Distinct galactose phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus lactis.
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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. (+info)
Influence of "energization" on the binding of M protein with p-nitrophenyl alpha-D-galactopyranoside.
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A specific binding of p-nitrophenyl alpha-D-galactopyranoside has been measured by flow dialysis with Escherichia coli ML 308225 membrane vesicles containing the lac carrier protein. The number of binding sites, 0.45 nmol/mg of membrane protein, remains unchanged in the presence or absence of energy. On the other hand, "energization" increases the M protein affinity for p-nitrophenyl alpha-D-galactopyranoside. The dissociation constant (Kd) is 4 and 21 microM in the presence and absence, respectively, of D-lactate. The same energization effects are found with E. coli A3245 membrane vesicles. p-Nitrophenyl alpha-D-galactopyranoside can be used as a substrate to study energization effect on binding to the lactose permease M protein. These results corroborate observations that energy increases the lac carrier protein affinity for its substrate, and they also confirm the concentration of the M protein, which corresponds to 1.4% of the membrane protein. (+info)
lac Thiogalactoside transacetylase of Escherichia coli K-12 and ML.
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The lac thiogalactoside transacetylase was purified from both a wild-type Escherichia coli K-12 strain (H3000) and an E. coli ML strain (ML308). These enzymes are indistinguishable by using several criteria. The subunit molecular weight of the enzyme is 24,800, which is significantly less than the previously reported value of 30,000. Although the function of the thiogalactoside transacetylase is unknown, it is suggested that this enzyme plays an important role in lactose utilization since its structure and enzymatic activity have been conserved. (+info)
Regulation of beta-galactoside phosphate accumulation in Streptococcus pyogenes by an expulsion mechanism.
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Streptococcus pyogenes pregrown on lactose took up glucose, lactose, or methyl beta-D-thiogalactopyranoside (MeSGal or TMG) by a phosphoenolpyruvate-dependent phosphotransferase system. MeSGal accumulated in the cell as MeSGal-phosphate (MeSGalP). Three effects were noted when various sugars were added to MeSGal preloaded cells: (i) no decrease in intracellular MeSGalP concentration after addition of fructose, sucrose, o-nitrophenyl-beta-D-galactoside, glycerol, 6-deoxyglucose, alpha-methyl D-glucoside, 2-deoxygalactose, glucose 1-phosphate, or glucose 6-phosphate; (ii) slow loss of preaccumulated MeSGalP evoked by lactose, 2-deoxy-D-glucose, or unlabeled MeSGal; and (iii) a short lag followed by extremely rapid expulsion of intracellular MeSGalP elicited by glucose or mannose and a slower expulsion elicited by glucosamine. The expelled compound was free MeSGal, indicating the involvement of dephosphorylation in the expulsion mechanism. Deoxyglucose inhibited the expulsion evoked by mannose, and prepoisoning of cells with fluoride or arsenate prevented the glucose-dependent expulsion. The expulsion is due to activation of an expulsion mechanism rather than to turnover of MeSGalP and leak of internal MeSGal with concomitant inhibition of MeSGal influx. The results suggest the need for phosphotransferase-dependent translocation of a preferential sugar or accumulation of the sugar catabolite for expulsion activation. The significance of the expulsion mechanism in synthesis regulation of enzymes involved in carbohydrate utilization is proposed. (+info)
Membrane biogenesis. Evidence that a soluble chimeric polypeptide can serve as a precursor of a mutant lac permease in Escherichia coli.
