Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an in vivo experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was paired with proteomics to assess how the proteome changes in response to the directed incorporation of exogenous FAs into the membrane of Bacillus subtilis. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phosphatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Changes in the abundance of enzymes involved in FA biosynthesis and degradation are observed; along with changes in abundance of cell wall enzymes and isoprenoid lipid production. The observed changes may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down-regulated in the labeled cells – as was the actin-like protein MreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored in regard to modulating cell membrane FA composition and its effects on lipid headgroup and raft/cytoskeletal associated proteins.

The effects of dietary antimicrobial removal and Bacillus subtilis supplementation on the growth and intestinal health of male broilers were investigated. Birds were fed either a control, antimicrobial, or a B. subtilis probiotic diet at different feeding phases. Birds were challenged with a 10 × dose of a coccidiosis vaccine. Supplementation of B. subtilis in for antimicrobials in the late grower and early finisher phases improved growth similar to birds fed antimicrobials until withdrawal, while antimicrobial removal without B. subtilis supplementation in those periods hindered growth. The improved growth suggests that the probiotic was able to alleviate the stress of the challenge compared to antimicrobial removal. Processing yields were improved with antimicrobial removal and B. subtilis supplementation in late grower and early finisher phase. Intestinal health was improved with lower intestinal lesions when antimicrobial were removed and B. subtilis supplemented suggesting the reduction of Eimeria species from colonizing the intestine.

The electrospray survivability of B. subtilis spores and E. coli was tested in atmospheric mobility experiments. E. coli did not survive electrospray charging and desolvation, but B. subtilis did. Experimental conditions ensured that any surviving bacteria were charged, desolvated, and de-agglomerated. B. subtilis was also found to survive both positive and negative electrospray and subsequent introduction into vacuum conditions. Attempts were made to measure the charge distribution of viable B. subtilis spores using electrostatic deflection. From those experiments, it was found that either the spores do not become highly charged under the electrospray conditions used or only spores in a low positive or negative charge state survive.

No description available.

No description available.

The process of sporulation in Bacillus subtilis serves as a paradigm for study of sporulation in all Bacillus and Clostridium species. During the process of sporulation there is a sequential and cell type specific activation of RNA polymerase sigma factors. The asymmetric sporulation septum forms, which divides the cell into the smaller prespore and the larger mother cell. Then, óF becomes active in the prespore and óE becomes active in the mother cell. Following completion of engulfment of the prespore by the mother cell, óG becomes active in the prespore and finally óK becomes active in the mother cell. At the time the sporulation septum forms only the 30% origin proximal portion of the chromosome is within the prespore. It is the responsibility of the SpoIIIE translocase to pump the remaining chromosome destined for the prespore into the prespore. We hypothesize that activation of óG does not occur until the complete chromosome has been translocated into the prespore, and the prespore has been engulfed by the mother cell. Our first method of investigation was to increase the time required to complete translocation of the chromosome into the prespore. Strains in which large inserts of foreign DNA have considerably increased the genome size were obtained and characterized. The strains have shown a delay in the completion of engulfment, which still occurred before the activation of óG. Activation was identified by visualization of GFP from a óG-directed promoter indicating translation of a product transcribed by óG. We have also shown that the terminus region of the chromosome entered the prespore shortly before the completion of engulfment. It was determined that the increased genome size did not result in a delay of the transcription of the structural gene for óG,spoIIIG. Using a strain in which the activity of the SpoIIIE translocase was reduced 2.5 fold we used a óG-directed GFP reporter to study óG activation under both time course and time lapse conditions. We again found that óG only became active following the completion of engulfment. Our second avenue of investigation was to change the site of replication termination. A strain was used in which several terminus associated genes have been relocated from their usual location near the terminus (172°) resulted in termination of replication occurring at 145° on the chromosome. In this strain, it has been found that, under both time course and time lapse microscopy conditions, óG activation still occurred only following completion of engulfment. The translocation of the chromosome in this strain was studied by time lapse microscopy using a óF- directed reporter and it was determined that the origin proximal region, the site of replication termination, and the traditional terminus region, were all translocated into the prespore prior to the completion of engulfment. The results support the hypothesis that there is a strong link requiring complete translocation of the chromosome, followed by engulfment of the prespore, before óG becomes active. / Microbiology and Immunology

