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General Description

Bacteria are prolific producers of a group of small molecules known as secondary metabolites that serve as a major source of drugs and drug candidates. Penicillin and erythromycin are but two examples of bacterial secondary metabolites that, since their discoveries, have been invaluable in a clinical setting. In addition to therapeutic applications, the complex structures of secondary metabolites serve as a source of inspiration for synthetic chemists, who attempt to recreate these molecules in the laboratory, and for biochemists, who investigate Nature’s biosynthetic strategies for concocting them. Thus bacterially-produced small molecules provide a common template for discovering potential therapeutics, finding novel, (bio)synthetically challenging structural scaffolds, investigating their physiological roles, understanding Nature’s biosynthetic repertoire as well as elucidating the corresponding enzymatic reaction mechanisms. We are engaged in examining and understanding these facets underlying bacterially-generated secondary metabolites. Below are examples of ongoing projects in our group.

Activating silent gene clusters

Figure 1. Our strategy for activating silent gene clusters. A reporter gene inside the gene cluster of interest affords a facile read-out for its activity while small molecule libraries provide candidate elicitors. High throughput screening identifies activators for the target cluster facilitating elucidation of the products structure and function.

Figure 1. Our strategy for activating silent gene clusters. A reporter gene inside the gene cluster of interest affords a facile read-out for its expression, while small molecule libraries provide candidate elicitors. High throughput screening identifies activators for the target cluster facilitating elucidation of the product’s structure and function.

Secondary metabolites are assembled by sets of contiguous genes, called biosynthetic gene clusters, which in a step-wise fashion generate these often complex molecules from simple building blocks. The beta-lactam penicillin, for example, is assembled from three natural amino acids that are condensed into a tripeptide and further tailored to give the final product. Recent genome sequencing efforts have revealed that the vast majority of biosynthetic gene clusters that can be identified in bacterial genomes are inactive under typical growth conditions. These so-called silent gene clusters represent a large and hidden reservoir of new molecules and potential therapeutics. We are currently devising new strategies to activate silent gene clusters in order to examine their products and biological activities. We recently reported one such strategy that allowed us to discover activators or elicitors of silent clusters (Fig. 1). Surprisingly, many of the elicitors we discovered turned out to be antibiotics, suggesting they play a role in modulating secondary metabolism in bacteria, and that old antibiotics may be used to find new ones.

Microbial Symbiotic Interactions

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Figure 2. Proposed model for the bi-phasic symbiotic interaction between P. gallaeciensis and E. huxleyi. In the mutualistic phase (green arrows), the algal host provides a food molecule, while the bacteria generate metabolites beneficial to the host. When the algae senesce, they release pCA, which triggers a mutualist-to-parasite switch. In the parasitic phase (red arrows), the bacteria produce the algaecidal roseobacticides, which kill the host.

An alternative strategy for the discovery of secondary metabolites that we are currently pursuing relies not on targeting biosynthetic gene clusters, as elaborated above, but rather on the physiological roles of these molecules. Because bacteria communicate using secondary metabolites, ‘listening in’ on these conversations provides an attractive search strategy. We are investigating a number of microbial interactions and the small molecules that mediate them. In one case, a naturally-occurring and wide-spread algal-bacterial symbiosis, we have discovered a novel family of small molecules, the roseobacticides, and a biphasic mode of interaction involving a mutualistic and a parasitic phase (Fig. 2). Under mutualistic conditions, the bacteria and algae exchange molecules beneficial to the symbiotic partner. However, when the algae begin to senesce, the bacteria produce the algaecidal roseobacticides, which kills the algal host. We have recently found that two molecules in this symbiosis are synthesized largely by the same gene cluster, the first example one gene cluster generating two different metabolites. Production of roseobacticides requires an unusual biosynthetic strategy, which we are examining further.

Novel Biosynthetic & Enzymatic Chemistries

Figure 3. Reaction catalyzed by StrB, a radical SAM enzyme involved in the biosynthesis of the natural product streptide. StrB contains multiple Fe-S clusters and installs a Lys-Trp crosslink using radical chemistry that is initiated by a 5′-deoxyadenosyl radical. Upon Lys-Trp crosslink formation, the product is proteolytically cleaved at the N- and C-termini to render the mature streptide product. Additional studies have allowed us to propose a mechanism for this unusual transformation.

The remarkable structures of secondary metabolites provide opportunities for examining their exotic biosynthetic pathways and discovering novel enzyme-catalyzed transformations.  The assembly can be grossly divided into two phases. The first involves production of the scaffold or backbone, and the second installs unique, pathway-specific alterations. While the canonical mechanisms for generating the peptide, polyketide, and terpene backbones of secondary metabolites have been largely elucidated, the tailoring enzymes that provide unique functionalities have received far less attention. Among these, metalloenzymes introduce often unusual, functionally essential, and mechanistically puzzling modifications. We are interested in elucidating the detailed mechanisms of these unusual enzyme-catalyzed transformations, especially those involving metallo-cofactors. We recently reported one such example, a carbon-carbon crosslink installed at unactivated positions between the side-chains of Lys and Trp by a radical SAM enzyme (Fig. 3). These studies are providing insights into the biosynthetic pathways of important secondary metabolites. They also suggest strategies for generating new analogs with orthogonal properties in a semi-synthetic fashion.