THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 11, Issue of April 15, pp. 7176-7181, 1991 Printed in U.S.A.

Molecular Cloning, Characterization, and Nucleotide Sequence of the Tagatose 6-Phosphate Pathway Gene Cluster of the Lactose Operon of Lactococcus Zactis*

(Received for publication, September 6, 1990)

Rutger J. van Rooijen, Saskia van Schalkwijk, and Willem M. de Vos* From the Molecular Genetics Grouo. DeDartment of BwDhysical Chemistry, Netherlands Institute for Dairy Research (NZZO), P. 0. Box 20, 6710 BA, Ede, The Nethehands

, 1

The tagatose 6-phosphate pathway gene cluster (lacABCD) encoding galactose-6-phosphate isomerase, tagatose-6-phosphate kinase, and tagatose- 1,6-diphos- phate aldolase of Lactococcus lactis subsp. lactis MGl820 has been characterized by cloning, nucleotide sequence analysis, and enzyme assays. Transcription studies showed that the four tagatose 6-phosphate pathway genes are the first genes of the lactose-induc- ible lactose-phosphotransferase operon consisting of the ZacABCDFEGX genes. Using a T7 expression sys- tem, it could be shown that the lacA, lacB, l a d , and 1acD genes code for proteins with apparent molecular masses of 15, 19, 33, and 36 kDa, respectively. Cell- free extracts of induced and noninduced Escherichia coli cells expressing the lacABCD genes were used to determine the functions of the encoded proteins. Expression of both lacA and lacB was required to ob- tain galactose-6-phosphate isomerase activity. The CacC gene codes for tagatose-6-phosphate kinase, the deduced amino sequence of which is similar to that of E. coli Pfk-2 phosphofructokinase, and Staphylococ- cus aureus LacC protein. The tagatose- 1,6-diphos- phate aldolase is encoded by the lacD gene, and its deduced primary sequence, which is homologous to that of the S. aureus LacD protein, predicts an amino acid composition which is virtually identical to that of the previously purified L. lactis E8 tagatose- 1,6-diphos- phate aldolase.

Lactose catabolism in lactic acid bacteria is initiated by either a lactose permease system (lac-PS'; Thompson, 1987) or a phosphoenolpyruvate-dependent lactose phosphotrans- ferase system (lac-PTS; Hengstenberg et al., 1989; McKay et al., 1970). In the lac-PS the intracellular lactose is hydrolyzed by the enzyme @-galactosidase into galactose and glucose, which are utilized in the Leloir (Maxwell et al., 1962) and Embden-Meyerhof-Parnas pathways, respectively. Lactococci that are used in industrial dairy fermentations, transport lactose exclusively via the lac-PTS, resulting in a rapid hom-

* This work was performed under Contract BAP-0477-NL of the Biotechnology Action Programme of the Commission of the European Communities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession numbeds) 505748.

$ To whom correspondence should be addressed. The abbreviations used are: lac-PS, lactose permease system;

IPTG, isopropyl-P-D-galactopyranoside; kb, kilobases; lac-PTS, lac- tose phosphotransferase system; SDS, sodium dodecyl sulfate.

olactic fermentation (de Vos and Simons, 1988). In this sys- tem, EnzymeII'"' (LacE) and EnzymeIII1"' (LacF) are the lactose-specific transport proteins. The resulting lactose 6- phosphate is hydrolyzed by phospho-@-galactosidase (LacG) yielding glucose and galactose 6-phosphate. Galactose 6-phos- phate is further metabolized in the tagatose 6-phosphate pathway by the enzymes galactose-6-phosphate isomerase, tagatose-6-phosphate kinase, and tagatose-1,6-diphosphate aldolase, respectively, as first described in Staphylococcus aureus by Bisset and Anderson (1973). The S. aureus tagatose 6-phosphate pathway enzymes have been partially purified and characterized (Bisset et al., 1980; Bisset and Anderson, 1980a, 1980b). Enzyme activities of the tagatose-6-phosphate pathway enzymes in various Lactococcus lactis strains have been determined and appeared to be induced during growth on lactose or galactose (Bisset and Anderson, 1974). The tagatose-1,6-diphosphate aldolase enzyme of L. lactis E8 has been purified and characterized (Crow and Thomas, 1982). In L. lactis H1 the genetic information for the tagatose 6-phos- phate pathway enzymes is plasmid-encoded (Crow et al., 1983). The tagatose-1,6-diphosphate aldolase gene from this strain has been localized on plasmid pDI-1 and, subsequently, cloned and expressed in E. coli (Limsowtin et al., 1986; Yu et al., 1988). In L. lactis MG1820 the lactose-PTS genes have been characterized and are located on the plasmid pMG820, where they are organized in an operon structure (designated lac-PTS operon) with the gene order: lacFEGX (de Vos et al., 1990; de Vos and Gasson, 1989; Maeda and Gasson, 1986). The lacFEG gene order is also found in the S. aureus lac operon, and these genes appear to be highly homologous to their L. lactis counterparts, although differences in the inter- cistronic regions have been described (de Vos et al., 1990; de Vos and Gasson, 1989). The L. lactis lacX gene, encoding a 34-kDa protein with unknown function, is not present in the S. aureus lac-operon. The L. lactis lac-PTS genes are tran- scribed as 6- and 8.5-kb polycistronic messengers and are induced 5- to 10-fold during growth on lactose as a sole energy source (de Vos et al., 1990). Regulation occurs at the transcrip- tional level and is mediated by the LacR repressor, the product of the divergently transcribed lacR gene (van Rooijen and de Vos, 1990). Transcription of the S. aureus lac-operon also appears to be mediated by a repressor (LacR; Oskouian and Stewart, 1990), which shows high homology (44% identity) to the L. lactis LacR (van Rooijen and de Vos, 1990). The main difference is that the S. aureus lacR gene has the same orientation as the structural genes of the lac-operon (0s- kouian and Stewart, 1990).

