74 research outputs found

    Characterization of the Shewanella oneidensis Fur gene: roles in iron and acid tolerance response

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    <p>Abstract</p> <p>Background</p> <p>Iron homeostasis is a key metabolism for most organisms. In many bacterial species, coordinate regulation of iron homeostasis depends on the protein product of a Fur gene. Fur also plays roles in virulence, acid tolerance, redox-stress responses, flagella chemotaxis and metabolic pathways.</p> <p>Results</p> <p>We conducted physiological and transcriptomic studies to characterize Fur in <it>Shewanella oneidensis</it>, with regard to its roles in iron and acid tolerance response. A <it>S. oneidensis</it><it>fur</it> deletion mutant was defective in growth under iron-abundant or acidic environment. However, it coped with iron depletion better than the wild-type strain MR-1. Further gene expression studies by microarray of the <it>fur</it> mutant confirmed previous findings that iron uptake genes were highly de-repressed in the mutant. Intriguingly, a large number of genes involved in energy metabolism were iron-responsive but Fur-independent, suggesting an intimate relationship of energy metabolism to iron response, but not to Fur. Further characterization of these genes in energy metabolism suggested that they might be controlled by transcriptional factor Crp, as shown by an enriched motif searching algorithm in the corresponding cluster of a gene co-expression network.</p> <p>Conclusion</p> <p>This work demonstrates that <it>S. oneidensis</it> Fur is involved in iron acquisition and acid tolerance response. In addition, analyzing genome-wide transcriptional profiles provides useful information for the characterization of Fur and iron response in <it>S. oneidensis</it>.</p

    Molecular Control of Extracellular DNA Release and Degradation in Shewanella oneidensis MR-1 Biofilms: The Role of Phages and Nucleases

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    Bakterien bilden unter natürlichen Bedingungen häufig oberflächen-assoziierte multizelluläre Gemeinschaften, welche allgemein als Biofilme bezeichnet werden. Die Bildung von Biofilmen ist ein komplexer und präzise regulierter Prozess, der es Bakterien ermöglicht, beinahe jede Art von Oberfläche zu besiedeln und dadurch physikalischen Stressfaktoren, Nährstoffmangel und Antibiotika standzuhalten. Des Weiteren kann oberflächenassoziiertes Wachstum die Virulenz von pathogenen Bakterien erhöhen und Umweltkeimen die Erschließung von Oberflächen als Nährstoff- und Energiequelle ermöglichen. Aus diesem Grund hat sich gezeigt, dass bakterielle Biofilmbildung von großer medizinischer, ökologischer und ökonomischer Relevanz ist. Ein wichtiger Bestandteil von Biofilmen ist die extrazelluläre polymere Matrix welche sich typischerweise aus Exopolysacchariden, Proteinen und extrazellulärer DNA (eDNA) zusammensetzt. Die Bedeutung der eDNA für Biofilme war lange unklar, jedoch konnte durch eine Reihe von Studien gezeigt werden, dass eDNA für die meisten Bakterienspezies, darunter Shewanella oneidensis MR-1, von essentieller Bedeutung für die strukturelle Entwicklung der Biofilme ist. Vielfach unbekannt sind jedoch Mechanismen, welche die Freisetzung von eDNA regulieren bzw. ausführen und solche, die an der Modulation und am Abbau (z.B. zur endogen induzierten Auflösung von Biofilmen oder zur Erschließung von eDNA als Nährstoffquelle) beteiligt sind. In der vorliegenden Arbeit wurde diese Mechanismen molekularbiologisch, mikroskopisch und biochemisch untersucht

    Integrating Mass Spectrometry Based Proteomics and Bioinformatics Technologies for the Molecular Level Characterization of \u3cem\u3eShewanella oneidensis\u3c/em\u3e to Chromate Exposure

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    The research outlined in this dissertation involves the development and demonstration of a mass spectrometry-based proteomics approach to characterize the global level molecular response of Shewanella oneidensis MR-1 to chromate exposure. The proteomics approach is centered on a high performance technique of multidimensional on-line liquid chromatographic separations with subsequent tandem mass spectrometric detection. Since very complex proteome samples are digested into peptides and then directly measured by MS, this technique is termed shotgun proteomics. This approach affords the identification and quantification of complex mixtures by directly analyzing their proteolytic peptides and then using computational techniques to reassemble the protein information. The research goals for this dissertation project were two-fold: (1) enhancement of the experimental and computational methodologies to permit deeper and more confident proteome characterizations, and (2) demonstration of this optimized approach for the comprehensive investigation of the molecular level response of the bacterium S. oneidensis to chromate insult. To address research needs, we developed a single-tube lysis method for cell lysis-proteome digestion to enable investigations of small amounts of cellular biomass, and identified suitable bioinformatic approaches to mine post-translational modifications from proteome datasets. These advancements were then utilized to examine the molecular level response of S. oneidensis to chromate insult, which was accomplished by varying chromate concentrations, dosages, and time points. These measurements provided the first global proteome-level observation of the dynamic changes of S. oneidensis in response to chromate insult

