Faithful DNA replication is essential for all organisms to maintain genetic integrity. During the DNA replication, replications forks are frequently stalled or collapsed due to the encounter of DNA lesions or blocking proteins. These events can occur anywhere on the chromosome which is away from the origin of replication. For survival, cells require a number of proteins to repair the damages and restart the replication near or at the damaged site. In Escherichia coli, a group of proteins called primosomal proteins consisting of PriA, PriB, PriC, Rep, DnaT, DnaC, DnaB and DnaG are required for directing DnaB replicative helicase back onto DNA substrates. The main difference between chromosomal replication at oriC and replication restart is the former process is initiated by DnaA recognizing a specific sequence of oriC while the latter process can be initiated by PriA recognizing a specific DNA structure. The mechanism of replication restart is highly ordered and well regulated, and to date, this mechanism has yet to be fully understood. This lab uses a genetic tool to understand replication restart in vivo and discovered that replication restart in E. coli can be explained by a multiple replication restart pathway model (Sandler, 2000). This dissertation presents work that advances our knowledge of replication restart by studying Rep and PriB using a genetic approach. Although it has been shown that Rep and PriB are important for replication restart, the contribution of these two proteins in vivo is still not clearly understood. In the case of Rep, this study provided the characterization of three rep mutants: a rep null mutant, a rep defective in a regulation of Rep monomer helicase activity and a rep ATP hydrolysis deficient mutant. Although these rep mutants showed a similar phenotype, there were some differences such as the phenotypes when combined with other mutations and spontaneous suppressors. In this study, a rep null mutant exhibited an unexpected phenotype including high basal levels of SOS expression and cell filamentation when combined with priB. The priB rep double mutant developed a spontaneous suppressor mapped in dnaC region. A rep mutant defective in an autoinhibition of Rep monomer helicase activity showed a more detrimental phenotype than a rep null mutant when combined with priB suggesting that this mutant Rep inappropriately removes PriC and completes with PriA. The other rep mutant, an ATPase deficient mutant, showed a similar phenotype to that of a rep null mutant as a single mutant and a double mutant with priB, however, spontaneous suppressors developed in the regions close to priC and dnaC, but not in the either of these genes. Lastly, PriB mutants that biochemical evidence has shown some effects on PriA, ssDNA and DnaT binding were tested for effects on replication restart in vivo. The studies of these PriB mutants lead to a model of the regulation of replication restart via PriA-PriB pathway which can be explained by a hand-off mechanism for primosomal assembly.

Bibliography: leaves [168]-207. / Metronidazole is a pro-drug which must be reduced to elicit a bactericidal effect. In the clostridia, some of the electron transport proteins that provide the source of electrons for the reductive activation of metronidazole play a key role in electron distribution, which in turn regulates the direction of carbon flow in the cell. The aim of this research project was to isolate electron transport gene(s) from the solvent-producing Clostridium acetobutylicum strain P262, using transposon-induced metronidazole resistance as a selection system. In the process, the feasibility of transposon mutagenesis in this strain, which lacks conventional systems for DNA delivery, was assessed, and the nature of metronidazole susceptibility in the C. acetobutylicum wild type was investigated. The metronidazole resistant transconjugant of interest, referred to as mutant 3R, was shown to harbour a single insertion of the Tn925: :Tn917 transposon cointegrate within a structural gene, designated sum (susceptibility to metronidazole).

