Desulfovibrio vulgaris Hildenborough (DvH) is a sulfate-reducing bacterium that has the potential to be an agent of bioremediation by aiding in the precipitation of heavy metals and radionuclides from contaminated environments via its natural metabolism. Because such environments are commonly associated with a variety of bacterial stressors, it is important to understand how Desulfovibrio regulates its response to changing conditions for successful environmental application. Very few types of regulation have been characterized in Desulfovibrio and many regulatory networks remain unknown. The importance of regulation by both cis and trans acting RNA molecules has become increasingly evident over the last decade. Both small RNAs and riboswitches have been shown in other bacteria to play key roles in sensing cellular conditions and eliciting the appropriate responses. A previous study in this laboratory identified several small regulatory RNAs, but their biological roles have remained largely unknown. Additionally, a few uncharacterized riboswitches have been predicted in DvH based on genome sequence analysis. This dissertation aimed to characterize the most conserved DvH sRNA and to investigate its potential dual role as a S-adenosylmethionine (SAM) sensing riboswitch. Expression of the sRNA, Dv SIC19, was confirmed during normal growth in lactate/sulfate medium via Northern blot analysis. Sequence and expression analyses also indicated that Dv SIC19 is located upstream of a small hypothetical protein DVU1170 and that the two genes are co-transcribed. Molecular techniques and computational analysis were also employed to determine a role for Dv SIC19 and to identify potential targets. Stress analysis using qRT-PCR suggested a potential role for Dv SIC19 under normal growth conditions as opposed to being involved in a specific stress response. While characterization of Dv SIC19 was ongoing, re-annotation of the DvH genome indicated that Dv SIC19 shared both sequence and structural similarity to the SAM-I class of riboswitches. Multiple techniques, both in vitro and in vivo were used to verify the riboswitch activity of Dv SIC19 and its response to SAM. Determining that Dv SIC19 played some role in the methionine biosynthesis pathway lead to the identification of a mRNA target encoding SahR, a predicted transcriptional regulator of methionine biosynthesis genes. Subsequent electrophoretic mobility shift assays confirmed the ability of Dv SIC19 to bind the sahR transcript and qRT-PCR analysis of a Dv SIC19 deletion strain suggested a negative regulatory role. This study presented the first regulatory role for a newly discovered sRNA in Desulfovibrio. Additionally, this study verified that Dv SIC19 acts not only as a trans regulatory molecule, but also as a cis regulatory element in the methionine biosynthesis pathway of DvH.

Desulfovibrio vulgaris strain Hildenborough is a key organism in the bioremediation of environments contaminated with anthropogenic sources of heavy metals and radionuclides. In order for D. vulgaris to be optimized for remediation of such sites in the environment, it is necessary to understand the organism's response to key stressors that are found in conjunction with both heavy metals and radionuclides. Few regulators have been discovered in this bacterium and even less is known about its genetic regulatory response to changing conditions in its environment. This dissertation project aimed to use computational, molecular, and culture-based methods to identify novel, small, regulatory RNAs (sRNAs)--an important class of cellular regulators that have yet to be studied in D. vulgaris--and to determine their effect on cellular processes within the organism. Computational and transcriptomic high-throughput sequencing resulted in over 200 high-quality putative sRNAs with the expression of sixteen of these sRNAs verified by Northern blot analysis. Eight of the confirmed sRNAs were selected and further characterized within this study resulting in three trans--or intergenic--encoded sRNA, three cis--or antisense--encoded sRNAs, and two sRNAs that may encode small proteins. These eight sRNAs included growth phase-specific sRNAs, differentially expressed sRNAs, as well as sRNAs processed into multiple forms. Molecular techniques such as quantitative reverse-transcriptase PCR (qRT-PCR) indicated differential expression of the confirmed sRNAs under a variety of stress conditions. Culture-based methods confirmed growth deficiencies in deletion and over-expression mutants of the sRNAs tested under stressors suggested during qRT-PCR analysis. Both of these tests suggested that the sRNAs discovered in this study participate within the cellular response of the bacterium to environmental stress. Computational methods were employed to reveal likely putative mRNA targets for the confirmed sRNAs in order to determine interactions and function within the regulatory networks. This study presents the first evidence of the existence and expression of sRNAs within D. vulgaris Hildenborough. Furthermore, the information gathered about the sRNAs analyzed in this work indicates that sRNAs are an important part of the regulatory response mechanism of D. vulgaris. Further research into target identification and interaction can close the loop of these regulatory networks and solidify the role of sRNAs in D. vulgaris.

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2008. / Title from second page of PDF document. Includes bibliographical references (p. 215-246).

