700 research outputs found
The ARC2 Response in Streptomyces coelicolor: Genetic and Physiological Changes Induced by a Chemical Probe
No full textStreptomyces are known for the production of biologically active secondary metabolites that are encoded in discrete gene clusters. Expression of these clusters is often controlled by factors that include growth rate, signaling molecules, metabolism, and physiological and environmental stresses. These diverse signals along with signal transduction proteins and transcription factors constitute a complex regulatory network that mediate various types of responses including the induction of secondary metabolism. In this work, I have taken a chemical genetic approach, using a chemical elicitor called ARC2, to investigate the regulatory network of Streptomyces coelicolor. I show that ARC2 alters the expression of primary metabolic genes associated with amino acid metabolism, carbohydrate metabolism, lipid metabolism and nucleic acid metabolism. I also show that ARC2 alters the expression of at least 16 different secondary metabolic gene clusters, including the gene cluster encoding the blue-pigmented secondary metabolite actinhorhodin. I go on to show that AfsR and AfsS, two regulators from the AfsK/AfsR/AfsS (AfsK/R/S) pleiotropic regulation system for secondary metabolism, are required for both the basal production of actinorhodin and the stimulation of actinorhodin production by the ARC2 elicitor. In addition, I show that the serine/threonine kinase AfsK is not required for actinorhodin production in S. coelicolor. Lastly, with the use of ARC2, I identified a functionally relevant repeat in the AfsS regulator that is required to activate secondary metabolism. This work demonstrates the use of a chemical tool to uncover novel aspects about the biology of secondary metabolism in the model organism S. coelicolor.Ph.D
Discovery and Characterization of Novel Tetrodecamycins
No full textAntibiotics are one of the most important groups of drugs ever discovered. In addition to their clinical use, these small molecules have also been essential for understanding the molecular details of numerous fundamental biological processes. As a result, the discovery and characterization of novel small molecules is of extraordinary value to both society and science. Streptomyces sp. strain WAC04657 is a wild-isolate bacterium that produces an antibiotic activity against multi-drug resistant Staphylococcus aureus. I purified the responsible molecule and characterized it as 13-deoxytetrodecamycin, a new antibiotic belonging to the tetrodecamycin-group of molecules. To study the biosynthesis of this molecule, I sequenced the genome of WAC04657 and, using bioinformatic methods, identified the biosynthetic gene cluster. I named this cluster the ted cluster. To confirm that these genes were responsible for producing 13-deoxytetrodecamycin, I genetically manipulated genes within the cluster with the goal of making an overproducer strain and a non-producer strain. In addition to affecting the expression of 13-deoxytetrodecamycin, these mutants show alterations in the expression of four other molecules. I purified two of these and solved their structures. One I identified as the known molecule tetrodecamycin, while the second was a novel molecule that I named W5.9. A search of public genome sequences revealed that the ted cluster was also found in three other bacteria: Streptomyces atroolivaceus, Streptomyces globisporus, and Streptomyces sp. LaPpAH-202. By genetically manipulating the ted cluster in S. atroolivaceus and S. globisporus, I was able to confirm that these bacteria were also producers of tetrodecamycin â group molecules. Specifically, they produced tetrodecamycin and dihydrotetrodecamycin. In summary, the work reported in this thesis describes the discovery of a novel antibiotic as well the biosynthetic gene cluster responsible for producing it. Future work will focus on assessing the therapeutic potential of the tetrodecamycin-group molecules and identifying their mechanism of action.Ph.D
Discovery and Characterization of Novel Tetrodecamycins
