Aspects of sugar transport via the phosphoenolpyruvate, sugar phosphotransferase system of streptococcus mutans

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Thevenot, Tracy Lynn
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In the first phase of my research, I was interested in the activity and regulation of (Ser)HPr kinase in whole cells and cell preparations. Early work with membrane preparations of Streptococcus mutans and S. salivarius indicated that (Ser)HPr kinase activity increased 2-fold with 5 mM fructose-1,6-bisphophate (FBP) and 0.5 mM ATP, and the enzyme was slightly inhibited by 1.0 mM ATP. To complement this work, the level of the four forms of HPr were assessed in steady state cells of S. mutans Ingbritt grown in continuous culture with limiting (10 and 50 mM) and excess glucose (100 and 200 mM) at a constant pH (7.0) and growth rate (dilution rate = 0.1 h$\sp{-1}).$ Crossed immunoelectrophores is detected all four forms of HPr in the four chemostat-grown cultures. The relative amount of P-(Ser)-HPr (50%) was highest for 100 mM glucose-grown and the level of FBP (1,114 $\mu$M) and ATP (1,416 $\mu$M) was highest for 200-mM glucose excess cells. The results indicated that the intracellular level of FBP was not high enough to promote activation of (Ser)HPr kinase and the level of ATP could be considere inhibitory. The second phase of my research involved altering the amino acid sequence of HPr by converting glycine-67, an amino acid which may be important for the recognition of P-(His)-HPr by the EII complexes, to an aspartate residue by site-directed mutagenesis of the ptsH gene. The gene from S. mutans NG5 was successfully mutated using single-stranded site-directed mutagenesis. The plasmid carrying the mutated gene also carried the gene for EI (ptsI) and the 5$\sp\prime$ portion of the glyceraldehyde-3-phosphate dehydrogenase gene (gapN). A kanamycin-resistance gene was integrated between the ptsI and gapN genes to provide convenient selection of mutants. Potential HPr mutants fermented phosphotransferase system (PTS) sugars indicating unsuccessful integration of the mutated gene. The third phase of my research focused on the genetics of sorbitol transport in S. mutans. A plasmid carrying Tn4001 was transformed into S. mutans LT11 generating the sorbitol-defective strain, S. mutans BH96. Examination of carbohydrate utilization indicated that S. mutans LT11 utilized glucose first, verifying catabolite repression by glucose. A long lag period was observed prior to growth by S. mutans LT11 on sorbitol, confirming the induction of sorbitol-related genes; S. mutans BH96 only grew only in the presence of glucose. The integrative plasmid p$\Omega$IS was used to recover a transposon/genome junction from S. mutans BH96. A 948-bp chromosomal fragment carried on p$\Omega$-SB was shown to consist of two partial open reading frames (ORFs). (Abstract shortened by UMI.)