Characterization of Streptococcus mutans strains deficient in EIIAB Man of the sugar phosphotransferase system

Abstract

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB(Man) (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159. The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB(Man) is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S. mutans EIIAB(Man) transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB(Man) controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIAB(Man)-deficient strains. In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB(Man) is required for optimal expression of gtfBC. No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB(Man)-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB(Man) has a pleiotropic effect on gene expression.

FIG. 1.

The arrangement of man gene clusters and flanking regions in S. mutans UA159 and other streptococci. The location of the insertion of a nonpolar Km cassette within the manL gene to generate the strain JAM1 is indicated. Patterned arrows represent similar genes. The websites or references from which the man gene clusters and flanking regions were obtained are indicated.

FIG. 2.

Sugar-specific PTS activity of S. mutans UA159 and JAM1 grown in glucose (A) and mannose (B). The glucose-, fructose-, and mannose-specific PTS activities are indicated as GLC, FRU, and MAN, respectively. The values are the means ± standard deviations (SD) from at least three individual experiments.

FIG. 3.

Sugar-specific PTS activity of S. mutans UA159 and EII^Fru− strains (TW30, TW31, and TW32) grown in glucose (A) and mannose (B). The glucose-, fructose-, and mannose-specific PTS activities are indicated as GLC, FRU, and MAN, respectively. The values are the means ± SD from at least three individual experiments.

FIG. 4.

Sugar-specific PTS activity of S. mutans UA159 and EIIAB^Man−/EII^Fru− strains (JAM11, JAM12, and JAM13) grown in glucose (A) and mannose (B). The glucose-, fructose-, and mannose-specific PTS activities are indicated as GLC, FRU, and MAN, respectively. The values are the means ± SD from at least three individual experiments.

FIG. 5.

Growth of UA159 and JAM1 in TV medium supplemented with 0.05% glucose and 0.5% inulin. The empty squares represent the wild-type strain UA159, whereas the filled triangles represent JAM1, the EIIAB^Man− strain. The results shown are representative of three independent experiments.

FIG. 6.

Silver stained 2-D gels of total cell lysates of UA159 (A) and JAM1 (B) grown in BHI. (A) EIIAB^Man (arrow) is indicated in a 2-D gel of proteins from the wild-type strain. (B) Thin arrowheads represent up-regulated proteins, whereas down-regulated proteins are represented by thin arrows. Tropomyosin, the internal control, is indicated by a wide arrowhead in each of the panels.

FIG. 7.

CAT-specific activity driven by gtfBC and ftf promoters in the wild-type and manL⁻ backgrounds under carbohydrate excess (200 mM glucose) and carbohydrate-limiting conditions (20 mM glucose) at pH 7, 6, and 5. Values shown are means ± SD from three independent chemostat runs. The results are expressed as nanomoles of Cm acetylated per minute per milligram of protein. (A) CAT activity driven by gtfBC promoter; (B) CAT activity driven by ftf promoter.