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 transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/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 Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and 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) 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 reverse transcriptase quantitative PCR (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 the 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.

Keywords: Bacillus subtilis; Bacillus thuringiensis; Clostridioides; Geobacillus; mRNA; spores.

FIG 1

Coverage of reads in RNA-seq for one high abundance (left) and one lower abundance mRNA (right) from among the 60 most abundant spore mRNAs in all six species studied as shown in Table S1 in the supplemental material. Coverage was determined as described in Materials and Methods, and the encoded protein and its location in the genome are shown above each panel. The left side of all panels gives the numbers of reads along the gene.

FIG 2

RT-qPCR analysis on cDNA libraries from B. subtilis and C. difficile spore mRNAs using primer pairs at both the 5′ and 3′ mRNA termini and in the mRNAs’ more internal regions. (A) Schematic for primer binding sites relative to the ribosomal binding site (RBS) and rho-independent terminator of the transcripts. Each condition was completed in biological and technical triplicate. However, biological replicate 2 with the ytzL internal forward and external reverse primer pair was excluded based on the negative control. The results represent mean values of normalized starting quantities from combined biological and technical triplicates. The error bars represent standard deviation (SD). Relative abundance of amplicons in B. subtilis (B) and C. difficile (C) produced using the four different primer sets. The relative abundance of each RT-qPCR amplicon was determined as described in Materials and Methods. P values were determined by a paired two-tailed t test comparing normalized starting quantities to the normalized full-length transcript; ns, not significant at P > 0.05; *, significant at P ≤ 0.05; **, significant at P ≤ 0.01; ***, significant at P ≤ 0.001; ****, significant at P ≤ 0.0001.