Soil microbial communities influencing organic phosphorus mineralization in a coastal dune chronosequence in New Zealand.

The Haast chronosequence in New Zealand is an ∼6500-year dune formation series, characterized by rapid podzol development, phosphorus (P) depletion and a decline in aboveground biomass. We examined bacterial and fungal community composition within mineral soil fractions using amplicon-based high-throughput sequencing (Illumina MiSeq). We targeted bacterial non-specific acid (class A, phoN/phoC) and alkaline (phoD) phosphomonoesterase genes and quantified specific genes and transcripts using real-time PCR. Soil bacterial diversity was greatest after 4000 years of ecosystem development and associated with an increased richness of phylotypes and a significant decline in previously dominant taxa (Firmicutes and Proteobacteria). Soil fungal communities transitioned from predominantly Basidiomycota to Ascomycota along the chronosequence and were most diverse in 290- to 392-year-old soils, coinciding with maximum tree basal area and organic P accumulation. The Bacteria:Fungi ratio decreased amid a competitive and interconnected soil community as determined by network analysis. Overall, soil microbial communities were associated with soil changes and declining P throughout pedogenesis and ecosystem succession. We identified an increased dependence on organic P mineralization, as found by the profiled acid phosphatase genes, soil acid phosphatase activity and function inference from predicted metagenomes (PICRUSt2).

Metagenomic Comparison of Antibiotic Resistance Genes Associated with Liquid and Dewatered Biosolids.

Municipal biosolids (MBs) that are land-applied in North America are known to possess an active microbial population that can include human pathogens. Activated sludge is a hotspot for the accumulation of antibiotics and has been shown to be a selective environment for microorganisms that contain antibiotic resistance genes (ARGs); however, the prevalence of ARGs in MBs is not well characterized. In this study, we enriched the plasmid metagenome from raw sewage sludge and two CP2 MBs, a mesophilic anaerobic digestate and a dewatered digestate, to evaluate the presence of ARGs in mobile genetic elements. The CP2-class biosolids are similar to Class B biosolids in the United States. The CP2 biosolids must meet a microbiological cut off of 2 × 10 colony-forming units (CFU) per dry gram or 100 mL of biosolids. The enriched plasmid DNA was sequenced (Illumina MiSeq). Sequence matching against databases, including the Comprehensive Antibiotic Resistance Database (CARD), MG-RAST, and INTEGRALL, identified potential genes of interest related to ARGs and their ability to transfer. The presence and abundance of different ARGs varied between treatments with heterogeneity observed among the same sample types. The MBs plasmid-enriched metagenomes contained ARGs associated with resistance to a variety of antibiotics, including ß-lactams, rifampicin, quinolone, and tetracycline as well as the detection of extended spectrum ß-lactamase genes. Cultured bacteria from CP2 MBs possessed antibiotic resistances consistent with the MBs metagenome data including multiantibiotic-resistant isolates. The results from this study provide a better understanding of the ARG and MGE profile of the plasmid-enriched metagenome of CP2 MBs.

The role of coral-associated bacterial communities in Australian Subtropical White Syndrome of Turbinaria mesenterina.

Australian Subtropical White Syndrome (ASWS) is an infectious, temperature dependent disease of the subtropical coral Turbinaria mesenterina involving a hitherto unknown transmissible causative agent. This report describes significant changes in the coral associated bacterial community as the disease progresses from the apparently healthy tissue of ASWS affected coral colonies, to areas of the colony affected by ASWS lesions, to the dead coral skeleton exposed by ASWS. In an effort to better understand the potential roles of bacteria in the formation of disease lesions, the effect of antibacterials on the rate of lesion progression was tested, and both culture based and culture independent techniques were used to investigate the bacterial communities associated with colonies of T. mesenterina. Culture-independent analysis was performed using the Oligonucleotide Fingerprinting of Ribosomal Genes (OFRG) technique, which allowed a library of 8094 cloned bacterial 16S ribosomal genes to be analysed. Interestingly, the bacterial communities associated with both healthy and disease affected corals were very diverse and ASWS associated communities were not characterized by a single dominant organism. Treatment with antibacterials had a significant effect on the rate of progress of disease lesions (p = 0.006), suggesting that bacteria may play direct roles as the causative agents of ASWS. A number of potential aetiological agents of ASWS were identified in both the culture-based and culture-independent studies. In the culture-independent study an Alphaproteobacterium closely related to Roseovarius crassostreae, the apparent aetiological agent of juvenile oyster disease, was found to be significantly associated with disease lesions. In the culture-based study Vibrio harveyi was consistently associated with ASWS affected coral colonies and was not isolated from any healthy colonies. The differing results of the culture based and culture-independent studies highlight the importance of using both approaches in the investigation of microbial communities.

