Effects of Sporulation Conditions on the Germination and Germination Protein Levels of Bacillus subtilis Spores

Abstract

Bacillus subtilis spores prepared in rich medium germinated faster with nutrient germinants than poor-medium spores as populations in liquid and multiple individual spores on a microscope slide. Poor-medium spores had longer average lag times between mixing of spores with nutrient germinants and initiation of Ca-dipicolinic acid (CaDPA) release. Rich-medium spores made at 37°C germinated slightly faster with nutrient germinants than 23°C spores in liquid, but not when spores germinated on a slide. The difference in germination characteristics of these spore populations in liquid was paralleled by changes in expression levels of a transcriptional lacZ fusion to the gerA operon, encoding a germinant receptor (GR). Levels of GR subunits were 3- to 8-fold lower in poor-medium spores than rich-medium spores and 1.6- to 2-fold lower in 23°C spores than 37°C spores, and levels of the auxiliary germination protein GerD were 3.5- to 4-fold lower in poor medium and 23°C spores. In contrast, levels of another likely germination protein, SpoVAD, were similar in all these spores. These different spores germinated similarly with CaDPA, and poor-medium and 23°C spores germinated faster than rich-medium and 37°C spores, respectively, with dodecylamine. Since spore germination with CaDPA and dodecylamine does not require GerD or GRs, these results indicate that determinants of rates of nutrient germination of spores prepared differently are primarily the levels of the GRs that bind nutrient germinants and trigger germination and secondarily the levels of GerD.