Identification of the operon for the sorbitol (Glucitol) Phosphoenolpyruvate:Sugar phosphotransferase system in Streptococcus mutans

Abstract

Transposon mutagenesis and marker rescue were used to isolate and identify an 8.5-kb contiguous region containing six open reading frames constituting the operon for the sorbitol P-enolpyruvate phosphotransferase transport system (PTS) of Streptococcus mutans LT11. The first gene, srlD, codes for sorbitol-6-phosphate dehydrogenase, followed downstream by srlR, coding for a transcriptional regulator; srlM, coding for a putative activator; and the srlA, srlE, and srlB genes, coding for the EIIC, EIIBC, and EIIA components of the sorbitol PTS, respectively. Among all sorbitol PTS operons characterized to date, the srlD gene is found after the genes coding for the EII components; thus, the location of the gene in S. mutans is unique. The SrlR protein is similar to several transcriptional regulators found in Bacillus spp. that contain PTS regulator domains (J. Stülke, M. Arnaud, G. Rapoport, and I. Martin-Verstraete, Mol. Microbiol. 28:865-874, 1998), and its gene overlaps the srlM gene by 1 bp. The arrangement of these two regulatory genes is unique, having not been reported for other bacteria.

FIG. 1

Schematic representation of the genetic characterization of the sorbitol transport operon in S. mutans LT11 obtained by sequencing. Inserts of plasmids used for sequencing, marker rescue, and/or complementation are indicated under the restriction map. A double-headed arrow represents the point of insertion of Tn4001 in S. mutans BH96SR. A putative hairpin structure between gpi and srlD is indicated. Restriction enzyme sites are BamHI (B), BglII (Bg), EcoRI (E), HindIII (H), MunI (M), NspV (N), and SstI (S). Gene symbols: orf1, unknown; gpi, glucose-6-phosphate isomerase; srlD, sorbitol-6-phosphate dehydrogenase; srlR and srlM, regulatory proteins; srlA to srlE, EIIBC; and srlB, EIIA.

FIG. 2

(A) Growth of the wild-type S. mutans LT11 on glucose (■), sorbitol (○), and glucose-sorbitol (▵). (B) Glucose (■) and sorbitol (○) utilization in the glucose-sorbitol culture.

FIG. 3

(A) Growth of the mutant S. mutans BH96SR on glucose (■), sorbitol (○), and glucose-sorbitol (▵). (B) Glucose (■) and sorbitol (○) utilization in the glucose-sorbitol culture.

FIG. 4

The structure of the sorbitol-PTS operons from S. mutans, C. beijerinckii, E. coli, and E. amylovora. The operons are shown arbritrarily aligned by their respective srlA and srlE genes.

FIG. 5

Alignment of the PRD-I and -II domains from the YdaA, LicR, and YjdC proteins from B. subtilis (Bs); the MtlR protein from B. stearothermophilus (Bt); and the SrlR proteins from S. mutans LT11 (Sm). Identical residues present in all domains at positions 14 and 61 are boxed, positions with similar residues in at least five domains are indicated by a period, and positions with identical residues in at least five domains are indicated by an asterisk. Positions 7, 14, 61, and 69 of the alignment (see text) are indicated at the top.