Despite years of intensive research, many of the fundamental aspects of two-component signal transduction pathways are not yet understood. Interestingly these systems are found throughout all domains of life including archaea, bacteria and eukaryotes and are known to regulate diverse cellular processes such as motility, pathogenesis, development, biofilm formation, and toxin production. Despite many groups working on two-component systems it is not yet appreciated whether these systems have conserved features, amino acid requirements, structures and specificity. By understanding the mechanisms by which signals propagate through these systems we could perhaps develop novel therapeutics targeting these pathways. In order to address these questions my thesis has focused on studying the signaling pathways which regulate multicellular development in the model soil bacterium Myxococcus xanthus. The developmental process is highlighted by large changes in gene expression patterns and motility resulting in the production of large macroscopic fruiting bodies composed of metabolically dormant myxospores. My initial work focused on characterizing the Che3 chemosensory pathway known to regulate time of aggregation required for fruiting body formation. I discovered an additional kinase CrdS which works with the Che3 system to regulate phosphorylation of the important developmental regulator CrdA. Additionally I performed mutagenesis on the kinase CrdS to demonstrate that specific residues in CrdS are required for both kinase and phosphatase activities. A conserved Thr/Asn was required for phosphatase activity while a conserved acidic residue was required for kinase activity. Importantly, these residues are highly conserved and when we made mutations in multiple other kinases, we saw similar requirements, indicating the importance of these residues. Further analysis focused on 26 other CrdA homologs found within the M. xanthus genome. Using phosphotransfer profiling and a newly created phosphatase profiling method we were able to demonstrate signaling specificity whereby each kinase was able to phosphorylate and dephosphorylate a single response regulator. Since phosphotransfer and phosphatase activities are predicated upon protein-protein interactions, we also determined that cognate pairs exhibited preferential binding. Cumulatively this research highlights some of the conserved mechanism governing the signal transduction pathways regulating multicellular development in M. xanthus.

Escherichia coli cell division is a complex process involving over 30 proteins that are recruited to the division site. One of the important division proteins is FtsN, which has a C-terminal peptidoglycan (PG) binding region called the SPOR domain. Most SPOR domain proteins are probably involved in bacterial cell division, but their precise role in this process is not known. Although the structure of the FtsN SPOR domain has been solved by NMR, nothing is known about how the domain binds PG. Understanding the SPOR:PG interaction is important because it could lead to novel insights into PG metabolism during cell division. We hypothesize that the SPOR domain from FtsN recognizes and binds septal PG. To test this hypothesis we have conducted a comprehensive mutagenesis of the FtsN SPOR domain to identify amino acid residues critical for septal localization and PG binding. We targeted 33 residues and made a total of 92 point mutants, all of which were tested for septal localization. Our results revealed four amino acids that are critical for septal localization. All four of these residues map in or near the â-sheet, which we now propose is the PG binding site. Further analysis of localization-defective proteins in a PG binding assay led to the realization that the assay measures nonspecific binding to bulk PG rather then specific binding to septal PG. An important priority for the future will be to develop a better PG binding assay.

