Carbon and phosphorus exchange rates in arbuscular mycorrhizas depend on environmental context and differ among co-occurring plants.

Phosphorus (P) for carbon (C) exchange is the pivotal function of arbuscular mycorrhiza (AM), but how this exchange varies with soil P availability and among co-occurring plants in complex communities is still largely unknown. We collected intact plant communities in two regions differing c. 10-fold in labile inorganic P. After a 2-month glasshouse incubation, we measured 32P transfer from AM fungi (AMF) to shoots and 13C transfer from shoots to AMF using an AMF-specific fatty acid. AMF communities were assessed using molecular methods. AMF delivered a larger proportion of total shoot P in communities from high-P soils despite similar 13C allocation to AMF in roots and soil. Within communities, 13C concentration in AMF was consistently higher in grass than in blanketflower (Gaillardia aristata Pursh) roots, that is P appeared more costly for grasses. This coincided with differences in AMF taxa composition and a trend of more vesicles (storage structures) but fewer arbuscules (exchange structures) in grass roots. Additionally, 32P-for-13C exchange ratios increased with soil P for blanketflower but not grasses. Contrary to predictions, AMF transferred proportionally more P to plants in communities from high-P soils. However, the 32P-for-13C exchange differed among co-occurring plants, suggesting differential regulation of the AM symbiosis.

High-resolution melt analysis for rapid comparison of bacterial community compositions.

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.

Bacterial endophytes enhance competition by invasive plants.

PREMISE OF THE STUDY: Invasive plants can alter soil microbial communities and profoundly alter ecosystem processes. In the invasive grass Sorghum halepense, these disruptions are consequences of rhizome-associated bacterial endophytes. We describe the effects of N2-fixing bacterial strains from S. halepense (Rout and Chrzanowski, 2009) on plant growth and show that bacteria interact with the plant to alter soil nutrient cycles, enabling persistence of the invasive. • METHODS: We assessed fluxes in soil nutrients for ∼4 yr across a site invaded by S. halepense. We assayed the N2-fixing bacteria in vitro for phosphate solubilization, iron chelation, and production of the plant-growth hormone indole-3-acetic acid (IAA). We assessed the plant's ability to recruit bacterial partners from substrates and vertically transmit endophytes to seeds and used an antibiotic approach to inhibit bacterial activity in planta and assess microbial contributions to plant growth. • KEY RESULTS: We found persistent alterations to eight biogeochemical cycles (including nitrogen, phosphorus, and iron) in soils invaded by S. halepense. In this context, three bacterial isolates solubilized phosphate, and all produced iron siderophores and IAA in vitro. In growth chamber experiments, bacteria were transmitted vertically, and molecular analysis of bacterial community fingerprints from rhizomes indicated that endophytes are also horizontally recruited. Inhibiting bacterial activity with antibiotics resulted in significant declines in plant growth rate and biomass, with pronounced rhizome reductions. • CONCLUSIONS: This work suggests a major role of endophytes on growth and resource allocation of an invasive plant. Indeed, bacterial isolate physiology is correlated with invader effects on biogeochemical cycles of nitrogen, phosphate, and iron.

Microbial degradation of 2,4-dichlorophenoxyacetic acid on the Greenland ice sheet.

The Greenland ice sheet (GrIS) receives organic carbon (OC) of anthropogenic origin, including pesticides, from the atmosphere and/or local sources, and the fate of these compounds in the ice is currently unknown. The ability of supraglacial heterotrophic microbes to mineralize different types of OC is likely a significant factor determining the fate of anthropogenic OC on the ice sheet. Here we determine the potential of the microbial community from the surface of the GrIS to mineralize the widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Surface ice cores were collected and incubated for up to 529 days in microcosms simulating in situ conditions. Mineralization of side chain- and ring-labeled [(14)C]2,4-D was measured in the samples, and quantitative PCR targeting the tfdA genes in total DNA extracted from the ice after the experiment was performed. We show that the supraglacial microbial community on the GrIS contains microbes that are capable of degrading 2,4-D and that they are likely present in very low numbers. They can mineralize 2,4-D at a rate of up to 1 nmol per m(2) per day, equivalent to ∼26 ng C m(-2) day(-1). Thus, the GrIS should not be considered a mere reservoir of all atmospheric contaminants, as it is likely that some deposited compounds will be removed from the system via biodegradation processes before their potential release due to the accelerated melting of the ice sheet.

E Unibus Plurum: genomic analysis of an experimentally evolved polymorphism in Escherichia coli.

