Isolation of novel cold-tolerance genes from rhizosphere microorganisms of Antarctic plants by functional metagenomics.

The microorganisms that thrive in Antarctica, one of the coldest environments on the planet, have developed diverse adaptation mechanisms to survive in these extreme conditions. Through functional metagenomics, in this work, 29 new genes related to cold tolerance have been isolated and characterized from metagenomic libraries of microorganisms from the rhizosphere of two Antarctic plants. Both libraries were hosted in two cold-sensitive strains of Escherichia coli: DH10B ΔcsdA and DH10B ΔcsdA Δrnr. The csdA gene encodes a DEAD-box RNA helicase and rnr gene encodes an exoribonuclease, both essential for cold-adaptation. Cold-tolerance tests have been carried out in solid and liquid media at 15°C. Among the cold-tolerance genes identified, 12 encode hypothetical and unknown proteins, and 17 encode a wide variety of different proteins previously related to other well-characterized ones involved in metabolism reactions, transport and membrane processes, or genetic information processes. Most of them have been connected to cold-tolerance mechanisms. Interestingly, 13 genes had no homologs in E. coli, thus potentially providing entirely new adaptation strategies for this bacterium. Moreover, ten genes also conferred resistance to UV-B radiation, another extreme condition in Antarctica.

Viroscope: Plant viral diagnosis from high-throughput sequencing data using biologically-informed genome assembly coverage.

High-throughput sequencing (HTS) methods are transforming our capacity to detect pathogens and perform disease diagnosis. Although sequencing advances have enabled accessible and point-of-care HTS, data analysis pipelines have yet to provide robust tools for precise and certain diagnosis, particularly in cases of low sequencing coverage. Lack of standardized metrics and harmonized detection thresholds confound the problem further, impeding the adoption and implementation of these solutions in real-world applications. In this work, we tackle these issues and propose biologically-informed viral genome assembly coverage as a method to improve diagnostic certainty. We use the identification of viral replicases, an essential function of viral life cycles, to define genome coverage thresholds in which biological functions can be described. We validate the analysis pipeline, Viroscope, using field samples, synthetic and published datasets, and demonstrate that it provides sensitive and specific viral detection. Furthermore, we developed Viroscope.io a web-service to provide on-demand HTS data viral diagnosis to facilitate adoption and implementation by phytosanitary agencies to enable precise viral diagnosis.

Atrazine pollution in groundwater and raw bovine milk: Water quality, bioaccumulation and human risk assessment.

Atrazine herbicide can bioaccumulate over time and thus affect humans for generations to come. However, scarce studies have evaluated its bioaccumulation potential in bovine milk, a nutritional staple for children and the elderly both domestically and internationally. This study aimed to determine its concentration in groundwater and bovine milk, as well as the risks it is likely to pose for human health. Eighteen dairy farms in the Pampean plain of Argentina were analyzed. A strong correlation was found between the chemical composition and the geomorphological characteristics of the plain. In addition, increased salinity was observed in the groundwater at greater distances from the aquifer's recharge area. Atrazine was quantified in 50 % of the groundwater samples (at values ranging from 0.07 to 1.40 µg/L), and in 89 % of the bovine milk samples (from 2.51 to 20.97 µg/L). Moreover, atrazine levels in 44.4 % of the groundwater samples and 11.1 % of the bovine milk samples (n = 18) exceeded the limits internationally established as safe for human consumption. The hazard quotient (HQ) values of the compound were negligible for children and adults, both in groundwater (child = 9.7E-4, adult = 4.5E-4) and in milk (child = 1.0E-2, adult = 1.6E-3). The estimated cancer risk (CR) values need further evaluation (child = 7.8E-6, adult = 3.6E-6 in groundwater; child = 6.6E-5, adult = 1.3E-5 in milk). In both types of samples, the HQ and CR of residual atrazine were higher for children than for adults. Nevertheless, bioaccumulation factors suggest that dairy cows have a moderate capacity to incorporate atrazine from abiotic matrices. This is the first report on residual atrazine in bovine milk in Argentina. The results presented here indicate that the status of atrazine contamination in the area should continue to be monitored in order to assess its long-term impact on public health.

