Year-Round Shotgun Metagenomes Reveal Stable Microbial Communities in Agricultural Soils and Novel Ammonia Oxidizers Responding to Fertilization.

The dynamics of individual microbial populations and their gene functions in agricultural soils, especially after major activities such as nitrogen (N) fertilization, remain elusive but are important for a better understanding of nutrient cycling. Here, we analyzed 20 short-read metagenomes collected at four time points during 1 year from two depths (0 to 5 and 20 to 30 cm) in two Midwestern agricultural sites representing contrasting soil textures (sandy versus silty loam) with similar cropping histories. Although the microbial community taxonomic and functional compositions differed between the two locations and depths, they were more stable within a depth/site throughout the year than communities in natural aquatic ecosystems. For example, among the 69 population genomes assembled from the metagenomes, 75% showed a less than 2-fold change in abundance between any two sampling points. Interestingly, six deep-branching Thaumarchaeota and three complete ammonia oxidizer (comammox) Nitrospira populations increased up to 5-fold in abundance upon the addition of N fertilizer. These results indicated that indigenous archaeal ammonia oxidizers may respond faster (are more copiotrophic) to N fertilization than previously thought. None of 29 recovered putative denitrifier genomes encoded the complete denitrification pathway, suggesting that denitrification is carried out by a collection of different populations. Altogether, our study identified novel microbial populations and genes responding to seasonal and human-induced perturbations in agricultural soils that should facilitate future monitoring efforts and N-related studies.IMPORTANCE Even though the impact of agricultural management on the microbial community structure has been recognized, an understanding of the dynamics of individual microbial populations and what functions each population carries are limited. Yet, this information is important for a better understanding of nutrient cycling, with potentially important implications for preserving nitrogen in soils and sustainability. Here, we show that reconstructed metagenome-assembled genomes (MAGs) are relatively stable in their abundance and functional gene content year round, and seasonal nitrogen fertilization has selected for novel Thaumarchaeota and comammox Nitrospira nitrifiers that are potentially less oligotrophic than their marine counterparts previously studied.

Detection and Diversity of Fungal Nitric Oxide Reductase Genes (p450nor) in Agricultural Soils.

UNLABELLED: Members of the Fungi convert nitrate (NO3 (-)) and nitrite (NO2 (-)) to gaseous nitrous oxide (N2O) (denitrification), but the fungal contributions to N loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations, and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N2O from added NO3 (-) or NO2 (-) in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, 151 of which produced N2O from NO2 (-) Novel PCR primers targeting the p450nor gene, which encodes the nitric oxide (NO) reductase responsible for N2O production in fungi, yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54 to 98% amino acid identity with reference P450nor sequences within the phylum Ascomycota and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N2O from NO2 (-), whereas nirK (encoding the NO-forming NO2 (-) reductase) was amplified in only 13 to 74% of the N2O-forming isolates using two separate nirK primer sets. Collectively, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation. IMPORTANCE: A comprehensive understanding of the microbiota controlling soil N loss and greenhouse gas (N2O) emissions is crucial for sustainable agricultural practices and addressing climate change concerns. We report the design and application of a novel PCR primer set targeting fungal p450nor, a biomarker for fungal N2O production, and demonstrate the utility of the new approach to assess fungal denitrification potential in fungal isolates and agricultural soils. These new PCR primers may find application in a variety of biomes to assess the fungal contributions to N loss and N2O emissions.

Unexpected nondenitrifier nitrous oxide reductase gene diversity and abundance in soils.