Microbial populations founded by a single clone and propagated under resource limitation can become polymorphic. We sought to elucidate genetic mechanisms whereby a polymorphism evolved in Escherichia coli under glucose limitation and persisted because of cross-feeding among multiple adaptive clones. Apart from a 29 kb deletion in the dominant clone, no large-scale genomic changes distinguished evolved clones from their common ancestor. Using transcriptional profiling on co-evolved clones cultured separately under glucose-limitation we identified 180 genes significantly altered in expression relative to the common ancestor grown under similar conditions. Ninety of these were similarly expressed in all clones, and many of the genes affected (e.g., mglBAC, mglD, and lamB) are in operons coordinately regulated by CRP and/or rpoS. While the remaining significant expression differences were clone-specific, 93% were exhibited by the majority clone, many of which are controlled by global regulators, CRP and CpxR. When transcriptional profiling was performed on adaptive clones cultured together, many expression differences that distinguished the majority clone cultured in isolation were absent, suggesting that CpxR may be activated by overflow metabolites removed by cross-feeding strains in co-culture. Relative to their common ancestor, shared expression differences among adaptive clones were partly attributable to early-arising shared mutations in the trans-acting global regulator, rpoS, and the cis-acting regulator, mglO. Gene expression differences that distinguished clones may in part be explained by mutations in trans-acting regulators malT and glpK, and in cis-acting sequences of acs. In the founder, a cis-regulatory mutation in acs (acetyl CoA synthetase) and a structural mutation in glpR (glycerol-3-phosphate repressor) likely favored evolution of specialists that thrive on overflow metabolites. Later-arising mutations that led to specialization emphasize the importance of compensatory rather than gain-of-function mutations in this system. Taken together, these findings underscore the importance of regulatory change, founder genotype, and the biotic environment in the adaptive evolution of microbes.

Non-invasive monitoring of multiple wildlife health factors by fecal microbiome analysis.

Fecal microbial biomarkers represent a less invasive alternative for acquiring information on wildlife populations than many traditional sampling methodologies. Our goal was to evaluate linkages between fecal microbiome communities in Rocky Mountain elk (Cervus canadensis) and four host factors including sex, age, population, and physical condition (body-fat). We paired a feature-selection algorithm with an LDA-classifier trained on elk differential bacterial abundance (16S-rRNA amplicon survey) to predict host health factors from 104 elk microbiomes across four elk populations. We validated the accuracy of the various classifier predictions with leave-one-out cross-validation using known measurements. We demonstrate that the elk fecal microbiome can predict the four host factors tested. Our results show that elk microbiomes respond to both the strong extrinsic factor of biogeography and simultaneously occurring, but more subtle, intrinsic forces of individual body-fat, sex, and age-class. Thus, we have developed and described herein a generalizable approach to disentangle microbiome responses attributed to multiple host factors of varying strength from the same bacterial sequence data set. Wildlife conservation and management presents many challenges, but we demonstrate that non-invasive microbiome surveys from scat samples can provide alternative options for wildlife population monitoring. We believe that, with further validation, this method could be broadly applicable in other species and potentially predict other measurements. Our study can help guide the future development of microbiome-based monitoring of wildlife populations and supports hypothetical expectations found in host-microbiome theory.

(±)-catechin, a root exudate of the invasive centaurea stoebe lam. (Spotted knapweed) exhibits bacteriostatic activity against multiple soil bacterial populations.

Understanding the effects of allelopathic plant chemicals on soil microorganisms is critical to understanding their ecological roles and importance in exotic plant invasion. Centaurea stoebe Lam. (spotted knapweed), an aggressive invasive weed in North America, secretes a racemic mixture of (±)-catechin as a root exudate. This enantiomeric, polyphenolic compound has been reported to have allelopathic effects on surrounding flora and microflora. To better understand how catechin affects microbial communities in the root zone of spotted knapweed, we assessed its impact on the total culturable bacterial component and numerous individual bacterial populations from Romanian (native range) and Montana (invaded range) soils. Catechin suppressed total culturable count numbers from the bacterial community and inhibited growth of some, but not all, soil bacterial populations tested. The native soil bacterial community was significantly more resistant to inhibitory effects of catechin than either the invaded or non-invaded soils. We further show that the inhibitory effect of catechin on nine different soil bacterial strains from seven genera was reversible, demonstrating that it acts via a bacteriostatic rather than bactericidal mechanism. These findings suggest that catechin might affect bacterial community composition and activity in the root zone.

Soil biota and exotic plant invasion.

