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Browsing by Subject "Streptomyces"

Now showing 1 - 8 of 8
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    Discovery, Dereplication, And Functional Assignment Of Natural Products In Streptomyces Coelicolor M145
    (2021-11) Marshall, Andrew
    Natural products are structurally complex molecules produced from numerous organisms such as fungi, plants, and bacteria. Sought after primarily for their therapeutic properties, especially as antibacterial agents, screening of natural product-producing organisms extracts from 1920-1960s resulted in a “golden age” of antibiotic discovery. However, this did not last as antibacterial resistance and rediscovery of known molecules became rampant. Re-invigoration of natural product screening efforts occurred as genome sequencing became commonplace and the biosynthetic potential of natural product producers was revealed to be much greater than previously thought. This has led to new efforts to exploit their biosynthetic potential. Key improvements in analytical tools, namely mass spectrometry, has accelerated efforts to sensitively examine natural product mixtures. Additionally, computational tools have enabled the analysis of the large datasets that are a result of these sensitive analytical tools, including quantitation and identification of many molecules, providing a more comprehensive view of natural product extracts than ever before. Application of these tools towards discovery and functional annotation of natural products enables greater insight into the production of these important molecules. This thesis is focused on developing and applying modern mass spectrometry-based approaches to investigate the natural products produced from Streptomyces coelicolor M145, the most well-studied streptomycete. First, ion mobility mass spectrometry is investigated and shown to be an effective approach to separate and distinguish isobaric cyclic-prodiginines that are produced by S. coelicolor. Second, discovery and characterization of a novel actinorhodin-related molecule, θ-actinorhodin, is accomplished using a combined metabolomics and proteomics approach. This represents the first known trimeric benzoisochromanequinone natural product and illustrates the effective use of metabolomics and proteomics on the same liquid chromatography-mass spectrometer. Third, a comprehensive analysis of the time-resolved relationship between gene expression and metabolite abundance of S. coelicolor is enabled by the use of mass spectrometry-based tools and correlation network analysis. This resulted in important conclusions about the concerted expression of biosynthetic gene clusters and the resulting production of the linked metabolite. Finally, several strategies are presented for future analysis of natural product extracts including the use of proteomics to identify putative Streptomyces using a collated list of natural product synthases and an approach combining modern dereplication tools and chemoselective probes for prioritization of natural products for purification.
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    Ecology of interspecies signaling among Streptomyces and its relationship to pathogen suppression
    (2013-05) Vaz Jauri, Patricia
    Interspecies signaling may be defined as the induced change in phenotype of one species by another that is not due to the metabolism of the signal. Although suggested to be a relatively widespread phenomenon, the role of signaling in natural soil communities has not been thoroughly studied. Within Streptomyces communities in soil, understanding the impacts of interspecies signaling on species interactions, and especially on nutrient competition and antagonism, may be key to effective Streptomyces-based suppression of plant pathogens. I evaluated the frequency of signaling interactions and their effect on inhibitory phenotypes of Streptomyces isolated from natural prairies. Signaling among Streptomyces was frequent, and observed in 35% of all interactions. Isolates from the same location in soil were more likely to signal one another than isolates from different locations, suggesting local selection for signaling interactions. Signaling was similarly more frequent between isolates that had similar nutrient use profiles. Finally, closely-related isolates were more likely to increase inhibition towards one another via signaling than distantly-related isolates. In chapter 2, subinhibitory concentrations of antibiotics were studied as signals, specifically in relation to their capacities to shift nutrient use among Streptomyces. We found that some antibiotics altered nutrient use by Streptomyces in ways that could reduce nutrient competition among isolates. Finally, pathogen suppression and signaling were evaluated in soils with different cropping histories. Pathogen suppression by Streptomyces varied significantly among soils, and suppressive activity was positively correlated with bacterial density. Among Streptomyces from these plots, shifts in inhibitory phenotypes in response to signaling by another isolate were very frequent (~ 50% of all interactions). Overall, signaling in Streptomyces is frequent and varies with spatial origin, nutrient overlap, antagonistic phenotype, and genetic relatedness among isolates, as well as soil cropping history. Moreover, some antibiotics have the potential to act as signals that can significantly alter nutrient competition among Streptomyces. Variation in signaling has significant potential to mediate pathogen suppression in soil communities.
