Thermostable alpha-galactosidase from Thermotoga neapolitana: cloning, sequencing and expression.

A gene encoding a thermostable alpha-galactosidase from the hyperthermophile Thermotoga neapolitana was cloned and sequenced. Sequence analysis showed that the 552-amino acid protein is similar to Escherichia coli Raf type alpha-galactosidase and belongs to Family 36 of the glycosyl hydrolases. Recombinant alpha-galactosidase expressed in E. coli has a molecular mass of ca. 61 kDa, and an optimum activity at 93 degrees C at pH 7.0. The enzyme is highly thermostable and retains 75% of activity after heating to 80 degrees C for 4 h. The potential application of the enzyme to high temperature processing of soy molasses has been demonstrated.

Repeated DNA sequence involved in mutations affecting transport of sucrose into Streptococcus mutans V403 via the phosphoenolpyruvate phosphotransferase system.

Mutants of Streptococcus mutans V403 defective in the intracellular sucrose-6-phosphate hydrolase (product of the scrB gene) are sensitive to sucrose because of the intracellular accumulation of the phosphorylated sugar. Using a scrB mutant prepared by allelic exchange, we have isolated and characterized a number of sucrose-resistant revertants. One such mutant was found to lack the ability to transport sucrose into the cell via the phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS). Genetic analysis of this strain revealed this lesion to be linked to the scrB gene. This was corroborated by the physical demonstration of an insertion mutation very near scrB. Taken together with DNA sequence information (Y. Sato, F. Poy, G. R. Jacobson, and H. K. Kuramitsu, J. Bacteriol. 171:263-271, 1989), our results indicated that all of the mutations characterized were located in the adjoining scrA gene which encodes the membrane-associated, sugar-specific enzyme II (EIIsucrose) component of the sucrose PTS in S. mutans. Biochemically, such a genetic lesion disables the sucrose PTS and prevents sucrose from entering the cell by this system. In this paper, we detail the nature of two independent insertion mutations and conclude them to be the result of duplicative transposition events into the scrA gene. This region of the chromosome was amplified and purified in large quantities by using the polymerase chain reaction. Examination of the amplified DNA revealed that the two independent insertion mutations were composed of sequences that were indistinguishable by size and by restriction site endonuclease maps. Their insertion points in the scrA gene were approximately 200 bp apart. The amplified DNA fragment was also used as a probe to demonstrate the presence of five copies of this element on the S. mutans V403 chromosome. A second strain, S. mutans V310, also was found to carry similarly arranged, multiple copies of this sequence on its chromosome, suggesting a clonal origin of V403 and V310. The small size of this sequence, its presence in multiple copies on the V403 chromosome, and its ability to duplicate itself semiconservatively into remote sites argue compellingly that it is an insertion sequence element. One such insertion mutant, with a defective sucrose PTS, was tested for virulence in rats and was found to cause caries at levels similar to those of the wild-type strain.

Molecular cloning and DNA sequence of lacE, the gene encoding the lactose-specific enzyme II of the phosphotransferase system of Lactobacillus casei. Evidence that a cysteine residue is essential for sugar phosphorylation.

The gene coding for the lactose-specific Enzyme II of the Lactobacillus casei phosphoenolpyruvate-dependent phosphotransferase system, lacE, has been isolated by molecular cloning and expressed in Escherichia coli. The DNA sequence of the lacE gene and the deduced amino acid sequence are presented. The putative translation product comprises a hydrophobic protein of 577 amino acids with a calculated molecular mass of 62,350 Da. The deduced polypeptide has a high degree of sequence similarity with the corresponding lactose-specific enzymes II of Staphylococcus aureus and Lactococcus lactis. The sequence surrounding cysteine 483 was strongly conserved in the three proteins. The identity of the lacE product as the Enzyme IIlacL.casei was demonstrated by in vitro lactose phosphorylation assays using the protein expressed in E. coli. Single replacement of each of the histidine and cysteine residues by site-directed mutagenesis pointed to cysteine 483 as an amino acid residue essential for the phosphoryl group transfer reaction.

Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA.

A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.

Evidence that Lactobacillus casei insertion element ISL1 has a narrow host range.

