Margaret F. Romine

Towards Environmental Systems Biology of Shewanella

Bacteria of the genus Shewanella are known for their versatile electron-accepting capacities, which allow them to couple the decomposition of organic matter to the reduction of the various terminal electron acceptors that they encounter in their stratified environments. Owing to their diverse metabolic capabilities, shewanellae are important for carbon cycling and have considerable potential for the remediation of contaminated environments and use in microbial fuel cells. Systems-level analysis of the model species Shewanella oneidensis MR-1 and other members of this genus has provided new insights into the signal-transduction proteins, regulators, and metabolic and respiratory subsystems that govern the remarkable versatility of the shewanellae.

Current Production and Metal Oxide Reduction by Shewanella oneidensis MR-1 Wild Type and Mutants

ABSTRACT Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.

Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organisms dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes : Sequence of tceA and substrate range characterization

ABSTRACT The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1,2-dibromoethane to ethene at rates of 7.5 and 30 μmol/min/mg, respectively, similar to the rates for TCE,cis-dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase,tceA, was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP)2. Direct N-terminal sequencing of the mature enzyme indicated that the first 42 amino acids constitute a signal sequence containing the twin-arginine motif, RRXFXK, associated with the Sec-independent membrane translocation system. This information coupled with membrane localization studies indicated that TCE-RDase is located on the exterior of the cytoplasmic membrane. Like the case for the two other RDases that have been cloned and sequenced, a small open reading frame, tceB, is proposed to be involved with membrane association of TCE-RDase and is predicted to be cotranscribed with tceA.

c-Type cytochrome-dependent formation of U(IV) nanoparticles by Shewanella oneidensis.

Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI) complexes in situ, the biomolecular mechanisms of U(VI) reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI) and formation of extracelluar UO 2 nanoparticles. In particular, the outer membrane (OM) decaheme cytochrome MtrC (metal reduction), previously implicated in Mn(IV) and Fe(III) reduction, directly transferred electrons to U(VI). Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI) reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO 2 nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS). In wild-type cells, this UO 2-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO 2 nanoparticles with MtrC and OmcA (outer membrane cytochrome). This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO 2 nanoparticles. In the environment, such association of UO 2 nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O 2 or transport in soils and sediments.

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

Significance Bacterial nanowires from Shewanella oneidensis MR-1 were previously shown to be conductive under nonphysiological conditions. Intense debate still surrounds the molecular makeup, identity of the charge carriers, and cellular respiratory impact of bacterial nanowires. In this work, using in vivo fluorescence measurements, immunolabeling, and quantitative gene expression analysis, we demonstrate that S. oneidensis MR-1 nanowires are extensions of the outer membrane and periplasm, rather than pilin-based structures, as previously thought. We also demonstrate that the outer membrane multiheme cytochromes MtrC and OmcA localize to these membrane extensions, directly supporting one of the two models of electron transport through the nanowires; consistent with this, production of bacterial nanowires correlates with an increase in cellular reductase activity. Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic–abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis 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 the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Global Analysis of Deinococcus Radiodurans Proteome by Csing Accurate Mass Tags

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organisms dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Geomicrobiology of High-Level Nuclear Waste-Contaminated Vadose Sediments at the Hanford Site, Washington State

ABSTRACT Sediments from a high-level nuclear waste plume were collected as part of investigations to evaluate the potential fate and migration of contaminants in the subsurface. The plume originated from a leak that occurred in 1962 from a waste tank consisting of high concentrations of alkali, nitrate, aluminate, Cr(VI), 137Cs, and 99Tc. Investigations were initiated to determine the distribution of viable microorganisms in the vadose sediment samples, probe the phylogeny of cultivated and uncultivated members, and evaluate the ability of the cultivated organisms to survive acute doses of ionizing radiation. The populations of viable aerobic heterotrophic bacteria were generally low, from below detection to ∼104 CFU g−1, but viable microorganisms were recovered from 11 of 16 samples, including several of the most radioactive ones (e.g., >10 μCi of 137Cs/g). The isolates from the contaminated sediments and clone libraries from sediment DNA extracts were dominated by members related to known gram-positive bacteria. Gram-positive bacteria most closely related to Arthrobacter species were the most common isolates among all samples, but other phyla high in G+C content were also represented, including Rhodococcus and Nocardia. Two isolates from the second-most radioactive sample (>20 μCi of 137Cs g−1) were closely related to Deinococcus radiodurans and were able to survive acute doses of ionizing radiation approaching 20 kGy. Many of the gram-positive isolates were resistant to lower levels of gamma radiation. These results demonstrate that gram-positive bacteria, predominantly from phyla high in G+C content, are indigenous to Hanford vadose sediments and that some are effective at surviving the extreme physical and chemical stress associated with radioactive waste.

