Toward a Census of Bacteria in Soil

For more than a century, microbiologists have sought to determine the species richness of bacteria in soil, but the extreme complexity and unknown structure of soil microbial communities have obscured the answer. We developed a statistical model that makes the problem of estimating richness statistically accessible by evaluating the characteristics of samples drawn from simulated communities with parametric community distributions. We identified simulated communities with rank-abundance distributions that followed a truncated lognormal distribution whose samples resembled the structure of 16S rRNA gene sequence collections made using Alaskan and Minnesotan soils. The simulated communities constructed based on the distribution of 16S rRNA gene sequences sampled from the Alaskan and Minnesotan soils had a richness of 5,000 and 2,000 operational taxonomic units (OTUs), respectively, where an OTU represents a collection of sequences not more than 3% distant from each other. To sample each of these OTUs in the Alaskan 16S rRNA gene library at least twice, 480,000 sequences would be required; however, to estimate the richness of the simulated communities using nonparametric richness estimators would require only 18,000 sequences. Quantifying the richness of complex environments such as soil is an important step in building an ecological framework. We have shown that generating sufficient sequence data to do so requires less sequencing effort than completely sequencing a bacterial genome

Alaskan soil carbon stocks: spatial variability and dependence on environmental factors

The direction and magnitude of soil organic carbon (SOC) changes in response to climate change depend on the spatial and vertical distributions of SOC. We estimated spatially resolved SOC stocks from surface to C horizon, distinguishing active-layer and permafrost-layer stocks, based on geospatial analysis of 472 soil profiles and spatially referenced environmental variables for Alaska. Total Alaska state-wide SOC stock was estimated to be 77 Pg, with 61% in the active-layer, 27% in permafrost, and 12% in non-permafrost soils. Prediction accuracy was highest for the active-layer as demonstrated by highest ratio of performance to deviation (1.5). Large spatial variability was predicted, with whole-profile, active-layer, and permafrost-layer stocks ranging from 1–296 kg C m<sup>−2</sup>, 2–166 kg m<sup>−2</sup>, and 0–232 kg m<sup>−2</sup>, respectively. Temperature and soil wetness were found to be primary controllers of whole-profile, active-layer, and permafrost-layer SOC stocks. Secondary controllers, in order of importance, were found to be land cover type, topographic attributes, and bedrock geology. The observed importance of soil wetness rather than precipitation on SOC stocks implies that the poor representation of high-latitude soil wetness in Earth system models may lead to large uncertainty in predicted SOC stocks under future climate change scenarios. Under strict caveats described in the text and assuming temperature changes from the A1B Intergovernmental Panel on Climate Change emissions scenario, our geospatial model indicates that the equilibrium average 2100 Alaska active-layer depth could deepen by 11 cm, resulting in a thawing of 13 Pg C currently in permafrost. The equilibrium SOC loss associated with this warming would be highest under continuous permafrost (31%), followed by discontinuous (28%), isolated (24.3%), and sporadic (23.6%) permafrost areas. Our high-resolution mapping of soil carbon stock reveals the potential vulnerability of high-latitude soil carbon and can be used as a basis for future studies of anthropogenic and climatic perturbations

A Response Regulator from a Soil Metagenome Enhances Resistance to the β-lactam Antibiotic Carbenicillin in \u3cem\u3eEscherichia Coli\u3c/em\u3e

Functional metagenomic analysis of soil metagenomes is a method for uncovering as-yet unidentified mechanisms for antibiotic resistance. Here we report an unconventional mode by which a response regulator derived from a soil metagenome confers resistance to the β-lactam antibiotic carbenicillin in Escherichia coli. A recombinant clone (βlr16) harboring a 5,169 bp DNA insert was selected from a metagenomic library previously constructed from a remote Alaskan soil. The βlr16 clone conferred specific resistance to carbenicillin, with limited increases in resistance to other tested antibiotics, including other β-lactams (penicillins and cephalosporins), rifampin, ciprofloxacin, erythromycin, chloramphenicol, nalidixic acid, fusidic acid, and gentamicin. Resistance was more pronounced at 24°C than at 37°C. Zone-of-inhibition assays suggested that the mechanism of carbenicillin resistance was not due to antibiotic inactivation. The DNA insert did not encode any genes known to confer antibiotic resistance, but did have two putative open reading frames (ORFs) that were annotated as a metallopeptidase and a two-component response regulator. Transposon mutagenesis and subcloning of the two ORFs followed by phenotypic assays showed that the response regulator gene was necessary and sufficient to confer the resistance phenotype. Quantitative reverse transcriptase PCR showed that the response regulator suppressed expression of the ompF porin gene, independently of the small RNA regulator micF, and enhanced expression of the acrD, mdtA, and mdtB efflux pump genes. This work demonstrates that antibiotic resistance can be achieved by the modulation of gene regulation by heterologous DNA. Functional analyses such as these can be important for making discoveries in antibiotic resistance gene biology and ecology

Mimicking climate warming effects on Alaskan soil microbial communities via gradual temperature increase