Groundwater quality and vulnerability in farms from agricultural-dairy basin of the Argentine Pampas.

Agricultural and livestock activities strongly influence groundwater quality and conditioning its use as water supply in rural areas. The aim was to determine the quality and suitability of the groundwater supply used in dairy farms of an agricultural area of Pampa plain of Córdoba (Argentina). Piper's diagram showed that the groundwater types were sodium bicarbonate, sodium bicarbonate-chloride, sodium chloride-sulfate, and sodium sulfate. Physicochemical parameters revealed that cations and anions showed a high and significant correlation in water samples, indicating a strong water-rock interaction. Nitrate (NO3-) content was significantly correlated with pH, water well depth, and distance from contamination sources. A high positive correlation between arsenic (As) and bicarbonate, sulfate, sodium, and chloride (p < 0.05) indicates a similar origin. Among the pesticides monitored, 2,4-D was detected in 25% of groundwater samples (0.4 to 0.8 µg/L) coinciding with the ordinary application practices. In general, most of the groundwater samples did not comply with national and international regulations for drinking water and dairy hygiene, due to the high content of As, NO3-, bacteria, and the presence of 2,4-D herbicide. However, the quality of water was suitable for livestock drink. The data obtained in this study contribute to a better understanding of the contamination processes taking place and improve the agricultural and livestock management for an efficient use of this resource by dairy farmers.

Monitoring of Atrazine Pollution and its Spatial-Seasonal Variation on Surface Water Sources of an Agricultural River Basin.

Surface water sources are greatly impacted in areas with major agricultural land use. The atrazine quantification in surface waters as well as the spatial-temporal patterns of this herbicide was studied to detect pollution hotspots and to understand the putative factors responsible of its occurrence at the Ctalamochita river basin. The samples were collected on the aeolian fluvial plain of the river basin during five consecutive years. The results showed the high ubiquity of this compound and several sites with hazardous concentration (exceeding 0.1 µg/L international guidelines). The frequencies of quantification range from 67 to 100% in spring and 33%-67% in autumn. The atrazine content in surface water increased during the warm-rainy season, as consequence of atrazine application events (coinciding to the prevalent crop type). Overall, the study highlights the factors that could have favored atrazine pollution in the river basin such as land use, transport by runoff processes and atmospheric deposition.

The microbial community from the early-plant colonizer (Baccharis linearis) is required for plant establishment on copper mine tailings.

Plants must deal with harsh environmental conditions when colonizing abandoned copper mine tailings. We hypothesized that the presence of a native microbial community can improve the colonization of the pioneer plant, Baccharis linearis, in soils from copper mining tailings. Plant growth and microbial community compositions and dynamics were determined in cultivation pots containing material from two abandoned copper mining tailings (Huana and Tambillos) and compared with pots containing fresh tailings or surrounding agricultural soil. Controls without plants or using irradiated microbe-free substrates, were also performed. Results indicated that bacteria (Actinobacteria, Gammaproteobacteria, and Firmicutes groups) and fungi (Glomus genus) are associated with B. linearis and may support plant acclimation, since growth parameters decreased in both irradiated (transiently without microbial community) and fresh tailing substrates (with a significantly different microbial community). Consistently, the composition of the bacterial community from abandoned copper mining tailings was more impacted by plant establishment than by differences in the physicochemical properties of the substrates. Bacteria located at B. linearis rhizoplane were clearly the most distinct bacterial community compared with those of fresh tailings, surrounding soil and non-rhizosphere abandoned tailings substrates. Beta diversity analyses showed that the rhizoplane bacterial community changed mainly through species replacement (turnover) than species loss (nestedness). In contrast, location/geographical conditions were more relevant than interaction with the plants, to explain fungal community differences.

Functional metagenomics of extreme environments.

The bioprospecting of enzymes that operate under extreme conditions is of particular interest for many biotechnological and industrial processes. Nevertheless, there is a considerable limitation to retrieve novel enzymes as only a small fraction of microorganisms derived from extreme environments can be cultured under standard laboratory conditions. Functional metagenomics has the advantage of not requiring the cultivation of microorganisms or previous sequence information to known genes, thus representing a valuable approach for mining enzymes with new features. In this review, we summarize studies showing how functional metagenomics was employed to retrieve genes encoding for proteins involved not only in molecular adaptation and resistance to extreme environmental conditions but also in other enzymatic activities of biotechnological interest.

