ 5∞-GTTCTGCACCCAAATCATGGAC-3∞ 5∞-GACCAGTAACTCCACGG-3∞ 5∞-CTTTTGCTGTTTCAGGG-3∞ 5∞-GGATTCGGATTCGACCC-3∞ 5∞-TTGGGTGCAGAACTTGCG-3∞ 5∞-ATCAAAATGACGTCTGTG-3∞ 5∞-GGGCGTGTTGTTATTTTTGCT-3∞ 5∞-TCAACTATACAACCCTCTGC-3∞ 5∞-CAATGGATAATCATATTCC-3∞ 5∞-TTCTTCAGCAACAGCTTTTGCTGTTTCAGGG-3∞ 5∞-CCGTTATCAAATTCTAAGCGTAAACCGACTCCCGGC-3∞ 5∞-GCTCACGTCAAAAGGGTTCATCAACAGATCAATTTGCTC-3∞ 5∞-GATTTTTTGGATTGCCGGA-3∞ 5∞-ATGTCAATAAACCAAGATGCCC-3∞ 5∞-CTACCAAAGGCTAGCCATTGCCCC-3∞ 5∞-CCTGCTGAGCTTCTTTGGG-3∞ 5∞-ATATGTTTTAAACTATGTTCCC-3∞ 5∞-GAATCATTGAGTAATAAATCCCC-3∞ 5∞-GCTCGTGATGTCGAAGCGGC-3∞ 5∞-NNNNNNNNNNGAATTC-3∞ 5∞-NNNNNNNNNNAAGCTT-3∞ 5∞-NNNNNNNNNNGATC-3∞

857–878 846–825 895–879 801–785 771–787 834–851 1186–1169 1083–1103 1128–1147 1324–1342 815–785 242–207 306–268 540–558 615–594 1528–1505 421–439 458–437 413–391 14–33

a Degeneracy is listed in accordance with the IUPAC recommendations. b Numbering refers to the total sequence submitted to the EMBL Data Library and assigned the Accession No. AJ011960.

that obtained with the third nested oligonucleotide. We believe this is partly due to the low stringency of the PCR-reaction conditions in this procedure, resulting in more products with the nested oligonucleotides than the desired oligonucleotides and partly to the observation that sequencing of PCR products is best carried out with an internal oligonucleotide. By using a fourth nested oligonucleotide for sequencing, we were able to read at least 350 bp of unknown sequence in such a run. With one additional round of Easy genome walking using primers pyrH4, SLLH11 and SLLH15 followed by sequencing, the total sequence of the presumed pyrH and flanking reading frames from MG1363 was deduced (Fig. 1). The GeneMark program (Lukashin and Borodovsky 1998) predicts that the motif 5∞-AGGTGC-3∞ (nucleotide 696–701) functions as a ribosomal binding site for pyrH. This Shine–Dalgarno motif is spaced three nucleotides upstream of the deduced translational initiation site of the pyrH reading frame, which is also predicted by GeneMark. A TAA translational stop codon terminates the 239-amino-acid polypeptide. The different PCR fragments used in this study covering different parts of the L. lactis subsp. cremoris MG1363 pyrH are shown in Fig. 2.

a pyrH mutant of E. coli was tried. Using the above deduced sequence of the L. lactis pyrH, a PCR fragment covering all translational signals thought to be necessary for expression in E. coli was obtained using the primers SLLH12 and SLLH14 and chromosomal DNA from MG1363, as indicated on Fig. 2. This fragment was cloned in the commercial cloning vector, pMOSBlue. The resulting construct, named pSH601, was transformed to the E. coli strain KUR1244 (Smallshaw and Kelln, 1992), which carries a temperature-sensitive pyrH mutation. The resulting transformants had lost the temperature sensitivity of KUR1244 and were able to grow at 42°C. Hence, the presumed pyrH gene from L. lactis is able to complement a pyrH mutation in E. coli. This provides additional evidence that the fragment cloned does indeed encode a L. lactis UMP kinase. It should be noted that activity of a L. lactis enzyme at 42°C compared to the cultivation temperature normally used for this organism, i.e. 30°C, is unusual but not unlikely, since L. lactis MG1363 can be grown at 37.5°C (Maguin et al., 1996).

