Vol. 174, No. 13

JOURNAL OF BACTERIOLOGY, JUlY 1992, p. 4475-4481 0021-9193/92/134475-07$02.00/0 Copyright X 1992, American Society for Microbiology

Leuconostoc lactis ,-Galactosidase Is Encoded by Two Overlapping Genes SILKE DAVID, HERMAN STEVENS, MAARTEN vAN RIEL, GUUS SIMONS, and WILLEM M. DE VOS* Molecular Genetics Group, Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), P.O. Box 20, 6710 BA Ede, The Netherlands Received 3 January 1992/Accepted 22 April 1992

A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional j-galactosidase. Deletion and complementation analysis showed that the coding region for ,-galactosidase was located on a 5.8-kb SalI-BamHI fragment. Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively. The L. lactis ,B-galactosidase was overproduced in E. coli by using a lambda PL expression system. Two new proteins with Mrs of 75,000 and 36,000 appeared upon induction of PL. The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional 1-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of ,-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral 13-galactosidase gene.

gram-positive cocci that are used for industrial milk and wine fermentations. We recently started the genetic characterization of Leuconostoc spp. (13) and focused on the plasmidlocated lac genes in L. lactis NZ6009 (12). Here we describe the molecular characterization of a DNA fragment from the lactose plasmid pNZ63 that encodes a functional ,-galactosidase. We demonstrate that the L. lactis ,B-galactosidase is encoded by two partially overlapping genes, lacL and lacM.

Two systems for lactose transport and hydrolysis among bacteria are known. The first system has only been found in gram-positive bacteria and involves a phosphoenolpyruvatedependent phosphotransferase system, by which lactose is phosphorylated during transport and subsequently hydrolyzed by a phospho-,B-galactosidase (for reviews, see references 14 and 22). In the second, more widespread system, lactose is transported across the cellular membrane by a galactoside permease, and the unmodified internalized sugar is hydrolyzed by a 13-galactosidase. Most research has focused on the lactose permease (lacY) and ,-galactosidase (lacZ) genes from Escherichia coli (for reviews, see references 3, 24, and 25), and its lacZ gene has been developed into a useful tool in molecular genetics. Similar lac genes located on chromosomal or plasmid DNA have been found in other gram-negative bacteria (20), and in one instance a lac transposon (Tn951) has been reported (11). Recently, lac genes have been characterized in lactic acid bacteria that are used as starter cultures in dairy fermentations and therefore are highly specialized lactose utilizers. Genetic studies have shown that the lactose-specific phosphotransferase system enzymes are homologous and plasmid encoded in Lactococcus lactis (14-16, 30) and Lactobacillus casei (1, 2, 38). In contrast, the homologous lac genes of Streptococcus thernophilus and Lactobacillus bulgaricus are chromosomally located and have been found to encode unique lactose permeases (28, 37) and ,B-galactosidases that show high similarity to those of gram-negative bacteria (42, 43). A plasmid-encoded P-galactosidase in Lactobacillus casei ATCC 393 has been reported (10). In addition, we showed recently that Leuconostoc lactis NZ6009 also contains a lactose plasmid that codes for a P-galactosidase (13). Bacteria of the genus Leuconostoc are heterofermentative, *

MATERIALS AND METHODS Bacterial strains, plasmids, and media. L. lactis NZ6009, previously designated Leuconostoc paramesenteroides (13), has been reclassified based on the hybridization of the V3 region of its 16S rRNA with probes specific for Leuconostoc spp. (26). Comparison of the plasmid complements of L. lactis NZ6009 and its lactose-deficient derivative NZ6091 revealed the absence of the 23-kb plasmid pNZ63 in strain NZ6091, which lacked 3-galactosidase activity (13). E. coli MC1061 (7) and TG1 (18) were used for routine cloning experiments with plasmid and M13 vectors, respectively. All P-galactosidase assays were performed in strain MC1061. E. coli strain AHlAtrp (39), carrying the thermosensitive cI857 repressor, was used in combination with the expression vector pNZ28 (46), a derivative of pPLc28 (39) containing a multiple cloning site. Other E. coli plasmids used were pACYC184 (8) and pAT153 (45). Leuconostoc strains were grown at 30°C in MRS broth (Difco Laboratories, Detroit, Mich.). E. coli strains were grown at 37°C in L broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl), supplemented when appropriate with chloramphenicol (10 ,ug/ml) or ampicillin (50 pg/ml). For selection of P-galactosidase-positive E. coli strains, L broth agar plates were supplemented with 40 ,ug of 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside (X-gal; Sigma Chemical Co., St. Louis, Mo.) per ml.

