Carbohydrate transport in Staphylococcus aureus. V. The accumulation of phosphorylated carbohydrate derivatives, and evidence for a new enzyme-splitting lactose phosphate.

Abstract

In previous papers, genetic and biochemical evidence has been presented which indicates a common step in the transport of carbohydrates across the staphylococcal membrane.1-3 At least ten carbohydrates4 utilize this common step, which can be lost by a single gene mutation. A study of uptake of radioactively labeled carbohydrates showed that several of the transported compounds occurred in the cell as derivatives. We wish now to report that the common step probably involves phosphorylation, since the derivatives have been found to be phosphates.In addition, the mystery of the labile f3-galactosidase of Staphylococcus has been clarified. While induced, intact cells of Staphylococcus hydrolyze orthonitrophenylgalactoside (ONPG), a fact which has been interpreted as indicating the presence of gl-galactosidase enzyme, nevertheless, efforts to obtain cell-free preparations of the enzyme have been uniformly unsuccessful.5-7 The present study now resolves the apparent discrepancy: Staphylococcus cannot hydrolyze lactose or ONPG, but possesses an enzyme which hydrolyzes the phosphorylated derivatives of those compounds that are probably formed in the passage across the cell membrane.Materials and Methods.-The cultures employed, all derivatives of Staphylococcus aureus NCTC 8511, and many of the techniques used have been described in the earlier publications.'-3 Special techniques are described where necessary.The radioactive chemicals employed, most of which were tested chromatographically for purity, were from the following sources: lactose-i-C14, 1.53 mc/mM, Calbiochem; maltose-C 4-UL, 5.95 mc/mM, Nuclear-Chicago; sucrose-C14-UL, 5.1 mc/mM, Nichem; a-methylglucoside-CL4-UL, 1.48 mc/mM, Nichem; and isopropyl-2-Cl4-thiogalactoside, 25 mc/mM, Calbiochem. Allolactose (6-$-Dgalactosyl-D-glucose) was labeled in the 1-position of the galactose moiety.8 Carrier-free phosphorus-32 was obtained from New England Nuclear Corporation. The Escherichia coli-purified j3-galactosidase employed was prepared in this department,9 and the gastric alkaline phosphatase obtained from the Sigma Chemical Company.Radioactivity measurements of chromatograms were made in an Atomic Accessories chromatogram scanner, with a Texas Instruments model RSC-5A recorder attached to a Baird Atomic model 432A ratemeter. Radioactive carbon measurements were made with a wide-open window. Radioactive phosphorus was measured with a 0.9-mg/cm2 gold-coated mylar screen across the window. This prevents counting of about 95 per cent of the C14 present in the chromatograms.In some experiments, the radioactivity in portions of the chromatograms was 274

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

“…Three years later, evidence for the presence of a PTS in a firmicute was obtained in the laboratory of Melvine Laurance Morse at the University of Colorado, Denver. They identified a PTS catalyzing the transport and phosphorylation of lactose in Staphylococcus aureus (201). In the following years, the proteins forming the glucose-and mannose-specific PTSs in E. coli (202,203) and the lactose-specific PTS in S. aureus (204) were identified, and their role in transport and phosphorylation of the two hexoses and the disaccharide was established.…”

Thiamine Limited Steady State Growth of the Yeast Cryptococcus albidus

“…If there is an initial common carrier (Hengstenberg, Egan & Morse, 1967) one can calculate a lower limit for the amount of carrier required to give a given flux at the reported concentrations of substrate (Amdur & Hammes, 1966). By using the upper limit for bimolecular reactions in liquids of I O~M -~ sec.-l and a carrier molecular weight of 30,000 the observed flux would require that the organism be composed of only 0.01 % of this carrier.…”

Phosphoenolpyruvate-dependent maltose:phosphotransferase activity in Fusobacterium mortiferum ATCC 25557: specificity, inducibility, and product analysis

“…However, maltose-6P hydrolase (P-a-glucosidase) activity was discernible in anaerobic extracts as well as in aerobic preparations supplemented with DTT. In both cases, the specific activity of maltose-6P hydrolase (-0.002 U/mg) was 25-to 100-fold lower than the activities of related disaccharide-P hydrolases which catalyze cleavage of lactose-6P (3,7,9,12,30), sucrose-6P (4,14,29,32,33), cellobiose-6P (18,19), trehalose-6P (1,15), and P-,B-glucosides (37) in other bacterial species. Parenthetically, it should be noted that P-ot-glucosidase activity in Streptococcus mutans OMZ 176 was described by Wursch and Koellreutter (38).…”