Improving oligonucleotide fingerprinting of rRNA genes by implementation of polony microarray technology.

Improvements to oligonucleotide fingerprinting of rRNA genes (OFRG) were obtained by implementing polony microarray technology. OFRG is an array-based method for analyzing microbial community composition. Polonies are discrete clusters of DNA, produced by solid-phase PCR in hydrogels, and derived from individual, spatially isolated DNA molecules. The advantages of a polony-based OFRG method include higher throughput and reductions in the PCR-induced errors and compositional skew inherent in all other PCR-based community composition methods, including high-throughput sequencing of rRNA genes. Given the similarities between polony microarrays and certain aspects of sequencing methods such as the Illumina platform, we suggest that if concepts presented in this study were implemented in high-throughput sequencing protocols, a reduction of PCR-induced errors and compositional skew may be realized.

Root-associated ectomycorrhizal fungi shared by various boreal forest seedlings naturally regenerating after a fire in interior alaska and correlation of different fungi with host growth responses.

The role of common mycorrhizal networks (CMNs) in postfire boreal forest successional trajectories is unknown. We investigated this issue by sampling a 50-m by 40-m area of naturally regenerating black spruce (Picea mariana), trembling aspen (Populus tremuloides), and paper birch (Betula papyrifera) seedlings at various distances from alder (Alnus viridis subsp. crispa), a nitrogen-fixing shrub, 5 years after wildfire in an Alaskan interior boreal forest. Shoot biomasses and stem diameters of 4-year-old seedlings were recorded, and the fungal community associated with ectomycorrhizal (ECM) root tips from each seedling was profiled using molecular techniques. We found distinct assemblages of fungi associated with alder compared with those associated with the other tree species, making the formation of CMNs between them unlikely. However, among the spruce, aspen, and birch seedlings, there were many shared fungi (including members of the Pezoloma ericae [Hymenoscyphus ericae] species aggregate, Thelephora terrestris, and Russula spp.), raising the possibility that these regenerating seedlings may form interspecies CMNs. Distance between samples did not influence how similar ECM root tip-associated fungal communities were, and of the fungal groups identified, only one of them was more likely to be shared between seedlings that were closer together, suggesting that the majority of fungi surveyed did not have a clumped distribution across the small scale of this study. The presence of some fungal ribotypes was associated with larger or smaller seedlings, suggesting that these fungi may play a role in the promotion or inhibition of seedling growth. The fungal ribotypes associated with larger seedlings were different between spruce, aspen, and birch, suggesting differential impacts of some host-fungus combinations. One may speculate that wildfire-induced shifts in a given soil fungal community could result in variation in the growth response of different plant species after fire and a shift in regenerating vegetation.

Functional diversity in resource use by fungi.

Fungi influence nutrient cycling in terrestrial ecosystems, as they are major regulators of decomposition and soil respiration. However, little is known about the substrate preferences of individual fungal species outside of laboratory culture studies. If active fungi differ in their substrate preferences in situ, then changes in fungal diversity due to global change may dramatically influence nutrient cycling in ecosystems. To test the responses of individual fungal taxa to specific substrates, we used a nucleotide-analogue procedure in the boreal forest of Alaska (USA). Specifically, we added four organic N compounds commonly found in plant litter (arginine, glutamate, lignocellulose, and tannin-protein) to litterbags filled with decomposed leaf litter (black spruce and aspen) and assessed the responses of active fungal species using qPCR (quantitative polymerase chain reaction), oligonucleotide fingerprinting of rRNA genes, and sequencing. We also compared the sequences from our experiment with a concurrent warming experiment to see if active fungi that targeted more recalcitrant compounds would respond more positively to soil warming. We found that individual fungal taxa responded differently to substrate additions and that active fungal communities were different across litter types (spruce vs. aspen). Active fungi that targeted lignocellulose also responded positively to experimental warming. Additionally, resource-use patterns in different fungal taxa were genetically correlated, suggesting that it may be possible to predict the ecological function of active fungal communities based on genetic information. Together, these results imply that fungi are functionally diverse and that reductions in fungal diversity may have consequences for ecosystem functioning.

