Transcription of the Bacillus subtilis gerK Operon, Which Encodes a Spore Germinant Receptor, and Comparison with That of Operons Encoding Other Germinant Receptors

Abstract

The gerA, gerB, and gerK operons, which encode germinant receptors in spores of Bacillus subtilis, were transcribed only in sporulation, and their mRNA levels peaked initially ϳ3 h before the initiation of accumulation of the spore's dipicolinic acid. After a rapid fall, levels of these mRNAs peaked again ϳ5 h later. In one wild-type strain (PS832), gerA mRNA was the most abundant, with levels of gerB and gerK mRNAs ϳ50% of that of gerA mRNA, whereas gerB mRNA was the most abundant in another wild-type strain (PY79). The synthesis of gerK mRNA in sporulation was abolished by loss of the forespore-specific RNA polymerase sigma factor, G , and induction of G synthesis in vegetative cells led to synthesis of gerK mRNA. SpoVT, a regulator of G -dependent gene expression, repressed gerK expression. The gerK promoter showed sequence similarities to G -dependent promoters, and deletion of elements of this putative promoter abolished gerK expression in sporulation.Spores of Bacillus species formed in sporulation are metabolically dormant and extremely resistant to environmental stresses (17, 27). As a consequence, these spores can survive for extremely long periods in the absence of nutrients (12,17,27). However, the spores can respond to nutrients in their environment, and when appropriate nutrients are present, the spore can rapidly return to life via spore germination followed by outgrowth (16,23,26). Spores sense nutrients via germinant receptors located in the spore's inner membrane (10,22). In Bacillus subtilis there are currently three known functional germinant receptors encoded by the gerA, gerB, and gerK operons, each of which contain three cistrons (16,23,26), and the three proteins encoded by each of these operons likely interact to form a functional germinant receptor (11,20). The GerA receptor responds to L-alanine alone, while the GerB receptor is required for the spore's response to a mixture of glucose, fructose, and K ϩ (GFK) plus either L-alanine or L-asparagine (16,23,26). The precise function of the GerK receptor is not known, but GerK is essential for the response of the GerB receptor to alanine or asparagine and GFK and for the stimulation of the GerA receptor's response to glucose (1,16,23,26). It has been suggested that GerK's major function is to respond to glucose (1).The regulation of expression of the gerA and gerB operons in B. subtilis was initially studied using transcriptional lacZ fusions to these operons (6, 9). These lacZ fusions are expressed only during sporulation in the developing forespore several hours before the forespore begins to accumulate its large depot (ϳ10% of spore dry weight) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]). Transcription of the gerA and gerB operons is largely (the gerA operon) or exclusively (the gerB operon) by RNA polymerase with the forespore-specific sigma factor, G (6, 7, 9, and see below), and the Ϫ10 and Ϫ35 sequences in the promoters of the gerA and gerB operons conform to those in G -dependent promoters. Studies of genes expressed ...

Numbers of Individual Nutrient Germinant Receptors and Other Germination Proteins in Spores of Bacillus subtilis

“…Together with the nearly 1:1 ratio of GerAA to GerAC proteins determined in the current work, these findings suggest that the A and C subunits of GRs are most likely present in a 1:1 ratio. The ϳ1.5-fold-higher levels of GerA GR subunits than GerB subunits or GerKA in spores are also consistent with the higher levels of ␤-galactosidase accumulated in spores from a transcriptional gerA-lacZ fusion than from a comparable gerB-lacZ fusion and also with the ϳ2-fold-higher levels of gerA mRNA than of gerB and gerK mRNAs (31)(32)(33). The finding that GerA is the most abundant GR is also consistent with the major effect that loss of this GR alone has on the germination of B. subtilis spores by a pressure of ϳ150 MPa, which triggers spore germination solely via direct activation of the spores' GRs (34).…”

Localization of the Germination Protein GerD to the Inner Membrane in Bacillus subtilis Spores

“…The GerD sequence also does not resemble that of any of the nutrient receptors. Transcription of gerD takes place only in the developing forespore compartment of the sporulating cell and is directed by the forespore-specific RNA polymerase sigma factor, G , as is transcription of the gerA, gerB, and gerK operons (9,10,15,30). Although components of the GerA and GerB receptors have been localized to the spore's inner membrane, the location of GerD is not yet known (7,23).…”

Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly

“…It is widely accepted that the germination commitment is irreversible once germinant receptors receive the signal from their germinants, and this commitment cannot be aborted by removing the germinants or by blocking the germinant receptors (42). It appears unlikely that these germinant receptors were still stored in the spore inner membrane since they are not required for vegetative growth and were expressed only under the control of the sporulation sigma factor SigG (47)(48)(49). We hypothesize that the GerKB germinant receptors were degraded as part of the commitment to germination, possibly by the AAA protease FtsH, which showed higher levels of detectability in germinated spores.…”