Thesis (M.S.)--Oregon State University, 2006. / Printout. Includes bibliographical references. Also available online.

Bioluminescence is a trait observed among different genera and families of bacteria. In this study part of the luxA gene was characterized from the new MS isolates and compared to luxA of other bacteria. The polymerase chain reaction (PCR) was used to amplify a fragment of the luxA gene of strain MS32 and Vibrio harveyi. These fragments were used as probes in hybridization experiments with luminous and nonluminous bacteria. The results from these experiments suggest that some nonluminous species may possess lux like regions in their chromosomal DNA and that luxA probes can demonstrate species identity. The MS32 luxA fragment was also sequenced and used in a phylogenetic analysis to identify the taxonomic affinities of MS strains. It was found that MS1 and MS32 were closely related, however, Shewanella hanedai was not. Thus there was a concordance between the phenotypic and genotypic approaches, which will help in establishing a consistent taxonomic affinity between these bacteria.

La bactérie aquatique Shewanella oneidensis est capable, en condition statique et en présence d'oxygène, de former un biofilm à l'interface air-liquide, appelé pellicule. Mon travail a porté sur la biogenèse de la pellicule.Il a été montré dans le groupe que le régulateur de réponse du système chimiotactique, la protéine CheY3, était impliqué dans la biogenèse de la pellicule. Cette protéine est essentielle dans les étapes précoces et tardives de sa formation alors que son partenaire habituel, CheA3, semble ne jouer un rôle que dans les étapes tardives. Mon travail s'est focalisé sur la recherche de partenaires de CheY3.J'ai introduit une banque d'ADN génomique de S. oneidensis dans la souche ΔcheY3 et j'ai cherché des gènes dont la surexpression permettait de restaurer la formation de la pellicule. Cette approche a révélé deux gènes pdgA et pdgB. J'ai montré que les protéines PdgA et PdgB étaient capables de synthétiser du di-GMPc, suggérant que ce messager secondaire est impliqué dans la biogenèse de la pellicule. L'hydrolyse du di-GMPc par des enzymes dédiées empêche en effet sa formation.J'ai montré que l'opéron mxd, contrôlant la synthèse d'exopolysaccharides dans les biofilms de surface, était impliqué dans la formation de la pellicule. La première protéine codée par cet opéron, MxdA, est capable de lier le di-GMPc. Des expériences de pontage chimique et de double hybride ont révélé que MxdA, CheY3, PdgA et PdgB, formaient un réseau de régulation gouvernant la biogenèse de la pellicule.J'ai montré que les systèmes à deux composants BarA/UvrY et ArcS/ArcA contrôlant la transcription de l'opéron mxd sont aussi impliqués dans la formation du biofilm flottant. / The aquatic bacterium Shewanella oneidensis is able to form, under static conditions and in the presence of oxygen, a biofilm at the air-liquid interface, called pellicle. My work was focused on the biogenesis of this pellicle.It was previously shown in the team that, surprisingly, the CheY3 protein, the response regulator of the chemotactic regulatory system, is involved in the biogenesis of the pellicle. This protein was shown to be essential both in early and late steps of pellicle formation whereas its usual partner, the kinase CheA3, seems to play a role in the late steps only. I was therefore looked for the partners of the CheY3 protein for pellicle formation.For this purpose, I have introduced a multi-copy genomic library in the ΔcheY3 strain and searched for genes whose overexpression allowed pellicle restoration. Strikingly, this approach revealed two genes pdgA and pdgB. Interestingly, we showed that PdgA and PdgB proteins are able to synthesize c-di-GMP, suggesting a role for this second messenger in pellicle biogenesis. Indeed, c-di-GMP hydrolysis by dedicated enzymes blocks pellicle formation.We also showed that the mxd operon, controlling the exopolysaccharides synthesis in biofilm associated with a solid surface, is also involved in pellicle formation. Moreover, the first protein encoded by this operon, MxdA, is able to bind c-di-GMP. Cross-linking and bacterial two-hybrid experiments revealed that MxdA, CheY3, PdgA and PdgB, form a complex regulatory pathway governing the biogenesis of the pellicle.Finally, we have shown that the two-component systems BarA/UvrY and ArcS/ArcA, controlling the mxd transcription, are also involved in pellicle formation.

