The stress-responsive dgk gene from Streptococcus mutans encodes a putative undecaprenol kinase activity

Abstract

We analyzed a previously constructed stress-sensitive Streptococcus mutans mutant Tn-1 strain resulting from disruption by transposon Tn916 of a gene encoding a protein exhibiting amino acid sequence similarity to the Escherichia coli diacylglycerol kinase. It was confirmed that the mutation led to significantly reduced lipid kinase activity, while expression of the intact gene on a plasmid restored both kinase activity and the wild-type phenotype. Further analysis revealed that the product of the dgk gene in S. mutans predominantly recognizes a lipid substrate other than diacylglycerol, most likely undecaprenol, as demonstrated by its efficient phosphorylation and the resistance of the product of the reaction to saponification. The physiological role of the product of the dgk gene as a putative undecaprenol kinase was further supported by a significantly higher sensitivity of the mutant to bacitracin compared with that of the parental strain.

FIG. 1.

Membrane preparations of S. mutans strains GS-5 and Tn-1 carrying vector pTS749 (GS-5 vector and Tn-1 vector) and Tn-1 carrying plasmid pDGK749 (Tn-1 dgk) were assayed for DGK activity in the presence of lipid extracts from S. mutans. Error bars represent standard errors of the means for reactions performed in duplicate.

FIG. 2.

DGK activities in membrane preparations of E. coli strain DH5α and DGK-deficient mutant RZ6 carrying vector pTrc99A and RZ6 expressing wild-type (wt) and mutated (mu) forms of dgk from S. mutans on plasmids pTrcDGK-1 and pTrcDGKTn-31, respectively. The reactions were performed in the presence of lipid extracts from S. mutans. Error bars represent standard errors of the means for reactions performed in duplicate.

FIG. 3.

Comparison of TLC separations of phospholipids generated by two different kinases. Lanes 1, 2, and 4, TLC separations of phospholipids generated by E. coli kinase from diacylglycerol (16:0), from S. mutans lipids, and from diacylglycerol (18:1), respectively; lane 3, separation of products of reactions catalyzed by membranes isolated from S. mutans and by purified DGK from E. coli by using S. mutans lipids as substrates. The products of both reactions were mixed prior to TLC separation.

FIG. 4.

Phosphorylation of the lipid extract from S. mutans (L) or of undecaprenol (U). The sources of lipid kinase activities used were isolated membranes of S. mutans strains: GS-5 (G) or Tn-1 expressing S. mutans dgk on a plasmid (T/D) or purified DGK from E. coli (E).

FIG. 5.

Saponification of phospholipids generated from undecaprenol by membranes isolated from S. mutans (S. m) or by purified DGK from E. coli (E. c).

FIG. 6.

Generation of phospholipids from undecaprenol (U) and diacylglycerol (DG) by S. mutans membranes (S), by purified kinase from S. mutans (pS), and by purified DGK from E. coli (pE).