Perchlorate-Coupled Carbon Monoxide (CO) Oxidation by Moorella glycerini, an Obligately Anaerobic, Thermophilic, Nickel-Dependent Carboxydotroph.

Many facultative and obligate anaerobes reduce perchlorate. Likewise, carbon monoxide (CO) oxidation has been documented in many aerobes, facultative anaerobes, and obligate anaerobes. A molybdenum-dependent CO dehydrogenase (Mo-CODH) and a nickel-dependent CO dehydrogenase (Ni-CODH) distinguish the former from the latter. Some Mo-dependent CO oxidizers (Mo-COX) couple CO oxidation to perchlorate reduction, but only at low concentrations of both under conditions that do not support growth in cultures. In contrast, CO-coupled perchlorate reduction has not been documented in Ni-dependent CO oxidizers (Ni-COX). To assess the potential for Ni-COX to reduce perchlorate, a model, obligately anaerobic homoacetogen, Moorella glycerini DSM 11254T, was cultivated with or without perchlorate, usiing CO or glycerol as its sole carbon and energy source. It grew with glycerol with or without perchlorate, and its maximum cell densities were only weakly affected by the perchlorate. However, when CO (at a 30% headspace concentration) was used as a carbon and energy source, perchlorate reduction supported greater cell densities and more rapid growth rates. The stoichiometry of CO uptake, perchlorate reduction, and chloride production were consistent with the cryptic pathway for perchlorate reduction with chlorite as an end product. Chloride production occurred abiologically in the medium due to a reaction between chlorite and the sulfide used as a reducing agent. These results provide the first demonstration of CO-coupled perchlorate reduction supporting growth in Ni-COX, and they provide constraints on the potential for perchlorate-coupled, anaerobic CO oxidation in engineered systems as well as terrestrial systems and hypothetical, sub-surface, serpentinite-hosted systems on Mars.

The conformations and basal conformational dynamics of translocation factor SecDF vary with translocon SecYEG interaction.

The general secretory, or Sec, system is a primary protein export pathway from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances polypeptide secretion, which is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. SecDF is thought to utilize a proton gradient to effectively pull precursor proteins from the cytoplasm into the periplasm. Working models have been developed to describe the structure and function of SecDF, but important mechanistic questions remain unanswered. Atomic force microscopy (AFM) is a powerful technique for studying the dynamics of single-molecule systems including membrane proteins in near-native conditions. The sharp tip of the AFM provides direct access to membrane-external protein conformations. Here, we acquired AFM images and kymographs (∼100 ms resolution) to visualize SecDF protrusions in near-native supported lipid bilayers and compared the experimental data to simulated AFM images based on static structures. When studied in isolation, SecDF exhibited a stable and compact conformation close to the lipid bilayer surface, indicative of a resting state. Interestingly, upon SecYEG introduction, we observed changes in both SecDF conformation and conformational dynamics. The population of periplasmic protrusions corresponding to an intermediate form of SecDF, which is thought to be active in precursor protein handling, increased more than ninefold. In conjunction, our dynamics measurements revealed an enhancement in the transition rate between distinct SecDF conformations when the translocon was present. Together, this work provides a novel vista of basal-level SecDF conformational dynamics in near-native conditions.

No Guts About It: Captivity, But Not Neophobia Phenotype, Influences the Cloacal Microbiome of House Sparrows (Passer domesticus).

Behavioral traits such as anxiety and depression have been linked to diversity of the gut microbiome in humans, domesticated animals, and lab-bred model species, but the extent to which this link exists in wild animals, and thus its ecological relevance, is poorly understood. We examined the relationship between a behavioral trait (neophobia) and the cloacal microbiome in wild house sparrows (Passer domesticus, n = 22) to determine whether gut microbial diversity is related to personality in a wild animal. We swabbed the cloaca immediately upon capture, assessed neophobia phenotypes in the lab, and then swabbed the cloaca again after several weeks in captivity to additionally test whether the microbiome of different personality types is affected disparately by captivity, and characterized gut microbiomes using 16S rRNA gene amplicon sequencing. We did not detect differences in cloacal alpha or beta microbial diversity between neophobic and non-neophobic house sparrows, and diversity for both phenotypes was negatively impacted by captivity. Although our results suggest that the adult cloacal microbiome and neophobia are not strongly linked in wild sparrows, we did detect specific OTUs that appeared more frequently and at higher abundances in neophobic sparrows, suggesting that links between the gut microbiome and behavior may occur at the level of specific taxa. Further investigations of personality and the gut microbiome are needed in more wild species to reveal how the microbiome-gut-brain axis and behavior interact in an ecological context.

