<p>The lipid A component of lipopolysaccharide (LPS) in the nitrogen-fixing plant endosymbionts <italic>Rhizobium leguminosarum</italic> and <italic>Rhizobium etli</italic> is strikingly different when compared to that of enteric bacteria such as <italic>Escherichia coli</italic>. The <italic>Rhizobium</italic> species produce several unique enzymes that process the lipid A biosynthetic intermediate Kdo₂-lipid IV_A. These enzymes include a 1-phosphatase (LpxE), a 4´-phosphatase (LpxF), a 3-O-deacylase (PagL), and a lipid A oxidase (LpxQ). The biological functions and enzymological properties of many of the modification enzymes have remained unconfirmed and/or unknown. The purpose of these studies was to confirm the activities of these enzymes and to explore the functional significance of the resulting lipid A modifications.</p> <p>To confirm the proposed biological functions of the enzymes <italic>in vivo</italic>, homologs of the lipid A phosphatases, LpxE and LpxF, from <italic>Francisella novicida</italic> and the lipid A oxidase LpxQ, were expressed heterologously in combination in <italic>E. coli</italic>. The resulting novel lipid A hybrids were analyzed by thin-layer chromatography (TLC) and electrospray ionization-mass spectrometry (ESI-MS). </p> <p>The lipid A oxidase LpxQ, was characterized further biochemically. Two new purification procedures and a new <italic>in vitro</italic> assay were developed to analyze the properties of the enzyme. Purified LpxQ was shown to be dependent on oxygen and divalent cations for activity. Hydrogen peroxide was found to be a product of lipid A oxidation. A new fluorescence-based assay based on the detection of hydrogen peroxide was developed to monitor oxidation. LpxQ did not co-purifiy with any discernable cofactors, suggesting that it may employ a unique mechanism for the oxidation of lipid A.</p> <p>The biological roles of LpxE and LpxF in plant nodulation were analyzed. Deletion mutants of the two phosphatases were generated in <italic>R. etli</italic>. The mutant strains accumulated the expected structures, confirming the specificity of the enzymes. Single and double phosphatase mutants were able to fix nitrogen <italic>in planta</italic>. Antimicrobial susceptibility testing indicated that dephosphorylation of lipid A increases resistance to cationic antimicrobials.</p> <p>The biological role of the 3-O-deacylase, PagL, was also investigated. The <italic>pagL</italic> gene was identified using systematic homology searches. PagL was shown to be stimulated by calcium. A deletion mutant of the enzyme in <italic>R. etli</italic> was constructed and analyzed. The deletion mutant was found to be viable and unaltered in its ability to fix nitrogen. In conclusion, these studies have confirmed the roles of LpxE, LpxF, PagL, and LpxQ in Rhizobium lipid A biosynthesis and contributed new knowledge regarding the biochemical properties of LpxQ.</p> / Dissertation

Thesis (Ph. D.)--Indiana University, Dept. of Biology, 2009. / Title from PDF t.p. (viewed Feb. 10, 2010). Source: Dissertation Abstracts International, Volume: 70-05, Section: B, page: 2697. Adviser: Yves V. Brun. Includes supplementary digital materials.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3298. Adviser: Steven R. Blanke. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

