Department of Molecular Biology and Biophysics, UConn Health, Farmington, Connecticut, USA.
Department of Medicine, UConn Health, Farmington, Connecticut, USA.
J Bacteriol. 2021 Jun 22;203(14):e0001721. doi: 10.1128/JB.00017-21.
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. 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 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.
芽孢杆菌物种的孢子中含有数百个 mRNA，其在枯草芽孢杆菌中的主要功能是在孢子发芽时为新 RNA 合成提供核苷酸。为了确定这是否是普遍现象，我们从多种芽孢杆菌物种的孢子中分离 RNA，并通过转录组测序 (RNA-seq) 确定相对 mRNA 水平。通过对单个孢子中的 RNA 水平进行测定，可以计算出 RNA 核苷酸/孢子，并假设 mRNA 占孢子 RNA 的 3%，这表明只有约 6%的孢子 mRNA 存在于 >1/孢子。枯草芽孢杆菌、萎缩芽孢杆菌和艰难梭菌孢子中 >1/孢子的 mRNA 分别为 49、42 和 51 个，而耐热脂肪芽孢杆菌和苏云金芽孢杆菌 Al Hakam 孢子中 >1/孢子的 mRNA 数量增加了 10%至 50%，而巨大芽孢杆菌孢子中的 mRNA 数量增加了约 4 倍。在所有物种中，一些到许多丰富的孢子 mRNA（i）由具有前孢子特异性 σ 因子的 RNA 聚合酶转录，（ii）编码与枯草芽孢杆菌丰富的孢子 mRNA 编码的蛋白质同源的蛋白质，并且是休眠孢子中的蛋白质，并且（iii）可能在产孢细胞的母细胞区室中转录。对来自所有物种的 RNA-seq reads 在 mRNA 上的覆盖度进行分析表明，丰富的孢子 mRNA 被碎片化，这通过对枯草芽孢杆菌和艰难梭菌丰富的孢子 mRNA 的逆转录定量 PCR (RT-qPCR) 分析得到证实。这些数据增加了证据表明，至少在所有芽孢杆菌孢子中的绝大多数 mRNA 的功能是在孢子发芽时降解以生成用于新 RNA 合成的核苷酸。六个芽孢杆菌物种的孢子中只有约 6%的 mRNA 存在于 ≥1 分子/孢子，许多丰富的孢子 mRNA 编码与枯草芽孢杆菌孢子蛋白相似的蛋白质，并且一些丰富的枯草芽孢杆菌和艰难梭菌孢子 mRNA 被碎片化。大多数丰富的枯草芽孢杆菌和其他孢子 mRNA 是在前孢子特异性 RNA 聚合酶 σ 因子 F 或 G 的控制下转录的，这些结果可能会刺激除枯草芽孢杆菌以外的物种的发育孢子中的转录分析。这些发现，加上孢子中缺乏关键核苷酸生物合成酶，表明芽孢杆菌孢子中的丰富 mRNA 不会在孢子发芽时翻译，而是在发芽的孢子中降解以产生用于新 RNA 合成的核苷酸。
