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. 2019 Nov 21;14(11):e0219332.
doi: 10.1371/journal.pone.0219332. eCollection 2019.

Protein:Protein Interactions in the Cytoplasmic Membrane Apparently Influencing Sugar Transport and Phosphorylation Activities of the E. Coli Phosphotransferase System

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Free PMC article

Protein:Protein Interactions in the Cytoplasmic Membrane Apparently Influencing Sugar Transport and Phosphorylation Activities of the E. Coli Phosphotransferase System

Mohammad Aboulwafa et al. PLoS One. .
Free PMC article

Abstract

The multicomponent phosphoenolpyruvate (PEP)-dependent sugar-transporting phosphotransferase system (PTS) in Escherichia coli takes up sugar substrates from the medium and concomitantly phosphorylates them, releasing sugar phosphates into the cytoplasm. We have recently provided evidence that many of the integral membrane PTS permeases interact with the fructose PTS (FruA/FruB) [1]. However, the biochemical and physiological significance of this finding was not known. We have carried out molecular genetic/biochemical/physiological studies that show that interactions of the fructose PTS often enhance, but sometimes inhibit the activities of other PTS transporters many fold, depending on the target PTS system under study. Thus, the glucose (Glc), mannose (Man), mannitol (Mtl) and N-acetylglucosamine (NAG) permeases exhibit enhanced in vivo sugar transport and sometimes in vitro PEP-dependent sugar phosphorylation activities while the galactitol (Gat) and trehalose (Tre) systems show inhibited activities. This is observed when the fructose system is induced to high levels and prevented when the fruA/fruB genes are deleted. Overexpression of the fruA and/or fruB genes in the absence of fructose induction during growth also enhances the rates of uptake of other hexoses. The β-galactosidase activities of man, mtl, and gat-lacZ transcriptional fusions and the sugar-specific transphosphorylation activities of these enzyme transporters were not affected either by frustose induction or by fruAB overexpression, showing that the rates of synthesis of the target PTS permeases were not altered. We thus suggest that specific protein-protein interactions within the cytoplasmic membrane regulate transport in vivo (and sometimes the PEP-dependent phosphorylation activities in vitro) of PTS permeases in a physiologically meaningful way that may help to provide a hierarchy of preferred PTS sugars. These observations appear to be applicable in principle to other types of transport systems as well.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
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.
Fig 2
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
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.
Fig 4
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).
Fig 5
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
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.

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