Dale A. Pelletier

Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris.

Rhodopseudomonas palustris is among the most metabolically versatile bacteria known. It uses light, inorganic compounds, or organic compounds, for energy. It acquires carbon from many types of green plant–derived compounds or by carbon dioxide fixation, and it fixes nitrogen. Here we describe the genome sequence of R. palustris, which consists of a 5,459,213-base-pair (bp) circular chromosome with 4,836 predicted genes and a plasmid of 8,427 bp. The sequence reveals genes that confer a remarkably large number of options within a given type of metabolism, including three nitrogenases, five benzene ring cleavage pathways and four light harvesting 2 systems. R. palustris encodes 63 signal transduction histidine kinases and 79 response regulator receiver domains. Almost 15% of the genome is devoted to transport. This genome sequence is a starting point to use R. palustris as a model to explore how organisms integrate metabolic modules in response to environmental perturbations.

Distinct microbial communities within the endosphere and rhizosphere of Populus deltoides roots across contrasting soil types.

ABSTRACT The root-rhizosphere interface of Populus is the nexus of a variety of associations between bacteria, fungi, and the host plant and an ideal model for studying interactions between plants and microorganisms. However, such studies have generally been confined to greenhouse and plantation systems. Here we analyze microbial communities from the root endophytic and rhizospheric habitats of Populus deltoides in mature natural trees from both upland and bottomland sites in central Tennessee. Community profiling utilized 454 pyrosequencing with separate primers targeting the V4 region for bacterial 16S rRNA and the D1/D2 region for fungal 28S rRNA genes. Rhizosphere bacteria were dominated by Acidobacteria (31%) and Alphaproteobacteria (30%), whereas most endophytes were from the Gammaproteobacteria (54%) as well as Alphaproteobacteria (23%). A single Pseudomonas-like operational taxonomic unit (OTU) accounted for 34% of endophytic bacterial sequences. Endophytic bacterial richness was also highly variable and 10-fold lower than in rhizosphere samples originating from the same roots. Fungal rhizosphere and endophyte samples had approximately equal amounts of the Pezizomycotina (40%), while the Agaricomycotina were more abundant in the rhizosphere (34%) than endosphere (17%). Both fungal and bacterial rhizosphere samples were highly clustered compared to the more variable endophyte samples in a UniFrac principal coordinates analysis, regardless of upland or bottomland site origin. Hierarchical clustering of OTU relative abundance patterns also showed that the most abundant bacterial and fungal OTUs tended to be dominant in either the endophyte or rhizosphere samples but not both. Together, these findings demonstrate that root endophytic communities are distinct assemblages rather than opportunistic subsets of the rhizosphere.

Silver nanocrystallites: biofabrication using Shewanella oneidensis, and an evaluation of their comparative toxicity on gram-negative and gram-positive bacteria.

Microorganisms have long been known to develop resistance to metal ions either by sequestering metals inside the cell or by effluxing them into the extracellular media. Here we report the biosynthesis of extracellular silver-based single nanocrystallites of well-defined composition and homogeneous morphology utilizing the gamma-proteobacterium, Shewanella oneidensis MR-1, upon incubation with aqueous silver nitrate solution. Further characterization of these particles revealed that the crystals consist of small, reasonably monodispersed spheres in the 2-11 nm size range (average of 4 +/- 1.5 nm). The bactericidal effect of these nanoparticles (biogenic-Ag) is compared to chemically synthesized silver nanoparticles (colloidal-Ag and oleate capped silver nanoparticles, oleate-Ag) and assessed using Gram-negative (E. coli and S. oneidensis) and Gram-positive (B. subtilis) bacteria. Relative toxicity was based on the diameter of inhibition zone in disk diffusion tests, minimum inhibitory concentrations, live/dead assays, and atomic force microscopy. From a toxicity perspective, strain-dependent inhibition depended on the synthesis procedure and the surface coat. Biogenic-Ag was found to be of higher toxicity compared to colloidal-Ag for all three strains tested, whereas E. coli and S. oneidensis were found to be more resistant to either of these nanoparticles than B. subtilis. In contrast, oleate-Ag was not toxic to any of the bacteria. These findings have implications for the potential uses of Ag nanomaterials and for their fate in biological and environmental systems.

