Analysis of bacterioplankton genes in an impaired Great Lakes harbour reveals seasonal metabolic shifts and a previously undetected cyanobacterium.

Hamilton Harbour is an impaired embayment of Lake Ontario that experiences seasonal algal blooms despite decades of remedial efforts. To study the harbour's cyanobacterial and heterotrophic bacterial communities, we extracted and sequenced community DNA from surface water samples collected biweekly from different sites during summer and fall. Assembled contigs were annotated at the phylum level, and Cyanobacteria were further characterized at order and species levels. Actinobacteria were most abundant in early summer, while Cyanobacteria were dominant in mid-summer. Microcystis aeruginosa and Limnoraphis robusta were most abundant throughout the sampling period, expanding the documented diversity of Cyanobacteria in Hamilton Harbour. Functional annotations were performed using the MG-RAST pipeline and SEED database, revealing that genes for photosynthesis, nitrogen metabolism, and aromatic compound metabolism varied in relative abundances over the season, while phosphorus metabolism was consistent, suggesting that these genes remained essential despite fluctuating environmental conditions and community succession. We observed seasonal shifts from anoxygenic to oxygenic phototrophy, and from ammonia assimilation to nitrogen fixation, coupled with decreasing heterotrophic bacteria and increasing Cyanobacteria relative abundances. Our data contribute important insights into bacterial taxa and functional potentials in Hamilton Harbour, revealing seasonal and spatial dynamics that can be used to inform ongoing remediation efforts.

The influence of biofertilizers on leaf economics spectrum traits in a herbaceous crop.

Microbial inoculations or 'biofertilizers' represent novel contributions to sustainable agriculture. While belowground mechanisms surrounding how biofertilizers enhance crop production are well described, their role in aboveground trait expression remains less well explored. We quantified infraspecific variation in leaf economics spectrum (LES) traits in response to 10 biofertilizer treatments in basil (Ocimum basiclicum) cultivated under hydroponic conditions. Multiple physiological (i.e. maximum photosynthesis rates (A), dark respiration (R), and leaf-level light compensation points) and morphological (i.e. leaf mass per area (LMA) and leaf thickness) traits varied significantly across microbial treatments. Following treatments, basil plants differentiated from one another along an infraspecific LES, with certain plants expressing more resource-acquiring LES trait values (i.e. high A, R, leaf N, and low LMA), versus others that expressed the opposite suite of resource-conserving LES trait values. Infraspecific trait covariation largely matched LES patterns observed among plants globally. Bivariate and multivariate trait analyses further revealed that certain treatments-namely those including closely related Bacillus and Brevibacillus species strains-increased leaf resource capture traits such as A and leaf N. Biofertilizers influence plant performance through a role in moderating infraspecific leaf trait variation, thereby suggesting aboveground leaf traits may be used to diagnose optimal biofertilizer formulations in basil and other crops.

Metagenomic Analysis of Virus Diversity and Relative Abundance in a Eutrophic Freshwater Harbour.

Aquatic viruses have been extensively studied over the past decade, yet fundamental aspects of freshwater virus communities remain poorly described. Our goal was to characterize virus communities captured in the >0.22 µm size-fraction seasonally and spatially in a freshwater harbour. Community DNA was extracted from water samples and sequenced on an Illumina HiSeq platform. Assembled contigs were annotated as belonging to the virus groups (i.e., order or family) Caudovirales, Mimiviridae, Phycodnaviridae, and virophages (Lavidaviridae), or to other groups of undefined viruses. Virophages were often the most abundant group, and discrete virophage taxa were remarkably stable across sites and dates despite fluctuations in Mimiviridae community composition. Diverse Mimiviridae contigs were detected in the samples and the two sites contained distinct Mimiviridae communities, suggesting that Mimiviridae are important algal viruses in this system. Caudovirales and Phycodnaviridae were present at low abundances in most samples. Of the 18 environmental parameters tested, only chlorophyll a explained the variation in the data at the order or family level of classification. Overall, our findings provide insight into freshwater virus community assemblages by expanding the documented diversity of freshwater virus communities, highlighting the potential ecological importance of virophages, and revealing distinct communities over small spatial scales.

