Leptospira enrichment culture followed by ONT metagenomic sequencing allows better detection of Leptospira presence and diversity in water and soil samplesMyranda Gorman - 2022
AbstractBackground
Leptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are the major transmission sources in humans and animals. Clusters of disease outbreaks are common during rainy seasons.
Methodology/Principal findings
In this study, to detect the presence of Leptospira, we applied PCR, direct metagenomic sequencing, and enrichment culture followed by PCR and metagenomic sequencing on water and soil samples. Direct sequencing and enrichment cultures followed by PCR or sequencing effectively detected pathogenic and nonpathogenic Leptospira compared to direct PCR and 16S amplification-based metagenomic sequencing in soil or water samples. Among multiple culture media evaluated, Ellinghausen-McCullough-Johnson-Harris (EMJH) media containing antimicrobial agents was superior in recovering and detecting Leptospira from the environmental samples. Our results show that enrichment culture followed by PCR can be used to confirm the presence of pathogenic Leptospira in environmental samples. Additionally, metagenomic sequencing on enrichment cultures effectively detects the abundance and diversity of Leptospira spp. from environmental samples.
Conclusions/Significance
The selection of methodology is critical when testing environmental samples for the presence of Leptospira. Selective enrichment culture improves Leptospira detection efficacy by PCR or metagenomic sequencing and can be used successfully to understand the presence and diversity of pathogenic Leptospira during environmental surveillance.
Biodegradation of microplastic in freshwaters: A long-lasting process affected by the lake microbiomeSami J. Taipale - 2022
AbstractPlastics have been produced for over a century, but definitive evidence of complete plastic biodegradation in different habitats, particularly freshwater ecosystems, is still missing. Using 13C-labelled polyethylene microplastics (PE-MP) and stable isotope analysis of produced gas and microbial membrane lipids, we determined the biodegradation rate and fate of carbon in PE-MP in different freshwater types. The biodegradation rate in the humic-lake waters was much higher (0.45% ± 0.21% per year) than in the clear-lake waters (0.07% ± 0.06% per year) or the artificial freshwater medium (0.02% ± 0.02% per year). Complete biodegradation of PE-MP was calculated to last 100–200 years in humic-lake waters, 300–4000 years in clear-lake waters, and 2000–20,000 years in the artificial freshwater medium. The concentration of 18:1ω7, characteristic phospholipid fatty acid in Alpha- and Gammaproteobacteria, was a predictor of faster biodegradation of PE. Uncultured Acetobacteraceae and Comamonadaceae among Alpha- and Gammaproteobacteria, respectively, were major bacteria related to the biodegradation of PE-MP. Overall, it appears that microorganisms in humic lakes with naturally occurring refractory polymers are more adept at decomposing PE than those in other waters.
Multigenerational Exposure to Heat Stress Induces Phenotypic Resilience, and Genetic and Epigenetic Variations in Arabidopsis thaliana OffspringNarendra Singh Yadav - 2022
AbstractPlants are sedentary organisms that constantly sense changes in their environment and react to various environmental cues. On a short-time scale, plants respond through alterations in their physiology, and on a long-time scale, plants alter their development and pass on the memory of stress to the progeny. The latter is controlled genetically and epigenetically and allows the progeny to be primed for future stress encounters, thus increasing the likelihood of survival. The current study intended to explore the effects of multigenerational heat stress in Arabidopsis thaliana. Twenty-five generations of Arabidopsis thaliana were propagated in the presence of heat stress. The multigenerational stressed lineage F25H exhibited a higher tolerance to heat stress and elevated frequency of homologous recombination, as compared to the parallel control progeny F25C. A comparison of genomic sequences revealed that the F25H lineage had a three-fold higher number of mutations [single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs)] as compared control lineages, suggesting that heat stress induced genetic variations in the heat-stressed progeny. The F25H stressed progeny showed a 7-fold higher number of non-synonymous mutations than the F25C line. Methylome analysis revealed that the F25H stressed progeny showed a lower global methylation level in the CHH context than the control progeny. The F25H and F25C lineages were different from the parental control lineage F2C by 66,491 and 80,464 differentially methylated positions (DMPs), respectively. F25H stressed progeny displayed higher frequency of methylation changes in the gene body and lower in the body of transposable elements (TEs). Gene Ontology analysis revealed that CG-DMRs were enriched in processes such as response to abiotic and biotic stimulus, cell organizations and biogenesis, and DNA or RNA metabolism. Hierarchical clustering of these epimutations separated the heat stressed and control parental progenies into distinct groups which revealed the non-random nature of epimutations. We observed an overall higher number of epigenetic variations than genetic variations in all comparison groups, indicating that epigenetic variations are more prevalent than genetic variations. The largest difference in epigenetic and genetic variations was observed between control plants comparison (F25C vs. F2C), which clearly indicated that the spontaneous nature of epigenetic variations and heat-inducible nature of genetic variations. Overall, our study showed that progenies derived from multigenerational heat stress displayed a notable adaption in context of phenotypic, genotypic and epigenotypic resilience.
