Bacillus subtilis strain 3610 is an ancestral undomesticated strain. It diers from the laboratory strain 168 in many aspects. One dierence in strain 3610 is the presence of plasmid pBS32 encoding the sigma factor N (σN). This σ factor is activated when DNA damage occurs and induces the bacteria's cell death. The aim of the Thesis was a systematic characterisation of σN-dependent transcription. First, I showed that plasmid-borne but not chromosome-borne predicted σN-dependent promoters were ac- tive in transcription in vitro. Next, the anities of RNAP with σN for DNA, initiating NTP (iNTP) were determined for both relaxed and supercoiled DNA templates. Sur- prisingly, the activity of RNAP on relaxed σN-dependent promoters was higher than on their supercoiled versions, an opposite trend than displayed by RNAP associated with other σ factors. This property of σN-dependent promoters was not encoded by the core promoter sequence. In summary, this Thesis contributed to our understanding of the bacterial transcription apparatus. 1

<p> Deletion in the chromosome of Bacillus subtilis strain Sp-H12-3 was demonstrated by Hg-Cs(2)SO(4) density gradient centrifugation. The base composition of the deleted DNA segments and transcription of m-RNA from these DNA segments were investigated. Physiological and biochemical studies of the mutant Sp-H12-3 yielded information on uridine derivatives which may be intimately associated with the process of sporulation. </p> / Thesis / Doctor of Philosophy (PhD)

To reduce reliance on fossil fuels, sustainable biofuels are being pursued, especially advanced biofuels like 1-butanol that have higher energy content and greater compatibility with existing infrastructure than ethanol. A persistent challenge is the yield-limiting toxicity of biofuels and process solvents, such as tetrahydrofuran, to the microbes that ferment biomass into biofuel. The cell membrane is a focal point of toxicity, and understanding how it interacts with fuels and solvents is key to improving yield. Phospholipid bilayers are the core of biomembranes, and model biomembranes of defined composition provide the ideal platform for biophysical studies. To this end, glycerophospholipids characteristic of Bacillus subtilis, a model producer organism, were synthesized. Two fatty acids (iso- and anteisopentadecanoic acids) characteristic of Bacilli were synthesized and incorporated into representative phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol lipids. The validated synthetic approach opens the door to future studies on the interaction of biofuels and solvents with biomembranes.

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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

No / With the general increase in the worldwide incidence of tuberculosis there is increasing interest in the use of upper room ultraviolet germicidal irradiation (UVGI) systems to disinfect air. A number of researchers have demonstrated experimentally the ability of such systems to inactivate airborne microorganisms. However, relatively little theoretical work has been done to explain the results observed and few models exist to describe the performance of upper room UVGI systems. This paper presents a new model, which can be used both to design such systems and to evaluate their germicidal effectiveness. A theoretical study is undertaken, which indicates that although upper room UVGI systems work well at lower ventilation rates, they are of limited benefit in highly ventilated applications. The paper also demonstrates and quantifies the relationship between inter-zonal air velocity and room ventilation rate. In particular, the paper shows that under steady-state conditions the number of passes made by bioaerosol particles through an upper room UV field is independent of the ventilation rate.