Plantibacter flavus, Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens Endophytes Provide Host-Specific Growth Promotion of Arabidopsis thaliana, Basil, Lettuce, and Bok Choy Plants.

A collection of bacterial endophytes isolated from stem tissues of plants growing in soils highly contaminated with petroleum hydrocarbons were screened for plant growth-promoting capabilities. Twenty-seven endophytic isolates significantly improved the growth of Arabidopsis thaliana plants in comparison to that of uninoculated control plants. The five most beneficial isolates, one strain each of Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens and two strains of Plantibacter flavus were further examined for growth promotion in Arabidopsis, lettuce, basil, and bok choy plants. Host-specific plant growth promotion was observed when plants were inoculated with the five bacterial strains. P. flavus strain M251 increased the total biomass and total root length of Arabidopsis plants by 4.7 and 5.8 times, respectively, over that of control plants and improved lettuce and basil root growth, while P. flavus strain M259 promoted Arabidopsis shoot and root growth, lettuce and basil root growth, and bok choy shoot growth. A genome comparison between P. flavus strains M251 and M259 showed that both genomes contain up to 70 actinobacterial putative plant-associated genes and genes involved in known plant-beneficial pathways, such as those for auxin and cytokinin biosynthesis and 1-aminocyclopropane-1-carboxylate deaminase production. This study provides evidence of direct plant growth promotion by Plantibacter flavusIMPORTANCE The discovery of new plant growth-promoting bacteria is necessary for the continued development of biofertilizers, which are environmentally friendly and cost-efficient alternatives to conventional chemical fertilizers. Biofertilizer effects on plant growth can be inconsistent due to the complexity of plant-microbe interactions, as the same bacteria can be beneficial to the growth of some plant species and neutral or detrimental to others. We examined a set of bacterial endophytes isolated from plants growing in a unique petroleum-contaminated environment to discover plant growth-promoting bacteria. We show that strains of Plantibacter flavus exhibit strain-specific plant growth-promoting effects on four different plant species.

Endophytic Bacterial Community Structure and Function of Herbaceous Plants From Petroleum Hydrocarbon Contaminated and Non-contaminated Sites.

Bacterial endophytes (BEs) are non-pathogenic residents of healthy plant tissues that can confer benefits to plants. Many Bacterial endophytes have been shown to contribute to plant growth and health, alleviation of plant stress and to in-planta contaminant-degradation. This study examined the endophytic bacterial communities of plants growing abundantly in a heavily hydrocarbon contaminated site, and compared them to those found in the same species at a non-contaminated. We used culture- dependent and independent methods to characterize the community structure, hydrocarbon degrading capabilities, and plant growth promoting traits of cultivable endophytes isolated from Achillea millefolium, Solidago Canadensis, and Daucus carota plants from these two sites. Culture- dependent and independent analyses revealed class Gammaproteobacteria predominated in all the plants regardless of the presence of petroleum hydrocarbon, with Pantoea spp. as largely dominant. It was interesting to note a >50% taxonomic overlap (genus level) of 16s rRNA high throughput amplicon sequences with cultivable endophytes. PERMANOVA analysis of TRFLP fragments revealed significant structural differences between endophytic bacterial communities from hydrocarbon-contaminated and non-contaminated soils-however, there was no marked difference in their functional capabilities. Pantoea spp. demonstrated plant beneficial characteristics, such as P solubilization, indole-3-acetic acid production and presence of 1-aminocyclopropane-1-carboxylate deaminase. Our findings reveal that functional capabilities of bacterial isolates being examined were not influenced by the presence of contamination; and that the stem endosphere supports ubiquitous BEs that were consistent throughout plant hosts and sites.