Agricultural and industrial practices more than doubled the intrinsic rate of terrestrial N fixation over the past century with drastic consequences, including increased atmospheric nitrous oxide (N(2)O) concentrations. N(2)O is a potent greenhouse gas and contributor to ozone layer destruction, and its release from fixed N is almost entirely controlled by microbial activities. Mitigation of N(2)O emissions to the atmosphere has been attributed exclusively to denitrifiers possessing NosZ, the enzyme system catalyzing N(2)O to N(2) reduction. We demonstrate that diverse microbial taxa possess divergent nos clusters with genes that are related yet evolutionarily distinct from the typical nos genes of denitirifers. nos clusters with atypical nosZ occur in Bacteria and Archaea that denitrify (44% of genomes), do not possess other denitrification genes (56%), or perform dissimilatory nitrate reduction to ammonium (DNRA; (31%). Experiments with the DNRA soil bacterium Anaeromyxobacter dehalogenans demonstrated that the atypical NosZ is an effective N(2)O reductase, and PCR-based surveys suggested that atypical nosZ are abundant in terrestrial environments. Bioinformatic analyses revealed that atypical nos clusters possess distinctive regulatory and functional components (e.g., Sec vs. Tat secretion pathway in typical nos), and that previous nosZ-targeted PCR primers do not capture the atypical nosZ diversity. Collectively, our results suggest that nondenitrifying populations with a broad range of metabolisms and habitats are potentially significant contributors to N(2)O consumption. Apparently, a large, previously unrecognized group of environmental nosZ has not been accounted for, and characterizing their contributions to N(2)O consumption will advance understanding of the ecological controls on N(2)O emissions and lead to refined greenhouse gas flux models.

Refined NrfA phylogeny improves PCR-based nrfA gene detection.

Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are contrasting microbial processes in the terrestrial nitrogen (N) cycle, in that the former promotes N retention and the latter leads to N loss (i.e., the formation of gaseous products). The nitrite reductase NrfA catalyzes nitrite reduction to ammonium, the enzyme associated with respiratory nitrite ammonification and the key step in DNRA. Although well studied biochemically, the diversity and phylogeny of this enzyme had not been rigorously analyzed. A phylogenetic analysis of 272 full-length NrfA protein sequences distinguished 18 NrfA clades with robust statistical support (>90% Bayesian posterior probabilities). Three clades possessed a CXXCH motif in the first heme-binding domain, whereas all other clades had a CXXCK motif in this location. The analysis further identified a KXRH or KXQH motif between the third and fourth heme-binding motifs as a conserved and diagnostic feature of all pentaheme NrfA proteins. PCR primers targeting a portion of the heme-binding motifs that flank this diagnostic region yielded the expected 250-bp-long amplicons with template DNA from eight pure cultures and 16 new nrfA-containing isolates. nrfA amplicons obtained with template DNA from two geomorphically distinct agricultural soils could be assigned to one of the 18 NrfA clades, providing support for this expanded classification. The extended NrfA phylogeny revealed novel diagnostic features of DNRA populations and will be useful to assess nitrate/nitrite fate in natural and engineered ecosystems.

Comparison of microbial community assemblages in the rhizosphere of three Amaranthus spp.

Weed management remains a major challenge in cropping systems worldwide, with rising interest in ecological based approaches that can be integrated with herbicide use. Soil microbial communities may play important, yet undiscovered, roles in weed success. Little is known about the rhizosphere communities associated with weeds like Amaranthus, commonly known as pigweeds, and considered some of the most problematic weeds in agricultural systems. In a greenhouse experiment that allowed controlled plant growth conditions and a high number of individual plant specimens to analyze for statistical robustness (n = 8 per species), we show that specific bacterial assemblages form in the rhizospheres of A. retroflexus L. (redroot pigweed), A. palmeri S. Watson (Palmer amaranth), and A. tuberculatus (Moq.) J. D. Sauer (waterhemp). Using a relatively rapid and easy approach of T-RFLP community profiling of the 16S rRNA genes, distinct assemblages corresponded to plant species (PERMANOVA F = 14.776, p = 0.001), and further within each species, similar communities (F = 11.449, p = 0.001) were associated with three rhizosphere soil fractions taken in increasing distances away from the root tissue. These results provide the first solid basis for distinct plant-microbe relationships within three closely related Amaranthus species, warranting closer examination of the identities and function of the microorganisms that appear to be selectively recruited from the extant soil community. More intensive efforts to obtain the microbial taxonomic identities via sequencing are underway that can lead to further detailed studies to elucidate important functional plant-microbe interactions that may associate with weed success. Such data provides underlying key information that may ultimately exploit weed-microbe interactions in development of new integrated weed control tactics.

