Biochemical and genetic analysis of Streptococcus mutans  -galactosidase

Aduse-Opoku, Joseph; Tao, L.; Ferretti, Joseph J.; Russell, R. R. B.

doi:10.1099/00221287-137-4-757

Cited by 36 publications

(25 citation statements)

References 10 publications

(13 reference statements)

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“…Melibiose is internalized in an unphosphorylated form by the multiple sugar metabolism (msm) pathway (41), which is a typical ABC transporter that transports a variety of galactosides, and is cleaved by a cytoplasmic ␣-galactosidase to release unphosphorylated glucose and galactose (5). The cells can then use the galactose via the Leloir pathway and the glucose could enter the Embden-Meyerhoff-Parnas pathway after phosphorylation by glucokinase.…”

Section: Resultsmentioning

confidence: 99%

Utilization of Lactose and Galactose by Streptococcus mutans : Transport, Toxicity, and Carbon Catabolite Repression

2010

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Abundant in milk and other dairy products, lactose is considered to have an important role in oral microbial ecology and can contribute to caries development in both adults and young children. To better understand the metabolism of lactose and galactose by Streptococcus mutans, the major etiological agent of human tooth decay, a genetic analysis of the tagatose-6-phosphate (lac) and Leloir (gal) pathways was performed in strain UA159. Deletion of each gene in the lac operon caused various alterations in expression of a P lacA -cat promoter fusion and defects in growth on either lactose (lacA, lacB, lacF, lacE, and lacG), galactose (lacA, lacB, lacD, and lacG) or both sugars (lacA, lacB, and lacG). Failure to grow in the presence of galactose or lactose by certain lac mutants appeared to arise from the accumulation of intermediates of galactose metabolism, particularly galatose-6-phosphate. The glucose-and lactose-PTS permeases, EII Man and EII Lac , respectively, were shown to be the only effective transporters of galactose in S. mutans. Furthermore, disruption of manL, encoding EIIAB Man , led to increased resistance to glucose-mediated CCR when lactose was used to induce the lac operon, but resulted in reduced lac gene expression in cells growing on galactose. Collectively, the results reveal a remarkably high degree of complexity in the regulation of lactose/galactose catabolism.Lactose, a ␤1,4-linked disaccharide of ␤-D-galactose and ␣/␤-D-glucose, is commonly found in the dairy-rich diets of most industrialized nations. Lactose is rapidly fermented by streptococci, including the cariogenic oral bacterium Streptococcus mutans (21), as well as by a variety of industrially important lactic acid bacteria (LAB) (19). Multiple pathways have been identified in bacteria for the utilization of lactose encountered in the environment. For example, Streptococcus salivarius strain 25975 (26) secretes a ␤-galactosidase that hydrolyzes extracellular lactose into galactose and glucose, although it is more common for lactose to be transported before cleavage (18). Most efficiently, and almost exclusively in Gram-positive bacteria, lactose is internalized by the phosphoenolpyruvate (PEP)-dependent sugar-phosphotransferase system (PTS), yielding lactose-6-phosphate (Lac-6-P) (36). The Lac-6-P is hydrolyzed to glucose and galactose-6-phosphate (Gal-6-P) by a cytoplasmic phospho-␤-galactosidase (LacG), and the Gal-6-P can be catabolized by the tagatose-6-phosphate pathway (18) (Fig. 1). Many bacteria, including Escherichia coli, Lactococcus lactis strain 7962, and S. salivarius strain 57.I, can internalize lactose through non-PTS transporters. Intracellular lactose is cleaved by a ␤-galactosidase enzyme and the D-galactose can directly enter the Leloir pathway ( Fig. 1) (18, 20).S. mutans has a functional lactose-specific PTS (14, 26) encoded by the lacF (EIIA) and lacE (EIIBC) genes (40). A phospho-␤-galactosidase (lacG) and the enzymes of the tagatose-6-phosphate pathway (Fig. 1B), including the two subunits of the heteromeri...