3.2. Complementation of an E. coli UMP kinase temperature-sensitive mutant

No obvious putative transcriptional or translational signals apart from the Shine–Dalgarno motif mentioned above can be deduced from the sequence immediately in front of pyrH. As can be seen from Figs. 1 and 2, pyrH was found to be flanked by two open reading

In order to determine whether the presumed pyrH sequence encoded a UMP kinase, complementation of

3.3. pyrH is transcribed as part of an operon

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Fig. 1. Total DNA sequence of pyrH encoding UMP kinase and flanking open reading frames from L. lactis subsp. cremoris MG1363. Written beneath the sequence is the amino acids of the open reading frame of UMP kinase, OrfA and ribosomal recycling factor 1. A consensus promoter sequence and consensus Shine–Dalgarno consensus sequences are marked in black boxes. An inverted repeat believed to function as a terminator is marked by underlining. The total sequence has been submitted to the EMBL database and given the Accession No. AJ011960.

frames, orfA and frr1, respectively. The downstream reading frame, frr1, shows extensive homology to several ribosomal recycling factors. This reading frame is also predicted by the GeneMark program (Lukashin and Borodovsky, 1998) and is most likely to be translated from the RBS 5∞-AGGAAA-3∞ (nucleotides 1589–1594) spaced 4 bp upstream of the translational start point of this open reading frame. The upstream reading frame orfA (147 aa, theoretical pI/MW: 5.15/16851.90) shows no significant homology to any known genes and is predicted by GeneMark to be translated from the RBS 5∞- AGGTGG-3∞ (nucleotides 158–163) spaced 5 bp upstream of the translational start point of this open

reading frame. Consensus −35 and −10 boxes with 17 bp spacing (nucleotides 117–145) are located immediately upstream of the putative translational start codon of orfA. Furthermore, a putative terminator structure followed by a run of U nucleotides and consisting of a 6 bp spaced 13 bp perfect inverted repeat with a stability of −12.4 kcal at 30°C, as predicted by the RNA-folding program written by Zucker (1989), is located immediately upstream of these consensus −10 and −35 boxes (nucleotides 46–78). In order to determine the transcriptional initiation site of the messenger including pyrH, a primer extension analysis was performed using two synthetic oligonucleotides, pyrHext1 and pyrHext2, and

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Fig. 2. Schematic representation of the PCR products and some of the plasmids used in this study. (A) pyrH with the two flanking open reading frames. (B) PCR fragment cloned in pSH60. (C ) PCR fragments obtained by easy gene walking using oligonucleotides pyrH3, pyrH2 and pyrH4 together with partly degenerate oligonucleotides containing either an EcoRI, HindIII or Sau3AI restriction site in the 3∞ end and cloned in pSH62 and pSH621. (D) PCR fragments obtained by easy gene walking using oligonucleotides pyrH5, pyrH6 and pyrH1 together with partly degenerate oligonucleotides containing either an EcoRI, HindIII or Sau3AI restriction site in the 3∞ end. (E ) PCR fragment obtained by easy gene walking using oligonucleotides pyrH4, SLLH11 and SLLH15 together with partly degenerate oligonucleotides containing either an EcoRI, HindIII or Sau3AI restriction site in the 3∞ end. (F ) PCR fragment obtained by easy gene walking as in (D) and cloned in pSH61 and pSH611. (G) PCR fragment obtained with oligonucleotides SLLH12 and SLLH14, cloned in pSH601 and used to complement a temperature-sensitive pyrH mutation in E. coli.