The 1.3-kilobase-pair insertion element ISL1, originally isolated from Lactobacillus casei S-1, was found to have an extremely restricted host range. By DNA-DNA hybrizations performed with Southern transfers by using a cloned internal fragment of ISL1 as a molecular probe, it was found that only 3 of 19 L. casei strains examined contained sequences that hybridized to the ISL1 probe. In two of these, the hybridizing sequences were found on lactose plasmids. No homologous sequences were detected in a survey of 14 other Lactobacillus strains (9 species) and 15 strains of other bacteria (8 genera, 12 species).

Strategies for the development of bacterial transformation systems.

An effective transformation system is a prerequisite for facile genetic manipulation of bacteria. Bacteria may be naturally competent for transformation or may be treated with various agents, such as Tris buffers or divalent metal ions, to induce competence. Transformation can also be accomplished by electroporation, or by fusion of protoplasts with PEG in the presence of transforming DNA. Unfortunately, the mechanism by which cells become permeable to DNA and the process by which DNA enters the cells is frequently unknown. In order to establish a transformation system for an untransformable bacterium, recipient strains and transforming DNA must be carefully selected. Since it is impossible to predict in advance which method of transformation will be successful with a particular bacterial strain, several techniques are usually evaluated. This review describes a number of factors that appear to be critical for developing a transformation system and presents a strategy for experimentation with novel bacteria.

Nucleotide sequence of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei: comparison to analogous pbg genes of other gram-positive organisms.

Lactose metabolism in Lactobacillus casei occurs via phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and subsequent cleavage of lactose-6-phosphate by beta-D-phosphogalactoside galactohydrolase (P-beta Gal). The genes for lactose uptake and P-beta Gal have been shown to be plasmid-associated in L. casei 64H [Chassy et al., Curr. Microbiol. 1 (1978) 141-144]. The cloned P-beta Gal-coding gene (pbg) previously described [Lee et al., J. Bacteriol. 152 (1982) 1138-1146] was subcloned on a 2.9-kb KpnI-Bg/II fragment isolated from pLZ605. Sequence analysis of this fragment revealed an open reading frame of 1422 bp capable of coding for a protein product containing 474 amino acids and having an Mr of 53,989. The L. casei protein showed a high degree of homology to the proteins whose sequence was deduced from the nucleotide sequence of the pbg genes of Staphylococcus aureus and Streptococcus lactis. Because of the significant homologies observed, as reflected in amino acid content as well as predicted structural characteristics of the three proteins, we suggest a common origin for the P-beta Gals of these three organisms.

Molecular cloning and nucleotide sequence of the factor IIIlac gene of Lactobacillus casei.

The lactose-specific factor III (FIIIlac of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Lactobacillus casei and purified to homogeneity by conventional protein purification methods. Its apparent native Mr, estimated from steric exclusion chromatography (approx. 39 kDa), and subunit Mr, estimated from sodium dodecyl sulfate-polyacrylamide gels, indicated that it exists as a trimer of identical subunits of 13 kDa. The gene for FIII L. casei lac was cloned into Escherichia coli using the vector pUC18. The coding sequences were contained on an 860-bp BglII-HindIII DNA fragment of the L. casei lactose plasmid, pLZ64. A protein identical in properties to FIII L. casei lac was isolated from clones of E. coli carrying this DNA insert. The nucleotide sequence of the FIII L. casei lac gene was determined by the dideoxy chain-termination technique. The 336-bp open reading frame for FIII L. casei lac was followed by a stem-loop structure, analogous to a Rho-independent terminator. We concluded that the FIII L. casei lac was the terminal gene in what appears to be an operon comprised of the lactose-PTS-P-beta Gal-coding genes. Comparison of the deduced amino acid sequence of FIII L. caseilac with the amino acid sequence of FIII S. aureus lac (derived from peptide sequencing) demonstrated a high degree of homology (49 identical residues and 21 conservative exchanges out of 103 total aa residues). The FIII L. casei lac lacked his82, previously identified as the phosphorylation site of FIII S. aureus. lac His80 was proposed to be the site of histidyl phosphorylation of FIII L. casei lac.

Molecular basis of bacterial adhesion in the oral cavity.

