Appl Microbiol Biotechnol (1990) 34:677-679
Applied Microbiology Biotechnology © Springer-Verlag 1990
In vitro expression of Lac-PTS and tagatose 1,6-bisphosphate aldolase genes from Lactococcus lactis subsp, cremoris plasmid pDI-21 Pak-Lam Yu, Robert A Hodge, and Xiao-Ping Li* Department of Biotechnology, Massey University, Palmerston North, New Zealand Received 10 October 1989/Accepted 11 May 1990
Summary. A 4.4-kb EcoR1-EcoR1 D N A fragment from the Lactococcus lactis subsp, cremoris plasmid pDI-21 encoded the tagatose 1,6-bisphosphate (TBP) aldolase gene and the Lac-PTS genes. In vitro transcriptiontranslation using Escherichia coli $30 extract showed the synthesis of 41000-, 23 000- and 12000-dalton proteins which correspond to the TBP-aldolase, Lac-PTS enzyme II, and factor III proteins respectively.
Introduction The primary function of lactic acid bacteria in dairy fermentations is the catabolism of lactose with the eventual production of lactic acid as a major or sole end-product. Considering the industrial importance of group N streptococci, now classified as the genus Lactococcus, characterization of the enzymes and genes responsible for lactose utilization is of considerable importance. The conjugative plasmid pDI-21 (63 kb) from L. lactis subsp, cremoris strain H2 encodes genes which are responsible for both lactose utilization and protein degradation (Yu et al. 1989). The tagatose-6-phosphate pathway genes, a LacPTS region and the phospho-fl-galactosidase gene have been mapped within a region of 12.5 kb (Inamine et al. 1986; Yu et al. 1989). Plasmid linkage of the genes responsible for lactose metabolism in lactic streptococci has been well established (Anderson and M c K a y 1977; Crow et al. 1983; Gasson 1983). In Streptococcus lactis 712, restriction enzyme mapping of deletion mutants established that lactose activity was determined by a 14.2-kb B c l I fragment of the plasmid pLP712 (Gasson 1983). The key lactose-splitting enzyme, p-fl-gal, was cloned and expressed as a 58000-dalton protein in Escherichia coli (Maeda and Gasson 1986). Harlander * Present address: Department of Chemical and Biochemical Engineering, Hua Chiao University, Quan Zhou, Fujian, People's Republic of China Offprint requests to: P.-L. Yu
et al. (1984) cloned a 23-kb K p n I fragment of S. lactis LM0230 into the vector plasmid pDB101 and used it to restore lactose activity upon transformation into an appropriate S. sangius mutant. The cloning and characterization of the p-fl-gal gene from S. lactis Z268 plasmid pUCL13 has been completed (Boizet et al. 1988). Sequencing data revealed the presence of two promoter-like structures in the 5' region of the p-fl-gal gene which has an open reading frame coding for polypeptides of 55096 or 54078 daltons. Recently, in vivo and in vitro expression of a S. lactis tagatose 1,6-bisphosphate aldolase gene in the E. coli transcription-translation system has shown a polypeptide of 37 500 daltons (Yu et al. 1988). However, expression of lac genes other than the TBP-aldolase gene and p-fl-gal gene has not yet been demonstrated in vitro. In this report, the localization and expression of lac-PTS and TBP-aldolase genes from the plasmid pDI-21 of L. lactis subSp, cremoris strain H2 are described.