In the present study, we describe the molecular cloning, nucleotide sequence, and characterization of the tagatose 6- phosphate pathway gene cluster (lacABCD) of the lactose- PTS operon of L. lactis MG1820. The lacAB, lacC, and hcD

7176

Tagatose 6-Phosphate Pathway Genes of L. lactis 7177

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- C T m T A M A T T T A f f i A G G T A G T C C A A A n ; G C T I \ T T G T T G T G T

M A I V V G A D L K G

ACGAGATTIVLAAGATGTTGTG~TTTTCTTGTAGAAGAAGGTTTTGAGGTAATTGAT T R L K D V V K N F L V E E G F E V I D

GTGACTAAGGACGGACAAGAmPGTTGTTGATGTTACCTTGGCAGTTGCCTCTGAAGT~T V T K D G Q D F V D V T L A V A S E V N

~ G A T G A G ~ C T T A G G T A ~ G T M T T G A T G C T T A T G G A G C T G G T C C A T T U T G G T A K D E Q N L G I V I D A Y G A G P F M V

G C M C T ~ ~ ~ G G T A T G G ~ C A G C A G M G T T T C A G A T G A A C G C T C A G C T T A T A T G A T K I K G M V A A E V S D E R S A Y ~

AUCGTGGACATMCAATGClrCGAATGATTACGGTTGGCGCTG~TCGTCGGTGATGAG T R G H N N A R M I T V G A E I V G D E

C T T A T G C T ~ C A T C G C T A C ~ C G T C M T G G T ~ T A T G A T G G C G G A C G T C A T C A A L A X N I A K A F V N G K Y D G G R H Q

G T C C G A G T A G A T A T G C T T A T M C G A T G T G T T M G ~ G G A G ~ ~ T G A G M T C G C V R V D M L N K M C ' M R I A

AATTCGATGTGACCACAWGTCACAGATGT-TGGCAGTATUGAATTCTTG~TC I G C D H I V T D V K M A V S E F L K S

IVLAAGGATATGAGGTC~GATTTCGGGACATATGACCACGTACGTACTCACTACCCTAT K G Y E V L D F G T Y D H V R T H Y P I

- CTATGGTAI\)LAMGTCGGTGAAGCAGTAGTAAGTGGTCGUGACTTGGGAGTATGTAT Y G K K V G E A V V S G Q A D L G V C I

C T G T G G T A C G G G T G T C G G C A T T M C A A T G C A G T C M T I \ G C C G T G V G I N N A V N K V P G V R S A

GTTGGTTCGTGATATGACGTCAGCTCTATATGCTAAGGAAGMCTTMTGCGAATG~AT L V R D M T S A L Y A K E E L N A N V I

CffimTGGTGGMTGATTACTGGTGGTCTTCTTATATGMTGACATCATTGMGCTTTCAT G F G G M I T G G L L H N D I I E A F I

T G M G C T G A G T A C ~ C C A A C A G M G ~ T ~ T T G A ~ G C ~ T C G M C A C G T E A E Y K P T E E N K K L I A K I E H V

T G A M U C A T M T G C A C A T C M G C A G A T G A G G M T T ~ A C A G M ~ C C T T G ~ T G E T H N A H Q A D E E F F T E F L E K W

GGACCGTGGTGAGTACCACGATTAAGGTATAACCAATGATTCTGACAGTCACACT~TC D R G E Y H D I M I L T V T L N P

C T T C A G T A G A C A T C T C T T A T C C T C T A G l r M C A T T O A A A A S V D I S Y P L E T L K I D T V N R V K

M G A T G T T A G T A A G A C A G C T G G T G G C T T A R T D V S K T A G G K G L N V T R V L Y E S

C T G G T G A T I T I U U G W A C T G C A C A G G T T T C T T A G G T A G D K V T A T G P L G G K I G E P I E S

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GCGAATTAGMCAATCTCCTGTTAGTCCAGCT~ATIVLAATCTClrGG~CACTAGAA ' E L E Q S P V S P A F Y K I S G N T R N

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A C T G T A T T G C T A T T T T A C A C G A G G G T A A C C ~ C A G A A A T T T C I A I L H E G N Q T E I L E Q G P T I

TTTCTCATGMGMGCAGMGGCmCPTGACCACUffATAGCAATCTTATTAAGCMTCAG S H E E A E G F L D H Y S N L I K Q S E

A A G T T G T C A C M T T T C A G G M G T T T G C ~ C A G G A C T T C C C ~ C G A T T A T T A T G ~ C V V T I S G S L P S G L P N D Y Y E K L

TTATCCMCTTGCTTCGGATGMGGAGTAGCAGTCG~GGA~TTCAGGTGCACCAC I Q L A S D E G V A V V L D C S G A P L

T T G A A A C A G T T C T ~ T C G T C G G C T ~ C C T A C T G C C l r T C ~ C C T M T M T G A G G A A C E T V L K S S A K P T A I K P N N E E L

T T T C A C A G T T G C T T A T G G I G M G T A A C ~ G A T A T T G A A G M C T C ~ G A C G ~ C ~ A S Q L L G K E V T K D I E E L K D V L K

M G A G T C A C P T T T T A G T G G T A T ~ M T G G A T A G T G G T C T C A T E S L F S G I E W I V V S L G R N G A F

TTGClUMCATGGTGATGmTCATAAGGTAGATATTCCTGATATCCCAGTTGTTAATC A K H G D V F Y K V D I P D I P V V N P

C A G T C f f i A T C A G G G G A C T C M C G G T ~ G ~ G G T A T T G C A T C A G ~ ~ T ~ G T ~ V G S G D S T V A G I A S A L N S K K S