total RNA isolated from L. lactis strain MG1363. Despite numerous attempts, this analysis failed to show a mRNA 5∞ end immediately upstream of the putative translational initiation site of pyrH using pyrHext1, whereas a mRNA 5∞ end immediately upstream of orfA at nucleotide 153 spaced nucleotides downstream of the putative −10 consensus box could be demonstrated using pyrHext2 (data not shown). An additional primer extension analysis using the synthetic oligonucleotide pyrHext3 confirmed the location of the mRNA terminus found upstream of orfA. To elucidate whether orfA, pyrH and frr1 are transcribed as part of an operon, we performed RT-PCR on RNA isolated from MG1363. As can be seen from Fig. 3, it is possible to obtain RT-PCR products spanning the intergenic regions of pyrH and frr1, orfA and pyrH and products spanning the reading frames of orfA, pyrH and frr1, respectively. However, it was not possible to obtain a RT-PCR product including both the putative rho-independent terminator upstream of orfA and the N-terminal end of the open reading frame encoded by orfA (Fig. 4B). This clearly indicates that in vivo, a RNA messenger spanning orfA, pyrH and frr1 is transcribed from a promoter located immediately upstream of orfA and hence that the three genes constitute an operon. In order to further exclude that significant transcription of pyrH is initiated by a cryptic promoter immediately in front of the pyrH open reading frame, one of the upstream PCR fragments obtained by easy gene walking (Fig. 2C ) was cloned in front of lacLM in the promoter probe vector pAK80 (Israelsen et al., 1995). The resulting plasmid pSH62 was transformed into MG1363, and the specific

b-galactosidase activity in exponentially growing cells was determined to be 0.002. Since the b-galactosidase activity of a strain harboring the vector pAK80 is less than 0.001 (Martinussen and Hammer, 1998), the result presented here indicates that no significant promoter activity is present immediately upstream of pyrH. Furthermore, the cloning of functional L. lactis promoters in pAK80 generally results in b-galactosidase activities several orders of magnitude higher than that measurable for the vector alone (Israelsen et al., 1995). 3.4. An attempt to construct pyrH mutants in L. lactis The PCR fragment cloned in pSH62 was cloned in the SmaI site of the pGHost4 vector (Maguin et al., 1992) and the construct named pSH621 (Fig 2). Also, one of the downstream PCR fragments obtained by easy gene walking (Fig. 2F ) was cloned in pGHost4 and the construct named pSH611. Since pGHost4 carries a temperature-sensitive origin of replication, pSH611 and pSH621 can only be maintained as plasmids in L. lactis at temperatures below 30°C. By transforming pSH611 or pSH621 to MG1363 and scoring for erythromycin resistance at the permissive temperature, the plasmids were established in MG1363. In order to still maintain erythromycin resistance at 37°C, pSH611 or pSH621 must integrate into the chromosome. This is most likely to take place by homologous recombination since pSH611 and pSH621 both carry part of the lactococcal pyrH. Despite numerous attempts, we were not able to isolate any mutants in which pSH611 or pSH621 had integrated on the chromosome by homologous recombi-

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Fig. 3. RT-PCR analysis of transcription of orfA, pyrH and frr1 in L. lactis. Numbering refers to the DNA sequence submitted to the EMBL data library and assigned the Accession No. AJ011960. Panel 1: pyrH with the two flanking open reading frames, orfA and frr1. (A) PCR fragment obtainable with oligonucleotides SLLH14 and SLLH11. (B) PCR fragment obtainable with oligonucleotides pyrH5 and pyrH7. (C ) PCR fragment obtainable with oligonucleotides SLLH20 and SLLH21. (D) PCR fragment obtainable with oligonucleotides SLLH22 and pyrHext2. ( E ) PCR fragment obtainable with oligonucleotides SLLH14 and pyrH7. ( F ) PCR fragment obtainable with oligonucleotides pyrH11 and SLLH21. Panel 2: result of a PCR reaction with DNA as template, a RT-PCR reaction and a PCR reaction with the RNA used in the RT-PCR reaction as template using oligonucleotides as in (A), (B), (C ), (D), ( E ) and ( F ) in panel 1. The marker is a 1 kb Plus DNA ladder from GibcoBRL (Order No. 10787-018 ).