Corresponding author. 4475

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pNZ634 I I in were cloned and its derivatives. 1. of pNZ63 Restriction map FIG. pNZ63 fragments pACYC184 (pNZ601), pAT153 (pNZ622, pNZ623, pNZ623AN, and pNZ627), or pNZ28 (pNZ633, pNZ633AN, and pNZ634). The orientation of the inserts with respect to vector-located promoters is illustrated by arrowheads on the left. The phenotypes of E. coli MC1061 transformed with these plasmids are indicated on the right: +, blue colonies on X-gal plates and P-galactosidase activity (more than 0.5 U/mg of protein); -, white colonies on X-gal plates and no 3-galactosidase activity (less than 0.01 U/mg of protein). Open boxes indicate insert DNA, and the solid triangles indicate a frameshift mutation created by filling in the NcoI site with Klenow polymerase. Striped arrows indicate the location and orientation of the lacL (left) and lacM (right) genes. B, BamHI; C, ClaI; H, HpaI; N, NcoI; P, PstI; Pv, PvuI; S, Sail; Sp, SphI.

Cloning procedures and sequence analysis. Plasmid DNA isolated from L. lactis by a modified alkaline lysis procedure (5) as described previously (13). Further purification of plasmid DNA by CsCI-ethidium bromide density gradient centrifugation, its cloning in E. coli, and subsequent manipulations were performed according to published procedures (40). Restriction endonucleases and other enzymes used for DNA manipulations were purchased from Bethesda Research Laboratories (Gaithersburg, Md.) or New England Biolabs Inc. (Beverly, Mass.) and used as specified by the suppliers. Sequence analysis by the dideoxy chain method (41) was performed on two strands by cascade sequencing on singlestranded DNA by cloning in bacteriophage M13mpl8 or M13mpl9 (33) with standard M13 or oligonucleotide primers and sequenase (U.S. Biochemical Corp., Cleveland, Ohio). All primers were synthesized on a Cyclone DNA synthesizer (Biosearch, San Rafael, Calif.). DNA sequence data were assembled with the PC/GENE programs (version 5.01; Genofit, Geneva, Switzerland). Amino acid comparisons were performed by screening the protein data bases SWISSPROT and NBRF/NEW, releases 12.0 and 23.0, respectively, with the facilities of the CAOS/CAMM Center (University of Nijmegen, The Netherlands). Cloning of the 13.galactosidase region. Total plasmid DNA from L. lactis was digested with BamHI and separated on a 0.7% agarose gel. The largest, 16-kb BamHI fragment derived from the lactose plasmid pNZ63 was isolated by using Geneclean (Bio 101, Inc., La Jolla, Calif.) and ligated to BamHI-linearized pAT153 or pACYC184. Ligation mixtures were used to transform E. coli MC1061, and 3-galactosidaseproficient transformants were selected. Appropriate subclones were made by using the E. coli vectors pAT153 and pNZ28. Overproduction and characterization of L. lactis 13-galacwas

tosidase. Restriction fragments of pNZ63 were cloned in E. coli AHlAtrp under control of the PL promoter with the expression vector pNZ28. Cells of AHlAtrp harboring the pNZ28 derivatives were grown at 28°C to an optical density at 600 nm of 0.5, at which time the culture was divided into two equal parts. One part was further incubated at 28°C, and the other part was incubated at 42°C to inactivate the host-encoded thermosensitive cI857 repressor. The expression products were analyzed on sodium dodecyl sulfate (SDS)-polyacrylamide gels (27). The N-terminal amino acid sequence of induced protein bands was determined with the aid of a gas phase sequenator (Applied Biosystems, Foster City, Calif.) after transfer of the protein to Problott paper (Applied Biosystems) as described before (47). Assay of 13-galactosidase activity. Cell extracts of exponentially growing cultures were prepared by ultrasonic disruption, and ,-galactosidase activities were determined at 37°C with the chromogenic substrate o-nitrophenyl-,B-D-galactopyranoside (ONPG; Sigma) (34). One unit of 0-galactosidase activity produced 1 nmol of o-nitrophenol per min. Protein concentrations were determined as described before (6). Nucleotide sequence accession number. The sequence reported here has been entered into GenBank under accession number M92281. RESULTS Location and cloning of an L. lactis DNA fragment encoding 3-galactosidase. A partial restriction map of the 23-kb lactose plasmid pNZ63 of L. lactis NZ6009 was constructed. The plasmid contained a single 16-kb BamHI fragment (Fig. 1). Insertion of this 16-kb BamHI fragment into the E. coli vector pAT153 resulted in two plasmids, pNZ620 and pNZ621, which represented both orientations of the fragment in the vector. After introduction into the Lac- E. coli