This study describes the characterisation and identification of two species of sulphate-reducing bacteria isolated from marine environments. The isolate coded Ind 1 was recovered from the heavily corroded hull of an oil storage vessel moored off the Indonesian coast. An isolate, referred to as Al 1, originated from a soured oil reservoir in Alaska. Observations using microscopy (light, scanning electron and atomic force) revealed that cells were Gram-negative, rod-shaped and very motile. Physiological characterisation, analysis of the fatty acid profiles and partial and full 16S rRNA sequencing demonstrated strong similarities between the two species and members of the Desulfovibrio genus. The position of the strains within phylogenetic trees showed Al 1 clustering closely with Desulfovibrio vietnamensis. Ind 1 revealed a high degree of similarity with both Desulfovibrio gigas and Desulfovibrio gabonensis and these three strains formed a separate cluster in the delta subdivision of the Proteobacteria. However, whole-cell protein profiles and Fourier-transform infrared spectroscopy studies showed that there is enough dissimilarity between the two isolates and the remaining species of the genus Desulfovibrio to consider Al 1 and Ind 1 as new separate species. Purification, physico-chemical and spectroscopic characterisation of the key enzymes involved in the sulphate metabolism was carried out for both isolates. Nuclear magnetic resonance and electron paramagnetic resonance studies revealed that the proteins of Al 1 and Ind 1 exhibited various features in common with their counterparts from other members of the genus Desulfovibrio. In particular, proteins from Ind 1 showed many similarities with the enzymes previously described for D. gigas. Based on the obtained results, the classification of Ind 1 as Desulfovibrio indonensiensis sp. nov. and Al 1 as Desulfovibrio alaskensis sp. nov. are proposed. The overall results highlight the complexity of the relationship between cell physiology and the organisms' environmental impact.

Il a été suggéré que les Desulfovibrio, qui sont des bactéries anaérobies sulfato-réductrices, pourraient être impliqués dans les parodontites. Nous avons évalué le pouvoir invasif de Desulfovibrio vis-à-vis de cellules épithéliales et leur capacité à induire la production de cytokines par ces cellules. Nous avons montré que Desulfovibrio desulfuricans et Desulfovibrio fairfieldensis sont capables d'envahir et de se multiplier dans les cellules épithéliales buccales (cellules KB). La localisation intracytoplasmique de ces deux bactéries a été confirmée par microscopie confocale et électronique à transmission. L'internalisation de ces souches était dépendante de la polymérisation des microtubules mais pas de celle de l'actine. L'infection avec Desulfovibrio était responsable d'une augmentation de la production d'IL-6 et d'IL-8 par les cellules KB. La capacité de D. desulfuricans et de D. fairfieldensis à survivre dans les cellules épithéliales et à moduler leur réponse immunitaire pourrait contribuer au développement des maladies parodontales. Desulfovibrio ainsi que d'autres parodontopathogènes peuvent produire des ß-lactamases et sont capables d'envahir les cellules épithéliales. Il a été suggéré que l'hydrolyse de l'amoxicilline pourrait être évitée grâce à l'utilisation d'un complexe amoxicilline-ß-cyclodextrine (ßCD) et que la diffusion intracellulaire d'agents antimicrobiens pourrait être améliorée après complexation avec des ßCD. Un complexe stable amoxicilline-ßCD, caractérisé après analyse spectrale et thermique, n'a ni amélioré l'activité de l'amoxicilline vis-à-vis de souches produisant des ß-lactamases ni augmenté la diffusion intracellulaire de ce composé. / It has been suggested that Desulfovibrio, which are sulfate-reducing anaerobic bacteria, may be involved in periodontitis. We investigated the capacity of Desulfovibrio to invade epithelial cells and induce cytokine secretion from these cells. We showed that Desulfovibrio desulfuricans and Desulfovibrio fairfieldensis were able to invade and to multiply within oral epithelial cells (KB cells). Intracytoplasmic location of both bacteria was confirmed by confocal laser scanning and transmission microscopy. Invasion of these strains involved microtubule but not microfilament polymerization. Infection by Desulfovibrio resulted in an increase of the production of IL-6 and IL-8 by KB cells. The ability of D. desulfuricans and D. fairfieldensis to survive within oral epithelial cells and to modulate the epithelial immune response may contribute to the initiation and progression of periodontal diseases. Desulfovibrio as well as other periodontopathogens may produce ß-lactamases and have the capacity to invade epithelial cells. It has been suggested that the hydrolysis of amoxicillin might be prevented by using an amoxicillin-ß-cyclodextrin (ßCD) complex and that intracellular diffusion of antimicrobial agents might be enhanced after complexation with ßCDs. A stable [1:1] amoxicillin-ßCD complex, characterized by spectroscopic and thermal analysis, did neither improve the activity of amoxicillin against ß-lactamase producing strains nor enhance the intracellular diffusion of this compound.