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Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Shewanella oneidensis is a facultative, gram-negative microbe that, in the absence of oxygen, can use a wide variety of terminal electron acceptors including iron, manganese, uranium, nitrite, nitrate, sulfate, fumarate, and DMSO. The anaerobic versatility is believed to be the result of a highly branched electron transfer pathway involving many redox-active proteins. Shewanella is capable of dissimilatory metal reduction (DMR) of insoluble iron and manganese oxides, in which electrons are transferred from the cell's interior to its exterior. Several multiheme c -type cytochromes comprise a pathway for this electron transfer. These cytochromes, specifically the tetraheme protein, CymA, and the decaheme protein, MtrA, are the primary focus of this thesis. The current model of electron transfer indicates that electrons originate in the cytoplasmic membrane from the menaquinol pool, and are transferred into the periplasm by CymA. From here the pathway branches and electrons are transferred into several potential periplasmic targets, including MtrA. MtrA may then transfer electrons directly or indirectly to MtrC and OmcA, which have been shown to reduce exogenous electron acceptors such as iron oxides. Recently, it has been suggested that MtrA and MtrC dock with 13-barrel protein, MtrB and transfer electrons through the porin sheath. Here, the DMR pathway has been studied with respect to four aims: (1) purification and characterization of the multiheme cytochromes through the use of non-catalytic protein film voltammetry (PFV), (2) structural analysis of MtrA by small angle X-ray scattering (SAXS), (3) investigation of protein-protein interactions via catalytic PFV and anaerobic affinity chromatography, and (4) exploration of heme cofactor function within the tetraheme cytochrome, CymA and MtrA by characterizing heme knockout mutants of the two proteins. We demonstrate that these proteins interact to form an electron transfer pathway from the cytoplasm to terminal electron acceptors on the outside of the cell through a "wire" of heme cofactors. Additionally, the data support the model that MtrA can span a large portion of the peri plasmic space to act as an intermediary by accepting electrons from CymA and subsequently docking with MtrB to transfer electrons to MtrC. / 2031-01-02

Microbial iron respiration contributes significantly to the biogeochemical cycling of metals and may be one of the earliest respiratory processes to have evolved on early earth. Metal-respiring microbes also hold great potential for use in microbial fuel cells for the generation of "green" energy and for remediation of radionuclides in contaminated environments. Despite its significance in global metal cycling processes, the molecular mechanism of Fe(III) respiration has yet to be determined. Unlike many other terminal electron acceptors, Fe(III) is a solid at circumneutral pH and, therefore, cannot come into direct contact with the microbial inner membrane: the site of terminal electron transfer in gram-negative bacteria. It is postulated that metal-respiring organisms have developed alternate strategies for the reduction of solid iron. One such strategy involves the production of an Fe(III)-solublizing ligand by the metal-respiring bacteria which solubilizes the Fe(III) prior to respiration, rendering the metal more easily accessible to the Fe(III) reductase complex. In this study, the genes involved in the solubilization of Fe(III) by the gram-negative dissimilatory metal reducing bacteria Shewanella oneidensis MR-1 were determined using random mutagenesis to generate mutations in the wild-type genome and high-throughput square-wave voltammetry to screen for the attenuation of Fe(III) production in the mutants. Two mutants unable to solubilize Fe(III) were identified and designated d29 and d64. After mutation complementation analysis, it was determined that the point mutations were both located in type II secretion genes: gspG and gspE respectively, indicating that the type II secretion system is required for Fe(III) solubilization prior to respiration. It was also hypothesized that the ligand produced for Fe(III) solubilization during dissimilatory Fe(III) respiration was a siderophore: a small Fe(III)-chelating molecule produced by the cells for the assimilation of Fe(III) for growth. A siderophore biosynthesis gene (SO3031) and a siderophore ferric reductase gene (SO3034) were deleted in frame and the resultant mutants screened to determine whether they were capable of Fe(III) solubilization and reduction during anaerobic Fe(III) respiration. Both mutants retained Fe(III) solubilization and reduction activity, indicating that the siderophore Fe(III) assimilatory system is distinct from the Fe(III) solubilization system utilized during Fe(III) respiration. The work presented here is significant in that it describes a rapid screening method for identifying Fe(III) solubilization mutants, reports on the involvement of the type II secretion system in Fe(III) solubilization during iron respiration, and finally demonstrates that a dissimilatory metal reducing bacteria synthesizes and secretes Fe(III)-chelating molecules which are distinct from Fe(III)-siderophores.