Invasive plants are an economic problem and a threat to the conservation of natural systems. Escape from natural enemies might contribute to successful invasion, with most work emphasizing the role of insect herbivores; however, microbial pathogens are attracting increased attention. Soil biota in some invaded ecosystems may promote 'exotic' invasion, and plant-soil feedback processes are also important. Thus, relatively rare species native to North America consistently demonstrate negative feedbacks with soil microbes that promote biological diversity, whereas abundant exotic and native species demonstrate positive feedbacks that reduce biological diversity. Here we report that soil microbes from the home range of the invasive exotic plant Centaurea maculosa L. have stronger inhibitory effects on its growth than soil microbes from where the weed has invaded in North America. Centaurea and soil microbes participate in different plant-soil feedback processes at home compared with outside Centaurea's home range. In native European soils, Centaurea cultivates soil biota with increasingly negative effects on the weed's growth, possibly leading to its control. But in soils from North America, Centaurea cultivates soil biota with increasingly positive effects on itself, which may contribute to the success of this exotic species in North America.

Empirical testing of 16S rRNA gene PCR primer pairs reveals variance in target specificity and efficacy not suggested by in silico analysis.

Phylogenetic and "fingerprinting" analyses of the 16S rRNA genes of prokaryotes have been a mainstay of microbial ecology during the last two decades. However, many methods and results from studies that rely on the 16S rRNA gene for detection and quantification of specific microbial taxa have seemingly received only cursory or even no validation. To directly examine the efficacy and specificity of 16S rRNA gene-based primers for phylum-, class-, and operational taxonomic unit-specific target amplification in quantitative PCR, we created a collection of primers based solely on an extensive soil bacterial 16S rRNA gene clone library containing approximately 5,000 sequences from a single soil sample (i.e., a closed site-specific library was used to create PCR primers for use at this site). These primers were initially tested in silico prior to empirical testing by PCR amplification of known target sequences and of controls based on disparate phylogenetic groups. Although all primers were highly specific according to the in silico analysis, the empirical analyses clearly exhibited a high degree of nonspecificity for many of the phyla or classes, while other primers proved to be highly specific. These findings suggest that significant care must be taken when interpreting studies whose results were obtained with target specific primers that were not adequately validated, especially where population densities or dynamics have been inferred from the data. Further, we suggest that the reliability of quantification of specific target abundance using 16S rRNA-based quantitative PCR is case specific and must be determined through rigorous empirical testing rather than solely in silico.

Extensive phylogenetic analysis of a soil bacterial community illustrates extreme taxon evenness and the effects of amplicon length, degree of coverage, and DNA fractionation on classification and ecological parameters.

To thoroughly investigate the bacterial community diversity present in a single composite sample from an agricultural soil and to examine potential biases resulting from data acquisition and analytical approaches, we examined the effects of percent G+C DNA fractionation, sequence length, and degree of coverage of bacterial diversity on several commonly used ecological parameters (species estimation, diversity indices, and evenness). We also examined variation in phylogenetic placement based on multiple commonly used approaches (ARB alignments and multiple RDP tools). The results demonstrate that this soil bacterial community is highly diverse, with 1,714 operational taxonomic units demonstrated and 3,555 estimated (based on the Chao1 richness estimation) at 97% sequence similarity using the 16S rRNA gene. The results also demonstrate a fundamental lack of dominance (i.e., a high degree of evenness), with 82% of phylotypes being encountered three times or less. The data also indicate that generally accepted cutoff values for phylum-level taxonomic classification might not be as applicable or as general as previously assumed and that such values likely vary between prokaryotic phyla or groups.

Habitat heterogeneity and associated microbial community structure in a small-scale floodplain hyporheic flow path.

The Nyack floodplain is located on the Middle Fork of the Flathead River, an unregulated, pristine, fifth-order stream in Montana, USA, bordering Glacier National Park. The hyporheic zone is a nutritionally heterogeneous floodplain component harboring a diverse array of microbial assemblages essential in fluvial biogeochemical cycling, riverine ecosystem productivity, and trophic interactions. Despite these functions, microbial community structure in pristine hyporheic systems is not well characterized. The current study was designed to assess whether physical habitat heterogeneity within the hyporheic zone of the Nyack floodplain was sufficient to drive bacterial beta diversity between three different hyporheic flow path locations. Habitat heterogeneity was assessed by measuring soluble reactive phosphorous, nitrate, dissolved organic carbon, dissolved oxygen, and soluble total nitrogen levels seasonally at surface water infiltration, advection, and exfiltration zones. Significant spatial differences were detected in dissolved oxygen and nitrate levels, and seasonal differences were detected in dissolved oxygen, nitrate, and dissolved organic carbon levels. Denaturing gradient gel electrophoresis (DGGE) and cell counts indicated that bacterial diversity increased with abundance, and DGGE fingerprints covaried with nitrate levels where water infiltrated the hyporheic zone. The ribosomal gene phylogeny revealed that hyporheic habitat heterogeneity was sufficient to drive beta diversity between bacterial assemblages. Phylogenetic (P) tests detected sequence disparity between the flow path locations. Small distinct lineages of Firmicutes, Actinomycetes, Planctomycetes, and Acidobacteria defined the infiltration zone and alpha- and beta-proteobacterial lineages delineated the exfiltration and advection zone communities. These data suggest that spatial habitat heterogeneity drives hyporheic microbial community development and that attempts to understand functional differences between bacteria inhabiting nutritionally heterogeneous hyporheic environments might begin by focusing on the biology of these taxa.