Tropical lacustrine sediment microbial community response to an extreme El Niño event.

Salinity can influence microbial communities and related functional groups in lacustrine sediments, but few studies have examined temporal variability in salinity and associated changes in lacustrine microbial communities and functional groups. To better understand how microbial communities and functional groups respond to salinity, we examined geochemistry and functional gene amplicon sequence data collected from 13 lakes located in Kiritimati, Republic of Kiribati (2° N, 157° W) in July 2014 and June 2019, dates which bracket the very large El Niño event of 2015-2016 and a period of extremely high precipitation rates. Lake water salinity values in 2019 were significantly reduced and covaried with ecological distances between microbial samples. Specifically, phylum- and family-level results indicate that more halophilic microorganisms occurred in 2014 samples, whereas more mesohaline, marine, or halotolerant microorganisms were detected in 2019 samples. Functional Annotation of Prokaryotic Taxa (FAPROTAX) and functional gene results (nifH, nrfA, aprA) suggest that salinity influences the relative abundance of key functional groups (chemoheterotrophs, phototrophs, nitrogen fixers, denitrifiers, sulfate reducers), as well as the microbial diversity within functional groups. Accordingly, we conclude that microbial community and functional gene groups in the lacustrine sediments of Kiritimati show dynamic changes and adaptations to the fluctuations in salinity driven by the El Niño-Southern Oscillation.

Stable carbon isotope values of syndepositional carbonate spherules and micrite record spatial and temporal changes in photosynthesis intensity.

Marine and lacustrine carbonate minerals preserve carbon cycle information, and their stable carbon isotope values (δ13 C) are frequently used to infer and reconstruct paleoenvironmental changes. However, multiple processes can influence the δ13 C values of bulk carbonates, confounding the interpretation of these values in terms of conditions at the time of mineral precipitation. Co-existing carbonate forms may represent different environmental conditions, yet few studies have analyzed δ13 C values of syndepositional carbonate grains of varying morphologies to investigate their origins. Here, we combine stable isotope analyses, metagenomics, and geochemical modeling to interpret δ13 C values of syndepositional carbonate spherules (>500 µm) and fine-grained micrite (<63 µm) from a ~1600-year-long sediment record of a hypersaline lake located on the coral atoll of Kiritimati, Republic of Kiribati (1.9°N, 157.4°W). Petrographic, mineralogic, and stable isotope results suggest that both carbonate fractions precipitate in situ with minor diagenetic alterations. The δ13 C values of spherules are high compared to the syndepositional micrite and cannot be explained by mineral differences or external perturbations, suggesting a role for local biological processes. We use geochemical modeling to test the hypothesis that the spherules form in the surface microbial mat during peak diurnal photosynthesis when the δ13 C value of dissolved inorganic carbon is elevated. In contrast, we hypothesize that the micrite may precipitate more continuously in the water as well as in sub-surface, heterotrophic layers of the microbial mat. Both metagenome and geochemical model results support a critical role for photosynthesis in influencing carbonate δ13 C values. The down-core spherule-micrite offset in δ13 C values also aligns with total organic carbon values, suggesting that the difference in the δ13 C values of spherules and micrite may be a more robust, inorganic indicator of variability in productivity and local biological processes through time than the δ13 C values of individual carbonate forms.

Molecular ecology of macrolide-lincosamide-streptogramin B methylases in waste lagoons and subsurface waters associated with swine production.

RNA methylase genes are common antibiotic resistance determinants for multiple drugs of the macrolide, lincosamide, and streptogramin B (MLS(B)) families. We used molecular methods to investigate the diversity, distribution, and abundance of MLS(B) methylases in waste lagoons and groundwater wells at two swine farms with a history of tylosin (a macrolide antibiotic structurally related to erythromycin) and tetracycline usage. Phylogenetic analysis guided primer design for quantification of MLS(B) resistance genes found in tylosin-producing Streptomyces (tlr(B), tlr(D)) and commensal/pathogenic bacteria (erm(A), erm(B), erm(C), erm(F), erm(G), erm(Q)). The near absence of tlr genes at these sites suggested a lack of native antibiotic-producing organisms. The gene combination erm(ABCF) was found in all lagoon samples analyzed. These four genes were also detected with high frequency in wells previously found to be contaminated by lagoon leakage. A weak correlation was found between the distribution of erm genes and previously reported patterns of tetracycline resistance determinants, suggesting that dissemination of these genes into the environment is not necessarily linked. Considerations of gene origins in history (i.e., phylogeny) and gene distributions in the landscape provide a useful "molecular ecology" framework for studying environmental spread of antibiotic resistance.

