The production of secondary metabolites is a fundamental way that bacteria interact with their environments, yet little is known about the fitness benefits conferred by specific molecules or the dynamics influencing the distribution of biosynthetic gene clusters among bacterial lineages. This dissertation consists of six chapters that address these topics in an unusual class of secondary metabolites called hybrid isoprenoids (HIs). The first chapter introduces microbial chemical ecology and secondary metabolism, followed by four research-oriented chapters and a summary chapter. Chapter 2 presents a literature review of the distribution of HI production within the actinobacterial genus Streptomyces. Reports of HI production were phylogenetically very scattered, with the exception of a putative marine Streptomyces lineage called the 'MAR4' clade. Following from this, Chapter 3 presents an in-depth study of the phylogenetic diversity of the MAR4 clade. Sequence data from a variety of sources led to a considerable expansion of the MAR4 clade, and representative strains were shown to produce 1-4 classes of HI molecules, an ability that is unmatched elsewhere in the streptomycetes. Chapter 4 presents an analysis of 120 Streptomyces genomes, including 12 MAR4 strains, which provides strong support for the hypothesis that this clade is enriched in gene clusters for HI production. Phylogenetic analysis of a HI biosynthesis gene suggest that vertical inheritance, horizontal gene transfer, duplication followed by functional divergence, and gene rearrangements have all contributed to the striking number of HI gene clusters found in MAR4 genomes. The final research chapter addresses the fact that none of these molecules have an experimentally characterized natural function, which is a major obstacle to understanding the unusual distribution of HI gene clusters. Because many HIs resemble characterized electron shuttles, the hypothesis that they function in redox cycling was explored. A redox- active pigment that was upregulated in low-oxygen conditions was isolated from a MAR4 strain. This compound was identified as an intermediate in a HI gene cluster, which largely goes to completion in aerobic culture. This provides an unusual example of how a single biosynthetic cluster can be used to generate products with distinct properties depending on culture conditions

Background

Considerable advances have been made in our understanding of the molecular genetics of secondary metabolite biosynthesis. Coupled with increased access to genome sequence data, new insight can be gained into the diversity and distributions of secondary metabolite biosynthetic gene clusters and the evolutionary processes that generate them. Here we examine the distribution of gene clusters predicted to encode the biosynthesis of a structurally diverse class of molecules called hybrid isoprenoids (HIs) in the genus Streptomyces. These compounds are derived from a mixed biosynthetic origin that is characterized by the incorporation of a terpene moiety onto a variety of chemical scaffolds and include many potent antibiotic and cytotoxic agents.

Results

One hundred and twenty Streptomyces genomes were searched for HI biosynthetic gene clusters using ABBA prenyltransferases (PTases) as queries. These enzymes are responsible for a key step in HI biosynthesis. The strains included 12 that belong to the 'MAR4' clade, a largely marine-derived lineage linked to the production of diverse HI secondary metabolites. We found ABBA PTase homologs in all of the MAR4 genomes, which averaged five copies per strain, compared with 21 % of the non-MAR4 genomes, which averaged one copy per strain. Phylogenetic analyses suggest that MAR4 PTase diversity has arisen by a combination of horizontal gene transfer and gene duplication. Furthermore, there is evidence that HI gene cluster diversity is generated by the horizontal exchange of orthologous PTases among clusters. Many putative HI gene clusters have not been linked to their secondary metabolic products, suggesting that MAR4 strains will yield additional new compounds in this structure class. Finally, we confirm that the mevalonate pathway is not always present in genomes that contain HI gene clusters and thus is not a reliable query for identifying strains with the potential to produce HI secondary metabolites.

Conclusions

We found that marine-derived MAR4 streptomycetes possess a relatively high genetic potential for HI biosynthesis. The combination of horizontal gene transfer, duplication, and rearrangement indicate that complex evolutionary processes account for the high level of HI gene cluster diversity in these bacteria, the products of which may provide a yet to be defined adaptation to the marine environment.

Streptomyces species dedicate a large portion of their genomes to secondary metabolite biosynthesis. A diverse and largely marine-derived lineage within this genus has been designated MAR4 and identified as a prolific source of hybrid isoprenoid (HI) secondary metabolites. These terpenoid-containing compounds are common in nature but rarely observed as bacterial secondary metabolites. To assess the phylogenetic diversity of the MAR4 lineage, complementary culture-based and culture-independent techniques were applied to marine sediment samples collected off the Channel Islands, CA. The results, including those from an analysis of publically available sequence data and strains isolated as part of prior studies, placed 40 new strains in the MAR4 clade, of which 32 originated from marine sources. When combined with sequences cloned from environmental DNA, 28 MAR4 operational taxonomic units (0.01% genetic distance) were identified. Of these, 82% consisted exclusively of either cloned sequences or cultured strains, supporting the complementarity of these two approaches. Chemical analyses of diverse MAR4 strains revealed the production of five different HI structure classes. All 21 MAR4 strains tested produced at least one HI class, with most strains producing from two to four classes. The two major clades within the MAR4 lineage displayed distinct patterns in the structural classes and the number and amount of HIs produced, suggesting a relationship between taxonomy and secondary metabolite production. The production of HI secondary metabolites appears to be a phenotypic trait of the MAR4 lineage, which represents an emerging model with which to study the ecology and evolution of HI biosynthesis.

Members of the actinomycete genus Streptomyces are non-motile, filamentous bacteria that are well-known for the production of biomedically relevant secondary metabolites. While considered obligate aerobes, little is known about how these bacteria respond to periods of reduced oxygen availability in their natural habitats, which include soils and ocean sediments. Here, we provide evidence that the marine streptomycete strain CNQ-525 can reduce MnO₂ via a diffusible mechanism. We investigated the effects of hypoxia on secondary metabolite production and observed a shift away from the antibiotic napyradiomycin towards 8-amino-flaviolin, an intermediate in the napyradiomycin biosynthetic pathway. We purified 8-amino-flaviolin and demonstrated that it is reversibly redox-active (midpoint potential -474.5 mV), indicating that it has the potential to function as an endogenous extracellular electron shuttle. This study provides evidence that environmentally triggered changes in secondary metabolite production may provide clues to the ecological functions of specific compounds, and that Gram-positive bacteria considered to be obligate aerobes may play previously unrecognized roles in biogeochemical cycling through mechanisms that include extracellular electron shuttling.