Induction of antimicrobial activities in heterologous streptomycetes using alleles of the Streptomyces coelicolor gene absA1.

The bacterial genus Streptomyces is endowed with a remarkable secondary metabolism that generates an enormous number of bioactive small molecules. Many of these genetically encoded small molecules are used as antibiotics, anticancer agents and as other clinically relevant therapeutics. The rise of resistant pathogens has led to calls for renewed efforts to identify antimicrobial activities, including expanded screening of streptomycetes. Indeed, it is known that most strains encode >20 secondary metabolites and that many, perhaps most of these, have not been considered for their possible therapeutic use. One roadblock is that many strains do not express their secondary metabolic gene clusters efficiently under laboratory conditions. As one approach to this problem, we have used alleles of a pleiotropic regulator of secondary metabolism from Streptomyces coelicolor to activate secondary biosynthetic gene clusters in heterologous streptomycetes. In one case, we demonstrate the activation of pulvomycin production in S. flavopersicus, a metabolite not previously attributed to this species. We find that the absA1-engineered strains produced sufficient material for purification and characterization. As a result, we identified new, broad-spectrum antimicrobial activities for pulvomycin, including a potent antimicrobial activity against highly antibiotic-resistant Gram-negative and Gram-positive pathogens.

Transmembrane topology of the AbsA1 sensor kinase of Streptomyces coelicolor.

The sensor kinase AbsA1 (SCO3225) phosphorylates the response regulator AbsA2 (SCO3226) and dephosphorylates AbsA2 approximately P. The phosphorylated response regulator represses antibiotic biosynthesis operons in Streptomyces coelicolor. AbsA1 was predicted to have an atypical transmembrane topology, and the location of its signal-sensing domain is not readily obvious. To better understand this protein and to gain insight into its signal response mechanism, we determined its transmembrane topology using fusions of absA1 to egfp, which is believed to be the first application of this approach to transmembrane topology in the actinomycetes. Our results are in agreement with the in silico topological predictions and demonstrate that AbsA1 has five transmembrane domains, four near the N terminus and one near the C terminus. Unlike most sensor kinases, the largest extracellular portion of AbsA1 is at the C terminus.

Chapter 5. Applying the genetics of secondary metabolism in model actinomycetes to the discovery of new antibiotics.

The actinomycetes, including in particular members of the filamentous genus Streptomyces, are the industrial source of a large number of bioactive small molecules employed as antibiotics and other drugs. They produce these molecules as part of their "secondary" or nonessential metabolism. The number and diversity of secondary metabolic pathways is enormous, with some estimates suggesting that this one genus can produce more than 100,000 distinct molecules. However, the discovery of new antimicrobials is hampered by the fact that many wild isolates fail to express all or sometimes any of their secondary metabolites under laboratory conditions. Furthermore, the use of previously successful screening strategies frequently results in the rediscovery of known molecules: the all-important novel structures have proven to be elusive. Mounting evidence suggests that streptomycetes possess many regulatory pathways that control the biosynthetic gene clusters for these secondary metabolic pathways and that cell metabolism plays a significant role in limiting or potentiating expression as well. In this article we explore the idea that manipulating metabolic conditions and regulatory pathways can "awaken" silent gene clusters and lead to the discovery of novel antimicrobial activities.

Phosphorylated AbsA2 negatively regulates antibiotic production in Streptomyces coelicolor through interactions with pathway-specific regulatory gene promoters.

The AbsA two-component signal transduction system, comprised of the sensor kinase AbsA1 and the response regulator AbsA2, acts as a negative regulator of antibiotic production in Streptomyces coelicolor, for which the phosphorylated form of AbsA2 (AbsA2 approximately P) is the agent of repression. In this study, we used chromatin immunoprecipitation to show that AbsA2 binds the promoter regions of actII-ORF4, cdaR, and redZ, which encode pathway-specific activators for actinorhodin, calcium-dependent antibiotic, and undecylprodigiosin, respectively. We confirm that these interactions also occur in vitro and that the binding of AbsA2 to each gene is enhanced by phosphorylation. Induced expression of actII-ORF4 and redZ in the hyperrepressive absA1 mutant (C542) brought about pathway-specific restoration of actinorhodin and undecylprodigiosin production, respectively. Our results suggest that AbsA2 approximately P interacts with as many as four sites in the region that includes the actII-ORF4 promoter. These data suggest that AbsA2 approximately P inhibits antibiotic production by directly interfering with the expression of pathway-specific regulators of antibiotic biosynthetic gene clusters.