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    Evaluating the Effects of Antagonistic Interactions on Pathogen Inhibition by Streptomyces Isolates from a Disease Suppressive Soil
    (2021-02) Pereyra, Matthew
    Diseases of plants threaten global food security and increase the cost of producing food and fiber. Synthetic pesticides have proven effective at suppressing many plant diseases but can require repeated applications at significant expense and cause harm to humans and the environment. Disease suppressive soils often support naturally-occurring soil microbial communities that inhibit plant pathogens and could be used to develop biocontrol or other plant disease management methods. However, high frequencies of resource competition and antagonistic interactions among naturally-occurring pathogen-suppressive populations represent a challenge for reproducing effective microbial disease suppression in agricultural settings. This work sought to further understanding of how the complex network of interactions that occur within disease suppressive soil microbial communities influences pathogen suppression by evaluating pairwise interactions of community members in vitro. Specifically, I characterized inhibition and nutrient competition among community members and their relationships to pathogen inhibition. Among a random collection of 75 Streptomyces isolates from the rhizosphere soil of potato plants grown in a naturally-occurring scab-suppressive soil in Grand Rapids, MN, 34 isolates were able to inhibit pathogenic Streptomyces scabies strain S87. I hypothesized that isolates would have decreased pathogen inhibition when grown in vitro with an inhibitory partner isolate relative to when grown alone. Similarly, isolates were hypothesized to have decreased pathogen inhibition when grown with highly nutrient competitive partners relative to when grown alone. However, when pathogen-inhibiting isolates were grown in pairs there were no consistent effects of partner inhibition or nutrient competition on pathogen inhibition. These results suggest that antagonistic and resource competitive interactions, while potentially important to the long-term establishment of disease suppressive soil microbiomes, may have limited effects on direct inhibition of pathogens. Moreover, this work suggests that successful biological control of plant diseases may not be limited solely to non-antagonistic inoculant mixtures.
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    Genomic analysis of regulatory mechanisms involved in secondary metabolite production.
    (2010-09) Castro-Melchor, Marlene
    Many secondary metabolites have beneficial uses for humans. In addition to their use as antibacterial and antifungal agents, secondary metabolites have been used as immunosuppressants, anti-tumor agents, and antiparasitics. Most of the secondary metabolites known today are produced by filamentous fungi or by members of the Streptomyces genus. Production of secondary metabolites by microorganisms involves a complex, dynamic system, with interconnected elements acting at different levels. Diverse tools were used in this work to explore regulation of secondary metabolite production, mostly in Streptomyces. The tools have a common characteristic: they either generate large amounts of data, or require large amounts of data. Regulation of secondary metabolite production in Streptomyces coelicolor was analyzed at the genome level, by using network modules inferred from a large transcriptome dataset. The upstream sequence of the elements in the network modules was searched for the presence of consensus sequences, and these results combined with information on known interactions, binding sites, and functional relatedness. The combination of this information resulted in a set of twenty networks that have a high likelihood of representing true interactions and represent a starting point for further experimental studies. The characteristics of high productivity were analyzed by comparing the genomes of two strains of the clavulanic acid producer Streptomyces clavuligerus. One of the strains is a high producer of clavulanic acid. Next generation sequence data was used to perform a genome-wide screening to identify all the differences between the two genomes. In addition to mutations in genes involved in β-lactam antibiotic production or their upstream region, structural differences were detected between the two strains. Next generation sequencing technologies were also used to assemble a draft genome for the curdlan producer Agrobacterium sp. ATCC 31749. Curdlan production mimics that of secondary metabolites, it is triggered under starvation conditions. These varied approaches exemplify some of the paths that can lead to a better understanding of secondary metabolism and its regulation.
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    Global biogeography and local adaptation of Streptomyces
    (2013-10) Schlatter, Daniel Cameron
    Streptomyces play crucial roles in key ecosystem processes including nutrient and plant disease suppression in natural and agricultural systems. Moreover, Streptomyces are major producers of clinically relevant antibiotic compounds. Despite the importance of Streptomyces in natural, agricultural, and clinical settings, we have a limited understanding of Streptomyces ecology and evolutionary biology in natural habitats. Here we characterize the function diversity and biogeography of Streptomyces to shed light on the roles of local adaptation and coevolution in structuring soil Streptomyces communities. Specifically, this work focuses on patterns of antibiotic inhibition, antibiotic resistance, resource use, and phylogeny among sympatric and allopatric Streptomyces communities from across the globe. This work documents the extensive functional diversity of Streptomyces antibiotic inhibitory, resistance, and resource use phenotypes and provides strong evidence that local adaptation, coevolution, and resource competition are crucial drivers of antibiotic inhibition and resistance among Streptomyces.
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    Interactions between plants and antagonistic streptomycetes.