Direct amplification of DNA from fresh and preserved ectomycorrhizal root tips.

Methods are described by which DNA can be amplified directly from ectomycorrhizal root tip homogenates of a variety of plant species (Picea mariana (black spruce), Betula papyrifera (paper birch), Populus tremuloides (trembling aspen) and Alnus sp.(alder)), including root tips that have been preserved in RNA Later (Ambion, Austin, TX). In most cases for extracts and homogenates diluted 10-fold prior to PCR, and in all cases for 100-fold dilutions, direct amplification of DNA from fresh root tip homogenates yielded as many or more ng of PCR amplicon (fungal ITS region) than amplification of DNA extracted from the same tips using a commercial kit or a manual ethanol precipitation-based method. For alder root tip extracts diluted 10-fold, the commercial kit method yielded more ng of PCR amplicon than 10-fold diluted, although direct use of homogenates still resulted in amplification in all tips tested. We also demonstrate consistent amplification of DNA from homogenates of birch, spruce and aspen ectomycorrhizal root tips preserved for 4months in RNA Later.

Investigations into peach seedling stunting caused by a replant soil.

Replant diseases often occur when pome and stone fruits are grown in soil that had previously been planted with the same or similar plant species. They typically lead to reductions in plant growth, crop yield and production duration. In this project, greenhouse assays were used to identify a peach orchard soil that caused replant disease symptoms. Biocidal treatments of this soil led to growth increases of Nemaguard peach seedlings. In addition, plants grown in as little as 1% of the replant soil exhibited reduced plant growth. These results suggest that the disease etiology has a biological component. Analysis of roots from plants exhibiting various levels of replant disease symptoms showed little difference in the amounts of PCR-amplified bacterial or fungal rRNA genes. However, analysis using a stramenopile-selective PCR assay showed that rRNA genes from this taxon were generally more abundant in plants with the smallest top weights. Nucleotide sequence analysis of these genes identified several phylotypes belonging to Bacillariophyta, Chrysophyceae, Eustigmatophyceae, Labyrinthulida, Oomycetes, Phaeophyceae and Synurophyceae. Sequence-selective quantitative PCR assays targeting four of the most abundant phylotypes showed that both diatoms (Sellaphora spp.) and an oomycete (Pythium ultimum) were negatively associated with plant top weights.

Bacteria and bacterial rRNA genes associated with the development of colitis in IL-10(-/-) mice.

BACKGROUND: Microorganisms appear to play important yet ill-defined roles in the etiology of inflammatory bowel disease (IBD). This study utilized a novel population-based approach to identify bacteria and bacterial rRNA genes associated with the development of colitis in IL-10(-/-) mice. METHODS: Mice were housed in 2 environments: a community mouse facility where the mice were fed nonsterile chow (Room 3) and a limited access facility where the mice were fed sterile chow (Room 4). Every month the disease activity levels were assessed and fecal bacterial compositions were analyzed. At the end of the experiments histological and bacterial analyses were performed on intestinal tissue. RESULTS: Although disease activity increased over time in both environments, it progressed at a faster rate in Room 3 than Room 4. Culture and culture-independent bacterial analyses identified several isolates and phylotypes associated with colitis. Two phylotypes (GpC2 and Gp66) were distinguished by their negative associations with disease activity in fecal and tissue samples. Notably, rRNA genes from these phylotypes had high sequence identity (99%) to an rRNA gene from a previously described flagellated Clostridium (Lachnospiraceae bacterium A4). CONCLUSIONS: The negative associations of these 2 phylotypes (GpC2 and Gp66) suggest that these bacteria were being immunologically targeted, consistent with prior findings that the Lachnospiraceae bacterium A4 bears a prevalent flagellar antigen for disease-associated immunity in murine immune colitis and human Crohn's disease. Identification of these associations suggests that the experimental approach used in this study will have considerable utility in elucidating the host-microbe interactions underlying IBD.