Climate change can trigger shifts in community structure and may therefore pose a severe threat to soil microbial communities, especially in high northern latitudes such as the Arctic. Arctic soils are covered by snow and ice throughout most of the year. This insulation shields them from high temperature variability and low surface temperatures. If this protective layer thaws, these soils are predicted to warm up at 1.5x to 4x the rate of other terrestrial biomes. In this study, we sampled arctic soils from sites with different elevations in Alaska, incubated them for 5 months with a simulated, gradual or abrupt temperature increase of +5 °C, and compared bacterial and fungal community compositions after the incubation. We hypothesized that the microbial communities would not significantly change with a gradual temperature treatment, whereas an abrupt temperature increase would decrease microbial diversity and shift community composition. The only differences in community composition that we observed were, however, related to the two elevations. The abrupt and gradual temperature increase treatments did not change the microbial community composition as compared to the control indicating resistance of the microbial community to changes in temperature. This points to the potential importance of microbial dormancy and resting stages in the formation of a “buffer” against elevated temperatures. Microbial resting stages might heavily contribute to microbial biomass and thus drive the responsiveness of arctic ecosystems to climate change

Novel resistance functions uncovered using functional metagenomic investigations of resistance reservoirs

Rates of infection with antibiotic-resistant bacteria have increased precipitously over the past several decades, with far-reaching healthcare and societal costs. Recent evidence has established a link between antibiotic resistance genes in human pathogens and those found in non-pathogenic, commensal, and environmental organisms, prompting deeper investigation of natural and human-associated reservoirs of antibiotic resistance. Functional metagenomic selections, in which shotgun-cloned DNA fragments are selected for their ability to confer survival to an indicator host, have been increasingly applied to the characterization of many antibiotic resistance reservoirs. These experiments have demonstrated that antibiotic resistance genes are highly diverse and widely distributed, many times bearing little to no similarity to known sequences. Through unbiased selections for survival to antibiotic exposure, functional metagenomics can improve annotations by reducing the discovery of false-positive resistance and by allowing for the identification of previously unrecognizable resistance genes. In this review, we summarize the novel resistance functions uncovered using functional metagenomic investigations of natural and human-impacted resistance reservoirs. Examples of novel antibiotic resistance genes include those highly divergent from known sequences, those for which sequence is entirely unable to predict resistance function, bifunctional resistance genes, and those with unconventional, atypical resistance mechanisms. Overcoming antibiotic resistance in the clinic will require a better understanding of existing resistance reservoirs and the dissemination networks that govern horizontal gene exchange, informing best practices to limit the spread of resistance-conferring genes to human pathogens

Functional metagenomics reveals diverse beta-lactamases in a remote Alaskan soil

Despite the threat posed by antibiotic resistance in infectious bacteria, little is known about the diversity, distribution and origins of resistance genes, particularly among the as yet unculturable environmental bacteria. One potentially rich but largely unstudied environmental reservoir is soil. The complexity of its microbial community coupled with its high density of antibiotic-producing bacteria makes the soil a likely origin for diverse antibiotic resistance determinants. To investigate antibiotic resistance genes among uncultured bacteria in an undisturbed soil environment, we undertook a functional metagenomic analysis of a remote Alaskan soil. We report that this soil is a reservoir for b-lactamases that function in Escherichia coli, including divergent b-lactamases and the first bifunctional b-lactamase. Our findings suggest that even in the absence of selective pressure imposed by anthropogenic activity, the soil microbial community in an unpolluted site harbors unique and ancient b-lactam resistance determinants. Moreover, despite their evolutionary distance from previously known genes, the Alaskan b-lactamases confer resistance on E. coli without manipulating its gene expression machinery, demonstrating the potential for soil resistance genes to compromise human health, if transferred to pathogens

Novel clinically relevant antibiotic resistance genes associated with sewage sludge and industrial waste streams revealed by functional metagenomic screening

A growing body of evidence indicates that anthropogenic activities can result in increased prevalence of antimicrobial resistance genes (ARGs) in bacteria in natural environments. Many environmental studies have used next-generation sequencing methods to sequence the metagenome. However, this approach is limited as it does not identify divergent uncharacterized genes or demonstrate activity. Characterization of ARGs in environmental metagenomes is important for understanding the evolution and dissemination of resistance, as there are several examples of clinically important resistance genes originating in environmental species. The current study employed a functional metagenomic approach to detect genes encoding resistance to extended spectrum β-lactams (ESBLs) and carbapenems in sewage sludge, sludge amended soil, quaternary ammonium compound (QAC) impacted reed bed sediment and less impacted long term curated grassland soil. ESBL and carbapenemase genes were detected in sewage sludge, sludge amended soils and QAC impacted soil with varying degrees of homology to clinically important β-lactamase genes. The flanking regions were sequenced to identify potential host background and genetic context. Novel β-lactamase genes were found in Gram negative bacteria, with one gene adjacent to an insertion sequence ISPme1, suggesting a recent mobilization event and/ the potential for future transfer. Sewage sludge and quaternary ammonium compound (QAC) rich industrial effluent appear to disseminate and/or select for ESBL genes which were not detected in long term curated grassland soils. This work confirms the natural environment as a reservoir of novel and mobilizable resistance genes, which may pose a threat to human and animal health.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.published version, accepted version, submitted versio