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Isolation and structural analysis of the phospho-β-galactosidase gene from Lactobacillus acidophilus
JOURNAL OF FERMENTATION AND BIOENGINEERING VO1.78, NO. 2, 123-129. 1994
Isolation and Structural Analysis of the Phospho-/%Galactosidase Gene from Lactobacillus acidophilus KAZUO KANATANI* AND MASAO OSHIMURA Research Laboratory, Tamon Sake Brewing Co. Ltd., 1-13-11 Higashi-cho, Nishinomiya-shi, Hyogo 662, Japan Received 13 April 1994/Accepted 23 May 1994
Lactobacillus acidophilus LTF42 metabolizes lactose by the phosphoenolpyruvate-dependent phosphotransferase system for lactose uptake and phospho-~-galactosidase (P-~-gal) that cleaves the lactose-6-phosphate formed. Strain LTF42 was found to carry a single 57-kb plasmid, pLA421. Since the Lac- strain LTF42-1, a pLA421-cured derivative of LTF42, has lost P-~-gal activity, pLA421 is likely to encode the gene for P-~-gal. A 4.8-kb Sall fragment of pLA421 containing the P-~-gal gene, designated pbg, was shotgun cloned in Escherichia coll. Further subcloning and deletion of this 4.8-kb Sall fragment allowed localization of the pbg gene on a minimal 1.9-kb Clal-Kpnl region. Analysis of the nucleotide sequence showed that the pbg gene was composed of 1,419 bp (473 amino acid residues) which can encode a protein with a molecular weight of 54,008. The deduced amino acid sequence of the pbg gene showed a degree of homology exceeding 6 0 ~ with the P-~-gal amino acid sequences of other Gram-positive bacteria. Transformants carrying the recombinant plasmid pULA5EP containing the pbg gene on pLA421 expressed P-~-gal activities comparable to those of the parent strains.
lation of the genes involved in lactose metabolism.
The primary function of lactic acid bacteria, which are used in starter cultures for fermented dairy products, is the rapid fermentative conversion of lactose into lactic acid. In view of the industrial importance of these bacteria, characterization of the enzymes and genes responsible for lactose metabolism is of considerable interest. Many dairy lactic acid bacteria are known to utilize the phosphoenolpyruvate-dependent lactose-specific phosphotransferase system (Lac-PTS) for lactose uptake with two lactose-specific membrane proteins, enzyme II ~¢ and factor IIP ac being used for the transport (1). Lactose appears intracellularly as lactose 6-phosphate which is cleaved into D-glucose and D-galactose 6-phosphate (Gal6P) in a reaction catalyzed by /3-D-phosphogalactoside galactohydrolate (phospho-/3-galactosidase; P-/3-gal). The Gal-6P thus formed is further metabolized to triose phosphates by the three enzymes (Gal-6P isomerase, D-tagatose 6-phosphate kinase, and D-tagatose 1,6-diphosphate aldolase) of the tagatose 6-phosphate (Tag-6P) pathway (2). Early studies of lactose metabolism suggested that genetic determinants for lactose metabolism are associated with plasmid DNA in lactic acid bacteria, since lactose metabolizing abilities are unstable and readily lost by treatment with plasmid-curing agents (3-7). For example, the genes for P-/%gal have been shown to be plasmid encoded in lactic lactococci (8, 9) and lactobacilli (10, 11). Recently, various lactose metabolism genes, encoding P-~-gal, the two lactose-specific PTS enzymes, and the three enzymes of the Tag-6P pathway, have been cloned and sequenced (12-18). We have isolated a L a c - mutant that cannot metabolize lactose from Lactobacillus acidophilus LTF42, which was isolated from a fermented milk product. Here we present the cloning of the P-~-gal gene residing on a single 57-kb plasmid, pLA421, found in strain LTF42, as a first step to characterizing the organization and regu-
MATERIALS AND METHODS Bacterial strains and media The L. acidophilus LTF42 strain used in this study was isolated from a fermented milk product, and identified by the Minitek system (BBL Microbiology Systems, Cockeysville, Md., USA). To complete the identification, the electrophoretic mobility of the lactic dehydrogenase was determined by the method of Uemura et al. (19). The L. acidophilus strain was grown in MRS medium (20) containing 2% lactose, or 2°//oovarious carbohydrates as carbon sources. Escherichia coli was grown in LB broth (Difco Laboratories, Detroit, MI, USA) at 37°C with shaking. When needed, antibiotics were used at the following concentrations: ampicillin, 50 pg/ml; erythromycin, 200/2g/ml for E. coli; erythromycin, 2.5/2g/ml for L. acidophilus. E n z y m e assays To assay the enzyme activities, cells grown to the stationary phase in MRS medium containing appropriate sugar were permeabilized with tolueneacetone (1 : 9) according to the method of LeBlanc et al. (21). P-/3-gal activity was assayed in a 500-/21 reaction mixture containing 50mM sodium-potassium phosphate buffer (pH 7.2), 1 mM o-nitrophenyl-/3-D-galactopyranoside 6-phosphate (ONPG6-P), and 50/21 of a permeabilized cell suspension. For the P-/3-gal of E. coli, cells grown to the mid-log phase in LB broth were assayed. The reaction mixtures were incubated for 30 min at 37°C and the reaction was stopped by adding 1 ml of 5% Na2CO3. After brief centrifugation to remove the cells, the P-/3-gal activity was measured from the amount of onitrophenol liberated. Lac-PTS activity was assayed as described previously (22) in a mixture containing 20/21 of a permeabilized cell suspension and 5 mM lactose. The activities of the three enzymes of the Tag-6P pathway were assayed as described previously (2). D N A isolation, analysis, and manipulations Plasmid DNA from L. acidophilus strains was prepared as
* Corresponding author. 123
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TABLE 1. P-/~-gal, Lac-PTS, and Tag-6P pathway activities in permeabilized cells of L. acidophilus LTF42 and LTF42-1 Strain
Energy source
LTF42
Lactose Galactose Glucose Galactose Glucose
LTF42-1
P-/~-gal"
Lac-PTSa
587 431 125 ND d ND
5.6 4.8 1.5 4.9 1.7
Specific activity Tag-6P pathway enzymesb Gal-6Pc isomerase Tag-6P ¢ kinase 1.005 0.480 0.841 0.472 0.316 0.171 0.905 0.468 0.356 0.215
TDP c aldolase 0.353 0.315 0.118 0.331 0.216
a p_t3.gal and Lac-PTS activities are expressed as nanomoles of o-nitrophenyl-/3-D-galactopyranoside 6-phosphate hydrolyzed and NADH oxidized per rain per mg dry cell weight, respectively. b All enzyme activities are defined in terms of micromoles of product formed per mg protein per rain. c Abbreviations: Gal-6P, D-galactose 6-phosphate; Tag-6P, D-tagatose 6-phosphate; TDP, D-tagatose 1,6-diphosphate. d ND, Not detected. described previously (7). Isolation o f plasmid D N A f r o m E. coli was p e r f o r m e d by the alkaline lysis m e t h o d described by Maniatis et al. (23). P l a s m i d D N A was purified by centrifugation in cesium chloride-ethidium b r o m i d e density gradients. Restriction endonucleases and D N A m o d i f y i n g enzymes were used in accordance with the m a n u f a c t u r e r ' s specifications. Restriction fragments for cloning were isolated and purified from 0.7% agarose gels with a P r e p - A - G e n e kit (Bio-Rad, R i c h m o n d , C A , USA). Other r e c o m b i n a n t D N A techniques were carried out as described by Maniatis et al. (23). T r a n s f o r m a t i o n o f E. coil with plasmid D N A was conducted according to the m e t h o d o f Inoue et al. (24). D o u b l e - s t r a n d e d D N A was sequenced by the dideoxynucleotide chain termination m e t h o d using a D N A Sequencer (Applied Biosystems, M o d e l 3 7 0 A ) . A series o f unidirectional deletions o f the 1.9-kb ClaI-KpnI fragment o f pCK19 was constructed in b o t h directions using a Kilo-sequencing kit ( T a k a r a Shuzou, Kyoto). Cloning and expression of the pbg gene Plasmid pLA421 f r o m L. acidophilus L T F 4 2 was digested with Sal I and shotgun-cloned in E. coli JM109 using pBluescript II S K + as the vector. The recombinants containing the inserts were treated at 37°C by tolueneacetone ( 1 : 9 ) vapors for 20rain, flooded with 5 m M O N P G 6 - P in 25 m M s o d i u m p h o s p h a t e buffer (pH 6.5), and incubated at 37°C for 30rain. Desired P-/3-gal + clones were isolated from yellow colonies. P l a s m i d
p U L A 1 0 5 E , an E. coli-Lactobacillus shuttle vector (25), was used in the expression studies o f the pbg gene. T r a n s f o r m a t i o n o f L. acidophilus strains by electroporation using a Gene Pulser a p p a r a t u s (Bio-Rad) was perf o r m e d as described previously (25). The P-/3-gal activities o f erythromycin-resistant (Em r) t r a n s f o r m a n t s obtained were assayed as described above. RESULTS
Characterization of L. acidophilus LTF42
The lactose-metabolizing abilities o f lactic lactobacilli have been shown to be related to the existence o f a plasmid in the bacterial cells (5-7). W h e n L. acidophilus LTF42 was cultured in the presence o f the plasmid-curing agents acriflavin and mitomycin C (final concentrations, 40 and 0.5/~g/ml, respectively), a non-lactose-metabolizing ( L a c - ) mutant, designated LTF42-1, was readily isolated. Mutants deficient in other carbohydrates, such as galactose, maltose, and sucrose, were not obtained under the same conditions. P l a s m i d analysis revealed that strain LTF42 had a single 57-kb plasmid, designated pLA421. However, this plasmid was missing in the L a c strain LTF42-1, indicating that the Lac ÷ p h e n o t y p e m a y be associated with pLA421. The specific activities o f P-/3-gal, Lac-PTS, and the enzymes o f the Tag-6P p a t h w a y were assayed in strains LTF42 and LTF42-1. As shown in Table 1, P-/3-gal activ-
p..8 -gal Specific activity [nmol / min / mg (protein)]
I (4.8 kb)
pbg
1.0 kb
Y pSS48
75
pBS40
78
pDS33
78
pNS27
80
pCS23
83
pNS18
>1
pCK19
83
pCD14
23
FIG. 1. Restriction map of the 4.8-kb pLA421 Sail fragment containing the P-/%gal gene and P-/3-gal activities of the deleted plasmids. The lines below the map show subcloned or successively deleted fragments in the indicated plasmids. The assay procedure for P-/3-gal activity in E. coli carrying the deleted plasmids is described in Materials and Methods.