Atomic force microscopy for quantitative understanding of peptide-induced lipid bilayer remodeling.

A number of peptides are known to bind lipid bilayer membranes and cause these natural barriers to leak in an uncontrolled manner. Though membrane permeabilizing peptides play critical roles in cellular activity and may have promising future applications in the therapeutic arena, significant questions remain about their mechanisms of action. The atomic force microscope (AFM) is a single molecule imaging tool capable of addressing lipid bilayers in near-native fluid conditions. The apparatus complements traditional assays by providing local topographic maps of bilayer remodeling induced by membrane permeabilizing peptides. The information garnered from the AFM includes direct visualization and statistical analyses of distinct bilayer remodeling modes such as highly localized pore-like voids in the bilayer and dispersed thinned membrane regions. Colocalization of distinct remodeling modes can be studied. Here we examine recent work in the field and outline methods used to achieve precise AFM image data. Experimental challenges and common pitfalls are discussed as well as techniques for unbiased analysis including the Hessian blob detection algorithm, bootstrapping, and the Bayesian information criterion. When coupled with robust statistical analyses, high precision AFM data is poised to advance understanding of an important family of peptides that cause poration of membrane bilayers.

Halobacterium bonnevillei sp. nov., Halobaculum saliterrae sp. nov. and Halovenus carboxidivorans sp. nov., three novel carbon monoxide-oxidizing Halobacteria from saline crusts and soils.

Three novel carbon monoxide-oxidizing Halobacteria were isolated from Bonneville Salt Flats (Utah, USA) salt crusts and nearby saline soils. Phylogenetic analysis of 16S rRNA gene sequences revealed that strains PCN9T, WSA2T and WSH3T belong to the genera Halobacterium, Halobaculum and Halovenus, respectively. Strains PCN9T, WSA2T and WSH3T grew optimally at 40 °C (PCN9T) or 50 °C (WSA2T, WSH3T). NaCl optima were 3 M (PCN9T, WSA2T) or 4 M NaCl (WSH3T). Carbon monoxide was oxidized by all isolates, each of which contained a molybdenum-dependent CO dehydrogenase. G+C contents for the three respective isolates were 66.75, 67.62, and 63.97 mol% as derived from genome analyses. The closest phylogenetic relatives for PCN9T, WSA2T and WSH3T were Halobacterium noricense A1T, Halobaculum roseum D90T and Halovenus aranensis EB27T with 98.71, 98.19 and 95.95 % 16S rRNA gene sequence similarities, respectively. Genome comparisons of PCN9T with Halobacterium noricense A1T yielded an average nucleotide identity (ANI) of 82.0% and a digital DNA-DNA hybridization (dDDH) value of 25.7 %; comparisons of WSA2T with Halobaculum roseum D90T yielded ANI and dDDH values of 86.34 and 31.1 %, respectively. The ANI value for a comparison of WSH3T with Halovenus aranensis EB27T was 75.2 %. Physiological, biochemical, genetic and genomic characteristics of PCN9T, WSA2T and WSH3T differentiated them from their closest phylogenetic neighbours and indicated that they represent novel species for which the names Halobaculum bonnevillei, Halobaculum saliterrae and Halovenus carboxidivorans are proposed, respectively. The type strains are PCN9T (=JCM 32472=LMG 31022=ATCC TSD-126), WSA2T (=JCM 32473=ATCC TSD-127) and WSH3T (=JCM 32474=ATCC TSD-128).

The effects of anthracycline drugs on the conformational distribution of mouse P-glycoprotein explains their transport rate differences.

