Distinct galactose phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus lactis.

Lactose-negative (Lac-) mutants were isolated from a variant of Streptococcus lactis C2 in which the lactose plasmid had become integrated into the chromosome. These mutants retained their parental growth characteristics on galactose (Lac- Gal+). This is in contrast to the Lac- variants obtained when the lactose plasmid is lost from S. lactis, which results in a slower growth rate on galactose (Lac- Gal+). The Lac- Gal+ mutants were defective in [14C]thiomethyl-beta-D-galactopyranoside accumulation, suggesting a defect in the lactose phosphoenolpyruvate-dependent phosphotransferase system, but still possessed the ability to form galactose-1-phosphate and galactose-6-phosphate from galactose in a ratio similar to that observed from the parental strain. The Lac- Gald variant formed only galactose-1-phosphate. The results imply that galactose is not translocated via the lactose phosphoenolpyruvate-dependent phosphotransferase system, but rather by a specific galactose phosphoenolpyruvate-dependent phosphotransferase system for which the genetic locus is also found on the lactose plasmid in S. lactis.     

Galactose fermentation by Streptococcus lactis and Streptococcus cremoris: pathways, products, and regulation

All of the lactic streptococci examined except Streptococcus lactis ML8 fermented galactose to lactate, formate, acetate, and ethanol. The levels of pyruvate-formate lyase and lactate dehydrogenase were elevated and reduced, respectively, in galactose-grown cells compared with glucose- or lactose-grown cells. Reduced intracellular levels of both the lactate dehydrogenase activator (fructose, 1,6-diphosphate) and pyruvate-formate lyase inhibitors (triose phosphates) appeared to be the main factors involved in the diversion of lactate to the other products. S. lactis ML8 produced only lactate from galactose, apparently due to the maintenance of high intracellular levels of fructose 1,6-diphosphate and triose phosphates. The growth rates of all 10 Streptococcus cremoris strains examined decreased markedly with galactose concentrations below about 30 mM. This effect appeared to be correlated with uptake predominantly by the low-affinity galactose phosphotransferase system and initial metabolism via the D-tagatose 6-phosphate pathway. In contrast, with four of the five S. lactis strains examined, galactose uptake and initial metabolism involved more extensive use of the high-affinity galactose permease and Leloir pathway. With these strains the relative flux of galactose through the alternate pathways would depend on the exogenous galactose concentration.     

Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway.

The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus.     

Influence of the lactose plasmid on the metabolism of galactose by Streptococcus lactis.

Streptococcus lactis strain DR1251 was capable of growth on lactose and galactose with generation times, at 30 degrees C, of 42 and 52 min, respectively. Phosphoenolpyruvate-dependent phosphotransferase activity for lactose and galactose was induced during growth on either substrate. This activity had an apparent K(m) of 5 x 10(-5) M for lactose and 2 x 10(-2) M for galactose. beta-d-Phosphogalactoside galactohydrolase activity was synthesized constitutively by these cells. Strain DR1251 lost the ability to grow on lactose at a high frequency when incubated at 37 degrees C with glucose as the growth substrate. Loss of ability to metabolize lactose was accompanied by the loss of a 32-megadalton plasmid, pDR(1), and Lac(-) isolates did not revert to a Lac(+) phenotype. Lac(-) strains were able to grow on galactose but with a longer generation time. Galactose-grown Lac(-) strains were deficient in beta-d-phosphogalactoside galactohydrolase activity and phosphoenolpyruvate phosphotransferase activity for both lactose and galactose. There was also a shift from a predominantly homolactic to a heterolactic fermentation and a fivefold increase in galactokinase activity, relative to the Lac(+) parent strain grown on galactose. These results suggest that S. lactis strain DR1251 metabolizes galactose primarily via the tagatose-6-phosphate pathway, using a lactose phosphoenolpyruvate phosphotransferase activity to transport this substrate into the cell. Lac(-) derivatives of strain DR1251, deficient in the lactose phosphoenolpyruvate phosphotransferase activity, appeared to utilize galactose via the Leloir pathway.     

Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster.

DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase. The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment. Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products. Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes. Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits. The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively. These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis.     

Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.     

