From soil to sequence: filling the critical gap in genome-resolved metagenomics is essential to the future of soil microbial ecology.

Soil microbiomes are heterogeneous, complex microbial communities. Metagenomic analysis is generating vast amounts of data, creating immense challenges in sequence assembly and analysis. Although advances in technology have resulted in the ability to easily collect large amounts of sequence data, soil samples containing thousands of unique taxa are often poorly characterized. These challenges reduce the usefulness of genome-resolved metagenomic (GRM) analysis seen in other fields of microbiology, such as the creation of high quality metagenomic assembled genomes and the adoption of genome scale modeling approaches. The absence of these resources restricts the scale of future research, limiting hypothesis generation and the predictive modeling of microbial communities. Creating publicly available databases of soil MAGs, similar to databases produced for other microbiomes, has the potential to transform scientific insights about soil microbiomes without requiring the computational resources and domain expertise for assembly and binning.

Biogeographic responses and niche occupancy of microbial communities following long-term land-use change.

Understanding the effects of forest-to-agriculture conversion on microbial diversity has been a major goal in soil ecological studies. However, linking community assembly to the ruling ecological processes at local and regional scales remains challenging. Here, we evaluated bacterial community assembly patterns and the ecological processes governing niche specialization in a gradient of geography, seasonality, and land-use change, totaling 324 soil samples, 43 habitat characteristics (abiotic factors), and 16 metabolic and co-occurrence patterns (biotic factors), in the Brazilian Atlantic Rainforest, a subtropical biome recognized as one the world's largest and most threatened hotspots of biodiversity. Pairwise beta diversities were lower in pastures than in forest and no-till soils. Pasture communities showed a predominantly neutral model, regarding stochastic processes, with moderate dispersion, leading to biotic homogenization. Most no-till and forest microbial communities followed a niche-based model, with low rates of dispersal and weak homogenizing selection, indicating niche specialization or variable selection. Historical and evolutionary contingencies, as represented by soil type, season, and dispersal limitation were the main drivers of microbial assembly and processes at the local scale, markedly correlated with the occurrence of endemic microbes. Our results indicate that the patterns of assembly and their governing processes are dependent on the niche occupancy of the taxa evaluated (generalists or specialists). They are also more correlated with historical and evolutionary contingencies and the interactions among taxa (i.e., co-occurrence patterns) than the land-use change itself.

Responses of Low-Cost Input Combinations on the Microbial Structure of the Maize Rhizosphere for Greenhouse Gas Mitigation and Plant Biomass Production.

The microbial composition of the rhizosphere and greenhouse gas (GHG) emissions under the most common input combinations in maize (Zea mays L.) cultivated in Brazil have not been characterized yet. In this study, we evaluated the influence of maize stover coverage (S), urea-topdressing fertilization (F), and the microbial inoculant Azospirillum brasilense (I) on soil GHG emissions and rhizosphere microbial communities during maize development. We conducted a greenhouse experiment and measured methane (CH4), carbon dioxide (CO2), and nitrous oxide (N2O) fluxes from soil cultivated with maize plants under factorial combinations of the inputs and a control treatment (F, I, S, FI, FS, IS, FIS, and control). Plant biomass was evaluated, and rhizosphere soil samples were collected at V5 and V15 stages and DNA was extracted. The abundance of functional genes (mcrA, pmoA, nifH, and nosZ) was determined by quantitative PCR (qPCR) and the structure of the microbial community was assessed through 16S rRNA amplicon sequencing. Our results corroborate with previous studies which used fewer input combinations and revealed different responses for the following three inputs: F increased N2O emissions around 1 week after application; I tended to reduce CH4 and CO2 emissions, acting as a plant growth stimulator through phytohormones; S showed an increment for CO2 emissions by increasing carbon-use efficiency. IS and FIS treatments presented significant gains in biomass that could be related to Actinobacteria (19.0%) and Bacilli (10.0%) in IS, and Bacilli (9.7%) in FIS, which are the microbial taxa commonly associated with lignocellulose degradation. Comparing all factors, the IS (inoculant + maize stover) treatment was considered the best option for plant biomass production and GHG mitigation since FIS provides small gains toward the management effort of F application.

Ecological Processes Shaping Bulk Soil and Rhizosphere Microbiome Assembly in a Long-Term Amazon Forest-to-Agriculture Conversion.

