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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 similar to 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 similar to 10(6) 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, sigma(G), 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 degrees 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
similar to 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 degrees C. These findings will have a major
impact on thinking about the roles that spore mRNAs may play in spore
revival.