Transcriptomes of Frankia sp. strain CcI3 in growth transitions

Background: Frankia sp. strains are actinobacteria that form N2-fixing root nodules on angiosperms. Several reference genome sequences are available enabling transcriptome studies in Frankia sp. Genomes from Frankia sp. strains differ markedly in size, a consequence proposed to be associated with a high number of indigenous transposases, more than 200 of which are found in Frankia sp. strain CcI3 used in this study. Because Frankia exhibits a high degree of cell heterogeneity as a consequence of its mycelial growth pattern, its transcriptome is likely to be quite sensitive to culture age. This study focuses on the behavior of the Frankia sp. strain CcI3 transcriptome as a function of nitrogen source and culture age. Results: To study global transcription in Frankia sp. CcI3 grown under different conditions, complete transcriptomes were determined using high throughput RNA deep sequencing. Samples varied by time (five days vs. three days) and by culture conditions (NH4+ added vs. N2 fixing). Assembly of millions of reads revealed more diversity of gene expression between five-day and three-day old cultures than between three day old cultures differing in nitrogen sources. Heat map analysis organized genes into groups that were expressed or repressed under the various conditions compared to median expression values. Twenty-one SNPs common to all three transcriptome samples were detected indicating culture heterogeneity in this slow-growing organism. Significantly higher expression of transposase ORFs was found in the five-day and N2-fixing cultures, suggesting that N starvation and culture aging provide conditions for on-going genome modification. Transposases have previously been proposed to participate in the creating the large number of gene duplication or deletion in host strains. Subsequent RT-qPCR experiments confirmed predicted elevated transposase expression levels indicated by the mRNA-seq data. Conclusions: The overall pattern of gene expression in aging cultures of CcI3 suggests significant cell heterogeneity even during normal growth on ammonia. The detection of abundant transcription of nif (nitrogen fixation) genes likely reflects the presence of anaerobic, N-depleted microsites in the growing mycelium of the culture, and the presence of significantly elevated transposase transcription during starvation indicates the continuing evolution of the Frankia sp. strain CcI3 genome, even in culture, especially under stressed conditions. These studies also sound a cautionary note when comparing the transcriptomes of Frankia grown in root nodules, where cell heterogeneity would be expected to be quite high. Overall design: Detection of gene expression variance among Frankia HfpCci3 (Cci3) cells grown in ammonium chloride for three days, five days and HfpCci3 cells grown in nitrogen fixing conditions for three days using mRNA-seq

背景：弗兰克氏菌属（Frankia sp.）菌株属于放线菌，能够在被子植物上形成固氮根瘤。目前已有多个参考基因组序列发布，为弗兰克氏菌的转录组研究提供了支撑。弗兰克氏菌的基因组尺寸差异显著，有研究推测这与其携带大量内源转座酶（transposase）有关——本研究使用的CcI3菌株中就含有超过200个转座酶编码基因。由于弗兰克氏菌以菌丝体方式生长，存在高度的细胞异质性，其转录组很可能对培养时长极为敏感。本研究聚焦于弗兰克氏菌CcI3菌株的转录组行为，探讨其与氮源类型及培养时长的关联。
结果：为研究不同培养条件下弗兰克氏菌CcI3的全局转录情况，本研究采用高通量RNA深度测序技术完成了完整转录组的测定。实验样本设置了两个变量：培养时长（5天 vs 3天）以及培养条件（添加NH4+ vs 固氮状态）。对百万级测序读长进行组装后发现，5天龄与3天龄培养物之间的基因表达差异，比不同氮源条件下3天龄培养物之间的差异更为显著。热图分析将基因按表达模式聚类，可分为与中位表达值相比，在各类条件下被诱导表达或被抑制的基因簇。在全部3个转录组样本中均检测到21个共有单核苷酸多态性（Single Nucleotide Polymorphism, SNP），提示这种生长缓慢的微生物存在培养物异质性。在5天龄培养物及固氮状态培养物中，转座酶开放阅读框（Open Reading Frame, ORF）的表达量显著升高，这表明氮饥饿与培养老化会为基因组持续修饰提供条件。此前已有研究提出，转座酶参与宿主菌株中大量基因重复或缺失事件的形成。后续的逆转录定量PCR（RT-qPCR）实验验证了mRNA-seq数据所预测的转座酶表达上调现象。
结论：CcI3老化培养物的整体基因表达模式表明，即便在以氨作为氮源的正常生长过程中，也存在显著的细胞异质性。固氮基因（nif）的大量转录现象，大概率反映了培养物菌丝体中存在厌氧、氮缺乏的微环境；而饥饿状态下转座酶转录水平显著升高，说明即便在实验室培养条件下，弗兰克氏菌CcI3菌株的基因组也在持续演化，尤其在胁迫环境中更为明显。本研究同时也为相关研究敲响了警钟：在比较从根瘤中分离培养的弗兰克氏菌转录组时，需考虑到根瘤内本身存在高度的细胞异质性。
整体实验设计：采用mRNA-seq技术，检测三组弗兰克氏菌HfpCci3（即Cci3）样本的基因表达差异：分别为在氯化铵培养基中培养3天、培养5天的菌体，以及在固氮条件下培养3天的菌体。