Phenotypic consequences resulting from a methionine-to-valine substitution at position 48 in the HPr protein of Streptococcus salivarius

Abstract

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His(15)) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser(46)) by an ATP-dependent protein kinase (His approximately P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His(15) by EI nor the phosphorylation at Ser(46) by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His approximately P) than the wild-type strain, while the levels of free HPr and HPr(His approximately P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of alpha-galactosidase, beta-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.

FIG. 1

Growth of mutants Ga3.78 and Ga3.14 in medium containing glucose and a non-PTS sugar. Cells were grown overnight in the presence of 0.1% glucose. One-milliliter aliquots of glucose-grown cells were used to inoculate 15-ml tubes containing a mixture of glucose and non-PTS sugars (lactose, galactose, and melibiose). The symbols represent the OD₆₆₀ (○) and the consumption of glucose (■), lactose (●), galactose (⧫), and melibiose (▾).

FIG. 2

Effect of glucose on lactose metabolism by growing cells. Cells were grown overnight in the presence of 0.2% lactose. A 0.75-ml aliquot was used to inoculate 15 ml of culture medium containing 0.2% lactose. When the culture reached mid-log phase, the medium was supplemented with 0.1% glucose. The symbols represent the OD₆₆₀ (○), the consumption of lactose (●), and the consumption of glucose (■).

FIG. 3

Sugar metabolism by resting cells. Cells were grown in the presence of 0.2% lactose, were harvested during the exponential phase of growth, were washed once, and were suspended in phosphate buffer at pH 7.0. Glucose and lactose (final concentrations, approximately 0.4%) were added at 0 min. The symbols represent the consumption of lactose (●) and the consumption of glucose (■).

FIG. 4

Sugar metabolism by energized resting cells. Cells were grown in the presence of 0.2% lactose, were harvested during the exponential phase of growth, were washed once, and were suspended in phosphate buffer at pH 7.0. At 0 min, approximately 0.4% lactose (panels A and C) or 0.4% glucose (panels B and D) was added. When the concentration of sugar in the medium was decreased approximately by half, glucose (panels A and C) or lactose (panels B and D) were added at a final concentration of 0.2%. The symbols represent the consumption of lactose (○) and the consumption of glucose (□).

FIG. 5

Effect of alkaline phosphatase on the modified form of Ga3.78 HPr. The modified form of HPr was purified to homogeneity from strain Ga3.78 and was incubated in the presence or absence of alkaline phosphatase for 17 h at 37°C. The proteins were separated by PAGE under native conditions that allow the separation of free HPr from phospho-HPr and the separation of HPr-1 (without N-terminal Met) from HPr-2 (with N-terminal Met). The proteins were revealed by staining with silver nitrate. Lane 1, 8 U of alkaline phosphatase; lane 2, 1.5 μg of free Ga3.78 HPr and 8 U of alkaline phosphatase; lane 3, 1.5 μg of free Ga3.78 HPr; lane 4, 1.5 μg of purified modified HPr from mutant Ga3.78; lane 5, 2 μg of purified modified HPr from mutant Ga3.78; lane 6, 1.5 μg of purified modified HPr from mutant Ga3.78 incubated with 2 U of alkaline phosphatase; lane 7, 1.5 μg of purified modified HPr from mutant Ga3.78 incubated with 8 U of alkaline phosphatase.

FIG. 6

In vitro phosphorylation of HPr by the ATP-dependent HPr(Ser) kinase. Mutant Ga3.78 was cultured in the presence of 0.5% glucose and was harvested at mid-log phase. A membrane-free cellular extract was obtained after the cells were ruptured by grinding with alumina and differential centrifugation. Purified HPr (2 μg) from the wild-type strain (lane 1) and cellular extract (8.25 μg of proteins) from mutant Ga3.78 (lane 2) were incubated in the presence of [γ-³²P]ATP and purified recombinant S. salivarius HPr(Ser) kinase (100 ng). Proteins were then separated by SDS-PAGE, and phosphoproteins were revealed by autoradiography.

FIG. 7

Western blot analyses with anti-IIAB_H^Man. Cellular extracts containing 25 μg of protein were electrophoresed in 10% acrylamide gels by the method of Laemmli (22) and were electrophoretically transferred to a nitrocellulose membrane. Lane 1, wild-type strain; lane 2, mutant Ga3.14; lane 3, mutant Ga3.78.