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Loss of compartmentalization of ?(E) activity need not prevent formation of spores by Bacillus subtilis.

ABSTRACT: Compartmentalization of the activities of RNA polymerase sigma factors is a hallmark of formation of spores by Bacillus subtilis. It is initiated soon after the asymmetrically located sporulation division takes place with the activation of ?(F) in the smaller cell, the prespore. ?(F) then directs a signal via the membrane protease SpoIIGA to activate ?(E) in the larger mother cell by processing of pro-?(E). Here, we show that ?(E) can be activated in the prespore with little effect on sporulation efficiency, implying that complete compartmentalization of ?(E) activity is not essential for spore formation. ?(E) activity in the prespore can be obtained by inducing transcription in the prespore of spoIIGA or of sigE*, which encodes a constitutively active form of ?(E), but not of spoIIGB, which encodes pro-?(E). We infer that ?(E) compartmentalization is partially attributed to a competition between the compartments for the activation signaling protein SpoIIR. Normally, SpoIIGA is predominantly located in the mother cell and as a consequence confines ?(E) activation to it. In addition, we find that CsfB, previously shown to inhibit ?(G), is independently inhibiting ?(E) activity in the prespore. CsfB thus appears to serve a gatekeeper function in blocking the action of two sigma factors in the prespore: it prevents ?(G) from becoming active before completion of engulfment and helps prevent ?(E) from becoming active at all.

SUBMITTER: Chary VK

PROVIDER: S-EPMC2953694 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Loss of compartmentalization of σ(E) activity need not prevent formation of spores by Bacillus subtilis.

Chary Vasant K VK Xenopoulos Panagiotis P Eldar Avigdor A Piggot Patrick J PJ

Journal of bacteriology 20100827 21

Compartmentalization of the activities of RNA polymerase sigma factors is a hallmark of formation of spores by Bacillus subtilis. It is initiated soon after the asymmetrically located sporulation division takes place with the activation of σ(F) in the smaller cell, the prespore. σ(F) then directs a signal via the membrane protease SpoIIGA to activate σ(E) in the larger mother cell by processing of pro-σ(E). Here, we show that σ(E) can be activated in the prespore with little effect on sporulatio ...[more]

PMID: 20802044

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Project description:Bacillus spores incubated on plates for 2 to 98 days at 37°C had identical Ca-dipicolinic acid contents, exhibited identical viability on rich- or poor-medium plates, germinated identically in liquid with all germinants tested, identically returned to vegetative growth in rich or minimal medium, and exhibited essentially identical resistance to dry heat and similar resistance to UV radiation. However, the oldest spores had a lower core water content and significantly higher wet heat and NaOCl resistance. In addition, 47- and 98-day spores had lost >98% of intact 16S and 23S rRNA and 97 to 99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 days. Levels of 3-phosphoglyceric acid (3PGA) also fell 30 to 60% in the oldest spores, but how the 3PGA was lost is not clear. These results indicate that (i) translation of dormant spore mRNA is not essential for completion of spore germination, nor is protein synthesis from any mRNA; (ii) in sporulation for up to 91 days at 37°C, the RNA broken down generates minimal levels of mononucleotides; and (iii) the lengths of time that spores are incubated in sporulation medium should be considered when determining conditions for spore inactivation by wet heat, in particular, in using spores to test for the efficacy of sterilization regimens.IMPORTANCE We show that spores incubated at 37°C on sporulation plates for up to 98 days have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2-day spores, and all these spores had identical viability. Thus, it is unlikely that spore mRNA, rRNA, or protein synthesis is important in spore germination. Spores incubated for 47 to 98 days also had much higher wet heat resistance than 2-day spores, suggesting that spore "age" should be considered in generating spores for tests of sterilization assurance. These data are the first to show complete survival of hydrated spores for ∼100 days, complementing published data showing dry-spore survival for years.

Project description:Large-scale shotgun sequencing (RNA-seq) analysis of mRNAs in dormant Bacillus subtilis spores prepared on plates or in liquid generally found the same ∼46 abundant mRNA species, with >250 mRNAs detected at much lower abundances. Knowledge of the amount of phosphate in a single B. subtilis spore allowed calculation of the amount of mRNA in an individual spore as ∼106 nucleotides (nt). Given the levels of abundant spore mRNAs compared to those of other mRNAs, it was calculated that the great majority of low-abundance mRNAs are present in only small fractions of spores in populations. Almost all of the most abundant spore mRNAs are encoded by genes expressed late in sporulation in the developing spore under the control of the forespore-specific RNA polymerase sigma factor, σG, and most of the encoded proteins are in spores. Levels of the most abundant spore mRNAs were also relatively stable for a week at 4°C after spore harvest. RNA-seq analysis of mRNAs in highly purified and less-well-purified spores made in liquid, as well as from spores that were chemically decoated to remove possible contaminating mRNA, indicated that low-abundance mRNAs in spores were not contaminants in purified spore preparations, and several sources of low-abundance mRNAs in spores are suggested. The function of at least the great majority of spore mRNAs seems most likely to be the generation of ribonucleotides for new RNA synthesis by their degradation early in spore revival.IMPORTANCE Previous work indicates that dormant Bacillus subtilis spores have many hundreds of mRNAs, some of which are suggested to play roles in spores' "return to life" or revival. The present work finds only ∼46 mRNAs at ≥1 molecule spore, with others in only fractions of spores in populations, often very small fractions. Less-abundant spore mRNAs are not contaminants in spore preparations, but how spores accumulate them is not clear. Almost all abundant spore mRNAs are synthesized in the developing spore late in its development, most encode proteins in spores, and abundant mRNAs in spores are relatively stable at 4°C. These findings will have a major impact on thinking about the roles that spore mRNAs may play in spore revival.

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Loss of compartmentalization of ?(E) activity need not prevent formation of spores by Bacillus subtilis.

Publications

Loss of compartmentalization of σ(E) activity need not prevent formation of spores by Bacillus subtilis.

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