P-glycoprotein (Pgp) is an ATP-dependent efflux transporter and plays a major role in anti-cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti-cancer drug efflux are not well understood. Even subtle differences in the anti-cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, this study focused on two closely-related anthracycline drugs, daunorubicin (DNR), and doxorubicin (DOX), with mouse Pgp. While only differing by a single hydroxyl functional group, DNR has a 4 to 5-fold higher transport rate than DOX. They both non-competitively inhibited Pgp-mediated ATP hydrolysis below basal levels. The Km of Pgp-mediated ATP hydrolysis extracted from the kinetics curves was lower for DOX than DNR. However, the dissociation constants (KDs) for these drugs determined by fluorescence quenching were virtually identical. Acrylamide quenching of Pgp tryptophan fluorescence to probe the tertiary structure of Pgp suggested that DNR shifts Pgp to a "closed" conformation, while DOX shifts Pgp to an "intermediate" conformation. The effects of these drugs on the Pgp conformational distributions in a lipid bilayer were also examined by atomic force microscopy (AFM). Analysis of AFM images revealed that DNR and DOX cause distinct and significant shifts in the conformational distribution of Pgp. The results were combined to build a conformational distribution model for anthracycline transport by Pgp.

The conformation and dynamics of P-glycoprotein in a lipid bilayer investigated by atomic force microscopy.

The membrane-bound P-glycoprotein (Pgp) transporter plays a major role in human disease and drug disposition because of its ability to efflux a chemically diverse range of drugs through ATP hydrolysis and ligand-induced conformational changes. Deciphering these structural changes is key to understanding the molecular basis of transport and to developing molecules that can modulate efflux. Here, atomic force microscopy (AFM) is used to directly image individual Pgp transporter molecules in a lipid bilayer under physiological pH and ambient temperature. Analysis of the Pgp AFM images revealed "small" and "large" protrusions from the lipid bilayer with significant differences in protrusion height and volume. The geometry of these "small" and "large" protrusions correlated to the predicted extracellular (EC) and cytosolic (C) domains of the Pgp X-ray crystal structure, respectively. To assign these protrusions, simulated AFM images were produced from the Pgp X-ray crystal structures with membrane planes defined by three computational approaches, and a simulated 80 ŠAFM cantilever tip. The theoretical AFM images of the EC and C domains had similar heights and volumes to the "small" and "large" protrusions in the experimental AFM images, respectively. The assignment of the protrusions in the AFM images to the EC and C domains was confirmed by changes in protrusion volume by Pgp-specific antibodies. The Pgp domains showed a considerable degree of conformational dynamics in time resolved AFM images. With this information, a model of Pgp conformational dynamics in a lipid bilayer is proposed within the context of the known Pgp X-ray crystal structures.

Isolation and characterization of Acidobacterium ailaaui sp. nov., a novel member of Acidobacteria subdivision 1, from a geothermally heated Hawaiian microbial mat.

A novel member of Acidobacteria was isolated from a microbial mat growing on a geothermally heated dead tree trunk in Hawai'i Volcanoes National Park (HI, USA). The rod-shaped, Gram-negative capsulated cells of strain PMMR2T were non-motile and catalase and oxidase negative. Growth occurred aerobically from 15 to 55 °C (optimum, 40 °C) and at pH values from 4.5 to 7.0 (optimum, 6.5). A limited range of sugars and organic acids supported growth. However, results of a genomic analysis suggested that various polysaccharides might be hydrolysed as carbon sources, and evidence for pectin degradation was observed in liquid cultures. A genomic analysis also revealed genes for a Group 1f uptake hydrogenase; assays with liquid cultures confirmed hydrogen consumption, including uptake at sub-atmospheric concentrations. Nitrate was not dissimilated to nitrite. Major membrane fatty acids included iso-C15 : 0 and iso-C17 : 0. The G+C content was 57.2mol%. A comparative genome analysis revealed an average nucleotide identity of 72.2 % between PMMR2T and its nearest cultured phylogenetic neighbour, Acidobacterium capsulatum ATCC 51196T (=JCM 7670T); analysis of the 16S rRNA gene revealed a 96.8 % sequence identity with Acidobacterium capsulatum ATCC 51196T. These results and other phenotypic differences indicated that strain PMMR2T represents a novel species in the genus Acidobacterium, for which the name Acidobacterium ailaaui sp. nov. is proposed. The type strain, PMMR2T (=DSM 27394T=LMG 28340T), is the second formal addition to the genus Acidobacterium.

