Properties of Lactose Plasmid ply101 in Lactobacillus casei

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1987, p. 2987-2991 0099-2240/87/122987-05$02.00/0 Copyright 1987, American Society for Microbiology Vol. 53, No. 12 Properties of Lactose Plasmid ply101 in Lactobacillus casei MARIKO SHIMIZU-KADOTA Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186, Japan Received 12 June 1987/Accepted 16 September 1987 A starter strain, Lactobacilus casei C257, was found to carry a lactose plasmid, ply101. Restriction mapping showed that ply101 DNA was 68.2 kilobases long. Since a non-lactose-utilizing variant of C257, MSK248, lost phospho-,-galactosidase (P-It-gal) activity and ply101 DNA had a sequence(s) homologous to the streptococcal fragment including a P-4-gal gene, ply101 is likely to encode a P-n-gal gene required for lactose metabolism in C257. MSK248 grew in galactose medium at a rate identical to that of C257 and retained phosphoenolpyruvate-dependent phosphotransferase system activity for lactose similar to that of C257. Therefore, the C257 chromosome appears to encode a complete set of genes for the lactose-phosphotransferase system and the predominant galactose metabolic pathway in C257. ply101 DNA had a sequence homologous to a lactobacillus insertion sequence, ISLI, which mapped more than 12 kilobases from the sequence homologous to the streptococcal P-Is-gal fragment. Lactose is a predominant carbohydrate in milk. Therefore, the ability to utilize lactose is important for the growth and acid production of lactic acid bacteria which are used in starter cultures for fermented dairy products. Two metabolic pathways for lactose utilization are known in lactic acid bacteria (3, 16, 21). (i) Lactose is transported into the cell and phosphorylated by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) (24), the resulting lactose 6-phosphate is hydrolyzed by phospho-p-galactosidase (P-pgal), and the galactose 6-phosphate moiety is further metabolized by the tagatose 6-phosphate (Tag-6P) pathway. (ii) Lactose is transported into the cell by permease and hydrolyzed by P-galactosidase, and the galactosyl moiety is metabolized by the Leloir pathway after phosphorylation to galactose 1-phosphate. Some Lactobacillus casei and lactic streptococci starter strains were reported to lose lactoseutilizing ability with concomitant loss of a plasmid (lactose plasmid; 2, 11, 18, 22). Since P-p-gal rather than p- galactosidase activity was found in many of these strains, the former pathway appears to be more common in these species (3, 15, 21, 25). Analyses by conjugation, transformation, and cloning showed that these lactose plasmids encode some of the genes involved in the former pathway, such as for P-p-gal (6, 10, 13, 18, 20), some components of the lactose- PTS (Lac-PTS) (6, 10; C. A. Alpert and B. M. Chassy, Gene, in press), and the enzymes of the Tag-6P pathway (6, 19). L. casei C257 (28) is a lactose-utilizing (Lac') starter strain used to produce lactic acid beverages and fermented milk products. Fast growth and high acid production in milk medium as well as stability of these properties are required for C257. However, non-lactose-utilizing (Lac-) variants of C257 were obtained from stock cultures grown on glucose medium, causing slow acid production. In this report, a 68.2-kilobase (kb) lactose plasmid designated ply101 was found in C257, which is likely to encode a P-p-gal gene. Also, lactose metabolism in C257 and functions of ply101 are discussed. L. casei strains were cultured at 37 C in Rogosa natural medium (8) containing 2% lactose, glucose, or galactose unless otherwise described. Doubling time was defined as the time required for a twofold increase of surviving cells in early log phase. Lac- variants of C257 were isolated by enrichment culture or by plasmid-curing agents. Enrichment culture with glucose involved