    (2011-06) Bakker, Matthew Gene
    This work explores a variety of mechanisms through which plants impact associated streptomycete communities. Streptomycete antibiotic-mediated antagonistic activity is a particular focus. Plant host species and plant richness are addressed as independent and interacting factors driving impacts on associated soil microbial communities. The possibility of modification to antagonistic phenotypes through chemical signaling between plants and streptomycetes is explored.
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    Nutrient competition and coevolutionary antagonistic interactions between Fusarium and Streptomyces in soil
    (2016-10) ESSARIOUI, Adil
    Plant impacts on nutrient use profiles among rhizosphere microbes are poorly understood. We examined the effects of plant host and plant species richness on nutrient use profiles of Streptomyces isolated from the rhizosphere of the prairie plants Andropogon gerardii (Ag) and Lespedeza capitata (Lc) growing in communities of 1 (monoculture) or 16 (polyculture) plant species. Growth on 95 carbon sources was assessed over time. Mean niche width and mean growth were significantly greater for isolates from polyculture vs. monoculture plots, and for Streptomyces from Lc vs. Ag. Isolates from high-carbon (polyculture) or high-nitrogen (Lc) soils had larger niche widths than isolates from low-C (monocultures) or low-N (Ag) soils. Isolates from polycultures were significantly more niche (nutrient) differentiated than isolates from monocultures. We also characterized antagonistic phenotypes and nutrient use among sympatric Streptomyces and Fusarium. Streptomyces from monocultures were more antagonistic against sympatric Fusarium populations than isolates from polycultures. In contrast, Fusarium isolates from polycultures were more inhibitory against sympatric Streptomyces than isolates from monocultures. Fusarium isolates from monocultures had greater niche overlap with Streptomyces than those from polycultures, suggesting greater potential for Fusarium to compete with Streptomyces in monoculture plant communities. In contrast, Streptomyces from polycultures had greater niche overlap with sympatric Fusarium than those from monocultures, suggesting that Fusarium experience greater competition from Streptomyces in polyculture than monoculture. These patterns are consistent with selection for Fusarium-antagonistic Streptomyces populations in the presence of strong Fusarium competition in monocultures, and selection for Streptomyces-inhibitory Fusarium populations in the presence of Streptomyces resource competition in polycultures. Finally, we found evidence for local adaptation between Fusarium and Streptomyces populations as evidenced by the presence of significantly greater inhibition among sympatric than allopatric populations in prairie soil. Additionally, for both taxa, there was a significant positive correlation between the strength of inhibition against each taxon and the intensity of resource competition from that taxon, supporting the hypothesis that antibiotics act as weapons in soil communities. Collectively, these results suggest that coevolutionary antagonistic interactions between Fusarium and Streptomyces in soil are driven by resource competition.
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    Rational Engineering of Expression Level of Multi-Gene Systems encoding Natural Product Biosynthesis in Streptomyces
    (2020-08) Hsu, Suzie
    It has been shown that genomes of bacteria, especially members of the Streptomyces genus, harbor unprecedented numbers of biosynthetic gene clusters (BGCs) potentially encoding novel compounds. However, cloning and controlled expression of these large BGCs in a heterologous host require tedious optimization on a case-by-case basis. This dissertation presents a synthetic biology platform to rapidly reconstitute BGCs by refactoring and physically piecing DNA fragments together in a hierarchical manner. The two core technologies are (i) a high-throughput DNA assembly pipeline for high GC organisms and (ii) synthetic genetic elements to control gene expression in Streptomyces. As a proof of concept, a small library of synthetic gene clusters was constructed to encode ent-atisanoic acid, a late-stage intermediate of the neuroprotectant serofendic acid. We successfully controlled the relative expression level of individual genes, identified the tailoring enzyme required for the oxidation as well as demonstrated the utility of this DNA assembly pipeline. Next, we rationally optimize isoprenoid biosynthesis by perturbing relative expression of eight enzymes in the methylerythritol phosphate (MEP) pathway. One of the Design of Experiment (DoE) methods called Plackett-Burman design was used to guide the optimization effort for this eight-gene system. A five-level Plackett-Burman design was used to guide the design of 125 unique synthetic gene clusters encoding the MEP pathway, which was required to fully screen the effects of expression of each gene on the output measured by isoprenoid titer. In the eight-gene pathway, each gene has one of five expression levels. Total screening of the entire pathway variants has revealed a surprising degree of robustness in actinobacterial secondary metabolism. In sum, the DNA assembly pipeline will become a powerful tool to fuel future rational optimization efforts of multi-gene systems, including large BGCs.