Timothy S. Magnuson

Ecophysiology and geochemistry of microbial arsenic oxidation within a high arsenic, circumneutral hot spring system of the Alvord Desert.

Microbial metabolism of arsenic has gained considerable interest, due to the potential of microorganisms to drive arsenic cycling and significantly influence the geochemistry of naturally arsenic-rich or anthropogenically arsenic-polluted environments. Alvord Hot Spring in southeastern Oregon is a circumneutral hot spring with an average arsenic concentration of 4.5 mg L(-1) (60 microM). Hydrogeochemical analyses indicated significant arsenite oxidation, increased pH and decreased temperature along the stream channels flowing into Alvord Hot Spring. The dynamic range of pH and temperature over the length of three stream channels were 6.76-7.06 and 69.5-78.2 degrees C, respectively. Biofilm samples showed As(III) oxidation ex situ. 16S rRNA gene studies of sparse upstream biofilm indicated a dominance of bacteria related to Sulfurihydrogenibium, Thermus, and Thermocrinis. The lush downstream biofilm community included these same three groups but was more diverse with sequences related to uncultured OP10 bacterial phylum, uncultured Bacteroidetes, and an uncultured clade. Isolation of an arsenite oxidizer was conducted with artificial hot spring medium and yielded the isolate A03C, which is closely related to Thermus aquaticus based on 16S rRNA gene analysis. Thus, this study demonstrated the bacterial diversity along geochemical gradients of temperature, pH and As(III): As(V), and provided evidence of microbial arsenite oxidation within the Alvord Hot Spring system.

Inhibition of Microorganisms on a Carrion Breeding Resource: The Antimicrobial Peptide Activity of Burying Beetle (Coleoptera: Silphidae) Oral and Anal Secretions

ABSTRACT Competition between scavengers and microorganisms for the nutrients within carrion is well documented. As a significant contributor to food web energetics, carrion serves not only as a food source for scavengers, but also as a reproductive resource for many insects. One example are the burying beetles of the Nicrophorus genus (Coleoptera: Silphidae) whose reproduction is dependent on locating and successfully sequestering vertebrate carrion. Throughout the cooperative preparation of carrion and feeding of the larval offspring, parental beetles coat the carrion with oral and anal secretions known to attenuate the growth of molds and bacteria in the laboratory. We test the hypotheses that Nicrophorus secretions attenuate the growth of naturally occurring microorganisms likely to be found colonizing the carrion resource, and that the active antimicrobial components of the secretions are small antimicrobial peptides (AMPs) similar to those produced by other insects.

Biogenic mineral production by a novel arsenic-metabolizing thermophilic bacterium from the Alvord Basin, Oregon.

ABSTRACT The Alvord Basin in southeast Oregon contains a variety of hydrothermal features which have never been microbiologically characterized. A sampling of Murky Pot (61°C; pH 7.1) led to the isolation of a novel arsenic-metabolizing organism (YeAs) which produces an arsenic sulfide mineral known as β-realgar, a mineral that has not previously been observed as a product of bacterial arsenic metabolism. YeAs was grown on a freshwater medium and utilized a variety of organic substrates, particularly carbohydrates and organic acids. The temperature range for growth was 37 to 75°C (optimum, 55°C), and the pH range for growth was 6.0 to 8.0 (optimum, pH 7.0 to 7.5). No growth was observed when YeAs was grown under aerobic conditions. The doubling time when the organism was grown with yeast extract and As(V) was 0.71 h. Microscopic examination revealed Gram stain-indeterminate, non-spore-forming, nonmotile, rod-shaped cells, with dimensions ranging from 0.1 to 0.2 μm wide by 3 to 10 μm long. Arsenic sulfide mineralization of cell walls and extracellular arsenic sulfide particulate deposition were observed with electron microscopy and elemental analysis. 16S rRNA gene analysis placed YeAs in the family Clostridiaceae and indicated that the organism is most closely related to the Caloramator and Thermobrachium species. The G+C content was 35%. YeAs showed no detectable respiratory arsenate reductase but did display significant detoxification arsenate reductase activity. The phylogenetic, physiological, and morphological characteristics of YeAs demonstrate that it is an anaerobic, moderately thermophilic, arsenic-reducing bacterium. This organism and its associated metabolism could have major implications in the search for innovative methods for arsenic waste management and in the search for novel biogenic mineral signatures.