Materials and methods All lactococci strains were grown in M17 broth (Terzaghi and Sandine 1975). E. coli strains were grown in Luria-Bertani (LB) broth. E. coli JM109, endA1, recA1, gyrA96, thi, hsdR17 (rk-, ink--), relA1, supE44- 2~-,A(lac-proAB), [F', traD36, proAB, laclqZ M15] (Yanisch-Perron et al. 1985) was used as the host for plasmid transformation. The plasmid vector GM3pE Blue (2743 bp, Ape) and the ExolII deletion system were supplied by Promega, Sydney, Australia. Recombinant clones were selected on LB agar supplemented with ampicillin (100 txg/ml), 5-bromo-4-chloro-3-indolyl-fl-D-galactopyranoside (X-gal, Bethesda Research Laboratories, Bethesda, Md, USA; 40 lxg/ml), and 0.2 mM isopropyl-fl-D-thiogalactopyranoside (IPTG, Boehringer, Auckland, New Zealand). Aldolase enzyme activity was assayed on E. coli extracts as described previously (Yu et al. 1988). Large-scale preparations of lactococcal plasmids were by the method of Anderson and McKay (1977), and E. coli plasmids by alkaline lysis (Maniatis et al. 1982). For ExolII deletion experiments, plasmid DNAs were further purified by ultracentrifugation in caesium chloride-ethidium bromide equilibrium gradients. The rapid boiling method (Holmes and Quigley 1981) was used for initial plasmid screen_ing of E. coli clones. The E. coli $30 in vitro transcription-translation
678 system was used as described by Pratt (1984). The $30 extract, [35S]methionine and [aaC]protein standards were from Amersham, Sydney, Australia. The gene products were separated on 15% sodium dodecyl sulphate-polyacrylamide gels (Laemmli 1970). Gels were treated for 30 min in 50% methanol and 4% glycerol before drying for 2 h at 80° C in a gel dryer (Bio-Rad, Richmond, Calif, USA). Autoradiography was for 3-4 days with Fuji Tokyo, Japan X-ray Film RX at room temperature.
Results and discussion
The 4.4 E c o R 1 - E c o R 1 fragment of pDI-21 which encodes the TBP-aldolase gene and Lac-PTS genes was cloned in both orientations by using the plasmid vector p G E M 3 Blue and is designated as pBH401 and p B H 5 0 1 (Fig. 1). The D N A insert was further characterized by restriction endonuclease mapping. In comparison to the TBP-aldolase gene from pDI-1 of S. lactis (Yu et al. 1988), no internal H i n d l I I sites were found in pBH401 and pBH501. For the localization of the Lac-PTS genes within the 4.4 EcoR1-EcoR1 fragment, purified pBH501 plasmid D N A was progressively shortened from one end by E x o l I I deletion. Two of the subclones pBH501-5.0 and pBH501-8.5 are shown in Fig. 1. In E. coli, pBH401 and pBH501 expressed TBPaldolase activity (1.6+0.10 Ixmol/min per milligram protein). Neither pBH501-5.0 nor pBH501-8.5 exhibited detectable aldolase activity. These plasmid DNAs were further isolated and assayed for the expression of the Lac-PTS genes in the E. coli in vitro transcriptiontranslation system. Both pBH401 and pBH501 expressed the TBP-aldolase gene in vitro and the size of the gene product was determined from 14C-labelled
I"---
E
H
II
I
H
I
A pBH401
S
H H
ill~
-----~Ii
H
H
!