G T G A C G C T G A C T T A T T A A I U C A T G C G A T G A C A ~ G G T A T G ~ ~ T G C T C A A G ~ C M D A D L L K H A M T L G ~ L N A Q E T M

T G A C A G G G C A T G T G A A T A G A C T A A ~ A C G I \ M C I G -

I T G H V N M T N Y E T L N S Q I G V K E

A G G T A T ~ T G G T A C A U G M C A G ~ C G ~ T ~ G G - ~ C A G A T M V I M V L T E Q K R K S L E K L S D K

A M C G G T T W A T C T C l r G C T T T f f i C A T T T G A C C M C G T G G T G ~ G ~ C G T T ~ A T G G C N G P I S A L A F D Q R G A L K R L M A

A C A G T A T C A A G A T A C l r G A C C A A C T G T A G C T C ~ T G G A A G M C T ~ G G ~ G G T T G C Q Y Q D T E P T V A Q M E E L K V L V A

TGATGAACTTAC~GTACGCTTCTTCAATGTTGCTTGACCCTGAATATGGAC~CCAGC D E L T K Y A S S M L L D P E Y G L P A

M C ~ G C A T T G G A T ~ G M G C A ~ T C T T ~ C T T G C ~ ~ C l r G G T T A T G A T X A L D K E A G L L L A F E K T G Y D

T A U T C G T A C I V L A A C G T T T G C C T G A C T G ~ G A T G ~ G T C T G C ~ G C G T A T C A A T S S T K R L P D C L D V W S A K R I K

AGAGCMGGCGCTGATGCCGTTMGTTCTTGCTTTACTATGATGTAGAClrGCTCAGATGA E Q G A D A V K F L L Y Y D V D S S D E

A C T T A A C C A A C ~ C A A G C T T A T A T A T T G T T G G L N Q Q X Q A Y I E R V G S E C V A E D

T A T T C C T T T C T T C T T G G I C ~ G C T T A T G A T G M G ~ T C T C A G A T G C A f f i ~ C C G T I P F F L E I L A Y D E E I S D A G S V

T G A A T A T G C ~ G T G I A A C C C G C l V U G T C A T T G M G C G A T G A T C C E Y A K V K P R K V I E A M K V F S D P

A C G ~ A A C A T T G A T G W C G T A G M G T A C C A G ~ M C G T T ~ T A T G T C G M G G R F N I D V L K V E V P V N V K Y V E G

T T T T G C T G A T G G T G A A G T G G T T T A C A G T ~ G C T G M G ~ G C T G A C T T C T T T ~ G C A C A F A D G E V V Y S K A E A A D F F K A Q

AGMGMGCGACTMCCTTATCCATACATCTACCTMGTGCAGGTGTATCTGCT~TTGTT E E A T N L P Y I Y L S A G V S A K L F

CCAAG~CATTGCMTTTGCTCACGA~CAGGTGCCGTTTMTGGTGTGCTTTGTGG Q E T L Q P A H D S G A K F N G V L C G

A C G T G C T A C T T G G G C A G G A T C T G T A G M C C T T A C A T C I I C G R A T W A G S V E P Y I K E G E K A A R

T G A A T G G T T G C G T A C T A C T G G A ~ C G ~ T A ~ G A T G ~ C T T M C ~ G T T C T T G T T A A E W L R T T G F E N I D E L N K V L V K

M C A G C T A G T C C A T G G A C T G A T ~ G T A T A G T ~ C A T ~ C G G A G G A T A ~ G T T G T T A S P W T D K V I H

- GAACAGAGMGAGATGACTCCTTAGGGTT 3250 N R E E M T L L G F

FIG. 2. Nucleotide sequence of the L. lactis lacABCD genes and deduced amino acid sequences of the encoded proteins. Translational stops (*) and putative ribosome binding sites (I) are indicated. The N-terminal amino acid sequences of the LacR and LacF proteins are also shown, position 1-38 and 3219-3250, respectively (van Rooijen and de Vos, 1990; de Vos et al., 1990).

genes appear to encode for the galactose-6-phosphate isomer- ase, tagatose-6-phosphate kinase, and tagatose-1,6-diphos- phate aldolase, respectively.

MATERIALS METHODS~

RESULTS AND DISCUSSION

Nucleotide Sequence and Transcriptional Analysis of the L. lactis lacABCD Genes-Fig. 2 shows the nucleotide sequence

Portions of this paper (including "Materials and Methods" and Fig. 1) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

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of the 3.2-kb DNA region between the lacR and lacF genes of pMG820. Four large open reading frames (designated lac- ABCD) are present that all show the same orientation as the lacFEG genes. All open reading frames contain an ATG start codon (position 508, 950, 1476, and 2211, respectively) and are preceded by potential lactococcal ribosome-binding sites (dG" values of complementarity to the L. lactis 16 S rRNA sequence: -16.6, -14.0, -8.4, and -9.8 kcal mol", respectively (Tinoco et al., 1973)) a t a distance that falls within the range (5-12 nucleotides) observed for L. lactis genes (de Vos, 1987). The deduced sizes of the proteins encoded by the lacABCD genes are 141, 171, 310, and 326 amino acids with calculated molecular sizes of 15,236, 18,926, 33,249, and 36,476 Da, respectively. Twenty-seven base pairs downstream of the lacD

7178 Tagatose 6-Phosphate Pathway Genes of L. lactis

1 2 1 2

A B

FIG. 3. Northern blot analysis of lacABCD gene expression i n L. lactis MG1820. Cells were grown on glucose (lane 1) or lactose (lane 2), and 50 pg of isolated RNA was separated on a 1% denaturing agarose gel that was either stained with ethidiumbromide ( A ) or after blotting, hybridized with a lacABC-specific probe (B). The positions of the 23 S (2.8 kb) and 16 S (1.5 kb) rRNAs are indicated, as is the estimated size (kilobases) of the lacABCD-specific transcripts.

