Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation

Abstract

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, enzyme I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose pernease. When strains were constructed which lacked the galactose permnease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are u'nable to carry out facilitated diffusion.The bacterial phosphoenolpyruvate (PEP): sugar phosphotranferase system (PTS) catalyzes the transport and concomitant phosphorylation of a number of sugars according to the following scheme (for a review, see reference 18): Eyme I HPr + PEP .' HPr-P + pyruvate HPr-P + sugarOt Enzyme m sugar phosphatei, + HPrThe phosphoryl group is transferred sequentially from PEP to enzyme I to HPr. The actual translocation step is catalyzed by a family of membrane-bound enzymes II, each of which is specific for one or a few sugars.Several lines of evidence, including studies with mutants lacking enzyme I or HPr or both, have suggested that phosphorylation and transport are tightly coupled processes. It is not clear, however, whether the enzymes II can catalyze the transport of their sugar substrates in the absence of phosphorylation. We will call this hypothetical process, which would result in the appearance of free sugar inside the cell instead t Present address:

Systematic genetic dissection of PTS in Vibrio cholerae uncovers a novel glucose transporter and a limited role for PTS during infection of a mammalian host

“…3C). These results suggest that phosphorylation of glucose may be uncoupled from transport through VC1821, which is contrary to the paradigm of PTS-dependent transport (Postma and Stock, 1980). Phosphorylation of glucose, however, would still be required to prime this carbohydrate for catabolism in the cell.…”

pH‐regulated gene expression in Salmonella: genetic analysis of aniG and cloning of the earA regulator

“…will grow on mannose as a carbon source while SF356, which lacks the permease, cannot. These strains were originally described by Postma and Stock (1980), who demonstrated a complete lack of detectable mannose transport in SF356. We transduced an/G::Mucyj into both of these strains in order to assess whether better induction would occur in the mannose transport-proficient strain.…”

Limits to inducer exclusion: inhibition of the bacterial phosphotransferase system by glycerol kinase

“…Secondly, MeGlc efflux follows first-order kinetics with respect to the intracellular MeGlc concentration (Hagihira et al, 1963). Consequently, efflux rates increase with increasing accumulation of intracellular MeGlc, and MeGlc uptake curves routinely bend off (for example, see Postma and Stock, 1980). The fact that inhibition of MeGlc uptake is already observed after 3 s in the linear part of the uptake curve ( Fig.…”

“…The radioactivity on the filters was determined by liquid scintillation counting as described previously (Postma, 1977). Because the MeGlc uptake curves levelled off quickly (see also Postma and Stock, 1980), uptake rates were calculated by linear regression of the data points in the initial linear range (typically four or five data points, including zero, taken within 12 s after MeGlc addition). In some cases (see text), the linear range was limited to 6 s, and only three data points (including zero) could be used for calculating the initial uptake rate.…”