The two varieties of fimbriae identified on oral strains of actinomyces have distinct functional properties. The type 1 fimbriae of Actinomyces viscosus T14V mediate attachment to saliva-treated hydroxyapatite. Type 2 fimbriae--on A. viscosus and the only fimbriae detected on A. naeslundii WVU45--are associated with lectin activity. Interaction of these fimbriae with complementary receptors initiates bacterial attachment to Streptococcus sanguis 34 and sialidase-treated epithelial cells and the killing of actinomyces by polymorphonuclear leukocytes (PMNs). Galactose, N-acetylgalactosamine (GalNAc), and related oligosaccharides inhibit these processes, and mutants lacking type 2 fimbriae do not participate in them. The actinomyces lectin is similar to lectins from Ricinus communis and Bauhinia purpurea that agglutinate certain strains of oral streptococci, block attachment of actinomyces to epithelial cells, and inhibit killing of actinomyces by PMNs. The S. sanguis receptor for the actinomyces lectin comprises repeating hexasaccharide units with GalNAc termini. Used as probes, the peanut agglutinin, with specificity for Gal(beta-3)GalNAc, and the lectin from B. purpurea detect a 160-kilodalton (kdal) band in SDS-PAGE-separated epithelial cell extracts and a 100-kdal band in PMN extracts. These may be receptors for type 2 fimbriae. A. viscosus genes encoding subunits of types 1 and 2 fimbriae have been cloned in Escherichia coli; the type 1 subunit is 65 kdal and the type 2 subunit is 59 kdal. Submandibular immunization of mice with a mixture of type 1 and type 2 fimbriae evokes the production of IgA and IgG antibodies in serum and saliva that inhibit in vitro adsorption of A. viscosus to SHA. These antibodies may modulate colonization of teeth by this organism.

Cloning and expression of a type 1 fimbrial subunit of Actinomyces viscosus T14V.

The type 1 fimbriae of Actinomyces viscosus mediate the adherence of this organism to saliva-treated hydroxyapatite. The gene encoding a putative subunit of this fimbrial adhesin was cloned in Escherichia coli, and its product was examined. A. viscosus T14V chromosomal DNA was partially restricted with Sau3AI and cloned into E. coli JM109 by using the plasmid vector pUC13. Two clones, each containing a different DNA insert with a common 4.1-kilobase region, reacted in colony immunoassays with specific polyclonal as well as monoclonal antibodies directed against A. viscosus T14V type 1 fimbriae. Western blot analysis revealed the expression of a 65-kilodalton protein that migrated slightly behind an antigenically similar protein from native type 1 fimbriae. Deletion analysis showed that the gene encoding the cloned protein was localized on a 1.9-kilobase PstI-BamHI fragment and that transcription was dependent on the lac promoter of the vector. The cloned fimbrial protein was purified from the E. coli cytoplasmic fraction by ion-exchange, immunoaffinity, and gel permeation chromatography. Rabbit antibodies prepared against the cloned protein and against purified A. viscosus type 1 fimbriae gave similar patterns with partially dissociated type 1 fimbriae after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The data therefore provide evidence that the gene cloned encodes a subunit of this fimbrial adhesin.

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.

Expression of Actinomyces viscosus antigens in Escherichia coli: cloning of a structural gene (fimA) for type 2 fimbriae.

A cosmid gene library of Actinomyces viscosus T14V was prepared in Escherichia coli to examine the expression of A. viscosus antigens and to gain insight into the structure of A. viscosus type 1 and type 2 fimbriae. Out of this library of 550 clones, 28 reacted in a colony immunoassay with antibodies against A. viscosus cells. The proteins responsible for these reactions were identified in three clones. Clones AV1209 and AV2009 displayed nonfimbrial antigens with subunits of 40 and 58 kilodaltons, respectively. Clone AV1402 showed a 59-kilodalton protein that reacted with monospecific antibody against type 2 fimbriae and that comigrated with a subunit of type 2 fimbriae during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that AV1402 expresses a gene (fimA) for a subunit of A. viscosus type 2 fimbriae.

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei.

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.

Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities.

A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose-phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of [14C]lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution 31P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM. We conclude from our data that phosphorylation of glucose by S. lactis 133 can be mediated by only two mechanisms: (i) via ATP-dependent glucokinase, and (ii) by the phosphoenolpyruvate-dependent mannose-PTS system.

