The purpose of this project was to explore the biomedical potential of a group of marine bacteria closely related to the medically prolific group of soil genomes of two marine actinomycetes.

Actinomycetes are a group of bacteria that live in soil and decompose organic matter such as cellulose. In the realm of drug discovery, these microorganisms are widely recognized for their ability to produce secondary metabolites (chemicals) with commercially viable antibiotic activity. Streptomycin, the first treatment for tuberculosis, was derived from the largest genus of these bacteria, Streptomyces. Erythromycin and tetracycline are two other examples of common medicines derived originally from these microorganisms’ metabolites.

Since the first studies looking into how plant specialized metabolites provide defense from herbivory, the field of chemical ecology has explored how organisms interact within their environment, and how these interactions structure the ecosystem. Over the last century, we have come to understand a lot about chemical signaling in plants and animals, but our understanding of bacterial chemical signaling is still in its infancy. This is partially because most of the focus has been placed on understanding bacteria potential for drug development as opposed to understanding their ecological roles. This thesis consists of five chapters and explores the interplay between bacterial specialized metabolites in marine sediments, their producers, and their ecological roles, by focusing on the model marine bacterial genus Salinispora. What follows are three research chapters preceded by an introduction to chemical ecology. The introduction focuses on our current understanding of marine bacterial chemical ecology, followed by a brief description of cutting edge mass spectrometry techniques that are now being exploited to further our understanding of the chemical landscape in the environment.

Chapter 2 summarizes a study concerning the identification of specialized metabolites in situ and the correlation between these metabolites and their potential producers. Heterotrophic marine sediment bacteria are prolific producers of natural products, but surprisingly little is known about the compounds they produce in the environment and their effects on co-occurring microbes. Using mass spectrometry and molecular networking, characterization of the sediment metabolome was undertaken from Belizean reef habitats. Dereplication results revealed numerous compounds could be detected directly from the sediments including synthetic, sponge, algal, and bacterial metabolites. Interestingly, one of these compounds, the cytotoxin staurosporine, was further quantified and found to occur in the sediments at abundances higher than those established to inhibit protein kinases as well as marine organisms. A 16S rRNA community analysis as well as culturing helped correlate the production of staurosporine to the obligate marine actinomycete species Salinispora arenicola. These results indicate that microbial organisms are likely capable of producing cytotoxins in situ at appreciable quantities that likely impact the community structure of the sediment biome.

Since the cytotoxin staurosporine, which is produced by Salinispora, was found to be abundant in the sediments, chapter 3 explored the potential for Salinispora to deter predatory eukaryotic organisms. S. arenicola and S. tropica co-occur in the Caribbean and share greater than 99% 16s rRNA. A recent sequencing effort revealed that each species maintains a unique set BGCs, with S. arenicola containing the BGC to produce staurosporine and S. tropica the BGCs to produce lomaiviticins, and salinosporamides. Numerous assays were developed to assess the ability of Salinispora spp. to deter feeding by the bacterivore C. elegans. Results indicated that S. tropica strains can produce a suite of lomaiviticins that deter C. elegans feeding at ecologically relevant concentrations, however; S. arenicola strains do not produce deterrent allelochemicals at ecologically relevant concentrations. Follow up studies using more ecologically relevant organisms indicated that this trend was still prevalent when tested against the marine polychaete Ophryotrocha n sp. as well as marine nematodes.

In chapter 3, I saw that Salinispora spp. exhibit different chemodeterrent strategies against bacterivorous eukaryotes. However, this study relied on constitutively produced specialized metabolites, even though there are numerous examples where microbial specialized metabolites can be induced in response to biotic stressors in the environment. To address this chapter 4 focuses on a high throughput method I developed to look at induction of specialized metabolites in co-cultures containing Salinispora and marine bacterial challengers. Results indicate that induction is prevalent in cocultures using all Salinispora strains tested. The induced mass features were also unique to cocultures indicating that induction is strain and not species specific. Efforts to identify compounds that were induced in cocultures resulted in the identification of a suite of desferrioxamines that were upregulated in some Salinispora strains in response to specific Streptomyces challengers, indicating that some Salinispora strains can modulate the production of iron scavenging specialized metabolites in response to stressors.

Chapter 5 provides conclusions related to the thesis and how it fits into the broader context of marine microbial chemical ecology.

Background

Results

Conclusions

Secondary metabolite production plays an important role in the interaction of bacteria and their environments. While many marine microbes have been found to produce bioactive molecules, relatively little is known about the diversity and distribution of biosynthetic gene clusters with known products. This dissertation includes five chapters that explore these topics in the genus Pseudoalteromonas. The first chapter introduces marine natural products and the group of bacteria, followed by three research chapters and a summary chapter.

Chapter 2 explores the evolution of the bmp gene cluster in the genus Pseudoalteromonas. A well-supported species phylogeny was generated and the distribution and diversity of the biosynthetic gene cluster was mapped onto the tree. Four distinct versions of the bmp cluster were found in the genus, with evidence supporting gene loss in certain cases. It is uncertain how many times the gene cluster was acquired within Pseudoalteromonas, but the conservation of the biosynthetic genes within some lineages suggests that the metabolites produced are relevant to the ecology of these bacteria.

Chapter 3 presents the results from the bacterial cytological profiling of pentabromopseudilin in an E. coli strain. The primary phenotype observed in treated cells was membrane permeability, which increases over time and concentration. Despite its apparent ability to disrupt the cell membrane of Gram-negative bacteria and increase the potency of two tested antibiotics, pentabromopseudilin is not likely a good candidate for therapeutic uses due its cytotoxicity.

The final research chapter investigates the bioactivity and biosynthetic potential of five Pseudoalteromonas species. The crude extracts and fractions of some of the strains included in the study were bioactive against Gram-positive and Gram-negative test strains. Preliminary genome mining results reveal that Pseudoalteromonas are rich in biosynthetic gene clusters, most of which do not have known products or similar clusters.