Effects of Engineered Cerium Oxide Nanoparticles on Bacterial Growth and Viability

ABSTRACT Interest in engineered nanostructures has risen in recent years due to their use in energy conservation strategies and biomedicine. To ensure prudent development and use of nanomaterials, the fate and effects of such engineered structures on the environment should be understood. Interactions of nanomaterials with environmental microorganisms are inevitable, but the general consequences of such interactions remain unclear, due to a lack of standard methods for assessing such interactions. Therefore, we have initiated a multianalytical approach to understand the interactions of synthesized nanoparticles with bacterial systems. These efforts are focused initially on cerium oxide nanoparticles and model bacteria in order to evaluate characterization procedures and the possible fate of such materials in the environment. The growth and viability of the Gram-negative species Escherichia coli and Shewanella oneidensis, a metal-reducing bacterium, and the Gram-positive species Bacillus subtilis were examined relative to cerium oxide particle size, growth media, pH, and dosage. A hydrothermal synthesis approach was used to prepare cerium oxide nanoparticles of defined sizes in order to eliminate complications originating from the use of organic solvents and surfactants. Bactericidal effects were determined from MIC and CFU measurements, disk diffusion tests, and live/dead assays. For E. coli and B. subtilis, clear strain- and size-dependent inhibition was observed, whereas S. oneidensis appeared to be unaffected by the particles. Transmission electron microscopy along with microarray-based transcriptional profiling was used to understand the response mechanism of the bacteria. Use of multiple analytical approaches adds confidence to toxicity assessments, while the use of different bacterial systems highlights the potential wide-ranging effects of nanomaterial interactions in the environment.

AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces

Immobilization of particulates, especially biomolecules and cells, onto surfaces is critical for imaging with the atomic force microscope (AFM). In this paper, gelatin coated mica surfaces are shown to be suitable for immobilizing and imaging both gram positive, Staphylococcus aureus, and gram negative, Escherichia coli, bacteria in both air and liquid environments. Gelatin coated surfaces are shown to be superior to poly-L-lysine coated surfaces that are commonly used for the immobilization of cells. This cell immobilization technique is being developed primarily for live cell imaging of Rhodopseudomonas palustris. The genome of R. palustris has been sequenced and the organism is the target of intensive studies aimed at understanding genome function. Images of R. palustris grown both aerobically and anaerobically in liquid media are presented. Images in liquid media show the bacteria is rod shaped and smooth while images in air show marked irregularity and folding of the surface. Significant differences in the vertical dimension are also apparent with the height of the bacteria in liquid being substantially greater than images taken in air. In air immobilized bacterial flagella are clearly seen while in liquid this structure is not visible. Additionally, significant morphological differences are observed that depend on the method of bacterial growth.

Cytotoxicity Induced by Engineered Silver Nanocrystallites Is Dependent on Surface Coatings and Cell Types

Due to their unique antimicrobial properties silver nanocrystallites have garnered substantial attention and are used extensively for biomedical applications as an additive to wound dressings, surgical instruments and bone substitute materials. They are also released into unintended locations such as the environment or biosphere. Therefore it is imperative to understand the potential interactions, fate and transport of nanoparticles with environmental biotic systems. Numerous factors including the composition, size, shape, surface charge, and capping molecule of nanoparticles are known to influence cell cytotoxicity. Our results demonstrate that the physical/chemical properties of the silver nanoparticles including surface charge, differential binding and aggregation potential, which are influenced by the surface coatings, are a major determining factor in eliciting cytotoxicity and in dictating potential cellular interactions. In the present investigation, silver nanocrystallites with nearly uniform size and shape distribution but with different surface coatings, imparting overall high negativity to high positivity, were synthesized. These nanoparticles included poly(diallyldimethylammonium) chloride-Ag, biogenic-Ag, colloidal-Ag (uncoated), and oleate-Ag with zeta potentials +45 ± 5, -12 ± 2, -42 ± 5, and -45 ± 5 mV, respectively; the particles were purified and thoroughly characterized so as to avoid false cytotoxicity interpretations. A systematic investigation on the cytotoxic effects, cellular response, and membrane damage caused by these four different silver nanoparticles was carried out using multiple toxicity measurements on mouse macrophage (RAW-264.7) and lung epithelial (C-10) cell lines. Our results clearly indicate that the cytotoxicity was dependent on various factors such as surface charge and coating materials used in the synthesis, particle aggregation, and the cell-type for the different silver nanoparticles that were investigated. Poly(diallyldimethylammonium)-coated Ag nanoparticles were found to be the most toxic, followed by biogenic-Ag and oleate-Ag nanoparticles, whereas uncoated or colloidal silver nanoparticles were found to be the least toxic to both macrophage and lung epithelial cells. Also, based on our cytotoxicity interpretations, lung epithelial cells were found to be more resistant to the silver nanoparticles than the macrophage cells, regardless of the surface coating.