Draft Genome Sequence of Bacillus sp. Strain UFRGS-B20, a Hydrocarbon Degrader.

Bacillus sp. strain UFRGS-B20 was isolated in 2012 from Brazilian land-farming soil contaminated with petrochemical oily sludge. This strain was subjected to hydrocarbon biodegradation tests, showing degradation rates of up to 60%. Here, we present the 6.82-Mb draft genome sequence of the strain, which contains 2,178 proteins with functional assignments.

Draft Genome Sequence of Microbacterium foliorum Strain 122 Isolated from a Plant Growing in a Chronically Hydrocarbon-Contaminated Site.

Microbacterium foliorum strain 122 is a bacterial endophyte isolated from a Dactylis glomerata plant growing in a natural oil seep soil located in Oil Springs, Ontario, Canada. We present here a draft genome sequence of an endophytic strain that has promising potential in hydrocarbon degradation and plant growth promotion.

Draft Genome Sequence of Plantibacterflavus Strain 251 Isolated from a Plant Growing in a Chronically Hydrocarbon-Contaminated Site.

Plantibacter flavus isolate 251 is a bacterial endophyte isolated from an Achillea millefolium plant growing in a natural oil seep soil located in Oil Springs, Ontario, Canada. We present here a draft genome sequence of an infrequently reported genus Plantibacter, highlighting an endophytic lifestyle and biotechnological potential.

Bacterial Endophytes Isolated from Plants in Natural Oil Seep Soils with Chronic Hydrocarbon Contamination.

The bacterial endophytic communities of four plants growing abundantly in soils highly contaminated by hydrocarbons were analyzed through culturable and culture-independent means. Given their tolerance to the high levels of petroleum contamination at our study site, we sought evidence that Achillea millefolium, Solidago canadensis, Trifolium aureum, and Dactylis glomerata support high levels of hydrocarbon degrading endophytes. A total of 190 isolates were isolated from four plant species. The isolates were identified by partial 16S rDNA sequence analysis, with class Actinobacteria as the dominant group in all species except S. canadensis, which was dominated by Gammaproteobacteria. Microbacterium foliorum and Plantibacter flavus were present in all the plants, with M. foliorum showing predominance in D. glomerata and both endophytic bacterial species dominated T. aureum. More than 50% of the isolates demonstrated degradative capabilities for octanol, toluene, naphthalene, kerosene, or motor oil based on sole carbon source growth screens involving the reduction of tetrazolium dye. P. flavus isolates from all the sampled plants showed growth on all the petroleum hydrocarbons (PHCs) substrates tested. Mineralization of toluene and naphthalene was confirmed using gas-chromatography. 16S based terminal restriction fragment length polymorphism analysis revealed significant differences between the endophytic bacterial communities showing them to be plant host specific at this site. To our knowledge, this is the first account of the degradation potential of bacterial endophytes in these commonly occurring pioneer plants that were not previously known as phytoremediating plants.

The microbiomes and metagenomes of forest biochars.

Biochar particles have been hypothesized to provide unique microhabitats for a portion of the soil microbial community, but few studies have systematically compared biochar communities to bulk soil communities. Here, we used a combination of sequencing techniques to assess the taxonomic and functional characteristics of microbial communities in four-year-old biochar particles and in adjacent soils across three forest environments. Though effects varied between sites, the microbial community living in and around the biochar particles had significantly lower prokaryotic diversity and higher eukaryotic diversity than the surrounding soil. In particular, the biochar bacterial community had proportionally lower abundance of Acidobacteria, Planctomycetes, and ß-Proteobacteria taxa, compared to the soil, while the eukaryotic biochar community had an 11% higher contribution of protists belonging to the Aveolata superphylum. Additionally, we were unable to detect a consistent biochar effect on the genetic functional potential of these microbial communities for the subset of the genetic data for which we were able to assign functions through MG-RAST. Overall, these results show that while biochar particles did select for a unique subset of the biota found in adjacent soils, effects on the microbial genetic functional potential appeared to be specific to contrasting forest soil environments.

