A two step mechanism for activation of actinorhodin export and resistance in Streptomyces coelicolor

Abstract

<p>Antibiotic producing organisms often bear cognate export and/or resistance mechanism encoded in the same cluster of antibiotic biosynthetic genes, but how expression of these protective genes associated with the cognate biosynthetic machinery was poorly understood. This work focuses on elucidating the mechanism by which the antibiotic producing bacteria initiate and sustain self-resistance in individual cells and across the colonies. Actinorhodin is a blue pigmented antibiotic produced by <em>Streptomyces coelicolor</em>. Within its biosynthetic gene cluster, an <em>actAB</em> operon encodes two putative efflux pumps and is regulated by the transcriptional regulator ActR. This work (1) identified that normal yields of actinorhodin require <em>actAB</em> expression and both actinorhodin and its 3-ring pathway intermediate (e.g. DHK) could relieve <em>actAB</em> expression from ActR repression <em>in vivo</em>; (2) created an ActR mutant that interacts productively with a 3-ring intermediate (e.g. (<em>S</em>)-DNPA) but not actinorhodin and responds to actinorhodin biosynthetic pathway with induction of <em>actAB</em> and normal actinorhodin production; (3) demonstrated that actinorhodin producing cells could induce <em>actAB</em> expression in non-producing cells and actinorhodin is the intercellular signal in this case; (4) showed <em>actAB</em> expression in “intermediate only” ActR mutant is short lived and in the absence of the actinorhodin-mediated signaling step the culture experiences widespread cell death. Based on these results, we proposed 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.</p>