Intracellular phosphorylation of glucose analogs via the phosphoenolpyruvate: mannose-phosphotransferase system in Streptococcus lactis.

The bacterial phosphoenolpyruvate:sugar-phosphotransferase system (PTS) mediates the vectorial translocation and concomitant phosphorylation of sugars. The question arises of whether the PTS can also mediate the phosphorylation of intracellular sugars. To investigate this possibility in Streptococcus lactis 133, lactose derivatives have been prepared containing 14C-labeled 2-deoxy-glucose (2DG), 2-deoxy-2-fluoro-D-glucose (2FG), or alpha-methylglucoside as the aglycon substituent of the disaccharide. Two of the compounds, beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-D-glucopyranose (2'D-lactose) and beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-2'-fluoro-D-glucopyranose (2'F-lactose), were high-affinity substrates of the lactose-PTS. After translocation, the radiolabeled 2'F-lactose 6-phosphate (2'F-lactose-6P) and 2'D-lactose-6P derivatives were hydrolyzed by P-beta-galactoside-galactohydrolase to galactose-6P and either [14C]2FG or [14C]2DG, respectively. Thereafter, the glucose analogs appeared in the medium, but the rates of sugar exit from mannose-PTS-defective mutants were greater than those determined in the parent strain. Unexpectedly, the results of kinetic studies and quantitative analyses of intracellular products in S. lactis 133 showed that initially (and before exit) the glucose analogs existed primarily in phosphorylated form. Furthermore, the production of intracellular [14C]2FG-6P and [14C]2DG-6P (during uptake of the lactose analogs) continued when the possibility for reentry of [14C]2FG and 2DG was precluded by addition of mannose-PTS inhibitors (N-acetylglucosamine or N-acetylmannosamine) to the medium. By contrast, (i) only [14C]2DG, [14C]2FG, and trace amounts of [14C]2FG-6P were found in cells of a mannose-PTS-defective mutant, and (ii) only [14C]2FG and [14C]2DG were present in cells of a double mutant lacking both mannose-PTS and glucokinase activities. We conclude from these data that the mannose-PTS can effect the intracellular phosphorylation of glucose and its analogs in S. lactis 133.

Production and Regeneration of Lactobacillus casei Protoplasts.

Methods for the production and regeneration of Lactobacillus casei protoplasts are described. Protoplasts of L. casei strains were obtained by treatment with mutanolysin or with mutanolysin and lysozyme together in a protoplast formation buffer containing 0.02 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.0), 1 mM MgCl(2), 0.5% gelatin, and 0.3 M raffinose. Cells were regenerated on a complex medium supplemented with bovine serum albumin, MgCl(2), CaCl(2), gelatin, and raffinose. Lengthy digestion with lytic enzymes inhibited the capacity of protoplasts to regenerate. The optimum conditions of protoplast formation varied from strain to strain. Using predetermined optimal conditions it was possible to prepare protoplasts of several L. casei strains and regenerate them with 10 to 40% efficiency. The methods were applicable to other species of lactobacilli as well.

Intracellular hexose-6-phosphate:phosphohydrolase from Streptococcus lactis: purification, properties, and function.

An intracellular hexose 6-phosphate:phosphohydrolase (EC 3.1.3.2) has been purified from Streptococcus lactis K1. Polyacrylamide disc gel electrophoresis of the purified enzyme revealed one major activity staining protein and one minor inactive band. The Mr determined by gel permeation chromatography was 36,500, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single polypeptide of apparent Mr 60,000. The enzyme exhibited a marked preference for hexose 6-phosphates, and the rate of substrate hydrolysis (at 5 mM concentration) decreased in the order, galactose 6-phosphate greater than 2-deoxy-D-glucose 6-phosphate greater than fructose 6-phosphate greater than mannose 6-phosphate greater than glucose 6-phosphate. Hexose 1-phosphates, p-nitrophenylphosphate, pyrophosphate, and nucleotides were not hydrolyzed at a significant rate. In addition, the glycolytic intermediates comprising the intracellular phosphoenolpyruvate potential in the starved cells (phosphoenolpyruvate and 2- and 3-phosphoglyceric acids) were not substrates for the phosphatase. Throughout the isolation, the hexose 6-phosphate:phosphohydrolase was stabilized by Mn2+ ion, and the purified enzyme was dependent upon Mn2+, Mg2+, Fe2+, or Co2+ for activation. Other divalent metal ions including Pb2+, Cu2+, Zn2+, Cd2+, Ca2+, Ba2+, Sr2+, and Ni2+ were unable to activate the enzyme, and the first four cations were potent inhibitors. Enzymatic hydrolysis of 2-deoxy-D-glucose 6-phosphate was inhibited by fluoride when Mg2+ was included in the assay, but only slight inhibition occurred in the presence of Mn2+, Fe2+, or Co2+. The inhibitory effect of Mg2+ plus fluoride was specifically and completely reversed by Fe2+ ion. The hexose 6-phosphate:phosphohydrolase catalyzes the in vivo hydrolysis of 2-deoxy-D-glucose 6-phosphate in stage II of the phosphoenolpyruvate-dependent futile cycle in S. lactis (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982).