Galactose Expulsion during Lactose Metabolism in Lactococcus lactis subsp. cremoris FD1 Due to Dephosphorylation of Intracellular Galactose 6-Phosphate

In Lactococcus lactis subsp. cremoris FD1, galactose and lactose are both transported and phosphorylated by phosphotransferase systems. Lactose 6-phosphate (lactose-6P) is hydrolyzed intracellularly to galactose-6P and glucose. Glucose enters glycolysis as glucose-6P, whereas galactose-6P is metabolized via the tagatose-6P pathway and enters glycolysis at the tagatose diphosphate and fructose diphosphate pool. Galactose would therefore be a gluconeogenic sugar in L. lactis subsp. cremoris FD1, but since fructose 1,6-diphosphatase is not present in this strain, galactose cannot serve as an essential biomass precursor (glucose-6P or fructose-6P) but only as an energy (ATP) source. Analysis of the growth energetics shows that transition from N limitation to limitation by glucose-6P or fructose-6P gives rise to a very high growth-related ATP consumption (152 mmol of ATP per g of biomass) compared with the value in cultures which are not limited by glucose-6P or fructose-6P (15 to 50 mmol of ATP per g of biomass). During lactose metabolism, the galactose flux through the tagatose-6P pathway (r(max) = 1.2 h) is lower than the glucose flux through glycolysis (r(max) = 1.5 h) and intracellular galactose-6P is dephosphorylated; this is followed by expulsion of galactose. Expulsion of a metabolizable sugar has not been reported previously, and the specific rate of galactose expulsion is up to 0.61 g of galactose g of biomass h depending on the lactose flux and the metabolic state of the bacteria. Galactose excreted during batch fermentation on lactose is reabsorbed and metabolized when lactose is depleted from the medium. In vitro incubation of galactose-6P (50 mM) and permeabilized cells (8 g/liter) gives a supernatant containing free galactose (50 mM) but no P(i) (less than 0.5 mM). No organic compound except the liberated galactose is present in sufficient concentration to bind the phosphate. Phosphate is quantitatively recovered in the supernatant as P(i) by hydrolysis with alkaline phosphatase (EC 3.1.3.1), whereas inorganic pyrophosphatase (EC 3.6.1.1) cannot hydrolyze the compound. The results indicate that the unknown phosphate-containing compound might be polyphosphate.     

Regulation of lactose fermentation in group N streptococci

Group N streptococci, which have the lactose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) and phospho-beta-d-galactosidase (beta-Pgal), grew rapidly on lactose and converted more than 90% of the sugar to l-lactate. In contrast, Streptococcus lactis 7962, which does not have a beta-Pgal, grew slowly on lactose and converted only 15% of the sugar to l-lactate. With glucose and galactose, this strain had growth rates and fermentation patterns similar to those of other S. lactis strains, suggesting that the rapid and homolactic fermentation of lactose that is characteristic of group N streptococci is dependent upon a functional PEP-dependent PTS and the presence of beta-Pgal. Seventeen strains of group N streptococci were examined for the activator specificities of pyruvate kinase and lactate dehydrogenase. The properties of each enzyme from all the strains, including S. lactis 7962, were similar. Pyruvate kinase had a broad activator specificity, whereas activation of lactate dehydrogenase was specific for ketohexose diphosphate. All intermediates of lactose metabolism from the hexose phosphates to the triose phosphates activated pyruvate kinase. No activation was obtained with adenosine 5'-monophosphate. K and Mg were required for pyruvate kinase activity but could be replaced by NH(4) and Mn, respectively. Lactate dehydrogenase was activated equally by fructose-1,6-diphosphate and tagatose-1,6-diphosphate, the activation characteristics being pH dependent. The roles of pyruvate kinase and lactate dehydrogenase in the regulation of lactose fermentation by group N streptococci are discussed.     

In vivo regulation of glycolysis and characterization of sugar: phosphotransferase systems in Streptococcus lactis.

Two novel procedures have been used to regulate, in vivo, the formation of phosphoenolpyruvate (PEP) from glycolysis in Streptococcus lactis ML3. In the first procedure, glucose metabolism was specifically inhibited by p-chloromercuribenzoate. Autoradiographic and enzymatic analyses showed that the cells contained glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-diphosphate, and triose phosphates.Dithiothreitol reversed the p-chloromercuribenzoate inhibition, and these intermediates were rapidly and quantitatively transformed into 3- and 2-phosphoglycerates plus PEP. The three intermediates were not further metabolized and constituted the intracellular PEP potential. The second procedure simply involved starvation of the organisms. The starved cells were devoid of glucose 6-phosphate, fructose 6-phosphate, fructose- 1,6-diphosphate, and triose phosphates but contained high levels of 3- and 2-phosphoglycerates and PEP (ca. 40 mM in total). The capacity to regulate PEP formation in vivo permitted the characterization of glucose and lactose phosphotransferase systems in physiologically intact cells. Evidence has been obtained for "feed forward" activation of pyruvate kinase in vivo by phosphorylated intermediates formed before the glyceraldehyde-3-phosphate dehydrogenase reaction in the glycolytic sequence. The data suggest that pyruvate kinase (an allosteric enzyme) plays a key role in the regulation of glycolysis and phosphotransferase system functions in S. lactis ML3.     