    The small RNA RyhB is a regulator of cytochrome expression in Shewanella oneidensis

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    Shewanella oneidensis produces an extensive electron transfer network that results in metabolic flexibility. A large number of c-type cytochromes are expressed by S. oneidensis and these function as the fundamental electron transport chain proteins. Although several S. oneidensis cytochromes have been well-characterized, little is known about how their expression is regulated. In this study, we investigate the role of the ferric uptake regulator (Fur) and the sRNA RyhB in regulation. Our results demonstrate that loss of Fur leads to diminished growth and an apparent decrease in heme-containing proteins. Remarkably, deleting the Fur-repressed ryhB gene almost completely reverses these physiological changes, indicating that the phenotypes resulting from loss of Fur are (at least partially) dependent on RyhB. RNA sequencing identified a number of possible RyhB repressed genes. A large fraction of these encode c-type cytochromes, among them two of the most abundant periplasmic cytochromes CctA (also known as STC) and ScyA. We show that RyhB destabilizes the mRNA of four of its target genes, cctA, scyA, omp35, and nrfA and this requires the presence of the RNA chaperone Hfq. Iron limitation decreases the expression of the RyhB target genes cctA and scyA and this regulation relies on the presence of both Fur and RyhB. Overall, this study suggests that controlling cytochrome expression is of importance to maintain iron homeostasis and that sRNAs molecules are important players in the regulation of fundamental processes in S. oneidensis MR-1

    Mass Spectrometry-Based Proteomics for Studying Microbial Physiology from Isolates to Communities

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    With the advent of whole genome sequencing, a new era of biology was ushered in allowing for “systems-biology” approaches to characterizing microbial systems. The field of systems biology aims to catalogue and understand all of the biological components, their functions, and all of their interactions in a living system as well as communities of living systems. Systems biology can be considered an attempt to measure all of the components of a living system and then produce a data-driven model of the system. This model can then be used to generate hypotheses about how the system will respond to perturbations, which can be tested experimentally. The first step in the process is the determination of a microbial genome. This process has, to a large extent, been fully developed, with hundreds of microbial genome sequences completed and hundreds more being characterized at a breathtaking pace. The developments of technologies to use this information and to further probe the functional components of microbes at a global level are currently being developed. The field of gene expression analysis at the transcript level is one example; it is now possible to simultaneously measure and compare the expression of thousands of mRNA products in a single experiment. The natural extension of these experiments is to simultaneously measure and compare the expression of all the proteins present in a microbial system. This is the field of proteomics. With the development of electrospray ionization, rapid tandem mass spectrometry and database-searching algorithms, mass spectrometry (MS) has become the leader in the attempts to decipher proteomes. This research effort is very young and many challenges still exist. The goal of the work described here was to build a state-of-the-art robust MS-based proteomics platform for the characterization of microbial proteomes from isolates to communities. The research presented here describes the successes and challenges of this objective. Proteome analyses of the metal-reducing bacteria Shewanella oneidensis and the metabolically versatile bacteria Rhodopseudomonas palustris are given as examples of the power of this technology to elucidate proteins important to different metabolic states at a global level. The analysis of microbial proteomes from isolates is only the first step of the challenge. In nature, microbial species do not act alone but are always found in mixtures with other species where their intricate interactions are critical for survival. These studies conclude with some of the first efforts to develop methodologies to measure proteomes of simple controlled mixtures of microbial species and then present the first attempt at measuring the proteome of a natural microbial community, a biofilm from an acid mine drainage system. This microbial system illustrates life at the extreme of nature where life not only exists but flourishes in very acidic conditions with high metal concentrations and high temperatures. The technologies developed through these studies were applied to the first deep characterization of a microbial community proteome, the deciphering of the expressed proteome of the acid mine drainage biofilm. The research presented here has led to development of a state-of-the-art robust proteome pipeline, which can now be applied to the proteome analysis of any microbial isolate for a sequenced species. The first steps have also been made toward developing methodologies to characterize microbial proteomes in their natural environments. These developments are key to integrating proteome technologies with genome and transcriptome technologies for global characterizations of microbial species at the systems level. This will lead to understanding of microbial physiology from a global view where instead of analyzing one gene or protein at a time, hundreds of genes/proteins will be interrogated in microbial species as the adapt and survive in the environment