Proteogenomic and functional analysis of chromate reduction in Acidiphilium cryptum JF-5, an Fe(III)-respiring acidophile.

Acidiphilium cryptum JF-5, an acidophilic iron-respiring Alphaproteobacterium, has the ability to reduce chromate under aerobic and anaerobic conditions, making it an intriguing and useful model organism for the study of extremophilic bacteria in bioremediation applications. Genome sequence annotation suggested two potential mechanisms of Cr(VI) reduction, namely, a number of c-type cytochromes, and a predicted NADPH-dependent Cr(VI) reductase. In laboratory studies using pure cultures of JF-5, an NADPH-dependent chromate reductase activity was detected primarily in soluble protein fractions, and a periplasmic c-type cytochrome (ApcA) was also present, representing two potential means of Cr(VI) reduction. Upon further examination, it was determined that the NADPH-dependent activity was not specific for Cr(VI), and the predicted proteins were not detected in Cr(VI)-grown cultures. Proteomic data did show measureable amounts of ApcA in cells grown with Cr(VI). Purified ApcA is reducible by menadiol, and in turn can reduce Cr(VI), suggesting a means to obtain electrons from the respiratory chain and divert them to Cr(VI). Electrochemical measurements confirm that Cr reduction by ApcA is pH dependent, with low pH being favored. Homology modeling of ApcA and comparison to a known Cr(VI)-reducing c-type cytochrome structure revealed basic amino acids which could interact with chromate ion. From these studies, it can be concluded that A. cryptum has the physiologic and genomic capability to reduce Cr(VI) to the less toxic Cr(III). However, the expected chromate reductase mechanism may not be the primary means of Cr(VI) reduction in this organism.

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

Complete Genome Sequence of Pelosinus sp. Strain UFO1 Assembled Using Single-Molecule Real-Time DNA Sequencing Technology

ABSTRACT Pelosinus species can reduce metals such as Fe(III), U(VI), and Cr(VI) and have been isolated from diverse geographical regions. Five draft genome sequences have been published. We report the complete genome sequence for Pelosinus sp. strain UFO1 using only PacBio DNA sequence data and without manual finishing.

