Genomic Insights into Evolution of AdpA Family Master Regulators of Morphological Differentiation and Secondary Metabolism in Streptomyces

Abstract

The AdpA protein from a streptomycin producer Streptomyces griseus is a founding member of the AdpA family of pleiotropic regulators, known to be ubiquitously present in streptomycetes. Functional genomic approaches revealed a huge number of AdpA targets, leading to the claim that the AdpA regulon is the largest one in bacteria. The expression of adpA is limited at the level of translation of the rare leucyl UUA codon. All known properties of AdpA regulators were discovered on a few streptomycete strains. There are open questions about the true abundance and diversity of AdpA across actinobacterial taxa (and beyond) and about the possible evolutionary forces that shape the AdpA orthologous group in Streptomyces. Here we show that, with respect to the TTA codon, streptomycete adpA is more diverse than has been previously thought, as the genes differ in presence/position of this codon. Reciprocal best hits to AdpA can be found in many actinobacterial orders, with a domain organization resembling that of the prototypical AdpA, but other configurations also exist. Diversifying positive selection was detected within the DNA-binding (AraC) domain in adpA of Streptomyces origin, most likely affecting residues enabling AdpA to recognize a degenerate operator. Sequence coding for putative glutamine amidotransferase (GATase-1) domain also shows signs of positive selection. The two-domain organization of AdpA most likely arose from a fusion of genes encoding separate GATase-1 and AraC domains. Indeed, we show that the AraC domain retains a biological function in the absence of the GATase-1 part. We suggest that acquisition of the regulatory role by TTA codon is a relatively recent event in the evolution of AdpA, which coincided with the rise of the Streptomycetales clade and, at present, is under relaxed selective constraints. Further experimental scrutiny of our findings is invited, which should provide new insights into the evolution and prospects for engineering of an AdpA-centered regulatory network.

Subtilisin-Involved Morphology Engineering for Improved Antibiotic Production in Actinomycetes

“…The recurrent fragmentation of A. pretiosum ATCC 31280 in early growth phase suggests an involvement of quorum sensing-like regulation ( Figure 1 B). The γ-butyrolactone system is the primary quorum sensing system in actinomycetes, and the widespread A-factor-dependent proteins (AdpA) mediate the regulatory signals of various γ-butyrolactone molecules and exert pleiotropic regulation on morphology differentiation and secondary metabolism [ 44 , 45 ]. Therefore, the upstream region of APASM_4178 was analyzed, and two conserved AdpA-binding motifs, 5′-TGACGGGGAG-3′ and 5′-CGCGCCGCCA-3′ (in reversed orientation), were identified ( Figure 3 A) [ 46 ].…”

Gene miaA for post‐transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces

“…A synthetic XNR_4181 gene was ordered (General Biosystems Inc., USA), where a TTA codon (673‐675 bp) was replaced with the synonymous CTC codon. Synthetic gene was amplified with primers xnr4181_XbaIup and xnr4181_EcoRIrp (Rabyk et al , ), digested with XbaI and EcoRI restriction endonucleases and subcloned into SpeI/EcoRI recognition sites of pmoeE5script, yielding pGM4181tta − . The plasmid was verified with restriction mapping and sequencing.…”

“…Both processes are under the control of pleiotropic TF AdpA (Higo et al , ). In a vast majority of Streptomyces strains the adpA gene carries TTA codon (Rabyk et al , ), and so translation of the adpA transcript might be a focal point of bldA ‐dependent control of morphogenesis and antibiotic production (Higo et al , ). However, it remains unknown what factors limit the accumulation of mature tRNA Leu UAA .…”

Eliciting the silent lucensomycin biosynthetic pathway in Streptomyces cyanogenus S136 via manipulation of the global regulatory gene adpA

“…The overexpression of such a promiscuous TF could enforce binding to numerous (probably unrecognizable by AdpA under physiological conditions) sites on the chromosome, impacting the expression of multiple genes and operons. The biosynthesis of many antibiotics in streptomycetes is AdpA-regulated and AdpA TFs are almost identical across different Streptomyces species and therefore likely display the same properties 35 . AdpA-binding sites are common in silent BGCs and overexpression of adpA genes could be used for high-throughput activation of such BGCs in strains of unknown genomic background.…”