The survival of all organisms depends on their ability to perform certain enzymatic activities and the ability to construct certain structures. In prokaryotes, enzymes are required for the final reactions of peptidoglycan (PG) synthesis, the structural element of the bacterial cell wall. These proteins, known as penicillin-binding proteins (PBPs), are identified through the presence of conserved motifs within their functional domains. The Class A high-molecular weight PBPs are bifunctional, performing the penicillin-sensitive transpeptidase activity and the glycosyl transferase (GT) activity required for the polymerization of the glycan strands. The Class A PBPs in Bacillus subtilis are PBP1, PBP4, PBP2c, and PBP2d (YwheE) and they are encoded by ponA, pbpD, pbpF, and pbpG (ywhE), respectively. These proteins appear to be somewhat functionally redundant because removal of one or more does not cause any noticeable change in phenotype. However, the loss of PBP1 has previously been demonstrated in B. subtilis to cause a decreased growth rate and changes in morphology of vegetative cells, both of which are increased upon the additional loss of PBP4. Furthermore, the loss of sporulation-expressed Class A PBPs, PBP2c and 2d, causes a 10,000-fold decrease in the production of heat resistant spores. This double mutant is shown to have changes in the structural parameters of cortex PG that appear minor when compared to other strains, but are coupled with a large defect on the deposition of cortex PG, apparently from the synthesis of an abnormal germ cell wall. The Class A PBPs are believed to be the only proteins capable of performing the GT activity and it is therefore believed that cell viability requires the presence of at least one functional Class A PBP. This requirement has been demonstrated in other organisms, but a B. subtilis strain lacking all Class A PBPs is viable. The phenotypical changes seen in the PBP1 mutant are exacerbated in this strain. The GT activity remaining in this strain is sensitive to the antibiotic moenomycin in vitro whereas it appears resistant in vivo. Identification of the protein(s) performing this novel GT activity will rely on the demonstration of the GT activity in vitro. / Ph. D.

Components of the bacterial spore germination apparatus are crucial for survival and for initiation of infection by some pathogens. While some components of the germination apparatus are well conserved in spore-forming species, such as the spoVA operon, each species may possess a different and possibly unique germinant recognition mechanism. The significance of several individual proteins in the germination process has been characterized. However, the mechanisms of how these proteins perform their functions and the network connecting these proteins in the complete germination process are still a mystery. In this study, we characterized a Bacillus subtilis superdormant spore population and investigated the abundance of 11 germination-related proteins. The relative quantities of these proteins in dormant, germinating and superdormant spores suggested that variation in the levels of proteins, other than germinant receptor proteins may result in superdormancy. Specifically, variation in the abundance of the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Spore membrane proteomes of Bacillus anthracis and B. subtilis were characterized to generate a candidate protein list that can be further investigated. Proteins that were not previously known to be spore-associated were identified, and many of these proteins shared great similarity in both Bacillus species. A significant number of these proteins are implicated in functions that play major roles in spore formation and germination, such as amino acid or inorganic ion transport and protein fate determination. By analyzing the in vivo and in vitro activity of HtrC, we proved that the protease is responsible for YpeB proteolytic processing at specific sites during germination. However, without HtrC present in the spore, other proteases appear to degrade YpeB at a reduced rate. The activity of purified HtrC in vitro was stimulated by a relatively high concentration of Mn²⁺ or Ca²⁺ ions, but the mechanism behind the stimulation is not clear. We also demonstrated that YpeB and SleB, in the absence of their partner protein, were degraded by unknown proteases other than HtrC during spore formation. Identification and characterization of these unknown proteases would be a future direction for revealing the roles of proteases in spore germination. / Ph. D.

Bacterial spore peptidoglycan (PG) is very loosely cross-linked relative to vegetative PG. Theories suggest that loosely cross-linked spore PG may have a flexibility which contributes to the attainment of spore core dehydration. The structure of the PG found in fully dormant spores has previously been examined in wild type and many mutant strains. These analyses showed little correlation between the degree of spore PG cross-linking and core dehydration. However, these studies only examined the structure of PG from dormant spores and did not allow for the structural analysis of spore PG during sporulation when actual spore PG synthesis and core dehydration occur. Structural analyses of developing spore PG from wild type Bacillus subtilis and eight mutant strains are included in this study. Structural analyses of developing spore PG suggest the following: a) the germ cell wall PG is synthesized first next to the inner forespore membrane; b) cross-linking is relatively high in the first 10% of spore PG synthesized; b) a rapid decrease in cross-linking is observed during synthesis of the next 20% of the spore PG; and c) this decrease is followed by an eightfold rise in the degree of cross-linking during synthesis of the final 70% of the spore PG. This increasing gradient of cross-linking was previously predicted to contribute to the attainment of spore core dehydration. However, analyses of mutant strains indicate this cross-linking gradient is not required for the attainment of spore dehydration. / Master of Science