Characterization of galactose catabolic pathways in Streptococcus agalactiae and identification of a major galactose: phosphotransferase importer

Abstract

We identified and characterized genomic regions of Streptococcus agalactiae that are involved in the Leloir and the tagatose-6-phosphate pathways for D-galactose catabolism. The accumulation of mutations in genes coding the Leloir pathway and the absence of these genes in a significant proportion of the strains suggest that this pathway may no longer be necessary for S. agalactiae and is heading toward extinction. In contrast, a genomic region containing genes coding for intermediates of the tagatose-6-phosphate pathway, a Gat family PTS transporter, and a DeoR/GlpR family regulator is present in the vast majority of strains. By deleting genes that code for intermediates of each of these two pathways in three selected strains, we demonstrated that the tagatose-6-phosphate pathway is their sole route for galactose catabolism. Furthermore, we showed that the Gat family PTS transporter acts as the primary importer of galactose in S. agalactiae. Finally, we proved that the DeoR/GlpR family regulator is a repressor of the tagatose-6-phosphate pathway and that galactose triggers the induction of this biochemical mechanism.IMPORTANCES. agalactiae, a significant pathogen for both humans and animals, encounters galactose and galactosylated components within its various ecological niches. We highlighted the capability of this bacterium to metabolize D-galactose and showed the role of the tagatose-6-phosphate pathway and of a PTS importer in this biochemical process. Since S. agalactiae relies on carbohydrate fermentation for energy production, its ability to uptake and metabolize D-galactose could enhance its persistence and its competitiveness within the microbiome.

Keywords: Leloir pathway; PTS Gat family; environmental adaptation; galactose catabolism; group B streptococcus; phosphotransferase system; sugar metabolism; tagatose-6-phosphate pathway.

Fig 1

Organization of the genes encoding intermediates of the Leloir pathway in Streptococcus agalactiae strains A909 and BM110. The genes encoding the enzymes of the Leloir pathway [galM (sak_0539/0542), aldose 1-epimerase; galK (sak_0536), galactose kinase; galT (sak_0537), galactose-1-phosphate uridyltransferase; galE (sak_0538), and UDP-galactose 4-epimerase] are found in an A909 genomic region of seventeen genes putatively involved in sugar metabolism (white arrows). This region, inserted between the sak_0522 and sak_0543 genes (gray arrows), is not present between the corresponding BM110_00578 and BM110_00579 genes of strain BM110. The BM110_00578/00579 intergenic region (IR) is composed by a fusion of 62% of the corresponding sak_0522/0523 IR and 93% of the sak_0539/0540 IR. ABC, ATP-binding cassette; Ara, arabinose; Gat, galactitol; IS, insertion sequence (black rectangle); MSD, membrane spanning domain; PRD, PTS regulation domain; PTS, phosphoenolpyruvate:carbohydrate phosphotransferase system; RPS5, ribosomal protein S5; SBP, substrate binding protein; hatched open arrow, pseudogene.

Fig 2

Organization of the genes encoding intermediates of the tagatose-6-phosphate pathway in Streptococcus agalactiae strain A909. The genes encoding the enzymes from the tagatose-6-phosphate pathway (LacAB, galactose-6-phosphate isomerase subunits A and B; LacC, tagatose-6-phosphate kinase; LacD, tagatose-1,6-diphosphate aldolase) are found in a genomic region of fourteen genes putatively involved in sugar metabolism (white arrows). The sak_1893–1896 genes encode a PTS transporter of the galactitol family. This fourteen gene region is bordered by genes (gray arrows) involved in zinc transport (sak_1897/1898; 29) and citrate transport (sak_1879–1881; 30). ABC, ATP-binding cassette; Cit, citrate; galE, putative UDP-glucose 4-epimerase gene; NBD, nucleotide binding domain; PTS, phosphoenolpyruvate:carbohydrate phosphotransferase system; Sht, streptococcal histidine triad; hatched open arrow, truncated gene.

Fig 3

Presence of genes encoding the enzymes of the two galactose catabolism pathways in Streptococcus agalactiae strains H36B, A909, and BM110. A schematic representation of the tagatose-6-phosphate and Leloir pathways is presented. Genes involved in these pathways are indicated in bold characters. The name of strain(s) possessing the corresponding non-pseudogenized genes are indicated within brackets. Enzymes encoded by these genes are galactose-6-phosphate isomerase for lacAB, tagatose-6-phosphate kinase for lacC, tagatose-1,6-diphosphate aldolase for lacD, galactose epimerase for galM, galactose kinase for galK, galactose-1-phosphate uridyltransferase for galT, and UDP-glucose-4-epimerase for galE. G3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate.

