Streptomyces coelicolor is a soil-dwelling prokaryote representative of an order of Gram-positive filamentous bacteria designated Actinomycetales, the richest known bacterial source of bioactive natural products. These specialized / secondary metabolites (including macrolides, aminoglycosides, tetracyclines, cephalosporins and polyketides) often have potent anti-microbial activity and, hence, have been a staple of modern medicine. Genome sequencing has revealed that a given actinomycete genome encodes the potential biosynthetic capacity to produce from 10 to 30, or even more, distinct natural products. S. coelicolor is a model organism for studying actinomycete development, genetics, and specialized metabolism. However, in its natural environment, S. coelicolor must engage with a heterogenous and complex collection of other microorganisms, as well as with abiotic factors in soil. Therefore, in the research described in this dissertation, I explored the role of interspecies interactions in regulating the production of specialized metabolites by S. coelicolor. To obtain insight about how production of these compounds is induced, I exploited the fact that S. coelicolor produces two, readily visualized, red-colored antibiotics (actinorhodin and undecyl-prodigiosin), but only when placed in close contact to certain other bacterial genera. I used extensive analysis of gene expression (RNA-seq) during interspecies interactions and, thereby, identified involvement of conservon8 in controlling induction of the red-colored antibiotics. A conservon is an operon so named because it encodes a characteristic set of four proteins and the S. coelicolor genome encodes 13 such cassettes that are highly homologous to each other. I constructed single-gene deletions in conservon 8 and determined, using transcriptome analysis, that CvnA8 and CvnF8 seem to function together. On the basis of my data, combined with bioinformatic analysis, I propose that CvnA8 is a membrane-bound signaling protein and CvnF8, a GAF domain-containing protein (an element that typically binds ligands and allosterically regulates proteins), serves as a sensor for CvnA8. From the gene expression patterns of deletion mutants, I deduce that CvnC8 and CvnD8 likely function together and hypothesize that CvnC8 is a transcription factor that is negatively regulated by CvnD8, which resembles a small RAS-like GTPase. Similarly, I observed that a ∆cvnB8 mutation has an intermediate effect on red pigment production and genome wide expression patterns, suggesting that it may connect CvnA8/F8 and CvnC8/D8, perhaps through regulation of the CvnD8 GTPase. Finally, my expression data additionally demonstrate that these conservon 8 factors also control expression of the gene clusters for at least two other specialized metabolites aside from the two red pigments, including a cryptic cluster that is predicted to encode a lanthipeptide.

Microbes occupy diverse habitats, forming interconnected, dynamic communities. Microbiomes associated with various plant structures often contain members with the potential to make specialized metabolites, e.g. molecules with antibacterial, antifungal, or siderophore activities. However, when and where microbes associated with plants produce specialized metabolites, and the potential role of these molecules in mediating intra-microbiome interactions, is not well understood. Root nodules of legume plants are organs devoted to hosting symbiotic bacteria that fix atmospheric nitrogen and have recently been shown to harbor a relatively simple accessory microbiome containing members with the ability to produce specialized metabolites in vitro. Based on these observations, we sought to develop a model nodule microbiome system for evaluating microbial specialized metabolism in planta. Starting with an inoculum derived from field-grown Medicago sativa nodules, serial passaging through gnotobiotic nodules yielded a simplified accessory community composed of four members: Brevibacillus brevis, Paenibacillus sp., Pantoea agglomerans, and Pseudomonas sp.. Some members of this community exhibited clear cooperation in planta, while others were antagonistic and capable of disrupting cooperation between other partners. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we found that metabolites associated with individual taxa had unique distributions, indicating that some members of the nodule community were spatially segregated, accounting for their ability to co-exist. Finally, we identified two families of molecules produced by B. brevis in planta as the antibacterial tyrocidines and a novel set of gramicidin-type molecules, which we term the britacidins. Collectively, these results indicate that in addition to nitrogen fixation, legume root nodules are likely also sites of active antimicrobial production.

Bacterial natural products have historically been a deep source of new medicines, but their slowed discovery in recent decades has put a premium on developing strategies that enhance the likelihood of capturing novel compounds. Here, we used a straightforward approach that capitalizes on the interactive ecology of ‘rare’ actinomycetes. Specifically, we screened for interactions that triggered the production of antimicrobials that inhibited the growth of a bacterial strain with exceptionally diverse natural antimicrobial resistance. This strategy led to the discovery of a family of antimicrobials we term the dynaplanins. Heterologous expression enabled identification of the dynaplanin biosynthetic gene cluster, which was missed by typical algorithms for natural product gene cluster detection. Genome sequencing of partially resistant mutants revealed a 2-oxo acid dehydrogenase E2 subunit as the likely molecular target of the dynaplanins, and this finding was supported by computational modeling of the dynaplanin scaffold within the active site of this enzyme. Thus, this simple strategy, which leverages microbial interactions and natural antibiotic resistance, can enable discovery of molecules with unique antimicrobial activity. We aim to adapt this strategy into a microfluidics platform to improve its throughput. In this regard, we have established a prototype of this device in its final stages, though some minor adjustments need to be made before undertaking a full screen. Overall, these results highlight the strength of this screening strategy and indicate that primary metabolism may be a direct target for inhibition via chemical interference in competitive microbial interactions.