P-~-GAL GENE FROM L. ACIDOPHILUS
VoL 78, 1994
125
C~I ATCC~TACAGCCCCAACTTCGCATATATGAAGCAAGTAAATATATCACTTAACTAACGCT
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180 240 300 360 420
480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320
TATC CGAATATTCATAAC43TTTACATCACAGAAAATC~TATTC-G'FFr ,'AAAGATAC C G T G Y P N I H K V Y I T E N G I G F K D T V C CGGATAATGAC43AAAC CGACAAAAC CGTTCATGATGATG CA~q'I~GAq'FATGq~GAAG P D N E E T D K T V H D D A R I D Y V K CAACA~'I~fC~3AAGTTAq'PGC'I~ATC41"TA~'I~GCGGA~CAAATGTCAAAGGq'PACTTC Q H L E V I A D A I A D G A N V K G Y F ATCTGGTCACTTATGGATGTGT'I'rACTTGGAC CAACGGq'~ACACTAAA~TATC-GTTTG I W S L M D V F T W T N G Y T K R Y G L TTCTATGTTG/~, T I - I ~ A T A C A C A A G A T C G C T A T C C T A G T A A A A C T G C CGACTC-G%"FgAAG F Y V D F D T Q D R Y P S K T A D W F K AATTTC-GCTGAAACTC~CATCATTGA~TAAAAAA'FFA-r CGAAA/JiTC~T~n~TTAGA N L A E T H I I E * TGAT~F~CATCTCATCAAAAAACG~AATAACTGATAAAGAAq'~A~CATTAA~ GGAAATGC CAG~CTGGCATACGCACTACC CGACTAAAACTAGATATAAAAq'~ATA
1380
GCTGC CATGTAATCATTAAAATACTAGCGACTTCTC-CCT~CAAAAGTAAAq'fATG TATCTATCTTATTACG~TTATA%~I~TAACTGCT~CATTAACTC43 CTATATGC
1860 1920
1440 1500 1560 1620 1680 1740 1800
Kpn I ATATGCATC-G~%A~TCq'I~TACC
1946
FIG. 2. Nucleotide sequence of the 1.9-kb ClaI-KpnI fragment of L. acidophilus plasmid pLA421 and protein sequence of the translated pbg gene. The deduced amino acid sequence (single letter code) is shown below the nucleotide sequence. A probable ribosome binding site and promoter ( - 3 5 and --10 regions) sequences are underlined and indicated as RBS, - 3 5 , and - 1 0 , respectively. The inverted repeat of the terminator is indicated by dashed arrows. The stop codon is marked with an asterisk.
126
KANATANI AND OSHIMURA
J. FERMENT. BIOENG.,
L. a c i d o D h i l u s L. c a s e i S. a u r e u s Lc. l a c t i s
20 40 M T K T L P KD F I F G G A T A A Y Q A E G A T K T D G K G R V A W D K F L E E N F W Y K G D MS KQL PQDFVMGGATAAYQVEGATKEDGKGRVLWDDFLDKQGRFKPD MTKTLPEDFI FGGATAAYQAEGATNTDGKGRVAWD TYL EENYWYTAE MTKTLPKDF IFGGATAAYQAEGATHTDGKGPVAWD KYLEDNYWYTAE *
*
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60 DFYHNYVEDLELAEKFGGNVI DFYHRYDEDLALAEKYGHQVI DFYNRYPVDLELS EKFGVNGI DFYHKYPVDLELAEEYGVNGI
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80 i00 RI S I A W S RI F P N G D G E V K P N G V D F Y H K L F A E C D A R H V E P F V T L H H R V S I A W SRI F P D G A G E V E P R G V A F Y H K L F A D C A A H H I E P ~ RI S I A W S R I F P N G Y G E V N P K G V E Y Y H K L F A E C H K R H V E P F V T L H H R IS IAWSRI FPTGYGEVNEKGVEFIq4KLFAECHKRHVEPFVTLHH
120 140 FDT PEGLHEDGDFLTHEKMDD FVEYADYCFKEF F D T P E R L H E A G D W L S QE2~/~DDFVAYAKFCFEEF FDTPEVLHKDGDFLNRKTIDYFVDYAEYCFKEF FDT PEALHSNGDFLNRENI EH F IDYAAFCFEEF *****
****
PAS PAA PAS PAS
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160 180 PEVKYW ITINEI RSVAVDQYI IGNFP PADTFGF S EVKYW ITINEPTSMAVQQYTTGTFPPAE SGRF P E V K Y W T T F N E IG P I G D G Q Y L V G K F P P G I K Y D F P E V N Y W T T F N E IG P I G D G Q Y L V G K F P P G I K Y D L **
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.0.
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200 220 240 D K M F Q T H H N Q M V G H A R A V K L F KI-IDGSK G E IGIVHALQTNYPFITESNPADI G A A E L E D L L D N K F L V D DKTFQAEHNQMVAHARIVNLYKSMQLGC-QIGIVHALQTVYPYSDS -A V D H H A A E L Q D A L E N R L Y L D EKVFQ S~AHARAVKLFKDC4~YKGEI G V V H A L P T K Y P F D P S N P E D V R A A E L E D I I H N K F I LD AKVFQ S~SHARAVKLYKDKGYKGEIGVVHALPTKYPYDPENPADVRAAELED I I H N K F I LD
260 280 300 G T F V G K Y P Q E T M E A V K D I L A A N H G G E F N I - E D E F K A I D A A K D V Q D F V G V D Y Y L S E W M R A Y D G K S EI GTLAGEYHQETLALVKEI LDANHQPMFQS TPQEMKAIDEAAHQLD FVGVNNYFSKWLRAYHGKS ET ATYI~3KYSRETMEGVQHI L S V N -G G K L N I T D E D Y A I L D A A K D L N D F L G I N Y Y M S D W M R G Y D G E S EI A T Y L G H Y S D K T M E G V N H I L A E N -G G E L D L R D E D F Q A L D A A K D L N D F L G I N Y Y M S D W M Q A F D G E T E I
320 340 360 THNGTGDKGTSKVQVKGVGEEKLPDGI ETTDWDWLI YPQGLYDKIMRVKNDYPNI HKVYI TENGIG I H N G D G T K G S S V A R L Q G V G E E KL PDGI E T T D W D W S I Y P R G M Y D I L M R I H N D Y P L V P V T Y V T E N G I G T H N A T G D K G G S K Y Q L K G V G Q R E F D V D V P R T D W D W M I Y P Q G L Y D Q I M R V V K D Y P N Y H K I YI T E N G L G I H N G K G E K G S S KYQI K G V G R R V A P D Y V P R T D W D W I I Y P E G L Y D Q I M R V E I ~ D Y P N Y K K I YI T E N G L G
380 400 420 440 F K D T V P D N E E T D K T V H D D A R I D Y V K Q H L E V I A D A I A D G A N V K G Y F IWSLMDV~"IgFfNGYTKRYGLF LKES L PENATPDTVI EDPKRIDYVKKYLSAMADAIHDGANVKGYF IWSLQDQFSWTNGYS KRYGLF Y K D E F I E S E - - -K T V H D D A R I D Y V R Q H L N V I A D A I I D G A N V K G Y F I W S L M D V F S W S N G Y E K R Y G L F Y K D E F V D ..... N T V Y D D G R I D Y V K Q H L E V L S D A I A D G A N V K G Y F I W S L M D V F S W S N G Y E K R Y G L F
460 YVDFDTQDRYPSKTADWF~THI I-E F V D F P T Q N R Y I K Q S A E W F K S V S E T H I I PD YVDFETQERYPKKSAYWYKELAETKE I - K YVDFDTQERYPKKSAHWYK~VI -E ***
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FIG. 3. Comparison of translated protein sequences of four different P-/~-gals: L. acidophilus (this study), t . casei (13), S. aureus (28), and Lactoc. lactis subsp, lactis (12, 14). The amino acid sequences have been aligned by introducing gaps to maximize identity. Identical and functionally related amino acids are indicated by an asterisk and dot, respectively.
