Production of Avilamycin A is regulated by AviC1 and AviC2, two transcriptional activators

Abstract

Streptomycetes are typical soil-dwelling bacteria with intricate morphological and biochemical differentiation of colonies, resulting in onset of secondary metabolite production. Compounds produced during these stages of differentiation comprise nearly two-thirds of bioactive molecules synthesized by microorganisms, including antibiotics, antitumor agents and immunosuppressants.1,2 The biosynthesis of antibiotics and other secondary metabolites is controlled by interactions of both global and pathway-specific regulators. However, in all cases, the influence of the environment is reflected by the activity of pathway-specific regulatory genes, which are located within a biosynthetic gene cluster and which control the expression of biosynthetic genes.3 On account of high structural and functional similarity, such regulators were grouped into two families, namely Streptomyces antibiotics regulatory proteins (SARP), mostly found within the aromatic polyketides clusters, and large ATP-binding regulators of LuxR family (LAL), controlling the production of macrolides and glycopeptides.4–6 Recent efforts in cloning and characterization of biosynthetic gene clusters for new groups of secondary metabolites revealed novel classes of transcriptional factors that differ from typical SARPs or LAL representatives.7,8 Avilamycins are secondary metabolites produced by Streptomyces viridochromogenes Tu57. They are active against Gram-positive bacteria. It is known that avilamycins bind to the 23S rRNA in the region proximal to the channel, where tRNA enters the A-site and blocks the protein synthesis.9 Avilamycin resistance is mediated by 23S rRNA methylation and active avilamycin transport.10 The avilamycin resistance genes together with the structural genes were identified and cloned as an entire biosynthetic gene cluster containing 54 open reading frames.11 The biosynthetic steps leading to the formation of the avilamycin molecule were established by the analysis of secondary metabolites produced by mutants.11,12 Within the cluster also, two putative regulatory genes named aviC1 and aviC2 were identified. Both genes are located in close proximity to each other and are transcribed in the same direction (Figure 1). Genes are separated by a 271-bp non-coding region. We succeeded in identifying the presence of putative bacterial promoter sequences upstream of aviC1 initiation codon by the use of BPROM bacterial promoter prediction server (Softberry Inc., Mount Kisco, NY, USA). It consists of a putative 10 box GGTTTTCAT (Score 34) and a 35 box ATGCGA (Score 12). Another putative promoter sequence is located upstream of the aviC2 translation start site consisting of a 10 box GCCCATGAT (Score 31) and a 35 box at TTTCTA (Score 34) similar to consensus Streptomycetes promoters.13 AviC1 consists of 206 amino acid (aa) residues. The C-terminal region of the putative aviC1 product is similar to response regulators, which contain a LuxR-type DNA-binding domain.14 Examples are CitB from Frankia sp. EAN1pec (53% identical aa) and NarQ, a nitrate/nitrite response regulator from Escherichia coli (43% identical aa).15 All these proteins possess common CheY-like N-terminal sensor domains that undergo phosphorylation by protein kinases and C-terminal LuxR-type Helix-Turn-Helix (HTH) DNA-binding domains.14 However, the AviC1 N terminus does not resemble CheY-like signal receiver domains, supposing that AviC1 responds to some other signals than phosphorylation. The product of aviC2 consists of 192 aa residues. The C-terminal region of AviC2 exhibits similarity to proteins involved in the control of nystatin production in S. noursei (NysRIV, 56% identical aa)7 and pimaricin production in S. natalensis (PimM, 53% identical aa).16 These proteins contain N-terminal PAS domains and sense internal and external signals, such as redox potential or light.17 The C-terminal part of these proteins represents a LuxR type of HTH DNA-binding motif, which is formed by two a-helixes (R145-E157 and R164-L174