In Streptomyces bacteria, the expression of many antibiotic biosynthetic clusters is controlled by both cluster-specific regulators and more globally-acting regulators; however, much remains unknown about the factors that govern antibiotic production. In Streptomyces venezuelae, we have discovered that the broadly-conserved nucleoid-associated protein Lsr2, plays a major role in repressing specialized metabolic cluster gene expression. To understand how Lsr2 exerts its gene silencing effects, we focused our attention on the well-studied, but transcriptionally silent, chloramphenicol cluster in S. venezuelae. We established that Lsr2 represses transcription of the chloramphenicol cluster by binding DNA both within the cluster and at distal positions. CmlR is a known activator of the chloramphenicol cluster, but expression of its associated gene is not under Lsr2 control. We discovered that CmlR functions to ‘counter-silence’ Lsr2 activity, alleviating Lsr2 repression and permitting chloramphenicol production, by recruiting RNA polymerase. Lsr2 plays a central role in controlling antibiotic production in Streptomyces; however, beyond this counter-silencing activity, little is known about how Lsr2 is regulated. We identified regulators that could control the expression of lsr2, and found that Lsr2 and LsrL, an Lsr2 homologue that is encoded by all streptomycetes, interact directly with each other, and that their respective DNA-binding activities are altered by the presence of the other protein. These data suggest that LsrL may impact Lsr2 activity in regulating antibiotic production in Streptomyces. Beyond Lsr2, we wanted to develop a comprehensive understanding of the regulatory proteins that impact biosynthetic gene cluster expression. To define the regulatory protein occupancy of antibiotic clusters, we developed ‘in vivo protein occupancy display-high resolution’ (IPOD-HR) technology for use in Streptomyces. This work will lay the foundation for establishing a comprehensive regulatory network map for biosynthetic clusters in Streptomyces, and guide future work aimed at stimulating the expression of metabolic clusters in any Streptomyces species. / Thesis / Doctor of Philosophy (PhD) / Streptomyces bacteria produce the majority of naturally-derived antibiotics, and they have the genetic potential to produce many more antibiotics and antibiotic-like compounds (‘specialized metabolites’). Specialized metabolism is controlled by multiple regulatory systems. In Streptomyces venezuelae, we have discovered that the nucleoid-associated protein, Lsr2, represses the expression of most specialized metabolic clusters, and manipulating Lsr2 activity can stimulate antibiotic production. To better understand how Lsr2 exerts its repressive effect, we explored how Lsr2 controlled the production of a known antibiotic. We ultimately identified multiple regulators that could impact the expression and/or activity of Lsr2. Building on the regulatory foundation provided by Lsr2, we then set out to establish a comprehensive regulatory network that governs biosynthetic gene cluster expression. Collectively, this work improves our understanding of antibiotic gene regulation in Streptomyces bacteria, and has the potential to guide novel strategies aimed at stimulating the production of new antibiotics in Streptomyces.

<p>The relatively recent discoveries of bacterial small RNAs (sRNAs) and their important regulatory functions prompted us to conduct a genome wide survey for sRNAs in <em>Streptomyces coelicolor</em>. We used a combined bioinformatics and experimental approach to identify and characterize six sRNAs. sRNA expression profiles were determined throughout <em>S. coelicolor</em> development, including vegetative and reproductive growth, during growth on minimal and rich media. Additionally, we also tested sRNA expression in various <em>S. coelicolor</em> developmental mutants. Two sRNAs were expressed exclusively during growth on one medium type and all but one were expressed constitutively throughout growth apart from the late sporulation timepoint. One of the identified sRNAs, scr1906, appeared to be closely associated with development. scr1906 was only expressed in nutrient limiting conditions just prior to aerial development and sporulation. Expression of scr1906 was abolished in a mutant that was defective in sporulation (due to a mutation in the sporulation sigma factor gene, <em>whiG</em>); however, expression was detected in mutants of both known σ^WhiG target genes, <em>whiH</em> and <em>whiI</em>, which encode sporulation transcription factors. Intriguingly, <em>in silico</em> analysis predicted <em>whiH</em> to be a direct target for scr1906-mediated regulation based on potential nucleotide binding sites. The effects of deletion and overexpression of <em>scr1906</em> on WhiH levels were tested, but require further experimentation.</p> <p>In a separate line of investigation, we sought to characterize a novel actinobacterial-specific protein named sIHF. The <em>sIHF</em> mutant strain revealed that sIHF influenced DNA compaction and segregation during <em>S. coelicolor</em> sporulation and also affected antibiotic production. sIHF associated with the nucleoid, and <em>in vitro</em>, it bound to DNA non-specifically in a length dependent manner, although it was determined to have a preference for three distinct DNA motifs. Like most nucleoid-associated proteins, sIHF affected gene expression indicating the potential for an additional role as a transcription factor. Interestingly, sIHF impacted the activity of topoisomerase. Leveraging information that we have gained from the sIHF-DNA co-crystal complex, studies aimed at characterizing the sIHF regions that are important for DNA interaction and topoisomerase modulation are currently underway.</p> / Doctor of Philosophy (PhD)