ABSTRACT
Dormant Bacillus subtilis spores can be induced to germinate by nutrients, as well as by nonmetabolizable chemicals, such as a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA). Nutrients bind receptors in the spore, and this binding triggers events in the spore core, including DPA excretion and rehydration, and also activates hydrolysis of the surrounding cortex through mechanisms that are largely unknown. As Ca(2+)-DPA does not require receptors to induce spore germination, we asked if this process utilizes other proteins, such as the putative cortex-lytic enzymes SleB and CwlJ, that are involved in nutrient-induced germination. We found that Ca(2+)-DPA triggers germination by first activating CwlJ-dependent cortex hydrolysis; this mechanism is different from nutrient-induced germination where cortex hydrolysis is not required for the early germination events in the spore core. Nevertheless, since nutrients can induce release of the spore's DPA before cortex hydrolysis, we examined if the DPA excreted from the core acts as a signal to activate CwlJ in the cortex. Indeed, endogenous DPA is required for nutrient-induced CwlJ activation and this requirement was partially remedied by exogenous Ca(2+)-DPA. Our findings thus define a mechanism for Ca(2+)-DPA-induced germination and also provide the first definitive evidence for a signaling pathway that activates cortex hydrolysis in response to nutrients.
Subject(s)
Amidohydrolases/metabolism , Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Calcium/pharmacology , Hydrolases/metabolism , Picolinic Acids/pharmacology , Amidohydrolases/genetics , Bacillus subtilis/drug effects , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Chelating Agents/pharmacology , Genotype , Hydrolases/genetics , Kinetics , Models, Biological , Spores, Bacterial/drug effects , Spores, Bacterial/physiologyABSTRACT
Sporulation of a Bacillus subtilis strain (termed alpha(-) beta(-)) lacking the majority of the alpha/beta-type small, acid-soluble spore proteins (SASP) that are synthesized in the developing forespore and saturate spore DNA exhibited a number of differences from that of the wild-type strain, including delayed forespore accumulation of dipicolinic acid, overexpression of forespore-specific genes, and delayed expression of at least one mother cell-specific gene turned on late in sporulation, although genes turned on earlier in the mother cell were expressed normally in alpha(-) beta(-) strains. The sporulation defects in alpha(-) beta(-) strains were corrected by synthesis of chromosome-saturating levels of either of two wild-type, alpha/beta-type SASP but not by a mutant SASP that binds DNA poorly. Spores from alpha(-) beta(-) strains also exhibited less glutaraldehyde resistance and slower outgrowth than did wild-type spores, but at least some of these defects in alpha(-) beta(-) spores were abolished by the synthesis of normal levels of alpha/beta-type SASP. These results indicate that alpha/beta-type SASP may well have global effects on gene expression during sporulation and spore outgrowth.
Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Sigma Factor , Transcription Factors , Bacillus subtilis/genetics , Bacterial Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial , Picolinic Acids/metabolism , Spores, Bacterial/genetics , Spores, Bacterial/physiologyABSTRACT
After a few minutes of germination, nucleoids in the great majority of spores of Bacillus subtilis and Bacillus megaterium were ring shaped. The major spore DNA binding proteins, the alpha/beta-type small, acid-soluble proteins (SASP), colocalized to these nucleoid rings early in spore germination, as did the B. megaterium homolog of the major B. subtilis chromosomal protein HBsu. The percentage of ring-shaped nucleoids was decreased in germinated spores with lower levels of alpha/beta-type SASP. As spore outgrowth proceeded, the ring-shaped nucleoids disappeared and the nucleoid became more compact. This change took place after degradation of most of the spores' pool of major alpha/beta-type SASP and was delayed when alpha/beta-type SASP degradation was delayed. Later in spore outgrowth, the shape of the nucleoid reverted to the diffuse lobular shape seen in growing cells.
Subject(s)
Bacillus megaterium/physiology , Bacillus subtilis/physiology , Sigma Factor , Transcription Factors , Bacillus megaterium/metabolism , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Microscopy, Fluorescence/methods , Spores, Bacterial/metabolism , Spores, Bacterial/physiologyABSTRACT
The Bacillus subtilis genome sequencing project [Kunst et al., Nature 390 (1997) 249-256] identified ywhE as a gene that potentially encodes a high-molecular-weight class A penicillin-binding protein. Analysis of the expression of a translational ywhE-lacZ fusion showed that ywhE expression is sporulation-specific, and is controlled predominantly by the forespore-specific sigma factor sigma(F), and to a lesser extent by sigma(G). Primer extension analysis identified two transcription start sites located 26 and 27 nucleotides upstream of the ywhE translational initiation codon. Sequences located in the -10 and -35 regions relative to the transcription start sites showed good homology to the consensus sequences for promoter elements of sigma(F)-dependent genes. An insertional mutation in ywhE had no significant effect on growth, morphology, and sporulation, and ywhE spores had normal heat-resistance, cortex structure, and germination and outgrowth properties. However, overexpression of ywhE in Escherichia coli resulted in cell lysis.