The family Arenaviridae consists of over 30 members, some of which can infect humans and cause severe hemorrhagic fever. The three arenaviruses studied in this thesis, Machupo virus (MACV), Junín virus (JUNV), and Lassa virus (LASV), are causative agents of Bolivian hemorrhagic fever, Argentine hemorrhagic fever, and Lassa fever, respectively. Epidemics of these diseases can carry high rates of morbidity and mortality, and due to a lack of available countermeasures, all three viruses are considered category A priority pathogens by the CDC. Arenavirus glycoproteins (GPCs) are considered class I viral fusion proteins, but in multiple regards, they are quite unusual for viral envelope proteins. The GP precursor is translated as a polypeptide that is proteolytically processed within the secretory pathway by two sequential cleavage events to produce a tripartite complex (heterotrimers) that assemble into homotrimers to form heterononamers. The three distinct units of the GPC structure are a receptor binding domain, GP1, and fusion domain, GP2, and most peculiarly, a stable signal peptide (SSP) that traverses the membrane twice and associates with the GP2. The glycoprotein complex (GPC) of MACV encodes nine putative sites for N-linked glycosylation. As N-glycans have been shown to be important for a multitude of factors in glycoprotein biology, we sought to investigate the potential roles of the N-glycans on MACV GPC expression, function, antigenicity, and immunogenicity. To do so, we used MACV GPC-VSVΔG-eGFP pseudovirions (MACV-VSV) to study the effects of both individual and combinatorial N-glycan losses. Our results demonstrate that loss of N-glycans at sites N178, N370, or N378 resulted in a loss of GPC proteolytic processing, while the accumulation of multiple N-glycans reduced total GPC expression and function. Replacement of the native proteolytic cleavage motif with those from LCMV or LASV or from furin reduced or eliminated such processing. While individual N-glycans did not themselves affect total GPC expression levels or translocation to the cell surface, those that allowed for efficient GP1-GP2 cleavage led to the favored incorporation of properly processed GPC, which strongly correlated with high virion production and transduction competence. Separate from these findings, we also discovered that loss of N-glycans dramatically increased the antigenicity of subsequent pseudovirions, and while N-glycan mutants retained immunogenicity, the resultant antisera was limited to autologous targets and was not able to inhibit WT MACV-VSV nor JUNV-VSV transduction. The GPC of arenaviruses is the only antigen correlated with antibody-mediated neutralization, but despite strong cross-reactivity of convalescent antisera between related species, weak or no cross-neutralization occurs. Two closely related arenaviruses, MACV and JUNV, have near identical overall GPC architecture and share a host receptor, transferrin receptor 1. Given their extensive likeness, it is not clear how these two viruses avoid cross-neutralization. To address this, a series of MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (mAbs). All mAbs targeted the GP1, and those that neutralized JUNV-VSV transduction competed with each other for binding to the receptor binding site (RBS), specifically. Interestingly, these mAbs did not recognize MACV GPC in its native conformation, despite detecting the protein in western blots. Mouse α-JUNV and α-MACV antisera were also evaluated for neutralization of the wild-type and chimeric JUNV- and MACV-VSV. These antisera neutralized pseudovirions containing the autologous wild-type GP1; however, removal of an RBS-adjacent small disulfide bonded loop unique to MACV GPC was sufficient to increase cross-neutralization with α-JUNV antisera. Our studies provide evidence that this additional loop in MACV GP1 is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV. To enter cells, LASV binds to O-linked glycans present on the cell surface receptor, α-dystroglycan (αDG). While αDG is ubiquitously expressed, glycosylation patterns on some cells restrict the ability of LASV to use this receptor. Multiple studies have suggested that alternative receptors exist on these cell types and have provided evidence that the phosphatidylserine (PtdSer)-binding receptor Axl, along with C-type lectins, play a role in DG-independent entry. In studies presented here, we demonstrate that TIM-1 functions as a receptor for LASV-VSV entry, mediating virion uptake in a PtdSer-dependent, mucin-like domain-independent, manner despite previous reports of the inability of TIM-1 to enhance LASV-VSV transduction. In cases of TIM-1-dependent entry, Ebola virus GP-VSV or LASV-VSV responded differentially to early entry inhibitors, Compound C and EIPA. This provides evidence that mechanism of virus internalization is viral glycoprotein-dependent and cell surface receptor-independent.

Biological Fe(III) reduction is an environmentally significant process, but the mechanisms of electron transfer to Fe(III) are poorly understood. While soluble electron acceptors like oxygen and nitrate diffuse into the cell, dissimilatory Fe(III) reducers transfer electrons onto an insoluble, and therefore extracellular, electron acceptor. Mechanisms of electron transfer in Geobacter species are of interest because these species are the predominant Fe(III)-reducing microorganisms in a variety of environments, including aquatic sediments, aquifers contaminated with organic pollutants or with toxic metals, and current-harvesting electrodes. Proteomic, genetic, and genomic approaches were used to identify components of the electron transport chains to Fe(III) and other electron acceptors in Geobacter sulfurreducens and Geobacter metallireducens. For G. sulfurreducens , the fumarate reductase was identified and its dual function as the succinate dehydrogenase was discovered. Growth by fumarate reduction was induced in G. metallireducens, which cannot naturally respire fumarate, by the expression of a dicarboxylate transporter that allowed the succinate dehydrogenase to function as a fumarate reductase. A c-type cytochrome that was abundant in Fe(III) but not fumarate reducing cells was partially purified and the encoding gene was identified. A strain lacking this gene was severely impaired in Fe(III) reduction, but was unaffected in fumarate reduction or growth under oxidative stress. The conservation of electron transport proteins between G. sulfurreducens and G. metallireducens was determined. All genes in the pathways of acetate oxidation were well conserved, as were genes involved in electron and proton transport within the inner membrane. However, those genes encoding cytochromes, which are the periplasmic and outer membrane electron carriers to Fe(III), were not well conserved, including those required for Fe(III) reduction in G. sulfurreducens. Finally, an 300 kb island present in the G. metallireducens but not the G. sulfurreducens genome was shown to encode genes for aromatics degradation, and the pathways for toluene, phenol, p-cresol, and benzoate oxidation were annotated. Genes encoding the aromatic ring reduction enzyme, the benzoyl-CoA reductase, could not be identified. Gene expression levels were compared between cells grown with acetate or benzoate as the electron donor, and were used to identify candidate genes for the missing enzyme.