Effects of stocking at the parr stage on the reproductive fitness and genetic diversity of a wild population of Atlantic salmon (Salmo salar L.)Raphael Bouchard - 2022
AbstractCaptive-breeding programs are among the most adopted conservation practices to mitigate the loss of biodiversity, including genetic diversity. However, both genetic and nongenetic changes occurring in captivity can reduce the fitness of supplemented individuals, which complicate rehabilitation efforts. In the case of Atlantic salmon, the intensity of changes that occur in captivity and their impact on fitness will vary with the stocking practice adopted. In this study, we test whether salmon stocked at the parr stage have reduced reproductive success compared with their wild conspecifics and whether they contribute to increase genetic diversity in the targeted population. To do so, we use high-throughput microsatellite sequencing of 38 loci to accurately assign 2381 offspring to a comprehensive set of possible parents from a supplemented
Atlantic salmon population in Québec, Canada. Captive-bred salmon stocked at the parr stage had fewer mates than their wild conspecifics, as well as a reduced relative reproductive success (RSS) compared with their wild counterparts. Nonetheless, in comparison with previous studies, stocking at the parr stage significantly improved RSS compared with salmon stocked as smolts and they displayed a reduction in reproductive success similar to salmon stocked as fry, which spend less time in captivity than parr. Moreover, supplementation of captive-bred salmon significantly contributed to increasing genetic diversity. These results should contribute to informing resource managers in determining the best stocking practice to enhance Atlantic salmon populations.
Genome evolution drives transcriptomic and phenotypic adaptation in Pseudomonas aeruginosa during 20 years of infectionSamuel J.T. Wardell - 2021
AbstractThe opportunistic pathogen Pseudomonas aeruginosa chronically infects the lungs of patients with cystic fibrosis (CF). During infection the bacteria evolve and adapt to the lung environment. Here we use genomic, transcriptomic and phenotypic approaches to compare multiple isolates of P. aeruginosa collected more than 20 years apart during a chronic infection in a CF patient. Complete genome sequencing of the isolates, using short- and long-read technologies, showed that a genetic bottleneck occurred during infection and was followed by diversification of the bacteria. A 125 kb deletion, an 0.9 Mb inversion and hundreds of smaller mutations occurred during evolution of the bacteria in the lung, with an average rate of 17 mutations per year. Many of the mutated genes are associated with infection or antibiotic resistance. RNA sequencing was used to compare the transcriptomes of an earlier and a later isolate. Substantial reprogramming of the transcriptional network had occurred, affecting multiple genes that contribute to continuing infection. Changes included greatly reduced expression of flagellar machinery and increased expression of genes for nutrient acquisition and biofilm formation, as well as altered expression of a large number of genes of unknown function. Phenotypic studies showed that most later isolates had increased cell adherence and antibiotic resistance, reduced motility, and reduced production of pyoverdine (an iron-scavenging siderophore), consistent with genomic and transcriptomic data. The approach of integrating genomic, transcriptomic and phenotypic analyses reveals, and helps to explain, the plethora of changes that P. aeruginosa undergoes to enable it to adapt to the environment of the CF lung during a chronic infection.
Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaksWanjuan Feng - 2021
AbstractGenome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays (‘PathSig-dPCR’) for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.
Nitrogen Fertilization and Native C4 Grass Species Alter Abundance, Activity, and Diversity of Soil Diazotrophic CommunitiesJialin Hu - 2021
AbstractNative C4 grasses have become the preferred species for native perennial pastures and bioenergy production due to their high productivity under low soil nitrogen (N) status. One reason for their low N requirement is that C4 grasses may benefit from soil diazotrophs and promote biological N fixation. Our objective was to evaluate the impact of N fertilization rates (0, 67, and 202 kg N ha–1) and grass species (switchgrass [Panicum virgatum] and big bluestem [Andropogon gerardii]) on the abundance, activity, diversity, and community composition of soil diazotrophs over three agricultural seasons (grass green-up, initial harvest, and second harvest) in a field experiment in East Tennessee, United States. Nitrogen fertilization rate had a stronger influence on diazotroph population size and activity (determined by nifH gene and transcript abundances) and community composition (determined by nifH gene amplicon sequencing) than agricultural season or grass species. Excessive fertilization (202 kg N ha–1) resulted in fewer nifH transcripts compared to moderate fertilization (67 kg N ha–1) and decreased both richness and evenness of diazotrophic community, reflecting an inhibitory effect of high N application rates on soil diazotrophic community. Overall, cluster I and cluster III diazotrophs were dominant in this native C4 grass system. Diazotroph population size and activity were directly related to soil water content (SWC) based on structural equation modeling. Soil pH, SWC, and C and N availability were related to the variability of diazotrophic community composition. Our results revealed relationships between soil diazotrophic community and associated soil properties, adding to our understanding of the response of soil diazotrophs to N fertilization and grass species in native C4 grass systems.
