Graduate Thesis Or Dissertation

Carbohydrate metabolism in lactic streptococci with special reference to galactose

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  • Phosphorylation of free galactose by lactic streptococci was mediated by an adenosine triphosphate (ATP) dependent kinase which was repressed by glucose. The phosphoenolypyruvate (PEP) phosphotransferase system (PTS) was not involved in transport of galactose, The conversion of free galactose to glucose was also demonstrated. A key Leloir pathway enzyme, uridine diphosphogalactose -4- epimerase, was found present with at least 0, 66 units/mg protein in cells grown in milk or broth containing galactose or lactose. A lower level of the enzyme (0, 14 units/mg protein) was found in glucose-grown cells. A modification of the standard technique for the assay of uridine diphosphogalactose-4-epimerase was introduced. Undialyzed crude extracts of lactic streptococcal cells could be used for the assay but only after incorporation of 0.2 M semicarbazide in the glycine buffer. In cell dry weight yield studies, it was shown that glucose was the best carbohydrate for growth, and galactose the poorest, It was, however, very difficult to show differences in growth rates of lactic streptococci using five carbohydrates. Also, in this group of organisms diauxic growth on a mixture of glucose and galactose could not be shown. Efforts were made to determine if lactic streptococci containing β-D-phosphogalactoside galactohydrolase (β -Pgal) could utilize glucose and galactose simultaneously. Results indicated that galactose induced cells of Streptococcus diacetilactis 18-16 could catabolize both sugars at the same time. In Streptococcus lactis 7962, however, an organism which possesses β-galactosidase (β-gal), growth curves employing both glucose and galactose revealed two distinct slopes with no lag between the time of glucose exhaustion and the time when galactose was utilized. In differential respirometry studies involving washed cells of S. diacetilactis 18-16, it was shown that less CO₂ (< 170μ,1/100 mg cell dry wt/hr. ) was evolved from galactose by glucose-induced cells, compared to that (916μl 1 / 100 mg cell dry wt/hr.) from glucose. The inability of glucose-induced cells to catabolize galactose and lactose to the same degree as glucose was apparently due to classical catabolite repression. Lactose-induced cells evolved CO₂ equally well from lactose or glucose, but the evolution of CO₂ from galactose and a mixture of glucose and galactose was somewhat repressed. Galactoseinduced cells required more oxygen (>1500 μl /100 mg cell dry wt/hr.) than did glucose or lactose-induced cells (<850 μ11/100 mg cell dry wt/hr.). Paper, thin layer, and column chromatographic techniques were employed in a search for galactose-6-phosphate from several wild type lactic streptococci. The compound was never detected nor was it possible to isolate it from a reaction mixture of o-nitrophenyl, β -D, galactopyranoside-6-phosphate (ONPG-6-P) and crude cellular extracts. A search for a mutant which transported and phosphorylated lactose but was defective in cleaving the phosphorylated derivative was undertaken. Of more than 60 lactose negative mutants examined, no such mutant was found. All mutants examined lacked any detectable β -P-gal and also the ability to transport and phosphorylate lactose. In metabolism studies involving galactose-6-PO₄, it was found that whole cells of lactic streptococci could not utilize the compound as a carbon source. Possible routes for the metabolism of this compound were hypothesized, and experiments were designed to test each route. The only pathway which appeared to function in its catabolism was the pentose pathway. However, it was later found that the galactose- 6-PO₄ used in these studies was contaminated with at least 0. 2 percent glucose-6-PO₄ and observed activity was believed due to the contaminant. Involvement of the PTS of transport in sugar utilization was investigated using ¹⁴C- labeled substrates and toluene-acetone-treated cells. Substrates and phosphorylated products were separated by ion-exchange chromatography and quantitated by liquid scintillation counting. The PTS was involved in the utilization of glucose, lactose, and mannose. In controlled experiments it was determined that ATP also acted as a phosphate donor for glucose, lactose and mannose. ATP was the exclusive phosphate donor for galactose, Two transport systems appeared to act for glucose, lactose and mannose. In a survey conducted on representatives of the four major groups of streptococci, it was shown that all lactic streptococci examined cleaved ONPG-6-P except S. lactis 7962, which cleaved 0-nitrophenyl β D-galactopyranoside (ONPG). This survey also showed that all of the viridans streptococci examined possessed β -gal and not β-Pgal activity.
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