Tri-trophic linkages in disease: pathogen transmission to rainbow trout through stonefly prey.

Relationships between macroinvertebrates and microorganisms in aquatic environments are only poorly understood despite the fact that many aquatic macroinvertebrates feed on microbial biofilms during some life stage. Better understanding of trophic interactions between microbial biofilms, macroinvertebrates, and fish may also help control fish diseases and loss of natural resources. It has also been suggested that pollution, habitat fragmentation, and poor water quality may contribute to increased pathogenesis and mortality in fish. Increased disease incidence is difficult to assess, however, in part because of the complexity of pathogen transmission dynamics. Several environmental pathogens exist whose reservoir(s) and means of transmission remain poorly understood, highlighting the need to study pathogen ecology and interactions with organisms other than susceptible hosts. Aeromonas salmonicida is rarely isolated from freshwater sediments. However, stonefly nymphs were found to frequently harbor A. salmonicida and were shown to preferentially feed on the bacterium. Rainbow trout juveniles were presented with different feeding regimes to determine the transmission capacity of nymphs, and all fish fed stoneflies harboring A. salmonicida expressed symptoms of disease. Although current rates of furunculosis in freshwater ecosystems are unknown, trout primarily feed on stoneflies when water oxygen levels are high and temperatures are low (winter months), which is presumed to correspond to high resistance to the pathogen. Given that furunculosis is associated with physiological stress and higher water temperatures, its natural incidence may change in response to global or regional climatological effects.

Ecology of sulfate-reducing bacteria in an iron-dominated, mining-impacted freshwater sediment.

A legacy of lead and silver mining in its headwaters left Lake Coeur d'Alene, Idaho with a sediment body that is highly reduced and contains up to 100 g kg(-1) iron and a smaller fraction of chemically active sulfide phases. The dynamic character of these sulfides and their importance for the sequestering of contaminating trace elements prompted this study of the sulfate-reducing bacteria (SRB) involved in their production. We estimated parameters indicative of the distribution and activity of SRB in relation to season, site, and depth. Most probable number estimates and quantitative PCR assays of an SRB-specific functional gene, alpha-adenosine 5'-phosphosulfate reductase, indicated 10(3) to 10(6) cultivable cells and 10(5) to 10(7) gene copy numbers g(-1) dry wt sediment, respectively. Although culture-based estimates of SRB abundance correlated poorly with site, season, depth, total S, or pore water SO(4), non-culture-based estimates of SRB abundance were markedly higher at contaminated sites and positively correlated with pore water SO(4). Ex situ estimates of (35)SO(4) respiration and acid volatile sulfides abundance also showed strong among-site effects, indicating elevated sulfidogenesis at contaminated sites. These observations support the view that biogenic sulfides may act in concert with reduced iron to retain soluble metal(loid)s in the solid phase.

Supplemental programs for enhanced recovery of data from the DOTUR application.

In order to retrieve phylogenetic information from distance matrices generated from large-scale clone libraries, and to explore OTU distribution among them, we have developed downstream applications for use with the already available DOTUR program. These programs enhance and ease data extraction, providing phylogeny to the already generated distance data.

Inter-laboratory testing of the effect of DNA blocking reagent G2 on DNA extraction from low-biomass clay samples.

Here we show that a commercial blocking reagent (G2) based on modified eukaryotic DNA significantly improved DNA extraction efficiency. We subjected G2 to an inter-laboratory testing, where DNA was extracted from the same clay subsoil using the same batch of kits. The inter-laboratory extraction campaign revealed large variation among the participating laboratories, but the reagent increased the number of PCR-amplified16S rRNA genes recovered from biomass naturally present in the soils by one log unit. An extensive sequencing approach demonstrated that the blocking reagent was free of contaminating DNA, and may therefore also be used in metagenomics studies that require direct sequencing.

Use of a PNA probe to block DNA-mediated PCR product formation in prokaryotic RT-PCR.

