POSITIVE SELECTION FOR RESISTANCE TO 2-DEOXYGLUCOSE GIVES RISE, IN STREPTOCOCCUS-SALIVARIUS, TO 7 CLASSES OF PLEIOTROPIC MUTANTS, INCLUDING PTSH AND PTSI MISSENSE MUTANTS

We have used the toxic non-metabolizable glucose/ mannose analogue 2-deoxyglucose to isolate a comprehensive collection of mutants of the phosphoenolpyruvate:sugar phosphotransferase system from Streptococcus salivarius. To increase the range of possible mutations, we isolated spontaneous mutants on different media containing P-deoxyglucose and various metabolizable sugars, either lactose, melibiose, galactose or fructose. We found that the frequency at which 2-deoxyglucose-resistant mutants were isolated varied according to the growth substrate. The highest frequency was obtained with the combination galactose and 5-deoxyglucose and was 15-fold higher than the rate observed with the mixture melibiose and P-deoxyglucose, the combination that gave the lowest frequency. By combining results from: (i) Western blot analysis of IIIMan, a specific component of the phosphaenolpyruvate:mannose phosphotransferase system in S. salivarius; (ii) rocket immunoelectrophoresis of HPr and El, the two general energy-coupling proteins of the phosphotransferase system; and (iii) from gene sequencing, mutants could be assigned to seven classes. Class 1 was composed of strains devoid of IIIMan, a low-molecular-weight form of IIIMan (35 200), class 2 was composed of strains exhibiting a reduced level of IIILMan, class 3 was composed of strains devoid of both forms of IIIMan (IIILMan as well as IIIHMan, the high-molecular-weight form of IIIMan (38900)), class 4 was composed of mutants bearing a mutation in ptsH, the gene encoding HPr, class 5 was composed of mutants bearing a mutation in ptsi, the gene encoding El, class 6 was composed of 2-deoxyglucose-resistant strains without any apparent defect in PTS components, and class 7 was composed of strains possessing both forms of IIIMan but abnormal levels of HPr and/or El without any mutation in the ptsH and/or the ptsI genes. Preliminary characterization of representative strains of each class is reported.