ity was fully induced in strain LTF42 grown on lactose or galactose, but the activity was not induced in strain LTF42-1 using any carbon sources. In contrast, both strains grown on galactose or glucose showed similar activities for the Lac-PTS and the enzymes of the Tag-6P pathway. Taken together, these results suggest that plasmid pLA421 encodes the gene for P-/9-gal responsible for lactose metabolism in L. acidophilus LTF42. Cloning and localization of the P-~-gal gene on pLA421 Since some P-/i-gal genes from lactic acid bacteria have been expressed in E. coli (8, 10, 11, 14), plasmid pLA421 was digested with SalI and shotguncloned in E. coli using pBluescript II SK + as the vector. Of approximately 500 clones containing inserts, four were found to display the P-/3-gal activity and all of
these contained a common 4.8-kb SalI fragment. The restriction map of the recombinant plasmid pSS48 from one of these is shown in Fig. I. To detect the P-/3-gal structural gene on the 4.8-kb SalI region of pLA421, a series of deletions and subclones of pSS48 was constructed, and then the P-~3-gal activity in E. coli carrying the derivative plasmid was assayed, pCK19 was found to have the shortest inserted fragment (1.9-kb) without any decrease of P-/3-gal activity. This observation suggested that an intact form of the P-/3-gal gene was located in the ClaI-KpnI region of pLA421. Hence, this region was used for D N A sequencing. Nucleotide sequencing The nucleotides of the 1.9kb ClaI-KpnI fragment were sequenced, as shown in Fig. 2. Expectedly, a large open reading frame, designated
VoL. 78, 1994
P-/~-GAL GENE FROM L. Sal I
J~
Acc I/C/aI EcoR I
ACIDOPHILUS
127
Kpn I
oaI
(pLAI05)
pULA105E7.8kb ~ (pUC19)
J
Emr
(pBluescfiptII~+~) -'~'~'-
\ KpnI
Hind III EcoR I I Kpn I
Y Sal I
EcoR I
Hind HI
V Hind III
FIG. 4. Construction and DNA structure of pULA5EP used in expressionof the p b g gene. Plasmid pULA5EP has the origins of replication of pUC19 and pLA105 and the gene coding for erythromycinresistance (Emr). The arrows indicate the direction of transcription and location of the p b g gene.
pbg, composed of 1,419bp corresponding to 473 amino acid residues, was observed. The molecular weight was estimated to be 54,008. A probable ribosome binding site (RBS), GGAGG, is located 8 bp upstream from the ATG initiation codon at nucleotide position 229. A putative promoter (coordinates - 3 5 and - 1 0 ) , showing considerable similarity to the typical prokaryotic promoter sequence (26, 27), is found directly upstream of the pbg gene. A 7-bp inverted repeat sequence that could form a stem-loop structure is present 95 bp downstream of the pbg stop codon. The deduced amino acid sequence of the protein coded in this pbg gene exhibited significant homology to the P-/~-gals found in Lactobacillus casei (13), Lactococcus lactis subsp, lactis (12, 14), and Staphylococcus aureus (28), which show 63.6, 66.9, and 69.3% identity at the amino acid level, respectively (Fig. 3). Phenotypic expression of the pbg gene in Lactobacillus strains L. acidophilus TKI-1 (7) and LTF42-1 had lost P-/~-gal activity and showed the Lac- phenotype. To examine pbg expression in these strains, the E. coli-Lactobacillus shuttle vector, pULA105E, was used to transform the P-/~-gal deficient strains. The 1.8kb EcoRI-KpnI region containing the entire pbg gene in pCK19 was ligated with the EcoRI-KpnI sites of pULA105E (Fig. 4), and the Tesulting plasmid, pULA5EP, was introduced via electroporation into L.
acidophilus recipients. Thus, the respective transformants carrying pULA5EP (strains LTF42-1T and TK11T) expressed P-~-gal activity comparable to those of parent strains, and showed the lactose-metabolizing phenotype (Table 2). DISCUSSION In this study, a gene involved in lactose metabolism from a 57-kb plasmid pLA421 in L. acidophilus LTF42 was cloned in E. coli. Sequencing analysis showed that this gene, designated pbg, was composed of 1,419bp, corresponding to 473 amino acid residues, and had a molecular weight of 54,008 (Fig. 2). A comparison of the sequencing data for this pbg gene with data for P-~-gals from other Gram-positive bacteria showed that these genes shared a high level of sequence homology and the protein products had structural similarities (Fig. 3). Furthermore, the P-~-gal- L. acidophilus strains had transformed to the P-/~-gal + phenotype after transformation with the recombinant plasmid pULA5EP containing the pbg gene (Table 2). Thus, this pbg gene could be encoded for P-~-gal, the key enzyme for lactose metabolism in this strain. Two different systems for lactose transport, the LacPTS system and the lactose permease system, have been found in lactic acid bacteria (29). L. acidophilus LTF42
128
KANATANI AND OSHIMURA TABLE 2. Relative P-/3-gal activities of strains used in the transformation experiment
Strain LTF42 LTF42-1 LTF42-1Tc TK8912d TKI-ld TKI-ITc
Energy source for growth Lactose Galactose Glucose 100a 73 21 NDb ND ND 92 69 20 104 100 ND ND ND ND 112 107 ND
The activity of strain LTF42 grown on lactose is taken as 100%. b Not detected. c Emr transformants of strains LTF42-1 and TKI-1. d L. acidophilus TK8912 (P-/~-gal+), strain TKI-I is a Lac (P-/% gal ) mutant of TK8912 (7). a
had both the Lac-PTS and P-/3-gal activities. However, P-/3-gal activity was missing in the L a c - m u t a n t , strain LTF42-1. A m a j o r c o n t r i b u t i o n of the lactose permease system seems to be very unlikely, since the Km value for /%galactosidase, the key enzyme for the lactose permease system, is approximately 100-fold higher than that for Pt3-gal (data not shown). Therefore, it is conceivable that strain LTF42 metabolizes lactose by the Lac-PTS, P-/3gal, and the Tag-6P pathway necessary for further metabolism of the Gal-6P produced by P-E-gal. In lactic lactococci, the genes that encode the enzymes of the Lac-PTS, P-~-gal, and the Tag-6P pathway are organized in a lactose operon comprising the IacABC D E F G X genes (9, 18). In the case of lactobacilli, a 35kb plasmid, pLZ64, in L. casei 64H was also shown to encode a similar lactose operon (15). However, plasmid pLA421 in strain LTF42 appears to encode only the gene for P-/3-gal, since (1) the L a c - m u t a n t , strain LTF42-1 retained the Lac-PTS and Tag-6P pathway activities (Table 1), and (2) operon-type structure sequences similar to the lactose operon genes are not f o u n d in the sequence upstream or downstream of the p b g gene. Thus, the genes for the Lac-PTS and the Tag-6P pathway are presumed to be chromosomally linked. A previous study showed that L . casei contained two P-~-gal genes; an inducible gene present o n the plasmid, and another cryptic gene located on the chromosome, which have been cloned and expressed in E. coli (11). In L . acidophilus LTF42, spontaneous Lac ÷ revertants from L a c - mutants LTF42-1 were not obtained. Furthermore, Southern blot hybridization analysis, using the 1.9-kb C l a I - K p n I fragment containing the p b g gene as a probe, showed the absence of a homologous sequence in the chromosome (data not shown). These results indicated that the only gene for P-/%gal in this strain is that residing on plasmid pLA421. ACKOWLEDGMENTS We wish to thank Dr. Satoshi Tomokiyo, Tanaka Farm, for providing the L. acidophilus strain. We express our thanks to Dr. Keiji Sano, School of Pharmacy, Kobe-Gakuin University, for his help in DNA analysis. REFERENCES 1. Thompson, J.: Lactic acid bacteria: model systems for in vivo studies of sugar transport and metabolism in Gram-positive organisms. Biochimie, 70, 325-336 (1988). 2. Blssett, D.L. and Anderson, R.L.: Lactose and D-galactose