Isolation and characterization of extremely halophilic CO-oxidizing Euryarchaeota from hypersaline cinders, sediments and soils and description of a novel CO oxidizer, Haloferax namakaokahaiae Mke2.3T, sp. nov.

The phylogenetic affiliations of organisms responsible for aerobic CO oxidation in hypersaline soils and sediments were assessed using media containing 3.8 M NaCl. CO-oxidizing strains of the euryarchaeotes, Haloarcula, Halorubrum, Haloterrigena and Natronorubrum, were isolated from the Bonneville Salt Flats (UT) and Atacama Desert salterns (Chile). A halophilic euryarchaeote, Haloferax strain Mke2.3(T), was isolated from Hawai'i Island saline cinders. Haloferax strain Mke2.3(T) was most closely related to Haloferax larsenii JCM 13917(T) (97.0% 16S rRNA sequence identity). It grew with a limited range of substrates, and oxidized CO at a headspace concentration of 0.1%. However, it did not grow with CO as a sole carbon and energy source. Its ability to oxidize CO, its polar lipid composition, substrate utilization and numerous other traits distinguished it from H. larsenii JCM 13917(T), and supported designation of the novel isolate as Haloferax namakaokahaiae Mke2.3(T), sp. nov (= DSM 29988, = LMG 29162). CO oxidation was also documented for 'Natronorubrum thiooxidans' HG1 (Sorokin, Tourova and Muyzer 2005), N. bangense (Xu, Zhou and Tian 1999) and N. sulfidifaciens AD2(T) (Cui et al. 2007). Collectively, these results established a previously unsuspected capacity for extremely halophilic aerobic CO oxidation, and indicated that the trait might be widespread among the Halobacteriaceae, and occur in a wide range of hypersaline habitats.

Microbial responses to the Deepwater Horizon oil spill: from coastal wetlands to the deep sea.

The Deepwater Horizon oil spill in the northern Gulf of Mexico represents the largest marine accidental oil spill in history. It is distinguished from past spills in that it occurred at the greatest depth (1,500 m), the amount of hydrocarbon gas (mostly methane) lost was equivalent to the mass of crude oil released, and dispersants were used for the first time in the deep sea in an attempt to remediate the spill. The spill is also unique in that it has been characterized with an unprecedented level of resolution using next-generation sequencing technologies, especially for the ubiquitous hydrocarbon-degrading microbial communities that appeared largely to consume the gases and to degrade a significant fraction of the petroleum. Results have shown an unexpectedly rapid response of deep-sea Gammaproteobacteria to oil and gas and documented a distinct succession correlated with the control of the oil flow and well shut-in. Similar successional events, also involving Gammaproteobacteria, have been observed in nearshore systems as well.

Thermomicrobium carboxidum sp. nov., and Thermorudis peleae gen. nov., sp. nov., carbon monoxide-oxidizing bacteria isolated from geothermally heated biofilms.