VOL. 174, 1992

strain MC1061, both plasmids gave rise to blue colonies on X-gal plates. However, after prolonged propagation, white colonies appeared. Analysis of the plasmid complement of these white colonies revealed that the large insert of pNZ620 or pNZ621 had been deleted. In order to circumvent possible instability due to the high copy number of pAT153, we repeated the cloning of the 16-kb BamHI fragment of pNZ63 with the lower-copy-number vector pACYC184. The cloned fragment could be stably maintained on pACYC184 in E. coli in only one orientation; the resulting plasmid was designated pNZ601 (Fig. 1). E. coli MC1061 transformed with pNZ601 gave blue colonies on X-gal plates and contained high ,-galactosidase activity (Fig. 1). Several subfragments of pNZ601 were cloned in E. coli and analyzed for ,-galactosidase production. The coding region for the L. lactis P-galactosidase was localized on a 5.8-kb SalI-BamHI fragment that was contained in pNZ623 (Fig. 1). A frameshift mutation was introduced into pNZ623 by filling in the unique NcoI site with Klenow polymerase. E. coli harboring the resulting plasmid, pNZ623AN, produced white colonies on X-gal plates, indicating that the NcoI site was located within the region encoding 3-galactosidase production. Nucleotide sequence of the L. lactis 13-galactosidase coding region. Determination of the nucleotide sequence of a 3.2-kb region of pNZ623 containing the NcoI site (Fig. 2) revealed the presence of two large open reading frames. Two ATG initiation codons (positions 196 and 277) were found at the start of the first open reading frame, which terminates with an ocher codon at position 2155. The N-terminal sequence of the expression product of this first open reading frame (described below) indicated that the second ATG (underlined in Fig. 2) is the start of a gene, encoding a protein of 626 amino acids (calculated molecular mass of 72,113 Da), that we designated lacL. The second open reading frame starts with an ATG at position 2141 and ends with an ocher termination codon at position 3105. This second open reading frame partially overlaps the 3' end of the lacL gene. The N-terminal sequence of its expression product (described below) indicated that the ATG at position 2141 (underlined in Fig. 2) is the start of a gene, designated lacM. It encodes a protein of 321 amino acids with a calculated molecular mass of 35,389 Da. A region of dyad symmetry followed by a T-rich region was found 16 bp 3' to the lacM stop codon (Fig. 2) and could represent a rho-independent terminator of transcription (36). No other open reading frames were detected immediately upstream or downstream of the lacLM genes. Overproduction of L. lactis 13-galactosidase in E. coli and identification of the LacL and LacM proteins. In order to analyze the expression products of the L. lactis lacL and lacM genes, we cloned a 9.0-kb SalI fragment comprising the 3-galactosidase coding region into the Sall-linearized expression vector pNZ28. This resulted in two plasmids, pNZ632 and pNZ633, representing the two orientations of the fragment within the vector, which both gave rise to blue colonies on X-gal plates after transformation into E. coli MC1061 (Fig. 1). Subsequently, both plasmids were introduced into E. coli AHlAtrp. The p-galactosidase activity observed after thermoinduction was determined. Inducible P-galactosidase activity was found only in E. coli AHlAtrp carrying pNZ633, and restriction analysis showed that the lacLM genes were under control of the PL promoter in this plasmid (Fig. 1). The P-galactosidase activity produced by strain AHlAtrp carrying pNZ633 increased from 1.8 U/mg of protein (after 45 min at 28°C) to 8.9 U/mg of protein (after 45

