Positive selection for resistance to 2‐deoxyglucose gives rise, in Streptococcus salivarius, to seven classes of pleiotropic mutants, including ptsH and ptsl missense mutants

Abstract

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 2-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 2-deoxyglucose and was 15-fold higher than the rate observed with the mixture melibiose and 2-deoxyglucose, the combination that gave the lowest frequency. By combining results from: (i) Western blot analysis of IIIMan, a specific component of the phosphoenolpyruvate:mannose phosphotransferase system in S. salivarius; (ii) rocket immunoelectrophoresis of HPr and EI, the two general energy-coupling proteins of the phosphotransferase system; and (iii) from gene sequencing, mutants could be assigned to seven classes.(ABSTRACT TRUNCATED AT 250 WORDS)

Diversity of Streptococcus salivarius ptsH Mutants That Can Be Isolated in the Presence of 2-Deoxyglucose and Galactose and Characterization of Two Mutants Synthesizing Reduced Levels of HPr, a Phosphocarrier of the Phosphoenolpyruvate:Sugar Phosphotransferase System

“…In a previous study, we reported that several types of PTS-negative mutants could be isolated by plating S. salivarius on a medium containing 2DG and a metabolizable sugar (11). We observed that the frequency at which mutants could be isolated was highest with the combination 2DG and galactose and that selection on galactose seemed to favor the isolation of ptsH mutants.…”

“…The nucleotide sequences of the ptsH and ptsI genes were determined as described by Gauthier et al (11). The sequence of the region upstream from the Ϫ35 box of the pts promoter was obtained after amplification of a 398-bp DNA fragment by PCR using two oligonucleotides (5Ј-TATCTTTACAGCTGACTTAG-3Ј and 5Ј-GCTGGACGTGCGTGGAT-3Ј) that annealed with a region of the idh gene located 252 bp upstream from the Ϫ35 region of the pts promoter and with a region in the ptsH gene.…”

“…In a previous study conducted with Streptococcus salivarius ATCC 25975, we observed that several types of PTS mutants, including ptsH mutants, could be obtained by positive selection on 2-deoxyglucose (2DG) in the presence of various metabolizable sugars (11). Preliminary data suggested that selection in the presence of galactose favored the isolation of ptsH mutants.…”

Replacement of isoleucine‐47 by threonine in the HPr protein of Streptococcus salivarius abrogates the preferential metabolism of glucose and fructose over lactose and melibiose but does not prevent the phosphorylation of HPr on serine‐46

“…Strain G22.4 is an isogenic spontaneous mutant isolated from S. salivarius that has a missense mutation in the ptsH gene, resulting in the substitution of the isoleucine at position 47 (Ile-47) in HPr by a threonine (Thr) (Gauthier et al, 1994). Cells were grown at 37ЊC in a medium containing 10 g of tryptone and 5 g of yeast extract (Difco Laboratories), 2.5 g of NaCl and 2.5 g of disodium phosphate per litre.…”

“…Mutant G22.4 is a spontaneous 2-deoxyglucose (2-DG)-resistant mutant isolated from S. salivarius 25975 that has a missense mutation in the HPr protein resulting in the replacement of the isoleucine at position 47 (Ile-47) by a threonine (I47 →T) (Gauthier et al, 1994). Ile-47 is adjacent to the serine residue (Ser-46) that is phosphorylated by the ATP-dependent HPr kinase in Gram-positive bacteria and is also believed to be involved in the interaction of HPr with EI of the PTS (Herzberg et al, 1992).…”