Dataset Information

Function of the SpoVAEa and SpoVAF proteins of Bacillus subtilis spores.

ABSTRACT: The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developing spores as members of the spoVA operon, which encodes proteins essential for the uptake and release of dipicolinic acid (DPA) during spore formation and germination. SpoVAF is likely an integral inner spore membrane protein and exhibits sequence identity to A subunits of the spore's nutrient germinant receptors (GRs), while SpoVAEa is a soluble protein with no obvious signals to allow its passage across a membrane. However, like SpoVAD, SpoVAEa is present on the outer surface of the spore's inner membrane, as SpoVAEa was accessible to an external biotinylation agent in spores and SpoVAEa disappeared in parallel with SpoVAD during proteinase K treatment of germinated spores. SpoVAEa and SpoVAD were also distributed similarly in fractions of disrupted dormant spores. Unlike spoVAD, spoVAEa is absent from the genomes of some spore-forming members of the Bacillales and Clostridiales orders, although SpoVAEa's amino acid sequence is conserved in species containing spoVAEa. B. subtilis strains lacking SpoVAF or SpoVAEa and SpoVAF sporulated normally, and the spores had normal DPA levels. Spores lacking SpoVAF or SpoVAEa and SpoVAF also germinated normally with non-GR-dependent germinants but more slowly than wild-type spores with GR-dependent germinants, and this germination defect was complemented by ectopic expression of the missing proteins.

SUBMITTER: Perez-Valdespino A

PROVIDER: S-EPMC4010985 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Function of the SpoVAEa and SpoVAF proteins of Bacillus subtilis spores.

Perez-Valdespino Abigail A Li Yunfeng Y Setlow Barbara B Ghosh Sonali S Pan David D Korza George G Feeherry Florence E FE Doona Christopher J CJ Li Yong-Qing YQ Hao Bing B Setlow Peter P

Journal of bacteriology 20140328 11

The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developing spores as members of the spoVA operon, which encodes proteins essential for the uptake and release of dipicolinic acid (DPA) during spore formation and germination. SpoVAF is likely an integral inner spore membrane protein and exhibits sequence identity to A subunits of the spore's nutrient germinant receptors (GRs), while SpoVAEa is a soluble protein with no obvious signals to allow its passage across a membrane. However ...[more]

PMID: 24682327

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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.

Dataset Information

Function of the SpoVAEa and SpoVAF proteins of Bacillus subtilis spores.

Publications

Function of the SpoVAEa and SpoVAF proteins of Bacillus subtilis spores.

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