PRISE (PRImer SElector): software for designing sequence-selective PCR primers.

This report presents PRImer Selector (PRISE), a new software package that implements several features that improve and streamline the design of sequence-selective PCR primers. The PRISE design process involves two main steps. In the first step, target and non-target DNA sequences are identified. In the second step, primers are designed to amplify target (but not non-target) sequences. One important feature of PRISE is that it automates the task of placing primer-template mismatches at the 3' end of the primers - a property that is crucial for sequence selectivity. Once a list of candidate primers has been produced, sorting tools in PRISE speed up the selection process by allowing a user to sort the primers by properties such as amplicon length, GC content and sequence selectivity. PRISE can be used to design primers with a range of specificities, targeting individual sequences as well as diverse assemblages of genes. PRISE also allows user-defined primers to be analyzed, enabling their properties to be examined in relation to target and non-target sequences. The utility of PRISE was demonstrated by using it to design sequence-selective PCR primers for an rRNA gene from the fungus Pochonia chlamydosporia.

Comparison of the cecal microbiota of domestic and wild turkeys.

The extent to which production methods alter intestinal microbial communities of livestock is currently unknown. As the intestinal microbiota may affect animal health, nutrition, and food safety, a baseline comparison of the cecal communities of domestic and wild turkeys was performed. Oligonucleotide fingerprinting of ribosomal RNA (rRNA) genes (OFRG) of 2,990 16S rRNA clones and dot blot quantification of dominant populations were used to identify the dominant bacterial taxa. Seventy-three percent of all the clones belonged to as yet uncultured genera. However, at a higher phylogenetic level, the OFRG library was composed of 54% Bacteroidetes clones (52% of the domestic library clones, 56% of the wild library clones), 30% Firmicutes clones (33% of the domestic library clones, 32% of the wild library clones), 3% Proteobacteria clones (5% domestic, 2% wild), and 3% Deferribacteres clones (4% domestic, 1% wild). Seven percent of the clones were unidentifiable (6% domestic, 9% wild). Bacteroidetes clones included the genera Alistipes, Prevotella, Megamonas, and Bacteroides. Of the Clostridiales clones, groups IV, IX, and XIV including genera Faecalibacterium, Megasphaera, Phascolarctobacterium, and Papillibacter were predominant. Lactobacillus, Enterococcus, and Streptococcus bacilli were also identified. beta- delta- and gamma-proteobacterial genera included Acinetobacter, Sutterella, and Escherichia. Deferribacteres clones showed high similarity to Mucispirillum schaedleri. Statistical comparison of the domestic and wild turkey clone libraries indicated similar levels of community richness and evenness despite the fact that the two libraries shared only 30% of the total clone operational taxonomic units. Together these results indicate that although high level taxonomic community structure is similar, high-density turkey production causes considerable divergence of the genera found in the ceca of commercial birds from those of their wild counterparts.

Robust spots finding in microarray images with distortions.

Microarray images, which allow the analysis of hybridization expressions of genes, have been one of the most widely used high-throughput technologies with many different applications. Accurate and automatic microarray image analysis is very important since researchers trust the information provided in these experiments and construct conclusions based on the results produced by the software responsible in analyzing the hybridized arrays. Every microarray image contains thousands of spots, so how to do the spots finding in microarray images accurately and automatically is very meaningful. There are always some problems, such as rotation and distortion, in a microarray image caused by mechanical errors and/or optical errors in the system. All these problems will hinder "doi"ng analysis automatically. Early research has worked out several algorithms to deal with the rotation problem, but those algorithms can not handle microarray images with distortions. In this paper, we propose a robust spots finding method to deal with both rotation and/or distortion in microarray images. The proposed method provides automatic gridding and can handle a microarray image with different type of rotation (global or sub-array rotation) and optical distortions.