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46-

30-

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14- 4

FIG. 4. Expression of the L. lactis lacAB genes in E. coli. Lanes 1-3, 4-6, 7-9, and 10-12 contain extracts from noninduced, induced, and induced + rifampicin, E. coli BLZl-pET& (control), BL21-LacA, BL21-LacB, and BL21-LacAB cells, respectively. Pro- teins were labeled and separated on a 12.5% polyacrylamide/SDS gel. Molecular size markers (in kilodaltons) are indicated. Arrows indicate position of induced proteins.

1 2 3 4 5 6 7 8 -~ ~

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30- *

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1 4-

FIG. 5. Expression of the L. lactis lacCD genes in E. coli. Lanes 1-3, E. coli BL21-LacC, induced + rifampicin, induced and noninduced, respectively. Lanes 4-6, E. coli BL21-LacD, induced + rifampicin, induced and noninduced, respectively. Lanes 7 and 8, control; E. coli BL21(DE3)lysS containing plasmid PET&, induced + rifampicin, noninduced, respectively. Proteins were labeled and separated on a 12.5% polyacrylamide/SDS gel. Molecular size mark- ers (in kilodaltons) are indicated. Arrow indicates position of induced proteins.

gene the lacF gene is initiated with an GTG start codon as described (de Vos et al., 1990). The h A B C D genes are pre- ceded by a nontranslated region containing the promoter and a large amount of direct and inverted repeats involved in regulation of the lac-PTS operon?

In order to investigate the transcriptional organization of the lacABCD genes, the 1.8-kb pMG820 insert of pNZ392 (containing the lacABC genes; see Fig. 1) was used as a probe. Fig. 3 shows the presence of 6- and 8-kb transcripts, the synthesis of which is induced during growth on lactose (lane 2 uersus lane I). These transcripts have the same sizes as those obtained with the h F E G X genes as a probe and are a

R. J. van Rooijen et al., manuscript in preparation.

TABLE I Specific activities (activities expressed as nanomoles. min” mg”) of enzymes in cell-free extracts prepared from induced or noninduced

E. coli strains carrying the L. lactis MG1820 lacABCD genes

Strain Galactose-6-P Tagatose-6-P Tagatose-l,g-diP isomerase kinase aldolase

BL21-LacAB +IPTG 330

48

+IPTG 10

-1PTG - - - -

BL21-LacA

-1PTG 8 - - - -

BL21-LacB +IPTG 11 -1PTG 13

- - - -

BL21-LacC +IPTG - 92 - -1PTG - 10 -

BL21-LacD +IPTG - - 110 -1PTG - - 25

BL2lpET& +IPTG 13 13 30 -1PTG 11 12 27

~ - Not determined.

consequence of the presence of an intercistronic terminator between the lacE and lacG genes (de Vos et al., 1990). There- fore, we conclude that the tagatose 6-phosphate pathway gene cluster and the lacFEGX genes of L. &tis are part of the same lac-PTS operon. In addition, since we have previously observed (van Rooijen and de Vos, 1990) that a lacR specific probe, upstream of the EcoRV site, did not hybridize with the lac-specific mRNA species, we conclude that the promoter of the lac-PTS operon must be located near the EcoRV (Fig. 1, position 500) site?

Expression of the L. lactis LacABCD Genes in E. coli-For expression of the lacABCD genes in E. coli the expression vector PET& was used, containing the 410 T7 RNA polym- erase promoter and its translation signals (Studier et al., 1990). Expression of the lacAB, lacA, and lacB genes is pre- sented in Fig. 4 and resulted in the synthesis of 15- and 19- kDa (lane 12), 15-kDa (lane 6 ) , and 19-kDa (lane 9 ) proteins, respectively. The 30-kDa protein in lanes 5 and 6 is the product of the bla gene of plasmid pNZ396, in which the $10

terminator has been deleted (see “Materials and Methods”). Fig. 5 shows the expression of the lacC and lacD genes (lanes 1 and 4, respectively) into 35-kDa proteins. In lane 4 the presence of an additional strongly labeled protein of 7-kDa is visible. This is the gene product of a small open reading frame (201 nucleotides) that during the cloning procedure has been generated in pNZ395 and is preceded by the efficient ribosome-binding and initiation site of the PET& expression vector. The inefficient labeling of the LacD protein suggests that it is poorly expressed, although it shows significant enzyme specific activity in E. coli (see below). However, the abundant labeling of the 7-kDa protein (predicted to contain 6 methionine residues) may explain the less efficient incor- poration of [35S]methionine in the LacD protein.

The molecular masses of all induced Lac proteins corre- spond closely to that predicted from the deduced amino acid sequences of LacA, LacB, LacC, and LacD.

LacAB Encodes Galactose-6-phosphate Isomerase-In order

tracts were prepared from induced and noninduced cells of E. coli BL21-LacAB9 BL21-LacA, BL21-LacB, respectively, fol- lowed by enzyme assays for galactose-6-phosphate isomerase (Table I). Induction of E. coli BL21-LacAB resulted in a 7- fold increase of the specific galactose-6-phosphate isomerase

Lo dekrn1ille Llle ~ U I I C L ~ U I I S of LncA nlld LncD, cell-flee ex-

Tagatose 6-Phosphate Pathway Genes of L. lactis

LacA (L. Lactis) I U I W G A D L K G T R L K D W K N F L V E E G F E V l D ~ K D W - D F V D E 9 N L G l V l D LacA (S. aureus) IUIIIGSDEAGKRLKEVIKSYLLDNKTDVVDVTEGPEVDFVDATLAVA~V9~EGNLGIVID

***"* * * ***-*-* _ _ ***** . - **** ****I -* -* *******

7179

Lac6 (L. Lactis) EFLEKYDRGEYHD Lac6 (S. aureus) EFLEKYDRGEYHD *************

FIG. 6. Homology between the deduced amino acid sequences of the N-terminal parts L. luctis LacA and S. uureus LacA (55%) and C-terminal parts of L. luctis LacB and S. uureus LacB (100%). The deduced partial amino acid sequences of the S. aureus LacA and LacB proteins have been published by Oskouian and Stewart (1990) and Rosey and Stewart (1989), respectively. In the LacA comparison one gap has been introduced to maximize identity. Identical and functionally related amino acids are indicated by an asterisk and dot, respectively (Higgins and Sharp, 1988).