Exploring the diversity of arsenic resistance genes from acid mine drainage microorganisms.

The microbial communities from the Tinto River, a natural acid mine drainage environment, were explored to search for novel genes involved in arsenic resistance using a functional metagenomic approach. Seven pentavalent arsenate resistance clones were selected and analysed to find the genes responsible for this phenotype. Insights about their possible mechanisms of resistance were obtained from sequence similarities and cellular arsenic concentration. A total of 19 individual open reading frames were analysed, and each one was individually cloned and assayed for its ability to confer arsenic resistance in Escherichia coli cells. A total of 13 functionally active genes involved in arsenic resistance were identified, and they could be classified into different global processes: transport, stress response, DNA damage repair, phospholipids biosynthesis, amino acid biosynthesis and RNA-modifying enzymes. Most genes (11) encode proteins not previously related to heavy metal resistance or hypothetical or unknown proteins. On the other hand, two genes were previously related to heavy metal resistance in microorganisms. In addition, the ClpB chaperone and the RNA-modifying enzymes retrieved in this work were shown to increase the cell survival under different stress conditions (heat shock, acid pH and UV radiation). Thus, these results reveal novel insights about unidentified mechanisms of arsenic resistance.

Novel acid resistance genes from the metagenome of the Tinto River, an extremely acidic environment.

Microorganisms that thrive in acidic environments are endowed with specialized molecular mechanisms to survive under this extremely harsh condition. In this work, we performed functional screening of six metagenomic libraries from planktonic and rhizosphere microbial communities of the Tinto River, an extremely acidic environment, to identify genes involved in acid resistance. This approach has revealed 15 different genes conferring acid resistance to Escherichia coli, most of which encoding putative proteins of unknown function or previously described proteins not known to be related to acid resistance. Moreover, we were able to assign function to one unknown and three hypothetical proteins. Among the recovered genes were the ClpXP protease, the transcriptional repressor LexA and nucleic acid-binding proteins such as an RNA-binding protein, HU and Dps. Furthermore, nine of the retrieved genes were cloned and expressed in Pseudomonas putida and Bacillus subtilis and, remarkably, most of them were able to expand the capability of these bacteria to survive under severe acid stress. From this set of genes, four presented a broad-host range as they enhance the acid resistance of the three different organisms tested. These results expand our knowledge about the different strategies used by microorganisms to survive under extremely acid conditions.

Bioaugmentation with Pseudomonas sp. strain MHP41 promotes simazine attenuation and bacterial community changes in agricultural soils.

Bioremediation is an important technology for the removal of persistent organic pollutants from the environment. Bioaugmentation with the encapsulated Pseudomonas sp. strain MHP41 of agricultural soils contaminated with the herbicide simazine was studied. The experiments were performed in microcosm trials using two soils: soil that had never been previously exposed to s-triazines (NS) and soil that had >20 years of s-triazine application (AS). The efficiency of the bioremediation process was assessed by monitoring simazine removal by HPLC. The simazine-degrading microbiota was estimated using an indicator for respiration combined with most-probable-number enumeration. The soil bacterial community structures and the effect of bioaugmentation on these communities were determined using 16S RNA gene clone libraries and FISH analysis. Bioaugmentation with MHP41 cells enhanced simazine degradation and increased the number of simazine-degrading microorganisms in the two soils. In highly contaminated NS soil, bioaugmentation with strain MHP41 was essential for simazine removal. Comparative analysis of 16S rRNA gene clone libraries from NS and AS soils revealed high bacterial diversity. Bioaugmentation with strain MHP41 promoted soil bacterial community shifts. FISH analysis revealed that bioaugmentation increased the relative abundances of two phylogenetic groups (Acidobacteria and Planctomycetes) in both soils. Although members of the Archaea were metabolically active in these soils, their relative abundance was not altered by bioaugmentation.

