Justin R. Nodwell

Chemical Perturbation of Secondary Metabolism Demonstrates Important Links to Primary Metabolism

Bacterially produced secondary metabolites are used as antibiotics, anticancer drugs, and for many other medicinal applications. The mechanisms that limit the production of these molecules in the laboratory are not well understood, and this has impeded the discovery of many important compounds. We have identified small molecules that remodel the yields of secondary metabolites in many actinomycetes and show that one set of these molecules does so by inhibiting fatty acid biosynthesis. This demonstrates a particularly intimate relationship between this primary metabolic pathway and secondary metabolism and suggests an approach to enhance the yields of metabolites for discovery and biochemical characterization.

Morphogenetic surfactants and their role in the formation of aerial hyphae in Streptomyces coelicolor

Withstanding environmental adversity and seeking optimal conditions for reproduction are basic requirements for the survival of all organisms. Filamentous bacteria of the genus Streptomyces produce a remarkable cell type called the aerial hyphae that is central to its ability to meet both of these challenges. Recent advances have brought about a major shift in our understanding of the cell surface proteins that play important roles in the generation of these cells. Here we review our current understanding of one of these groups of proteins, the morphogenetic surfactants, with emphasis on the SapB protein of Streptomyces coelicolor.

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.

The ramC gene is required for morphogenesis in Streptomyces coelicolor and expressed in a cell type-specific manner under the direct control of RamR

The bacterium Streptomyces coelicolor produces two cell types during the course of its life cycle: the aerial hyphae, which metamorphose into spores, and the substrate hyphae, which synthesize antibiotics. We show that the genes ramC and ramR are required for the production of the aerial hyphae but are dispensable for vegetative growth and antibiotic synthesis. We find that ramC is expressed in the substrate hyphae and shut off in the aerial hyphae by the time visible signs of sporulation‐associated septation are evident. Production of RamC requires the developmental regulators bldD, cprA and ramR, but not bldM or bldN, and we show that the RamR protein interacts directly with DNA in the ramC promoter region sug‐gesting that it is, at least in part, responsible for regulating ramC expression.

A synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria

The luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens has proven to be a superb transcriptional reporter. It encodes a luciferase (LuxA and LuxB) and the enzymes that produce its substrate (LuxC, LuxD and LuxE) so cells that express the cluster emit the 490-nm light spontaneously. The sequence of these genes is AT-rich (>69%) and for this and other reasons, they are not expressed efficiently in high-GC bacteria like Streptomyces coelicolor. We therefore constructed a synthetic luxCDABE operon encoding the P. luminescens Lux proteins optimized for expression in high-GC bacteria. We tested the genes using transcriptional fusions to S. coelicolor promoters having well-established expression profiles during this organisms life cycle. The hrdB gene encodes a housekeeping sigma factor; while ramC is important for the formation of the spore-forming cells called aerial hyphae and whiE is required for the production of a grey, spore-associated pigment that is deposited in the walls of developing spores. Using these fusions we demonstrated that our synthetic lux genes are functional in S. coelicolor and that they accurately report complex developmental gene expression patterns. We suggest that this lux operon and our procedure for generating synthetic high-GC genes will be widely useful for research on high-GC bacteria.

Towards a new science of secondary metabolism

Secondary metabolites are a reliable and very important source of medicinal compounds. While these molecules have been mined extensively, genome sequencing has suggested that there is a great deal of chemical diversity and bioactivity that remains to be discovered and characterized. A central challenge to the field is that many of the novel or poorly understood molecules are expressed at low levels in the laboratory—such molecules are often described as the ‘cryptic’ secondary metabolites. In this review, we will discuss evidence that research in this field has provided us with sufficient knowledge and tools to express and purify any secondary metabolite of interest. We will describe ‘unselective’ strategies that bring about global changes in secondary metabolite output as well as ‘selective’ strategies where a specific biosynthetic gene cluster of interest is manipulated to enhance the yield of a single product.

Biochemical Activities of the absA Two-Component System of Streptomyces coelicolor

The AbsA1 sensor kinase and its cognate response regulator AbsA2 are important regulators of antibiotic synthesis in Streptomyces coelicolor. While certain point mutations in absA1 reduce or eliminate the synthesis of several antibiotics, null mutations in these genes bring about enhanced antibiotic synthesis. We show here that AbsA1, which is unusual in sequence and structure, is both an AbsA2 kinase and an AbsA2 approximately P phosphatase. The half-life of AbsA2 approximately P in solution is 68.6 min, consistent with a role in maintaining a relatively stable state of transcriptional repression or activation. We find that mutations in the absA locus that enhance antibiotic synthesis impair AbsA2 kinase activity and that mutations that repress antibiotic synthesis impair AbsA2 approximately P phosphatase activity. These results support a model in which the phosphorylation state of AbsA2 is determined by the balance of the kinase and phosphatase activities of AbsA1 and where AbsA2 approximately P represses antibiotic biosynthetic genes either directly or indirectly.

Crystal structures of the Streptomyces coelicolor TetR-like protein ActR alone and in complex with actinorhodin or the actinorhodin biosynthetic precursor (S)-DNPA.

Actinorhodin, an antibiotic produced by Streptomyces coelicolor, is exported from the cell by the ActA efflux pump. actA is divergently transcribed from actR, which encodes a TetR-like transcriptional repressor. We showed previously that ActR represses transcription by binding to an operator from the actA/actR intergenic region. Importantly, actinorhodin itself or various actinorhodin biosynthetic intermediates can cause ActR to dissociate from its operator, leading to derepression. This suggests that ActR may mediate timely self-resistance to an endogenously produced antibiotic by responding to one of its biosynthetic precursors. Here, we report the structural basis for this precursor-mediated derepression with crystal structures of homodimeric ActR by itself and in complex with either actinorhodin or the actinorhodin biosynthetic intermediate (S)-DNPA [4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho-[2,3-c]-pyran-3-(S)-acetic acid]. The ligand-binding tunnel in each ActR monomer has a striking hydrophilic/hydrophobic/hydrophilic arrangement of surface residues that accommodate either one hexacyclic actinorhodin molecule or two back-to-back tricyclic (S)-DNPA molecules. Moreover, our work also reveals the strongest structural evidence to date that TetR-mediated antibiotic resistance may have been acquired from an antibiotic-producer organism.

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.

Ligand recognition by ActR, a TetR-like regulator of actinorhodin export.

TetR-like transcriptional repressors interact with small-molecule ligands to control many facets of prokaryotic biology, including clinical antibiotic resistance. ActR is a TetR-like protein encoded in the biosynthetic gene cluster for the antibiotic actinorhodin and controls the expression of two actinorhodin exporters. We showed previously that actinorhodin and its precursor 4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho-[2,3-c]-pyran-3-(S)-acetic acid can bind ActR and prevent its interaction with DNA. Here, we compare ActRs interaction with naturally occurring and synthetic molecules to show that pathway intermediates bind to ActR 5- to 10-fold more tightly than actinorhodin itself, consistent with our suggestion that they are the biologically relevant triggers for actinorhodin export. We also find that the ligand-binding cavity of this protein can accommodate a surprisingly large diversity of ligands, many of which can release ActR from DNA in vitro and in vivo. These data suggest that the actR locus could be activated by, and perhaps adapted to confer resistance to other antibiotics.