J. FERMENT.BIOENG., metabolism in Staphylococcus aureus: pathway of D-galactose 6-phosphate degradation. Biochem. Biophys. Res. Commun., 52, 641-647 (1973). 3. Efstathlon, J.D. and MeKay, L. L.: Plasmids in Streptococcus lactis: evidence that lactose metabolism and protease activity are plasmid linked. Appl. Environ. Microbiol., 32, 38-44 (1976). 4. Anderson, D.G. and MeKay, L.L.: Plasmids, loss of lactose metabolism, and appearance of partial and full lactose-fermenting revertants in Streptococcus cremoris BI. J. Bacteriol., 129, 367-377 (1977). 5. Chassy, B. M., Gibson, E. M., and Giuffrida, A.: Evidence for plasmid-associated lactose metabolism in Lactobacillus casei subsp, casei. Cuur. Microbiol., 1, 141-144 (1978). 6. Shimizu-Kadota, M.: Properties of lactose plasmid pLY101 in Lactobacillus casei. Appl. Environ. Microbiol., 53, 2987-2991 (1987). 7. Kanatani, K., Yoshida, K., Tahara, T., Miura, H., Sakamoto, M., and Oshimura, M.: Isolation and characterization of plasmid DNA in Lactobacillus acidophilus. Agric. Biol. Chem., 55, 2051-2056 (1991). 8. Inamine, J. M., Lee, L. N., and LeBlanc, D.J.: Molecular and genetic characterization of lactose-metabolic genes of Streptococcus cremoris. J. Bacteriol., 167, 855-862 (1986). 9. Yu, P-L., Appleby, R.D., Pritehard, G.G., and Limsowtin, G. K. Y.: Restriction mapping and localization of the lactosemetabolizing genes of Streptococcus cremoris pDI-21. Appl. Microbiol. Biotechnol., 30, 71-74 (1989). 10. Lee, L-J., Hansen, J.B., Jagusztyn-Krynieka, E.K., and Chassy, B.M.: Cloning and expression of the ~-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12. J. Bacteriol., 152, 1138-1146 (1982). 11. Shimizu-Kadota, M.: Cloning and expression of the phospho/%galactosidase genes on the lactose plasmid and the chromosome of Lactobacillus casei C257 in Escherichia coil Biochimie, 70, 523-529 (1988). 12. Boizet, B., Villeval, D., Slos, P., Novel, M., Novel, G., and Mercenier, A.: Isolation and structural analysis of the phospho-//-galactosidase gene from Streptococcus lactis Z268. Gene, 62, 249-261 (1988). 13. Porter, E. V. and Chassy, B. M.: Nucleotide sequence of the//D-phosphogalactoside galactohydrolase gene of Lactobacillus casei: comparison to analogous pbg genes of other Gram-positive organisms. Gene, 62, 263-276 (1988). 14. De Vos, W. M. and Gasson, M.J.: Structure and expression of the Lactococcus lactis gene for phospho-/~-galactosidase (lacG) in Escherichia coli and L. lactis. J. Gen. Microbiol., 135, 1833-1846 (1989). 15. Alpert, C-A. and Chassy, B. M.: Molecular cloning and nucleotide sequence of the factor IIItac gene of Lactobacillus casei. Gene, 62, 277-288 (1988). 16. De Vos, W.M., Boerrigter, I., Van Rooyen, R.J., Reiche, B., and Hengstenberg, W.: Characterization of the lactose-specific enzymes of the phosphotransferase system in Laetococcus lactis. J. Biol. Chem., 265, 22554--22560 (1990). 17. AIpert, C-A. and Chassy, B.M.: 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 (1990). 18. Van Rooijen, R.J., Van SchalkwiJk, S., and De Vos, W.M.: Molecular cloning, characterization, and nucleotide sequence of the tagatose 6-phosphate pathway gene cluster of the lactose operon of Lactococcus lactis. J. Biol. Chem., 266, 7176-7181 (1991). 19. Uemura, J., Toba, T., Fujisawa, T., and Itoh, T.: Identification of species among the Lactobacillus acidophilus group by electrophoresis of lactate dehydrogenases. Anim. Sci. Technol. (Jpn.), 65, 112-119 (1994). 20. De Man, J. C., Rogosa, M., and Sharpe, M. E.: A medium for the cultivation of lactobacilli. J. Appl. Bacteriol., 23, 130-135 (1960). 21. LeBlan¢, D.J., Crow, V.L., Lee, L.N., and Garon, C.F.: Influence of the lactose plasmid on the metabolism of galactose
VOL. 78, 1994 by Streptococcus lactis. J. Bacteriol., 137, 878-884 (1979). 22. Crow, V.L., Davey, G.P., Pearce, L. E., and Thomas, T. D.: Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism. J. Bacteriol., 153, 76-83 (1983). 23. Maniatis, T., Fritsch, E.F., and Sambrook, J.: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). 24. Inoue, H., No|ima, H., and Okayama, H.: High efficiency transformation of Escherichia coli with plasmids. Gene, 96, 23-28 (1990). 25. Kanatani, K., Yoshida, K., Tahara, T., Yamada, K., Miura, H., Sakamoto, M., and Oshimura, M.: Transformation of Lactobacillus acidophilus TK8912 by electroporation with
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pULAI05E plasmid. J. Ferment. Bioeng., 74, 358-362 (1992). 26. Natori, Y., Kano, Y., and Imamoto, F.: Characterization and promoter selectivity of Lactobacillus acidophilus RNA polymerase. Biochimie, 70, 1765-1774 (1988). 27. Koivula, T., Sibakov, M., and Palva, I.: Isolation and characterization of Lactococcus lactis subsp, lactis promoters. Appl. Environ. Microbiol., 57, 333-340 (1991). 28. Breidt, F. and Stewart, G.C.: Nucleotide and deduced amino acid sequences of the Staphylococcus aureus phospho-/~-galactosidase gene. Appl. Environ. Microbiol., 53, 969-973 (1987). 29. De Vos, W.M. and Simons, G.: Molecular cloning of lactose genes in dairy lactic streptococci: the phospho-/5-galactosidase and /~-galactosidase genes and their expression products. Biochimie, 70, 461-473 (1988).
Isolation and Structural Analysis of the Phospho-/%Galactosidase Gene from Lactobacillus acidophilus KAZUO KANATANI* AND MASAO OSHIMURA Research Laboratory, Tamon Sake Brewing Co. Ltd., 1-13-11 Higashi-cho, Nishinomiya-shi, Hyogo 662, Japan Received 13 April 1994/Accepted 23 May 1994
Lactobacillus acidophilus LTF42 metabolizes lactose by the phosphoenolpyruvate-dependent phosphotransferase system for lactose uptake and phospho-~-galactosidase (P-~-gal) that cleaves the lactose-6-phosphate formed. Strain LTF42 was found to carry a single 57-kb plasmid, pLA421. Since the Lac- strain LTF42-1, a pLA421-cured derivative of LTF42, has lost P-~-gal activity, pLA421 is likely to encode the gene for P-~-gal. A 4.8-kb Sall fragment of pLA421 containing the P-~-gal gene, designated pbg, was shotgun cloned in Escherichia coll. Further subcloning and deletion of this 4.8-kb Sall fragment allowed localization of the pbg gene on a minimal 1.9-kb Clal-Kpnl region. Analysis of the nucleotide sequence showed that the pbg gene was composed of 1,419 bp (473 amino acid residues) which can encode a protein with a molecular weight of 54,008. The deduced amino acid sequence of the pbg gene showed a degree of homology exceeding 6 0 ~ with the P-~-gal amino acid sequences of other Gram-positive bacteria. Transformants carrying the recombinant plasmid pULA5EP containing the pbg gene on pLA421 expressed P-~-gal activities comparable to those of the parent strains.
lation of the genes involved in lactose metabolism.