The aim of my project was to compare representatives of each of seven genomovars/species comprising the “Burkholderia cepacia complex” with respect to overall genome size and chromosome number and also to examine their capacity to form N-acyl homoserine lactones (AHLs). A major goal was to determine whether there were significant differences between clinical and non-clinical isolates of these bacteria. My working hypothesis was that clinical isolates (primarily members of genomovar III) might have reduced catabolic and biosynthetic potential and correspondingly smaller genomes than non-clinical isolates. It also seemed reasonable that AHL-dependent quorum sensing, a mechanism that commonly governs expression of genes important for host colonization, would be more prominent in clinical isolates. A survey of 34 B. cepacia complex isolates including representatives of all seven genomovars indicated that all had large multichromosomal genomes. Randomly linearized replicons from preparations of intact chromosomal DNA were resolved by pulsed-field gel electrophoresis and their sizes estimated by comparison of their electrophoretic mobilities with yeast DNA size markers. Overall genome size was confirmed by determining the sums of the molecular weights of macro restrict ion fragments obtained by digestion of chromosomal DNA with enzymes such as CeuI and SwaI. Analysis of a larger group of 62 isolates indicated that all of the strains were prototrophs which exhibited a high degree of nutritional versatility. Thus the notion that clinical isolates had lost DNA and functions important for survival as free-living bacteria proved incorrect. A survey for ability to produce AHLs indicated that members of genomovars I–IV formed low levels of these compounds compared to members of genomovars V–VII. I subsequently isolated mutant derivatives of representatives of genomovars II and III which produced extremely high levels of AHL. I characterized a genetic locus, bmuIR from strain 17616 which contained genes encoding AHL synthase and AHL-binding transcriptional activator. The mutations leading to increased AHL formation were located outside of the bmuIR locus, indicating that other regulatory genes influence AHL formation. An important result of my studies of AHL formation was the development of a rapid procedure for concentration of AHLs from culture supernatants, which involved their adsorption to a copolymer of divinylbenzene and N-vinylpyrrolidone.

The bacterial family of Geobacteraceae is comprised of many members including both Geobacter and Pelobacter species. The Geobacteraceae are the predominant Fe(III) reducing organisms in the subsurface due to their capacity for extracellular electron transfer, and play an important role in both the carbon and iron cycles in sedimentary environments. Their metal reducing capabilities can be applied to groundwater bioremediation and to the production of electrical current in microbial fuel cells. Although many members of this family are well known for their novel electron transfer mechanisms, there are also species that are capable of syntrophic growth, coupling the oxidation of certain organics with the production of byproducts, which in turn support the growth of partner microbes. It is a rare occurrence that a pure culture is applied for use in a large-scale bioreactor or in the sediment during in situ bioremediation. That being true, the study of the Geobacteraceae in pure culture and in microbial communities has far reaching significance. Laboratory evolution techniques were used to determine whether Geobacter species could evolve enhanced fitness in novel environments within a laboratory setting, offering insight into how these versatile microbes change with their environments. G. sulfurreducens was adapted for enhanced growth on lactate as a novel carbon and energy source, and the metabolic, regulatory, and genomic changes due to this adaptation were documented. Enhanced growth on lactate could be applied to the bioremediation of harmful metal contaminants by offering a more efficient and cost effective growth substrate relative to the current one used to stimulate the growth of Geobacter species in the subsurface. Laboratory adaptation techniques were also used to determine whether two different Geobacter species could grow together in coculture, and by what mechanisms the two species would interact. This latter study advances the field of interspecies electron transfer, by offering a novel mechanism of electron transfer between microbial cells. This is relevant to the mechanisms that may be used in situ, such as in biofilms, microbial mats, or wastewater granules.

