Analysis of temporal gene expression during Bacillus subtilis spore germination and outgrowth

Abstract

Bacillus subtilis forms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites, and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, the results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30% of the B. subtilis genome.

FIG. 1.

Spore germination and outgrowth. (A) After the addition of Bacillus subtilis spores to prewarmed germination medium (see Materials and Methods), germination and outgrowth were monitored by measuring changes in the optical density at 600 nm (OD₆₀₀) (○). In addition, the release of dipicolinic acid (DPA) (▴) was monitored as a measure for the efficiency of spore germination. (B) Extracellular glucose, pyruvate, and acetate levels were monitored during germination and outgrowth. The switch from an endogenous metabolism to the use of extracellular metabolites was indicated by the decrease of the glucose concentration and increase of the acetate concentration in the medium. Pyruvate levels were found to decrease rapidly late in outgrowth. (C) Morphological changes during spore germination and outgrowth were investigated by microscopic analysis. Cells harvested at various time points during germination and outgrowth were fixed and monitored by phase-contrast microscopy (top row) and fluorescence microscopy following DNA DAPI (4′,6′-diamidino-2-phenylindole) straining (bottom row).

FIG. 2.

Gene expression profiles during spore germination and outgrowth. By K-means clustering, genes were grouped in 12 clusters according to their gene expression pattern. Plotted is the mean log₂ ratio of the individual genes in the 12 K-means clusters (I to XII) at the various time points (minutes into outgrowth) over the average value. Indicated by the bars are the standard deviations of the individual genes in the 12 K-means clusters.

FIG. 3.

Comparison of microarray signal intensity levels of genes of the sigma G regulon during late sporulation, derived from reference , and those found in dormant spores. The scatter plot shows the microarray signal intensity values of late-sporulation transcripts of the prespore after 6.5 h of sporulation and the microarray signal intensity derived from the spore transcripts. Indicated are the early (•) and late (○) subclasses of σ^G, expressed at relatively early and late stages, respectively, of prespore gene expression.

FIG. 4.

Analysis of overrepresented gene ontology (GO) groups during germination and outgrowth. Significantly overrepresented gene ontology groups are indicated in black.

FIG. 5.

Hierarchic clustering of transcriptional profiles of genes associated with cell division, cell wall functions, and membrane biosynthesis (left) and DNA replication/repair (right). Rows represent time points from 0 to 100 min. Red and green indicate genes that are induced and repressed, respectively.