## 2 Results
### Expression of ncRNAs in S. ambefaciens ATCC 23877, and Derivative _CpirA_ and _CpirA_
ncRNA levels were compared in w.t., \(\Omega\)pirA, and \(\Omega\)pirA rpoB(R) at two corresponding time points: 48 h, RG1 and 120 h, stationary phase. Results demonstrated that 21 (out of 45, i.e., about 47%) sRNAs and 72 (out of 119, i.e., about 70%) asRNAs (asRNAs I) were differentially expressed at least in one strain when compared to the w.t. (Supplementary Table S13). The plots in Figure 7 show log2 fold changes (log2 FC) of the mutants compared to w.t. at 48 and 120 h. The data indicated that at 48 h more ncRNAs were differentially expressed in \(\Omega\)pirA rpoB(R) with respect to 120 h (42 and 16, respectively), while for \(\Omega\)pirA the number was similar (55 and 52, respectively). At 48 h the cloud was shifted up toward \(\Omega\)pirA rpoB(R) indicating that, in general, the delta in expression level was higher for this strain, while at 120 h this was not true anymore. At both time points we could notice that the greatest part of differentially expressed genes was not propor  
Figure 6: Time-course cluster analysis of RNAseq data in _S. ambofaciens_ ATCC 23877 (w.t., red), and derivative \(\Omega\)pirA (green), and \(\Omega\)pirA rpoB(R) (blue) mutants. maSigPro identified the expression pattern of 1166 variable genes during the time course subdivided into 9 clusters (Supplementary Table S12) and median profile of each cluster was inferred from the expression patterns. tional between the two mutants (not within the red lines) but tended to be differentially expressed only in \(\Omega\)pirA genotype. sRNAs are generally equally distributed between differentially up and downregulated features, apart from \(\Omega\)pirA rpoB(R) at 120 h which shows only 3 upregulated sRNAs (Supplementary Table S13). asRNAs showed clear trends of downregulation in mutant strains (85% to 97% of the differentially expressed features, Supplementary Table S13). Furthermore, \(\Omega\)pirA rpoB(R) had an opposite trend at 48 h with 62% of upregulated sRNAs. In the \(\Omega\)pirA strain, a marked downregulation of all asRNAs I targeting genes involved in translation, ribosomal structure and biogenesis (i.e, asRNA #14, asRNA #20, asRNA #27, asRNA #28, asRNA #65, asRNA #66, asRNA #67, asRNA #68, asRNA #82, and asRNA #84) may be noted, while in \(\Omega\)pirA rpoB(R) expression of these asRNAs was restored to w.t. levels (Supplementary Table S13). asRNAs that are transcribed complementary to tRNAMet (CAU) (asRNA #63) were also downregulated in the \(\Omega\)pirA strain, and their expression was restored to w.t. levels in \(\Omega\)pirA rpoB(R). In contrast, asRNA #94, which is transcribed complementary to tRNACys (GCA), tRNALeu (CAG), and tRNAPro (UGG), was more expressed in \(\Omega\)pirA with respect to the w.t. strain. As for asRNAs I targeting genes involved in secondary metabolism, we found that asRNA #119 was less expressed in \(\Omega\)pirA than in both w.t. and \(\Omega\)pirA rpoB(R). This asRNA is transcribed complementary to _afsA_ coding for A-factor biosynthesis protein AfsA. Expression of asRNA #76, which was transcribed complementary to _whiB_, exhibited a similar trend. This asRNA was more expressed in w.t. than in \(\Omega\)pirA, and even more in \(\Omega\)pirA rpoB(R). In addition, asRNA #87, which is transcribed complementary to _smrB_ in the spiramycin gene cluster, was also more expressed in w.t. than in \(\Omega\)pirA, even more in \(\Omega\)pirA rpoB(R) at earlier time point. _smrB_ encodes for ribosomal protection factor that is involved in spiramycin resistance [60; 61]. Therefore, it would be interesting to understand if there is a link between spiramycin production and expression of asRNA #87.