Two thermophilic, Gram-stain-positive, rod-shaped, non-spore-forming bacteria (strains KI3(T) and KI4(T)) were isolated from geothermally heated biofilms growing on a tumulus in the Kilauea Iki pit crater on the flank of Kilauea Volcano (Hawai'i, USA). Strain KI3(T) grew over an examined temperature range of 50-70 °C (no growth at 80 °C) and a pH range of 6.0-9.0, with optimum growth at 70 °C and pH 7.0. Strain KI4(T) grew at temperatures of 55-70 °C and a pH range of 5.8-8.0, with optimum growth at 65 °C and pH 6.7-7.1. The DNA G+C contents of strains KI3(T) and KI4(T) were 66.0 and 60.7 mol%, respectively. The major fatty acid for both strains was 12-methyl C(18 : 0). Polar lipids in strain KI3(T) were dominated by glycolipids and phosphatidylinositol, while phosphatidylinositol and phosphoglycolipids dominated in strain KI4(T). Strain KI3(T) oxidized carbon monoxide [6.7±0.8 nmol CO h(-1) (mg protein)(-1)], but strain KI4(T) did not. 16S rRNA gene sequence analyses determined that the strains belong to the class Thermomicrobia, and that strains KI3(T) and KI4(T) are related most closely to Thermomicrobium roseum DSM 5159(T) (96.5 and 91.1% similarity, respectively). 16S rRNA gene sequence similarity between strain KI3(T) and strain KI4(T) was 91.4%. Phenotypic features and phylogenetic analyses supported the affiliation of strain KI3(T) to the genus Thermomicrobium, while results of chemotaxonomic, physiological and biochemical assays differentiated strains KI3(T) and KI4(T) from Thermomicrobium roseum. Strain KI3(T) ( = DSM 27067(T) = ATCC BAA-2535(T)) is thus considered to be the type strain of a novel species, for which the name Thermomicrobium carboxidum sp. nov. is proposed. Additionally, the characterization and phylogenetic position of strain KI4(T) showed that it represents a novel species of a new genus, for which the name Thermorudis peleae gen. nov., sp. nov. is proposed. The type strain of Thermorudis peleae is KI4(T) ( = DSM 27169(T) = ATCC BAA-2536(T)).

Description of Thermogemmatispora carboxidivorans sp. nov., a carbon-monoxide-oxidizing member of the class Ktedonobacteria isolated from a geothermally heated biofilm, and analysis of carbon monoxide oxidation by members of the class Ktedonobacteria.

A thermophilic, aerobic, Gram-stain-positive bacterium (strain PM5(T)), which formed mycelia of irregularly branched filaments and produced multiple exospores per cell, was isolated from a geothermally heated biofilm. Strain PM5(T) grew at 40-65 °C and pH 4.1-8.0, with optimal growth at 55 °C and pH 6.0. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain PM5(T) belonged to the class Ktedonobacteria, and was related most closely to Thermogemmatispora onikobensis ONI-1(T) (97.7 % similarity) and Thermogemmatispora foliorum ONI-5(T) (96.1 %). Morphological features and fatty acid profiles (major fatty acids: iso-C17 : 0, iso-C19 : 0 and 12,17-dimethyl C18 : 0) supported the affiliation of strain PM5(T) to the genus Thermogemmatispora. Strain PM5(T) oxidized carbon monoxide [CO; 10±1 nmol h(-1) (mg protein)(-1)], but did not grow with CO as a sole carbon and energy source. Results from analyses of related strains indicated that the capacity for CO uptake occurred commonly among the members of the class Ktedonobacteria; 13 of 14 strains tested consumed CO or harboured coxL genes that potentially enabled CO oxidation. The results of DNA-DNA hybridization and physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain PM5(T) from the two recognized species of the genus Thermogemmatispora. Strain PM5(T) differed from Thermogemmatispora onikobensis ONI-1(T) in its production of orange pigment, lower temperature optimum, hydrolysis of casein and starch, inability to grow with mannitol, xylose or rhamnose as sole carbon sources, and utilization of organic acids and amino acids. Strain PM5(T) is therefore considered to represent a novel species, for which the name Thermogemmatispora carboxidivorans sp. nov. is proposed. The type strain is PM5(T) ( = DSM 45816(T) = ATCC BAA-2534(T)).

Distribution and diversity of carbon monoxide-oxidizing bacteria and bulk bacterial communities across a succession gradient on a Hawaiian volcanic deposit.

Relatively little is known about the distribution and diversity of CO-oxidizing bacteria during succession on volcanic deposits even though they are among the primary colonists. We surveyed CO-oxidizing communities across a vegetation gradient on a 1959 cinder deposit using coxL (large subunit gene of carbon monoxide dehydrogenase) sequences. Sequences most closely related to a coxL sequence from Ktedonobacter racemifer, dominated unvegetated cinders, while Proteobacteria-like sequences dominated vegetated sites. The number of coxL operational taxonomic units (OTUs) increased threefold with increased vegetation, and correlated most strongly with the increased beta-Proteobacteria richness (r = 0.987). These compositional shifts were also reflected in overall bacterial community compositions as determined by 16S rRNA gene analysis. Notably, coxL OTU:16S rRNA OTU ratios increased with increased vegetation, indicating that CO oxidizers became a larger fraction of total bacterial richness during succession. Results from most probable number estimates and maximum potential CO uptake activity assays indicate that increased richness is paralleled by increased CO oxidizer abundance, which likely results from increased vegetation and organic carbon content. Collectively, results suggest that in contrast to patterns observed for plant succession, a versatile bacterial functional group that is important during early colonization and succession can remain important in later stages of succession, irrespective of dramatic environmental changes.

