## 3 Discussion and Conclusions  
In this study, the redefinition of monocistronic and polycistronic transcription units, and other structural elements including 5'- and 3'-UTR and intercistronic regions of the _S. ambofaciens_ ATCC 23877 transcriptome was preliminary to the identification of novel sRNAs and asRNAs. We identified 45 sRNAs and 119 asRNAs (asRNAs I) that are predicted  
Figure 7: Differential expression of sRNAs and asRNAs of class I in S. _ambofaciens_ ATCC 23877 (w.t. ), and derivative \(\Omega\)pirA and \(\Omega\)pirA rpoB(R) mutants at 48 h (left panel) and 120 h (right panel). (Supplementary Table S13). Log2 fold changes of the two mutants compared to the w.t. strain were reported in the plots as indicated by the labels. to be transcribed from dedicated promoters. Some sRNAs were previously identified in _Streptomyces_. Other sRNAs are novel, and bioinformatic analysis predicted several mRNAs encoding proteins involved in the control of mycelial growth, morphological differentiation, secondary metabolism, and stress responses, which may be targeted by these sRNAs. Indeed, most putative sRNA targets code for key proteins involved in transcription control including sigma and transcriptional factors. Among the sRNAs that were predicted to target genes encoding transcriptional factors, it may be worth noting the cases of the sRNAs targeting the pleiotropic Lrp and AcrR paralogs. Specifically, the Lrp RS15615 was predicted to be targeted by 3 sRNAs. Lrp RS15615 is the homolog of SCO3361 in _S. coelicolor_ A3(2). SCO3361 functions as a pleiotropic regulator controlling secondary metabolism and morphological development [62]. In particular, it activates actinorhodin (Act) production by directly binding to actII-ORF4 promoter, and it stimulates the expression of _amf_C, _whiB_, and _ssgB_ thus promoting hyphae formation and sporulation [62]. Phenylalanine and cysteine were identified as the effector molecules of SCO3361, with phenylalanine reducing the binding affinity, whereas cysteine increasing it [62]. In this regard, one might note that 3 Hfq-dependent sRNAs, namely, DsrA, MicF, and GcvB, each independently downregulate the _lrp_ transcript in _E. coli_[63]. MicF and DsrA interact with an overlapping site early in the _lrp_ ORF, while GcvB acts upstream in the long lrp 5\({}^{\prime}\)-UTR [63]. In particular, GcvB was responsible for _lrp_ downregulation in response to oxidative stress. Four AcrR paralogs appeared to be, instead, each targeted by a distinct sRNA. AcrR is a one-component allosteric repressor of the genes associated with lipid transport and antibiotic resistance. AcrR contains a C-terminal ligand-binding domain and an N-terminal operator-binding region. When fatty acid ligands bind to the C-terminal domain, a conformational change in the N-terminal domain is triggered, which releases the repressed DNA and initiates transcription [64]. Among the sRNAs that were predicted to target genes involved in the biosynthesis of cellular alarmones, one might note sRNA #2 targeting two paralogs coding for diadenosine tetraphosphate (Ap4A) hydrolase (RS00910 and RS35245). Ap4A is a product of the back reaction of the amino acid activation catalyzed by some aminoacyl-tRNA [65]. While on one hand, Ap4A may be considered an unavoidable and toxic by-product of protein synthesis that has to be cleared from the cell, on the other hand, it can function as a signal molecule and be deeply involved in the regulation of DNA replication, cell division, and stress response [66]. Ap4A hydrolases catalyzes the hydrolysis of Ap4A into two ADP and are involved in heat shock and oxidative stress responses in bacteria by regulating the intracellular Ap4A concentration [67]. Regarding the asRNAs that we detected in our study, some of them are also well conserved in _Streptomyces_, including the subgroup transcribed complementary to mRNAs coding for several subunits of the respiratory complex I. In particular, the asRNAs targeting _muoF_, _muoL2_, and _muoM2_ are evolutionarily conserved in streptomyces [20].