Forest-to-agriculture conversion has been identified as a major threat to soil biodiversity and soil processes resilience, although the consequences of long-term land use change to microbial community assembly and ecological processes have been often neglected. Here, we combined metagenomic approach with a large environmental dataset, to (i) identify the microbial assembly patterns and, (ii) to evaluate the ecological processes governing microbial assembly, in bulk soil and soybean rhizosphere, along a long-term forest-to-agriculture conversion chronosequence, in Eastern Amazon. We hypothesized that (i) microbial communities in bulk soil and rhizosphere have different assembly patterns and (ii) the weight of the four ecological processes governing assembly differs between bulk soil and rhizosphere and along the chronosequence in the same fraction. Community assembly in bulk soil fitted most the zero-sum multinomial (ZSM) neutral-based model, regardless of time. Low to intermediate dispersal was observed. Decreasing influence of abiotic factors was counterbalanced by increasing influence of biotic factors, as the chronosequence advanced. Undominated ecological processes of dispersal limitation and variable selection governing community assembly were observed in this soil fraction. For soybean rhizosphere, community assembly fitted most the lognormal niche-based model in all chronosequence areas. High dispersal and an increasing influence of abiotic factors coupled with a decreasing influence of biotic factors were found along the chronosequence. Thus, we found a dominant role of dispersal process governing microbial assembly with a secondary effect of homogeneous selection process, mainly driven by decreasing aluminum and increased cations saturation in soil solution, due to long-term no-till cropping. Together, our results indicate that long-term no-till lead community abundances in bulk soil to be in a transient and conditional state, while for soybean rhizosphere, community abundances reach a periodic and permanent distribution state. Dominant dispersal process in rhizosphere, coupled with homogeneous selection, brings evidences that soybean root system selects microbial taxa via trade-offs in order to keep functional resilience of soil processes.

A Novel Multifunctional ß-N-Acetylhexosaminidase Revealed through Metagenomics of an Oil-Spilled Mangrove.

The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a ß-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a ß-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-ß-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-ß-d-galactopyranoside, and 4-Nitrophenyl ß-d-glucopyranoside, presenting ß-N-acetylglucosaminidase, ß-glucosidase, and ß-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel ß-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product.

Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries.

A significant proportion of oral bacteria are unable to undergo cultivation by existing techniques. In this regard, the microbiota from root canals still requires complementary characterization. The present study aimed at the identification of bacteria by sequence analysis of 16S rDNA clone libraries from seven endodontically infected teeth. Samples were collected from the root canals, subjected to the PCR with universal 16S rDNA primers, cloned and partially sequenced. Clones were clustered into groups of closely related sequences (phylotypes) and identification to the species level was performed by comparative analysis with the GenBank, EMBL and DDBJ databases, according to a 98% minimum identity. All samples were positive for bacteria and the number of phylotypes detected per subject varied from two to 14. The majority of taxa (65.2%) belonged to the phylum Firmicutes of the Gram-positive bacteria, followed by Proteobacteria (10.9%), Spirochaetes (4.3%), Bacteroidetes (6.5%), Actinobacteria (2.2%) and Deferribacteres (2.2%). A total of 46 distinct taxonomic units was identified. Four clones with low similarity to sequences previously deposited in the databases were sequenced to nearly full extent and were classified taxonomically as novel representatives of the order Clostridiales, including a putative novel species of Mogibacterium. The identification of novel phylotypes associated with endodontic infections suggests that the endodontium may still harbour a relevant proportion of uncharacterized taxa.

Growth and siderophore production of Xylella fastidiosa under iron-limited conditions.

In this study, the production of siderophores by Xylella fastidiosa from the citrus bacteria isolate 31b9a5c (FAPESP - ONSA, Brazil) was investigated. The preliminary evidence supporting the existence of siderophore in X. fastidiosa was found during the evaluation of sequencing data generated in our lab using the BLAST-X tool, which indicated putative open reading frames (ORFs) associated with iron-binding proteins. In an iron-limited medium siderophores were detected in the supernatant of X. fastidiosa cultures. The endophytic bacterium Methylobacterium extorquens was also evaluated. Capillary electrophoresis was used to separate putative siderophores produced by X. fastidiosa. The bacterial culture supernatants of X. fastidiosa were identified negative for hydroxamate and catechol and positive for M. extorquens that secreted hydroxamate-type siderophores.