Control of EpsE, the phosphoglycosyltransferase initiating exopolysaccharide synthesis in Streptococcus thermophilus, by EpsD tyrosine kinase.
We studied the roles of Streptococcus thermophilus phosphogalactosyltransferase (EpsE) (the priming enzyme), tyrosine kinase (EpsD), phosphatase (EpsB), and a membrane-associated protein with no known biochemical function (EpsC) in exopolysaccharide (EPS) synthesis. These proteins are well-conserved among bacteria and are usually encoded by clustered genes. Exopolysaccharide synthesis took place in the wild-type strain and a mutant lacking EpsB but not in mutants lacking EpsC, EpsD, or EpsE. The three mutants unable to synthesize EPS lacked the EpsE phosphogalactosyltransferase activity, while the two EPS-synthesizing strains possessed this activity, showing that EpsC and EpsD are required for EpsE function. An EpsD phosphorylated form was found in all strains except the epsC mutant, indicating that EpsC is necessary for EpsD phosphorylation. Moreover, the phosphorylated form of EpsD, a supposedly cytoplasmic protein, was found to be associated with the plasma membrane, possibly due to interaction with EpsC. Finally, the EpsD and EpsE elution profiles in a gel filtration chromatography assay were similar, suggesting that these two proteins colocalize in the membrane. Mutation of Tyr200, predicted to be a phosphorylation site and present in a conserved motif in bacterial phosphoglycosyltransferases, led to EpsE inactivation. In contrast, mutation of Tyr162 or Tyr199 had no effect. Taken together, these data show that EpsD controls EpsE activity. Possible mechanisms for this control are discussed.
Minic
Zoran
Z
Génétique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas cedex, France.
Marie
Corinne
C
Delorme
Christine
C
Faurie
Jean-Michel
JM
Mercier
Gérald
G
Ehrlich
Dusko
D
Renault
Pierre
P
D000595:Amino Acid Sequence; D017353:Gene Deletion; D016695:Glycosyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D048249:Streptococcus thermophilus / Q000201:enzymology* / Q000378:metabolism*
D011135:Polysaccharides, Bacterial; C098632:exopolysaccharide, Streptococcus; D016695:Glycosyltransferases; D011505:Protein-Tyrosine Kinases