LacC (4.Lact is) I I - - ILTYTLYPSI ID ISYPLETLKlDTVWR~VSKTAG~GLNVTRVLYES~~TATGF

PfkB (m) W V I I I Y T L T L ~ S L D Y I T I T W I Y P E E N ~ ~ H R C S Y H L G G S A T A I F P LacC (S.aureus) II--ILTLTLYPSM)ISYPLTALKLDDVWRMEVSTAG~GLW\nllVLAPVEPVLASGF

*.**.**.* . . . . . ....e= *.**:.. . *. . * LacC (L.Lactis) LVJIIGEFIESELEOSWSPAFYKISUITRYCIAILHE--GYPTEILEPGPTISHEEAEG LacC (S.aureus) I U ; E L W F I A I ( K L D H A D I K ~ F Y N I K ~ T R N C I A I L H E - - ~ T E I L E O ~ E I D N ~ M G PfkB (m) A ~ T C E H L V S L L A D E Y W V A T ~ ~ ~ R ~ L H V H V O Y R F V I I P C M L N E D E F R P

** *. . . *. ... * ... -9. . . 9.9 .. .= ...... LacC (L.Lactis) FU)HYSYLIKPSEVVTIU;SLPSGLPY)YYEKLIPLASDEGVAWLDCSGAPLETVLKSS LacC (S.aureus) FIKHFEPLLEYMAVAISGSLPXGLWQ)YYAPIIERCOWKCVPVILDCSMTLPTVLEWP PfkB (m) LEEPVLE- IESGAILVISGSLPP~LEKLTOLISLRKNKG~SSTVLWGLS~LAIG

. . . . . . . . . **-** *. . . . .* . .* . . . *. . : LacC (L. lact is) AKPTAIKPYYEELSPLL~EVTI(DIEELIDVLKESLFSG-IEUIWSLGRYWIFAKHcDV LacC (s.aureus) YI(PTVIKPYISELYPLLYPPLDESLESLKMV~LFEG-lEUIIVSLGAOWIFAKHNHT PfkB (m) -WIELVYPW~ELSALVWRELTPP-DDVRIMPEIVYSGVIKRVWSLGWGALGWSEW

. . .*** .** .*... ........... .* ....**** .e*.. ... LacC (L.LactiS) FY~IPDIPWWPVGS(;DSTVAGlAYILNSKKSDADLLKH~TL~LNAOETMTGHVNM

PfkB (m) CIOYVPPALKSPSTVM(ORLV~TLKL~NASLEEWVIIFGVMG---SMTLNPGTRL LacC (S.aureus) FYRVWIPTISVLYPVGS(DSTVAGlTYI ILNHEYDHDLLK~TLG~LNA~AOTGY~L

.* 9.. .. =*.**. *".. . . . . . . . . . . ........ LacC (L-Lact is) TYYETLYSPIGVKEV LacC (S.aureus) YYYDDLFYPlEVLEV PfkB C m ) CSHDDTQKIYAYLSR

. . . . . . . FIG. 7. Homology between the deduced amino acid se-

quences of L. luctis LacC, S. aureus LacC, and E. coli Pfk-2. The amino acid sequences have been aligned by introducing gaps to maximize identity. Percentage identity for pairwise comparisons are 61,26, and 25% for L. lactis LacC and S. aureus LacC, L. lactis LacC and E. coli Pfk-2, and S. aurew LacC and E. coli Pfk-2, respectively. Identical and functionally related amino acids, present in all three proteins, are indicated by an asterisk and dot, respectively (Higgins and Sharp, 1988).

activity. The slight activity in extracts of noninduced E. coli BL21-LacAB cells (3.5 times higher than background activity in BL21-pET8c cells) could be attributed to the incomplete repression of the T7 polymerase-dependent gene expression that we occasionally observe. No galactose-6-phosphate iso- merase activity was detected in extracts of induced or nonin- duced E. coli BL21-LacA or BL21-LacB. Therefore, we con- clude that the galactose-6-phosphate isomerase activity is mediated by LacA and LacB. The galactose-6-phosphate iso- merase of S. aureus has been partially purified and its native molecular mass has been estimated at 100-kDa (Bisset et al., 1980). Since the deduced amino acid sequences of the L. lactis LacA and LacB proteins are highly homologous to the S. aureus LacA and LacB proteins (Fig. 6), we assume that the L. lactis native galactose-6-phosphate isomerase is a multimer consisting of two subunits (LacA and LacB; 15- and 19-kDa, respectively). The nature of the interactions between these subunits awaits further investigation. Attempts to visualize a native enzyme on a SDS-polyacrylamide gel by omitting p- mercaptoethanol during the preparation of the protein Sam- ples were unsuccessful (not shown). Combined extracts pre- pared from induced E. coli BL21-LacA and BL21-LacB cells (by incubating equal amounts of protein on ice for 30 min)

TABLE I1 Comparison between the amino acid composition of purified tagtose- l,&diphosphate aldolase (TDP-A) from L. lactis subsp. cremoris E8

(Crow and Thomas, 1982) and the deduced amino acid composition of L. lactis subsp. &tis MG1820 LacD

TDP-A LacD (L. crernoris) (L. la&)

Asp + Asn 30 31 Thr 17 15 Ser 25 20 Glu + Gln 46 44 Pro 13 11 GlY 19 17 Ala 34 35 CYS ND" 3 Val 25 2 1 Met 4 5 Ile 9 11 Leu 30 32 TY 11 14 Phe 11 15 His 3 1 LY s 28 30 '4% 11 11 Trp 4

Amino acid

3-s - 4 326

ND, not determined.

did not result in detectable quantities of galactose-6-phos- phate isomerase activity (results not shown). This could be due to an inefficient formation of the multimer from its subunits LacA and LacB. Alternatively, the presence of trun- cated LacA (49 amino acids) and LacB (32 amino acids) proteins in extracts of E. coli BL21-LacB and BL21-LacA could interfere with an efficient multimer formation.