The primary function of lactic acid bacteria, which are used in starter cultures for fermented dairy products, is the rapid fermentative conversion of lactose into lactic acid. In view of the industrial importance of these bacteria, characterization of the enzymes and genes responsible for lactose metabolism is of considerable interest. Many dairy lactic acid bacteria are known to utilize the phosphoenolpyruvate-dependent lactose-specific phosphotransferase system (Lac-PTS) for lactose uptake with two lactose-specific membrane proteins, enzyme II ~¢ and factor IIP ac being used for the transport (1). Lactose appears intracellularly as lactose 6-phosphate which is cleaved into D-glucose and D-galactose 6-phosphate (Gal6P) in a reaction catalyzed by /3-D-phosphogalactoside galactohydrolate (phospho-/3-galactosidase; P-/3-gal). The Gal-6P thus formed is further metabolized to triose phosphates by the three enzymes (Gal-6P isomerase, D-tagatose 6-phosphate kinase, and D-tagatose 1,6-diphosphate aldolase) of the tagatose 6-phosphate (Tag-6P) pathway (2). Early studies of lactose metabolism suggested that genetic determinants for lactose metabolism are associated with plasmid DNA in lactic acid bacteria, since lactose metabolizing abilities are unstable and readily lost by treatment with plasmid-curing agents (3-7). For example, the genes for P-/%gal have been shown to be plasmid encoded in lactic lactococci (8, 9) and lactobacilli (10, 11). Recently, various lactose metabolism genes, encoding P-~-gal, the two lactose-specific PTS enzymes, and the three enzymes of the Tag-6P pathway, have been cloned and sequenced (12-18). We have isolated a L a c - mutant that cannot metabolize lactose from Lactobacillus acidophilus LTF42, which was isolated from a fermented milk product. Here we present the cloning of the P-~-gal gene residing on a single 57-kb plasmid, pLA421, found in strain LTF42, as a first step to characterizing the organization and regu-
MATERIALS AND METHODS Bacterial strains and media The L. acidophilus LTF42 strain used in this study was isolated from a fermented milk product, and identified by the Minitek system (BBL Microbiology Systems, Cockeysville, Md., USA). To complete the identification, the electrophoretic mobility of the lactic dehydrogenase was determined by the method of Uemura et al. (19). The L. acidophilus strain was grown in MRS medium (20) containing 2% lactose, or 2°//oovarious carbohydrates as carbon sources. Escherichia coli was grown in LB broth (Difco Laboratories, Detroit, MI, USA) at 37°C with shaking. When needed, antibiotics were used at the following concentrations: ampicillin, 50 pg/ml; erythromycin, 200/2g/ml for E. coli; erythromycin, 2.5/2g/ml for L. acidophilus. E n z y m e assays To assay the enzyme activities, cells grown to the stationary phase in MRS medium containing appropriate sugar were permeabilized with tolueneacetone (1 : 9) according to the method of LeBlanc et al. (21). P-/3-gal activity was assayed in a 500-/21 reaction mixture containing 50mM sodium-potassium phosphate buffer (pH 7.2), 1 mM o-nitrophenyl-/3-D-galactopyranoside 6-phosphate (ONPG6-P), and 50/21 of a permeabilized cell suspension. For the P-/3-gal of E. coli, cells grown to the mid-log phase in LB broth were assayed. The reaction mixtures were incubated for 30 min at 37°C and the reaction was stopped by adding 1 ml of 5% Na2CO3. After brief centrifugation to remove the cells, the P-/3-gal activity was measured from the amount of onitrophenol liberated. Lac-PTS activity was assayed as described previously (22) in a mixture containing 20/21 of a permeabilized cell suspension and 5 mM lactose. The activities of the three enzymes of the Tag-6P pathway were assayed as described previously (2). D N A isolation, analysis, and manipulations Plasmid DNA from L. acidophilus strains was prepared as
* Corresponding author. 123
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TABLE 1. P-/~-gal, Lac-PTS, and Tag-6P pathway activities in permeabilized cells of L. acidophilus LTF42 and LTF42-1 Strain
Energy source
LTF42
Lactose Galactose Glucose Galactose Glucose
LTF42-1
P-/~-gal"
Lac-PTSa
587 431 125 ND d ND
5.6 4.8 1.5 4.9 1.7
Specific activity Tag-6P pathway enzymesb Gal-6Pc isomerase Tag-6P ¢ kinase 1.005 0.480 0.841 0.472 0.316 0.171 0.905 0.468 0.356 0.215
TDP c aldolase 0.353 0.315 0.118 0.331 0.216
a p_t3.gal and Lac-PTS activities are expressed as nanomoles of o-nitrophenyl-/3-D-galactopyranoside 6-phosphate hydrolyzed and NADH oxidized per rain per mg dry cell weight, respectively. b All enzyme activities are defined in terms of micromoles of product formed per mg protein per rain. c Abbreviations: Gal-6P, D-galactose 6-phosphate; Tag-6P, D-tagatose 6-phosphate; TDP, D-tagatose 1,6-diphosphate. d ND, Not detected. described previously (7). Isolation o f plasmid D N A f r o m E. coli was p e r f o r m e d by the alkaline lysis m e t h o d described by Maniatis et al. (23). P l a s m i d D N A was purified by centrifugation in cesium chloride-ethidium b r o m i d e density gradients. Restriction endonucleases and D N A m o d i f y i n g enzymes were used in accordance with the m a n u f a c t u r e r ' s specifications. Restriction fragments for cloning were isolated and purified from 0.7% agarose gels with a P r e p - A - G e n e kit (Bio-Rad, R i c h m o n d , C A , USA). Other r e c o m b i n a n t D N A techniques were carried out as described by Maniatis et al. (23). T r a n s f o r m a t i o n o f E. coil with plasmid D N A was conducted according to the m e t h o d o f Inoue et al. (24). D o u b l e - s t r a n d e d D N A was sequenced by the dideoxynucleotide chain termination m e t h o d using a D N A Sequencer (Applied Biosystems, M o d e l 3 7 0 A ) . A series o f unidirectional deletions o f the 1.9-kb ClaI-KpnI fragment o f pCK19 was constructed in b o t h directions using a Kilo-sequencing kit ( T a k a r a Shuzou, Kyoto). Cloning and expression of the pbg gene Plasmid pLA421 f r o m L. acidophilus L T F 4 2 was digested with Sal I and shotgun-cloned in E. coli JM109 using pBluescript II S K + as the vector. The recombinants containing the inserts were treated at 37°C by tolueneacetone ( 1 : 9 ) vapors for 20rain, flooded with 5 m M O N P G 6 - P in 25 m M s o d i u m p h o s p h a t e buffer (pH 6.5), and incubated at 37°C for 30rain. Desired P-/3-gal + clones were isolated from yellow colonies. P l a s m i d
p U L A 1 0 5 E , an E. coli-Lactobacillus shuttle vector (25), was used in the expression studies o f the pbg gene. T r a n s f o r m a t i o n o f L. acidophilus strains by electroporation using a Gene Pulser a p p a r a t u s (Bio-Rad) was perf o r m e d as described previously (25). The P-/3-gal activities o f erythromycin-resistant (Em r) t r a n s f o r m a n t s obtained were assayed as described above. RESULTS
Characterization of L. acidophilus LTF42
The lactose-metabolizing abilities o f lactic lactobacilli have been shown to be related to the existence o f a plasmid in the bacterial cells (5-7). W h e n L. acidophilus LTF42 was cultured in the presence o f the plasmid-curing agents acriflavin and mitomycin C (final concentrations, 40 and 0.5/~g/ml, respectively), a non-lactose-metabolizing ( L a c - ) mutant, designated LTF42-1, was readily isolated. Mutants deficient in other carbohydrates, such as galactose, maltose, and sucrose, were not obtained under the same conditions. P l a s m i d analysis revealed that strain LTF42 had a single 57-kb plasmid, designated pLA421. However, this plasmid was missing in the L a c strain LTF42-1, indicating that the Lac ÷ p h e n o t y p e m a y be associated with pLA421. The specific activities o f P-/3-gal, Lac-PTS, and the enzymes o f the Tag-6P p a t h w a y were assayed in strains LTF42 and LTF42-1. As shown in Table 1, P-/3-gal activ-
p..8 -gal Specific activity [nmol / min / mg (protein)]
I (4.8 kb)
pbg
1.0 kb
Y pSS48
75
pBS40
78
pDS33
78
pNS27
80
pCS23
83
pNS18
>1
pCK19
83
pCD14
23
FIG. 1. Restriction map of the 4.8-kb pLA421 Sail fragment containing the P-/%gal gene and P-/3-gal activities of the deleted plasmids. The lines below the map show subcloned or successively deleted fragments in the indicated plasmids. The assay procedure for P-/3-gal activity in E. coli carrying the deleted plasmids is described in Materials and Methods.