Algorithmic approaches to selecting control clones in DNA array hybridization experiments.

We study the problem of selecting control clones in DNA array hybridization experiments. The problem arises in the OFRG method for analyzing microbial communities. The OFRG method performs classification of rRNA gene clones using binary fingerprints created from a series of hybridization experiments, where each experiment consists of hybridizing a collection of arrayed clones with a single oligonucleotide probe. This experiment produces analog signals, one for each clone, which then need to be classified, that is, converted into binary values 1 and 0 that represent hybridization and non-hybridization events. In addition to the sample rRNA gene clones, the array contains a number of control clones needed to calibrate the classification procedure of the hybridization signals. These control clones must be selected with care to optimize the classification process. We formulate this as a combinatorial optimization problem called Balanced Covering. We prove that the problem is NP-hard, and we show some results on hardness of approximation. We propose approximation algorithms based on randomized rounding, and we show that, with high probability, our algorithms approximate well the optimum solution. The experimental results confirm that the algorithms find high quality control clones. The algorithms have been implemented and are publicly available as part of the software package called CloneTools.

Development of a 9600-clone procedure for oligonucleotide fingerprinting of rRNA genes: utilization to identify soil bacterial rRNA genes that correlate in abundance with the development of avocado root rot.

Oligonucleotide fingerprinting of rRNA genes (OFRG) is an array-based method that generates microbial community profiles through analysis of rRNA gene clone libraries. The original OFRG method allowed 1536 clones to be analyzed per experiment. This report describes a procedure for analyzing 9600 clones per experiment, including a new probe set for bacterial analysis, and improved data processing and statistical analysis tools. The software tools are available at the OFRG website (). Use of the 9600-clone procedure was demonstrated by examining the bacterial rRNA gene compositions of soils subjected to various temperature treatments. These treatments produced a series of soils with a range of abilities to suppress avocado root rot, enabling the identification of bacterial rRNA genes that correlate in abundance with root rot suppressiveness. OFRG analysis of these soils produced 8876 bacterial rRNA gene fingerprints grouped into 5123 clusters, or operational taxonomic units (OTUs). Eleven OTUs exhibited a positive correlation between the number of clones and the percentage of healthy roots. An in silico analysis was performed to examine the relationship between the number of rRNA genes analyzed and the number of correlates (rRNA gene-avocado root rot symptoms) identified. As the number of clones decreased, fewer correlates were identified. To further increase the throughput of the OFRG method, use of a glass slide-fluorescent probe microarray format was also explored.

Abundant and diverse fungal microbiota in the murine intestine.

Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. The largest assemblages of fungal rRNA sequences were related to the genera Acremonium, Monilinia, Fusarium, Cryptococcus/Filobasidium, Scleroderma, Catenomyces, Spizellomyces, Neocallimastix, Powellomyces, Entophlyctis, Mortierella, and Smittium and the order Mucorales. The majority of bacterial rRNA gene clones were affiliated with the taxa Bacteroidetes, Firmicutes, Acinetobacter, and Lactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4',6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.

Potential for misidentification of a spore-forming Paenibacillus polymyxa isolate as an endophyte by using culture-based methods.

While Paenibacillus polymyxa strain Pw-2 has been identified as an endophyte of lodgepole pine (M. Shishido, B. M. Loeb, and C. P. Chanway, Can. J. Microbiol. 41:707-713, 1995), P. polymyxa strain L6 has not, a distinction that could be explained by the differential abilities of these isolates to form spores, rather than the differential abilities to colonize the interior tissues of lodgepole pine. Chemical disinfection was used to destroy bacteria on the root exterior, but bacterial endospores are known for their ability to withstand chemical disinfection, and strain Pw-2 was found to produce 300 to 11,000 times more germinating endospores than strain L6 under the experimental conditions used by Shishido et al. (Can. J. Microbiol. 41:707-713, 1995). Attempts to identify strain Pw-2 within lodgepole pine root tissues by using confocal microscopy techniques failed. We discuss the possibility that spore-forming bacteria can be mistakenly identified as endophytes when culture-based methods alone are used.