successive daily culturing of 2987 1% inocula in Rogosa medium (glucose) and assaying of the lactose-utilizing abilities of cells by growth on Rogosa agar plates (lactose) including 0.004% bromocresol purple. For isolation by plasmid-curing agents, cells were grown to mid-log phase in Rogosa medium (glucose), diluted to a density of 103 CFU/ml in Rogosa medium (glucose) containing 8,ug of acridine orange per ml or 4,ug of acriflavine per ml, and cultured at room temperature for 48 h to a density of 8 x 106 CFU/ml, and Lac- variants were selected by using bromocresol purple. For preparation of plasmid DNA, protoplasts were first formed by the method of Iwata et al. (14) with slight modifications as follows. Cells were grown to early stationary phase at 34 C in 500 ml of Rogosa medium containing 0.1% glucose and harvested. After two washes, the cells were suspended in 500 ml of 10 mm potassium phosphate buffer (ph 6.9) containing 1.0 M sucrose and treated with lysozyme (Sigma Chemical Co., St. Louis, Mo.) and N-acetylmuramidase SG (Seikagaku Kogyo, Tokyo, Japan) (final concentrations of 40 and 2 p.g/ml, respectively) at 37 C for 20 min to form protoplasts. After being collected by centrifugation at 2,000 x g for 20 min, protoplasts were burst by suspension in 20 ml of 50 mm Tris hydrochloride (ph 8.0)-20 mm sodium EDTA and completely lysed by treatment with sodium dodecyl sulfate (final concentration of 0.1%) at 60 C for 10 min. Plasmid DNA was extracted from the lysate by the method of Anderson and McKay (1). For restriction mapping, plasmid DNA was purified by two successive centrifugations in cesium chloride-ethidium bromide density gradients and analyzed as described previously (30). To assay sugar-pts and P-p-gal activities, fresh cells grown to stationary phase in Rogosa medium containing the appropriate sugar were permeabilized and measured for sugar-pts and P-p-gal activities by the methods of Chassy and Thompson (3). The dry cell weight per milliliter was estimated from the optical density at 660 nm of the equivalent cell suspension according to the following formula by Kodaira (unpublished): dry cell concentration (milligrams per milliliter) 0.277 = x optical density at 600 nm. As the ISLI probe for hybridization, the 525-base-pair fragment between the unique ApaI and Sacl sites of ISLI DNA isolated from pmsk126 (27) was used. The 4.4-kb XhoI fragment from recombinant plasmid pmq012, which was provided by M. Kiwaki, was used as the P-p-gal probe.

2988 NOTES TABLE 1. Doubling time of C257 and MSK248 in Rogosa medium (natural) with or without an appropriate sugar (2%) at 37'C Strain Doubling time (h) in Rogosa medium containing: Lactose Galactose Glucose No sugar C257 1.9 1.7 1.5 4.2 MSK248 4.5 1.7 1.5 4.2 pmq012 contains DNA derived from the lactose plasmid of Streptococcus lactis NCDO763 (M. Kiwaki, unpublished data). S. lactis NCDO763 and NCDO712 were derived from the same strain (7). The P-4-gal gene of NCDO712 is located on a 4.4-kb XhoI fragment of lactose plasmid plp712 (20), and the 4.4-kb XhoI fragment of pmq012 used as the probe was confirmed to contain a P-4-gal gene by cloning and assaying expression of P-4-gal (unpublished data). Bacteriophage lambda DNA was also used as a probe for detection of HindlIl-digested lambda phage DNA as size markers. Probe fragments were labeled by using the Multiprime DNA labeling system kit (Amersham, Buckinghamshire, United Kingdom) (9) and [ot-32p]dctp. Unincorporated nucleotides were removed by passage through a Sephadex G-50 column. For dot hybridization, solutions of alkaline-denatured DNA were spotted onto a nitrocellulose filter and dried. For Southern filter hybridization (31), filters were prepared as described previously (29). For hybridization, conditions described previously (29) were applied as stringent conditions. Relaxed conditions involved the following changes. The