<p> In this thesis, we discuss three topics concerning extracellular electron transfer in the Dissimilatory Metal Reducing Bacterium (DMRB) <i>Shewanella oneidensis</i> MR-1. One proposed strategy to accomplish extracellular charge transfer in <i>Shewanella</i> involves forming a conductive pathway to electrodes by incorporating redox components on outer cell membranes and along extracellular appendages known as bacterial nanowires within biofilms. In the first part of this dissertation, to describe extracellular charge transfer in microbial redox chains, we employed a model based on incoherent hopping between sites in the chain and an interfacial treatment of electrochemical interactions with the surrounding electrodes. Based on this model, we calculated the current-voltage (I-V) characteristics and found the results to be in good agreement with I-V measurements across and along individual microbial nanowires produced by the bacterium <i>S. oneidensis</i> MR-1. Based on our analysis, we propose that multistep hopping in redox chains constitutes a viable strategy for extracellular charge transfer in microbial biofilms. </p><p> In the second part, we report the first <i>in vivo</i> observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe <i>S. oneidensis</i> MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to <i> S. oneidensis</i> MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by our proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution. </p><p> In addition, to elucidate the membranous nature of <i>Shewanella </i> nanowires, we imaged these filaments using Transmission Electron Microscopy. The TEM images reported in this thesis also provide the most accurate estimates of bacterial nanowire dimensions to date. Future TEM and cryo-TEM imaging can establish the specific alignment and configuration of outer membrane cytochromes that facilitate electron transport along bacterial nanowires. </p><p> In the third part of this thesis, we focus on the molecular conductance of MtrF, the first decaheme outer membrane cytochrome with a solved crystal structure. Decaheme outer membrane cytochromes of <i>Shewanella</i> play a crucial role in all the suggested pathways of extracellular electron transfer. An understanding of the electron transfer properties in MtrF will therefore impact all aspects of extracellular electron transfer research. In this thesis, using purified MtrF, we form monolayers of the protein on atomically flat gold substrates and address the dry monolayer with a Scanning Tunneling Microscope (STM) tip. This technique can be used in the future to examine the conductivity of individual MtrF molecules within the monolayer in the form of I-V curves. This methodology will allow experimental comparison with recently developed simulations of MtrF conductance.</p>

Giardia lamblia is an early branching eukaryote and although distinctly eukaryotic in its cell and molecular biology, transcription in G. lamblia demonstrates important differences from these processes in higher eukaryotes. α-amanitin is a relatively selective inhibitor of eukaryotic RNA Polymerase II (RNAP II), and is commonly used to study RNAP II transcription. Therefore, we measured the sensitivity of G. lamblia RNAP II transcription to α-amanitin and found that unlike most other eukaryotes, RNAP II transcription in Giardia is resistant to 1 mg/ml amanitin. To better understand transcription in G. lamblia, we identified 10 out of the 12 known eukaryotic rph genes, including all ten that are required for viability in Saccharomyces cerevisiae. The amanitin motif (amanitin binding site) of Rpbl from G. lamblia differs from other eukaryotes at six highly conserved sites in which substitutions have been associated with amanitin resistance in other organisms. These observations of amanitin resistance provide a molecular framework for the development of novel drugs with selective activity against G. lamblia. Giardia trophozoites exhibit antigenic variation of a surface protein encoded by a family of genes known as the vsp genes. A single trophozoite expresses only one vsp at a time and it has been previously determined that steady state mRNA of only the expressed vsp is detectable in Northern blots. Our nuclear run-on assays indicated transcription of only the expressed vsp genes, suggesting that control is primarily at the level of transcription rather than post-transcription. In order to better understand vsp gene control mechanisms, we used a luciferase reporter to determine the vsp core promoter, which is present within 100 bp upstream of the ORF in the case of vspCS and vspA6. The fact that the vsp promoter is able to drive expression irrespective of the antigenic variant type indicates that control requires a chromosomal context as do most epigenetic mechanisms of control. Based on the existing data, we provide a privileged site model for the control of vsp gene expression, in which vsp transcription takes place in a set nuclear location which is occupied by a singe vsp locus at a time.

This dissertation is concerned with studying aspects of the ecology of microorganisms from a functional perspective using different microbial populations in soils. In the first study, an investigation was done on the distribution of biosurfactant producing microorganisms. In the second study, temporal changes were observed in an indigenous phenanthrene degrading community following a long-term pulse of phenanthrene.