LacC and lacD Encode Tagatose-6-phosphate Kinase and Tagatose-l,6-diphosphnte Aldolase, Respectively-The func- tions of LacC and LacD were determined by testing cell-free extracts, prepared from induced and noninduced cells of E. coli BL21-LacC and BL21-LacD, for tagatose-6-phosphate kinase and tagatose-1,6-diphosphate aldolase activity, respec- tively (Table I). Induction of E. coli BL21-LacC resulted in a &fold increase of the specific activity of tagatose-6-phosphate kinase. Therefore, we conclude that lacC encodes tagatose-6- phosphate kinase. It is conceivable that the observed E. coli background activity is due to the E. coli Pfk-2 protein that acts as a type I1 phosphofructokinase catalyzing the phos- phorylation of tagatose 6-phosphate into tagatose 1,6-diphos- phate in the galactitol metabolism (Lengeler, 1977). In a protein database search significant homology was found be- tween the L. lactis LacC, E. coli Pfk-2 (Daldal, 1984), and S. aureus LacC (Rosey and Stewart, 1989) proteins (Fig. 7). The function of the S. aureus LacC protein has not yet been reported. The homology between the L. lactis LacC and E.

7180 Tagatose 6-Phosphate Pathway Genes of L. lactis

LacD (L. tac t is ) I I V L T E P W R K S L E K L S D K N G F l S A L A F D P R G A L K R L l U Q r L L D LacD (S. aureus) MSKSNQKIASIEPLSNNEGIISALAFDPRCALKRmAKHPTEEPTVAPlEPLYVLVAEELTPYASSILLD

-H *" *" ""******H*****"* *H***""*****~*** ****"**

FIG. 8. Homology between the de- duced amino acid sequences of L. luctis LacD and S. uureus LacD. Identical and functionally related amino acids are indicated by an asterisk and dot, respectively (Higgins and Sharp, 1988). Percentage identity is 73%.

LacD (L. [ ac t is ) PEYGLPATKALDKEAGLLLAFEKTGYDTSSTKRLPOCLDVVSAKRIKEPGADAVKFLLYYDVDSSDELNP LacD (S. aureus) PEYGLPASDARNKDCGLLLAYEKTGYD~AKGRL~CLVE~RLKEPCANAVKFLLYYDVDDAEEINI

******* "- *****"***** ****** *****"****"********** **-*

LacD (L. [ac t is ) PKQAYIERVGSECVAEDIPFFLElLAYDEEISDAGSMYALCVKPRKVIEKVFSDPRFNIDVLwMVPV LacD (S. aureus) PKKAYIER1GSECVAEDIPFFLEVLTYDDNIPDNGSMFAKVKPRYVWEAllKLFSEPRFNVDVLKVEVPV ** **H*"*H*H********* **"* *"****H** ****~**"***"********

LacD (L. tac t is ) NWMGFADGEVWSKAEMDFFKAQEEATNLPYIYLUGVSAKLFQETLPFAHDSCAKFNGVLCGRAT LacD (S. aureus) NMl(rVEGFAEGEVWTKEEMAPHFKWDMTHLPY1YLSAGVSAELFeETLKFAHEAGAKFlGVLCGRAT

*""~n*** * **** ** *- H"H**HHH* "*** **" *************

LacD (L. t ac t is ) UAGSVEPYIKEGEKMREYLRTTGFENIDELNI(VLVKTASPVTDKV LacD (S. aureus) USCAWWIEPCEDMREYLRTTGFKNIDDLNKVLKDTATSUKPRK

* *- ** -** *********** ***-***** ** _ _

coli Pfk-2 enzymes, both catalyzing the same reaction (i.e. phosphorylation of tagatose 6-phosphate), indicates that these enzymes have evolved from a common ancestor.

Induction of E. coli BL21-LacD resulted in the 4-fold in- crease of the specific tagatose-1,6-diphosphate aldolase activ- ity. The tagatose-l,6-&phosphate aldolase gene of L. lactis H1 has been previously located on a 2.2-kb EcoRI-AuaI restriction fragment of plasmid pDI-1 (Yu et al., 1988). The lacD gene of L. lactis MG1820 is located on a similar sized EcoRI-AuaI plasmid fragment (extending from position 1425 to 3700, Fig. 1). A comparison between the derived amino acid composition of the L. lactis LacD protein and that of the purified tagatose-1,6-diphosphate aldolase from L. &tis E8 (Crow and Thomas, 1982) showed that these proteins have an almost identical amino acid composition (Table 11). From these data, the E. coli expression studies and enzyme assay, and the homology at the restriction map level between the L. lactis MG1820 lacD gene and the tagatose-1,6-diphosphate gene of L. lactis H1, we conclude that lacD encodes tagatose- 1,6-diphosphate aldolase. Very strong homology was found between the L. lactis and S. aureus LacD proteins (73% identity, Fig. 8). No biological function for the S. aureus LacD protein has yet been published.

The high degree of homology between the deduced amino acid sequences of the L. lactis and S. aureus LacABCD pro- teins strongly suggests that the lacABCD genes of s. aureus also code for the tagatose-6-phosphate pathway enzymes.