Initial Phases of Biofilm Formation in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative Fe(III)- and Mn(IV)-reducing microorganism and serves as a model for studying microbially induced dissolution of Fe or Mn oxide minerals as well as biogeochemical cycles. In soil and sediment environments, S. oneidensis biofilms form on mineral surfaces and are critical for mediating the metabolic interaction between this microbe and insoluble metal oxide phases. In order to develop an understanding of the molecular basis of biofilm formation, we investigated S. oneidensis biofilms developing on glass surfaces in a hydrodynamic flow chamber system. After initial attachment, growth of microcolonies and lateral spreading of biofilm cells on the surface occurred simultaneously within the first 24 h. Once surface coverage was almost complete, biofilm development proceeded with extensive vertical growth, resulting in formation of towering structures giving rise to pronounced three-dimensional architecture. Biofilm development was found to be dependent on the nutrient conditions, suggesting a metabolic control. In global transposon mutagenesis, 173 insertion mutants out of 15,000 mutants screened were identified carrying defects in initial attachment and/or early stages in biofilm formation. Seventy-one of those mutants exhibited a nonswimming phenotype, suggesting a role of swimming motility or motility elements in biofilm formation. Disruption mutations in motility genes (flhB, fliK, and pomA), however, did not affect initial attachment but affected progression of biofilm development into pronounced three-dimensional architecture. In contrast, mutants defective in mannose-sensitive hemagglutinin type IV pilus biosynthesis and in pilus retraction (pilT) showed severe defects in adhesion to abiotic surfaces and biofilm formation, respectively. The results provide a basis for understanding microbe-mineral interactions in natural environments.

Biofabrication of discrete spherical gold nanoparticles using the metal-reducing bacterium, Shewanella oneidensis

Nanocrystallites have garnered substantial interest due to their various applications, including catalysis and medical research. Consequently important aspects of synthesis related to control of shape and size through economical and non-hazardous means are desirable. Highly efficient bioreduction-based fabrication approaches that utilize microbes and/or plant extracts are poised to meet these needs. Here we show that the γ-proteobacterium Shewanella oneidensis can reduce tetrachloroaurate (III) ions to produce discrete extracellular spherical gold nanocrystallites. The particles were homogeneously shaped with multiple size distributions and produced under ambient conditions at high yield, 88% theoretical maximum. Further characterization revealed that the particles consist of spheres in the size range of ∼2-50 nm, with an average size of 12±5 nm. The nanoparticles were hydrophilic and resisted aggregation even after several months. Based on our experiments, the particles are likely fabricated by the aid of reducing agents present in the bacterial cell membrane and are capped by a detachable protein/peptide coat. Ultraviolet-visible and Fourier transform infrared spectroscopy, X-ray diffraction, energy dispersive X-ray spectra and transmission electron microscopy measurements confirmed the formation, surface characteristics and crystalline nature of the nanoparticles. The antibacterial activity of these gold nanoparticles was assessed using Gram-negative (Escherichia coli and S. oneidensis) and Gram-positive (Bacillus subtilis) bacterial species. Toxicity assessments showed that the particles were neither toxic nor inhibitory to any of these bacteria.

Isolation and Characterization of Anaerobic Ethylbenzene Dehydrogenase, a Novel Mo-Fe-S Enzyme

The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(-)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (alphabetagamma) with subunit masses of 100 kDa (alpha), 35 kDa (beta), and 25 kDa (gamma). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the alpha subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the beta subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the gamma subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes.

Twenty-One Genome Sequences from Pseudomonas Species and 19 Genome Sequences from Diverse Bacteria Isolated from the Rhizosphere and Endosphere of Populus deltoides

To aid in the investigation of the Populus deltoides microbiome, we generated draft genome sequences for 21 Pseudomonas strains and 19 other diverse bacteria isolated from Populus deltoides roots. Genome sequences for isolates similar to Acidovorax, Bradyrhizobium, Brevibacillus, Caulobacter, Chryseobacterium, Flavobacterium, Herbaspirillum, Novosphingobium, Pantoea, Phyllobacterium, Polaromonas, Rhizobium, Sphingobium, and Variovorax were generated.