Analysis of the mRNAs in Spores of Bacillus subtilis

Abstract

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.

Integrative analysis of proteome and transcriptome dynamics duringBacillus subtilisspore revival

“…We have identified 36 transcripts present abundantly in the dormant spores. This number is in close agreement with the previous findings 1,24 . These transcripts mainly belong to the genes encoding small acid soluble proteins ( sspE, sspO, sspI, sspK, sspF ) and proteins with uncharacterized functions.…”

“…Therefore, along with transporters 1 , the genes related to the processes of transcription regulation, translation, DNA replication and repair, rRNA processing, ribosome as well as inosine and uridine monophosphate (IMP-UMP) biosynthesis, cell shape and cell division are evidently upregulated in this first phase of transcription initiation. At the same time, some spore transcripts as well as the transcripts of spore associated hypothetical protein genes are seen to be downregulated (broken down) which may serve as the source of nucleotides (for the de novo nucleotide synthesis) as suggested previously 24 . In the next phase of transcription, genes belonging to the amino acid biosynthesis, protein/peptide transport are upregulated, which may later facilitate the transport of newly synthesized proteins and peptides.…”

“…In agreement with this observation, our data confirms that the transcription machinery is highly active during and beyond the phase transition period with the expression of ∼ 2400 mRNA transcripts being upregulated. This is necessary as for many transcripts <1 molecule is present in a dormant spore 24 . Therefore, along with transporters 1 , the genes related to the processes of transcription regulation, translation, DNA replication and repair, rRNA processing, ribosome as well as inosine and uridine monophosphate (IMP-UMP) biosynthesis, cell shape and cell division are evidently upregulated in this first phase of transcription initiation.…”

Woeseiales transcriptional response in Arctic fjord surface sediment

“…The transcriptional behavior observed for the Woeseiales MAGs 375 is similar to quiescence strategies noted for other organisms, such as B. subtilis and 376 Mycobacterium tuberculosis[44]. For example, the overall high transcription of a single protein 377 coupled to much lower overall mRNA diversity and abundance agrees with a recent RNA-seq 378 study on B. subtilis spores conducted byKorza et al (2019). B. subtilis spores had overall low wherein growth and metabolism are extremely depressed or put on hold entirely 399 until conditions again become optimal, has been proposed as a key strategy of life in marine 400 sediments[6, 57].…”

Properties of Aged Spores of Bacillus subtilis

“…(iv) What is the fate of the spore rRNA, which is known to be cleaved during incubation of highly purified spores at 37 to 50°C for extended periods of time? (v) Last, what are the effects of extended incubation on the stability of the dormant spore mRNAs (8)(9)(10)(11)(12)(13)(14)(15)(16)? Notably, there is one report that incubation of sporulating cultures at various temperatures following spore release from sporangia results in spores with very different rates of germination, although with essentially identical viability (8).…”