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A mutant in the Escherichia coli lac permease, called Yf, appears to be defective in the biogenesis and proper assembly of this membrane protein. It was proposed that this defect led to the accumulation of a precursor of the mutant permease (Fried, V. A. (1977) J. Mol Biol. 114, 477-490). In this communication, evidence is presented that the lacYf mutant accumulates a novel lac-specific soluble polypeptide with a molecular weight of 87,000. Detected by double-label analysis on sodium dodecyl sulfate gels, and identified as a lac-specific polypeptide on a two-dimensional gel system, this polypeptide is immunoprecipitated by anti-transacetylase antibody. Pulse-chase experiments are consistent with the hypothesis that it is converted in vivo into a lac-specific membrane protein with an apparent molecular weight of 28,000, which appears to be the mutant lac permease. The results suggest that the 87,000-dalton soluble protein is a precursor of the mutant lac permease. It is proposed that this precursor is a polyprotein chimera containing both the lacY and lacA gene products. (+info)
Dependence on pH of parameters of lactose transport in Escherichia coli. Evidence for an essential protonated group of the carrier.
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The kinetic parameters Km and V of transported by the lactose permease of Escherichia coli have been explored in the pH range 4.8--9.2. Besides uphill transport of methylthiogalactoside, two other criteria have been used. Downhill transport of o-nitrophenylgalactoside and substrate protection of the carrier against thiol reagents have both been explored in normal aerated cells and in cells inhibited by cyanide plus azide, therefore unable to build up a proton-motive force. V of the transport processes did not exhibit a major pH dependence that would support an essential protonation. Ktransport for methylthiogalactoside and for o-nitrophenylgalactoside in the energized and in the inhibited state did not show a sharp pH dependence between pH 4.8 and 8.0, but increased between pH 8 and 9, as would be expected if there were an essential protonated group with a pK of 8--8.4, depending on the test utilized. Substrate protection allowed the calculation of a Kprotection which was close to the corresponding Ktransport and was also largely independent of pH between 5 and 8 and independent of energy supply. The role of energization in substrate-carrier binding and the role of the essential protonation in the context of the proton symptom model are discussed. (+info)
Regulation of the lactose phosphotransferase system of Streptococcus bovis by glucose: independence of inducer exclusion and expulsion mechanisms.
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Streptococcus bovis had a diauxic pattern of glucose and lactose utilization, and both of these sugars were transported by the sugar phosphotransferase system (PTS). Lactose catabolism was inducible, and S. bovis used the tagatose pathway to ferment lactose. Since a mutant that was deficient in glucose PTS activity transported lactose as fast as the wild-type, it appeared that S. bovis has separate enzyme IIs for glucose and lactose. The nonmetabolizable glucose analogue 2-deoxyglucose (2-DG) was a noncompetitive inhibitor of methyl beta-D-thiogalactopyranoside (TMG) transport, and cells that were provided with either glucose or 2-DG were unable to transport TMG or lactose. Because the glucose-PTS-deficient mutant could ferment glucose, but could not exclude TMG, it appeared that enzyme IIGlc rather than glucose catabolism per se was the critical feature of inducer exclusion. Cells that had accumulated TMG as TMG 6-phosphate expelled free TMG when glucose was added, but 2-DG was unable to cause TMG expulsion. The glucose-PTS-deficient mutant could still expel TMG in the presence of exogenous glucose. Membrane vesicles also exhibited glucose-dependent TMG exclusion and TMG expulsion. Membrane vesicles that were electroporated with phosphoenolpyruvate (PEP) and HPr retained TMG for more than 3 min, but vesicles that were electroporated with PEP plus HPr and fructose 1,6-diphosphate (FDP) (or glycerate 2-phosphate) lost their ability to retain TMG. Because FDP was able to trigger the ATP-dependent phosphorylation of HPr, it appeared that inducer expulsion was mediated by an FDP-activated protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
Properties of two sugar phosphate phosphatases from Streptococcus bovis and their potential involvement in inducer expulsion.
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Streptococcus bovis possesses two sugar phosphate phosphatases (Pases). Pase I is a soluble enzyme that is inhibited by the membrane fractions from lactose-grown cells and is insensitive to activation by S46D HPr, an analog of HPr(ser-P) of the sugar phosphotransferase system. Pase II is a membrane-associated enzyme that can be activated 10-fold by S46D HPr, and it appears to play a role in inducer expulsion. (+info)