How the xap Locus Put Electrical “Zap” in Geobacter sulfurreducens Biofilms

Investigation of microbial mineral respiration remains an experimental challenge. In this issue of Journal of Bacteriology, Rollefson et al. (11) present a foundational study on the functionality of the biofilm matrix in Geobacter sulfurreducens, a model dissimilatory metal respiring bacterium (DMRB). In this study, the investigators identify an extracellular polysaccharide scaffold or network that entraps redox-active proteins, thus positioning these proteins for optimal electron transfer from the membrane-bound respiratory supercomplexes to a mineral phase electron acceptor. The distinguishing feature of this study is the perspective, in that the team examined specifically exopolysaccharide formation and how it enables entrapment and tethering of redox proteins in the vicinity of the cell. Previous studies on Geobacter (10) and Shewanella (4) have focused primarily on the presence and functionality of conductive pili and nanowires, proteinaceous structures that also enable and enhance extracellular electron transfer. Rollefson et al. remind investigators in this field that many microbial systems have redundancy in essential functions, and in the case of DMRB, it is clearly critical that more than one mechanism exists to ensure vectoral electron transport to mineral phase electron acceptors. The major findings of Rollefson et al. (11) were (i) identification of a biofilm locus in G. sulfurreducens that harbors exopolysaccharide synthesis and export genes; (ii) detection of c-type cytochromes in exopolysaccharide materials; (iii) genetic mutation of a gene (xapD [GSU1501], an ATP-dependent ABC transporter) in the locus which results in reduced functionality of the biofilm matrix, i.e., reduced agglutination and attachment, and less matrix-bound cytochrome; and (iv) microscopic confirmation of exopolysaccharide materials in the biofilm matrix. The research employed a combination of traditional microbiological techniques, genetic manipulation and mutation, and fluorescence and electron microscopy to obtain an integrated view of biofilm matrix composition and functionality in G. sulfurreducens. The elegance of the study is in its relative simplicity. For example, while agglutination has been examined in bacteria for decades, basic assessment of this phenomenon has not been made in many DMRB models, thus showing that classical microbiology still can reveal a great deal about DMRB biofilms. For example, Fig. 2 and 3 of the Rollefson et al. paper clearly reveal both macroscopic and microscopic differences in culture and biofilm morphology. Differential extraction procedures purify pili and exopolysaccharide and demonstrate binding of redox proteins and positioning of an extracellular biomolecular conductive network, and although one can argue the efficacy and resolution of these methods, the results seem to suggest that protein-exopolysaccharide complexes can be successfully isolated from biofilm cultures, and these approaches can be reliably used on other model DMRB systems. Unique experiments using polysaccharide binding stains coupled with scanning electron microscopy (SEM) reveal that exopolysaccharide is a key component for attachment and colonization of mineral and electrode surfaces. Indeed, the exopolysaccharide appears as a network of strands wiring cells together and to the substrate. Blocking techniques with safranin O work to strengthen the interpretation of the SEM images by better visualization of the extracellular matrix material.

Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100‐bp insertion in helix 6

Two different versions of the 16S rRNA gene, one of which contained an unusual 100-bp insertion in helix 6, were detected in isolate UFO1 acquired from the Oak Ridge Integrated Field-Research Challenge (ORIFRC) site in Tennessee. rRNA was extracted from UFO1 and analyzed by reverse transcriptase-quantitative PCR with insert- and non-insert-specific primers; only the noninsert 16S rRNA gene sequence was detected. Similarly, PCR-based screening of a cDNA library (190 clones) constructed from reverse-transcribed rRNA from UFO1 did not detect any clones containing the 100-bp insert. Examination of cDNA with primers specific to the insert-bearing 16S rRNA gene, but downstream of the insert, suggests that the insert was excised from rRNA. Inspection of other 16S rRNA genes in the GenBank database revealed that a homologous insert sequence, also found in helix 6, has been reported in other environmental clones, including those acquired from ORIFRC enrichments. These findings demonstrate the existence of widely divergent copies of the 16S rRNA gene within the same organism, which may confound 16S rRNA gene-based methods of estimating microbial diversity in environmental samples.

In situ measurement of Fe(III) reduction activity of Geobacter pelophilus by simultaneous in situ RT-PCR and XPS analysis

Geobacter pelophilus is capable of dissimilatory Fe(III)-reduction on solid phase Fe(III)-oxides by means of surface attachment and direct electron transport to Fe(III), in part mediated by outer membrane c-type cytochromes. A study was undertaken to characterize surface colonization patterns, gene expression, and mineral transformation by this organism. The gene ferA (Geobacter sulfurreducens outer membrane Fe(III) reductase cytochrome c) was used as a target for PCR based molecular detection methods for visualizing G. pelophilus surface colonization. Protein extracts were prepared from solid-phase cultures, and cytochrome c content assessed. Mineral transformations were followed by X-ray photoelectron spectroscopy (XPS). Results of in situ (IS) RT-PCR experiments demonstrate that G. pelophilus attaches and grows at ferrihydrite mineral surfaces. Fluorescently-labeled cells were observed after IS-RT-PCR experiments, suggesting that G. pelophilus contains a cytochrome c sequence similar to ferA in G. sulfurreducens which is expressed in the presence of ferrihydrite. Protein extracts possessed high mass c-type cytochromes of similar size to those found in G. sulfurreducens. In addition, unique high-mass c-type cytochromes were also detected. XPS analysis demonstrated mineral transformation to occur, mediated by the surface associated population. This study demonstrates that G. pelophilus attaches to Fe(III)-oxide surfaces, reduces the Fe(III) oxides at the surface, produces c-type cytochromes under these growth conditions, and expresses cytochrome c-encoding genes as measured by in situ molecular detection techniques.

