Utilization of lactose and galactose by Streptococcus mutans: transport, toxicity, and carbon catabolite repression

Abstract

Abundant in milk and other dairy products, lactose is considered to have an important role in oral microbial ecology and can contribute to caries development in both adults and young children. To better understand the metabolism of lactose and galactose by Streptococcus mutans, the major etiological agent of human tooth decay, a genetic analysis of the tagatose-6-phosphate (lac) and Leloir (gal) pathways was performed in strain UA159. Deletion of each gene in the lac operon caused various alterations in expression of a P(lacA)-cat promoter fusion and defects in growth on either lactose (lacA, lacB, lacF, lacE, and lacG), galactose (lacA, lacB, lacD, and lacG) or both sugars (lacA, lacB, and lacG). Failure to grow in the presence of galactose or lactose by certain lac mutants appeared to arise from the accumulation of intermediates of galactose metabolism, particularly galatose-6-phosphate. The glucose- and lactose-PTS permeases, EII(Man) and EII(Lac), respectively, were shown to be the only effective transporters of galactose in S. mutans. Furthermore, disruption of manL, encoding EIIAB(Man), led to increased resistance to glucose-mediated CCR when lactose was used to induce the lac operon, but resulted in reduced lac gene expression in cells growing on galactose. Collectively, the results reveal a remarkably high degree of complexity in the regulation of lactose/galactose catabolism.

FIG. 1.

Genetic organization of the lac operon in S. mutans UA159 (A) and the pathways for metabolism of galactose and lactose (B). (A) The encoding sequences of all eight genes are depicted as filled arrows: lacR, the DeoR-like negative transcriptional regulator; lacA and lacB, the A and B subunits of the galactose-6-P isomerase; lacC, the tagatose-6-P kinase; lacD, tagatose-1,6-bP adolase; lacF and lacE, the A and BC components of the lactose-PTS enzyme II; and lacG, the phospho-β-galactosidase. Below the genes are the antibiotic-resistance-encoding elements used in the allelic exchange mutagenesis of each open reading frame, with em, km and sp. representing the erythromycin, kanamycin, and spectinomycin resistance cassette, respectively. Locations of three point mutations are indicated by vertical arrows. (B) Schematics of the Leloir (left) and tagatose-6-phosphate (right) pathways. Gal-6-P, galactose-6-phosphate; Gal-1-P, galactose-1-phosphate; UDP-Gal, UDP-galactose; UDP-Glc, UDP-glucose; Glc-1-P, glucose-1-phosphate.

FIG. 2.

Growth curves generated using a Bioscreen C while monitoring the growth of strains UA159, JAM1 (manL), JAM2 (galK), lacF/manL double mutant, and the deletion mutant of ptsI (strain EI). Cells were incubated in TV medium supplemented with 0.5% (A) or 2% (B) of galactose.

FIG. 3.

Growth curves of the lacA:em mutant generated using TV medium containing 0.5% of glucose (Glc), or the combination of 0.5% glucose and 0.5% galactose (Gal), 2% galactose, or 0.5% lactose (Lac).

FIG. 4.

Growth curves of UA159, lacG and various lacG derived mutants on (A) 0.5% galactose or (B) the combination of 0.5% sorbitol and 0.5% of galactose.