CONCLUSIONS

In this paper, we present the nucleotide sequence of the genes (lacABCD) encoding the enzymes involved in the ta- gatose-6-phosphate pathway. The lacAB, l a d , and &D genes code for the multimeric galactose-6-phosphate isomerase (15- and 19-kDa subunits), tagatose-6-phosphate kinase (33-kDa), and tagatose-1,6-diphosphate aldolase (36-kDa), respectively, and are located in between the L. lactis lacR and lacFEGX genes. Transcription studies showed that the lacABCD genes are transcribed as 6- and 8-kb polycistronic messengers to- gether with the lacFEGX genes and, therefore, are part of the lac-PTS operon. To our knowledge this is the first molecular analysis of the tagatose 6-phosphate pathway, which has a pivotal role in the lactose and galactose metabolism in several Gram-positive bacteria.

Since it has been shown that a distinct galactose-PTS exists in L. lactis (Park and McKay, 1982; Crow et al., 1983; LeBlanc et al., 1979), it remains to be determined what the location is of the gal-PTS genes and whether they are coupled to the

From this study and earlier studies (de Vos and Gasson, 1986; van Rooijen and de Vos, 1990; de Vos et al., 1990), we conclude that the L. lactis lac-PTS regulon includes two distinct transcriptional units with the following gene order: lacR-lacABCDFEGX, which are followed by a iso-ISS1 ele- ment.

Acknowledgments-We thank Gaetan K. Y. Limsowtin, New Zea- land Dairy Institute, for his kind gift of tagatose 6-phosphate and tagatose 1,6-diphosphate and Karin Merck, University of Nijmegen, who provided us with the strains for the E. coli expression studies. We are also grateful to Mike J. Gasson (Institute of Food Research, Norwich) for strains and stimulating discussions. Finally, we thank Joop Mondria, Simon van der Laan, and Henny van Bake1 for artwork and photography and Roland Siezen for critically reading this man- uscript.

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HOLECOLIR C W N I H D , CKLRICTLRXPATION, .ulD NUCLEOTIDE SEQUENCE OP THE TAQATOSE 6-PHOSPHATE GENE CLUSTER OF THE UCTOSE OPERON OF LACTOCOCCUS w. Rutger J. van Rooijen. Sasklil van Schalkwljk, and Wlllem M. de Vos

MATERIALS UID METHODS

Bacterial strains, meaia, and plasmids. E. u strains TGl (Gibson. 1984). MC1061 (Casabadan and Cohen. 1980). JM83 IVleira and Messing.

cloning experlnents. For overproductioo of the Lac proteins, E. toll 1982). and "745174 (Campell et a l . , 1978) were used as reclplents in the

K12 lysogen BL21(DE3]- vas used. The I,. Lactis "Ybsp. "train used was MC1820. containing the lactose miniplasmld pMC820 (Maeda and Gasson. 19861. Medxa based On 1117 broth (Difco) contalning 0.5 i (v/v) glucose or lactose, and L-broth (1% tryptone, 0 . 5 % yeast extract. 0 . 5 % NaC11, were used for' the growth of L. lactis and L m, respectively. hmpiclllin (Amp) and chloranphenlcol (cm) were purchased from Sigma and were Used in L toll at a flnal concentration of 5 0 ug/ml and 10 pg/mi,

ptiC16 (Yanisch-Perron et al.. 1985). and pET8c (Studler et al.. 1989). respectively. Plasmids used in the clonlng experiments were pMG820,

.Molecular cloning, reagents, and enzymes. Isolation of DNA from E_ toll and L. lactls was performed by the alkallne lysis method (Blrnbom and ooly, 1979) and a modifred alkallne iysls method (de Vos and Gasson, 1989) . respectAuely. A I 1 mnnlpulatLons 111 vitro and in E. were performed as described by naniatis et a i (19891. AIL enzymes. IF'TG. and

or Boehrinqer. and used dCC0rdln9 to the inStrUCtionS of the rlfamplcln were purchased from Bethesda Research Laboratories. Biolabs,

manufacturers. Sequenase was purchased from Sophar Biochem., and [ e - "P]dliTP and 'SS-methionlnc from Amershdm. 011gonucieotldes Were syntheslied on a Blosearch Cyclone DNA Synthesizer.

Construction Of plasmids. Plasmids PNZ390 and pNZ391 Contaln the 1.0 kb &I-&&EII fragment (fllled i n with Xlenov DNA polymerase) and 2.9 kb &gRI-&CI TeStTlCtlOn fragment Of PMG820 cloned lnto the &I and ECORI-&l of pUC18. respectlvely (Fig. 1 ) . Plasmid pNZ39O contains the

pNZ391 contains the l a c C and h P genes. Plasmid pNZ3Ol consists of the l a c R (van Roaijen and de Do%, 1990). W, and iacs genes. and plasmid

cloned into the HlncII site Of pUC7, and contains the -D gene and 2 . 6 kb & t E I I frnqncnt (filled in wlth Klenod DNA polymerase) Of pM6820

parts Of the k C and k F genes (Fig. 11. In plasmid pNZ392 the 1 . 8 kb m R V restrlctlon fragment of pMG820 (see F l g . 1 1 was cloned into

genes (Fig. I ) the S m ~ l s i t e of pllC18. and confalns Che IacR. lacs. and (part of) L C

~~

Nucleotide s*quY.no. analpis. DNA fragments Were cloned into the multlple clonlng Ilte of Hl3mpi8 and Nllmpl9 (Yanrsch-Perron et a l . ,

dldeoxy chain termination method (Sanger et al.. 1977) using either MI3 1 9 8 5 1 . Nucleotide Sequences of both sfrands Were determined by the

YnlVersP1 prlmer Or synthesized prlrner. Samples were electrophoresed on a 68 polyacrylamide. 7.5 M urea sequencing g e l . The sequencing strategy is presented in FLg. 1. Sequence data were assembled and analyzed u s i n g the PC/CENE program Version 5.01 (Cenofit Geneva). The

