Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing.
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ABSTRACT:
SUBMITTER: Waschulin V
PROVIDER: S-EPMC8692599 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Antibiotic resistance genes (ARGs) as a novel type of environmental pollutant pose a health risk to humans. Oxazolidinones are one of the most important antibiotics for the treatment of Gram-positive bacterial infections in humans. Although oxazolidinones are not utilized in the livestock industry, florfenicol is commonly used on farms to treat bacterial infections, which may contribute to the spread of the cfr, optrA, and poxtA genes on farms. Using metagenomics sequencing, we looked into the antibiotic resistome context of florfenicol and oxazolidinone in 10 large-scale commercial farms in China. We identified 490 different resistance genes and 1,515 bacterial genera in the fecal samples obtained from 10 farms. Florfenicol-resistant Kurthia, Escherichia, and Proteus were widely present in these samples. The situation of florfenicol and oxazolidone resistance in pig farms is even more severe. The total number of genes and the abundance of drug resistance genes were higher in pigs than in chickens, including optrA and poxtA. All the samples we collected had a high abundance of fexA and floR. Through nanopore metagenomic analysis of the genetic environment, we found that plasmids, integrative and conjugative element (ICE), and transposons (Tn7-like and Tn558) may play an important role in the spread of floR, cfr, and optrA. Our findings suggest that florfenicol and oxazolidinone resistance genes have diverse genetic environments and are at risk of co-transmission with, for example, tetracycline and aminoglycoside resistance genes. The spread of florfenicol- and oxazolidinone-resistant bacteria on animal farms should be continuously monitored.
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Project description:BackgroundLong reads have gained popularity in the analysis of metagenomics data. Therefore, we comprehensively assessed metagenomics classification tools on the species taxonomic level. We analysed kmer-based tools, mapping-based tools and two general-purpose long reads mappers. We evaluated more than 20 pipelines which use either nucleotide or protein databases and selected 13 for an extensive benchmark. We prepared seven synthetic datasets to test various scenarios, including the presence of a host, unknown species and related species. Moreover, we used available sequencing data from three well-defined mock communities, including a dataset with abundance varying from 0.0001 to 20% and six real gut microbiomes.ResultsGeneral-purpose mappers Minimap2 and Ram achieved similar or better accuracy on most testing metrics than best-performing classification tools. They were up to ten times slower than the fastest kmer-based tools requiring up to four times less RAM. All tested tools were prone to report organisms not present in datasets, except CLARK-S, and they underperformed in the case of the high presence of the host's genetic material. Tools which use a protein database performed worse than those based on a nucleotide database. Longer read lengths made classification easier, but due to the difference in read length distributions among species, the usage of only the longest reads reduced the accuracy. The comparison of real gut microbiome datasets shows a similar abundance profiles for the same type of tools but discordance in the number of reported organisms and abundances between types. Most assessments showed the influence of database completeness on the reports.ConclusionThe findings indicate that kmer-based tools are well-suited for rapid analysis of long reads data. However, when heightened accuracy is essential, mappers demonstrate slightly superior performance, albeit at a considerably slower pace. Nevertheless, a combination of diverse categories of tools and databases will likely be necessary to analyse complex samples. Discrepancies observed among tools when applied to real gut datasets, as well as a reduced performance in cases where unknown species or a significant proportion of the host genome is present in the sample, highlight the need for continuous improvement of existing tools. Additionally, regular updates and curation of databases are important to ensure their effectiveness.
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Project description:Here, a new medium, named intensive soil extract medium (ISEM), based on new soil extract (NSE) using 80% methanol, was used to efficiently isolate previously uncultured bacteria and new taxonomic candidates, which accounted for 49% and 55% of the total isolates examined (n = 258), respectively. The new isolates were affiliated with seven phyla (Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes, and Bacteroidetes). The result of chemical analysis showed that NSE included more diverse components of low-molecular-weight organic substances than two conventional soil extracts made using distilled water. Cultivation of previously uncultured bacteria is expected to extend knowledge through the discovery of new phenotypic, physiological, and functional properties and even roles of unknown genes.IMPORTANCE Both metagenomics and single-cell sequencing can detect unknown genes from uncultured microbial strains in environments, and either method may find the significant potential metabolites and roles of these strains. However, such gene/genome-based techniques do not allow detailed investigations that are possible with cultures. To solve this problem, various approaches for cultivation of uncultured bacteria have been developed, but there are still difficulties in maintaining pure cultures by subculture.
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Project description:Regulation of transcript structure generates transcript diversity and plays an important role in human disease1-7. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure8-16. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns.
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Project description:Our current understanding of Antarctic soils is derived from direct culture on selective media, biodiversity studies based on clone library construction and analysis, quantitative PCR amplification of specific gene sequences and the application of generic microarrays for microbial community analysis. Here, we investigated the biodiversity and functional potential of a soil community at Mars Oasis on Alexander Island in the southern Maritime Antarctic, by applying 454 pyrosequencing technology to a metagenomic library constructed from soil genomic DNA. The results suggest that the commonly cited range of phylotypes used in clone library construction and analysis of 78-730?OTUs (de-replicated to 30-140) provides low coverage of the major groups present (?5%). The vast majority of functional genes (>77%) were for structure, carbohydrate metabolism, and DNA/RNA processing and modification. This study suggests that prokaryotic diversity in Antarctic terrestrial environments appears to be limited at the generic level, with Proteobacteria, Actinobacteria being common. Cyanobacteria were surprisingly under-represented at 3.4% of sequences, although ?1% of the genes identified were involved in CO(2) fixation. At the sequence level there appeared to be much greater heterogeneity, and this might be due to high divergence within the relatively restricted lineages which have successfully colonized Antarctic terrestrial environments.
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2022-05-31 | PXD031213 | Pride
Project description:Antarctica, being the coldest, driest, and windiest continent on Earth, represents the most extreme environment in which a living organism can survive. Under constant exposure to harsh environmental threats, terrestrial Antarctica remains home to a great diversity of microorganisms, indicating that the soil bacteria must have adapted a range of survival strategies that require cell-to-cell communication. Survival strategies include secondary metabolite production, biofilm formation, bioluminescence, symbiosis, conjugation, sporulation, and motility, all of which are often regulated by quorum sensing (QS), a type of bacterial communication. Until now, such mechanisms have not been explored in terrestrial Antarctica. In this study, LuxI/LuxR-based quorum sensing (QS) activity was delineated in soil bacterial isolates recovered from Adams Flat, in the Vestfold Hills region of East Antarctica. Interestingly, we identified the production of potential homoserine lactones (HSLs) with chain lengths ranging from medium to long in 19 bacterial species using three biosensors, namely, Agrobacterium tumefaciens NTL4, Chromobacterium violaceum CV026, and Escherichia coli MT102, in conjunction with thin-layer chromatography (TLC). The majority of detectable HSLs were from Gram-positive species not previously known to produce HSLs. This discovery further expands our understanding of the microbial community capable of this type of communication, as well as provides insights into physiological adaptations of microorganisms that allow them to survive in the harsh Antarctic environment.IMPORTANCE Quorum sensing, a type of bacterial communication, is widely known to regulate many processes, including those that confer a survival advantage. However, little is known about communication by bacteria residing within Antarctic soils. Employing a combination of bacterial biosensors, analytical techniques, and genome mining, we found a variety of Antarctic soil bacteria speaking a common language, via LuxI/LuxR-based quorum sensing, thus potentially supporting survival in a mixed microbial community. This study reports potential quorum sensing activity in Antarctic soils and has provided a platform for studying physiological adaptations of microorganisms that allow them to survive in the harsh Antarctic environment.
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