Lactic streptococci, classically regarded as homolactic fermenters of glucose and lactose, became heterolactic when grown with limiting carbohydrate concentrations in a chemostat. At high dilution rates (D) with excess glucose present, about 95% of the fermented sugar was converted to L-lactate. However, as D was lowered and glucose became limiting, five of the six strains tested changed to a heterolactic fermentation such that at D = 0.1 h-1 as little as 1% of the glucose was converted to L-lactate. The products formed after this phenotypic change in fermentation pattern were formate, acetate, and ethanol. The level of lactate dehydrogenase, which is dependent upon ketohexose diphosphate for activity, decreased as fermentation became heterolactic with Streptococcus lactis ML3. Transfer of heterolactic cells from the chemostat to buffer containing glucose resulted in the nongrowing cells converting nearly 80% of the glucose to L-lactate, indicating that fine control of enzyme activity is an important factor in the fermentation change. These nongrowing cells metabolizing glucose had elevated (ca. twofold) intracellular fructose 1,6-diphosphate concentrations ([FDP]m.) compared with those in the glucose-limited heterolactic cells in the chemostat. [FDP]ui was monitored during the change in fermentation pattern observed in the chemostat when glucose became limiting. Cells converting 95 and 1% of the glucose to L-lactate contained 25 and 10 mM [FDP]in, respectively. It is suggested that factors involved in the change to heterolactic fermentation include both [FDP]in and the level of lactate dehydrogenase. Group N streptococci (Streptococcus cremoris, S. lactis, and S. diacetylactis) play a vital role in many commercial milk fermentations, in which their primary function is to convert lactose to lactic acid (18). Lactic streptococci are useful because they possess limited metabolic diversity and usually convert about 95% of the fermented sugar to L-lactate (23, 26). This homolactic fermentation of either lactose or glucose occurs in batch culture when organisms are grown anaerobically near pH 7 at 30°C. In contrast, heterolactic fermentation was observed during growth on galactose (26) and during growth on lactose of variants defective in either lactate dehydrogenase (LDH; 21) or the lactose phosphotransferase system and/or phospho-fB-D-galactosidase (7, 26), suggesting that these organisms have pathways which are not normally expressed. The alternative products reported include acetate, acetoin, C02, ethanol, formate, and glycerol. Unsuccessful attempts

The synthesis of proteolytic enzymes by starter bacteria is a fundamental requirement for rapid acid production in milk fermentations. These organisms possess a number of proteinases and peptidases which act in concert to hydrolyse milk protein to the free amino acids required for cell growth. The same enzymes have an important secondary role in cheese ripening contributing to rheological and organoleptic changes. A highly complex mixture of both enzymes and substrates is present. The strategic location of these enzymes, in the cell wall and membrane structures and in the cytoplasm, governs enzyme access to the substrates and is central to both roles. An overview of the above topics is presented.

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.

In the absence of an exogenous energy source, galactose-grown cells of Streptococcus lactis Mb3 rapidly accumulated thiomethyl-,8-galactopyranoside (TMG) and 2-deoxyglucose to intracellular concentrations of 40 to 50 mM. Starved cells maintained the capacity for TMG uptake for many hours, and accumulation of the f8-galactoside was insensitive to proton-conducting ionophores (tetrachlorosalicylanilide and carbonylcyanide-m-chlorophenyl hydrazone) and sulfydryl group reagents including iodoacetate and N-ethylmaleimide. Fluorimetric analysis of glycolytic intermediates in extracts prepared from starved cells revealed (a) high intracellular levels of phosphoenolpyruvate (13 mM; PEP) and 2-phosphoglycerate (-39 mM; 2-PG), but (b) an absence of other metabolites including glucose 6-phosphate, fructose 6-phosphate, fructose 1,6diphosphate, and triosephosphates. The following criteria showed PEP (and 2-PG) to be the endogenous energy source for TMG accumulation by the phosphotransferase system: (i) the intracellular concentrations of PEP and 2-PG decreased with concomitant uptake of TMG, and a close correlation was observed between maximum accumulation of the B-galactoside and the total available concentration of the two intermediates; (ii) TMG accumulated as an anionic derivative, which after extraction and incubation with alkaline phosphatase (EC 3.1.3.1) formed the original analogue; (iii) fluoride inhibition of 2-phospho-Dglycerate hydrolyase (EC 4.2.1.11) prevented the conversion of 2-PG to PEP, and uptake of TMG by the starved cells was reduced by 80%; and (iv) the stoichiometric ratio [TMG] accumulated/[PEP] consumed was almost unity (0.93). In cells metabolizing glucose, all intermediates listed in (a) and (b) were found. Upon exhaustion of glucose from the medium, the metabolites in (b) were not longer detectable, while the intracellular concentrations of PEP and 2-PG increased to the levels previously observed in starved cells. The glycolytic intermediates in (b) are all in vitro heterotropic effectors of pyruvate kinase (adenosine 5'triphosphate:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from S. lactis ML3. It is suggested that the capacity of starved cells to maintain high intracellular concentrations of PEP and 2-PG is a consequence of decreased in vivo activity of this key regulatory enzyme of glycolysis. Streptococcus lactis and S. faecalis possess certain physiological characteristics (10, 11, 14) that have proved extremely useful in the study of energy coupling to solute accumulation by microorganisms (9, 10, 34). For example, under normal growth conditions these organisms lack a functional cytochrome system and so are unable to produce adenosine 5'-triphosphate (ATP) via oxidative phosphorylation. S. lactis and S. faecalis usually rely upon glycolysis or arginine catabolism for the generation of the compounds that serve directly (phosphoenolpyruvate, PEP) or indirectly (ATP) as energy

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