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min at 42°C). However, the activity decreased severalfold after incubation at 42°C for 3 h, and no P-galactosidase activity could be detected after overnight incubation at 42°C. Analysis of the total cellular proteins of E. coli AHlAtrp cells harboring pNZ633 showed that after induction at 42°C, two major proteins of 75 and 36 kDa were produced. These two polypeptides were not found in AHlAtbp harboring the vector pNZ28 alone (Fig. 3). In addition, lesser amounts of a protein of 58 kDa also appeared to be induced. This 58-kDa protein was not found in induced lysates of strain AHlAtrp harboring pNZ633AN, which contains a frameshift in the unique NcoI site within the lacL gene (Fig. 3). Therefore, this 58-kDa protein is encoded by (part of the) lacL gene and could be a proteolytic degradation product of the 75-kDa protein or represent a truncated derivative of the lacL gene product caused by premature transcriptional or translational termination. The N-terminal sequences of the major inducible proteins encoded by pNZ633 were determined with a gas phase sequencer. The sequence of the first six residues obtained with the 75-kDa protein was Met-Gln-Ala-Asn-Leu-Gln. This sequence is identical to the N-terminal sequence predicted for the lacL gene product (underlined in Fig. 2). Ten amino acid residues were determined from the smaller band of 36 kDa, and the most prominent sequence was Ala-TyrThr-Asn-Asn-Gln-Leu-His-Val-Ile. With the exception of the N-terminal Met residue, the removal of which is not unusual in E. coli (4), this sequence was identical to the N-terminal sequence deduced from the lacM gene. These results demonstrate that the products of the lacL and lacM genes are the 75- and 36-kDa proteins, respectively. lacL and lacM gene products both required for functional 13-galactosidase activity. In order to determine whether the lacL gene alone or in combination with the lacM gene was encoding an active 3-galactosidase, derivatives of pNZ623 or pNZ633 (Fig. 1) were introduced into E. coli strains that contained a frameshift mutation in the lacL gene (pNZ623AN or pNZ633AN) or in which the major part of the lacM gene had been deleted (pNZ627 or pNZ634). E. coli MC1061 harboring one of the mutated plasmids showed white colonies on X-gal plates and no detectable ,-galactosidase activity (Fig. 1), indicating that both the lacL and lacM genes are required for the formation of a functional P-galactosidase. Expression of lacL required for LacM production. To further analyze the expression of the lacL and lacM genes, which overlap in a 14-bp region (Fig. 1), we compared the proteins present in lysates from induced and noninduced cells of strain AHlAtrp harboring either pNZ633AN or pNZ634 with those present in cells harboring pNZ633 (Fig. 4). Deletion of the last 0.3 kb from the lacM coding region, as in pNZ634, results in the formation of a truncated LacM of approximately 20 kDa, while the production of the lacL gene product remains unaffected. In contrast, introduction of a frameshift mutation into the lacL gene at the NcoI site (Fig. 2) affects the production of both LacL and LacM (see also Fig. 3). These results indicate that expression of the lacL gene is required for LacM production. DISCUSSION From the expression, mutation, and sequencing studies described here, we conclude that the plasmid-encoded ,-galactosidase of L. lactis NZ6009 is encoded by two overlapping genes, lacL and lacM, that are both required for the production of a functional 13-galactosidase in E. coli. The N-terminal sequences of the 75-kDa LacL and 36-kDa LacM

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SaIL 1

GTCGACCGAAGGTCCGTATTCATCGGATAGATCAGTATATGAACGGCCTTGATTGTGCATCTCAACAAGAGACTGCTTAAATTCTTTTTTGTAGCGAATCATATTAAAAAATGTCTCCTA

121

TGCGATTAGTTTACAGGGCAGAAGAAAATCTGTCCGTAAATCTAGCATAAGAGCATTTAGTTAAAATTTAACTAAATGACATATATAAATTTAATATTTTGTTTTATAATAATTGTAAGC

2 41

GTTTTTTATTTATGTAACT,TTGAAAGGATCTTCCTCATGCAAGCTAATCTTCAATGGTTAGATGACCCAGAAGTCTTCCGGGTCAACCAATTACCTGCACATAGTGATCACCATTATTAT