i
A
TBP-aldolase structural gene and its promoter sequences. This is indicated by the synthesis of the 41000 dalton polypeptide in either orientation. This result was also obtained by Yu et al. (1988) for the in vitro expression of the TBP-aldolase gene from S. lactis plasmid pDI-1, which has a lower molecular weight of 37500 daltons. The plasmid pBH501-5.0 expressed two proteins of 23 000 and 12 000 daltons. The level of expression of the 23000-dalton protein was much higher than that of 12000-daltons. The subclone pBH501-8.5 was 390 bp shorter than pBH501-5.0 in the left end of the insert. This deletion resulted in no expression of either of the Lac-PTS proteins, which could indicate the position of a regulatory region to the left of the two ClaI sites (Fig. 2). The presence of a lactose-inducible promoter which controls the expression of the Lac-PTS and p-fl-gal
E
I
~
CpC
TBP-Aldolase gene
E
I
I
standards as 41000 daltons (Fig. 2). The 4.4-kb EcoR1EcoR1 fragment of pDI-21 appears to carry both the
I
S
Lac-PTS genes
H H
E
r~i~ ~
I
C ~C
pBH501
H H
I
E
-----1%, ,,d
I
,
,
P
pBH501-5.0
H
i
~ l pBH501-8.5
i ,.J
~' CC P
H
I
E
;
I 1 kb
Fig. 1. Schematic representation of linearized forms of recombinant plasmids pBH401, pBH501, pBH501-5.0, and pBH501-8.5. The positions of the TBP-aldolase gene and Lac-PTS genes are shown. The small arrow within the pGEM3 Blue vector indicates the direction of transcription and position of the Lac promoter: A (AvaI), C (ClaI), E (EcoR1), H (HindlI), P (PstI) and S (SphI)
Fig. 2. Autoradiogram of [35S]methionine-labelled polypeptides from in vitro transcription-translation run on a 15% sodium dodecyl sulphate-polyacrylamide gel: lane 1, pBH501-8.5 DNA; lane 2, pBH501-5.0 DNA; lane 3, pBH501 DNA; lane 4, pBH401 DNA
679 genes have been postulated in lactose plasmids of L. lactis and Lactobacillus casei (Chassy 1987; Porter and Chassy 1988, De Vos and Gasson 1989). In the case of Lactococcus lactis plasmid pMG820, the lactose genes are organized in an operon-type structure with the gene order L a c F (Lac-PTS FactorlII), L a c E (Lac-PTS Enzyme II), L a c G (p-fl-gal) (De Vos, personal communication). The nucleotide and deduced amino-acid sequences o f the lactose genes are highly homologous to those from Staphylococcus aureus and Lactobacillus casei (Porter and Chassy 1988; De Vos and Gakson 1989). The lactose-specific factor I I I has been isolated from L. casei and has the structure of a trimer of identical subunits of 13 000 daltons, which is similar in size to the S. aureus factor I I I subunit (mol. w t . = 11367) (StiJber et al. 1985, Alpert and Chassy 1988). Our data from the in vitro expression system showed the expression of a 12000-dalton protein which corresponds to the soluble lactose factor I I I protein subunit of L. casei and S. aureus. Purification and characterization of the galactoside-specific membrane-bound protein enzyme II from S. aureus showed a protein of 50 000 mol. wt. u p o n induction o f the staphylococcal lac operon (Sch/afer et al. 1981). The size of enzyme II protein from S. aureus is considerably bigger than the 23 000-dalton protein that we have obtained in the in vitro system, which could indicate the presence of a dimeric form of this protein in Lactococcus lactis. Sequence analyses o f these genes are in progress and the comparison of their primary sequences with other Gram-positive microorganisms will be made. The presence of a regulatory region and operon structure will then be established. Acknowledgement. We thank V. L. Crow, New Zealand Dairy Research Institute, for the assay of aldolase enzyme activity.