Seasonal variation of bacterial endophytes in urban trees.

Bacterial endophytes, non-pathogenic bacteria residing within plants, contribute to the growth and development of plants and their ability to adapt to adverse conditions. In order to fully exploit the capabilities of these bacteria, it is necessary to understand the extent to which endophytic communities vary between species and over time. The endophytes of Acer negundo, Ulmus pumila, and Ulmus parvifolia were sampled over three seasons and analyzed using culture dependent and independent methods (culture on two media, terminal restriction fragment length polymorphism, and tagged pyrosequencing of 16S ribosomal amplicons). The majority of culturable endophytes isolated were Actinobacteria, and all the samples harbored Bacillus, Curtobacterium, Frigoribacterium, Methylobacterium, Paenibacilllus, and Sphingomonas species. Regardless of culture medium used, only the culturable communities obtained in the winter for A. negundo could be distinguished from those of Ulmus spp. In contrast, the nonculturable communities were dominated by Proteobacteria and Actinobacteria, particularly Erwinia, Ralstonia, and Sanguibacter spp. The presence and abundance of various bacterial classes and phyla changed with the changing seasons. Multivariate analysis on the culture independent data revealed significant community differences between the endophytic communities of A. negundo and Ulmus spp., but overall season was the main determinant of endophytic community structure. This study suggests studies on endophytic populations of urban trees should expect to find significant seasonal and species-specific community differences and sampling should proceed accordingly.

Rapid degradation of Deepwater Horizon spilled oil by indigenous microbial communities in Louisiana saltmarsh sediments.

The Deepwater Horizon oil spill led to the severe contamination of coastal environments in the Gulf of Mexico. A previous study detailed coastal saltmarsh erosion and recovery in a number of oil-impacted and nonimpacted reference sites in Barataria Bay, Louisiana over the first 18 months after the spill. Concentrations of alkanes and polyaromatic hydrocarbons (PAHs) at oil-impacted sites significantly decreased over this time period. Here, a combination of DNA, lipid, and isotopic approaches confirm that microbial biodegradation was contributing to the observed petroleum mass loss. Natural abundance (14)C analysis of microbial phospholipid fatty acids (PLFA) reveals that petroleum-derived carbon was a primary carbon source for microbial communities at impacted sites several months following oil intrusion when the highest concentrations of oil were present. Also at this time, microbial community analysis suggests that community structure of all three domains has shifted with the intrusion of oil. These results suggest that Gulf of Mexico marsh sediments have considerable biodegradation potential and that natural attenuation is playing a role in impacted sites.

Probing the functional diversity of global pristine soil communities with 3-chlorobenzoate reveals that communities of generalists dominate catabolic transformation.

Understanding of functional diversity of microbial populations has lagged description of their molecular diversity. Differences in substrate specificity, kinetics, products, and regulation can dramatically influence phenotypic variation among closely related strains, features that are missed when the strains studied are the fastest-growing and most easily isolated from serial enrichments. To investigate the broader bacterial diversity underlying degradation of anthropogenic chemicals in nature, we studied the 3-chlorobenzoate (3-CBA) degradation rate in a collection of aerobic 3-CBA degraders previously isolated from undisturbed soils in two representative ecosystems: (i) Mediterranean sclerophyllous woodlands in California, Chile, South Africa, and Australia and (ii) boreal forests in Canada and Russia. The majority of isolates degraded 3-CBA slowly and did not completely mineralize 1.0 mM 3-CBA within 1 week. Those with intermediate degradation rates had incomplete degradation pathways and produced colored intermediates indicative of chlorocatechol, a product likely metabolized by other members of the community. About 10% of the isolates grew rapidly and mineralized greater than 90% of the 3-CBA, but because of population heterogeneity in soil, they are likely not large contributors to a soil's total transformation capacity. This suggests that xenobiotic degradation in nature is carried out by a community of cometabolic generalists and not by the efficient specialists that have been traditionally studied in the laboratory. A subset of 58 genotypically distinct strains able to degrade >80% of the 3-CBA was examined for their catabolic versatility using 45 different compounds: mono- and dichlorinated benzoates, phenols, anilines, toluenes, nitrobenzenes, chlorobenzenes, and 2,4-dichlorophenoxyacetic acid. The isolates degraded from 2 to more than 30 compounds with a median of 7, but there was no correlation to habitat of isolation or 3-CBA activity. However, these findings were indicative of finer-scale functional diversity.

