From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate͞glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (Km for growth Ϸ8 mM but with Vmax for generation time Ϸ1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme II Glc of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG ؉ from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (Vmax Ϸ7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposonmediated inactivations both of ptsG and of the genes specifying enzyme II Man (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl ␣-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.E scherichia coli grow readily on fructose as the sole source of carbon. When this sugar is present in the growth medium at concentrations Յ2 mM, it is taken up and phosphorylated predominantly to fructose 1-phosphate (1) via two membraneassociated proteins specified by fruA (2) and fruB (3); at concentrations Ͼ2 mM, it also is taken up and phosphorylated (but to fructose 6-phosphate) via the membrane-associated uptake system for mannose, specified by manXYZ (4, 5). Strains of E. coli impaired in both these systems have been shown to mutate further and to take up and phosphorylate fructose to fructose 6-phosphate via derepression of glucitol transport proteins (6). In all of these instances, the simultaneous translocation and phosphorylation of fructose requires the action of the phosphoenolpyruvate͞glycose phosphotransferase system (PTS) (7-9).It has been shown that Salmonella mutants lacking one or more of the cytosolic proteins of the PTS, which consequently are unable to use any of the sugars normally taken up via the PTS, can give rise to further mutants that are able to grow on fructose as the sole carbon source (9). Such mutants also had elevated levels of fructo(manno)kinase (Mak) (10), which implies that fructose entered the cells in an unphosphorylated form and only thereafter received a phosphate group from ATP. But it was not known how fructose was translocated across the cell envelope: the available evidence was interpreted (8) as r...

Mutants of Escherichia coli unable to use fructose by means of the phosphoenolpyruvate͞glycose phosphotransferase system mutate further to permit growth on that ketose by derepression of a manno(fructo)kinase (Mak ؉ phenotype) present in only trace amounts in the parent organisms (Mak-o phenotype). The mak gene was located at min 8.8 on the E. coli linkage map as an ORF designated yajF, of hitherto unknown function; it specifies a deduced polypeptide of 344 aa. The derepression of Mak activity was associated with a single base change at position 71 (codon 24) of the gene, where GCC (alanine) in Mak-o has been changed to GAC (aspartate) in Mak ؉ . By cloning selected portions of the total 1,032-bp mak gene into a plasmid that also carried a temperaturesensitive promoter, we showed that the mutation resided in a 117-bp region that does not specify sequences necessary for Mak activity but was located 46 bp upstream of a 915-bp portion that does. Mak ؉ and Mak-o strains differ greatly in the heat stability of the enzyme: at 61°C, mak-o cloned into a mak-o recipient loses 50% of its activity in approximately 6 min, whereas it takes over 30 min to achieve a similar reduction in the activity of mak ؉ cloned into a mak-o strain. However, the Mak activity of the cloned fragment specifying the enzyme without the regulatory region lost activity with a half-life of 29 min irrespective of whether it was derived from a mak ؉ or a mak-o donor, which indicates that the A24D mutation contributes to the high enzyme activity of Mak ؉ mutants by serving to protect Mak from denaturation.

Although each of the membrane-spanning proteins of the PTS is sugar-specific and will effect the transport and concomitant phosphorylation of only a fairly limited range of hexoses, the cytoplasmic proteins that catalyze the transfer of the phosphate from PEP to the hexoses exhibit no such specificity. In each case, an enzyme (enzyme I) catalyzes the transfer of phosphate to a histidine residue in a carrier protein, from which that phosphate is subsequently transferred, directly or via additional carrier proteins, to the incoming hexose. The simultaneous provision oftwo hexoses to E. coli thus imposes a need upon the cells to choose whether to direct the flux of phosphate equally to the uptake of each hexose, as happens when fructose and glucitol are utilized simultaneously (4), or to channel that flux so that one substrate is taken up in preference to another, as happens when glucose is used in preference to other substrates of the PTS (5-8). The competition for uptake between hexoses transported respectively via IIGIc and TIME has been elegantly studied by Scholte and Postma (9), who concluded that uptake of the preferred sugar resulted in a partial dephosphorylation of one of the carrier proteins so that the less favored hexose received an inadequate supply of phosphate.In their experiments, insufficiency of PEP was ruled out as imposing a rate-limiting step.Not only glucose, which is transported via II0c, but also its noncatabolizable analog 2DG, which is taken up via IIUm, are utilized in preference to fructose; in consequence, 2DG powerfully inhibits the growth ofE. coli on fructose (6-8). By using 21) in concentrations below 0.5 mM, we have obtained evidence that, contrary to the earlier view (9), the extent to which each of two competing sugars is taken up by the cells can be determined by the availability of PEP. MATERIALS AND METHODSAll sugars were of the D configuration. Chemicals and enzymes were purchased from Boehringer Mannheim and from Sigma, 14C-labeled fructose and 2DG were from Amersham. The strains of E. coli used were wild type for the uptake ofmost sugars; strain HK 1711 also carried a mutation in the gene (pps) specifying PEP synthase. The introduction of the normal (pps+) allele into this strain by P1 phagemediated transduction, to yield strain HK 1719, was performed as described (10), transductants being selected for their ability to grow on lactate as sole carbon source. Cultures of bacteria were grown with shaking in liquid medium containing the appropriate carbon source at 10 mM, the amino acids required by these strains (histidine, arine, threonine, and leucine) at 40 ug/ml, and salts (11); 2DG was added to such cultures as specified in the text. Measurements of growth and of the uptake of 14C-labeled substrates have been described (8). For determination of the incorporation and retention of [14CJ2DG by E. coli growing on fructose, the labeled material was added (0.05 mM) to cells, grown on 10 mM fructose and 0.2 mM 21)G, suspended in 5 ml of 10 mM fructose growth medium at a...

Mutants of Escherichia coli devoid of the membrane-spanning proteins PtsG and PtsMP, which are components of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and which normally effect the transport into the cells of glucose and mannose, do not grow upon or take up either sugar. Pseudorevertants are described that take up, and grow upon, mannose at rates strongly dependent on the mannose concentration in the medium (apparent Km > 5 mM); such mutants do not grow upon glucose but are derepressed for the components of the fructose operon. Evidence is presented that mannose is now taken up via the fructose-PTS to form mannose 6-phosphate, which is further utilized for growth via fructose 6-phosphate and fructose 1,6-bisphosphate.