Shewanella putrefaciens is a Gram-negative bacillus and marine pathogen that rarely causes disease in humans. We report a case of osteomyelitis by this organism in a 48-year-old male patient, who presented with pain and erythema of the right foot that was initially diagnosed as cellulitis and did not revert despite treatment. He was transferred to Lima where osteomyelitis was diagnosed and started on empirical treatment with partial regression. A biopsy and culture of the compromised area found S. putrefaciens. The infection was treated according to the antibiotic sensitivity profile of the pathogen. S. putrefaciens infection represents a rare opportunistic infection of devitalized or exposed areas of the body. It is associated with residence in coastal areas and commonly affects the skin and soft tissues. Exceptional cases of osteomyelitis have been reported, but this is the first that involves the metatarsal bones. Shewanella putrefaciens es un bacilo Gram negativo, patógeno marino que rara vez ocasiona enfermedad en humanos. Se presenta un caso de osteomielitis por este microorganismo en un paciente varón de 48 años, procedente de Chimbote. Presentó dolor y eritema en el pie derecho, inicialmente diagnosticado como celulitis, pero que no revirtió pese al tratamiento. Fue transferido a Lima donde se diagnosticó osteomielitis e inició tratamiento empírico con escasa mejoría. Por ello, se realizó una biopsia y cultivo de la zona comprometida, el metatarso, en el cual se aisló Shewanella putrefaciens. Se trató de acuerdo al perfil de sensibilidad. La infección por Shewanella putrefaciens representa una rara infección oportunista, que se localiza en áreas desvitalizadas o expuestas del cuerpo. Se asocia a vivir en zonas costeras, afectando comúnmente piel y tejidos blandos. Se han reportado casos excepcionales de osteomielitis. Este es el primero que involucra metatarso.

Dissimilatory iron reduction (DIR) is significant to the biogeochemical cycling of iron, carbon and other elements, and may be applied to bioremediation of organic pollutants, toxic metals, and radionuclides; however, the mechanism(s) of DIR and factors controlling its kinetics are still unclear. To provide insights into these questions, the interaction between a common dissimilatory iron reducing bacterium (DIRB)was studied, Shewanella oneidensis MR-1, and ferric iron (Fe(III)) (oxy)(hydr)oxide mineral surfaces. Firstly, atomic force microscopy was used to study how S. oneidensis MR-1 dissolved Fe(III) (oxy)(hydr)oxides and compared it to two other cases where Fe(III) (oxy)(hydr)oxides were either dissolved by a chemical reductant or by a mutant with an electron shuttling compound. Without the electron shuttling compound, the mutant is unable to respire on Fe(III) (oxy)(hydr)oxides, but with the electron shuttling compound, it can. It was found that the cells of S. oneidensis MR-1 formed microcolonies on mineral surfaces and dissolved the minerals in a non-uniform way which was consistent with the shape of microcolonies, whereas Fe(III) (oxy)(hydr)oxides were uniformly dissolved in both of the other cases. Secondly, confocal microscopy was used to study the adhesion behavior of S. oneidensis MR-1 cells on Fe(III) (oxy)(hydr)oxide surfaces across a broad range of bulk cell densities. While the cells were evenly distributed under low bulk cell densities, microcolonies were observed at high bulk cell densities. This adhesion behavior was modeled by a new, two-step adhesion isotherm which fit better than a simple Langmuir or Freundlich isotherm. The results of these studies suggest that DIR is in-part transport limited and the surface cell density may control DIR.

The g-proteobacterium Shewanella oneidensis MR-1 reduces a wide range of terminal electron acceptors, including solid Fe(III) oxides. Pathways for Fe(III) oxide reduction by S. oneidensis include non-reductive (organic ligand-promoted) solubilization reactions, and either direct enzymatic, or indirect electron shuttling pathways. Results of the present study expand the spectrum of electron acceptors reduced by S. oneidensis to include the naturally occurring disulfide compounds cystine, oxidized glutathione, dithiodiglycolate, dithoidiproponiate and cystamine. Subsequent electron shuttling experiments demonstrated that S. oneidensis employs the reduced (thiol) form of the disulfide compounds (cysteine, reduced glutathione, mercaptoacetate, mercaptopropionate, and 2-nitro-5-thiobenzoate, cystamine) as electron shuttles to transfer electrons to extracellular Fe(III) oxides. The results of the present study indicate that microbial disulfide reduction may represent an important electron-shuttling pathway for electron transfer to Fe(III) oxides in anaerobic marine and freshwater environments.

Thèse de doctorat : Pharmacie. Chimie et microbiologie de l'eau : Nancy 1 : 2006. / Titre provenant de l'écran-titre.