Acquisition of iron contributes to bacterial pathogenesis. In C. jejuni, there are more than five systems which contribute to ferric iron uptake, mainly consisting of siderophore receptor systems. In vivo, C. jejuni encounters the anaerobic environment of the intestine where the predominant state of iron is likely the ferrous ion. A homolog of FeoB, a high-affinity ferrous iron transporter, is encoded in the C. jejuni genome. Initial experiments with C. jejuni demonstrated low levels of ⁵⁵Fe²⁺ uptake. Furthermore, mutations in feoB failed to inhibit ferrous iron uptake when compared to the parental isolate. No differences were noted between the wild type and mutant strains in epithelial cell invasion, macrophage survival, or peroxide sensitivity. The feo locus also failed to complement an E. coli ΔfeoB mutant. Sequence analysis indicated various mutations in C. jejuni strains. Notably, strains RM1221 and F38011 contained frameshift mutations that caused early termination of the protein. M129 had an amino acid substitution in a critical region of the protein involved in nucleotide binding. While the presence of magnesium significantly inhibited⁵⁵Fe²⁺ uptake (> 3-fold), inhibition of CorA activity did not affect uptake of ⁵⁵Fe²⁺. Viability of C. jejuni required the function of CorA and mutants at this locus were lethal. When compared to wild-type, a putative fhuA mutant was attenuated in epithelial cell invasion (∼1000-fold) and intra-macrophage survival (∼10-fold) but not in a piglet model of the disease. The putative mutant failed to produce a protein with similar size to FhuA and an immunoblot reacted with a protein of the size of FhuA in the parent but not in the mutant. In summary, ferrous iron uptake is not mediated by FeoB or CorA in C. jejuni. Homologs of feoB in different strains of C. jejuni appear to have acquired various mutations resulting in non-functional proteins. The corA locus is absolutely required for growth of C. jejuni since it is likely the only magnesium transporter in this species. The FhuABD system is required for the optimal invasion of epithelial cells and intra-macrophage survival of C. jejuni. However, mutants in fhuABD do not attenuate the organism in vivo.

The assembly of viral proteins and nucleic acids into mature and biologically active virions involves a diverse spectrum of macromolecular interactions. After capsid formation, structural and packaging proteins must interact with viral nucleic acids. These interactions may confer packaging specificity, spatially organize the genome, enhance particle stability, or contribute directly to capsid quaternary structure. In the Microviridae, packaging and capsid proteins are tightly associated, tethering the genome to the inner surface and guiding it into the overall icosahedral symmetry of the particle. All of these factors may influence the final stages of maturation, which involves an inward collapse of coat proteins around the packaged genome. These packaging parameters were altered in three ways. (1) The DNA binding residues of the DNA binding protein were altered. Although the genome and protein are in the interior of the capsid, alterations were expressed on the capsid's outer surfaces. The results of second site genetic analyses illustrate how coat protein modifications can compensate for defective phenotypes. (2) Non-DNA binding amino acid residues believed to be of structural importance were mutated. The results of these analyses elucidate the function of these residues in optimizing DNA-protein interactions and organizing the DNA into the capsid's symmetry. Again, the results of second site genetic analyses demonstrate the inherent evolutionary plasticity of the system. (3) Packaged DNA was changed by altering base composition and folding parameters. The experimental results support a model in which the secondary structure of the packaged genome acts in a scaffold-like manner during the final stage of virion morphogenesis, affecting the biophysical and biological properties of the mature virion. Finally a chimeric particle was constructed by placing a wild type DNA binding protein in a wild type, but foreign environment. The biophysical characterization of these particles is consistent with the mentioned model. A structural analysis was performed, in collaboration with Dr. Rossmann's group (Purdue University), to provide a structural context in which to interpret the observed biophysical effects. While not all questions were answered or hypotheses verified, the results elucidate the limitations and interpretation of structural analyses, a current debate in the structural biology field.