Hierarchical detection of diverse Clade II (atypical) nosZ genes using new primer sets for classical- and multiplex PCR array applications.

The reduction of nitrous oxide (N2O) to N2 represents the key terminal step in canonical denitrification. Nitrous oxide reductase (NosZ), the enzyme associated with this biological step, however, is not always affiliated with denitrifying microorganisms. Such organisms were shown recently to possess a Clade II (atypical) nosZ gene, in contrast to Clade I (typical) nosZ harbored in more commonly studied denitrifiers. Subsequent phylogenetic analyses have shown that Clade II NosZ are affiliated with a much broader diversity of microorganisms than those with Clade I NosZ, the former including both non-denitrifiers and denitrifiers. Most studies attempting to characterize the nosZ gene diversity using DNA-based PCR approaches have only focused on Clade I nosZ, despite recent metagenomic sequencing studies that have demonstrated the dominance of Clade II nosZ genes in many ecosystems, particularly soil. As a result, these studies have greatly underestimated the genetic potential for N2O reduction present in ecosystems. Because the high diversity of Clade II NosZ makes it impossible to design a universal primer set that would effectively amplify all nosZ genes in this clade, we developed a suite of primer sets to specifically target seven of ten designated subclades of Clade II nosZ genes. The new primer sets yield suitable product sizes for paired end amplicon sequencing and qPCR, demonstrated here in their use for both conventional single-reaction and multiplex array platforms. In addition, we show the utility of these primers for detecting nosZ gene transcripts from mRNA extracted from soil.

Fate and transport of antibiotic residues and antibiotic resistance genes following land application of manure waste.

Antibiotics are used in animal livestock production for therapeutic treatment of disease and at subtherapeutic levels for growth promotion and improvement of feed efficiency. It is estimated that approximately 75% of antibiotics are not absorbed by animals and are excreted in waste. Antibiotic resistance selection occurs among gastrointestinal bacteria, which are also excreted in manure and stored in waste holding systems. Land application of animal waste is a common disposal method used in the United States and is a means for environmental entry of both antibiotics and genetic resistance determinants. Concerns for bacterial resistance gene selection and dissemination of resistance genes have prompted interest about the concentrations and biological activity of drug residues and break-down metabolites, and their fate and transport. Fecal bacteria can survive for weeks to months in the environment, depending on species and temperature, however, genetic elements can persist regardless of cell viability. Phylogenetic analyses indicate antibiotic resistance genes have evolved, although some genes have been maintained in bacteria before the modern antibiotic era. Quantitative measurements of drug residues and levels of resistance genes are needed, in addition to understanding the environmental mechanisms of genetic selection, gene acquisition, and the spatiotemporal dynamics of these resistance genes and their bacterial hosts. This review article discusses an accumulation of findings that address aspects of the fate, transport, and persistence of antibiotics and antibiotic resistance genes in natural environments, with emphasis on mechanisms pertaining to soil environments following land application of animal waste effluent.

Water-soluble exudates from seeds of Kochia scoparia exhibit antifungal activity against Colletotrichum graminicola.

