Choline starvation induces the gene licD2 in Streptococcus pneumoniae

Abstract

Mutant strains of Streptococcus pneumoniae were constructed to monitor the regulation of three dispersed genes known or predicted to act in choline metabolism. One gene (licD2) was regulated in response to choline deprivation over a 30-fold range. The other two (SP1860 and licC) responded little if at all to the same challenge.

FIG. 1.

Gene tagging strategy. Genes are designated as in Tettelin et al. (12) or by previously assigned names. (A and B) Loci selected for mutagenesis; (C) organization of the vector pEVP3. The 3′ region of each target gene, indicated by an open arrow, was inserted into pEVP3 at its multiple cloning site (MCS) to target insertion of the chimeric plasmid in S. pneumoniae by homologous recombination.

FIG. 2.

Effects of choline limitation on the activity of lacZ reporters inserted at three chromosomal sites. Two independent transformants with a lacZ reporter inserted at each target site were cultured in CDM with the specified amounts of choline until optical densities at 550 nm of 0.05 to 0.1 were reached, and then they were lysed and assayed for beta-galactosidase activity.

FIG. 3.

Effects of choline limitation on growth and on gene expression of licD2 tagged strains. During culture of CP1250 and two licD2::lacZ reporter transformants in CDM with the indicated amounts of choline, optical density was monitored at 550 nm and samples were harvested at indicated times for beta-galactosidase assay. (A and D) Beta-galactosidase negative parental strain CP1250; (B and E) licD2::lacZ clone 1; (C and F) licD2::lacZ clone 2. Upper panels (A, B, and C) show growth curves of the CDM cultures; lower panels (D, E, and F) show their LacZ activity.