P-~-GAL GENE FROM L. ACIDOPHILUS
VoL 78, 1994
125
C~I ATCC~TACAGCCCCAACTTCGCATATATGAAGCAAGTAAATATATCACTTAACTAACGCT
60
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TCTTAAAC-CTAATAAq'IX~ CAA~IT~AATT C T T C Gq'I~TAATGGTTT C
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120
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q'PC CTGTGATCACAATAAATCACAAAGAAA'I'FI'I'AA%~I~GAAATTATAACGAATAGAC TAT RBS ~ I ~ 3 T A G T A A T A T T G T T A A A A A A ' I ' F F I'CGAAAAATC-GGAGGTAAq~PA~ATGACTAAA CA M T K T T T A C C T A A A G ~ I T A " rA'i'FA'I'IC-GTGGTC-CAACCGCCGC C T A T C A A G ~ T G C G A C C L P K D F I F G G A T A A Y Q A E G A T AAGAC~F_~ATC-GTAAAGGTCC-CGTAC-CATC~3GATAAG'FFFI-IC-GAAGAAAA'FA'Iu'IC-GTAC K T D G K G R V A W D K F L E E N F W Y . ~ C C CAC~TGA'I •F F F , ' A T C A T ~ A T G q ' I ~ _ G G A q ' I ' F A ( 3 A A T T A G C G G A A K G D P A S D F Y H N Y V E D L E L A E Nn~l AAATTTGGTC-GTAATGTGA~'PCC-GATq'TCAATTC-C CTGGTCGCGAA', ",',TAC C A A A ~ T K F G G N V I R I S I A W S R I F P N G GATC-GCGA~I~TTAAGCCAAATGGTGTTGI~FFrA'rACCATAAAC'rt-,-,-A~CGGAA~AT D G E V K P N G V D F Y H K L F A E C D C-CC C G T C A T G T T G A A C C A T I ' I ~ T G A C G T T G C A T C ~ I ' F F I G A T A C G C -C A R H V E P F V T L H H F D T P E G L H GAAGATC~A.r FFr FrAACTCATGAAAAGATGGACGAL'FFA~TGGAATATG CCGAq'PAT E D G D F L T H E K M D D F V E Y A D Y TG'FFFrAA~AATTTC CGGA~/~TC~TACTC-GATTACCAq'FAATGAAAq'FCGATCTG~ C F K E F P E V K Y W I T I N E I R S V GC CGq'I~ATC~TATATAATAC~TTTCCCGC CAC~C(3ATACATTTC-G'FFFI~ATAAA A V D Q Y I I G N F P P A D T F G F D K ATG~'r C C A A A C C C A T C A T A A T C A A A T G G T C ~ 3 G C C A T G C C C G T G C C G T T A A A T T G T T T A A G M F Q T H H N Q M V G H A R A V K L F K CATGATGGq'f CAAAAGGTGAAA~'I~3GAA%~I~3'I'FCATGCGTTGCAAACTAAq'PATC C C%~TC H D G S K G E I G I V H A L Q T N Y P F AATGAGAC~TC C A G CC43ATATTC-GTGCCGCCGAATTC-GAGGA%~FI~3%~rGGATAATAAA N E S N P A D I G A A E L E D L L D N K q~I~CTCGTC-GATC-GCACC-r F I G T I ~ T A A G T A T C CACAAGAAACGATGGAAG CCGTGAAA F L V D G T F V G K Y P Q E T M E A V K GATATI~AGCTGCTAATCATC-GTGGTGAAq'I'~AATATTGAGGATGAATTTAAAGCTATT D I L A A N H G G E F N I E D E F K A I G A T G C G C 4 2 G A A A G A T G TC-CAAGAC"I'I'I~TTC-GTGTCGA'I'FAq~fACTTGAGTGAGTC-GA~ D A A K D V Q D F V G V D Y Y L S E W M CGTGCCTATGATC~TCTGAAATCACC-CATAATGGC3%CCGGCGATAAAGGCACCTCT R A Y D G K S E I T H N G T G D K G T S AAAGTC-CAC~TGAAA~TTC~CGAAGAAAAAC%~TCCAGATGGCATTGA~CGACAGAT K V Q V K G V G E E K L P D G I E T T D TC~A~3G~(3Aq*I~fATC CACAAGGATTATATGACAAAATCATGCC-GGTGAAAAATGAT W D W L I Y P Q G L Y D K I M R V K N D
180 240 300 360 420
480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320
TATC CGAATATTCATAAC43TTTACATCACAGAAAATC~TATTC-G'FFr ,'AAAGATAC C G T G Y P N I H K V Y I T E N G I G F K D T V C CGGATAATGAC43AAAC CGACAAAAC CGTTCATGATGATG CA~q'I~GAq'FATGq~GAAG P D N E E T D K T V H D D A R I D Y V K CAACA~'I~fC~3AAGTTAq'PGC'I~ATC41"TA~'I~GCGGA~CAAATGTCAAAGGq'PACTTC Q H L E V I A D A I A D G A N V K G Y F ATCTGGTCACTTATGGATGTGT'I'rACTTGGAC CAACGGq'~ACACTAAA~TATC-GTTTG I W S L M D V F T W T N G Y T K R Y G L TTCTATGTTG/~, T I - I ~ A T A C A C A A G A T C G C T A T C C T A G T A A A A C T G C CGACTC-G%"FgAAG F Y V D F D T Q D R Y P S K T A D W F K AATTTC-GCTGAAACTC~CATCATTGA~TAAAAAA'FFA-r CGAAA/JiTC~T~n~TTAGA N L A E T H I I E * TGAT~F~CATCTCATCAAAAAACG~AATAACTGATAAAGAAq'~A~CATTAA~ GGAAATGC CAG~CTGGCATACGCACTACC CGACTAAAACTAGATATAAAAq'~ATA
1380
GCTGC CATGTAATCATTAAAATACTAGCGACTTCTC-CCT~CAAAAGTAAAq'fATG TATCTATCTTATTACG~TTATA%~I~TAACTGCT~CATTAACTC43 CTATATGC
1860 1920
1440 1500 1560 1620 1680 1740 1800
Kpn I ATATGCATC-G~%A~TCq'I~TACC
1946
FIG. 2. Nucleotide sequence of the 1.9-kb ClaI-KpnI fragment of L. acidophilus plasmid pLA421 and protein sequence of the translated pbg gene. The deduced amino acid sequence (single letter code) is shown below the nucleotide sequence. A probable ribosome binding site and promoter ( - 3 5 and --10 regions) sequences are underlined and indicated as RBS, - 3 5 , and - 1 0 , respectively. The inverted repeat of the terminator is indicated by dashed arrows. The stop codon is marked with an asterisk.