Plant seed exudates are composed of complex mixtures of chemicals with potential for bioactive compounds with antimicrobial properties. This study focused on kochia (Kochia scoparia), one of many weedy plant species considered invasive in many agricultural systems. Extraction of compounds in water yielded an exudate mass equivalent to 7% of the original seed mass used. Water-soluble exudates were tested against 16 known plant pathogens in disk diffusion assays and kochia exudates were found to inhibit Colletotrichum graminicola, the fungal causative agent of anthracnose and stalk rot in maize. The narrow range of fungi found as targets suggested the mechanism of inhibition may be specific rather than broadly antifungal. A decline in viability of cells over four orders of magnitude occurred within six hours of exposure to exudate. The minimum inhibitory concentration was 3.125 mg L-1. Hyphae formation in C. graminicola appeared inhibited following exposure to the exudate. Small molecular weight compounds as determined by GC/MS analysis showed high relative amounts of the sugars fructose, galactopyranose, glucose, and sorbitol, along with moderate proportions of organic acids and amino acids. Protein content averaged 0.7% in the standard concentration (100 mg mL-1) used for inhibition assays. Size fractionation of the exudate and subsequent disk diffusion assays revealed bioactive fractions with compounds in the MW range <5 kDa. To the best of our knowledge, this study is the first to show promising bioactivity against C. graminicola that was associated with water-extractable compounds from a common weed species. The results suggest that seeds of persistent plant species with long-lived seed banks like kochia may have potential for use in the discovery of compounds active in inhibiting fungal pathogens.

Influence of the geophagous earthworm Aporrectodea sp. on fate of bisphenol A and a branched 4-nonylphenol isomer in soil.

Large amounts of endocrine disrupting chemicals (EDCs) including bisphenol A (BPA) and nonylphenol (NP) are released into the soil due to the application of biosolids. Earthworms are the predominant biomass in many terrestrial ecosystems and profoundly influence the physico-chemical and biological properties of soils. However, information about the effects of earthworm activities on the behaviors of EDCs in soil is still limited. Here, the effects of earthworms on mineralization, degradation, and bound residue formation of BPA and NP were investigated using the 14C tracer technique. The results showed that earthworms did not affect mineralization of BPA, but significantly inhibited bound residue formation of BPA and changed the size distribution of BPA residues within humic substances. Regarding NP, earthworms significantly inhibited mineralization and bound residue formation, and thus significantly promoted the degradation of NP and NP's metabolites in soil. After nine days of incubation, 75% and 46% of the initially applied 14C-BPA and 14C-NP were already present in bound residues, respectively, indicating that the major route of degradation of BPA and NP in soil was bound-residue formation. Among total 14C-BPA or 14C-NP residues accumulated in earthworms, bound residues were also predominant (>50%), implying that risk assessment of EDCs based on their concentrations of free form in earthworms might be significantly underestimated. Taken together, our results suggest that fate of EDCs in soil not only depended on their physico-chemical properties but also was intensively affected by earthworm activities, underlining that effects of earthworms should be considered when evaluating environmental behavior and potential risk of EDCs in soil.

Effects of triclosan and triclocarban on denitrification and N2O emissions in paddy soil.

Triclosan (TCS) and triclocarban (TCC) are two common antimicrobial compounds, which are widely used as ingredients in pharmaceuticals and personal care products. They occur ubiquitously in soil due to biosolid application as agricultural fertilizers, but their influence on microbially mediated soil biogeochemical processes is poorly understood. We tested the effects of varying concentrations of TCS and TCC applied both individually and together on denitrification and N2O emissions in paddy soil. We also quantified denitrification functional gene abundances by q-PCR to elucidate the microbial mechanisms of TCS and TCC's effects. Our results showed that TCS and TCC exposure both individually and together significantly (p < 0.05) inhibited denitrification (7.0-36.7%) and N2O emissions (15.4-86.4%) except for the 0.01 mg kg-1 TCC treatment in which denitrification was slightly but significantly (p < 0.05) stimulated. The inhibitory effects of TCS and TCC exposure were mainly attributed to their negative net effects on denitrifying bacteria as suggested by the decrease in abundances of 16S rRNA, narG, nirK and clade I nosZ genes in the TCS and TCC treatments. Overall, we found that TCS and TCC exposure in paddy soil could substantially alter nitrogen cycling in rice paddy ecosystems by inhibiting denitrification and N2O emissions. These effects should be taken into consideration when evaluating the environmental impacts of TCS and TCC.

Comparing DNA, RNA and protein levels for measuring microbial dynamics in soil microcosms amended with nitrogen fertilizer.