The overall goal of my scientific pursuits has been to understand how the environment influences ecological systems and organismal biology and vice versa. Over the past two centuries humans have placed unprecedented demands on the Earth’s natural resources. As a result of these pressures, we have witnessed an accelerated loss of biodiversity and a dramatic alteration of ecological patterns and processes. These changes have potentially devastating consequences in the long-term and are a cause for instigating novel scientific investigation and ecological mediation. Thus, the aim of my doctoral research was to understand community dynamics and primary metabolisms of soil-dwelling microbes following a fire disturbance.

Prescribed fire is a critical strategy for mitigating the effects of catastrophic wildfires. While the above-ground response to fire has been well-documented, fewer studies have addressed the effect of prescribed fire on soil microorganisms. As documented in Chapter 2, we set out to understand how soil microbial communities respond to prescribed fire. For this work we extensively sampled four plots (two burned, two controls) for 17 months in a mixed conifer forest in northern California, USA. Using amplicon sequencing, we found that prescribed fire significantly altered both fungal and bacterial community structure. We found that most differentially abundant fungal taxa had a positive fold-change, while differentially abundant bacterial taxa generally had a negative fold-change in burned plots. We tested the null hypothesis that these communities assembled due to neutral processes (i.e., drift and/or dispersal), finding that >90% of taxa fit this neutral prediction. However, a dynamic subcommunity composed of burn-associated indicator taxa that were positively differentially abundant was enriched for non-neutral ASVs, suggesting assembly via deterministic processes. In synthesizing these results, we identified 15 pyrophilous taxa with a significant and positive response to prescribed burns. Together, these results lay the foundation for building a process-driven understanding of microbial community assembly in the context of the classical disturbance regime of fire.

Fires have a significant impact on soil carbon stocks because combustion transforms carbon into either CO2 or in the production of recalcitrant pyrogenic organic matter (PyOM). PyOM is chemically heterogeneous and composed of a spectrum of fixed organic carbon (C), ranging from living plant biomass to completely charred material and soot. Pyrogenic carbon can modify microbial activity via promotion of microbial metabolism of PyOM as an energy or nutrient source, alteration of physical and chemical properties of the soil, or by interference of microbial signaling. As documented in Chapter 3, we were interested in bacterial species whose metabolic activities were promoted by the PyOM generated by fire. We targeted and isolated post-fire bacteria which can utilize the complex carbon compounds, found in PyOM, from fire-affected soils collected from wildfire sites across Northern California, in addition to soil collected from our prescribed burn sites. We find two bacterial genera, Streptomyces sp. and Paraburkholderia sp., were the dominate species in our post-fire isolate collection, suggesting they have the metabolic potential to utilize carbon substrates found in PyOM. Further, preliminary experiments reveal that a post-fire isolate from our prescribed burn sites, Paraburkholderia caledonica F3, can grow on aromatic carbon substrates in the lab. Finally, genomic analyses reveal that Paraburkholderia caledonica F3 possess the metabolic potential to degrade a variety of complex aromatic carbon compounds found in PyOM. These findings provide an exciting and promising outlook for the application of these post-fire bacteria for bioremediation efforts following fire.

Bioremediation utilizes the naturally occurring abilities of microorganisms (or plants) to break down harmful compounds and detoxify a polluted environment. Many of these organisms are native to these ecosystems, thus researchers are particularly interested in uncovering who these microorganisms are and how they can metabolically catabolize toxic compounds, such as aromatics and PAHs. While isolating organisms which are native to environments of interest poses its own challenges, we are still able to strategically and successful isolated these specialists in the laboratory. The arduous task arises in interpreting gene functions which have a phenotype under laboratory conditions. However, recent innovations in high-throughput analysis of mutant phenotypes have improved the rate at which researchers can predict gene functions. As documented in Chapter 4, we applied Random-Barcoded Transposon Sequencing to characterize the functional pathways of aromatic degradation in our laboratory isolate, Paraburkholderia caledonica F3. Here we validate the success of the generation of our Paraburkholderia caledonica F3 barcoded transposon insertion mutant library and propose fitness experiments with compounds of interest which are to follow.