3 61

CACGACACAGCAGAATTCAAAACGGGTAGTCGCTTCATCAAGAGTCTCAATGGCGCTTGGCGTTTTAACTTCGCCAAGACACCGGCTGAACGCCCAGTTGATTTTTATCAACCCGATTTC

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481~~~~~~~~~~~~~~~~~~~~~~H2 GATGCAACCGACTTTGATACGATTCAAGTTCCCGGTCATATTGAACTAGCCGGCTATGGTCAAATTCAATACATTAACACGCTATACCCATGGGAAGGTAAAATTTACCGTCGCCCACCG

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TATACCCTCAATCAAGATCAATTAACACCAGGCCTATTCAGCGACGCTGCGGACAACACCGTCGGCTCGTACCTCAAAACCTTCGATCTCGACGATGTTTTTAAAGGGCAACGTATTATC

721

ATTCAGTTCCAAGGGGTAGAAGAAGCCCTGTACGTCTGGTTAAATGGCCATTTTATTGGCTACTCTGAAGATAGTTTcACCCTTCAGAATTTGATGACGCCGTATATTCAGGACCAA

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GGTAACGTTTTAGCGGTTCGGGTCTAcAAAcACAGTACTGCTGCCATTGAAGAccAAGATATGTTccGTcTGGTATTTTccGTGAcGTcAATATAcTGGcGGAGccTGCTAGC

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CATATTACTGATTTGGACATCCGACCAGTTCCAAATGCCAATCTCAAAAGTGGTGAGCTCAACATCACTACTAAAGTAACCGGCGAACCAGCCACTTTAGCGCTGACCGTTAAAGACCAT H I T D L D I R P V P N A N L K S G E L N I T T K V T G E P A T L A L T V K D H

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1081 GACGGGCGAGTACTGACGAGTCAAACGCAAACCGGTAGTGGCAGTGTAACCTTTGATACTATGTTATTCGACCAACTGCACTTGTGGTCACCACAAACGCCGTATCTCTATCAATTGACA D G R V L T S Q T Q T G S G S V T F D T M L F D Q L H L W S P Q T P Y L Y Q L T 1201 ATTGAAGTTTACGATGCTGATCACCAACTCTTGGAAGTCGTCCCATATCAGTTTGGGTTCCGGACGGTCGAGCTGCGCGATGACAAAGTCATTACGTCAACAATAAACGGTTGGTGATC I E V Y D A D H Q L L E V V P Y Q F G F R T V E L R D D K V I Y V N N K R L V I 1321 AACGGGGTTAACCGGCACGAATGGAACGCCCACACCGGTCGCGTTATCAGTATGGCTGATATGCGCGCTGATATCCAAACCATGTTAGCTAACAATATCAATGCCGATCGGACCTGCCAT N G V N R H E W N A H T G R V I S M A D M R A D I Q T M L A N N I N A D R T C H 1441 TATCCTGATCAATTACCTTGGTATCAATTATGTGACGAGGCCGGTATCTACCTAATGGCCGAAACCAACCTCGAATCGCACGGGTCATGGCAAMAGATGGGGGCTATCGAGCCTTCTTAC Y P D Q L P W Y Q L C D E A G I Y L H A E T N L E S H G S W Q K M G A I E P S Y 1561 AATGTTCCTGGCGATAATCCACACTGGCCAGCAGCGGTGATCGACCGGGCCCGTTCAAACTACGAATGGTTAAAAACCACCCATCAATCAmTTTTGGTCACTTGGCAATGAATCGTAT N V P G D N P H W P A A V I D R A R S N Y E W F K N H P S I I F W S L G N E S Y 1681

GCTGGCGAAGATATCGCGGCGATGCAGGCTTTTTATAAAGAACACGATGATTCACGACTCGTCCACTACGAAGGCGTTTTCTACACACCAGAATTAAAAGATCGCATTCTGATGTTGAA A

