## 2 Results
### Expression of ncRNAs in S. ambefaciens ATCC 23877, and Derivative _CpirA_ and _CpirA_  
_rpoB(R) Mutants_  
With the aim of gaining some insight into the possible biological effects of predicted ncRNAs, we analyzed their expression levels in three _S. ambefaciens_ strains: the wild type ATCC 23877 strain (hereafter indicated as w.t. ), an isogenic _pirA_-defective mutant (here referred to as \(\Omega\)pirA) that is characterized by central carbon and energy metabolism imbalance, high sensitivity to oxidative injury, and repression of polyketide antibiotic production [35], and a _pirA_-defective derivative strain harboring a stringent RNA polymerase exhibiting partial suppression of _pirA_-defective phenotypes (here referred to as \(\Omega\)pirA rpoB(R)). Specifically, in the \(\Omega\)pirA strain the integrative plasmid pTYM-18 (a \(\Phi\)C31 Att/Int system-based vector) inactivates _pirA_, encoding a redox-sensitive negative modulator of very long-chain acyl-CoA dehydrogenase, which catalyzes the first committed step of the beta-oxidation pathway [35]. In \(\Omega\)pirA rpoB(R) the integrative plasmid pTYM-18 was replaced by pTYM-rpoB(R), a pTYM-18 derivative harboring the _rpoB_(R) gene of _Nonomuraa gerenzanensis_ ATCC 39727. _N. gerenzanensis_ possess two RNA polymerase beta-chain genes, _rpoB(S)_ (the wild type _rpoB_ gene), and _rpoB(R)_ (the mutant type _rpoB_ gene) [59]. RpoB(R) harbors a specific histidine-to-asparagine substitution in the rifampicin resistance cluster I, which was believed to be essential for the activation of the secondary metabolism by mimicking a \"stringent\" phenotype [36; 37], and, indeed, we found that _rpoB(R)_ in _S. ambofaciens_\(\Omega\)pirA increased spiramycin (in YS medium) (Figure 5a,b) and antimycin (in SFM medium) (Figure 5c,d) production. This effect may be a consequence of _rpoB(R)_-mediated phenotypic suppression of the \"relaxed\" phenotype exhibited by \(\Omega\)pirA strain that was characterized by upregulation of genes involved in translation, ribosomal structure and biogenesis, and amino acid biosynthesis, and downregulations of _spoT_, encoding the enzyme involved in (p)ppGpp biosynthesis [35]. Partial suppression of _pirA_-associated phenotypes was also evident at the transcriptome level (Supplementary Table S12). In these experiments, w.t., \(\Omega\)pirA, and \(\Omega\)pirA rpoB(R) were cultivated in YS broth. In this medium, growth rates during rapid growth phase 1 (RG1), and final biomass values of \(\Omega\)pirA were slightly lower as compared to w.t., while in \(\Omega\)pirA rpoB(R) and w.t. these parameters were similar (Figure 5a). This finding was also supported by maSigPro analysis of RNA-Seq time series dataset that identified the expression pattern of 1166 of the most variable genes during the time course subdivided into 9 clusters (Supplementary Table S12). The median profile of each cluster  
Figure 5: Growth curves and antibiotic production in _S. ambofaciens_ ATCC 23877 (w.t. ), and derivative \(\Omega\)pirA, and \(\Omega\)pirA rpoB(R) mutants. (**a–d**) Strains were cultivated in either YS (**a,b**) or SFM (**c,d**) broths, and biomass (**a,c**), spiramycin (**b**), and antimycin (**d**) values were determined at different time points. The values were calculated as the mean of three or more experiments. The errors bar indicates the standard deviation. was inferred from the expression patterns and was used to represent the expression profile of the strains (Figure 6). For most of the clusters, the expression profile of \(\Omega\)pirA rpoB(R) shows an intermediate profile between w.t. and \(\Omega\)pirA if not a complete rescue of either the expression levels, time course profile, or both, as in cluster 8 and in cluster 4, supporting the experimental data. The growth of _Streptomyces_ follows a biphasic curve in which two phases of rapid growth (RG1, RG2), interspersed by a transition phase (T), precede the stationary phase (S).