Formation of spores by Bacillus subtilis is a primitive system of differentiation that has become a paradigm for studying cell differentiation in prokaryotes. Differential gene expression commences soon after the single, asymmetric sporulation division through the activation of different RNA polymerase sigma factors, sigma F in the smaller prespore and sigma E in the larger mother cell. sigma E activation relies on an inter-cellular signaling emanating from sigma F-directed gene expression. Formation of the asymmetric division septum and compartmentalized activity of both sigma factors occur prior to chromosome partitioning. At the time of septation, only 30% of the chromosome destined to be in the prespore is actually present in that compartment and the remaining 70% is in the mother cell. Thus, both cell types contain unequal DNA content. This study focused on the effect of this genetic asymmetry on sigma F-directed gene expression, and exploited this effect in order to study aspects of sigma F to sigma E inter-compartmental signaling. Perturbed signaling resulted in the discovery of a novel twin-spore forming morphology, which was further characterized. A DNA translocase is required to translocate the remaining portion of the chromosome from the mother cell to the prespore. The replication terminus region of the chromosome was observed to be the last to enter the prespore and thus, sigma F-directed genes showed delayed and reduced expression when moved to a terminus-proximal location. The studies indicate that this positional regulation of sigma F-directed gene expression is attributed to both delayed entry and inhibition in sigma F activity at late stages of sporulation. Moreover, the next prespore-specific sigma factor, sigma G, could have a role in inhibiting sigma F. The link between sigma F and sigma E activation is the spoIIR locus, which is transcribed in the prespore from a sigma F-directed promoter soon after the formation of the asymmetric septum. Inactivation of the structural genes for sigma F or sigma E or SpoIIR results in the formation of a second septum at the opposite pole; development proceeds no further, resulting in an "abortively disporic" phenotype. The second septum is formed about 20 min after the first, and sigma E activity is required to prevent its formation. As a sigma F-directed gene, spoIIR is subject to `positional regulation': a delay in spoIIR expression caused by moving it from its origin proximal position to the chromosome terminus, is sufficient to delay sigma E activation and block spore formation, giving the abortively disporic phenotype. The effects of delaying and enhancing spoIIR expression were tested. The changes delayed sigma E activation, and many organisms formed a septum at both ends. However, both prespores in these organisms were able to develop into mature spores (twins). Extra rounds of chromosome replication occured during twin formation, so that each twin had a chromosome and the mother cell had either one or two chromosomes. This over-initiation of chromosome replication is a prerequisite for twin spore formation. Moreover, the studies showed that mother cells of twin forming organisms were longer than those containing single spores; image analysis showed that mother cell length correlates with chromosome content. In contrast to twin spore formation, during normal spore development, there is usually one copy of the chromosome in the prespore and one in the mother cell, with no growth of either compartment. Therefore, the system allowed investigating regulation of chromosome replication and growth of the mother cell. The studies showed that replication and growth are permitted because of the absence of active sigma E and of reduced levels of transcription directed by the master regulator for entrance to spore formation, Spo0A. The results indicate that the burst of Spo0A-directed expression along with activation of sigma E provide mechanisms to block replication and growth of the mother cell. / Microbiology and Immunology

<p> The increase in microbes resistant to a wide array of antibiotics has led to the need for the development of novel antimicrobials. However in order to develop new antimicrobials, novel pathways need to be targeted. Teichoic acid is an anionic polymer covalently attached to the cell wall of Gram-positive bacteria. Recent research has demonstrated that teichoic acid genes are indispensable to the viability of Bacillus subtilis. This makes teichoic acid biosynthetic proteins ideal candidates for the development of a new antimicrobial. Of the teichoic acid glycerol phosphate (tag) genes involved in the biosynthesis of teichoic acid in B. subtilis 168, a conditional deletion mutant of tagD, whose protein product encodes the proposed glycerol-3-phosphatecytidylyltransferase, has been previously constructed and was shown to have a lethal phenotype upon depletion of TagD. This was used in a microarray analysis to find genes that were transcriptionally up-regulated upon the depletion of TagD in B. subtilis 168. Ten candidate genes were selected from those up-regulated and used in the design of a novel, real-time, cell-based luminescent reporter system that responds to lesions in wall biosynthesis. Characterization of these reporter systems in tag gene deletion backgrounds and an examination of their response to antibiotics of various mechanism of action led to the identification of our candidate reporter system P ywac, a robust reporter of both lesions in teichoic acid and peptidoglycan synthesis. In a proof-of-principle screen, the use of Pywac as a reporter of lesions in the cell wall was validated. This reporter system is unique in that it combines conventional genetics with a high throughput capacity. It will not only be amenable for screening small molecules to find inhibitors that impinge on teichoic acid biosynthesis, but it can also be used to probe genetic interactions in B. subtilis. </p> / Thesis / Master of Science (MSc)

