FIG 6.
Expression of iolT is regulated by RpoS and the availability of serine. Exponentially growing cultures of L. pneumophila strain JR32 (A), the ΔlqsR mutant (B), or the ΔrpoS mutant (C) harboring plasmid pCM007 [unstable GFP(ASV) under the control of Piol] were diluted to a starting OD600 of 0.1 in AYE broth. Bacteria were grown at 37°C with 10 mM inositol or 6 mM serine or without the addition of additional nutrients. Optical density at 600 nm and GFP fluorescence were measured every hour for 24 h, and the results were plotted with relative fluorescent units (rfu) as a function of OD600 over time. (D) L. pneumophila JR32 or the ΔrpoS mutant was grown to an OD600 of 0.5, 1.0, 2.0, and 3.0. At these points, samples were taken, 10 mM inositol mixed with 1% [U-14C6]inositol was added, and the bacteria were further incubated for 20 min. Cells were spun onto cellulose acetate filter disks and washed, and filter-associated radioactivity was determined in a liquid scintillation counter. The mean and SD of triplicates are shown (Student's t test; A and B [serine versus none], >14 h, P < 0.05; D, OD600, >1.0, P < 0.05). The data are representative of the results from three independent experiments.