Quantification of burkholderia coxL genes in Hawaiian volcanic deposits.

Isolation of multiple carbon monoxide (CO)-oxidizing Burkholderia strains and detection by culture-independent approaches suggest that Burkholderia may be an important component of CO-oxidizing communities in Hawaiian volcanic deposits. The absolute and relative abundance of the bacteria in these communities remains unknown, however. In this study, a quantitative PCR (Q-PCR) approach has been developed to enumerate Burkholderia coxL genes (large subunit of carbon monoxide dehydrogenase). This represents the first attempt to enumerate coxL genes from CO oxidizers in environmental samples. coxL copy numbers have been determined for samples from three sites representing a vegetation gradient on a 1959 volcanic deposit that included unvegetated cinders (bare), edges of vegetated sites (edge), and sites within tree stands (canopy). Q-PCR has also been used to estimate copy numbers of Betaproteobacteria 16S rRNA gene copy numbers and total Bacteria 16S rRNA. coxL genes could not be detected in the bare site (detection limit, > or = 4.7 x 10(3) copies per reaction) but average 1.0 x 10(8) + or - 2.4 x 10(7) and 8.6 x 10(8) + or - 7.6 x 10(7) copies g(-1) (dry weight) in edge and canopy sites, respectively, which differ statistically (P = 0.0007). Average Burkholderia coxL gene copy numbers, expressed as a percentage of total Bacteria 16S rRNA gene copy numbers, are 6.2 and 0.7% for the edge and canopy sites, respectively. Although the percentage of Burkholderia coxL is lower in the canopy site, significantly greater gene copy numbers demonstrate that absolute abundance of coxL increases in vegetated sites and contributes to the expansion of CO oxidizer communities during biological succession on volcanic deposits.

Nitrate-dependent anaerobic carbon monoxide oxidation by aerobic CO-oxidizing bacteria.

Two dissimilatory nitrate-reducing (Burkholderia xenovorans LB400 and Xanthobacter sp. str. COX) and two denitrifying isolates (Stappia aggregata IAM 12614 and Bradyrhizobium sp. str. CPP), previously characterized as aerobic CO oxidizers, consumed CO at ecologically relevant levels (<100 ppm) under anaerobic conditions in the presence, but not absence, of nitrate. None of the isolates were able to grow anaerobically with CO as a carbon or energy source, however, and nitrate-dependent anaerobic CO oxidation was inhibited by headspace concentrations >100-1000 ppm. Surface soils collected from temperate, subtropical and tropical forests also oxidized CO under anaerobic conditions with no lag. The observed activity was 25-60% less than aerobic CO oxidation rates, and did not appear to depend on nitrate. Chloroform inhibited anaerobic but not aerobic activity, which suggested that acetogenic bacteria may have played a significant role in forest soil anaerobic CO uptake.

Diversity and structure of bacterial chemolithotrophic communities in pine forest and agroecosystem soils.