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1801 AGTCGGATGTACGAAAAGCCCCAAAATATTGTAGCTTACTTGGAAGATAACCCAACCAAACCTTTCCTAAATTGTGAATATATGCATGACATGGGGAATTCTCTGGGCGGTATGCAATCA H D H G N S L G G M Q S S R M Y E K P Q N I V A Y L E D N P T K P F L N C E YV -- -_ ---------__ - *M^Prltrsrs|rrr * __ _ vsvv nww_Xoosss o 1921 TATAATGATTTGATCGACAAGTATCCAATGTATCAAGGTGGCTTTATTTGGGACTTTATTGATCAAGCACTCTTCATGACCCATCACCACCAAGACGTGCTCCGGTAuw>, Y N D L I D K Y P M Y Q G G F I W D F I Q D A L F V H D P I T D Q D V L R Y G G ~~~~~~~~~~~~~~~~~~-

2041

GATTTCGACGAACGCCACTCCGATTATGCATTCTCCGGTAACGGCTTAATGTTTGCCGACCGGACACCAAAACCAGCAATGCAAGAGGTGAAATATTATTATGGCTTACACAAATAATCA

D F D E R H S D Y A F S G N G L M F A D R T P K P A M Q E V K Y Y Y G L H K LacM M A Y T N N 0 216 1 ACTACACGTTATTTACGGCGACGGGAGCTTAGGACTACAGGGGGCTAATTTCCACTACCTCTTTAGCTACGAACGTGGGGGACTTGAATCACTCGTCGTCAACGATAAAGAGTGGCTCTA L H V I Y G D G S L G L Q G A N F H Y L F S Y E R G G L E S L V V N D K E W L Y 2 281 TCGTACACCCACGCCCATGTTTTGGCGGGCGACAACCGATAATGATCACGGTAGCGGCTTTTCAGTCAAATCCGCACAGTGGTACGCGGCCGATAAGTTCTCAACTTGTCAAGATATCGA R T P T P M F W R A T T D N D H G S G F S V K S A Q W Y A A D K F S T C Q D I E 2 401 ATTGACGGTTGACGACCAACCAGTCACACCGTTACCAATCGCGCCACTCAATAACAAATACACGGATCACGAAATCGCCACGAAAGTCTCTCTGGCTTACCACTTCGTTACCACGACCGT L T V D D Q P V T P L P I A P L N N K Y T D H E I A T K V S L A Y H F V T T T V

2 521 TCCTAGTACCATCGTCACAGTGACTTATACGGTGACAGCAGACGGTCAGATCAATATCGCCACCCATTATAGCGGTCAGTCTGATTTGCCAGAGCTACCCGCATTTGGTCTGCGGTTTAT P S T I V T V T Y T V T A D G Q I N I A T H Y S G Q S D L P E L P A F G L R F I 2641

CATGCCAACTACCGCGACCGGCTTCGACTATACCGGTTTGTCCGGTGAGACTTATCCTGACCGGCTGGCTGGCGCAACACACGGGCAATTCCACGTTGACAGTCTGCCAGTCACACCATA H

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2761 CTTGGTCCCACAAGAATGCGGCATGCACATGCAAACTGAACAAGTGACAGTAACGCGATCAACAACACAAAATAACGCTGACCACGACAACACACCATTCAGTTTAACATTTAGCCAAAC L V P Q E C G M H M Q T E Q V T V T R S T T Q N N A D H D N T P F S L T F S Q T 2881

CGATGCACCATTCGCCTTCAGCTGCCTTCCCTATACCGCTGCTGAACTAGAAAACGCAACACACATGGAAGAATTACCATTAGCACGGCGAACGGTCTTATCAATCTACGGTGCCGTTCG D

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3001 TGGGGTCGGTGGCATTGACAGTTGGGGAACGGACGTAGAAGCCCCATATCATATCCTCGCTAATCAAGACATTGACTTCAGCTTTAACATTCATTTCTAAAAATTAATTTGATTTCAAAA G V G G I D S W G T D V E A P Y H I L A N Q D I D F S F N I H F 3121

GAATCGCTCCGGCAGTTATTTGCCAGAGCGTTr,=AATATGm TATGGCTGAGCTTAGTCCrlGAAGTGA

FIG. 2. Nucleotide sequence of the L. lactis NZ6009 P-galactosidase coding region and flanking sequences. Numbering starts at the SalI site (see Fig. 1); relevant restriction sites are indicated. The start and stop codons for lacL and lacM are underlined. The putative ribosome-binding site in front of the lacL gene is marked by stars. A region of dyad symmetry following the lacLM genes, representing a putative terminator of transcription, is underlined.

