Levels and Characteristics of mRNAs in Dormant Spores of Firmicute Species

Spores of Bacillales and Clostridiales species contain 100s of different mRNAs, and their major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. In new work, RNA was isolated from spores of five Bacillales and one Clostridioides species and relative spore mRNA levels were 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 allowed calculation 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, respectively. Numbers of mRNAs at ≥1/spore were ~10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores, respectively, and ~ 4-fold higher in Bacillus megaterium spores. Notably, in all species: i) many of the 60 most abundant spore mRNAs were transcribed by RNA polymerase with forespore-specific s factors; ii) some to many of the most abundant spore mRNAs encoded  orthologs of those encoded by abundant B. subtilis spore mRNAs and proteins present in dormant spores ; and iii) some spore mRNAs were likely transcribed in the mother cell compartment of the sporulating cell. Indeed , analysis of the coverage of RNA-seq reads on mRNAs from all six species suggested that abundant spore mRNAs were at least somewhat fragmented. This observation was confirmed by RT-qPCR analysis of three abundant mRNAs each from B. subtilis and C. difficile spores. These data add to a growing body of evidence indicating that the great majority of mRNAs in spores of Firmicutes are degradation and function as a ribonucleotide depot for new RNA synthesis when spores germinate. RNA from spores of 5 different Bacillales and 1 Clostridiales were isolated and submitted for next generation sequencing via illumina HiSeq.

芽孢杆菌目（Bacillales）与梭菌目（Clostridiales）物种的芽孢内含有数百种不同的信使RNA（mRNA），在枯草芽孢杆菌（Bacillus subtilis）中，这类RNA的核心功能是在芽孢萌发时为新生RNA的合成提供核糖核苷酸。在本项最新研究中，研究人员从5株芽孢杆菌目菌株和1株艰难梭菌（Clostridioides difficile）的芽孢中分离得到总RNA，并通过RNA测序（RNA-seq）测定了芽孢内mRNA的相对丰度。通过对单个芽孢的RNA水平进行定量，可计算出每个芽孢所含的RNA核苷酸总量；若假设mRNA占芽孢总RNA的3%，则可推算出仅有约6%的芽孢mRNA在每个芽孢内的拷贝数≥1。枯草芽孢杆菌、萎缩芽孢杆菌（Bacillus atrophaeus）和艰难梭菌的芽孢中，拷贝数＞1的mRNA分别有49、42和51种。嗜热脂肪地芽孢杆菌（Geobacillus stearothermophilus）与苏云金芽孢杆菌阿尔哈姆株（Bacillus thuringiensis Al Hakam）的芽孢中，拷贝数≥1的mRNA数量分别高出约10%至50%，而巨大芽孢杆菌（Bacillus megaterium）芽孢中的该类mRNA数量则提升约4倍。值得注意的是，在所有受试物种中：其一，60种丰度最高的芽孢mRNA中，多数由识别前芽孢特异性σ因子的RNA聚合酶转录；其二，部分乃至多数丰度最高的芽孢mRNA所编码的蛋白，与枯草芽孢杆菌芽孢中高丰度mRNA及休眠芽孢内蛋白的同源物存在对应关系；其三，部分芽孢mRNA可能在产芽孢细胞的母细胞区室中完成转录。事实上，对全部6个物种的mRNA上RNA测序读段覆盖度的分析显示，丰度较高的芽孢mRNA均存在一定程度的片段化。这一结论通过对枯草芽孢杆菌和艰难梭菌芽孢各3种高丰度mRNA进行逆转录定量PCR（RT-qPCR）分析得到了验证。本研究数据进一步佐证了日益增多的研究证据：厚壁菌门（Firmicutes）芽孢中的绝大多数mRNA都会发生降解，并作为核糖核苷酸储备库，在芽孢萌发时为新生RNA的合成提供原料。研究人员已从5株不同的芽孢杆菌目菌株和1株梭菌目菌株的芽孢中分离得到总RNA，并通过Illumina HiSeq平台提交了下一代测序（NGS）样本。