Adsorption studies of the herbicide simazine in agricultural soils of the Aconcagua valley, central Chile.

Simazine is a s-triazine herbicide that has been applied worldwide for agriculture. This herbicide is the second most commonly detected pesticide in surface and groundwater in the United States, Europe and Australia. In this study, simazine adsorption behaviour was studied in two agricultural soils of the Aconcagua valley, central Chile. The two studied soils were soil A (loam, 8.5% organic matter content) and soil B (clay-loam, 3.5% organic matter content). Three times higher simazine adsorption capacity was observed in soil A (68.03 mg kg(-1)) compared to soil B (22.03 mg kg(-1)). The simazine adsorption distribution coefficients (K(d)) were 9.32 L kg(-1) for soil A and 7.74 L kg(-1) for soil B. The simazine adsorption enthalpy in soil A was -21.0 kJ mol(-1) while in soil B the adsorption enthalpy value was -11.5 kJ mol(-1). These results indicate that simazine adsorption process in these soils is exothermic, governing H bonds the adsorption process of simazine in both the loam and clay-loam soils. These results and the potentiometric profiles of both soils, suggest that simazine adsorption in soil A is mainly governed by simazine-organic matter interactions and in soil B by simazine-clay interactions. The understanding of simazine sorption-desorption processes is essential to determine the pesticide fate and availability in soil for pest control, biodegradation, runoff and leaching.

Novel s-triazine-degrading bacteria isolated from agricultural soils of central Chile for herbicide bioremediation

s-Triazine-degrading bacterial strains were isolated from long-term simazine-treated agricultural soils of central Chile. The number of culturable heterotrophic bacteria of these agricultural soils (7 x 10(6) CFU/g of dry soil) was not affected by simazine application on field. The simazine-degrading bacterial strains P51, P52 and C53 were isolated by enrichment in minimal medium using simazine as the sole nitrogen source. Resting cells of strains P51 and P52 degraded >80 percent of simazine within 48 hrs, whereas strain C53 was able to remove >60 percent of the herbicide. The atzA and atzD genes of the s-triazine upper and lower catabolic pathways were detected in strains P51 and C53, while only atzD gene was observed in strain P52. To compare the bacterial 16S rRNA gene sequence structure, ARDRA were performed using the restriction enzymes Msp1 and Hha1. ARDRA indicated that strain P52 was a different ribotype than C53 and P51 strains. For further characterization the novel isolates were identified by 16S rRNA gene sequencing. Strains C53 and P51 belong to the genus Stenotrophomonas and the strain P52 belongs to the genus Arthrobacter . s -Triazine-degrading bacterial strains isolated from contaminated soils could be used as biocatalysts for bioremediation of these herbicides.

Isolation and characterization of a novel simazine-degrading bacterium from agricultural soil of central Chile, Pseudomonas sp. MHP41.

s-Triazine herbicides are used extensively in South America in agriculture and forestry. In this study, a bacterium designated as strain MHP41, capable of degrading simazine and atrazine, was isolated from agricultural soil in the Quillota valley, central Chile. Strain MHP41 is able to grow in minimal medium, using simazine as the sole nitrogen source. In this medium, the bacterium exhibited a growth rate of mu=0.10 h(-1), yielding a high biomass of 4.2 x 10(8) CFU mL(-1). Resting cells of strain MHP41 degrade more than 80% of simazine within 60 min. The atzA, atzB, atzC, atzD, atzE and atzF genes encoding the enzymes of the simazine upper and lower pathways were detected in strain MHP41. The motile Gram-negative bacterium was identified as a Pseudomonas sp., based on the Biolog microplate system and comparative sequence analyses of the 16S rRNA gene. Amplified ribosomal DNA restriction analysis allowed the differentiation of strain MHP41 from Pseudomonas sp. ADP. The comparative 16S rRNA gene sequence analyses suggested that strain MHP41 is closely related to Pseudomonas nitroreducens and Pseudomonas multiresinovorans. This is the first s-triazine-degrading bacterium isolated in South America. Strain MHP41 is a potential biocatalyst for the remediation of s-triazine-contaminated environments.