References Alpert C, Chassy BM (1988) Molecular cloning and nucleotide sequence of the factor III ~acgene of Lactobacillus casei. Gene 62:277-288 Anderson DG, McKay LL (1977) Plasmids, loss of lactose metabolism and appearance of partial and full lactose-fermenting revertants in Streptococcus crernoris B1. J Bacteriol 129:367377 Boizet B, Villeral D, Slos P, Novel M, Novel G, Mercenier A (1988) Isolation and structural analysis of the phospho-fl-galactosidase gene from Streptococcus lactis Z 268. Gene 62:249261
Chassy BM (1987) Prospects for genetic manipulation in lactobacilli. FEMS Microbiol Lett 46:297-312 Crow VL, Davey GP, Pearce LE, Thomas TD (1983) Plasmid linkage of the tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism. J Bacteriol 153:76-83 De Vos WM, Gasson MJ (1989) Structure and expression of the Lactococcus lactis gene for phospho-fl-galactosidase (lacG) in Escherichia coli and L. lactis. J Gen Microbiol 135:1833-1846 Gasson MJ (1983) Plasmid complements of Streptococcus lactis NCD0712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154:1-9 Harlander SK, McKay LL, Schactele CF (1984) Molecular cloning of the lactose-metabolizing genes from Streptococcus lactis. Appl Environ Microbiol 48:347-351 Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193-197 Inamine JM, Lee LN, LeBlanc DJ (1986) Molecular and genetic characterization of lactose-metabolic genes of Streptococcus cremoris. J Bacteriol 167:855-862 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Maeda S, Gasson MJ (1986) Cloning, expression and location of the Streptococcus lactis gene for phospho-fl-D-galactosidase. J Gen Microbiol 132:331-340 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Porter EV, Chassy BM (1988) Nucleotide sequence of the fl-Dphosphogalactoside galactohydrolase gene of Lactobacillus casei: comparison to analogous pbg genes of other Gram-positive organisms. Gene 62:263-276 Pratt JM (1984) Coupled transcription-translation in prokaryotic cell-free systems. In: Hames BD, Higgins SJ (eds) Transcription and translation - a practical approach. IRL Press, Oxford, pp 179-209 Schafer A, Schrecker O, Hengstenberg W (1981) The staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Eur J Biochem 113:289-294 Stiiber K, Deutscher J, Sobek HM, Hengstenberg W, Beyreuther K (1985) Amino acid sequence of the amphiphilic phosphocarrier protein factor III L~c of the lactose-specific phosphotransferase system of Staphylococcus aureus. Biochemistry 24:1164-1168 Terzaghi BE, Sandine WE (1975) Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol 29:807-813 Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mpl8 and pUC19 vectors. Gene 33:103-119 Yu PL, Limsowtin GKY, Crow VL, Pearce LE (1988) In vivo and invitro expression of a Streptococcus lactis tagatose, 1,6-bisphosphate aldolase gene in Escherichia coli. Appl Microbiol Biotechnol 28: 471-473 Yu PL, Appleby RD, Pritchard GG, Limsowtin GKY (1989) Restriction mapping and localization of the lactose-metabolizing genes of Streptococcus cremoris plasmid pDI-21. Appl Microbiol Biotechnol 30:71-74
Applied Microbiology Biotechnology © Springer-Verlag 1990
In vitro expression of Lac-PTS and tagatose 1,6-bisphosphate aldolase genes from Lactococcus lactis subsp, cremoris plasmid pDI-21 Pak-Lam Yu, Robert A Hodge, and Xiao-Ping Li* Department of Biotechnology, Massey University, Palmerston North, New Zealand Received 10 October 1989/Accepted 11 May 1990
Summary. A 4.4-kb EcoR1-EcoR1 D N A fragment from the Lactococcus lactis subsp, cremoris plasmid pDI-21 encoded the tagatose 1,6-bisphosphate (TBP) aldolase gene and the Lac-PTS genes. In vitro transcriptiontranslation using Escherichia coli $30 extract showed the synthesis of 41000-, 23 000- and 12000-dalton proteins which correspond to the TBP-aldolase, Lac-PTS enzyme II, and factor III proteins respectively.