    Comparative Proteomics Reveals Core vs. Unique Molecular Signatures for Dissimilatory Metal Reducing Bacteria Grown with Various Electron Acceptors

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    Dissimilatory metal reducing bacteria (DMRB) are probably one of the most respiratory versatile microorganisms on earth. Their ability to use metals as terminal electron acceptor allows them to survive in severe environments (e.g. radionuclide contaminated soil). In addition to metals, many other organic and inorganic substrates can be utilized as electron acceptors for DMRB respiration, including fumarate, nitrate, oxygen, etc. Genome information for many DMRB species is available, which reveals large numbers of c-type cytochrome encoding genes present in their genomes. For example, the genomes of three DMRBs, Anaeromyxobacter dehalogenans strain 2CP-C, Shewanella oneidensis strain MR-1, and Geobacter daltonii strain FRC-32, contain 69, 40, and 72 putative c-type cytochrome genes, respectively. Although mutagenesis techniques have determined the respiratory roles of several c-type cytochromes, gene disruption for majorities of the putative c-type cytochromes does not generate visible phenotypical alterations, and is not able to functionally link them to specific respirational activities. Thus, comprehensive proteome characterization for DMRBs is needed to elucidate the molecular mechanisms underlying their respirational versatilities. In this dissertation, a mass spectrometry-based proteomics approach was used to interrogate the proteomes of A. dehalogenans strain 2CP-C, S. oneidensis strain MR-1, and G. daltonii strain FRC-32. The proteomic responses of DMRBs to a wide range of electron acceptors were tested in this dissertation, including soluble and insoluble ferric iron, manganese oxide, fumarate, nitrate, oxygen, and nitrous oxide. The in-depth proteomic characterizations comparatively revealed the c-type cytochrome profiles of DMRBs, providing evidence for the identities and expressions of putative c-type cytochromes, and established the linkage between specific electron acceptor and individual c-type cytochromes. The entire proteome complements of DMRBs were also characterized, generating metabolic maps reflecting pathway-level activities responding to various electron acceptors. The results identified the core proteome carrying out the essential cellular machineries for each tested DMRB, and demonstrated clearly elevated energy metabolism for A. dehalogenans strain 2CP-C during respiration of metal electron acceptors. Comparative proteomics analysis between tested DMRB strains revealed the commonalities and differences of proteomic phenotypes displayed by different strains, and shed light into deeper understandings for DMRB metabolic activities

    Steering biogas performance by implementation of bioelectrochemical cell (BEC) technology

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    A new concept for integrating conventional anaerobic digester (AD) with bioelectrochemical cell (BEC) technology was investigated in the current study. The BEC technology can convert energy stored in organic matter directly into bioelectricity. Coupling AD with BEC could be a profitable approach that could lead to overcoming limiting factors in AD, such as hydrogen partial pressure and accumulation of volatile fatty acids, inhibiting the methanogenesis

    Analysis of Genes Involved in Metal Resistance and Cytochrome C Maturation in Shewanella Oneidensis MR-1

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    Metals play crucial roles in many cellular processes where they form active centers of metabolic enzymes or participate in electron transfer reactions during respiration. At high concentrations, metals can be toxic and result in the formation of reactive oxygen species and protein denaturation. Bacteria have evolved homeostasis systems to maintain intracellular concentrations of various metals and avoid their toxic effects. The aim of this project is to identify and characterize metal homeostasis systems in the metal reducer Shewanella oneidensis MR-1. This bacterium can use metals and radionuclides as electron acceptors during anaerobic respiration and is therefore a good candidate for bioremediation of metal-contaminated environments. Furthermore, this bacterium is able to maintain low internal levels of heavy metals through the use of multiple efflux pumps such as the P-type ATPase - CopA, and the HME RND efflux pump – CzcCBA. This study aims to understand the role of these efflux pumps and their regulators in metal resistance. Shewanella oneidensis also expresses a large number of c-type cytochromes, many of which function as terminal reductases. All of these proteins contain the typical heme-binding motif CXXCH and require the Ccm proteins for maturation. SirA, the terminal sulfite reductase, also possesses an atypical heme binding site CX15CH which requires a specialized system for heme attachment. S. oneidensis MR-1 encodes two cytochrome c synthetases (CcmF and SirE) and two apocytochrome c chaperones (CcmI and SirG). In this study we show that both apocytochrome c chaperones, CcmI and SirG, are required for the maturation of SirA and they each interact with the terminal sulfite reductase independently of each other, even in the absence of other components of the cytochrome c maturation system

