## 3 Discussion and Conclusions
Expression levels of these genes are strictly dependent on tRNA\({}^{\text{Leu}}\) (UAA) levels, which increase during the stringent response [74]. In _S. ambofaciens_, UUA codons are present in _srmR_ (RS26685) (containing 1 UUA codon) and _srmS_ (RS26595) (containing 3 UUA codons) genes encoding two pathway-specific regulators of the spiramycin biosynthetic cluster. It is presumable that tRNA\({}^{\text{Pro}}\) (UGG) may have a similar regulatory function on secondary metabolism and morphological differentiation. This hypothesis is supported by a considerable higher frequency of this codon in some CDSs for proteins involved in secondary metabolism and morphological development control, including _bldG_ (RS24355) (Supplementary Figure S2). The existence of an asRNA that is transcribed complementary to the tRNA\({}^{\text{Pro}}\) (UGG) gene could add complexity to this putative regulatory level. The association between antisense transcription and _Streptomyces_ developmental cycle seems to be supported by the evidence of many putative asRNAs that are transcribed complementary to genes coding for regulatory proteins that oversee morphological and biochemical differentiation. In particular, two asRNAs are transcribed complementary to _phoR_ and _whiB_, respectively. PhoR is the sensor kinase of the two-component PhoR-PhoP system that controls both primary and secondary metabolism in _Streptomyces_ in response to phosphate availability [52], while the regulatory protein WhiB, which belongs to the Wbl family of proteins that are characterized by an (4 Fe-4S) iron-sulfur cluster, is essential for sporulation septation. By acting in concert with WhiA, WhiB halts the aerial growth to initiate the septation event at the aerial mycelial tip, and chromosome partition into the spores [53; 54]. In _S. coelicolor_ A3(2) WhiA and WhiB function cooperatively to control the expression of a common set of genes organized in about 240 transcriptional units [54]. Both _whiA_ and _whiB_ transcription is subject to negative repression by BldD when this regulatory protein is associated with c-di-GMP, whose levels are sustained high during vegetative growth [75]. Some asRNAs appear to also target genes coding for proteins involved in the control of secondary metabolite biosynthesis, and, in particular, spiramycin production and resistance. An asRNA is transcribed complementary to _smrR_ mRNA. SmrR is a key transcriptional activator of the spiramycin gene cluster [55; 56]. SmrR acts by activating, in turn, the pathway-specific transcriptional activator SrmS that controls most of the spiramycin biosynthetic genes [56]. SrmR is characterized by Hsp70 superfamily and GAF superfamily domain at the N-terminus, and PucR-like helix-turn-helix domain (HTH_30) at the C-terminus. The GAF domain is a type of protein domain that was so named after its initial discovery in cGMP-specific phosphodiesterase, adenylyl cyclases, and EthA (formate hydrogen lyase transcriptional activator). This universal domain is responsible for binding allosteric regulatory molecules such as the second messenger cyclic nucleotides cGMP and cAMP [76], although in some bacterial proteins the GAF domain was shown to contain haem [77] or a non-haem mononuclear iron center [78] enabling them to sense molecular oxygen or nitric oxide, a second messenger gaseous compound whose importance in the physiological transitions that led to morphological differentiation in _Streptomyces_ is increasingly apparent [79]. Another asRNA is transcribed complementary to _smrB_ mRNA _smrB_ gene (RS26680) is adjacent to _smrR_ (RS26685) and transcribed in convergent direction and encodes an ABC-F type ribosomal protection protein [61], SrmB, that is involved in spiramycin resistance [60]. Further work is required to understand if and to which extent these asRNAs, targeting _smrR_ and _smrB_, may contribute to either regulation of spiramycin production, resistance, or both. The availability of a catalog of sRNAs and asRNAs in _S.