A novel method eliminating DNA-mediated PCR product formation in reverse transcription PCR (RT-PCR) amplification of specific RNA sequences is described. The method exploits the higher melting temperature values of peptide nucleic acid (PNA)/DNA duplexes compared with DNA/DNA duplexes by binding a sequence-specific PNA probe to a genomic sequence immediately overlapping one of the PCR-primer attachment sites within the sequence of interest. Hybridization of the blocking probe precludes primer attachment to DNA without affecting attachment of the same primer to the reverse transcription-generated cDNA sequence. A four-step PCR cycle is used that allows the PNA probe to hybridize to the DNA strand at a higher temperature just prior to the primer annealing step.

Quantification of mRNA in Salmonella sp. seeded soil and chicken manure using magnetic capture hybridization RT-PCR.

Direct quantification of mRNA from Salmonella sp. seeded for 1 h to soil and chicken manure was accomplished using magnetic capture hybridization as a purification technique. This detection strategy targeted the invA gene present in Salmonella sp. After cell lysis, phenol/chloroform purification and isopropanol precipitation, the RNA extract was combined with the hybridization probe conjugated to paramagnetic beads. After hybridization, the captured nucleic acids were released by denaturation and purified of contaminating DNA using DNase. The resulting RNA was of high purity and there was no need for dilution of the samples prior to RT-PCR. The developed procedure was reproducibly used to quantify Salmonella sp. in high organic agricultural soil. The detection limit for mRNA using ordinary quantitative PCR (employing SYBRgreen-based detection) was 5 x 10(4)Salmonella sp. cells per gram of soil. Chicken manure amended into soil (1:4 w/w) did not reduce the ability to quantify Salmonella sp. mRNA in soil. Pasteurization (65 degrees C, 30 min) of chicken manure containing Salmonella sp. dramatically reduced the detection of invA mRNA (requiring 42 qPCR cycles for detection versus 26 cycles in unpasteurized manure), presumably due to degradation of the invA mRNA in Salmonella sp. cells killed by pasteurization. By contrast, DNA-based qPCR still detected Salmonella sp. in the pasteurized manure. Thus, in this case using samples seeded with fresh Salmonella sp. the mRNA-based detection appears to be superior to minimizing false-positive detection which was prevalent with DNA-based qPCR.

DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community.

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 x 10 cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1-mug DNA sample.

Functional response of a near-surface soil microbial community to a simulated underground CO2 storage leak.

Understanding the impacts of leaks from geologic carbon sequestration, also known as carbon capture and storage, is key to developing effective strategies for carbon dioxide (CO2) emissions management and mitigation of potential negative effects. Here, we provide the first report on the potential effects of leaks from carbon capture and storage sites on microbial functional groups in surface and near-surface soils. Using a simulated subsurface CO2 storage leak scenario, we demonstrate how CO2 flow upward through the soil column altered both the abundance (DNA) and activity (mRNA) of microbial functional groups mediating carbon and nitrogen transformations. These microbial responses were found to be seasonally dependent and correlated to shifts in atmospheric conditions. While both DNA and mRNA levels were affected by elevated CO2, they did not react equally, suggesting two separate mechanisms for soil microbial community response to high CO2 levels. The results did not always agree with previous studies on elevated atmospheric (rather than subsurface) CO2 using FACE (Free-Air CO2 Enrichment) systems, suggesting that microbial community response to CO2 seepage from the subsurface might differ from its response to atmospheric CO2 increases.

Yersinia enterocolitica: an unlikely cause of positive brucellosis tests in greater yellowstone ecosystem bison (Bison bison).

Yersinia enterocolitica serotype O:9 has identical O-antigens to those of Brucella abortus and has apparently caused false-positive reactions in numerous brucellosis serologic tests in elk (Cervus canadensis) from southwest Montana. We investigated whether a similar phenomenon was occurring in brucellosis antibody-positive bison (Bison bison) using Y. enterocolitica culturing techniques and multiplex PCR of four diagnostic loci. Feces from 53 Yellowstone bison culled from the population and 113 free-roaming bison from throughout the Greater Yellowstone Ecosystem (GYE) were tested. Yersinia enterocolitica O:9 was not detected in any of 53 the bison samples collected at slaughter facilities or in any of the 113 fecal samples from free-ranging bison. One other Y. enterocolitica serotype was isolated; however, it is not known to cause cross-reaction on B. abortus serologic assays because it lacks the perosamine synthetase gene and thus the O-antigens. These findings suggest that Y. enterocolitica O:9 cross-reactivity with B. abortus antigens is unlikely to have been a cause of false-positive serology tests in GYE bison and that Y. enterocolitica prevalence was low in bison in the GYE during this study.