126
KANATANI AND OSHIMURA
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L. a c i d o D h i l u s L. c a s e i S. a u r e u s Lc. l a c t i s
20 40 M T K T L P KD F I F G G A T A A Y Q A E G A T K T D G K G R V A W D K F L E E N F W Y K G D MS KQL PQDFVMGGATAAYQVEGATKEDGKGRVLWDDFLDKQGRFKPD MTKTLPEDFI FGGATAAYQAEGATNTDGKGRVAWD TYL EENYWYTAE MTKTLPKDF IFGGATAAYQAEGATHTDGKGPVAWD KYLEDNYWYTAE *
*
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60 DFYHNYVEDLELAEKFGGNVI DFYHRYDEDLALAEKYGHQVI DFYNRYPVDLELS EKFGVNGI DFYHKYPVDLELAEEYGVNGI
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120 140 FDT PEGLHEDGDFLTHEKMDD FVEYADYCFKEF F D T P E R L H E A G D W L S QE2~/~DDFVAYAKFCFEEF FDTPEVLHKDGDFLNRKTIDYFVDYAEYCFKEF FDT PEALHSNGDFLNRENI EH F IDYAAFCFEEF *****
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160 180 PEVKYW ITINEI RSVAVDQYI IGNFP PADTFGF S EVKYW ITINEPTSMAVQQYTTGTFPPAE SGRF P E V K Y W T T F N E IG P I G D G Q Y L V G K F P P G I K Y D F P E V N Y W T T F N E IG P I G D G Q Y L V G K F P P G I K Y D L **
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200 220 240 D K M F Q T H H N Q M V G H A R A V K L F KI-IDGSK G E IGIVHALQTNYPFITESNPADI G A A E L E D L L D N K F L V D DKTFQAEHNQMVAHARIVNLYKSMQLGC-QIGIVHALQTVYPYSDS -A V D H H A A E L Q D A L E N R L Y L D EKVFQ S~AHARAVKLFKDC4~YKGEI G V V H A L P T K Y P F D P S N P E D V R A A E L E D I I H N K F I LD AKVFQ S~SHARAVKLYKDKGYKGEIGVVHALPTKYPYDPENPADVRAAELED I I H N K F I LD
260 280 300 G T F V G K Y P Q E T M E A V K D I L A A N H G G E F N I - E D E F K A I D A A K D V Q D F V G V D Y Y L S E W M R A Y D G K S EI GTLAGEYHQETLALVKEI LDANHQPMFQS TPQEMKAIDEAAHQLD FVGVNNYFSKWLRAYHGKS ET ATYI~3KYSRETMEGVQHI L S V N -G G K L N I T D E D Y A I L D A A K D L N D F L G I N Y Y M S D W M R G Y D G E S EI A T Y L G H Y S D K T M E G V N H I L A E N -G G E L D L R D E D F Q A L D A A K D L N D F L G I N Y Y M S D W M Q A F D G E T E I
320 340 360 THNGTGDKGTSKVQVKGVGEEKLPDGI ETTDWDWLI YPQGLYDKIMRVKNDYPNI HKVYI TENGIG I H N G D G T K G S S V A R L Q G V G E E KL PDGI E T T D W D W S I Y P R G M Y D I L M R I H N D Y P L V P V T Y V T E N G I G T H N A T G D K G G S K Y Q L K G V G Q R E F D V D V P R T D W D W M I Y P Q G L Y D Q I M R V V K D Y P N Y H K I YI T E N G L G I H N G K G E K G S S KYQI K G V G R R V A P D Y V P R T D W D W I I Y P E G L Y D Q I M R V E I ~ D Y P N Y K K I YI T E N G L G
380 400 420 440 F K D T V P D N E E T D K T V H D D A R I D Y V K Q H L E V I A D A I A D G A N V K G Y F IWSLMDV~"IgFfNGYTKRYGLF LKES L PENATPDTVI EDPKRIDYVKKYLSAMADAIHDGANVKGYF IWSLQDQFSWTNGYS KRYGLF Y K D E F I E S E - - -K T V H D D A R I D Y V R Q H L N V I A D A I I D G A N V K G Y F I W S L M D V F S W S N G Y E K R Y G L F Y K D E F V D ..... N T V Y D D G R I D Y V K Q H L E V L S D A I A D G A N V K G Y F I W S L M D V F S W S N G Y E K R Y G L F
460 YVDFDTQDRYPSKTADWF~THI I-E F V D F P T Q N R Y I K Q S A E W F K S V S E T H I I PD YVDFETQERYPKKSAYWYKELAETKE I - K YVDFDTQERYPKKSAHWYK~VI -E ***
**.**
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FIG. 3. Comparison of translated protein sequences of four different P-/~-gals: L. acidophilus (this study), t . casei (13), S. aureus (28), and Lactoc. lactis subsp, lactis (12, 14). The amino acid sequences have been aligned by introducing gaps to maximize identity. Identical and functionally related amino acids are indicated by an asterisk and dot, respectively.
ity was fully induced in strain LTF42 grown on lactose or galactose, but the activity was not induced in strain LTF42-1 using any carbon sources. In contrast, both strains grown on galactose or glucose showed similar activities for the Lac-PTS and the enzymes of the Tag-6P pathway. Taken together, these results suggest that plasmid pLA421 encodes the gene for P-/9-gal responsible for lactose metabolism in L. acidophilus LTF42. Cloning and localization of the P-~-gal gene on pLA421 Since some P-/i-gal genes from lactic acid bacteria have been expressed in E. coli (8, 10, 11, 14), plasmid pLA421 was digested with SalI and shotguncloned in E. coli using pBluescript II SK + as the vector. Of approximately 500 clones containing inserts, four were found to display the P-/3-gal activity and all of
these contained a common 4.8-kb SalI fragment. The restriction map of the recombinant plasmid pSS48 from one of these is shown in Fig. I. To detect the P-/3-gal structural gene on the 4.8-kb SalI region of pLA421, a series of deletions and subclones of pSS48 was constructed, and then the P-~3-gal activity in E. coli carrying the derivative plasmid was assayed, pCK19 was found to have the shortest inserted fragment (1.9-kb) without any decrease of P-/3-gal activity. This observation suggested that an intact form of the P-/3-gal gene was located in the ClaI-KpnI region of pLA421. Hence, this region was used for D N A sequencing. Nucleotide sequencing The nucleotides of the 1.9kb ClaI-KpnI fragment were sequenced, as shown in Fig. 2. Expectedly, a large open reading frame, designated
VoL. 78, 1994
P-/~-GAL GENE FROM L. Sal I
J~
Acc I/C/aI EcoR I
ACIDOPHILUS
127
Kpn I
oaI
(pLAI05)
pULA105E7.8kb ~ (pUC19)
J
Emr
(pBluescfiptII~+~) -'~'~'-
\ KpnI
Hind III EcoR I I Kpn I
Y Sal I
EcoR I
Hind HI
V Hind III
FIG. 4. Construction and DNA structure of pULA5EP used in expressionof the p b g gene. Plasmid pULA5EP has the origins of replication of pUC19 and pLA105 and the gene coding for erythromycinresistance (Emr). The arrows indicate the direction of transcription and location of the p b g gene.
pbg, composed of 1,419bp corresponding to 473 amino acid residues, was observed. The molecular weight was estimated to be 54,008. A probable ribosome binding site (RBS), GGAGG, is located 8 bp upstream from the ATG initiation codon at nucleotide position 229. A putative promoter (coordinates - 3 5 and - 1 0 ) , showing considerable similarity to the typical prokaryotic promoter sequence (26, 27), is found directly upstream of the pbg gene. A 7-bp inverted repeat sequence that could form a stem-loop structure is present 95 bp downstream of the pbg stop codon. The deduced amino acid sequence of the protein coded in this pbg gene exhibited significant homology to the P-/~-gals found in Lactobacillus casei (13), Lactococcus lactis subsp, lactis (12, 14), and Staphylococcus aureus (28), which show 63.6, 66.9, and 69.3% identity at the amino acid level, respectively (Fig. 3). Phenotypic expression of the pbg gene in Lactobacillus strains L. acidophilus TKI-1 (7) and LTF42-1 had lost P-/~-gal activity and showed the Lac- phenotype. To examine pbg expression in these strains, the E. coli-Lactobacillus shuttle vector, pULA105E, was used to transform the P-/~-gal deficient strains. The 1.8kb EcoRI-KpnI region containing the entire pbg gene in pCK19 was ligated with the EcoRI-KpnI sites of pULA105E (Fig. 4), and the Tesulting plasmid, pULA5EP, was introduced via electroporation into L.
