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.

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.

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 organism's 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.

The secondary metabolites produced by bacterial species serve many clinically useful purposes, and Streptomyces have been an abundant source of such compounds. However, a poor understanding of their regulatory cascades leads to an inability to isolate all of the secondary metabolites this genus is capable of producing. This study focuses on comparing synthetic small molecules that were found to alter the production of secondary metabolites in Streptomyces coelicolor. A survey of these molecules suggests that each has a distinct mechanism of action, and hence, could be used as a unique probe of secondary metabolism. A comparative analysis of two of these molecules, ARC2 and ARC6, confirmed that they modulate secondary metabolites in different ways. In a separate study, ARC2 was shown to give rise to a different phenotype through the inhibition of a target in fatty acid biosynthesis. The results of this study suggest that ARC6 does not have the same target, although it might target the same metabolic system. Furthermore, the results demonstrate that ARC2 and ARC6 act through distinct mechanisms and further suggest that chemical probes can be important tools in enhancing our understanding of secondary metabolism and the streptomycete life cycle.

Antibiotic-resistant bacteria are a significant public health concern and motivate efforts to develop new classes of antibiotics. One such class of antibiotics is the arylomycins, which target type I signal peptidase (SPase), the enzyme responsible for the release of secreted proteins from their N-terminal leader sequences. Despite the essentiality, conservation, and relative accessibility of SPase, the activity of the arylomycins is limited against some bacteria, including the important human pathogen Staphylococcus aureus. To understand the origins of the limited activity against S. aureus, we characterized the susceptibility of a panel of strains to two arylomycin derivatives, arylomycin A-C 16 and its more potent analog arylomycin M131. We observed a wide range of susceptibilities to the two arylomycins and found that resistant strains were sensitized by cotreatment with tunicamycin, which inhibits the first step of wall teichoic acid synthesis. To further understand how S. aureus responds to the arylomycins, we profiled the transcriptional response of S. aureus NCTC 8325 to growth-inhibitory concentrations of arylomycin M131 and found that it upregulates the cell wall stress stimulon (CWSS) and an operon consisting of a putative transcriptional regulator and three hypothetical proteins. Interestingly, we found that mutations in the putative transcriptional regulator are correlated with resistance, and selection for resistance ex vivo demonstrated that mutations in this gene are sufficient for resistance. The results begin to elucidate how S. aureus copes with secretion stress and how it evolves resistance to the inhibition of SPase.T he widespread use of antibiotics imposes a relentless selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens, and novel classes of antibiotics are needed (1, 2). Staphylococcus aureus has emerged as a particular health concern due to its ability to cause a wide range of infections, ranging from superficial lesions such as skin abscesses and wound infections to more systemic and life-threatening conditions such as osteomyelitis, endocarditis, pneumonia, septicemia, and toxinoses. To infect different tissues, S. aureus relies on the secretion of virulence factors that facilitate adhesion and colonization, promote dissemination, facilitate the evasion of the host immune response, and scavenge nutrients and minerals from the environment (3-6). The majority of proteins destined for export out of the cytoplasm are synthesized as preproteins with N-terminal signal peptide sequences, which target them to the general secretory (Sec) pathway (7-9), and these leader peptides ultimately must be removed by the proteolytic activity of type I signal peptidase (SPase) (10).SPase has long been appreciated as a promising target for antibiotic therapy, and a search for inhibitors led to the identification in 2002 of the arylomycin family of natural product lipopeptide antibiotics from a strain of Streptomyces (Fig. 1) (11-13). Despite the essentiali...