proteins produced at high levels in E. coli were identical to those predicted from the DNA sequences. Although trans complementation of the E. coli M15 3-galactosidase by the a-fragment is well known and exploited widely (3, 40, 48), to our knowledge this is the first example of a naturally occurring P-galactosidase consisting of two nonidentical subunits. In spite of the phylogenetic analysis based on Leuconostoc 16S rRNA sequences (32), to date the sequence of the 3'

ends is not known. However, 8 bp preceding the start codon of the lacL gene (Fig. 2), a potential ribosome-binding site was found, with the sequence 5'-GAAAGGA-3' (positions 263 to 269), that shows high complementarity to the 3' end of the 16S RNAs from the phylogenetically related Lactococcus lactis (29) and Bacillus subtilis (19). In contrast, the lacM gene is preceded by a stretch of 10 pyrimidines, which are unlikely to act as a ribosome-binding site. The introduction of a frameshift mutation at the NcoI site in the lacL gene,

L. LACTIS 3-GALACTOSIDASE GENES

VOL. 174, 1992 1

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FIG. 3. Expression products of the lacL and lacM genes. SDSpolyacrylamide gel electrophoresis followed by Coomassie blue staining of whole-cell lysates of E. coli AH1Atrp containing pNZ28 (lanes 1 and 4), pNZ633AN (lanes 2 and 5), or pNZ633 (lanes 3 and 6). Lanes 1 to 3 show the lysates of cells grown at 28°C; lanes 4 to 6 show lysates of cells after induction at 42°C for 45 min. The molecular masses (in kilodaltons) of induced proteins are indicated at the right.

generating premature translation termination at position 579, results in the loss of production of both the LacL and LacM proteins (Fig. 3 and 4). Assuming that this mutation does not cause polar effects, this would imply that the lacM gene is translationally coupled to the lacL gene, which is in agreement with the absence of a good ribosome-binding site for the lacM gene and its overlap with the lacL gene (44). Translational coupling is an efficient and simple way to regulate gene expression. In this case, translational coupling could ensure a constant ratio between the amounts of LacL and LacM proteins synthesized (44). Preliminary estimations of the apparent molecular weight of the L. lactis 3-galactosidase indicated that it is a heterodimer. This would suggest that the translational coupling should be highly efficient, resulting in the synthesis of equal molar amounts of LacL and LacM proteins. The instability of the L. lactis P-galactosidase that is apparent after prolonged incubation at 42°C might be due to dissociation of LacL and LacM at elevated temperatures. 1

2

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FIG. 4. Expression products of mutated lacLM genes. SDSpolyacrylamide gel electrophoresis followed by Coomassie blue staining of whole-cell lysates of E. coli AHlAttp containing pNZ634 (lanes 1 and 2), pNZ633AN (lanes 3 and 4), or pNZ633 (lanes 5 and 6). Lanes 1, 3, and 5, lysates of cells grown at 28°C; lanes 2, 4, and 6, lysates of cells after induction at 42°C for 45 min. The molecular masses (in kilodaltons) of induced proteins are indicated at the right. An induced protein also found in the control (AHlAtp harboring pNZ28; not shown) is indicated by a star.