    Ecophysiology and diversity of anaeromyxobacter spp. and implications for uranium bioremediation

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    Uranium has been released into the environment due to improper practices associated with mining and refinement for energy and weapons production. Soluble U(VI) species such as uranyl carbonate can be reduced to form the insoluble U(IV) mineral uraninite (UO2) via microbial respiratory processes. Formation of UO2 diminishes uranium mobility and prevents uranium-laden groundwater from being discharged into surface water; however, oxygen and other oxidants re-solubilize UO2. Many organisms have been shown to reduce uranium, but variations in microbial physiology change the dynamics of microbial uranium reduction in situ and affect uraninite stability. Anaeromyxobacter dehalogenans is a metal-reducing delta-Proteobacterium in the myxobacteria family that displays remarkable respiratory versatility and efficiently reduces U(VI). The approach of this research was to enhance characterization of A. dehalogenans by identifying unique genetic traits, describing variability within the species, and examining the environmental distribution of A. dehalogenans strains. Genome analysis revealed that A. dehalogenans shares many traits with the myxobacteria including type IV pilus-based motility and an aerobic-like electron transport chain. In addition, the genome revealed genes that share sequence similarity with strict anaerobes and other metal-reducing organisms. Physiological examination of microaerophilism in A. dehalogenans strain 2CP-C revealed growth at sub-atmospheric oxygen partial pressure. Physiological characterization of novel isolates demonstrated that strain-level variation in the 16S rRNA gene coincides with metabolic changes that can be linked to the loss of specific gene homologs. Anaeromyxobacter spp. were present at the Oak Ridge Integrated Field-scale Subsurface Research Challenge (IFC) site and multiplex qPCR tools designed using a minor-groove binding probe gave insights into strain and species differences in the community. Finally, 16S rRNA gene sequences were identified which suggest a novel Anaeromyxobacter species that is responsible for uranium reduction at the Oak Ridge IFC site. This research contributes new knowledge of the ecophysiology of a widely distributed, metal-reducing bacterial group capable of uranium immobilization. The characterization of Anaeromyxobacter spp. helps to elucidate the dynamics of biological cycling of metals at oxic-anoxic interfaces, like those at the Oak Ridge IFC, and contributes to the broader study of microbial ecology in groundwater and sediment environments.Ph.D.Committee Chair: Dr. Frank E. Löffler; Committee Member: Dr. Joseph B. Hughes; Committee Member: Dr. Kurt D. Pennell; Committee Member: Dr. Lawrence J. Shimkets; Committee Member: Dr. Robert A. Sanford; Committee Member: Dr. Thomas DiChristin

    Expanding the role of FurA as essential global regulator in cyanobacteria

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    In the nitrogen-fixing heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, the ferric uptake regulator FurA plays a global regulatory role. Failures to eliminate wild-type copies of furA gene from the polyploid genome suggest essential functions. In the present study, we developed a selectively regulated furA expression system by the replacement of furA promoter in the Anabaena sp. chromosomes with the Co2+/Zn2+ inducible coaT promoter from Synechocystis sp. PCC 6803. By removing Co2+ and Zn2+ from the medium and shutting off furA expression, we showed that FurA was absolutely required for cyanobacterial growth. RNA-seq based comparative transcriptome analyses of the furA-turning off strain and its parental wild-type in conjunction with subsequent electrophoretic mobility shift assays and semi-quantitative RT-PCR were carried out in order to identify direct transcriptional targets and unravel new biological roles of FurA. The results of such approaches led us to identify 15 novel direct iron-dependent transcriptional targets belonging to different functional categories including detoxification and defences against oxidative stress, phycobilisome degradation, chlorophyll catabolism and programmed cell death, light sensing and response, heterocyst differentiation, exopolysaccharide biosynthesis, among others. Our analyses evidence novel interactions in the complex regulatory network orchestrated by FurA in cyanobacteria
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