No full textAntibiotics are one of the most important groups of drugs ever discovered. In addition to their clinical use, these small molecules have also been essential for understanding the molecular details of numerous fundamental biological processes. As a result, the discovery and characterization of novel small molecules is of extraordinary value to both society and science. Streptomyces sp. strain WAC04657 is a wild-isolate bacterium that produces an antibiotic activity against multi-drug resistant Staphylococcus aureus. I purified the responsible molecule and characterized it as 13-deoxytetrodecamycin, a new antibiotic belonging to the tetrodecamycin-group of molecules. To study the biosynthesis of this molecule, I sequenced the genome of WAC04657 and, using bioinformatic methods, identified the biosynthetic gene cluster. I named this cluster the ted cluster. To confirm that these genes were responsible for producing 13-deoxytetrodecamycin, I genetically manipulated genes within the cluster with the goal of making an overproducer strain and a non-producer strain. In addition to affecting the expression of 13-deoxytetrodecamycin, these mutants show alterations in the expression of four other molecules. I purified two of these and solved their structures. One I identified as the known molecule tetrodecamycin, while the second was a novel molecule that I named W5.9. A search of public genome sequences revealed that the ted cluster was also found in three other bacteria: Streptomyces atroolivaceus, Streptomyces globisporus, and Streptomyces sp. LaPpAH-202. By genetically manipulating the ted cluster in S. atroolivaceus and S. globisporus, I was able to confirm that these bacteria were also producers of tetrodecamycin â group molecules. Specifically, they produced tetrodecamycin and dihydrotetrodecamycin. In summary, the work reported in this thesis describes the discovery of a novel antibiotic as well the biosynthetic gene cluster responsible for producing it. Future work will focus on assessing the therapeutic potential of the tetrodecamycin-group molecules and identifying their mechanism of action.Ph.D
The Molecular Action of Actinorhodin, an Antibiotic Produced by Streptomyces coelicolor
No full textActinorhodin is a blue-pigmented, redox-active secondary metabolite that is produced by the bacterium Streptomyces coelicolor. Although actinorhodin has been used as a model compound for studying secondary metabolism, its biological activity is not well understood. Indeed, redox-active antibiotics in general have not been widely investigated at the mechanistic level. In this work, I have conducted a comprehensive chemical genetic investigation of actinorhodinâ s antibacterial effect on target organisms. I show that actinorhodin is a potent, bacteriostatic, pH-responsive antibiotic and that its redox activity, but not its in vitro organocatalyst activity, is important for its antibacterial activity. In the presence of actinorhodin, cells activate at least three stress responses, including those responsible for managing oxidative damage, protein damage, and selected forms of DNA damage. I find that mutations in the Staphylococcus aureus walRKHI operon can confer low level resistance to actinorhodin, indicating possible targeting of the cell envelope. This study indicates a complex mechanism of action for actinorhodin that is distinct from other redox-active compounds.Ph.D
Discovery of Novel Antibiotics via Streptomyces sporulation
No full textStreptomyces are filamentous bacteria known for producing a wide range of bioactive molecules. In addition to being nature’s chemists they have a unique multicellular life-cycle that ends with the sporulation of aerial hyphae. I used this unique Streptomyces development cycle, specifically sporulation, as a screening platform to discover novel biologically active molecules and characterized their mechanisms of action. Min-1 inhibits the growth of a range of Gram-positive bacteria, is active against the cell envelope, and induces a short cell phenotype in Bacillus subtilis. Another molecule, EN-7, inhibits bacterial gyrase and is active against extensively resistant Staphylococcus aureus in addition to other Gram-positive pathogens. In addition to investigating these specific molecules, I developed a high-throughput screen to identify molecules that disrupt the Gram-positive bacterial membrane and found that Streptomyces venezuelae sporulation is highly sensitive to multiple forms of DNA damage.Ph.D
Stress Responses Affecting the Sporulation and Specialized Metabolism Programs of Streptomycetes
No full textAs the global population grows simultaneously with increases in multi-drug resistance for infectious diseases and various cancers, there is a dire need for the discovery of novel cures and therapeutics. Secondary metabolites from the bacterial genus Streptomyces have classically provided medicines such as antibiotics and chemotherapeutics. However, decades of research have yielded high rates of compound rediscovery. Fortunately, it is known that Streptomyces genomes encode the capacity to produce many more secondary metabolites than we presently observe under standard laboratory conditions. Therefore, to take advantage of their inherent chemical potential, it is imperative that we understand the basic physiology of Streptomyces.