nation. Furthermore, in both cases, mutants could only be isolated with a frequency of approximately 10−6 integrations per cell (ipc) corresponding to that obtainable by non-specific integration (Maguin et al., 1992, 1996; Biswas et al., 1993). In the case of pSH621, integration would lead to disruption of pyrH and most likely influence the transcription of any genes downstream of, and in operon with, pyrH. In the case of pSH611, integration would not lead to disruption of pyrH but, again, would most likely influence the transcription of any genes downstream of, and in operon with, pyrH. The results obtained with pSH611 and pSH621 strongly support the conclusion based on the RT-PCR experiments described above that pyrH is transcribed as part of an operon with putative downstream reading frames, of which at least one is needed for cell growth. In this case, the above described recombination event for as well pSH611 as pSH621 would result in an operon disruption making the cell devoid of any distal gene product of the operon and hence not capable of growth. The fact that the lactococcal pyrH is transcribed as the middle part of an operon and that one of the downstream located genes, presumably frr1, is vital for the cell, makes it less trivial to create a mutation in pyrH. Hence, it is not straightforward, to test whether the cloned pyrH is the only enzyme capable of UDP synthesis in lactococci and thus a vital enzyme for these bacteria. 3.5. Resemblance to other UMP kinases The function and regulation of UMP kinase has been subject to very intensive studies in primarily E. coli and Saccharomyces cerevisiae. The UMP kinase of E. coli is

known to function as a homohexamer, with GTP and UTP being allosteric effectors (Serina et al., 1995). Apart from catalyzing the conversion of UMP to UDP, it has recently been reported, that the UMP kinase of E. coli serves a regulatory function in pyrimidine de-novo synthesis, namely in the regulation of the carAB operon encoding carbamoylphosphate synthetase ( Kholti et al., 1998). In contrast to the enzyme, very little is known about the regulation of pyrH expression, but the existence of a mRNA 5∞ end 43 bp upstream of the start codon of UMP kinase, which can be correlated with a putative promoter, has been demonstrated in E. coli ( Kholti et al., 1998). The UMP kinase of the Gram-positive Bacillus subtilis has been given very little attention as a consequence of the original findings that kinase activities for CMP and UMP could be copurified during ammonium sulfate fractionation and several chromatographic steps ( Waleh and Ingraham 1976) and that the cmk-encoded CMP kinase catalyzes the conversion of UMP to UDP to a greater extent than the E. coli enzyme (Shultz et al., 1997). Early work on cmk (alias jofC ) in B. subtilis seems to indicate that a disruption of the cmk gene of B. subtilis is deleterious (Sorokin et al 1995), while a gene disruption of the jofD (alias rpsA) gene encoding ribosomal protein S1 is not deleterious. In conclusion, it has been generally accepted, that the phosphorylation of UMP and CMP in B. subtilis is carried out by the same enzyme, namely that encoded by cmk. However, the total genome sequence of this organism shows that an smbA (alias pyrH ) homologue is present on the chromosome ( Kunst et al., 1997). It remains unclear whether this open reading frame is transcribed into a functional UMP kinase in vivo.

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Fig. 4. Clustal W alignment of deduced amino acid sequences of the pyrH encoded UMP kinases of L. lactis, Synechocystis ( Kaneko et al., 1996), A. aeolicus (Deckert et al., 1998) and E. coli (Smallshaw and Kelln, 1992) and the smbA (alias pyrH ) encoded open reading frame of B. subtilis ( Kunst et al., 1997). Identical amino acid residues are indicated by an asterisk, and similarity between amino acid residues is shown by a dot. Positions where only one of the aligned UMP kinases deviates from the remaining are indicated by two dots. The aspartate residue (D -172) L.l believed to be essential for binding UMP and the aspartate residue (D -75) believed to be essential for interaction with UTP and GTP are L.l indicated in bold.

A ClustalW ( Thompson et al., 1994) alignment of all known UMP kinases, including the smbA encoded open reading frame, using default parameters reveals that those most closely resembling the Lactococcus lactis enzyme are the B. subtilis ( Kunst et al., 1997), Aquifex aeolicus (Deckert et al., 1998) and Synechocystis ( Kaneko et al., 1996) enzymes with similarity scores from 58 to 50. An alignment of these four enzymes and the E. coli enzyme, showing a similarity score of 43, is shown in Fig. 4. The lactococcal enzyme appears to have conserved the aspartate residue (D -172) essential L.l for binding UMP and the aspartate residue (D -75) L.l essential for interaction with the allosteric effectors GTP and UTP in E. coli UMP kinase (Bucurenci et al 1998). This, and the fact that these residues are also conserved in the B. subtilis ( Kunst et al., 1997), Aquifex aeolicus (Deckert et al., 1998) and Synechocystis ( Kaneko et al., 1996) enzymes (Fig. 4), strengthens the conclusions drawn from the mutational analysis carried out on the E. coli enzyme (Bucurenci et al., 1998).