Obligate lithotrophs (e.g., ammonia oxidizers) and facultative lithotrophs (e.g., CO and hydrogen oxidizers) collectively comprise a phylogenetically diverse functional group that contributes significantly to carbon and nitrogen cycles in soils and plays important roles in trace gas dynamics (e.g., carbon monoxide and nitrous and nitric oxides) that affect tropospheric chemistry and radiative forcing. In spite of their diverse physiologies, facultative and obligate lithotrophs typically possess the Calvin-Benson-Bassham cycle enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisCO). In an effort designed to understand the structure of lithotrophic communities in soil, genomic DNA extracts from surface (0 to 2 cm) and subsurface (5 to 7 cm) soils have been obtained from two sites in a Georgia agroecosystem (peanut and cotton plots) and an unmanaged pine stand (>50 years old). The extracts have been used in PCR amplifications of the cbbL gene for the rubisCO large subunit protein. cbbL PCR products were cloned, sequenced, and subjected to phylogenetic and statistical analyses. Numerous novel lineages affiliated with the form IC clade (one of four form I rubisCO clades), which is typified by facultative lithotrophs, comprised lithotrophic communities from all soils. One of the form IC clone sequences clustered with a form IC clade of ammonia-oxidizing Nitrosospira. Distinct assemblages were obtained from each of the sites and from surface and subsurface soils. The results suggest that lithotrophic populations respond differentially to plant type and land use, perhaps forming characteristic associations. The paucity of clone sequences attributed to ammonia-oxidizing bacteria indicates that even though ammonia oxidation occurs in the various soils, the relevant populations are small compared to those of facultative lithotrophs.

Probing nanotube-nanopore interactions.

We demonstrate a new nanoscale system consisting of a nanotube threaded through a nanopore in aqueous solution. Its electrical and mechanical properties are sensitive to experimentally controllable conformational changes on sub-Angstrom length scales. Ionic current transport through a nanopore is significantly suppressed by the threading nanotube and the mechanical interactions between the nanotube and pore are accounted for by a folding geometry. The experiments provide first measurements of the longitudinal resolution and metrology of a solid-state nanopore "microscope." This new nanostructure provides a means to study molecule-nanotube interactions in conducting ionic solutions as well as geometrical and surface properties of nanopores and nanotubes.

Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase.

A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.

Isolation, characterization, and polyaromatic hydrocarbon degradation potential of aerobic bacteria from marine macrofaunal burrow sediments and description of Lutibacterium anuloederans gen. nov., sp. nov., and Cycloclasticus spirillensus sp. nov.

Two new polyaromatic hydrocarbon-degrading marine bacteria have been isolated from burrow wall sediments of benthic macrofauna by using enrichments on phenanthrene. Strain LC8 (from a polychaete) and strain M4-6 (from a mollusc) are aerobic and gram negative and require sodium chloride (>1%) for growth. Both strains can use 2- and 3-ring polycyclic aromatic hydrocarbons as their sole carbon and energy sources, but they are nutritionally versatile. Physiological and phylogenetic analyses based on 16S ribosomal DNA sequences suggest that strain M4-6 belongs to the genus Cycloclasticus and represents a new species, Cycloclasticus spirillensus sp. nov. Strain LC8 appears to represent a new genus and species, Lutibacterium anuloederans gen. nov., sp. nov., within the Sphingomonadaceae. However, when inoculated into sediment slurries with or without exogenous phenanthrene, only L. anuloederans appeared to sustain a significant phenanthrene uptake potential throughout a 35-day incubation. In addition, only L. anuloederans appeared to enhance phenanthrene degradation in heavily contaminated sediment from Little Mystic Cove, Boston Harbor, Boston, Mass.

Enrichment of high-affinity CO oxidizers in Maine forest soil.

Carboxydotrophic activity in forest soils was enriched by incubation in a flowthrough system with elevated concentrations of headspace CO (40 to 400 ppm). CO uptake increased substantially over time, while the apparent K(m) ((app)K(m)) for uptake remained similar to that of unenriched soils (<10 to 20 ppm). Carboxydotrophic activity was transferred to and further enriched in sterile sand and forest soil. The (app)K(m)s for secondary and tertiary enrichments remained similar to values for unenriched soils. CO uptake by enriched soil and freshly collected forest soil was inhibited at headspace CO concentrations greater than about 1%. A novel isolate, COX1, obtained from the enrichments was inhibited similarly. However, in contrast to extant carboxydotrophs, COX1 consumed CO with an (app)K(m) of about 15 ppm, a value comparable to that of fresh soils. Phylogenetic analysis based on approximately 1,200 bp of its 16S rRNA gene sequence suggested that the isolate is an alpha-proteobacterium most closely related to the genera Pseudaminobacter, Aminobacter, and Chelatobacter (98.1 to 98.3% sequence identity).