Global Transcriptome Analysis of Shewanella oneidensis MR-1 Exposed to Different Terminal Electron Acceptors

To gain insight into the complex structure of the energy-generating networks in the dissimilatory metal reducer Shewanella oneidensis MR-1, global mRNA patterns were examined in cells exposed to a wide range of metal and non-metal electron acceptors. Gene expression patterns were similar irrespective of which metal ion was used as electron acceptor, with 60% of the differentially expressed genes showing similar induction or repression relative to fumarate-respiring conditions. Several groups of genes exhibited elevated expression levels in the presence of metals, including those encoding putative multidrug efflux transporters, detoxification proteins, extracytoplasmic sigma factors and PAS-domain regulators. Only one of the 42 predicted c-type cytochromes in MR-1, SO3300, displayed significantly elevated transcript levels across all metal-reducing conditions. Genes encoding decaheme cytochromes MtrC and MtrA that were previously linked to the reduction of different forms of Fe(III) and Mn(IV), exhibited only slight decreases in relative mRNA abundances under metal-reducing conditions. In contrast, specific transcriptome responses were displayed to individual non-metal electron acceptors resulting in the identification of unique groups of nitrate-, thiosulfate- and TMAO-induced genes including previously uncharacterized multi-cytochrome gene clusters. Collectively, the gene expression results reflect the fundamental differences between metal and non-metal respiratory pathways of S. oneidensis MR-1, where the coordinate induction of detoxification and stress response genes play a key role in adaptation of this organism under metal-reducing conditions. Moreover, the relative paucity and/or the constitutive nature of genes involved in electron transfer to metals is likely due to the low-specificity and the opportunistic nature of the metal-reducing electron transport pathways.

Taxonomic Study of Aromatic-Degrading Bacteria from Deep- Terrestrial-Subsurface Sediments and Description of Sphingomonas aromaticivorans sp. nov., Sphingomonas subterranea sp. nov., and Sphingomonas stygia sp. nov.

Phylogenetic analyses of 16S rRNA gene sequences by distance matrix and parsimony methods indicated that six strains of bacteria isolated from deep saturated Atlantic coastal plain sediments were closely related to the genus Sphingomonas. Five of the strains clustered with, but were distinct from, Sphingomonas capsulata, whereas the sixth strain was most closely related to Blastobacter natatorius. The five strains that clustered with S. capsulata, all of which could degrade aromatic compounds, were gram-negative, non-spore-forming, non-motile, rod-shaped organisms that produced small, yellow colonies on complex media. Their G + C contents ranged from 60.0 to 65.4 mol%, and the predominant isoprenoid quinone was ubiquinone Q-10. All of the strains were aerobic and catalase positive. Indole, urease, and arginine dihydrolase were not produced. Gelatin was not liquified, and glucose was not fermented. Sphingolipids were present in all strains; 2OH14:0 was the major hydroxy fatty acid, and 18:1 was a major constituent of cellular lipids. Acid was produced oxidatively from pentoses, hexoses, and disaccharides, but not from polyalcohols and indole. All of these characteristics indicate that the five aromatic-degrading strains should be placed in the genus Sphingomonas as currently defined. Phylogenetic analysis of 16S rRNA gene sequences, DNA-DNA reassociation values, BOX-PCR genomic fingerprinting, differences in cellular lipid composition, and differences in physiological traits all indicated that the five strains represent three previously undescribed Sphingomonas species. Therefore, we propose the following new species: Sphingomonas aromaticivorans (type strain, SMCC F199), Sphingomonas subterranea (type strain, SMCC B0478), and Sphingomonas stygia (type strain, SMCC B0712).