Involvement of Phosphoenolpyruvate in Lactose Utilization by Group N Streptococci

Abstract

The effect of sodium fluoride on lactose metabolism and o-nitrophenyl-,B-Dgalactopyranoside (ONPG) hydrolysis by Streptococcus lactis strains 7962 and C2F suggested that different mechanisms of lactose utilization existed in the two strains. Sodium fluoride prevented lactose utilization and ONPG hydrolysis by whole cells of S. lactis C2F but had no effect on S. lactis 7962. Although hydrolysis of ONPG by toluene-treated cells of S. lactis 7962 occurred without addition of phosphoenolpyruvate (PEP), toluene-treated cells of S. lactis C2F required the presence of this cofactor. Concentrated cell extracts of S. lactis C2F hydrolyzed ONPG; this hydrolysis was inhibited by NaF, but the addition of PEP, in the presence of NaF, restored maximal activity. Addition of acetyl-phosphate, carbamyl-phosphate, adenosine-5'-triphosphate, guanosine-5'-triphosphate, or uridine-5'-triphosphate did not stimulate activity. The presence of cofactors did not stimulate and NaF did not inhibit the hydrolysis in extracts of S. lactis 7962. To confirm the operation of two mechanisms, S. lactis 7962 was shown to hydrolyze lactose to glucose and galactose, whereas S. lactis C2F was unable to split the disaccharide. In addition, whole cells of S. lactis C2F rapidly accumulated a phosphorylated derivative of thiomethyl-3-D-galactoside (TMG) which behaved chromatographically and electrophoretically like TMG-PO4. Unexpectedly, S. lactis 7962 also accumulated a TMG derivative, although the rate was extremely low. These data indicate that different mechanisms of lactose utilization exist in the two strains, with a phosphorylation step dependent on PEP involved in S. lactis C2F.

Genetics of lactose utilization in lactic acid bacteria

“…A lactose PTS is found in industrially used strains of Lactococcus lactis and Lactobacillus casei [35,36]. These mesophilic bacteria have been selected for rapid conversion of lactose and the presence of a lactose PTS can be explained by its prominent biochemical and bioenergetic efficiency.…”

“…These mesophilic bacteria have been selected for rapid conversion of lactose and the presence of a lactose PTS can be explained by its prominent biochemical and bioenergetic efficiency. The affinity for lactose is high in Lactobacillus casei (Km of 14 /xM) and Lactococcus lactis (Km of 15 /xM), while the V, .... can reach 117 nmol/min/mg protein [35][36][37]. In addition, a lactose molecule is translocated and simultane- ously phosphorylated at the expense of one ATP equivalent.…”

“…In contrast, galactose entering via a galactose PTS is converted into galactose 6-phosphate that is further degraded via the tagatose 6-phosphate pathway [34]. In the latter case tagatose 6-phosphate is formed, which explains the induction of the lac operon during growth on galactose [35]. Thc galactose permease system is highly specific for galactose and has little or no affinity for lactose [60,61].…”

Regulation of sugar transport and metabolism in lactic acid bacteria

“…In 1969-1970 McKay and coworkers [18,19] first described the lac-PTS in the lactic strepto- cocci by in vitro complementation of soluble components (EI, HPr, EIII l"~) with a sugar-specific membrane fraction (Eli la~) prepared from S. lactis. However, it was not until 1977 following the discovery of the endogenous PEP-potential in starved cells of S. lactis [20] that the importance of PEP, and the highly efficient coupling of sugar transport to phosphorylation via the lac-PTS, were demonstrated in vivo.…”

Characterization of Lactose-Fermenting Revertants from Lactose-Negative Streptococcus lactis C2 Mutants

“…Group N streptococci use lactose via the phosphoenolpyruvate (PEP) transferase system (14,15) similar to that observed in Staphylococcus aureus (7,17). The mechanism of this reaction is described as follows, where HPr is a heat-stable soluble protein, EI (enzyme I) is a soluble protein, Ell-lac (enzyme II) is a lactosespecific membrane-bound component, FIII-lac (factor III) is a lactose-specific factor found in the soluble fraction of the cell, and P-fl-gal is phospho-f-D-galactosidase: PEP + HPr E P-HPr + pyruvate EI and HPr are constitutive components and are produced regardless of the type of carbohydrate present in the growth medium.…”