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<p>Bacterial small RNAs have emerged as a class of molecules having important regulatory roles. Accumulating numbers of <em>cis</em>-encoded sRNAs (antisense RNAs) have been recently discovered to be transcribed from the chromosomal DNA of many bacterial species, including the streptomycetes. Here, we investigate potential regulatory roles for two <em>S. coelicolor</em> antisense RNAs, scr4677 and α-abeA.</p> <p>The scr4677 antisense RNA is transcribed from the intergenic region between <em>SCO4676</em> (a gene encoding a conserved protein of unknown function) and <em>SCO4677</em>, encoding a regulatory protein with proposed anti-sigma factor activity. Transcription profiling revealed that scr4677 may not only interact with <em>SCO4676</em> mRNA but also with <em>SCO4677-4676</em> read-through transcripts. Our study suggested that scr4677 functioned to destabilize <em>SCO4676</em> mRNA, at the same time that it stabilized the <em>SCO4677-4676</em> read-through transcript. The potential role for scr4677 in destabilizing <em>SCO4676</em> mRNA was not mediated by the double stranded ribonuclease RNase III. Genetic analysis showed <em>scr4677</em> transcription was affected by SCO4677, and the transcription was apparently dependent on an unknown protein binding to the <em>SCO4676 </em>coding sequence.</p> <p>A second independent study focused on investigating the regulation of a previously uncharacterized genetic region, <em>SCO3287-3290</em>, since renamed <em>abeABCD</em>. This region contains an antisense RNA (α-abeA)-encoding gene, and is adjacent to the downstream <em>SCO3291</em> (<em>abeR</em>) gene, which encodes a putative regulatory protein. Genetic analysis revealed that overexpression of <em>abeR </em>or <em>abeABCD</em> stimulated the production of the blue-pigmented antibiotic actinorhodin, and deletion of <em>abeR</em> impaired actinorhodin production. Transcription analysis revealed the <em>abe</em> genes (including α-<em>abeA</em>) to be subject to multiple levels of regulation. We found an internal promoter within the <em>abeA</em> coding sequence and that required AbeR for expression. Furthermore, biochemical experiments demonstrated that AbeR regulated <em>abeBCD</em> directly, by binding to four heptameric repeats in its promoter region. The expression of α-<em>abeA</em> and other <em>abe</em> genes were differentially affected by RNase III.</p> / Doctor of Science (PhD)

<p>Bacteria demonstrate an extraordinary capacity to survive and adapt to changing environments. In part, this ability to adapt can be attributed to horizontal gene transfer, a phenomenon which introduces novel genetic information that can be appropriated for use in particular niches. Nowhere is this more relevant than in pathogenic bacteria, whose acquisition of virulence genes have provided an arsenal that permits them to thrive within their selected host. Regulatory evolution is necessary for timely regulation of these acquired virulence genes in the host environment. <em>Salmonella enterica</em> serovar Typhimurium is an intracellular pathogen which possesses numerous horizontally-acquired genomic islands encoding pathogenic determinants that facilitate its host lifestyle. One island, <em>Salmonella</em> Pathogenicity Island (SPI)-2, encodes a type-III secretion system (T3SS) which is regulated by the two-component regulatory system SsrA-SsrB. This system coordinates expression of the SPI-2 T3SS as well as an array of virulence effectors encoded in horizontally-acquired regions throughout the <em>Salmonella</em> genome. The studies presented here investigated the mechanisms in which the transcription factor SsrB functions to integrate virulence processes through regulatory adaptation. This work identified the regulatory logic controlling SsrB and defined the associated SsrB regulon. Furthermore, SsrB was found to induce a regulatory cascade responsible for the expression of bacteriophage genes encoded within SPI-12, an island that also contributes to <em>Salmonella</em> virulence. These findings demonstrate the important contribution of regulatory evolution in pathogen adaptation to the host, and show that horizontally-acquired genes, once integrated into appropriate regulatory networks, can contribute to pathogen fitness in specific niche environments.</p> / Doctor of Philosophy (PhD)

Streptococcus mutans is a facultative member of the oral plaque and is associated with dental caries. It is able to survive long periods of sugar starvation. The purpose of this project was to explore specific avenues that S. mutans may use in order to cope with carbohydrate deprivation. Intracellular polysaccharide (IPS) is accumulated by S. mutans when grown in excess sugar, and can contribute towards the cariogenicity of S. mutans. Inactivation of the glgA gene, encoding a putative glycogen synthase, prevented accumulation of IPS in batch cultures grown with excess glucose or sucrose. Inactivation of the pul gene, encoding a putative pullulanase which is thought to be involved in IPS catabolism, did not prevent IPS accumulation. IPS was found to be important for the persistence of S. mutans grown in batch culture with excess glucose, and then starved of glucose. In these conditions, the IPS was largely used up within one day of starvation, and yet survival of the parental strain was extended by at least 15 days beyond that of the glgA and pul mutants; potentially, some feature of IPS metabolism, distinct from providing nutrients, is important for persistence. IPS was not needed for persistence when sucrose was carbon source or when mucin was present in batch cultures. IPS accumulation was not clearly demonstrated in biofilm conditions. When grown in condition permissive for IPS accumulation, biofilms of the glgA and pul mutants did not show decreased survival, compared to the parental strain. It is plausible that, within a biofilm, S. mutans can use alternative sources of energy (like the extracellular matrix) to compensate for the lack of IPS. To look at specific genes upregulated by sugar starvation, microarrays analysis was performed on S. mutans batch cultures. Some of the genes upregulated by starved, stationary phase bacteria, appeared to be organized in an operon, thought to encode components of the pyruvate dehydrogenase (PDH) complex. Northern Blot analysis showed that pdhD and the downstream genes, pdhA, pdhB and pdhC, form an operon that is transcribed predominantly in stationary phase. Inactivation of pdhD impaired survival of both batch cultures and biofilms. Analysis with fluorescent reporters revealed a distinct expression pattern for the pdh promoter, with less than 1% of stationary phase bacteria displaying pdh expression. When first detected, after one day of sugar starvation, expression was in individual bacteria. At later times, expressing bacteria were often in chains. The lengths of chains increased with time suggesting growth and division. It is likely that the pdh-expressing sub-population is able to persist for extend times in stationary phase. / Microbiology and Immunology

Poor reproductive performance by turkey hens compared with chickens is partially due to the early cessation of egg production associated with the onset of incubation behavior. Prolactin (Prl) is involved in the induction and maintenance of incubation behavior in birds, and understanding the regulatory mechanism(s) of Prl gene expression will provide fundamental information to manipulate Prl production for better reproductive performance in turkey hens. / Ph. D.