Assessing microbial carbon sources and potential PAH degradation using natural abundance 14C analysis.

Natural abundance (14)C analysis was applied to PLFAs collected from an industrial site in southern Ontario in order to assess microbial carbon sources and potential PAH biodegradation in soils. Δ(14)C of microbial phospholipid fatty acids (PLFA) at the site ranged from +54‰ to -697‰. Comparison of these values to surrounding carbon sources found that microbial carbon sources were derived primarily from vegetation and/or natural organic matter present in the soils rather than PAHs. This study highlights that microbes are able to utilize almost all available pools of organic matter including older pools which are thought to contain recalcitrant compounds. Furthermore, it shows that even with the presence of an active microbial community, there may be little biodegradation of PAHs. This study illustrates challenges in assessing microbial activity in the environment and the advantage of using natural abundance (14)C analysis as a tool to elucidate microbial carbon sources.

Land-use change and soil type are drivers of fungal and archaeal communities in the Pampa biome.

The current study aimed to test the hypothesis that both land-use change and soil type are responsible for the major changes in the fungal and archaeal community structure and functioning of the soil microbial community in Brazilian Pampa biome. Soil samples were collected at sites with different land-uses (native grassland, native forest, Eucalyptus and Acacia plantation, soybean and watermelon field) and in a typical toposequence in Pampa biome formed by Paleudult, Albaqualf and alluvial soils. The structure of soil microbial community (archaeal and fungal) was evaluated by ribosomal intergenic spacer analysis and soil functional capabilities were measured by microbial biomass carbon and metabolic quotient. We detected different patterns in microbial community driven by land-use change and soil type, showing that both factors are significant drivers of fungal and archaeal community structure and biomass and microbial activity. Fungal community structure was more affected by land-use and archaeal community was more affected by soil type. Irrespective of the land-use or soil type, a large percentage of operational taxonomic unit were shared among the soils. We accepted the hypothesis that both land-use change and soil type are drivers of archaeal and fungal community structure and soil functional capabilities. Moreover, we also suggest the existence of a soil microbial core.

Changes in bacterial community structure after exposure to silver nanoparticles in natural waters.

Silver nanoparticles (AgNPs) are widely used in commercial products as antibacterial agents, but AgNPs might be hazardous to the environment and natural aquatic bacterial communities. Our recent research demonstrated that AgNPs rapidly but temporarily inhibited natural bacterioplankton production. The current study investigates the mechanism for the observed bacterial reaction to AgNPs by examining how AgNPs impact bacterial abundance, metabolic activity (5-cyano-2,3-ditolyl tetrazolium chloride (CTC+) cells), and 16S rRNA community composition. Natural bacterioplankton communities were dosed with carboxy-functionalized AgNPs at four concentrations (0.01-1 mg-Ag/L), incubated in triplicate, and monitored over 5 days. Ionic silver (AgNO(3)) and Milli-Q water treatments were used as a positive and negative control, respectively. Four general AgNP exposure responses, relative to the negative control, were observed: (1) intolerant, (2) impacted but recovering, (3) tolerant, and (4) stimulated phylotypes. Relationships between cell activity indicators and bacterial phylotypes, suggested that tolerant and recovering bacteria contributed the most to the community's productivity and rare bacteria phylotypes stimulated by AgNPs did not appear to contribute much to cell activity. Overall, natural bacterial communities tolerated single, low level AgNP doses and had similar activity levels to the negative control within five days of exposure, but bacterial community composition was different from that of the control.

