A Two-Step Mechanism for the Activation of Actinorhodin Export and Resistance in Streptomyces coelicolor

Abstract

Many microorganisms produce secondary metabolites that have antibiotic activity. To avoid self-inhibition, the producing cells often encode cognate export and/or resistance mechanisms in the biosynthetic gene clusters for these molecules. Actinorhodin is a blue-pigmented antibiotic produced by Streptomyces coelicolor. The actAB operon, carried in the actinorhodin biosynthetic gene cluster, encodes two putative export pumps and is regulated by the transcriptional repressor protein ActR. In this work, we show that normal actinorhodin yields require actAB expression. Consistent with previous in vitro work, we show that both actinorhodin and its 3-ring biosynthetic intermediates [e.g., (S)-DNPA] can relieve repression of actAB by ActR in vivo. Importantly, an ActR mutant that interacts productively with (S)-DNPA but not with actinorhodin responds to the actinorhodin biosynthetic pathway with the induction of actAB and normal yields of actinorhodin. This suggests that the intermediates are sufficient to trigger the export genes in actinorhodin-producing cells. We further show that actinorhodin-producing cells can induce actAB expression in nonproducing cells; however, in this case actinorhodin is the most important signal. Finally, while the “intermediate-only” ActR mutant permits sufficient actAB expression for normal actinorhodin yields, this expression is short-lived. Sustained culture-wide expression requires a subsequent actinorhodin-mediated signaling step, and the defect in this response causes widespread cell death. These results are consistent with a two-step model for actinorhodin export and resistance where intermediates trigger initial expression for export from producing cells and actinorhodin then triggers sustained export gene expression that confers culture-wide resistance.

The TetR Family of Regulators

“…Genetic evidence suggests that in actinorhodin-producing cells (S)-DNPA and/or other 3-ring intermediates serve to activate the expression of efflux genes, the only known self-resistance mechanism, before the final product is synthesized (81). Furthermore, there are now several reports that the export proteins are required for efficient, high-yield biosynthesis of actinorhodin (81,82). The biochemical basis for reduced actinorhodin biosynthesis in cells defective in the actAB operon is not well understood, but it has been interpreted as evidence that initial activation of the actinorhodin export genes is primarily dependent on intermediates.…”

“…The biochemical basis for reduced actinorhodin biosynthesis in cells defective in the actAB operon is not well understood, but it has been interpreted as evidence that initial activation of the actinorhodin export genes is primarily dependent on intermediates. However, it is also clear that sustained expression of the actinorhodin efflux pumps throughout the culture (i.e., including cells that produce actinorhodin and those that do not) requires the actinorhodin final product (81). Thus, actinorhodin is believed to act as a cell-cell signal to trigger export and resistance in nonproducing cells.…”

GouR, a TetR Family Transcriptional Regulator, Coordinates the Biosynthesis and Export of Gougerotin in Streptomyces graminearus

“…The production of antibiotics is subject to hierarchical control of regulators, including cluster-situated regulators, pleiotropic regulators, and global regulators (1). Among them, TetR family transcriptional regulators constitute some of the most frequently occurring transcriptional regulators and serve as both repressors (2)(3)(4)(5) and activators (6,7). TetR, the founder member of this family of regulators, is a repressor of divergently transcribed tetA, which encodes a tetracycline efflux pump.…”

The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development

“…In some Gram-positive Streptomyces species, known for their production of small-molecule antibiotics and pigments, genes for antibiotic biosynthetic enzymes are found in large clusters, which can also contain genes encoding efflux pumps and regulatory proteins that control expression of adjacent loci. Efflux pumps have been implicated in the transport of, and resistance to, antibiotics such as actinorhodin, daunorubicin, and oxytetracycline in their streptomycete producers (3)(4)(5)(6). In several cases, either the antibiotic itself, an intermediate in antibiotic synthesis, or compounds with structural similarities to the antibiotic have been demonstrated or inferred to be inducers of pump expression (3,4,7,8).…”