Spore photoproduct lyase (SP lyase), is one of two DNA repair enzyme systems active during germination of Bacillus subtilis endospores. The presence of this enzyme is partially responsible for the high resistance of endospores to killing by ultraviolet (UV) radiation. Amino acid sequence analysis revealed that SplB contains three closely-spaced cysteine residues located in the amino-terminal one-third of the deduced sequence, which led to the hypothesis that SP lyase contains an iron-sulfur center. Chemical analysis and UV-visible spectra of purified SplB overproduced in E. coli supported this hypothesis. SP lyase activity requires an anaerobic/reducing environment and S-adenosylmethionine (AdoMet) as a cofactor. Identification of the catalytic requirements led to the formulation of the hypothesis that SP lyase effects catalysis via an adenosyl radical, which is generated by reduction of AdoMet by the iron-sulfur center. The electron paramagnetic resonance (EPR) spectrum of SplB purified under aerobic conditions was characteristic of a protein containing a partially degraded [3Fe-4S] center. When SplB was incubated with dithionite, the spectrum shifted to that of a reduced [4Fe-4S] center. When the enzyme was treated with AdoMet, there was a significant decrease in EPR signal intensity. These results indicate that the [4Fe-4S] center is capable of acquiring electrons and transferring them to AdoMet. Analysis of the reactions via high-pressure liquid chromatography and electrospray ionization mass spectroscopy showed that SP lyase generates 5'-deoxyadenosine during the reaction. Analysis of SplB via SDS-PAGE after treatment with the protein cross-linking agent dimethyl suberimidate and size exclusion chromatography under native conditions, revealed the presence of a protein species of approximately two times the molecular weight of SplB demonstrating that SplB is capable of forming dimers. Based on the evidence presented here, the following model has been generated: (i) two SplB units form a dimer that is coordinated via a [4Fe-4S] center; (ii) the [4Fe-4S] center becomes reduced and in turn reduces a molecule of AdoMet; (iii) AdoMet is split to generate an adenosyl radical which can then participate in the in situ reversal of SP into two intact thymines.

Chlorinated solvents are pollutants found frequently in the environment. Whereas lower chlorinated solvents are easily degraded under aerobic conditions, degradation of higher chlorinated solvents is favored under anaerobic conditions. It is known that some compounds can act as redox mediators assisting the electron exchange required for the anaerobic dechlorination reactions to occur. Redox mediators are compounds that can assist the anaerobic dechlorination reactions either abiotically or biologically by accelerating the degradation rates. The objective of this study is to evaluate the role of different redox mediators on the anaerobic degradation of higher chlorinated solvents such as chloroform (CF), carbon tetrachloride (CT) and perchloroethylene (PCE). The redox mediators studied are vitamin B12 (cyanocobalamin CNB12-hydroxocobalamin HOB12), riboflavin (RF) and a model of humic compound anthraquinone-2,6-disulfonate (AQDS). The study includes abiotic and biological redox mediated dechlorination. The biological studies involve two varieties of unadapted methanogenic sludge. The results obtained demonstrate that redox mediators greatly enhance the degradation rates of CF and CT when utilized at substoichiometric molar ratios. At the same time, the results also highlight the importance of the role of the microorganisms during the dechlorination process. Vitamin B12 and riboflavin presented larger impact than AQDS on the degradation rates of CF and CT. Since vitamin B12 had the highest positive impact on the biodegradation rates of CF and CT, an attempt was done to stimulate its biosynthesis by the methanogenic sludge during the CT dechlorination. The approach used to promote the biosynthesis of vitamin B12 involved different vitamin B12 precursors such as porphobilinogen, and some primary substrates such as methanol and 1,2-propanediol. The results obtained show that the formation of the corrin ring may be the limiting step during vitamin B12 biosynthesis. One carbon substrates such as methanol, combined with porphobilinogen had a positive impact on the biodegradation rates of CT. The study suggests that the combination of methanol and vitamin B12 precursors could be a good alternative to stimulate the vitamin B12 biosynthesis by methanogens and therefore, the enhancement of the biodegradation rates of chlorinated solvents. This study presents the lowest molar ratio of vitamins that enhanced the dechlorination rates reported so far for biodegradation of chlorinated solvents involving methanogens. Also, this is the first report on the use of riboflavin as a redox mediator during dechlorination processes.