Novel phosphoenolpyruvate-dependent futile cycle in Streptococcus lactis: 2-deoxy-D-glucose uncouples energy production from growth.

The addition of 2-deoxy-D-glucose to cultures of Streptococcus lactis 133 that were growing exponentially on sucrose or lactose reduced the growth rate by ca. 95%. Inhibition did not occur with glucose or mannose as the growth sugar. The reduction in growth rate was concomitant with rapid accumulation of the analog in phosphorylated form (2-deoxy-D-glucose 6-phosphate) via the phosphoenolpyruvate-dependent mannose:phosphotransferase system. Within 5 min the intracellular 2-deoxy-D-glucose 6-phosphate concentration reached a steady-state level of greater than 100 mM. After maximum accumulation of the sugar phosphate, the rate of sucrose metabolism (glycolysis) decreased by only 30%, but the cells were depleted of fructose-1,6-diphosphate. The addition of glucose to 2-deoxy-D-glucose 6-phosphate preloaded cells caused expulsion of 2-deoxy-D-glucose and a resumption of normal growth. S. lactis 133 contained an intracellular Mg2+-dependent, fluoride-sensitive phosphatase which hydrolyzed 2-deoxy-D-glucose 6-phosphate (and glucose 6-phosphate) to free sugar and inorganic phosphate. Because of continued dephosphorylation and efflux of the non-metabolizable analog, the maintenance of the intracellular 2-deoxy-D-glucose 6-phosphate pool during growth stasis was dependent upon continued glycolysis. This steady-state condition represented a dynamic equilibrium of: (i) phosphoenolpyruvate-dependent accumulation of 2-deoxy-D-glucose 6-phosphate, (ii) intracellular dephosphorylation, and (iii) efflux of free 2-deoxy-D-glucose. This sequence of events constitutes a futile cycle which promotes the dissipation of phosphoenolpyruvate. We conclude that 2-deoxy-D-glucose functions as an uncoupler by dissociating energy production from growth in S. lactis 133.     

Characterization of lactose-fermenting revertants from lactose-negative Streptococcus lactis C2 mutants

Partial lactose-fermenting revertants from lactose-negative (lac(-)) mutants of Streptococcus lactis C2 appeared on a lawn of lac(-) cells after 3 to 5 days of incubation at 25 C. The revertants grew slowly on lactose with a growth response similar to that for cryptic cells. In contrast to lac(+)S. lactis C2, the revertants were defective in the accumulation of [(14)C]thiomethyl-beta-d-galactoside, indicating that they were devoid of a transport system. Hydrolysis of o-nitrophenyl-beta-d-galactoside-6-phosphate by toluene-treated cells confirmed the presence of phospho-beta-d-galactosidase (P-beta-gal) in the revertant. However, this enzyme was induced only when the cells were grown in the presence of lactose; galactose was not an inducer. In lac(+)S. lactis C2, enzyme induction occurred in lactose- or galactose-grown cells. The revertants were defective in EII-lactose and FIII-lactose of the phosphoenolpyruvate-dependent phosphotransferase system. Galactokinase activity was detected in cell extracts of lac(+)S. lactis C2, but the activity was 9 to 13 times higher in extracts from the revertant and lac(-), respectively. This suggested that the lac(-) and the revertants use the Leloir pathway for galactose metabolism and that galactose-1-phosphate rather than galactose-6-phosphate was being formed. This may explain why lactose, but not galactose, induced P-beta-gal in the revertants. Because the revertant was unable to form galactose-6-phosphate, induction could not occur. This compound would be formed on hydrolysis of lactose phosphate. The data also indicate that galactose-6-phosphate may serve not only as an inducer of the lactose genes in S. lactis C2, but also as a repressor of the Leloir pathway for galactose metabolism.     

Inorganic salts resistance associated with a lactose-fermenting plasmid in Streptococcus lactis.