Introduction The primary function of lactic acid bacteria in dairy fermentations is the catabolism of lactose with the eventual production of lactic acid as a major or sole end-product. Considering the industrial importance of group N streptococci, now classified as the genus Lactococcus, characterization of the enzymes and genes responsible for lactose utilization is of considerable importance. The conjugative plasmid pDI-21 (63 kb) from L. lactis subsp, cremoris strain H2 encodes genes which are responsible for both lactose utilization and protein degradation (Yu et al. 1989). The tagatose-6-phosphate pathway genes, a LacPTS region and the phospho-fl-galactosidase gene have been mapped within a region of 12.5 kb (Inamine et al. 1986; Yu et al. 1989). Plasmid linkage of the genes responsible for lactose metabolism in lactic streptococci has been well established (Anderson and M c K a y 1977; Crow et al. 1983; Gasson 1983). In Streptococcus lactis 712, restriction enzyme mapping of deletion mutants established that lactose activity was determined by a 14.2-kb B c l I fragment of the plasmid pLP712 (Gasson 1983). The key lactose-splitting enzyme, p-fl-gal, was cloned and expressed as a 58000-dalton protein in Escherichia coli (Maeda and Gasson 1986). Harlander * Present address: Department of Chemical and Biochemical Engineering, Hua Chiao University, Quan Zhou, Fujian, People's Republic of China Offprint requests to: P.-L. Yu
et al. (1984) cloned a 23-kb K p n I fragment of S. lactis LM0230 into the vector plasmid pDB101 and used it to restore lactose activity upon transformation into an appropriate S. sangius mutant. The cloning and characterization of the p-fl-gal gene from S. lactis Z268 plasmid pUCL13 has been completed (Boizet et al. 1988). Sequencing data revealed the presence of two promoter-like structures in the 5' region of the p-fl-gal gene which has an open reading frame coding for polypeptides of 55096 or 54078 daltons. Recently, in vivo and in vitro expression of a S. lactis tagatose 1,6-bisphosphate aldolase gene in the E. coli transcription-translation system has shown a polypeptide of 37 500 daltons (Yu et al. 1988). However, expression of lac genes other than the TBP-aldolase gene and p-fl-gal gene has not yet been demonstrated in vitro. In this report, the localization and expression of lac-PTS and TBP-aldolase genes from the plasmid pDI-21 of L. lactis subSp, cremoris strain H2 are described.
Materials and methods All lactococci strains were grown in M17 broth (Terzaghi and Sandine 1975). E. coli strains were grown in Luria-Bertani (LB) broth. E. coli JM109, endA1, recA1, gyrA96, thi, hsdR17 (rk-, ink--), relA1, supE44- 2~-,A(lac-proAB), [F', traD36, proAB, laclqZ M15] (Yanisch-Perron et al. 1985) was used as the host for plasmid transformation. The plasmid vector GM3pE Blue (2743 bp, Ape) and the ExolII deletion system were supplied by Promega, Sydney, Australia. Recombinant clones were selected on LB agar supplemented with ampicillin (100 txg/ml), 5-bromo-4-chloro-3-indolyl-fl-D-galactopyranoside (X-gal, Bethesda Research Laboratories, Bethesda, Md, USA; 40 lxg/ml), and 0.2 mM isopropyl-fl-D-thiogalactopyranoside (IPTG, Boehringer, Auckland, New Zealand). Aldolase enzyme activity was assayed on E. coli extracts as described previously (Yu et al. 1988). Large-scale preparations of lactococcal plasmids were by the method of Anderson and McKay (1977), and E. coli plasmids by alkaline lysis (Maniatis et al. 1982). For ExolII deletion experiments, plasmid DNAs were further purified by ultracentrifugation in caesium chloride-ethidium bromide equilibrium gradients. The rapid boiling method (Holmes and Quigley 1981) was used for initial plasmid screen_ing of E. coli clones. The E. coli $30 in vitro transcription-translation
678 system was used as described by Pratt (1984). The $30 extract, [35S]methionine and [aaC]protein standards were from Amersham, Sydney, Australia. The gene products were separated on 15% sodium dodecyl sulphate-polyacrylamide gels (Laemmli 1970). Gels were treated for 30 min in 50% methanol and 4% glycerol before drying for 2 h at 80° C in a gel dryer (Bio-Rad, Richmond, Calif, USA). Autoradiography was for 3-4 days with Fuji Tokyo, Japan X-ray Film RX at room temperature.