Teaching Cellular Respiration & Alternate Energy Sources with a Laboratory Exercise Developed by a Scientist-Teacher Partnership

[ILLUSTRATION OMITTED] Students often resort to memorization and recall when learning about cellular respiration. The concepts of glycolysis, Krebs cycle, and the electron transfer chain are abstract with multiple steps that are difficult to follow. The electron transport chain is the major workhorse for creating ATP in living organisms, and yet there are very few ways to clearly illustrate the electron transport chain in the laboratory. The above comment started a conversation between a high school biology teacher and scientists from the local university who were participants in a National Science Foundation (NSF)-funded teacher-scientist partnership program. This conversation led to a collaboration that developed this laboratory exercise demonstrating cellular respiration. Cellular respiration is the process of obtaining biochemical energy (stored as ATP) from fuel molecules (sugars). There are three major reactions that occur in cellular respiration: glycolysis, the Krebs cycle, and the electron transport chain (ETC). The ETC is the final step in cellular respiration and produces the most ATE In eukaryotes, the ETC is on the mitochondrial membrane; however, prokaryotes do not have a mitochondria and thus the ETC is on the plasma membrane. In addition, eukaryotes are only capable of respiring on oxygen (glucose + [O.sub.2] [right arrow] C[O.sub.2] + [H.sub.2]O), called aerobic respiration. When oxygen is not present, eukaryotes can perform the less efficient fermentation reactions. Fermentation produces less ATP than aerobic respiration because it does not use the Krebs cycle and the ETC. However, in the absence of oxygen, prokaryotes have the ability to ferment as well as use the ETC (anaerobic respiration). For example, some bacteria are able to respire on solid phase iron (glucose + [Fe.sup.+3] [right arrow] C[O.sub.2] + [Fe.sup.+2]). Respiration on multiple elements gives microbes an advantage in harsh environments where oxygen is not present. In addition, microbial respiration on solid phase compounds can be exploited to produce electricity. Microbial fuel ceils are a current research area that harvests electricity from bacteria capable of anaerobic respiration (Holmes et al., 2004; Liu et al., 2004; Logan et al., 2005). Graphite is an electrically conductive material that bacteria can respire on, thus it can be used to capture electrons from bacteria. When bacteria transfer electrons to graphite, an electrical potential is created that can produce electricity when in a circuit. A sediment battery is a simple circuit that uses graphite and anaerobic bacteria naturally found in dirt. The electrical potential produced by bacterial respiration on the graphite can be measured on a voltmeter and thus can be used as a visual aid for teaching cellular respiration. The combination of the need for a new learning tool and the expertise of the scientists led to the development of the laboratory exercise described here. It uses student-designed sediment batteries to better visualize and measure electron transfer in living cells. This exercise satisfies National Science Education Teaching Standards A and B, and Content Standards A, B, and C. * Background Chemical Batteries A battery uses chemicals to produce electrons. One common battery is a zinc/carbon battery, which has two terminals: a positive (cathode) and negative (anode). At the negative terminal, a zinc rod is placed in sulfuric acid. The sulfuric acid dissolves the zinc rod at the surface. A zinc atom will leave the rod as a [Zn.sup.+2] ion leaving two electrons on the rod; thus electrons are built up at the anode. When the battery is incorporated into a circuit, the electrons are allowed to travel from the anode to the cathode. In the cathode, the electrons travel through the carbon into sulfuric acid to produce hydrogen gas. The production and movement of electrons in a battery can power a device. …