Present evidence indicates that lactose metabolism in group N streptococci is linked to plasmid deoxyribonucleic acid. Lactose-positive (Lac+) Streptococcus lactis and lactose-negative (Lac-) derivatives were examined for their resistance to various inorganic ions. Lac+ S. lactis strains ML3, M18, and C2 were found more resistant to arsenate (7.5- to 60.2-fold), arsenite (2.25- to 3.0-fold), and chromate (6.6- to 9.4-fold), but more sensitive to copper (10.0- to 13.3-fold) than their Lac- derivatives. These results suggested that genetic information for resistance and/or sensitivity to these ions resides on the "lactose plasmid." Kinetics of ultraviolet irradiation inactivation of transducing ability for lactose metabolism and arsenate resistance confirmed the plasmid location of the two markers. Lac+ transductants from S. lactis C2 received genetic determinants for resistance to arsenate, arsenite, and chromate but not for copper sensitivity. In this case, resistance markers were lost when the transductants became Lac- but the derivatives remained copper resistant. The resistant markers for arsenate and arsenite could not be identified as separate genetic loci, but chromate resistance and copper sensitivity markers were found to be independent genetic loci. The "lactose plasmid" from S. lactis C10 possessed the genetic loci for arsenate and arsenite resistance but not for chromate resistance or copper sensitivity.     

Expression of a beta-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis

A beta-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp. lactis strains H1 and 7962. When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact. lactis subsp. lactis strain H1, the resulting construct had high beta-galactosidase activity but utilized lactose only slightly faster than the recipient. beta-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the beta-galactosidase gene. Growth rates of Lac+ H1 and 7962 derivatives were not affected after introduction of the clostridial beta-galactosidase, even though beta-galactosidase activity in a 7962 construct was more than double that of the wild-type strain. When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-beta-galactosidase activity and a growth rate equal to that of the H1 wild-type strain. The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-beta-galactosidase activity. We suggest that beta-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact. lactis subsp. lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-beta-galactosidase genes.     

Lactose and D0galactose metabolism in Staphylococcus aureus: pathway of D-galactose 6-phosphate degradation

The pathway by which D-galactose 6-phosphate is degraded in Staphylococcus aureus has been elucidated. Galactose 6-phosphate is isomerized to tagatose 6-phosphate, which is phosphorylated with adenosine 5′-triphosphate, and the resulting tagatose 1,6-diphosphate is cleaved to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The isomerase, kinase, and aldolase that catalyze these reactions are inducible and are distinct from the corresponding enzymes of glucose 6-phosphate metabolism.     

Expression of a β-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis

A β-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp. lactis strains H1 and 7962. When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact. lactis subsp. lactis strain H1, the resulting construct had high β-galactosidase activity but utilized lactose only slightly faster than the recipient. β-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the β-galactosidase gene. Growth rates of Lac⁺ H1 and 7962 derivatives were not affected after introduction of the clostridial β-galactosidase, even though β-galactosidase activity in a 7962 construct was more than double that of the wild-type strain. When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-β-galactosidase activity and a growth rate equal to that of the H1 wild-type strain. The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-β-galactosidase activity. We suggest that β-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact. lactis subsp. lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-β-galactosidase genes.     

Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12

Summary We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli. Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS). We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues). In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively. This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate. Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel.Abbreviations G1P glucose-1-phosphate - G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1-6DP fructose-1,6-diphosphate - PEP phosphoenol pyruvate     

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.     

Molecular cloning of the lactose-metabolizing genes from Streptococcus lactis

Restriction endonucleases and agarose gel electrophoresis were used to analyze plasmid pLM2001, which is required for lactose metabolism by Streptococcus lactis LM0232. The enzymes XhoI, SstI, BamHI, and KpnI each cleaved the plasmid into two fragments, whereas EcoRI and BglII cleaved the plasmid into seven and five fragments, respectively. Sizing of fragments and multiple digestions allowed construction of a composite restriction map. The KpnI fragments of pLM2001 were cloned into the KpnI cleavage site of the vector plasmid pDB101. A recombinant plasmid (pSH3) obtained from a lactose-fermenting, erythromycin-resistant (Lac+ Eryr) transformant of Streptococcus sanguis Challis was analyzed by enzyme digestion and agarose gel electrophoresis. Plasmid pSH3 contained 7 of the 11 KpnI-HindIII fragments from pLM2001 and 5 of the 7 fragments from pDB101. It was determined that a 23-kilobase (kb) KpnI-generated fragment from pLM2001 had been cloned into pDB101 with deletion of part of the vector plasmid. The recombinant plasmid could be transformed with high frequency into several Lac- strains of S. sanguis, conferring the ability to ferment lactose and erythromycin resistance. The presence of pSH3 allowed a strain deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-dependent phosphotransferase system to efficiently ferment lactose. Under conditions designed to maximize curing of plasmid DNA with acriflavin, no Lac- derivatives could be isolated from cells transformed with pSH3. Seven of the 40 Lac+ colonies isolated after 10 transfers in acriflavin were shown to be sensitive to erythromycin and did not appear to harbor plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS)