RNA polymerase (RNAP) is a key multi-subunit enzyme of gene expression that, together with the σ factor, forms a holoenzyme and transcribes genetic information from DNA to RNA. RNAP from Bacillus subtilis and its primary factor σA were studied in this thesis. The σA factor determines the specificity for the promoters to which the holoenzyme binds. Part of its structure is domain 1.1, which is likely to prevent binding of σA to the promoter by itself (unless it is part of the holoenzyme) by binding to domains 2 and 4. The first part of the thesis verifies the hypothesis that domain 1.1 binds domains 2 and 4 and thus prevents binding of σA to the promoter. To this end, various domain constructs have been created and their interactions have been tested. Domain interaction was tested by Nitrocellulose Filter Binding Assay, EMSA, and in vitro transcription. The results did not show significant interaction between domains. The second part of the thesis deals with the creation of a tool for the study of the enzymatology of RNAP from B. subtilis - recombinant RNAP (rRNAP). First, a plasmid construct for expression of rRNAP in Escherichia coli was constructed by a series of cloning steps, followed by protein isolation and characterization. Isolation was achieved without contamination by σ factors (this...

Microorganisms are capable of producing advanced biofuels that can be used as ‘drop-in’ alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms for improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. First, using genomic DNA from Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance. Additionally, we used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP. putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP. putida induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoDP. putida, while others provide tolerance across a wide range of rpoDP. putida levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries. This dissertation describes a method of effectively screening genomic DNA from multiple organisms for genes to mitigate biofuel stress and shows how tolerance genes can improve bacterial growth in the presence of toxic biofuel compounds. These identified genes can be targeted in future studies as candidates for use in biofuel production strains to increase biofuel yields.

(ENGLISH) The bacterial cell needs to regulate its gene expression in response to changing environmental conditions. RNA polymerase (RNAP) is the pivotal enzyme of this process and its activity is controlled by a number of auxiliary factors. Here I focus on RNAP-associating factors involved in regulation of transcription in G+ bacteria:  factors, initiating nucleoside triphosphates (iNTPs), HelD, δ and small RNA Ms1. The main emphasis is on σ factors from Bacillus subtilis. σ factors allow RNAP to specifically recognize promoter DNA. In my first project I set up in vitro transcription systems with purified alternative σ factors, σB , σD , σH , σI from B. subtilis. Using these systems, I studied the effect of initiating NTP concentration ([iNTP]) on transcription initiation. I showed that promoters of alternative  factors are often regulated by [iNTP]. In the next project I comprehensively characterized one of the least explored alternative  factors from B. subtilis, I . I identified ~130 genes affected by I , though only 16 of them were directly affected. Moreover, I discovered that I is involved in iron metabolism. Finally, I showed that I binding requires not only the conserved -35 and -10 hexamers, but also extended -35 and -10 elements located in the spacer region. In collaboration with...

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Cyanobacteria are prokaryotes capable of oxygenic photosynthesis, and for many species, nitrogen fixation, giving cyanobacteria an important role in global carbon and nitrogen cycles. Furthermore, multicellular filamentous cyanobacteria are developmentally complex, capable of differentiation into different cell types, including cells capable of nitrogen fixation and cells for motility, making them an ideal platform for studying development, as well as for practical use in biotechnology. Understanding how developmental programmes are activated require an understanding of the role of alternative sigma factors, which are required for transcriptional activation in bacteria. In order to investigate the gene regulatory network and to determine the role of alternative sigma factors in hormogonium development, real time PCR and Next Generation RNA-seq were used to measure expression levels of genes involved in hormogonium development and to further characterise the nature of the hormogonium developmental programme in the filamentous cyanobacterium Nostoc punctiforme. The results support a model where a hierarchal sigma factor cascade activates hormogonium development, in which expression of sigJ activates expression of the sigma factors sigC and sigF, as well as a wide range of other genes, including those involved in the type IV pilus (T4P), chemotaxis-like systems, and cell architecture. SigC and SigF have more limited roles: cell division genes are dependent on SigC and pilA expression was stringently SigF-dependent. Interestingly, SigC was also found to enhance expression of sigJ during hormogonium development, implying a potential positive feedback loop between sigJ and sigC.

Bacteria, the most dominant organisms on Earth, are an everyday presence in our lives. Symbiotic bacteria, which are present in the digestive tract of animals, usually have a beneficial effect on the body. On the opposite side of the spectrum are pathogenic species that cause more or less serious diseases around the world. In order to fight pathogens effectively, it is necessary to learn as much as possible about the molecular mechanisms by which bacteria respond to their environment, and also about the types of communication within bacterial populations that allow them to react to environmental changes as "multicellular" organisms. This Thesis consists of two main parts. In the first part, selected aspects of bacterial gene expression are characterized, using Bacillus subtilis and Mycobacterium smegmatis as model organisms. DNA-dependent RNA polymerase (RNAP) is the enzyme that is responsible for transcription of DNA into RNA, and thus plays a key role in gene expression. This Thesis deals with the structure of bacterial RNAP and important auxiliary factors (proteins and RNA) that associate with this enzyme and modulate its function. In the second part, the focus is on cell-to-cell communication, revealing which factors/mechanisms, including gene expression, affect this process in B. subtilis....

<p><h1>Abstract</h1></p> <p><strong> </strong></p> <p><strong> </strong> Understanding the adaptive mechanisms of large regulatory networks can provide insight into long-term survival of bacterial populations in nature. The RpoS master stress regulator found in <em>E. coli</em> controls the expression of nearly 10% of the genome when cells enter stationary phase or in response to general stress conditions. Despite its important role in stress protection, mutations in the <em>rpoS </em>gene are frequently selected in laboratory strains, pathogenic strains and natural isolates. Loss-of-function mutations are beneficial in long-term stationary phase cultures and in steady state glucose- limited chemostat cultures. Although these mutants have increased utilization of an extensive set of substrates, selection for loss of RpoS function occurs at the cost of reduced stress resistance. Previous studies have demonstrated that highly reversible mutations occur within the <em>rpoS</em> gene (for example, transversions and nonsense mutations) when selected on succinate minimal media; however, no study has yet identified a natural compound that can select for restoration of RpoS function. In this study we identify a natural compound allowing restoration of RpoS⁺ cells from a succinate selected RpoS^- culture. Using an RpoS-dependent <em>osmY-lacZ</em> fusion reporter strain carrying a loss-of-function point mutation in the <em>rpoS</em> gene, we demonstrate that growth on 6% NaCl results in selection of mutants with restored RpoS function occurring at a mutation frequency of 10^-9 mutants per cell plated. This is confirmed by RpoS protein detection, and <em>rpoS</em> sequencing results show transversion or transition mutations. These results are the first to demonstrate that selection for restoration of RpoS function can be mediated by a single condition/compound and are consistent with the idea that mutations in the <em>rpoS</em> gene may act as a physiological molecular switch to control the expression of the RpoS regulon.</p> <p><strong> </strong></p> / Master of Science (MSc)

Sporulation in Bacillus subtilis is triggered by starvation for carbon and nitrogen sources. The process of endospore formation involves a highly orchestrated program of gene expression resulting in morphological change. A key early event is the asymmetric sporulation division, which yields the smaller prespore and larger mother cell. The transcription factor σF becomes active in the prespore, and directs the transcription of approximately 50 genes. One of those genes, paramount to this study, is spoIIR. The SpoIIR protein is exported, and localizes to the inter-membrane space of the sporulation septum, which may be mediated directly by SpoIIGA. SpoIIR and SpoIIGA are essential for the activation of σE from its inactive precursor, pro-σE. Pro-σE, encoded by spoIIGB, is part of a two-gene operon that also includes spoIIGA located immediately upstream. SpoIIGA is an integral membrane protein, and is an aspartyl protease that cleaves 27 residues from the N-terminus of pro-σE to yield active σE. The N-terminal portion of SpoIIGA contains five membrane-spanning hydrophobic domains. The C-terminal portion lies within the mother cell cytoplasm, and contains the proteolytic domain with residue D183 acting as the catalytic aspartate. Activation of the proteolytic domain of SpoIIGA is dependent on signaling through SpoIIR at the septum. The interaction with SpoIIR is thought to cause a conformational change in the proteolytic domain of SpoIIGA, which activates it. Normally, σE only becomes active in the mother cell. The current model for σE compartmentalization suggests that before septation SpoIIGA is evenly distributed throughout the entire cell membrane. Once septation occurs, there is a higher amount of SpoIIGA in the mother cell than in the prespore. SpoIIGA is then concentrated in the septum and the mother cell outcompetes the prespore for the limited amount of SpoIIR available. We are investigating the role of SpoIIGA in compartmentalization of σE. To accomplish this, a spoIIGA-gfp translational fusion was used. The fusion was introduced into a B. subtilis SpoIIGA mutant (SpoIIGA49) that lacks functional SpoIIGA because of a point mutation, G100R. The mutation is located within the fourth membrane-spanning domain. The spoIIGA-gfp fusion was also introduced into a spoIIGA knockout strain (spoIIGA-null) in order to assess the effect of SpoIIGA-GFP on sporulation without influence from SpoIIGA-G100R. The SpoIIGA49 strain that expressed spoIIGA-gfp in the prespore from the σF-directed promoter, PspoIIQ showed a weak GFP signal in the prespore, and restored sporulation to parental levels. The same fusion also showed a weak prespore GFP signal in the spoIIGA-null background, however sporulation was not restored. This result suggests that the fusion protein could interact with SpoIIGA-G100R across the septum through SpoIIR, restoring proteolytic activity to SpoIIGA-G100R. In both cases, fluorescence was only detected after σE had become active. Expression of spoIIGA-gfp from its natural promoter also largely complemented SpoIIGA49, but only partially complemented spoIIGA-null. Again, GFP fluorescence was weak, and was only detected after σE had become active. Possible explanations for the poor fluorescence are: 1, GFP function is impaired. 2, SpoIIGA-GFP is present at low levels. To assess the amount of protein present, western blot analysis was performed using anti-GFP antibodies. The results indicated weak expression. When spoIIGA-gfp was expressed from PspoIIG, protein was detected two hours after entry into stationary phase (T2), which was before GFP fluorescence was detected, with or without SpoIIGA-G100R. Detection of SpoIIGA-GFP from PspoIIQ occurred by T4 in with and without influence from SpoIIGA-G100R. Because PspoIIQ requires σF to be active and PspoIIG is active prior to septation, it was expected that SpoIIGA-GFP expressed from PspoIIG would be detected earlier. Weak bands representing SpoIIGA-GFP were observed which suggests low levels of SpoIIGA-GFP. Overall, the PspoIIG promoter appeared to drive more expression of spoIIGA-gfp before septation than PspoIIQ in the prespore alone. / Microbiology and Immunology

Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of stresses in its environment and during metabolism. The oxidative an pH stresses of four ECF (extracytoplasmic function) σ-factors are investigated. Three of these, σ0550, σ1813, and σ1819 show responses to light-generated singlet oxygen and respiration-generated superoxide reactive oxygen species (ROS). The EcfG homolog, σ4225, shows a high response to superoxide and acid stress. Two proteins, one containing the EcfG regulatory sequence, and an alternative exported catalase, KatE, are presented to be regulated by σ4225. Transcripts of both genes show similar responses to oxidative stress compared to σ4225, indicating it is the EcfG-like σ-factor homolog and controls the global stress response in R. palustris.

The genome sequencing projects have generated a wealth of genomic data and the analysis of this data has provided many interesting findings. However, genome wide analysis of bacteria for promoters has lagged behind, because it has been difficult to accurately predict the promoters with so much background noise that are found in bacterial genomes. One approach to overcome this problem is to predict phylogenetically conserved promoters across multiple genomes of different bacteria, thus filtering out many of the false positives, which are predicted by the current methods. However, there are no programmes capable of doing this. Therefore, the work presented in this thesis has developed a position weight matrix (PWM) based programme called Multiscan that predicts conserved promoters across multiple bacterial genomes. Since Chlamydia is one of the most sequenced bacterial genera and has a high level of conservation of genes and large-scale conservation of gene order between species, Multiscan was developed and tested on Chlamydia. When Multiscan analysed a genome wide dataset of equivalent non-coding regions (NCRs) upstream of genes, from Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia caviae for σ66 promoters that are phylogenetically conserved, Multiscan predicted 42 promoters. Since only one of the 42 promoters predicted by Multiscan had previously available biological data to confirm its prediction, an additional subset of 10 of the remaining 41 σ66 promoters were analysed in C. trachomatis by mapping the 5' end of the transcripts. The primer extension assay synthesised cDNA products of the correct length for seven of the 10 genes chosen. When the performance of Multiscan was compared to one of the accepted method for genome wide prediction of promoters in bacteria, the &quotstandard PWM method", Multiscan predicted 32 more promoters than the &quotstandard PWM method" in Chlamydia. Furthermore, the promoters predicted by Multiscan were up to three more mismatches from the Escherichia coli σ70 consensus sequence than the promoters predicted by the standard PWM method. Although Multiscan predicted 42 promoters that were well conserved across the three chlamydial species, the analysis was unable to identify the 14 known σ66 promoters in C. trachomatis. These promoters were missed (1) because they were dissimilar to the E. coli σ70 consensus sequence and/or (2) because the promoters were poorly conserved across the three chlamydial species. To address the second possibility, the 14 false negatives were analysed by another phylogenetic footprinting method. Fourteen sets of equivalent NCRs located upstream of the homologous genes from the three chlamydiae were aligned with the computer programme Clustal W and the alignment analysed &quotby eye" for evidence of phylogenetic footprints containing the 14 false negatives. The analysis identified that seven of the 14 false negatives were poorly conserved across the chlamydial species. Analysis of two of the seven promoters that could not be footprinted, the promoters of ltuA and ltuB, by mapping the transcriptional start sites in C. caviae, confirmed their poor conservation across C. trachomatis and C. caviae. This analysis showed that substantial differences exist in chlamydial σ66 promoters from equivalent NCRs upstream of genes. This study has developed a new computer programme for genome wide prediction of promoters that are phylogenetically conserved and has shown the value of this programme by identifying seven new well conserved promoters and seven candidate poorly conserved promoters in Chlamydia.