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.

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

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.

Aquacultured products assist the human demands for seafood so that foodfish supplies can remain sustainable and consistent. Although the fish-farming industry has seen dramatic growth and intensification in recent years, the latter has led to an increase in bacterial diseases and fish health management problems, resulting in major economic losses around the world. In addition to the lack of understanding of fish physiology, these complications are exacerbated by the inappropriate and controversial use of antibiotics. This work addressed these issues in striped catfish (Pangasius hypophthalmus) and Nile tilapia (Oreochromis niloticus), two economically important foodfish, by investigating alternative, more cost-effective options to promote fish health. The first two studies established reference intervals for immunology, hematology and plasma chemistry analytes in striped catfish in a recirculating aquaculture system (RAS). In a third study, the immunomodulatory effects after directly feeding probiotic strains of Bacillus subtilis NZ86 and O14VRQ in Nile tilapia were ascertained. This last study revealed that supplementation with both of the probiotic strains for 51 days stimulated several local and systemic innate immune responses of tilapia. When these transient probiotic bacteria were present in the gut, a pro-inflammatory environment was developed as evidenced by the localized higher expression of the cytokines tumor necrosis factor (TNF) – α and interleukin (IL) – 1β. Significant increases (p < 0.05) were noted differentially by both probiotic strains throughout the trial in plasma lysozyme content, alternative complement activity, and in the peripheral blood leukocyte profiles. Additionally, there were trends for increased levels of phagocytosis and respiratory burst in leukocytes of the anterior kidney and spleen at the end of the trial, suggesting the potential use of these probiotic strains for improved immune-competence. These findings help to understand and clarify the potential mechanism of action associated with the increased disease resistance recorded in preliminary studies with the same probiotic strains. Implementation of the tools established and validated in this work could be useful in evaluating fish welfare situations involving striped catfish grown in RAS conditions, and also show promise for a healthier foodfish supply where antibiotic applications practices could be minimized. / Ph. D. / Aquaculture, or fish farming, is one of the most prosperous production sectors of animal-derived food. Despite the success story of aquaculture, the fish industry is heavily plagued by bacterial diseases, which cause losses in billions of dollars annually around the world, and directly contribute to increases in human food insecurity. Since the options to cost-effectively address diseases are limited, I explored alternative ways to more safely monitor and also ensure optimal health in striped catfish and tilapia, two globally important aquaculture fishes. I investigated the values of different cellular and chemical components of the blood to monitor the health of striped catfish when grown in indoor recirculating conditions, in order to understand normal catfish physiology. The values of these blood components were comparable to those of other freshwater fishes. As part of another study, I supplemented probiotics in the diet of the tilapia for 51 days, and assessed the effects of these on the immune system of the fish. Dietary supplementation of the probiotics resulted in the presence of the probiotics in the gut of the fish. Furthermore, the presence of these microbes was tightly linked to elevated values of numerous functions of the immune system. These functions included levels of lysozyme, alternative complement, and percentage of neutrophils, which are all related with a state of heightened immunity in the animal host. The tools that I established and validated in this study are promising alternatives to optimize the health of these two important foodfish. Moreover, they could be useful for the fish farmer because of their greater cost-effectiveness, and can potentially lead to a safer foodfish supply by decreasing the reliance on antibiotics.

Penicillin-binding proteins (PBPs) are required in the synthesis of the cell wall of bacteria. In Bacillus subtilis, PBPs play important roles in the life cycle, including both vegetative growth and sporulation, and contribute to the formation of the different structures of vegetative cell wall and spore cortex. The B. subtilis genome sequencing project revealed there were two uncharacterized genes, ykuA and yrrR, with extensive sequence similarity to class B PBPs. These two genes are renamed and referred to henceforth as pbpH and pbpI, respectively. A sequence alignment of the predicted product of pbpH against the microbial protein database demonstrated that the most similar protein in B. subtilis is PBP2A and in E. coli is PBP2. This suggested that PbpH belongs to a group of the genes required for maintaining the rod shape of the cell. Study of a pbpH-lacZ fusion showed that pbpH was expressed weakly during vegetative growth and the expression reached the highest level at the transition from exponential phase to stationary phase. The combination of a pbpA deletion and the pbpH deletion was lethal and double mutant strains lacking pbpH and pbpC or pbpI (also named yrrR) were viable. The viable mutants were indistinguishable from the wild-type except that the vegetative PG of the pbpC pbpH strain had a slightly slightly lower amount of disaccharide tetrapeptide with 1 amidation and higher amount of disaccharide tripeptide tetrapeptide with 2 amidations when compared to others strains. This suggests that PbpC (PBP3) is involved in vegetative PG synthesis but only affects the PG structure with a very low efficiency. A pbpA pbpH double mutant containing a xylose-regulated pbpH gene inserted into the chromosome at the amyE locus was constructed. Depletion of PbpH resulted in an arrest in cell growth and a dramatic morphological change in both vegetative cells and outgrowing spores. Vegetative cells lacking pbpA and pbpH expression swelled and cell elongation was arrested, leading to the formation of pleiomorphic spherical cells and eventual lysis. In these cells, cell septations were randomly localized, cell walls and septa were thicker than those seen in wild type cells, and the average cell width and volume were larger than those of cells expressing pbpA or pbpH. The vegetative PG had an increased abundance of one unidentified muropeptide. Spores produced by the pbpA pbpH double mutant were able to initiate germination but the transition of the oval-shaped spores to rod-shape cells was blocked. The outgrowing cells were spherical, gradually enlarged, and eventually lysed. Outgrowth of these spores in the presence of xylose led to the formation of helical cells. Thus, PbpH is apparently required for maintenance of cell shape, specifically for cell elongation. PbpH and PBP2a play a redundant role homologous to that of PBP2 in E. coli. A sequence alignment of the predicted product of pbpI against the microbial protein database demonstrated that the most similar protein in B. subtilis is SpoVD and in E. coli is PBP3. This suggested that PbpI belongs to the group of the genes required for synthesis of the spore or septum PG. PbpI was identified using radio-labeled penicillin and found to run underneath PBP4 on SDS-PAGE. PbpI is therefore renamed PBP4b. Study of a pbpI-lacZ fusion showed that pbpI was expressed predominantly during early sporulation. A putative sigma F recognition site is present in the region upstream of pbpI and studies using mutant strains lacking sporulation-specific sigma factors demonstrated that the expression of pbpI is mainly dependent on sigma factor F. A pbpI single mutant, a pbpI pbpG double mutant, and a pbpI pbpF double mutant were indistinguishable from the wild-type. The sporulation defect of a pbpI pbpF pbpG triple mutant was indistinguishable from that of a pbpF pbpG double mutant. Structure parameters of the forespore PG in a pbpI spoVD strain are similar to that of a spoVD strain. These results indicate that PBP4b plays a unknown redundant role. / Master of Science