Microbial diversity, tolerance, and biodegradation potential of urban wetlands with different input regimes.

Though microbial transformations are the primary mechanism of contaminant attenuation in wetlands, much remains to be known about microbial communities in urban wetlands. In this study, the microbial communities from urban wetlands with different runoff regimes (i.e., a contaminated remnant wetland, a constructed wetland, and a remnant wetland) were assessed for their capacity to attenuate and tolerate typical urban runoff pollutants. Results from denaturing gradient gel electrophoresis of 16S rRNA genes showed relatively high similarity in community composition among the wetlands. Community-level physiological profiles had similar results but exhibited within-site variation in both the contaminated remnant and remnant wetlands. All wetland communities were less tolerant to copper than 2,4-dichlorophenoxyacetic acid; however, the contaminated remnant wetland had the highest tolerance. All study wetlands had a limited capacity to biodegrade model chlorinated aromatic compounds (e.g., 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate). Though having different input regimes and contaminant exposure histories, the study wetlands were generally similar with respect to microbial community diversity and function. Additionally, the generally low capacity for these wetlands to biodegrade mobile chlorinated organic contaminants offers preliminary insight into the limited ecosystem services these wetlands may provide in urban environments.

Low sequencing efforts bias analyses of shared taxa in microbial communities.

The potential for comparing microbial community population structures has been greatly enhanced by developments in next generation sequencing methods that can generate hundreds of thousands to millions of reads in a single run. Conversely, many microbial community comparisons have been published with no more than 1,000 sequences per sample. These studies have presented data on levels of shared operational taxonomic units (OTUs) between communities. Due to lack of coverage, that approach might compromise the conclusions about microbial diversity and the degree of difference between environments. In this study, we present data from recent studies that highlight this problem. Also, we analyzed datasets of 16 rRNA sequences with small and high sequence coverage from different environments to demonstrate that the level of sequencing effort used for analyzing microbial communities biases the results. We randomly sampled pyrosequencing-generated 16S rRNA gene libraries with increasing sequence effort. Sequences were used to calculate Good's coverage, the percentage of shared OTUs, and phylogenetic distance measures. Our data showed that simple counts of presence/absence of taxonomic unities do not reflect the real similarity in membership and structure of the bacterial communities and that community comparisons based on phylogenetic tests provide a way to test statistically significant differences between two or more environments without need an exhaustive sampling effort.

Bacterial community dynamics in the hyporheic zone of an intermittent stream.

The dynamics of in situ bacterial communities in the hyporheic zone of an intermittent stream were described in high spatiotemporal detail. We assessed community dynamics in stream sediments and interstitial pore water over a two-year period using terminal-restriction fragment length polymorphism. Here, we show that sediments remained saturated despite months of drought and limited hydrologic connectivity. The intermittency of stream surface water affected interstitial pore water communities more than hyporheic sediment communities. Seasonal changes in bacterial community composition was significantly associated with water intermittency, phosphate concentrations, temperature, nitrate and dissolved organic carbon (DOC) concentrations. During periods of low- to no-surface water, communities changed from being rich in operational taxonomic units (OTUs) in isolated surface pools, to a few OTUs overall, including an overall decline in both common and rare taxa. Individual OTUs were compared between porewater and sediments. A total of 19% of identified OTUs existed in both porewater and sediment samples, suggesting that bacteria use hyporheic sediments as a type of refuge from dessication, transported through hydrologically connected pore spaces. Stream intermittency impacted bacterial diversity on rapid timescales (that is, within days), below-ground and in the hyporheic zone. Owing to the coupling of intermittent streams to the surrounding watershed, we stress the importance of understanding connectivity at the pore scale, consequences for below-ground and above-ground biodiversity and nutrient processing, and across both short- and long-time periods (that is, days to months to years).

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.