4479

Alternatively, it may be attributed to proteolysis. The generation of a 58-kDa protein that is encoded by the lacL gene (Fig. 3) is in favor of the latter possibility. The amino acid sequence deduced from the L. lactis lacL gene was compared with that of the ,-galactosidases from E. coli (25), Streptococcus thermophilus (43), Lactobacillus bulgaricus (42), and Clostridium acetobutylicum (21). A computer alignment of these sequences is given in Fig. 5 and shows that LacL has high sequence similarity with the N-terminal region of approximately 700 residues of the other ,B-galactosidases. In addition, the deduced LacM amino acid sequence showed homology to the C-terminal part of approximately 300 residues of the ,B-galactosidases of the other bacteria. Several regions of high similarity were detected, and the average percent identity between the P-galactosidase sequences varied between 22 and 45% (Table 1). The highest identity was obtained between the gram-positive bacteria, and unexpectedly, the L. lactis 3-galactosidase showed higher identity with the C. acetobutylicum sequence than with the 3-galactosidases from the other lactic acid bacteria. These results strongly suggest that these enzymes have evolved from a common ancestral ,-galactosidase gene. The putative active-site residue (Glu-461) of the E. coli ,-galactosidase (23) and the two residues (Leu-316 and Pro-429), substitution of which increased the cold sensitivity of the Lactobacillus bulgaricus P-galactosidase (31), were all conserved in the L. lactis P-galactosidase (Fig. 5). The alignment shows that, in spite of the small insertions at the N terminus of LacL and the C terminus of LacM, the L. lactis LacL and LacM proteins combined are smaller than the other ,-galactosidases. A specific domain, located in a region of low homology (amino acid positions 675 to 775; Fig. 5), is not found in either LacL or LacM and hence does not seem to be required for the activity of the L. lactis 3-galactosidase. In the E. coli ,B-galactosidase, there is an elastase cleavage site just a few residues C-terminal from this region (positions 779 to 780; Fig. 5). It has been shown that cleavage of this site, generating N- and C-terminal fragments that are no longer covalently linked, does not affect the activity of the E. coli enzyme (17). The present results with the L. lactis ,B-galactosidase support this finding and further indicate that the C-terminal fragment (LacM) is required for a functional enzyme. It is very likely that the L. lactis lacL and lacM genes have been generated by one or more deletion events within an ancestral ,B-galactosidase gene that included the fragment coding for the dispensable domain. Spontaneous deletions within the P-galactosidase gene of Lactobacillus bulgaricus NCDO1489 have been found to occur frequently, and in one case (lac1S0) included the fragment lacking in the L. lactis P-galactosidase coding region (35). In Streptococcus thermophilus and Lactobacillus bulgaricus, the genes encoding the ,3-galactosidase and the lactose permease are organized in an operon structure (28, 37), in which the lactose permease gene is located immediately upstream of the ,B-galactosidase gene, separated by only 3 bp. This organization is different from that of the lac genes in E. coli, which have the gene order lacZ-lacY. We could detect no open reading frames in the sequences immediately upstream or downstream of the L. lactis lacLM genes (Fig. 2). Recent results (12) have shown that lactose transport is also encoded by the lactose plasmid pNZ61 in L. lactis NZ6009, and the lac genes have been located approximately 4 kb upstream of the lacL gene. The organization of the plasmid-encoded genes involved in lactose metabolism in L.

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lactis is therefore considerably different from that described for the lac genes of other bacteria. The nucleotide and deduced amino acid sequences of the plasmid-encoded P-galactosidase genes from Lactobacillus casei ATCC 393 have recently been reported (10). The L. casei P-galactosidase has a heterodimeric subunit structure similar to that of L. lactis. Comparison of the L. lactis and L. casei lacL and lacM genes reveals almost complete identity (99%) on the DNA and deduced protein levels, indicating that they not only have a common ancestor but diverged only very recently. Plausible routes for the dissemination of the lacL TABLE 1. Amino acid identity (based on the comparison shown in Fig. 5) between pairs of 0-galactosidases from L. lactis, S. thermophilus, L. bulgancus, C. acetobutylicum, and E. coli 13-Galactosidase

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and lacM genes include horizontal plasmid transfer followed by transposition or recombination into endogenous plasmids. ACKNOWLEDGMENTS We thank Bruce Chassy for exchanging results prior to publication. We are grateful to Roland Siezen and Bruce Chassy for critically reading the manuscript and helpful discussions. This work was partially funded by the Programme Committee on Agricultural Biotechnology (PcLB) and the BRIDGE Programme of the Commision of European Communities (contract BIOT-CT91-

0263). REFERENCES 1. Alpert, C. A., and B. M. Chassy. 1988. Molecular cloning and nucleotide sequence of the factor III'ac gene of Lactobacillus casei. Gene 62:277-288. 2. Alpert, C. A., and B. M. Chassy. 1990. Molecular cloning and DNA sequence of lacE, the gene encoding the lactose-specific enzyme II of the phosphotransferase system of Lactobacillus casei. J. Biol. Chem. 265:22561-22568. 3. Beckwith, J. 1983. Lac: the genetic system, p. 11-30. In J. H. Miller and W. S. Reznikoff (ed.), The operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 4. Ben-Bassat, A., K. Bauer, S. Y. Chang, K. Myambo, A. Boosman, and S. Chang. 1987. Processing the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure. J. Bacteriol. 169:751757.

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