3.6. pyrH is part of an evolutionary highly conserved chromosomal gene cluster Surprisingly, we found that the organization of the pyrH and frr1 genes in L. lactis corresponds exactly to what has also been found for the UMP kinase and the ribosomal recycling factor encoded by frr1 in B. subtilis, A. aeolicus and Synechocystis ( Kaneko et al., 1996; Kunst et al., 1997; Deckert et al., 1998). As stated above, these organisms also harbors the UMP kinases most closely resembling the Lactococcus lactis enzyme, but when reviewing the available sequences in the databases, one finds that this chromosomal arrangement of the pyrH and frr1 genes is conserved in (apart from that mentioned above) such diverse organisms as E. coli, Chlamydia trachomatis, Aquifex aeolicus, Synechocystis, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Mycoplasma genetalium and Rickettsia prowazekii (data not shown). A phylogenetic tree representing the mutual relationship of all available

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Fig. 5. Phylogenetic tree showing the evolutionary relationship of all known UMP kinases, including the smbA encoded open reading frame of B. subtilis. Organisms in which the same arrangement of pyrH and fr1 as in L. lactis has been found are underlined.

UMP kinase aa sequences, based on a ClustalW alignment ( Thompson et al., 1994), shows a high correspondence between phylogenetic relationship and the conservation of this chromosomal organization of pyrH and frr1, as indicated in Fig. 5. The reading frame found upstream of pyrH in L. lactis, orfA, does not show homology to any genes of known function as determined by a BLAST search. Since in this study there is no experimental evidence demonstrating that orfA is actually translated into mature protein, it might also be possible that the messenger including pyrH and frr1 is transcribed with an approximately 550 bp long leader sequence. On the B. subtilis, A. aeolicus and Synechocystis chromosomes, the reading frame corresponding to elongation factor Ts encoded by tsf is found upstream of pyrH. In fact, the chromosomal placement of tsf seems to be highly conserved from an evolutionary point of view since it is found immediately upstream of pyrH in E. coli, C. trachomatis, A. aeolicus, Synechocystis, M. pneumoniae, M. genetalium and Thermus thermophilus. Furthermore, in the case of M. tuberculosis, it appears that an insertion event has displaced tsf since this gene is placed approximately 5 kb upstream of pyrH in this organism. A similar event could have taken place in L. lactis. The fact that pyrH in all the organisms mentioned above is part of highly conserved chromosomal region could imply that the described organization of pyrH and adjacent genes is of great importance for cell survival since it has been conserved through evolution. This, combined with the observation that pyrH in L. lactis is transcribed as part of an operon with at least one of these adjacent genes (i.e. frr1), and the recently reported

role of pyrH as a regulatory factor in E. coli ( Kholti et al 1998), could imply that the role of pyrH is more complex than the mere conversion of UMP to UDP. This might point to the importance of keeping genes encoding key proteins from ribosomes and nucleotide metabolism closely linked on the chromosomal level, possibly due to regulatory advantages of such a genetic arrangement. 4. Conclusions 1. The pyrH gene from L. lactis has been cloned and sequenced. 2. The encoded protein can complement an UMPkinase-deficient E. coli strain, proving that the pyrH gene of L. lactis encodes an UMP kinase. 3. The pyrH gene from L. lactis encoding UMP kinase is transcribed as part of an operon including the frr1 gene, most likely encoding Ribosomal Recycling Factor 1. 4. The disruption of this operon is deleterious to L. lactis. 5. The pyrH gene from L. lactis encoding UMP kinase belongs to an evolutionary, highly conserved gene cluster since the organization of pyrH on the chromosomal level shows high resemblance to that found in Bacillus subtilis as well as in Escherichia coli and several other prokaryotes. Acknowledgements This work was supported by grants from the Danish government program for food science and technology

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(FØTEK ) through the Center for Advanced Food Studies. We thank Dr Paal Skyt Andersen for the original PCR fragment. We acknowledge the excellent technical assistance of Janni Juul Jørgensen. We acknowledge Zeynep Vuralhan for partial confirmation of the pyrH sequence.

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