Results and discussion
The 4.4 E c o R 1 - E c o R 1 fragment of pDI-21 which encodes the TBP-aldolase gene and Lac-PTS genes was cloned in both orientations by using the plasmid vector p G E M 3 Blue and is designated as pBH401 and p B H 5 0 1 (Fig. 1). The D N A insert was further characterized by restriction endonuclease mapping. In comparison to the TBP-aldolase gene from pDI-1 of S. lactis (Yu et al. 1988), no internal H i n d l I I sites were found in pBH401 and pBH501. For the localization of the Lac-PTS genes within the 4.4 EcoR1-EcoR1 fragment, purified pBH501 plasmid D N A was progressively shortened from one end by E x o l I I deletion. Two of the subclones pBH501-5.0 and pBH501-8.5 are shown in Fig. 1. In E. coli, pBH401 and pBH501 expressed TBPaldolase activity (1.6+0.10 Ixmol/min per milligram protein). Neither pBH501-5.0 nor pBH501-8.5 exhibited detectable aldolase activity. These plasmid DNAs were further isolated and assayed for the expression of the Lac-PTS genes in the E. coli in vitro transcriptiontranslation system. Both pBH401 and pBH501 expressed the TBP-aldolase gene in vitro and the size of the gene product was determined from 14C-labelled
I"---
E
H
II
I
H
I
A pBH401
S
H H
ill~
-----~Ii
H
H
!
i
A
TBP-aldolase structural gene and its promoter sequences. This is indicated by the synthesis of the 41000 dalton polypeptide in either orientation. This result was also obtained by Yu et al. (1988) for the in vitro expression of the TBP-aldolase gene from S. lactis plasmid pDI-1, which has a lower molecular weight of 37500 daltons. The plasmid pBH501-5.0 expressed two proteins of 23 000 and 12 000 daltons. The level of expression of the 23000-dalton protein was much higher than that of 12000-daltons. The subclone pBH501-8.5 was 390 bp shorter than pBH501-5.0 in the left end of the insert. This deletion resulted in no expression of either of the Lac-PTS proteins, which could indicate the position of a regulatory region to the left of the two ClaI sites (Fig. 2). The presence of a lactose-inducible promoter which controls the expression of the Lac-PTS and p-fl-gal
E
I
~
CpC
TBP-Aldolase gene
E
I
I
standards as 41000 daltons (Fig. 2). The 4.4-kb EcoR1EcoR1 fragment of pDI-21 appears to carry both the
I
S
Lac-PTS genes
H H
E
r~i~ ~
I
C ~C
pBH501
H H
I
E
-----1%, ,,d
I
,
,
P
pBH501-5.0
H
i
~ l pBH501-8.5
i ,.J
~' CC P
H
I
E
;
I 1 kb
Fig. 1. Schematic representation of linearized forms of recombinant plasmids pBH401, pBH501, pBH501-5.0, and pBH501-8.5. The positions of the TBP-aldolase gene and Lac-PTS genes are shown. The small arrow within the pGEM3 Blue vector indicates the direction of transcription and position of the Lac promoter: A (AvaI), C (ClaI), E (EcoR1), H (HindlI), P (PstI) and S (SphI)
Fig. 2. Autoradiogram of [35S]methionine-labelled polypeptides from in vitro transcription-translation run on a 15% sodium dodecyl sulphate-polyacrylamide gel: lane 1, pBH501-8.5 DNA; lane 2, pBH501-5.0 DNA; lane 3, pBH501 DNA; lane 4, pBH401 DNA
679 genes have been postulated in lactose plasmids of L. lactis and Lactobacillus casei (Chassy 1987; Porter and Chassy 1988, De Vos and Gasson 1989). In the case of Lactococcus lactis plasmid pMG820, the lactose genes are organized in an operon-type structure with the gene order L a c F (Lac-PTS FactorlII), L a c E (Lac-PTS Enzyme II), L a c G (p-fl-gal) (De Vos, personal communication). The nucleotide and deduced amino-acid sequences o f the lactose genes are highly homologous to those from Staphylococcus aureus and Lactobacillus casei (Porter and Chassy 1988; De Vos and Gakson 1989). The lactose-specific factor I I I has been isolated from L. casei and has the structure of a trimer of identical subunits of 13 000 daltons, which is similar in size to the S. aureus factor I I I subunit (mol. w t . = 11367) (StiJber et al. 1985, Alpert and Chassy 1988). Our data from the in vitro expression system showed the expression of a 12000-dalton protein which corresponds to the soluble lactose factor I I I protein subunit of L. casei and S. aureus. Purification and characterization of the galactoside-specific membrane-bound protein enzyme II from S. aureus showed a protein of 50 000 mol. wt. u p o n induction o f the staphylococcal lac operon (Sch/afer et al. 1981). The size of enzyme II protein from S. aureus is considerably bigger than the 23 000-dalton protein that we have obtained in the in vitro system, which could indicate the presence of a dimeric form of this protein in Lactococcus lactis. Sequence analyses o f these genes are in progress and the comparison of their primary sequences with other Gram-positive microorganisms will be made. The presence of a regulatory region and operon structure will then be established. Acknowledgement. We thank V. L. Crow, New Zealand Dairy Research Institute, for the assay of aldolase enzyme activity.