In this work, I leveraged a chemical biology approach to understand how Streptomyces grows and produces bioactive small molecules. First, I show how DNA damage affects the Streptomyces developmental cycle as a means of understanding its growth and cell division under stressful conditions with a focus on the cell division gene ssgB. Second, I show how a Streptomyces-specific small protein called AfsS may have a function related to linking stress responses with secondary metabolism. This study highlights the importance for pursuing basic research as it relates to the potential discovery of new medicines. The full chemical potential of Streptomyces can only be unlocked once there is a fulsome understanding of all factors which guide the production of potentially life-saving molecules.Ph.D
The Molecular Action of Actinorhodin, an Antibiotic Produced by Streptomyces coelicolor
No full textActinorhodin is a blue-pigmented, redox-active secondary metabolite that is produced by the bacterium Streptomyces coelicolor. Although actinorhodin has been used as a model compound for studying secondary metabolism, its biological activity is not well understood. Indeed, redox-active antibiotics in general have not been widely investigated at the mechanistic level. In this work, I have conducted a comprehensive chemical genetic investigation of actinorhodinâ s antibacterial effect on target organisms. I show that actinorhodin is a potent, bacteriostatic, pH-responsive antibiotic and that its redox activity, but not its in vitro organocatalyst activity, is important for its antibacterial activity. In the presence of actinorhodin, cells activate at least three stress responses, including those responsible for managing oxidative damage, protein damage, and selected forms of DNA damage. I find that mutations in the Staphylococcus aureus walRKHI operon can confer low level resistance to actinorhodin, indicating possible targeting of the cell envelope. This study indicates a complex mechanism of action for actinorhodin that is distinct from other redox-active compounds.Ph.D
Book Review: Ground Combat: Puncturing the Myths of Modern War
No full textAuthor: Ben Connable
Reviewed by: Justin R. Lynch, lecturer, Georgetown University
Ground Combat: Puncturing the Myths of Modern War by Ben Connable dismantles popular assumptions about the future of warfare by grounding its analysis in over 400 real-world battles. Rather than relying on hype around technology and precision, Connable reveals the enduring, gritty realities of land combat. This thought-provoking study challenges military planners and strategists to rethink how wars are truly fought—and won.https://press.armywarcollege.edu/parameters_bookshelf/1107/thumbnail.jp
Discovery of Novel Antibiotics via Streptomyces sporulation
No full textStreptomyces are filamentous bacteria known for producing a wide range of bioactive molecules. In addition to being nature’s chemists they have a unique multicellular life-cycle that ends with the sporulation of aerial hyphae. I used this unique Streptomyces development cycle, specifically sporulation, as a screening platform to discover novel biologically active molecules and characterized their mechanisms of action. Min-1 inhibits the growth of a range of Gram-positive bacteria, is active against the cell envelope, and induces a short cell phenotype in Bacillus subtilis. Another molecule, EN-7, inhibits bacterial gyrase and is active against extensively resistant Staphylococcus aureus in addition to other Gram-positive pathogens. In addition to investigating these specific molecules, I developed a high-throughput screen to identify molecules that disrupt the Gram-positive bacterial membrane and found that Streptomyces venezuelae sporulation is highly sensitive to multiple forms of DNA damage.Ph.D
Development Towards a CRISPR-Cas3 Genome Editing System for Streptomyces
No full textStreptomyces sp. have unusual linear chromosomes that are large compared to those of most other bacteria and archaea. However genetic research of this bacterial genus is hampered by the lack of effective gene editing tools that are capable of producing large, undefined, targeted deletions. The Bondy-Denomy lab has shown that CRISPR-Cas3 in Pseudomonas aeruginosa is capable of creating large, undefined, targeted deletions. The aim of this thesis was to generate a map of putative essential and dispensable regions in a Streptomycete chromosome and work towards adapting CRISPR-Cas3 from P. aeruginosa for use in Streptomyces venezuelae. The plan was to delete the whiE gene, which generates a visibly apparent spore pigment as a proof of concept. Despite successful expression of CRISPR-Cas3 in S. venezuelae, whiE was not successfully deleted. However, data in this thesis highlights the similarities in Streptomyces species which should support the possibility of a CRISRP-Cas3 system that is compatible with all Streptomyces. Recent research has shown that S. avermitilis has a functional CRISPR-Cas3 system and can be adapted for gene editing in Streptomyces.M.Sc
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