Were Used to Screen the PIDteln databases SWISS-PROT and NBRF/NEW. facllltlee of the Netherlands CAOS/CAIM Center (Universlt; of Nijnegen)

releases 1 4 . 0 and 2 5 . 0 , renpectlvely.

m a isolation ana northern blot analysis. L lactie 1161820 cells grovlng on glucose or lactose (100 mll Were harvested and total RNA Was ,solated 65 previously descrlbed (van RDOllen and de V O I , 1990). RNA (50 09) was glyoxylated, size fcactlandted. and blotted to a membrane

were performed a s descrlbed (van Roollen and de VOS, 19901. A 1.8 kb (Gene Screen: New England Nuclearl. Prehybridizatlon and hybridization

EcoRI-BamHI restlictlon fragment of pNZl92 Vas labelled by K k t r E l a t l o n (Manlatls et al.. 1489) and used as a hybridization probe.

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2, 3.6 p i of 1 M glycine/PiaOH buffer (pH 10,51, 1 lil a l k d l l n c phosphatase (1 U), and 7.4 p1 H20. were added to the hea t - t r ea fe : l reaction mixture. followed by incubation far 60 mln at 25 - C . The I l n l l . step lnvolved the enlymatlc determinatlon of galactose a s deicr~lwci 1))

performed as described by Bisset and Anderson ( I l r U a ) , t h e re ,7c?>v,> Kura and Wallenfels (1974). The tagatose 6-phosph*te kinase ds."dy'. . e r v

glycylglycine-Na0H buffer (pH 8.5). 6.7 mM M9C1,. 1 . 3 mM ATP, 2 . ; # P I , mlxture ( 0 . 2 5 ml) contained cell-free exrract (l-=, ,111, r , . 01

phosphoenolpyruvate. 0 . 3 3 mM NADH, 0.33 mM D-tagatose 6-phosphafe. ,381.1

non-linitmg amounts of pyruvate klnase ( 1 . 6 F) and lmc .>tc dehydrogenase (NH.'-sdlt, 10 U J . Tagatose 1.6-dlphosphate dldul i isc assays ( 0 . 2 5 ml) were performed a s described (Crow and Thomas, l C e l l , and contalned cell-free extract, 50 mM trlethanalamlne-HCl buffer (til 7 . 8 ) . 0.25 m NADH, non-limiting amounts of the coupling enzyrnes 0 -

glycerolphosphate dehydrogenase (1.5 U ) and triose phosphate isonerasc

monltored at 340 nm Wlth a CARY 219 (Varlun) a h s o r h a n c c - r e c o r ~ l l n r ] (4.5 U ) . and 0.16 mM tagatose 1.6-dlphosphate. The reactions .:PI('

soectrofatometer thermostated at 2 5 C. A correction for NAliH O Y I J , < ~ ?

2, 3.6 p i of 1 M glycine/PiaOH buffer (pH 10,51, 1 lil a l k d l l n c phosphatase (1 U), and 7.4 p1 H20. were added to the hea t - t r ea fe : l reaction mixture. followed by incubation far 60 mln at 25 - C . The I l n l l . step lnvolved the enlymatlc determinatlon of galactose a s deicr~lwci 1))

performed as described by Bisset and Anderson ( I l r U a ) , t h e re ,7c?>v,> Kura and Wallenfels (1974). The tagatose 6-phosph*te kinase ds."dy'. . e r v

glycylglycine-Na0H buffer (pH 8.5). 6.7 mM M9C1,. 1 . 3 mM ATP, 2 . ; # P I , mlxture ( 0 . 2 5 ml) contained cell-free exrract (l-=, ,111, r , . 01

phosphoenolpyruvate. 0 . 3 3 mM NADH, 0.33 mM D-tagatose 6-phosphafe. ,381.1

non-linitmg amounts of pyruvate klnase ( 1 . 6 F) and lmc .>tc dehydrogenase (NH.'-sdlt, 10 U J . Tagatose 1.6-dlphosphate dldul i isc assays ( 0 . 2 5 ml) were performed a s described (Crow and Thomas, l C e l l , and contalned cell-free extract, 50 mM trlethanalamlne-HCl buffer (til 7 . 8 ) . 0.25 m NADH, non-limiting amounts of the coupling enzyrnes 0 -

glycerolphosphate dehydrogenase (1.5 U ) and triose phosphate isonerasc

monltored at 340 nm Wlth a CARY 219 (Varlun) a h s o r h a n c c - r e c o r ~ l l n r ] (4.5 U ) . and 0.16 mM tagatose 1.6-dlphosphate. The reactions .:PI('

soectrofatometer thermostated at 2 5 C. A correction for NAliH O Y I J , < ~ ?

Fig. 1. Physical map and sequencing strategy of the r e g i o n contalninq

bars and the arrows abave indicate the coding reqlons and the direction the lacl\BCO genes of the L. lactis MG1820 plasmid pMG820. The hatched

h Q Z and l a c F genes are also presented' (Van Raoijen and De "0% 1440). of the lacl\BCO genes. respectlvely. Parts of the codlng regions of the

The positions of the eestrlction enzyme cleavaqe sites used 'in DNA- sequencing and cloning experiments are indicated: A , -1: 0 . m E I I : E, m R I : H , U I I : H I , &I: X . W I : s . =I: SI. =I: V . m R V : x , XbaI. The arrow5 indicate the origin. directlo", and extent of the

sequence derived oligonucleotide primer. Open bars represent the DNA individual sequencing reactions. A black box indicates the use of a

* A frameshift in the U coding r 'eqlon has been introduced by fllling fragments used In the ConStr-UCtiDns of the V d e l ~ u s plasmlda.

in the restriction site of plasrnld p N Z 3 9 3 with KlenOY polymerase (TI .

-