formamide concentration in the prehybridization and hybridization mixtures was reduced from 50% (vol/vol) to 30% (vol/vol), and after hybridization filters were washed twice with 50% (vol/vol) formamide solution containing 5x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at room temperature. The lambda probe did not detect any ply101 sequence even under the relaxed conditions. When enrichment culture was done in C257, the 12th culture included 2% Lac- variants. When the plasmid DNA fraction was prepared from C257 cells and electrophoresed in an agarose gel, a single plasmid was observed. However, this plasmid was missing in a Lac- variant designated MSK248 as well as from other Lac- variants isolated from enrichment culture and those from cultures exposed to acridine orange and acriflavine (data not shown). These results suggest that the plasmid harbored in C257, designated ply101, is a lactose plasmid. Growth of C257 and MSK248 in medium containing lactose or its moieties, galactose or glucose, was examined (Table 1). C257 was able to grow on Rogosa medium containing any of the three sugars. Final cell densities were more than 3 x 109 CFU/ml, and doubling times were less than 2 h at 37 C. MSK248 grew similarly to C257 in the medium with galactose or glucose. However, the doubling time of MSK248 grown on lactose was extended to 4.5 h at 37 C and the final cell density was 108 CFU/ml, which was similar to growth on Rogosa medium without added sugar. The activities of P-4-gal and the Lac-, galactose (Gal)-, and glucose (Glu)-PTSs were measured in C257 and MSK248 cells (Table 2). C257 cells grown on glucose had P-4-gal activity, which increased when the cells were grown on lactose or galactose. However, no P-4-gal activity was detected in MSK248 cells. These results show that ply101 is involved in expression of P-4-gal activity in C257. In contrast, cells of both strains displayed detectable activity TABLE 2. APPL. ENVIRON. MICROBIOL. Activities of P-p-gal and sugar-ptss in C257 and MSK248 Activity in Rogosa medium plus sugar of straina: Activity being C257 MSK248 measured Lactose Galactose Glucose Galactose Glucose P-p-gal 521 378 110 NDb ND Lac-PTS 40 45 19 49 25 Gal-PTS 7 5 ND 7 ND Glu-PTS 47 59 33 93 43 afor P-a-gal, expressed as nanomoles of o-nitrophenyl-,3-d-galactoside 6-phosphate hydrolyzed per minute per milligram of dry cells. For others, expressed as micromoles of sugar phosphorylated per minute per milligram of dry cells. b ND, Not detected. for all three PTSs when grown on galactose, although Gal-PTS activity was low. Similarly, C257 cells grown on lactose showed activities of all three PTSs at levels comparable to those of cells grown on galactose. However, the PTS activities in cells of both strains grown on glucose were decreased compared with those of cells grown on galactose, and Gal-PTS activity was not detectable. Since no plasmid was found in MSK248, the C257 chromosome appears to encode at least a complete set of genes for each of the three PTS activities. A restriction map of ply101 DNA was constructed with ApaI, BamHI, Sacl, SalI, SmaI, and XhoI (Fig. 1). The molecular size of ply101 DNA was calculated to be 68.2 kb. ply101 DNA was probed by Southern filter hybridization for the presence of ISLI (Fig. 2), since L. casei S-1, the parental strain of C257, was reported to carry insertion sequence ISLJ (26, 27), and many insertion sequences are found in plasmids (12). When the ISLI probe was used, the ply101 XhoI B, Sacl B, SmaI A, SalI A, and ApaI A BamHI FIG. 1. Restriction map of ply101 DNA with ApaI, BamHI, Sacl, SalI, SmaI, and XhoI. Fragments produced by digestion with each restriction endonuclease were designated A to D or E in order of decreasing molecular size. The numerical scale indicates the distance (in kilobases) clockwise from the BamHI site between the BamHI A and BamHI D fragments.

