## 2 Results  
### S. ambofaciens Transcriptome: Structure and Organization  
The transcriptome of _S. ambofaciens_ ATCC 23877 linear chromosome was redefined from RNAseq data of bacteria growing in YS broth at different time points (48 h, 72 h, 96 h, and 120 h) based on Rockhopper software [38; 39; 40] predictions. The original 7229 genes located on the linear chromosome were structured in 5587 transcriptional units (TUs). Furthermore, 4433 TUs were monocistronic while 1154 were polycistronic, with a maximum of 23 genes included in a single unit (Table 1, and Supplementary Table S1). Only 53% of the originally defined genes (2944) retained the exactly original annotation while, for the other genes, either new 5\({}^{\prime}\)-UTR, 3\({}^{\prime}\)-UTR, or both, sequences, or polycistronic arrangements were individuated. The new annotation increased the portion of linear chromosome nucleotides included in the transcriptome from 87% to 90.8%. This 3.8% increase leads to a doubling of the mapped reads falling into annotated transcripts, which increase from 45% to 94% on average on _S. ambofaciens_ ATCC 23877 strain. This gain is even more impressive looking at the 48 h timepoint (Supplementary Table S2). Both polycistronic and monocistronic transcription units appeared to be uniformly distributed along the linear chromosome of _S. ambofaciens_ as shown in Figure 1 that illustrates the transcription units as a function of their lengths and locations in the genome map. The median and length range of these transcription units are reported in Table 1. Some monocistronic and polycistronic transcription units largely exceeding the average length were located near the chromosome \"right arm\" (Figure 1). Other large monocistronic transcription units mapped to a specific \"core\" region of the _S. ambofaciens_ chromosome where the spiramycin gene cluster and additional gene clusters for secondary metabolites are located. Computational analysis also reveals 162 new transcripts putatively originated from dedicated promoters. We classified them into sRNAs (Supplementary Table S3) and asRNAs (herein referred as asRNAs I) according to the fact that, based on our re-annotation, they overlap on the opposite strand of another gene (Figure 1, and Supplementary Table S4). Furthermore, we identified asRNAs resulting from divergent transcription (herein referred to asRNAs II) (Supplementary Table S5) and asRNAs resulting from the overlap of the terminal region of convergently oriented genes (cutoRNAs, herein referred to as \begin{table}
\begin{tabular}{c c c} \hline \hline
**Transcriptional Units** & **Number** & **Length (Median)** \\ \hline Monocistronic & 4433 & 902 \\ Polycistronic & 1154 & 2177 \\ sRNA & 45 & 100 \\ asRNA I & 119 & 166 \\ \hline
**Transcriptional Units** & **Number** & **Overlap Length (Median)** \\ \hline asRNA I & 119 & 150 \\ asRNA II & 83 & 113 \\ asRNA III (cutoRNA) & 507 & 167 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Transcriptional unit classification in _S. ambofaciens_ genome. asRNAs III) (Supplementary Table S6). A scheme of sRNA (Figure 2a), asRNA I (Figure 2b), asRNA II (Figure 2c), and asRNA III (Figure 2d) is depicted in Figure 2. Abundances and median plus length ranges of these three classes of asRNAs are reported in Table 1, while Figure 1 illustrates the asRNAs I, II, and III as a function of their lengths and locations in the genome map. For asRNA II and asRNA III, only the total overlaps between the two involved genes were considered. Most sRNAs and asRNAs I and II were mapped in the \"core\" region of the _S. ambofaciens_ linear chromosome, at variance with asRNAs III that were uniformly distributed along the chromosome. The high number of asRNA III (also known as cutoRNAs) is noteworthy, a typical feature of streptomycetes that have genomes with high GC content and weak transcription termination [20].