Characterization of mutant histidine-containing proteins of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and Salmonella typhimurium

Abstract

Histidine-containing phosphocarrier protein (HPr) is common to all of the phosphoenolpyruvate:sugar phosphotransferase systems (PTS) in Escherichia coli and SalmoneUa typhimurium, except the fructose-specific PTS. Strains which lack HPr activity (ptsH) have been characterized in the past, and it has proved difficult to delineate between tight and leaky mutants. In this study four different parameters of ptsH strains were measured: in vitro sugar phosphorylation activity of the mutant HPr; detection of 32P-labeled P-HPr; ability of monoclonal antibodies to bind mutant HPr; and sensitivity of ptsH strains to fosfomycin. Tight ptsH strains could be defined; they were fosfomycin resistant and produced no HPr protein or completely inactive mutant HPr. All leaky ptsH strains were fosfomycin sensitive, usually produced normal amounts of mutant HPr protein, and had low but measurable activity, and HPr was detectable as a phosphoprotein. This indicates that the regulatory functions of the PTS require a very low level of HPr activity (about 1%). The antibodies used to detect mutant HPr in crude extracts were two monoclonal immunoglobulin G antibodies Jel42 and Jel44. Both antibodies, which have different pls, inhibited PTS sugar phosphorylation assays, but the antibody-HPr complex could still be phosphorylated by enzyme I. Preliminary evidence suggests that the antibodies bind to two different epitopes which are in part located in a B-sheet structure.

Identification of a site in the phosphocarrier protein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specific mutants

“…1), were constructed and inserted into the low-expression vector, plasmid pT7-4 (43). An HPr-negative E. coli strain, LBG1650 (2,45), was then transformed with these plasmids, and the fermentation responses were recorded (Table 1). Replacement of Asp-69 with Tyr (D69Y) resulted in a marked decrease in growth rate and fermentation response on plates containing glucose, mannitol, N-acetylglucosamine, or any one of a number of other sugar substrates of the PTS.…”

“…Two examples of mutant HPrs with hydrolysis rates that are 50% of the wild-type E. coli HPr rate have been reported. The first example is a mutant E. coli HPr (Term-85) that lacks E-85 and possesses L-84 at the C terminus, while the second example is the Enterococcus faecalis HPr, which has a C-terminal glutamine (11)(12)(13)(44)(45)(46)(47). In the case of Term-85, determination of its structure by NMR revealed an adjustment such that E-83 contributes its carboxyl group to the active site (1,11), presumably in a less effective arrangement.…”

“…An HPr-negative strain, LBG1650 (2,45) . The Biorad Muta-Gene M13 in vitro mutagenesis kit was used.…”

“…Cells of mutant LBG1650 (ptsH) (2,45) bearing the wildtype or mutant forms of the E. coli ptsH gene in plasmid pT7-4 (43) were grown with shaking at 30ЊC in LB medium (18) containing ampicillin (50 g/ml) and the indicated sugar (1%). Cells were harvested by centrifugation, washed twice with medium 63, and resuspended in the same medium to an absorbance of 20 Klett units (filter 66) (0.08 mg [dry weight] of cells per ml).…”

Substitution of Aspartate and Glutamate for Active Center Histidines in the Escherichia coliPhosphoenolpyruvate:Sugar Phosphotransferase System Maintain Phosphotransfer Potential

“…The proteins and enzymes were expressed in strains that contained suitable mutations. HPr mutants were overproduced in a ptsH strain in which a terminator codon occurs at residue 71 4 and in which no HPr activity or protein can be detected (38). HPr preparations could not be phosphorylated without the addition of enzyme I.…”