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Section: Resultsmentioning

confidence: 99%

Utilization of Lactose and Galactose by Streptococcus mutans : Transport, Toxicity, and Carbon Catabolite Repression

2010

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“…The a-galactosidase RafA of E. coli (family 36) is contained in a transmissible plasmid as part of an operon responsible for raffinose utilization (Schmid and Schmitt, 1976). It has been suggested that E. coli might have acquired RafA from gram-positive bacteria, because it has high amino acid similarity with the enzymes of S. rnutans and Bacillus steurothermoyhilus and is different from the a-galactosidase coded by the chromosomal E. coli MelA gene (family 4; Aslanidis et al, 1989;Aduse-Opoku et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…Genes encoding a-galactosidases have been isolated from several sources, such as from human (Bishop et al, 1988), plant seeds (Overbeeke eta!., 1989; Zhu and Goldstein, 19941, bacte-ria (Liljestrom and Liljestrom, 1987;Koyama et al, 1990;Russell et al, 1992;Aduse-Opoku et al, 1991), yeast (SumnerSmith et al, 1985) and filamentous fungi (den Herder et al, 1992;Shibuya et al, 1995). Based on similarities in primary structure and hydrophobic cluster analyses they have been grouped into three well conserved families in the general classification of glycosyl hydrolases (Henrissat and Bairoch, 1993).…”

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confidence: 99%

Three α‐Galactosidase Genes of Trichoderma reesei Cloned by Expression in Yeast

Margolles-Clark

Tenkanen

Luonteri

et al. 1996

European Journal of Biochemistry

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Three cx-galactosidase genes, agll, ag12 and ag13, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-a-D-gnlactopyranoside. The genes ugll, ag12 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the a-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial a-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-a-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(g1uco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-l,4-/?-mannanase of T reesei. AGLII and AGLIII showed synergy in galacto(g1uco)mannan hydrolysis with the endo-l,4-/?-mannanase of 7: reesei and a B-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the a-galactosidase previously purified from 7: reesei.

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“…14 ) In addition, the cDNA for a-galactosidase I from M. vinacea has also been cloned and its primary structure analyzed. 15) Here we report the purification and characterization of a-galactosidase II from M. vinacea, and its primary structure from the nucleotide sequence of the eDNA.…”

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confidence: 99%

Purification, Characterization, and cDNA Cloning of a Novelα-Galactosidase fromMortievella vinacea

Shibuya

Kobayashi

Sato

et al. 1997

Bioscience, Biotechnology, and Biochemistry

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A novel alpha-galactosidase, designated alpha-galactosidase II, was isolated from the culture filtrate of Mortierella vinacea. The molecular size of the purified enzyme estimated by gel filtration was 60 kDa, which agreed with that, 51-62 kDa, estimated by SDS-PAGE. The enzyme was thermolabile at neutral pH, but the addition of BSA to the enzyme solution at the concentration of 0.01% increased its stability considerably. The enzyme appears to be novel because it showed a distinct substrate specificity from other microbial alpha-galactosidases on galactomanno-oligosaccharides, prepared from galactomannan, that is, the enzyme liberated not only side-chain alpha-galactosyl residue from 6(3)-mono-alpha-D-galactopyranosyl-beta-1,4-D-mannotetraose but also terminal alpha-galactosyl residue from 6(3)-mono-alpha-D-galactopyranosyl-beta-1,4-D-mannotriose. In addition, the enzyme acted on galactomannans effectively. alpha-Galactosidase II cDNA was cloned and its nucleotides sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 376 amino acid residues with a molecular mass of 41,334 Da. The derived amino acid sequence of the enzyme showed 31-49% sequence similarity with those of alpha-galactosidases from other origins.

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Biochemical and genetic analysis of Streptococcus mutans -galactosidase

Cited by 36 publications

References 10 publications

Utilization of Lactose and Galactose by Streptococcus mutans : Transport, Toxicity, and Carbon Catabolite Repression

Utilization of Lactose and Galactose by Streptococcus mutans : Transport, Toxicity, and Carbon Catabolite Repression

Three α‐Galactosidase Genes of Trichoderma reesei Cloned by Expression in Yeast

Purification, Characterization, and cDNA Cloning of a Novelα-Galactosidase fromMortievella vinacea

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