Many spore forming bacteria are the causative agents of severe disease, such as Bacillus anthracis and anthrax. In these cases, the spore often acts as the infectious agent. Spores boast extreme resistance to chemical and UV damage among other bactericidal conditions. This is problematic due to the difficulty and economic costs of decontaminating exposure sites. The present work focuses on identifying and characterizing proteins active within spore germination, with a focus towards understanding the triggering of the major stages of germination. Understanding how each stage is initiated could allow for development of methods that induce these processes to efficiently germinate spores, thus facilitating cheap and effective decontamination. Sequencing of a spore transposon insertion library after exposure to germinants led to the identification of 42 genes with previously uncharacterized roles in spore germination. Fourteen of the genes, encoding proteins associated with the inner spore membrane, were further characterized. Mutants lacking these genes portrayed phenotypes consistent with failure of a GerA receptor-mediated germination response, and these genes affect the earliest stages of germination. Chemical cross-linking was used to characterize protein interactions important for stage II of spore germination. Site-directed in vivo crosslinking indicated that YpeB may exist as a multimer within the dormant spore. Further investigation of individual protein domains using bacterial two-hybrid analysis suggested that both N- and C-terminal domains of YpeB contribute to the formation of a multimer. In addition, the uncharacterized YpeB N-terminal domain was demonstrated to have strong self-association and may mediate self-association within the dormant spore. Additional genes that contribute to efficient initiation of spore germination in a GerA-dependent manner were identified via TnSeq. Chemical cross-linking of dormant spores was implemented to characterize protein interactions leading to stabilization and activation of an important enzyme that contributes to cortex degradation in stage II of germination. The presented studies employed a variety of techniques to provide additional insight into both stages of spore germination with a goal of furthering understanding of specific events that contribute to a loss of spore dormancy. / Doctor of Philosophy / Few bacterial species can undergo a specialized division process leading to the generation of a bacterial endospore. Endospores are dormant cells that boast resistance to a variety of environmental conditions that would otherwise cause bacterial cell death. These resistance traits make endospores immune to traditional bactericidal methods, making decontamination a nontrivial task. Further complicating the matter, spores are often the infectious particle of the associated disease, including hospital acquired diarrhea, infant botulism, anthrax, and many others. Presented work focuses on furthering understanding the process by which a dormant spore returns to a typical growing bacteria cell. Comprehension of major steps in this process may lead to novel methods for spore cleanup in which mechanisms within the spore are subverted to force a return to a typical bacterial cell state.

The physiological role of the teichoic acid polymers found in Gram-positive bacterial cell walls is not known. Studies of Bacillus subtilis hybrid strains implicate a defined chromosomal region, which includes the tag-1 locus, as necessary for teichoic acid biosynthesis. A set of ten mutants carrying lesions in this region was identified from among forty-four temperature-sensitive (ts) mutants generated by nitrosoguanidine mutagenesis and bacteriophage 029 selection. This protocol gave a population enriched for ts, versus auxotrophic, mutants. For each of the ten mutants, the frequency of genetic reconstruction, or correction, of the ts phenotype indicated that it was due to change(s) in a single gene. Results of two-factor transformation crosses sorted the mutants into three complementation groups; all ten could complement tag-1. Mutants in two complementation groups were transformed to ts⁺ with cloned rodC DNA. The map order of the newly isolated ts markers was determined from the results of two factor crosses. Orientation with respect to the hisA marker was inferred from transduction experiments. The newly isolated strains were shown to be conditional rod⁻ mutants. Growth at 48°C resulted in reduced growth rates and spherically shaped cells. Additional phenotypes seen for some mutants, namely 029 phage resistance and ts spore outgrowth, appeared closely associated with the ts rod⁻ mutation. Wall phosphate content for two of the mutants, following growth at 48°C, was found to be reduced in comparison to the wild-type control. Taken together these results lend support to the argument that the tag-1 region of the chromosome, which most likely directs teichoic acid biosynthesis, is important for establishment and maintenance of the normal bacillary morphology seen for B. subtilis. The importance of other gene products to the organization of newly synthesized wall was examined using B. subtilis macrofibers. Left- and right-handed macrofibers were converted to spheroplasts and the multi-celled structures regenerated under the two sets of conditions conducive for production of the original, and inverse hand. The helix hands observed for the regenerated structures always corresponded to those expected on the basis of the parental genotype.