Characterization of a Galactokinase-Positive Recombinant Strain of Streptococcus thermophilus

Abstract

The lactic acid bacterium Streptococcus thermophilus is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid. Unlike the phylogenetically related species Streptococcus salivarius, S. thermophilus is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose. This metabolic property may result from the inability of S. thermophilus to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate. In this work, we report the complementation of Gal ؊ strain S. thermophilus SMQ-301 with S. salivarius galK, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01. The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing S. salivarius galK, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose. Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of S. salivarius galK and (ii) transcription of the plasmid-borne galK gene did not require GalR, a transcriptional regulator of the gal and lac operons, and did not interfere with the transcription of these operons. Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05%. Thus, unlike S. salivarius, the ability to metabolize galactose was not sufficient for S. thermophilus to simultaneously metabolize the glucose and galactose moieties of lactose. Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal ؉ strain grew and produced acid more rapidly than the Gal ؊ wild-type strain.Streptococcus thermophilus is widely used in yoghurt fermentation and cheese making. Specific S. thermophilus strains may also have the potential to improve human health as probiotics. For instance, they may interfere with the adhesion of indigenous cariogenic bacteria to teeth (9) and may prolong the useful life of indwelling voice prostheses by preventing colonization by Candida spp. (8).The ability of S. thermophilus to rapidly take up sugars from the environment is a prerequisite for these applications. Unlike several other lactic acid bacteria, S. thermophilus only uses a few sugars, with most strains showing a marked preference for saccharose and lactose, while glucose and fructose are more slowly fermented, if they are fermented at all (15,19). S. thermophilus takes up lactose via the membrane protein LacS, a member of the glycoside-pentoside-hexuronide:cation symporter family (29). Inside the cell, the disaccharide is hydrolyzed into glucose and galactose by the enzyme ␤-galactosidase. While all strains metabolize glucose via the EmbdenMeyerhof-Parnas pathway, most are unabl...

The Doubly Phosphorylated Form of HPr, HPr(Ser-P)(His∼P), Is Abundant in Exponentially Growing Cells ofStreptococcus thermophilusand Phosphorylates the Lactose Transporter LacS as Efficiently as HPr(His∼P)

“…This is consistent with the findings that S. salivarius ␤-galactosidase is repressed 24-to 75-fold by PTS sugars (44), while the S. thermophilus enzyme is repressed less than twofold (51). Lastly, the lower levels of HPr(Ser-P) in S. thermophilus also agree with the observation that the lac operon is expressed at higher levels in lactose-grown S. thermophilus cells than in S. salivarius cells grown under the same conditions even though the lac promoter regions of both strains are virtually the same (48,49).…”

“…In most bacteria that use this mode of transport, internalized lactose is hydrolyzed by ␤-galactosidase into glucose and galactose, which are metabolized via the Embden-Meyerhof-Parnas and Leloir pathways, respectively (15,20). However, most strains of S. thermophilus are unable to metabolize galactose due to insufficient expression levels of the galactokinase-encoding galK gene (29,48,49) and release the hexose into the external medium (19). The galactose expulsion phenomenon is closely associated with S. thermophilus LacS, which is able to catalyze two modes of transport: a ⌬p-driven lactose uptake in symport with protons and a lactose-galactose exchange (32-34).…”

Molecular and Biochemical Analysis of the Galactose Phenotype of Dairy Streptococcus thermophilus Strains Reveals Four Different Fermentation Profiles

“…However, Galpositive strains had a tendency to consume galactose faster than Gal-negative strains and to completion. IMDOST40 was the only strain that was capable of metabolizing the glucose and the galactose moieties of lactose simultaneously, a feature that is commonly found in S. salivarius (27). Although the gal promoter played an important role in the Gal-positive phenotype, it did not determine the Gal-positive phenotype exclusively.…”

High genetic and phenotypic variability of Streptococcus thermophilus strains isolated from artisanal Yuruk yoghurts

“…The authors explained this finding by characterizing the mutant galactose positive S. thermophilus strain and demonstrated the point mutations in the promoter region of Gal operon, which enhances the strength of the promoter. In another study, the low level of galactokinase activity in Gal (À) strains with respect to Gal (þ) strains indicated a poor galK translation, which was related to nucleotide differences in ribosome binding site (De Vin, Radstörm, Herman & de Vuyst, 2005;Vaillancourt et al, 2004). The galactose positive phenotype of our isolated strains could also be due to similar mutations in the promoter region or Shine Dalgarno sequences of Gal operon, and it requires further genetic characterization.…”