## 2 Results
### Antisense Transcription in S. ambfaciens  
Although it was initially considered non-functional transcriptional noise, antisense transcription is increasingly considered important in regulating gene expression [10]. This assumption is corroborated by the high phylogenetic conservation of several asRNAs. In _S. ambfaciens_ we detected 119 asRNAs putatively expressed from a dedicated promoter (asRNAs I) targeting 146 mRNAs (Supplementary Table S4). Two of them were antisense to tRNAs, seven to pseudogenes, three to other ncRNA, and two to mixed feature types. The remaining 133 targeted newly defined gene extensions (UTR and within_operon regions) (19), CDS (83), and a mix of the three (31). Only 21 antisense_ncRNA accounted for less than 90% of their length for the target TUs. Functional enrichment analysis by Clusters of Orthologous Groups (COGs) demonstrated that asRNA-targeted CDSs, with respects to whole-genome CDSs, showed a prevalence in the COGs J (translation, ribosomal structure, and biogenesis), F (nucleotide transport and metabolism), and C (energy production and conversion) (Figure 4). GlassGO analysis revealed that about 30% of class I asRNAs were specific to _S. ambefaciens_: 10% of the sequences (12 out of 119) were identified only in _S. ambefaciens_ ATCC 23877 strain while 20% of the sequences (24 out of 119) were identified in both ATCC 23877 and DSM 40697 strains. The remaining 70% of the sequences appeared to be evolutionarily conserved: 56.3% were present in the _Streptomyces_ genus, and 13.4% in both _Streptomyces_ and other genera (Supplementary Table S11). Although this tool does not allow assess whether an ncRNA is expressed or not, the results are accurate (see Section 4.7 of the Materials and Methods). Among asRNA-targeted mRNAs of genes involved in energy production and conversion, we found _gltA_ (RS15735) coding for citrate synthase 2, _nuoF_ (RS20780), _nuoL2_ (RS21040), and _nuoM2_ (RS21045). _nuoF_, _nuoL2_, and _nuoM2_ code for subunits of the NADH-quinone oxidoreductase multi-protein complex (the respiratory complex I) (Supplementary Table S4). Like other streptomyces, _S. ambefaciens_ possesses a complete 14-subunit encoding _nuo_ operon (_nuoA-N_), and an additional copy of many _nuo_ genes (_nuoA2_, _nuoB2_, _nuoD2_, _nuoH2_, _nuoL2_, _nuoK2_, _nuoM2_, and _nuoN2_). While _nuoD2_ is separated from the other _nuo_ genes, the other ones, i.e., _nuoA2_, _nuoB2_, _nuoH2_, _nuoD2_, _(_Ynuo)  
Figure 4: Functional classification of CDSs arranged in mononcistronic or polyistronic transcripts and CDSs targeted by asRNAs (I, II, and III). CDSs that are predicted targets of asRNAs I, asRNAs II, and asRNAs III are classified according to Clusters of Orthologous Groups (COGs). As references, COGs classification of whole genome CDSs, and CDSs in mononcistronic and polyistronic transcriptional units are also shown. Genes with unknown functions are excluded from the representation. _nuoK2_, _nuoL2_, _muoM2_, and _nuoN2_ are clustered together in an operon (_nuo2_). Interestingly, the asRNAs targeting _nuoF_, _nuoL2_, and _nuoM2_ are evolutionarily conserved in streptomycetes [20]. Two asRNAs that appear to be conserved in _Streptomyces_, albeit not described so far in this genus, map in the biosynthetic gene cluster for aminobacteriahopanetriol (a C35 homenoid), while another asRNA is transcribed complementary to _dnAK_ mRNA. We found a remarkable number of asRNAs having as putative targets, mRNAs of genes involved in translation, ribosomal structure, and biogenesis. Overall, these genes account for 16% of all targets, including: i.) ten 305 ribosomal protein-encoding genes _rpsA_ (S1) (RS09750), _rpsF_ (S6) (RS17780), _rpsH_ (S8) (RS21400), _rpsI_ (S9) (RS21495), _rpsL_ (S12) (RS21225), _rpsM_ (S13) (RS21455), _rpsM2_ (S13) RS07055, _rpsO_ (S15) (RS25835), _rpsP_ (S16) (RS25395), and _rpsT_ (S20) (RS12280); ii.)