To fulfil global energy demand and to mitigate economical, geopolitical and ecological challenges associated with fossil fuel utilisation, the energy sector is moving towards greater use of sustainable and environmentally friendly energy sources, including biofuels. The ideal transport biofuel would be hydrocarbons that are identical to fossil petroleum. However, to date characterised hydrocarbon biosynthetic pathways include a decarbonylation or decarboxylation reaction, which involves the loss of one carbon resulting in odd-numbered carbon chain hydrocarbons. This carbon loss decreases carbon efficiency for alkane production, which reduces microbial fuel economic competitiveness. Therefore, it is key that new pathways for alkane production are identified. The sulphate-reducing bacteria genus Desulfovibrio was previously reported to synthesise even-numbered carbon chain alkanes, which suggests an alternative route for alkane production without carbon loss. This investigation aimed to verify Desulfovibrio alkane biosynthesis and characterise the possible synthetic pathway. Ten Desulfovibrio strains, representing seven species, were screened for alkane synthesis using isotopically labelled growth media. The ability to produce alkanes within the Desulfovibrio genus was confirmed and was shown to be strain-specific under a set of culture conditions. The biogenic alkanes detected were octadecane (C18), nonadecane (C19) and eicosane (C20), with a predominance of even-numbered carbon chain alkanes. Fatty acid analysis of Desulfovibrio strains showed an alkane biosynthetic pathway was unlikely to involve a decarbonylation or decarboxylation step. A novel hypothesis was therefore proposed that alkane biosynthesis by Desulfovibrio follows a metabolic route, which has not previously been characterised, involving a series of reduction reactions from the fatty acid pool. The characterisation of the putative Desulfovibrio hydrogenation pathway for alkane biosynthesis was undertaken via a target-directed genome mining approach. The genomic DNA of nine Desulfovibrio spp. was purified, sequenced, de novo assembled and annotated. Seven of these nine genomes are unpublished to date. No homologs of previously characterised alkane biosynthetic enzymes from bacteria were in silico identified in the genomes and proteomes of alkane producing Desulfovibrio spp., suggesting that Desulfovibrio alkane biosynthetic pathway is likely to be catalysed by currently uncharacterised enzymes. The 16S rRNA-based phylogeny of Desulfovibrio spp. supported the hypothesis that the Desulfovibrio alkane biosynthetic pathway was acquired by a common ancestral strain via horizontal gene transfer. The ability of Desulfovibrio to produce alkanes was therefore hypothesised to be due to the presence of recruited genes encoding enzymes involved in alkane synthesis. A comparative genomic analysis intersecting six-alkane producing and four non-alkane producing Desulfovibrio genomes resulted in the in silico identification of 33 hypothetical proteins considered with high confidence to be exclusive to alkane producing Desulfovibrio strains. A novel hypothetical Desulfovibrio alkane biosynthetic pathway was proposed involving a V-type ATPase, an uncharacterised protein, named as a putative reductase in this investigation, and a putative methyltransferase, which were predicted to be exclusive to alkane producing Desulfovibrio spp. The inorganic phosphates resulting from the ATP hydrolysis catalysed by the V-type ATPase would be involved in a reaction with fatty alcohols to form alkyl phosphates, which are putative activated intermediates required for the hydrogenation route from fatty alcohols to alkanes. The putative reductase and the methyltransferase, predicted to share similar structural features with known alkane-binding proteins, would subsequently reduce alkyl phosphates to alkanes and to iso-alkanes respectively. Empirical investigation of the candidate molecular basis function in Desulfovibrio alkane biosynthesis was undertaken. The Desulfovibrio alkane biosynthetic pathway remains to be fully characterised.

Chromium has been used extensively in the industry process of metal refinishing and electroplating. It is also a byproduct of the processing of fissionable materials at United States Department of Energy facilities. Chromate (Cr (VI)) is soluble and readily absorbed by cells, while the reduced form of chromium, Cr (III), is insoluble. Thus means of reducing Cr (VI) to Cr (III) in the environment is a potential means of remediation. Desulfovibrio desulfuricans strain 27774 is a sulfate reducing bacterium that can reduce Cr (VI). It also can respire nitrate to ammonia. As some sites of chromium contamination also contain high concentrations of nitrate, an investigation of Cr (VI) reduction under nitrate reducing growth conditions by D. desulfuricans strain 27774 was conducted. A growth medium that was compatible with the colorimetric assay for Cr (VI) and did not itself reduce Cr (VI) was formulated. Cell assays determined that Cr (VI) reduction was primarily in the supernatant, catalyzed by a secreted secondary metabolite. A genomics investigation identified two pathways as possible mechanisms. / Bayer School of Natural and Environmental Sciences / Environmental Science and Management (ESM);= / MS / Thesis

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 174-176). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 174-176). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / Title from title screen of research.pdf file (viewed on December 22, 2006). The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "May 2005" Vita. Includes bibliographical references.