Bacillus anthracis harbors two plasmids, designated pXO1 and pXO2, which are involved in the synthesis of toxin and capsule. Proof that pXO1 and pXO2 are involved in the synthesis of toxin and capsule, respectively, came from experiments in which the plasmids were transferred by the Bacillus thuringiensis subsp. thuringiensis fertility plasmid, pXO12. The number of transcipients acquiring either pXO1 or pXO2 was low when compared to the number of transcipients that acquired pBC16. Plasmids pXO1 and pXO2 that had been transferred were shown to have acquired a 4.2-kb transposon which is related to the B. thuringiensis transposon Tn4430. The transposon, Tn4430, appears to be responsible for the formation of cointegrate plasmids necessary for pXO12-mediated transfer of pXO1 and pXO2. Putative cointegrate plasmids were obtained between pXO12 and the B. anthracis plasmids, pXO1 and pXO2. Strains harboring cointegrate plasmids were shown to be capable of transferring pBC16, and also transferred the PA$\sp+$ and Cap$\sp+$ phenotypes at high frequencies. Experiments using a pXO12 plasmid tagged with the Streptococcus faecalis transposon, Tn917, showed that this transposon was also capable of forming a cointegrate between pXO2 and pXO12::Tn917. In contrast, pBC16 was unchanged after transfer, and showed no homology to pXO12. Mobilization of the resident B. anthracis plasmids was shown to occur by conduction, while mobilization of the small tetracycline-resistance plasmid pBC16 appeared to occur by donation.

Geobacter metallireducens is the first microorganism in pure culture shown to be able to completely oxidize the aromatic hydrocarbon toluene to carbon dioxide coupled to the reduction of iron. Two parallel routes involving sequential oxidation in the upper pathway for toluene oxidation are proposed. One is hydroxylation of the aromatic ring to form p-cresol, with subsequent oxidation to produce p-hydroxybenzylalcohol, p-hydroxybenzaldehyde and p-hydroxybenzoate. The other is the sequential oxidation of the methyl group to form benzylalcohol, benzaldehyde and benzoate. G. metallireducens can utilize all of the intermediates in the putative upper pathway. The presence of toluene enhances the utilization of p-cresol, p-hydroxybenzylalcohol and benzaldehyde. Some of the intermediates are found to accumulate in the growth medium during the simultaneous utilization experiments. The activity of the catabolic enzyme representing each step in the upper pathway has been demonstrated. The data suggest that the expression of these enzymes is regulated dependent upon the growth substrates. The reactions of p-hydroxybenzylalcohol dehydrogenase and benzylalcohol dehydrogenase are coupled to NAD$\sp+$ but not NADP$\sp+$. The reactions of p-hydroxybenzaldehyde dehydrogenase and benzaldehyde dehydrogenase are coupled to NADP$\sp+$ but not NAD$\sp+$. All of the catabolic enzyme activities exist in the soluble protein fraction of the cells. The demonstration of benzaldehyde hydroxylase activity and several lines of evidence suggest that at least one connection is present between two parallel routes in the upper pathway. The results from the simultaneous utilization experiments suggest that the primary transformation product of toluene is benzylalcohol rather than p-cresol. The soluble protein fraction of the cells exhibits benzoyl-CoA ligase, p-hydroxybenzoyl-CoA ligase, cyclohexanecarboxyl-CoA ligase and pimelyl-CoA ligase activities. The reactions of these enzymes are ATP and Mg$\sp{++}$ dependent. Three activation mechanisms are proposed to be involved in the metabolism of aromatic compounds in G. metallireducens. G. metallireducens can utilize cyclohexanecarboxylate and pimelate as the growth substrates. Both were detected as the intermediates during the radioisotope dilution experiments. This suggests that G. metallireducens may carry out the reductive pathway for the cleavage of the aromatic ring.