VOL. 53, 1987 1 2 3 4 5 6 1No mm tow 600 u -23.1 9.4 6.6 4.4 2.3-2.0 0.-56 FIG. 2. Location of the ISLI sequence on ply101 determined by Southern filter hybridization. ply101 DNA was digested with XhoI (lane 1), Sacl (lane 2), SmaI (lane 3), Sall (lane 4), ApaI (lane 5), and ApaI plus Sacl (lane 6), electrophoresed in a 0.7% agarose gel, transferred to a nitrocellulose filter, and hybridized to the ISLI probe under stringent conditions. fragments hybridized to the probe under stringent conditions. The same probe also detected a 0.5-kb fragment in the ApaI-SacI double-digested DNA. From the positions of the ApaI A fragment (6.2 to 41.0 kb) and the Sacl B fragment (40.5 to 50.5 kb) on the restriction map, the sequence(s) highly homologous to the ISLI probe was localized between 40.5 and 41.0 kb on ply101. ply101 DNA was probed for the P-3-gal sequence by dot hybridization. When the P-,-gal probe (containing the P-,- gal gene from S. lactis) was used and hybridized to ply101 DNA under the stringent conditions, no hybridization was observed. The same probe, however, was able to detect a sequence(s) on ply101 DNA under the relaxed conditions (data not shown). Therefore, the location of the detected sequence(s) was defined by Southern filter hybridization. The ply101 XhoI C, Sacl C, SmaI A, and Sall C fragments hybridized to the probe under relaxed conditions (Fig. 3). The common region was localized between the XhoI site at 53.8 kb and the Sacl site was at 60.4 kb on the restriction map of ply101. Cells of C257 have both P-n-gal and Lac-PTS activities. However,,-galactosidase activity (ability to hydrolyze o- nitrophenyl-p-d-galactopyranoside) was low in the absence of phosphoenolpyruvate (unpublished data), and P-,-gal activity was missing in the Lac- variant MSK248. Therefore, in C257 lactose is probably metabolized by the Lac- PTS, P-p-gal, and the enzymes constituting the Tag-6P pathway as in most other L. casei strains. It should be noted, however, that C257 cells grown on glucose had 21% and 48% P-,-gal and Lac-PTS activities, respectively, in comparison with cells grown on lactose. These activities were repressed by glucose in most other L. casei strains (3). NOTES 2989 C257 was found to contain a 68.2-kb lactose plasmid, ply101. Since the Lac- variant MSK248 lost both ply101 and cellular P-p-gal activity and ply101 DNA has a sequence(s) weakly homologous to the streptococcal fragment including a P-n-gal gene, a P-,-gal gene is likely to reside on ply101 and is responsible for lactose metabolism in C257. MSK248, however, retained Lac-PTS activity, suggesting that the genes necessary for expression of this activity are on the chromosome. This does not exclude the possibility that some of the Lac-PTS genes are duplicated on the plasmid. Chassy and his colleagues found that the lactose plasmid plz64 in L. casei 64H encoded P-a-gal (18) and FactorIlllac, one of the Lac-PTS components (Alpert and Chassy, in press). The phosphorylated form of galactose and the galactosyl moiety of lactose are metabolized by either the Leloir pathway or the Tag-6P pathway in both L. casei and S. lactis (4, 32, 33). Since some lactose plasmids in S. lactis were reported to encode genes for the Gal-PTS (5, 6, 17) and, in some cases, also the Tag-6P pathway (5, 6) in addition to genes for P-3-gal and the Lac-PTS, curing of such plasmids from S. lactis resulted in the Lac- Gald (slow growth on galactose medium) phenotype (17, 23) in the strains. In the case of L. casei C257 and 64H (4), neither the Gal- nor the Gald phenotype was observed when the lactose plasmid was cured. Therefore, the genes for whichever pathway serves as the predominant pathway for galactose metabolism must be encoded on the chromosome. Since Gal-PTS activity in C257 was low, galactose and the galactosyl moiety of lactose are likely to be metabolized differently in C257, via the Leloir pathway and the Tag-6P pathway, respectively. Therefore, the location of the genes for the Tag-6P pathway was not 1 2 3 4 --23.1 _ 9.4-6.6 4.4-2.3-2.0 FIG. 3. Detection and location of a sequence(s) homologous to the streptococcal P-a-gal gene by Southern filter hybridization. ply101 DNA was digested with XhoI (lane 1), SacI (lane 2), SmaI (lane 3), and Sall (lane 4), electrophoresed in a 1.0% agarose gel, transferred to a nitrocellulose filter, and hybridized to the P-3-gal probe under relaxed conditions.

