Levels and Characteristics of mRNAs in Spores of Firmicute Species

Abstract

Spores of Firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple Firmicute species and relative mRNA levels determined by RNA-seq. Determination of RNA levels in single spores allowed calculation of RNA nt/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at > 1/spore. Bacillus subtilis, Bacillus atrophaeus and Clostridiodes difficile spores had 49, 42 and 51 mRNAs at > 1/spore, numbers of mRNAs at ≥ 1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores, and ∼4-fold higher in Bacillus megaterium spores. In all species some to many abundant spore mRNAs: i) were transcribed by RNA polymerase with forespore-specific σ factors; ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores; and iii) a few were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by RT-qPCR analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all Firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. Importance Only ∼6% of mRNAs in spores of six Firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that Firmicute spores’ abundant mRNAs are not translated when spores germinate, but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.