Fig 1.
Phosphoryl transfer pathway for the bacterial phosphotransferase system (PTS).
The figure shows the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to enzyme I (EI), then to HPr, then to the sugar-specific enzyme IIAs and then to the enzyme IIBs before transfer to the incoming sugar via the enzyme IIC, which catalyzes both transport and phosphoryl transfer in a coupled process. The PTS also catalyzes the group translocation of many other sugars. As indicated, the three extracellular sugars represented are mannitol (Mtl), fructose (Fru), and N-acetylglucosamine (NAG), and their phosphorylated derivatives are released into the cytoplasm. Arrows indicate the pathways of phosphoryl transfer.
Table 1.
Proteins of the PTS in E. coli, relevant to the study reported here.
References for these systems can be found in TCDB.
Table 2.
Selected high scoring PTS transporter (Enzyme II complex) interactions.
The data were derived from Babu et al., 2018 [1]. The higher the score, the stronger the interaction should be, although these scores are also dependent on the protein concentrations.
Fig 2.
Ratios of apparent transport activities for various sugars (PTS substrates and galactose) by wild type E. coli BW25113 (WT) and its fruBKA isogenic mutants (BW25113ΔfruA, BW25113ΔfruB and BW25113-fruBKA:kn (triple mutant, TM)) using cells grown in LB with (WT or mutant + Fru) and without 0.2% fructose.
(A) Relative apparent sugar uptake by wild type E. coli BW25113 (WT) grown in the presence and absence of fructose. (B) Relative apparent sugar uptake by the fruBKA triple E. coli BW25113 mutant (TM) grown in the presence and absence of fructose. (C&D) Relative apparent sugar uptake by the triple mutant (TM) relative to that of the wild type E. coli BW25113 strain when grown in LB or LB plus 0.2% fructose, respectively, as indicated. (E&F) Relative apparent sugar uptake by a fruA mutant relative to that of the wild type E. coli BW25113 strain when grown in LB and LB plus 0.2% fructose, respectively. (G&H) Relative apparent sugar uptake by a fruB mutant relative to that of the wild type E. coli BW25113 strain when grown in LB or LB plus 0.2% fructose, respectively. 1, Fructose; 2, Mannitol; 3, N-acetylglucosamine; 4, Methyl alpha glucoside; 5, 2-Deoxyglucose; 6, Trehalose; 7, Galactitol and 8, Galactose. The raw data for these plots are shown in supplementary materials (S1, S2, S3, S3B, S4, S4B, S5 and S5B Tables). Note: In this Figure and elsewhere, αMG and 2DG uptake values represent accumulation levels, while for other sugars, a combination of uptake + metabolic rates were estimated. Galactose is taken up via the GalP secondary carrier, not by the PTS.
Fig 3.
Ratios of apparent transport activities for various sugars (PTS substrates and galactose) by wild type E. coli BW25113 (WT) and its fruBKA triple mutant (BW25113-fruBKA:kn (TM) over-expressing individual or combined fruBKA operon genes.
Ratios of apparent transport rates for [14C]sugar uptake in wild type (WT) and ΔfruBKA mutant backgrounds over-expressing individual or combined fruBKA operon genes as presented at the tops of the figures: A. fruA in the TM strain. B. fruA in the WT parental strain. C. fruB in the TM strain. D. fruB in the WT parental strain. E. fruA and fruB in the TM strain. F. fruA and fruB in the WT parental strain. 1, Fructose; 2, Mannitol; 3, N-acetylglucosamine; 4, Methyl alpha glucoside; 5, 2-Deoxyglucose; 6, Trehalose; 7, Galactitol and 8, Galactose. The raw data for these plots are provided in supplementary materials (S6–S12 Tables). ND, not determined.
Table 3.
Ratios of the responses of lacZ fusion-bearing strains to various conditions and genetic backgrounds (S13–S15 Tables).
WT = wild type, BW25113; OE = overexpression; ΔfruBKA = deletion of the entire fruBKA operon, also called triple mutant, TM. All strains were grown in LB medium to which 0.2% fructose was added, only for the first two entries as indicated. The remaining entries reveal the consequences of the overexpression of specific fru genes or gene combinations on lacZ-fusion gene expression. In no case were the changes significant, suggesting that the presence of fructose in the growth medium or the overexpression (OE) of specific fru genes, or the entire fru operon, did not influence expression of the mtl, gat, or man operons.
Table 4.
Effects of overexpressing fruA and varying amounts of purified FruB on the transphosphorylation activities of tested enzyme II complexes.
Results are expressed as the ratios of enzyme activities in the presence relative to the absence of purified FruB for the different preparations. The raw data are presented in S25 and S26 Tables. In all cases, neither FruA nor FruB influenced the transphosphorylation activities for the 5 sugar-specific Enzyme II complexes assayed.
Fig 4.
PEP-dependent phosphorylation of various radioactive PTS sugars by crude extracts of wild type and ΔfruBKA strains of E. coli following either induction with fructose or overexpression of fruBKA or fruA and fruB.
Ratios of the PEP-dependent phosphorylation rates for various radioactive PTS sugars by crude extracts of wild type and ΔfruBKA strains of E. coli. A. wild type cells induced by growth in LB + fructose (0.2%) compared to LB grown cells. B. Effect of the overexpression of the entire fruBKA operon on in vitro PEP-dependent sugar phosphorylation rates when cells were grown in LB medium. C. The consequences of the simultaneous overexpression of fruA and fruB in the WT background. D. The same as C except that the ΔfruBKA strain was used. 1, Fructose; 2, Mannitol; 3, N-acetylglucosamine; 4, Methyl alpha glucoside; 5, 2-Deoxyglucose; 6, Trehalose and 7, Galactitol. The raw data for these plots are presented in S16–S21 Tables. Other conditions and combinations of gene overexpression did not result in appreciable changes in activities (see S16–S21 Tables).
Table 5.
Effect of purified FruB or HPr on the PEP-dependent phosphorylation of [14C]mannitol using membrane pellets (MP) of E. coli strain BW25113-pMAL-fruA and BW25113-fruBKA:kn-pMAL-fruA or crude extracts (Cr.Ext.) of strain BW25113-fruBKA:kn-pMAL-fruA as compared to their corresponding control strains BW25113-pMAL and BW25113-fruBKA:kn-pMAL.
Fig 5.
Effect of purified FruB on PEP-dependent phosphorylation activities of crude extracts of the recombinant triple mutant E. coli strain BW25113-fruBKA:kn-pMAL-fruA (TM-pMAL-fruA, closed symbols) as compared to the BW25113-fruBKA:kn-pMAL (TM-pMAL, open symbols), assaying phosphorylation of [14C]PTS sugars, mannitol (Mtl, squares) and N-acetylglucosamine (NAG, triangles).
The raw data are presented in S23 Table.
Fig 6.
Effects of using a HSS from lysed E. coli BW25113 chs kn:T:Ptet-fruBKA cells, overexpressing the fruBKA operon, on the PEP-dependent phosphorylation activities of enzymes II in membrane pellets (MP) of E. coli BW25113 overexpressing fruA (A) or galP (B) for PTS sugars: 1, Fructose; 2, Mannitol; 3, N-acetylglucosamine and 4, Galactitol. An aliquot of an 8 h HSS from E. coli BW25113 chs kn:T:Ptet-fruBKA (WT OE fruBKA operon) was used as a source of soluble PTS enzymes for the measurement of PEP-dependent phosphorylation activities. The raw data are presented in S24 Table.