Regulation of lactose-phosphoenolpyruvate-dependent phosphotransferase system and beta-D-phosphogalactoside galactohydrolase activities in Lactobacillus casei.

The lactose-phosphoenolpyruvate-dependent phosphotransferase system (lac-PTS) and beta-D-phosphogalactoside galactohydrolase (P-beta-gal) mediate the metabolism of lactose by Lactobacillus casei. Starved cells of L. casei contained a high intracellular concentration of phosphoenolpyruvate, and this endogenous energy reserve facilitated characterization of phosphotransferase system activities in physiologically intact cells. Data obtained from transport studies with whole cells and from in vitro phosphotransferase system assays with permeabilized cells revealed that the lac-PTS had a high affinity for beta-galactosides (e.g., lactose, lactulose, lactobionic acid, and arabinosyl-beta-D-galactoside). lac-PTS and P-beta-gal activities were determined in wild-type strains and strains defective in the glucose-phosphoenolpyruvate-dependent phosphotransferase system after growth on various sugars and in the presence of potential inducers. We found that (i) the lac genes (i.e., the genes coding for the lac-PTS proteins and P-beta-gal) were induced by metabolizable and non-metabolizable beta-galactosides (presumably acting as their phosphorylated derivatives), (ii) galactose 6-phosphate was not an inducer in most strains, (iii) the ratio of lac-PTS activity to P-beta-gal activity in a given strain was not constant, and (iv) inhibition of lac gene expression during growth on glucose was a consequence of glucose-phosphoenolpyruvate-dependent phosphotransferase system-mediated inducer exclusion, repressive effects of a functional glucose-phosphoenolpyruvate-dependent phosphotransferase system and glucose-derived metabolites. The expression of the lac-PTS structural genes and the expression of the P-beta-gal gene are independently regulated and may be subject to both positive control and negative control.

Regulation and characterization of the galactose-phosphoenolpyruvate-dependent phosphotransferase system in Lactobacillus casei.

Cells of Lactobacillus casei grown in media containing galactose or a metabolizable beta-galactoside (lactose, lactulose, or arabinosyl-beta-D-galactoside) were induced for a galactose-phosphoenolpyruvate-dependent phosphotransferase system (gal-PTS). This high-affinity system (Km for galactose, 11 microM) was inducible in eight strains examined, which were representative of all five subspecies of L. casei. The gal-PTS was also induced in strains defective in glucose- and lactose-phosphoenolpyruvate-dependent phosphotransferase systems during growth on galactose. Galactose 6-phosphate appeared to be the intracellular inducer of the gal-PTS. The gal-PTS was quite specific for D-galactose, and neither glucose, lactose, nor a variety of structural analogs of galactose caused significant inhibition of phosphotransferase system-mediated galactose transport in intact cells. The phosphoenolpyruvate-dependent phosphorylation of galactose in vitro required specific membrane and cytoplasmic components (including enzyme IIIgal), which were induced only by growth of the cells on galactose or beta-galactosides. Extracts prepared from such cells also contained an ATP-dependent galactokinase which converted galactose to galactose 1-phosphate. Our results demonstrate the separate identities of the gal-PTS and the lactose-phosphoenol-pyruvate-dependent phosphotransferase system in L. casei.