acidophilus recipients. Thus, the respective transformants carrying pULA5EP (strains LTF42-1T and TK11T) expressed P-~-gal activity comparable to those of parent strains, and showed the lactose-metabolizing phenotype (Table 2). DISCUSSION In this study, a gene involved in lactose metabolism from a 57-kb plasmid pLA421 in L. acidophilus LTF42 was cloned in E. coli. Sequencing analysis showed that this gene, designated pbg, was composed of 1,419bp, corresponding to 473 amino acid residues, and had a molecular weight of 54,008 (Fig. 2). A comparison of the sequencing data for this pbg gene with data for P-~-gals from other Gram-positive bacteria showed that these genes shared a high level of sequence homology and the protein products had structural similarities (Fig. 3). Furthermore, the P-~-gal- L. acidophilus strains had transformed to the P-/~-gal + phenotype after transformation with the recombinant plasmid pULA5EP containing the pbg gene (Table 2). Thus, this pbg gene could be encoded for P-~-gal, the key enzyme for lactose metabolism in this strain. Two different systems for lactose transport, the LacPTS system and the lactose permease system, have been found in lactic acid bacteria (29). L. acidophilus LTF42
128
KANATANI AND OSHIMURA TABLE 2. Relative P-/3-gal activities of strains used in the transformation experiment
Strain LTF42 LTF42-1 LTF42-1Tc TK8912d TKI-ld TKI-ITc
Energy source for growth Lactose Galactose Glucose 100a 73 21 NDb ND ND 92 69 20 104 100 ND ND ND ND 112 107 ND
The activity of strain LTF42 grown on lactose is taken as 100%. b Not detected. c Emr transformants of strains LTF42-1 and TKI-1. d L. acidophilus TK8912 (P-/~-gal+), strain TKI-I is a Lac (P-/% gal ) mutant of TK8912 (7). a
had both the Lac-PTS and P-/3-gal activities. However, P-/3-gal activity was missing in the L a c - m u t a n t , strain LTF42-1. A m a j o r c o n t r i b u t i o n of the lactose permease system seems to be very unlikely, since the Km value for /%galactosidase, the key enzyme for the lactose permease system, is approximately 100-fold higher than that for Pt3-gal (data not shown). Therefore, it is conceivable that strain LTF42 metabolizes lactose by the Lac-PTS, P-/3gal, and the Tag-6P pathway necessary for further metabolism of the Gal-6P produced by P-E-gal. In lactic lactococci, the genes that encode the enzymes of the Lac-PTS, P-~-gal, and the Tag-6P pathway are organized in a lactose operon comprising the IacABC D E F G X genes (9, 18). In the case of lactobacilli, a 35kb plasmid, pLZ64, in L. casei 64H was also shown to encode a similar lactose operon (15). However, plasmid pLA421 in strain LTF42 appears to encode only the gene for P-/3-gal, since (1) the L a c - m u t a n t , strain LTF42-1 retained the Lac-PTS and Tag-6P pathway activities (Table 1), and (2) operon-type structure sequences similar to the lactose operon genes are not f o u n d in the sequence upstream or downstream of the p b g gene. Thus, the genes for the Lac-PTS and the Tag-6P pathway are presumed to be chromosomally linked. A previous study showed that L . casei contained two P-~-gal genes; an inducible gene present o n the plasmid, and another cryptic gene located on the chromosome, which have been cloned and expressed in E. coli (11). In L . acidophilus LTF42, spontaneous Lac ÷ revertants from L a c - mutants LTF42-1 were not obtained. Furthermore, Southern blot hybridization analysis, using the 1.9-kb C l a I - K p n I fragment containing the p b g gene as a probe, showed the absence of a homologous sequence in the chromosome (data not shown). These results indicated that the only gene for P-/%gal in this strain is that residing on plasmid pLA421. ACKOWLEDGMENTS We wish to thank Dr. Satoshi Tomokiyo, Tanaka Farm, for providing the L. acidophilus strain. We express our thanks to Dr. Keiji Sano, School of Pharmacy, Kobe-Gakuin University, for his help in DNA analysis. REFERENCES 1. Thompson, J.: Lactic acid bacteria: model systems for in vivo studies of sugar transport and metabolism in Gram-positive organisms. Biochimie, 70, 325-336 (1988). 2. Blssett, D.L. and Anderson, R.L.: Lactose and D-galactose
J. FERMENT.BIOENG., metabolism in Staphylococcus aureus: pathway of D-galactose 6-phosphate degradation. Biochem. Biophys. Res. Commun., 52, 641-647 (1973). 3. Efstathlon, J.D. and MeKay, L. L.: Plasmids in Streptococcus lactis: evidence that lactose metabolism and protease activity are plasmid linked. Appl. Environ. Microbiol., 32, 38-44 (1976). 4. Anderson, D.G. and MeKay, L.L.: Plasmids, loss of lactose metabolism, and appearance of partial and full lactose-fermenting revertants in Streptococcus cremoris BI. J. Bacteriol., 129, 367-377 (1977). 5. Chassy, B. M., Gibson, E. M., and Giuffrida, A.: Evidence for plasmid-associated lactose metabolism in Lactobacillus casei subsp, casei. Cuur. Microbiol., 1, 141-144 (1978). 6. Shimizu-Kadota, M.: Properties of lactose plasmid pLY101 in Lactobacillus casei. Appl. Environ. Microbiol., 53, 2987-2991 (1987). 7. Kanatani, K., Yoshida, K., Tahara, T., Miura, H., Sakamoto, M., and Oshimura, M.: Isolation and characterization of plasmid DNA in Lactobacillus acidophilus. Agric. Biol. Chem., 55, 2051-2056 (1991). 8. Inamine, J. M., Lee, L. N., and LeBlanc, D.J.: Molecular and genetic characterization of lactose-metabolic genes of Streptococcus cremoris. J. Bacteriol., 167, 855-862 (1986). 9. Yu, P-L., Appleby, R.D., Pritehard, G.G., and Limsowtin, G. K. Y.: Restriction mapping and localization of the lactosemetabolizing genes of Streptococcus cremoris pDI-21. Appl. Microbiol. Biotechnol., 30, 71-74 (1989). 10. Lee, L-J., Hansen, J.B., Jagusztyn-Krynieka, E.K., and Chassy, B.M.: Cloning and expression of the ~-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12. J. Bacteriol., 152, 1138-1146 (1982). 11. Shimizu-Kadota, M.: Cloning and expression of the phospho/%galactosidase genes on the lactose plasmid and the chromosome of Lactobacillus casei C257 in Escherichia coil Biochimie, 70, 523-529 (1988). 12. Boizet, B., Villeval, D., Slos, P., Novel, M., Novel, G., and Mercenier, A.: Isolation and structural analysis of the phospho-//-galactosidase gene from Streptococcus lactis Z268. Gene, 62, 249-261 (1988). 13. Porter, E. V. and Chassy, B. M.: Nucleotide sequence of the//D-phosphogalactoside galactohydrolase gene of Lactobacillus casei: comparison to analogous pbg genes of other Gram-positive organisms. Gene, 62, 263-276 (1988). 14. De Vos, W. M. and Gasson, M.J.: Structure and expression of the Lactococcus lactis gene for phospho-/~-galactosidase (lacG) in Escherichia coli and L. lactis. J. Gen. Microbiol., 135, 1833-1846 (1989). 15. Alpert, C-A. and Chassy, B. M.: Molecular cloning and nucleotide sequence of the factor IIItac gene of Lactobacillus casei. Gene, 62, 277-288 (1988). 16. De Vos, W.M., Boerrigter, I., Van Rooyen, R.J., Reiche, B., and Hengstenberg, W.: Characterization of the lactose-specific enzymes of the phosphotransferase system in Laetococcus lactis. J. Biol. Chem., 265, 22554--22560 (1990). 17. AIpert, C-A. and Chassy, B.M.: 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 (1990). 18. Van Rooijen, R.J., Van SchalkwiJk, S., and De Vos, W.M.: Molecular cloning, characterization, and nucleotide sequence of the tagatose 6-phosphate pathway gene cluster of the lactose operon of Lactococcus lactis. J. Biol. Chem., 266, 7176-7181 (1991). 19. Uemura, J., Toba, T., Fujisawa, T., and Itoh, T.: Identification of species among the Lactobacillus acidophilus group by electrophoresis of lactate dehydrogenases. Anim. Sci. Technol. (Jpn.), 65, 112-119 (1994). 20. De Man, J. C., Rogosa, M., and Sharpe, M. E.: A medium for the cultivation of lactobacilli. J. Appl. Bacteriol., 23, 130-135 (1960). 21. LeBlan¢, D.J., Crow, V.L., Lee, L.N., and Garon, C.F.: Influence of the lactose plasmid on the metabolism of galactose
VOL. 78, 1994 by Streptococcus lactis. J. Bacteriol., 137, 878-884 (1979). 22. Crow, V.L., Davey, G.P., Pearce, L. E., and Thomas, T. D.: Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism. J. Bacteriol., 153, 76-83 (1983). 23. Maniatis, T., Fritsch, E.F., and Sambrook, J.: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). 24. Inoue, H., No|ima, H., and Okayama, H.: High efficiency transformation of Escherichia coli with plasmids. Gene, 96, 23-28 (1990). 25. Kanatani, K., Yoshida, K., Tahara, T., Yamada, K., Miura, H., Sakamoto, M., and Oshimura, M.: Transformation of Lactobacillus acidophilus TK8912 by electroporation with
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pULAI05E plasmid. J. Ferment. Bioeng., 74, 358-362 (1992). 26. Natori, Y., Kano, Y., and Imamoto, F.: Characterization and promoter selectivity of Lactobacillus acidophilus RNA polymerase. Biochimie, 70, 1765-1774 (1988). 27. Koivula, T., Sibakov, M., and Palva, I.: Isolation and characterization of Lactococcus lactis subsp, lactis promoters. Appl. Environ. Microbiol., 57, 333-340 (1991). 28. Breidt, F. and Stewart, G.C.: Nucleotide and deduced amino acid sequences of the Staphylococcus aureus phospho-/~-galactosidase gene. Appl. Environ. Microbiol., 53, 969-973 (1987). 29. De Vos, W.M. and Simons, G.: Molecular cloning of lactose genes in dairy lactic streptococci: the phospho-/5-galactosidase and /~-galactosidase genes and their expression products. Biochimie, 70, 461-473 (1988).