To what extent multi-omic techniques could reflect in situ microbial process rates remains unclear, especially for highly diverse habitats like soils. Here, we performed microcosm incubations using sandy soil from an agricultural site in Midwest USA. Microcosms amended with isotopically labeled ammonium and urea to simulate a fertilization event showed nitrification (up to 4.1 ± 0.87 µg N-NO3- g-1 dry soil d-1) and accumulation of N2O after 192 hours of incubation. Nitrification activity (NH4+ → NH2OH → NO → NO2- → NO3-) was accompanied by a 6-fold increase in relative expression of the 16S rRNA gene (RNA/DNA) between 10 and 192 hours of incubation for ammonia-oxidizing bacteria Nitrosomonas and Nitrosospira, unlike archaea and comammox bacteria, which showed stable gene expression. A strong relationship between nitrification activity and betaproteobacterial ammonia monooxygenase and nitrite oxidoreductase transcript abundances revealed that mRNA quantitatively reflected measured activity and was generally more sensitive than DNA under these conditions. Although peptides related to housekeeping proteins from nitrite-oxidizing microorganisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. Altogether, these findings underscore the strengths and limitations of multi-omic approaches for assessing diverse microbial communities in soils and provide new insights into nitrification.

DNA buoyant density shifts during 15N-DNA stable isotope probing.

DNA-based stable isotope probing (SIP) is a novel technique for the identification of organisms actively assimilating isotopically labeled compounds. Herein, we define the limitations to using 15N-labeled substrates for SIP and propose modifications to compensate for these shortcomings. Changes in DNA buoyant density (BD) resulting from 15N incorporation were determined using cultures of disparate GC content (Escherichia coli and Micrococcus luteus). Incorporation of 15N into DNA increased BD by 0.015+/-0.002 g mL(-1) for E. coli and 0.013+/-0.002 g mL(-1) for M. luteus. The DNA BD shift was greatly increased (0.045 g mL(-1)) when dual isotope (13C plus 15N) labeling was employed. Despite the limited DNA BD shift following 15N enrichment, we found the use of gradient fractionation, followed by a comparison of T-RFLP profiles from fractions of labeled and control treatments, facilitated detection of enrichment in DNA samples from either cultures or soil.

Detection of tetracycline resistance genes by PCR methods.

Rapid, accurate, and sensitive determination of antibiotic resistance profiles of various human and animal pathogens becomes a vital prerequisite for successful therapeutic intervention in the face of the increased occurrences of drug-resistant bacterial infections. The current methods, which are dependent on cultivation of pathogens and phenotypic expression of antibiotic resistance, usually require excessive time, special microbiological equipment, and qualified personnel. However, even with all these requisites, for example, no bacteria can be grown from more than 80% of all clinical samples sent to clinical microbiology laboratories. Besides the cultivation limitations, the cultivation-based determination of an antibiotic resistance profile lacks the genotypic information, which is essential for understanding the epidemiology and routes of transmission of antibiotic resistance genes. These genes often reside on mobile genetic elements and can move freely between commensal and pathogenic microbiota, occurring even between taxonomically distant clinical and environmental microbiota. Therefore, development of genotyping methods for detection of antibiotic resistance genes is highly desirable for fast, accurate, and sensitive detection of antibiotic resistance genes in a broad range of pathogenic and commensal bacteria in both clinical and environmental samples. As a model for our studies we have chosen the genes conferring resistance to tetracyclines. Tetracyclines belong to a family of broad-spectrum antibiotics that include tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, and a number of other semisynthetic derivatives. These antibiotics inhibit protein synthesis in Gram-positive and Gram-negative bacteria by preventing the binding of aminoacyl-tRNA molecules to the 30S ribosomal subunit. The antibiotics of this group were introduced in the late 1950s and since then have been widely used in clinical and veterinary medicine, as well as for prophylaxis and growth promotion in food animals. Because of the possible misuse and overuse of these drugs, resistance to this class of antibiotics is widespread among many clinical isolates, thus limiting the utility of tetracyclines in treating infections. Despite this shortcoming, antibiotics of this class still remain in the active arsenal for dermatologists to treat skin infections such as acne and rosacea.