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Vaillancourt, Katy

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Vaillancourt

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Katy

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Faculté de médecine dentaire, Université Laval

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ncf12003962

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  • PublicationRestreint
    Characterization of a galactokinase-positive recombinant strain of Streptococcus thermophilus
    (American Society for Microbiology, 2004-08-04) Vadeboncoeur, Christian; Lamoureux, Maryse.; Frenette, Michel; Moineau, Sylvain; LeMay, Jean-Dominique.; Vaillancourt, Katy
    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.
  • PublicationRestreint
    Role of galK and galM in galactose metabolism by Streptococcus thermophilus
    (American Society for Microbiology, 2008-02-07) Vadeboncoeur, Christian; Frenette, Michel; Moineau, Sylvain; Vaillancourt, Katy; Robitaille, Gilles; Turgeon, Nathalie; Bédard, Nathalie; Bart, Christian
    Streptococcus thermophilus is unable to metabolize the galactose moiety of lactose. In this paper, we show that a transformant of S. thermophilus SMQ-301 expressing Streptococcus salivarius galK and galM was able to grow on galactose and expelled at least twofold less galactose into the medium during growth on lactose.
  • PublicationRestreint
    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
    (American Society for Microbiology, 2002-02-01) Vadeboncoeur, Christian; Frenette, Michel; Lessard, Christian; Moineau, Sylvain; Vaillancourt, Katy
    Streptococcus salivarius is a lactose- and galactose-positive bacterium that is phylogenetically closelyrelated 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 geneclusters. 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-Puridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactosetransporter), and lacZ (β-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 genecluster. 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, where as the ribosome binding site of S. thermophilus galK differed from that of S. salivariusby two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities.
  • PublicationRestreint
    Phosphorylation of Streptococcus salivarius lactose permease (LacS) by HPr(HisP) and HPr(Ser-P)(HisP) and effects on growth
    (American Society for Microbiology, 2003-11-14) Vadeboncoeur, Christian; Frenette, Michel; Lessard, Christian; Cochu, Armelle; Moineau, Sylvain; LeMay, Jean-Dominique.; Vaillancourt, Katy; Roy, Denis
    The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(HisP), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIALacS) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIALacS was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(HisP) but also by HPr(Ser-P)(HisP), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIABL Man and IIABH Man, were unable to phosphorylate IIALacS. The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(HisP) or HPr(SerP)(HisP). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.
  • PublicationRestreint
    The doubly phosphorylated form of HPr, HPr(Ser-P)(HisP), is abundant in exponentially growing cells of streptococcus thermophilus and phosphorylates the lactose transporter LacS as efficiently as HPr(HisP)
    (American Society for Microbiology, 2005-03-03) Vadeboncoeur, Christian; Frenette, Michel; Cochu, Armelle; Casabon, Israël; Moineau, Sylvain; LeMay, Jean-Dominique.; Vaillancourt, Katy; Roy, Denis
    In Streptococcus thermophilus, lactose is taken up by LacS, a transporter that comprises a membrane translocator domain and a hydrophilic regulatory domain homologous to the IIA proteins and protein domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The IIA domain of LacS (IIALacS) possesses a histidine residue that can be phosphorylated by HPr(His∼P), a protein component of the PTS. However, determination of the cellular levels of the different forms of HPr, namely, HPr, HPr(His∼P), HPr(Ser-P), and HPr(Ser-P)(His∼P), in exponentially lactose-growing cells revealed that the doubly phosphorylated form of HPr represented 75% and 25% of the total HPr in S. thermophilus ATCC 19258 and S. thermophilus SMQ-301, respectively. Experiments conducted with [32P]PEP and purified recombinant S. thermophilus ATCC 19258 proteins (EI, HPr, and IIALacS) showed that IIALacS was reversibly phosphorylated by HPr(Ser-P)(His∼P) at a rate similar to that measured with HPr(His∼P). Sequence analysis of the IIALacS protein domains from several S. thermophilus strains indicated that they can be divided into two groups on the basis of their amino acid sequences. The amino acid sequence of IIALacS from group I, to which strain 19258 belongs, differed from that of group II at 11 to 12 positions. To ascertain whether IIALacS from group II could also be phosphorylated by HPr(His∼P) and HPr(Ser-P)(His∼P), in vitro phosphorylation experiments were conducted with purified proteins from Streptococcus salivarius ATCC 25975, which possesses a IIALacS very similar to group II S. thermophilus IIALacS. The results indicated that S. salivarius IIALacS was phosphorylated by HPr(Ser-P)(His∼P) at a higher rate than that observed with HPr(His∼P). Our results suggest that the reversible phosphorylation of IIALacS in S. thermophilus is accomplished by HPr(Ser-P)(His∼P) as well as by HPr(His∼P).