References Alpert C, Chassy BM (1988) Molecular cloning and nucleotide sequence of the factor III ~acgene of Lactobacillus casei. Gene 62:277-288 Anderson DG, McKay LL (1977) Plasmids, loss of lactose metabolism and appearance of partial and full lactose-fermenting revertants in Streptococcus crernoris B1. J Bacteriol 129:367377 Boizet B, Villeral D, Slos P, Novel M, Novel G, Mercenier A (1988) Isolation and structural analysis of the phospho-fl-galactosidase gene from Streptococcus lactis Z 268. Gene 62:249261
Chassy BM (1987) Prospects for genetic manipulation in lactobacilli. FEMS Microbiol Lett 46:297-312 Crow VL, Davey GP, Pearce LE, Thomas TD (1983) Plasmid linkage of the tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism. J Bacteriol 153:76-83 De Vos WM, Gasson MJ (1989) Structure and expression of the Lactococcus lactis gene for phospho-fl-galactosidase (lacG) in Escherichia coli and L. lactis. J Gen Microbiol 135:1833-1846 Gasson MJ (1983) Plasmid complements of Streptococcus lactis NCD0712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154:1-9 Harlander SK, McKay LL, Schactele CF (1984) Molecular cloning of the lactose-metabolizing genes from Streptococcus lactis. Appl Environ Microbiol 48:347-351 Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193-197 Inamine JM, Lee LN, LeBlanc DJ (1986) Molecular and genetic characterization of lactose-metabolic genes of Streptococcus cremoris. J Bacteriol 167:855-862 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Maeda S, Gasson MJ (1986) Cloning, expression and location of the Streptococcus lactis gene for phospho-fl-D-galactosidase. J Gen Microbiol 132:331-340 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Porter EV, Chassy BM (1988) Nucleotide sequence of the fl-Dphosphogalactoside galactohydrolase gene of Lactobacillus casei: comparison to analogous pbg genes of other Gram-positive organisms. Gene 62:263-276 Pratt JM (1984) Coupled transcription-translation in prokaryotic cell-free systems. In: Hames BD, Higgins SJ (eds) Transcription and translation - a practical approach. IRL Press, Oxford, pp 179-209 Schafer A, Schrecker O, Hengstenberg W (1981) The staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Eur J Biochem 113:289-294 Stiiber K, Deutscher J, Sobek HM, Hengstenberg W, Beyreuther K (1985) Amino acid sequence of the amphiphilic phosphocarrier protein factor III L~c of the lactose-specific phosphotransferase system of Staphylococcus aureus. Biochemistry 24:1164-1168 Terzaghi BE, Sandine WE (1975) Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol 29:807-813 Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mpl8 and pUC19 vectors. Gene 33:103-119 Yu PL, Limsowtin GKY, Crow VL, Pearce LE (1988) In vivo and invitro expression of a Streptococcus lactis tagatose, 1,6-bisphosphate aldolase gene in Escherichia coli. Appl Microbiol Biotechnol 28: 471-473 Yu PL, Appleby RD, Pritchard GG, Limsowtin GKY (1989) Restriction mapping and localization of the lactose-metabolizing genes of Streptococcus cremoris plasmid pDI-21. Appl Microbiol Biotechnol 30:71-74