2990 NOTES able to be inferred from this experiment. In contrast, the lactose plasmid-cured strain of L. casei 64H retained high Gal-PTS ability, and 4.7 times more galactose 6-phosphate was detected than galactose 1-phosphate in cells grown with galactose (4). Therefore, the Tag-6P pathway is likely to be the main pathway used for metabolism of both galactose and the galactosyl moiety of lactose in this strain and the genes for this pathway are probably on the chromosome. The P-p-gal probe fragment was derived from S. lactis NCDO763, which has the same ancestor strain as NCDO712 (7), and was able to hybridize to ply101 DNA under relaxed conditions. The P-p-gal genes on the lactose plasmids of S. lactis NCDO712 and S. cremoris H2 have been cloned into Escherichia coli, and their restriction maps with respect to Clal, EcoRI, HindIII, and PstI are identical (13, 20). Furthermore, a DNA fragment including the cloned P-a-gal gene from S. lactis NCDO712 hybridized to plasmids from all 22 S. lactis, 7 5. cremoris, and 3 S. lactis subsp. diacetylactis starter strains tested (34), and that from S. cremoris H2 hybridized to cellular DNAs from S. lactis, S. sanguis, S. mutans, and S. faecalis strains (13). Therefore, this particular P-n-gal gene may be widespread. Although the S. cremoris H2 probe did not hybridize to DNA from L. casei 64H under the stringent conditions (13), hybridization under relaxed conditions is yet to be performed. ply101 DNA had a sequence(s) that hybridized to the ISLI probe (internal fragment of ISLI) between 40.5 and 41.0 kb on the restriction map. Since this region and the probe fragment were both between ApaI and Sacl sites and their lengths were identical, an intact copy of ISLI possibly resides on ply101. In preliminary experiments, the same probe hybridized to another sequence in the chromosomal DNA of MSK248 (unpublished data). Insertion sequences are proposed to contribute to adaptation and evolution by transposition and amplification of genes (12). The fact that the ISLJ sequence was found on lactose plasmid ply101 raises the possibility that ISLI was involved in the adaptation of S-1 to lactose-rich environments. I am grateful to M. Kiwaki for providing pmq012 and to M. Iwata for helpful suggestions as to protoplast formation and the sugar-pts assay. I am also indebted to K. M. Polzin for critical reading of the manuscript. Finally, I thank A. Hirashima and M. Mutai for their support and encouragement. APPL. ENVIRON. MICROBIOL. LITERATURE CITED 1. Anderson, D. G., and L. L. McKay. 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46:549-552. 2. Chassy, B. M., E. M. Gibson, and A. Giuffrida. 1978. 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VOL. 53, 1987 NOTES 2991 DNA fragments separated by gel electrophoresis. J. Mol. Biol. 33. Thompson, J. 1980. Galactose transport systems in Streptococ- 98:503-517. cus lactis. J. Bacteriol. 144:683-691. 32. Thomas, T. D., K. W. Turner, and V. L. Crow. 1980. Galac- 34. von Wright, A., M. Suominen, and S. Sivela. 1986. Identification tose fermentation by Streptococcus lactis and Streptococcus of lactose fermentation plasmids of streptococcal dairy starter cremoris: pathways, products, and regulation. J. Bacteriol. strains by Southern hybridization. Lett. Appl. Microbiol. 2: 144:672-682. 73-76.