Abstract
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.