Fig 4

Growth of selected Streptococcus agalactiae strains on single carbon sources. S. agalactiae A909 (black curve), BM110 (green curve), and H36B (red curve) strains were grown in a chemically defined medium containing 1% galactose (A) or 1% glucose (B) as a single carbon source. These cultures were incubated for 40 hours at 37°C without agitation in a microtiter plate (200 μL-culture volume per well) in an Eon thermoregulated spectrophotometer plate reader. The OD_{600 nm} was measured every hour after double orbital shaking of the plate for 5 seconds. Three independent experiments were realized, and the standard deviation is indicated.

Fig 5

The tagatose-6-phosphate pathway is the unique galactose catabolic pathway in Streptococcus agalactiae strains A909 and H36B. Wild-type (WT) S. agalactiae A909 (black curves) and its isogenic mutants A909ΔlacAB (red curves) or A909ΔgaltT (green curves) were grown in a chemically defined medium containing 1% galactose (A) or 1% glucose (B) as a single carbon source. Similarly, wild-type strain H36B (black curves) and its isogenic mutants H36BΔlacAB (red curves) or H36ΔgaltT (green curves) were grown in the presence of 1% galactose (C) or 1% glucose (D) as a single carbon source. These cultures were incubated for 40 hours at 37°C without agitation in a microtiter plate (200 μL-culture volume per well) in an Eon thermoregulated spectrophotometer plate reader. The OD_{600 nm} was measured every hour after double orbital shaking of the plate for 5 seconds. Three independent experiments were realized, and the standard deviation is indicated.

Fig 6

Strength of the lacA promoter of Streptococus agalactiae strains A909, H36B, and BM110. In panel A, the nucleotide sequence of the intergenic region upstream of lacA of S. agalactiae strains A909, H36B, and BM110 is shown. Minus 35 and minus 10 boxes of a predicted σ⁷⁰ promoter are framed. Direct repeats (DR) are underlined, and a ribosome binding site (RBS) is shown in bold. In panel B, the strength of the lacA promoter is reported. To quantify the strength of this promoter in each stain, their lacA intergenic region were cloned in front of the β-galactosidase gene (a spoVG-lacZ fusion) of the promoter probe plasmid pTCV-lacZ. The recombinant plasmids were then inserted in their respective wild-type strains A909, H36B, and BM110. The strains were all grown to the same OD in CDM containing glucose. The strength of the lacA promoter was then quantified by measuring β-galactosidase specific activity after a 1-h incubation of the bacteria in a chemical defined medium containing 1% glucose (white rectangle) or 1% galactose (black rectangles) as a sole carbon source. Results are presented as the mean and standard deviation of three independent cultures.

Fig 7

SAK_1896 is a repressor of the tagatose-6-phosphate pathway. The intergenic region upstream of the lacA transcription start of strain A909 was cloned in front of the β-galactosidase gene (a spoVG-lacZ fusion) of the promoter probe plasmid pTCV-lacZ. The recombinant plasmid was then inserted into the wild-type strain A909 and in the deletion mutant A909Δsak_1896. The strength of the lacA promoter was quantified by measuring β-galactosidase specific activity during the mid-exponential growth phase of both strains in a chemically defined medium containing either 1% glucose (white rectangle) or 1% galactose (black rectangle) as a sole carbon source. Results are presented as the mean and standard deviation of three independent cultures. The statistical significance of the differences in the activity of the lacA promoter in the presence of glucose or galactose and in the presence or absence of the SAK_1896 regulator was determined by an unpaired Student t‐test (*, P < 0.05; *** , P < 0.005).

Fig 8

The PTS transporter SAK_1893–1895 is involved in galactose transport in Streptococcus agalactiae strain A909. S. agalactiae A909 (black curve), A909Δsak_0524–530 (red curve), A909Δsak_1893–1895 (blue curve), and A909 Δsak_0524–530Δsak_1893–1895 (yellow curve) strains were grown in a chemically defined medium containing 0.1% galactose (A) or 1% galactose (B) as a single carbon source. These cultures were incubated for 40 hours at 37°C without agitation in microtiter plates (300 μL-culture volume per well) in an Eon thermoregulated spectrophotometer plate reader. The OD_{600 nm} was measured every hour after double orbital shaking of the plate for 5 seconds. Three independent experiments were realized, and the standard deviation is indicated.