Analysis of Temporal Gene Expression duringBacillus subtilisSpore 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.A number of bacterial species such as bacilli and clostridia have the ability to form dormant spores. The spore has a specialized and complex structure, enabling the organism to survive for a long time under harsh environmental conditions and in the absence of nutrients. When triggered by specific nutrients, the spore is capable of breaking dormancy (germination) and initiating vegetative growth (34, 52). The Bacillus subtilis spore is composed of a dehydrated central compartment (the spore core) engulfed by two protective outer layers: a thick spore-specific peptidoglycan layer known as the spore cortex and a multilayered protein structure known as the coat (12).The process of endospore formation in Bacillus subtilis has been studied in great detail. Studies have revealed a highly ordered and strictly regulated program ensuring the correct coordination of various aspects of the sporulation process, such as asymmetric cell division, prespore engulfment, spore maturation, and mother cell lysis (22). The sporulation program involves the timed activation of several mother cell and forespore compartment-and sporulation stage-dependent RNA polymerase sigma factors that transcribe specific sets of sporulation genes. Eventually, the sporulation program results in the lysis of the mother cell and the release of a dormant spore (22).The process of spore germination and outgrowth has been studies in less detail. Spore germination is initiated when the spore senses the appropriate trigger molecules, often simple sugars and/or amino acids. The germinant molecules are sensed by germination receptors. This, by an unknown mechanism, leads to an irreversible commitment of a spore to germination. The germinating spore initially releases Zn 2ϩ and H ϩ (65). Simultaneously (and probably as a consequence), the pH of the spore core rises from 6.5 to 7.7. In a second stage, the germinating spore releases the spore core's large supply of dipicolinic acid (pyridine-2,6-dicarboxylic acid), and the spore core is rehydrated. Subsequently, cortex lytic enzymes are activated and the protective spore peptidoglycan cortex is degraded. This enables the germinating spore to hydrate the spore core further and to swell. These germination events coincide with a loss of...

Transcriptome Analysis of Sorbic Acid-Stressed Bacillus subtilis Reveals a Nutrient Limitation Response and Indicates Plasma Membrane Remodeling

“…The efficiency of labeling was monitored spectrometrically on a Nanodrop apparatus (Isogen Life Science). The B. subtilis DNA microarrays were constructed as described previously by Keijser et al (40). Each constructed array contained spots in duplicate with 4,100 gene-specific 65-mer oligonucleotides representing 4,100 of the 4,106 protein-coding genes in B. subtilis (as reported for the B. subtilis genome at http://genolist.pasteur.fr/SubtiList/).…”

“…The whole procedure took no longer than 50 s. Two biologically independent experiments were performed. Total RNA was isolated as described previously (40).…”

“…Each constructed array contained spots in duplicate with 4,100 gene-specific 65-mer oligonucleotides representing 4,100 of the 4,106 protein-coding genes in B. subtilis (as reported for the B. subtilis genome at http://genolist.pasteur.fr/SubtiList/). Hybridization and scanning was performed as described previously (40).…”

Identification of genes involved in radioresistance of nasopharyngeal carcinoma by integrating gene ontology and protein-protein interaction networks

“…Differential gene screening criteria: the experimental group or control group contain effective genes (p<0.01) and single sample differscore value in the experimental group was <-20 or >+20. Significant difference of genes were analyzed by GEPAS (17)(18)(19). A t-test was used for the difference in mean expression between the two groups of arrays.…”

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

“…In contrast, Tlp (Cac1487) did not show essential features of a/b-type SASPs. Although a s F promoter (present study), transcription data (Jones et al, 2008) and tlp mRNA transcripts in dormant spores of B. subtilis (Keijser et al, 2007) indicate a spore-related function, the role of the Tlp remains unknown. Its assignment as a thioredoxin-like protein is confusing because it implies a function as a thioredoxin, and thioredoxins usually serve as small, ubiquitous redox proteins characterized by an active centre (C-G-P-C) with two redox-active cysteine residues (Holmgren, 1995;Qi & Grishin, 2005).…”