Galactose and lactose genes from the galactose-positive bacterium Streptococcus salivarius and the phylogenetically related galactose-negative bacterium Streptococcus thermophilus: organization, sequence, transcription, and activity of the gal gene products

Abstract

Streptococcus salivarius is a lactose- and galactose-positive bacterium that is phylogenetically closely related to Streptococcus thermophilus, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the S. salivarius and S. thermophilus gal-lac gene clusters. The clusters have the same organization with the order galR (codes for a transcriptional regulator and is transcribed in the opposite direction), galK (galactokinase), galT (galactose-1-P uridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactose transporter), and lacZ (beta-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from galR, the gal operon, galM, and the lac operon of S. salivarius. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the S. thermophilus gal-lac gene cluster. An additional putative internal promoter at the 3' end of galT was found in S. thermophilus but not in S. salivarius. The results clearly indicated that the gal-lac gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of galT and galE were identical in both species, whereas the ribosome binding site of S. thermophilus galK differed from that of S. salivarius by two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities.

FIG. 1.

Scheme for cloning galR, galK, galT, galE, and the 5′ end of galM of S. salivarius. The procedure is detailed in Materials and Methods. MCS, multiple cloning site.

FIG. 2.

Growth of S. salivarius and S. thermophilus on galactose or lactose. (A) Tubes containing 10 ml of M17 culture medium supplemented with 0.1% galactose were inoculated with one colony of either S. salivarius or S. thermophilus and incubated overnight at either 37°C for S. salivarius or 42°C for S. thermophilus. These cultures (100 μl) were used to inoculate 10 ml of fresh media containing 0.2% galactose. Open circles, growth of S. salivarius at 37°C; solid circles, growth of S. thermophilus at 42°C. (B and C) Cells were grown overnight in the presence of 0.1% lactose. Tubes containing M17 medium supplemented with 0.15% lactose were then inoculated with 100 μl of the overnight cultures. Growth of S. salivarius at 37°C (B; open circles) and of S. thermophilus at 42°C (C; solid circles) is shown. Solid diamonds, concentration of lactose in the medium; open diamonds, concentration of galactose in the medium.

FIG. 3.

The gal-lac gene cluster of S. salivarius. Shaded boxes, genes (the direction of transcription is indicated). The genes code for the following proteins: galR, putative transcriptional regulator; galK, galactokinase; galT, galactose-1-P uridylyltransferase; galE, UDP-glucose 4-epimerase; galM, galactose mutarotase; lacS, lactose transporter; lacZ, β-galactosidase. Terminators are indicated by stem-loop structures. Arrows, promoters. The sequences of the terminators, promoters (−10 and −35 boxes), and ribosome binding sites (SD) are indicated. The promoter upstream from galM is indicated within the sequence of the terminator located downstream from galE.

FIG. 4.

Organization of the galactose and lactose genes in various gram-positive bacteria. Abbreviations: S., Streptococcus; Lc. cremoris, Lactococcus lactis subsp. cremoris; Lc. lactis, Lactococcus lactis subsp. lactis; Lb., Lactobacillus. The genes code for the following proteins: dexB, dextran glucosidase; galR, putative transcriptional regulator; galK, galactokinase; galT, galactose-1-P uridylyltransferase; galE, UDP-glucose 4-epimerase; galM, galactose mutarotase; lacS, lactose transporter; lacZ, β-galactosidase; galA, galactose permease; lacY, lactose permease; lacA, galactoside acetyltransferase.

FIG. 5.

Nucleotide sequence alignment of the promoter regions of the S. salivarius and S. thermophilus gal-lac gene clusters. The ribosome binding sites (SD); the −10 and −35 boxes of the promoters; the initiation codons of galR, galK, and lacS; and the stop codons of galE and galM are in boldface. +1, transcriptional start sites determined by primer extension. The CRE sequences are boxed. Two putative GalR binding sites are underlined. Arrows, terminators.

FIG. 6.

Northern blot analysis of S. salivarius and S. thermophilus. The lanes contained total RNA isolated from cells grown on 0.5% lactose. S. salivarius was harvested at an OD₆₆₀ of 0.45, and S. thermophilus was harvested at an OD₆₆₀ of 0.75. Lane 1, S. salivarius; lane 2, S. thermophilus. (A) Experiments performed with a probe specific to galE. (B) Experiments performed with a probe specific to galM. Both lanes contain total RNA isolated from S. thermophilus. (C) Experiments performed with a probe specific to lacS. RNA molecular weight markers were used to determine the sizes of the transcripts. (Bottom) Map of the S. salivarius and S. thermophilus gal-lac gene clusters. The symbols are the same as those described in the legend of Fig. 3. Black boxes, probes used in Northern blot experiments; thick lines, major transcripts; thin lines, transcripts found at low levels.

FIG. 7.

Mapping of the 5′ ends of the galE and the galEM lacSZ transcripts by primer extension analysis. For lane galE, oligonucleotide 5′-GTGAGAACCGATATAACCAGCT-3′ was annealed to 20 μg of S. thermophilus RNA and extended using murine leukemia virus reverse transcriptase. The nucleotide sequence was determined with an amplicon encompassing the DNA region of interest by using the same oligonucleotide as a primer (lanes T, G, C, and A). Samples of both reaction mixtures were loaded on the same gel, and the final figure is a composite. The exposure time for the primer extension reaction was 7.5 h, and that for the sequence reaction was 21.5 h. +1, position of the primer extension products.