Prophage-Cured Derivatives of Streptococcus lactis and Streptococcus cremoris

Prophage curing was achieved in Streptococcus lactis and Streptococcus cremoris, and the cured derivatives were shown to be indicators for their temperate bacteriophages. Relysogenization of these cured derivatives completed the first formal demonstration of the lysogenic state in lactic streptococci.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

The effect of low temperature on Streptococcus lactis

Summary When milk cultures of Streptococcus lactis are held near 0⁰, the number of living cells decreases rather slowly for about one month. Even after 3 months, viable cells are still present, though reduced to a few thousands. The death is partly due to accumulation of acid, but mainly to oxygen. Neutralization and replacement of the free oxygen by nitrogen in the culture prolong the viability.The early cessation of fermentation when large numbers of cells are kept in milk at 0⁰ (or at any temperature below the growth minimum) is due to some damaging effect of oxygen which the inactivated growth mechanism can not prevent or repair.The enzyme content of the cells, measured by their fermenting capacity (mg lactic acid per cell per hour) slowly decreases at temperatures below the growth minimum. After restoration of higher temperatures, the cells recover their enzyme content very slowly, and require several generations for complete recovery.The damage caused by oxygen is not the cause, but the result of the minimal temperature of growth.     

Conversion of methionine to methional by Lactococcus lactis

Lactic acid bacteria were screened for methional production from 4-methylthio-2-ketobutanoate. Only Lactococcus lactis IFPL730 produced high amounts of methional. It was demonstrated that production of this compound was an exclusively enzymatic reaction. The present work describes for the first time that L. lactis can convert enzymatically methionine to methional in a process mediated by aminotransferase and alpha-ketoacid decarboxylase activities. The activity seems to be strain dependent.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

Galactose transport systems in Streptococcus lactis

Galactose-grown cells of Streptococcus lactis ML3 have the capacity to transport the growth sugar by two separate systems: (i) the phosphoenolpyruvate-dependent phosphotransferase system and (ii) an adenosine 5'-triphosphate-energized permease system. Proton-conducting uncouplers (tetrachlorosalicylanilide and carbonyl cyanide-m-chlorophenyl hydrazone) inhibited galactose uptake by the permease system, but had no effect on phosphotransferase activity. Inhibition and efflux experiments conducted using beta-galactoside analogs showed that the galactose permease had a high affinity for galactose, methyl-beta-D-thiogalactopyranoside, and methyl-beta-D-galactopyranoside, but possessed little or no affinity for glucose and lactose. The spatial configurations of hydroxyl groups at C-2, C-4, and C-6 were structurally important in facilitating interaction between the carrier and the sugar analog. Iodoacetate had no inhibitory effect on accumulation of galactose, methyl-beta-D-thiogalactopyranoside, or lactose via the phosphotransferase system. However, after exposure of the cells to p-chloromercuribenzoate, phosphoenolpyruvate-dependent uptake of lactose and methyl-beta-D-thiogalactopyranoside were reduced by 75 and 100%, respectively, whereas galactose phosphotransferase activity remained unchanged. The independent kinetic analysis of each transport system was achieved by the selective generation of the appropriate energy source (adenosine 5'-triphosphate or phosphoenolpyruvate) in vivo. The maximum rates of galactose transport by the two systems were similar, but the permease system exhibited a 10-fold greater affinity for sugar than did the phosphotransferase system.