|COOK GREGORY M - CORNELL UNIVERSITY
|KEARNS DANIEL B - CORNELL UNIVERSITY
|ZEIZER JONATHAN - UNIV OF CA, SAN DIEGO
|SAIER MILTON H - UNIV OF CA, SAN DIEGO
Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/30/1995
Publication Date: N/A
Interpretive Summary: Streptococcus bovis is an opportunistic bacterium that outgrows other ruminal and colonic bacteria when there is an abundance of carbohydrate. This overgrowth causes acute indigestion, ruminal acidosis, and even the death of cattle. In humans, S. bovis has been implicated in the etiology of colon cancer. We undertook research to study the regulation of carbohydrate uptake by S. bovis and showed that it had two distinctly different regulatory mechanisms for regulating uptake of the twelve-carbon sugar, lactose. These two mechanisms for regulating sugar uptake have been termed "inducer exclusion" and "inducer expulsion". Our experiments showed that inducer exclusion was mediated at the level of the cell membrane and did not entail the metabolism of glucose, a six carbon sugar. Conversely, inducer expulsion was mediated by the metabolism of glucose and involved an intermediate compound (fructose 1,6 bis phosphate), an enzyme (protein kinase) and the phosphorylation of a protein previously designated as HPr. Information on the regulation of carbohydrate utilization may provide ways of inhibiting S. bovis, thus alleviating ruminal acidosis, improving the economics of animal production and decreasing colon cancer.
Technical Abstract: Streptococcus bovis had a diauxic pattern of glucose & lactose utilization & both were transported by the sugar phosphotransferase system (PTS). Lactose catabolism by the tagatose pathway was inducible. A glucose-PTS- deficient mutant transported lactose as fast as the wild-type suggesting separate enzyme II's for glucose & lactose. The non-metabolizable glucose analog, 2-deoxyglucose (2-DG) inhibited methyl-B-D-thiogalactopyranoside (TMG) transport, non-competitively & cells provided either glucose or 2-DG were unable to transport TMG or lactose. A glucose PTS-deficient mutant fermented glucose but did not exclude TMG, suggesting that enzyme IIglc, rather than glucose catabolism was critical to inducer exclusion. Cells that had accumulated TMG-6-phosphate, expelled free TMG when glucose was added but not when 2-DG was added. The glucose PTS-deficient mutant could still expel TMG in the presence of exogenous glucose. Membrane vesicles also exhibited glucose-dependent TMG exclusion & expulsion. Vesicles electroporated with PEP & HPr retained TMG more than 3 min, but vesicles electroporated with PEP plus HPr & FDP (or glycerate-2-phosphate) did not retain TMG. FDP triggered ATP-dependent phosphorylation of HPr suggesting that inducer expulsion was mediated by an FDP-activated protein kinase. This conclusion was supported by the observation that mutant forms of HPr differed in their ability to facilitate inducer expulsion. S46DHPr, a mutant HPr with aspartate substituted for serine at position 46, promoted TMG expulsion from membrane vesicles in the absence of FDP better than wild-type HPr or S46AHPr, a mutant with alanine substituted for serine at position 46. These results suggest that glucose catabolism was needed for inducer expulsion, but not inducer exclusion in S. bovis.