SOX9 reprograms endothelial cells by altering the chromatin landscapeBettina M Fuglerud - 2022
AbstractThe transcription factor SOX9 is activated at the onset of endothelial-to-mesenchymal transition (EndMT) during embryonic development and in pathological conditions. Its roles in regulating these processes, however, are not clear. Using human umbilical vein endothelial cells (HUVECs) as an EndMT model, we show that SOX9 expression alone is sufficient to activate mesenchymal genes and steer endothelial cells towards a mesenchymal fate. By genome-wide mapping of the chromatin landscape, we show that SOX9 displays features of a pioneer transcription factor, such as opening of chromatin and leading to deposition of active histone modifications at silent chromatin regions, guided by SOX dimer motifs and H2A.Z enrichment. We further observe highly transient and dynamic SOX9 binding, possibly promoted through its eviction by histone phosphorylation. However, while SOX9 binding is dynamic, changes in the chromatin landscape and cell fate induced by SOX9 are persistent. Finally, our analysis of single-cell chromatin accessibility indicates that SOX9 opens chromatin to drive EndMT in atherosclerotic lesions in vivo. This study provides new insight into key molecular functions of SOX9 and mechanisms of EndMT and highlights the crucial developmental role of SOX9 and relevance to human disease.
Stem traits, compartments and tree species affectfungal communities on decaying woodShanshan Yang - 2022
AbstractDead wood quantity and quality is important for for-est biodiversity, by determining wood-inhabiting fun-gal assemblages. We therefore evaluated how fungalcommunities were regulated by stem traits and com-partments (i.e. bark, outer- and inner wood) of14 common temperate tree species. Fresh logs wereincubated in a common garden experiment in a forestsite in the Netherlands. After 1 and 4 years of decay,the fungal composition of different compartmentswas assessed using Internal Transcribed Spaceramplicon sequencing. We found that fungal alphadiversity differed significantly across tree speciesand stem compartments, with bark showing signifi-cantly higher fungal diversity than wood. Gymno-sperms and Angiosperms hold different fungalcommunities, and distinct fungi were found betweeninner wood and other compartments. Stem traitsshowed significant afterlife effects on fungal commu-nities; traits associated with accessibility (e.g. con-duit diameter), stem chemistry (e.g. C, N, lignin) andphysical defence (e.g. density) were important factorsshaping fungal community structure in decayingstems. Overall, stem traits vary substantially acrossstem compartments and tree species, thus regulatingfungal communities and the long-term carbondynamics of dead trees.
Ammonia-oxidizing bacterial communities are affected by nitrogen fertilization and grass species in native C4 grassland soilsJialin Hu - 2021
AbstractBackground
Fertilizer addition can contribute to nitrogen (N) losses from soil by affecting microbial populations responsible for nitrification. However, the effects of N fertilization on ammonia oxidizing bacteria under C4 perennial grasses in nutrient-poor grasslands are not well studied.
Methods
In this study, a field experiment was used to assess the effects of N fertilization rate (0, 67, and 202 kg N ha−1) and grass species (switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii)) on ammonia-oxidizing bacterial (AOB) communities in C4 grassland soils using quantitative PCR, quantitative reverse transcription-PCR, and high-throughput amplicon sequencing of amoA genes.
Results
Nitrosospira were dominant AOB in the C4 grassland soil throughout the growing season. N fertilization rate had a stronger influence on AOB community composition than C4 grass species. Elevated N fertilizer application increased the abundance, activity, and alpha-diversity of AOB communities as well as nitrification potential, nitrous oxide (N2O) emission and soil acidity. The abundance and species richness of AOB were higher under switchgrass compared to big bluestem. Soil pH, nitrate, nitrification potential, and N2O emission were significantly related to the variability in AOB community structures (p < 0.05).
Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)Xi Wang - 2020
AbstractPlasmid DNA (pDNA) isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research. Almost all pDNA purification involves disruption of bacteria, removal of membrane lipids, proteins and genomic DNA, purification of pDNA from bulk lysate, and concentration of pDNA for downstream applications. While many liquid-phase and solid-phase pDNA purification methods are used, the final pDNA preparations are usually contaminated with varied degrees of host RNA, which cannot be completely digested by RNase A. To develop a simple, cost-effective, and yet effective method for RNA depletion, we investigated whether commercially available size selection magnetic beads (SSMBs), such as Mag-Bind® TotalPure NGS Kit (or Mag-Bind), can completely deplete bacterial RNA in pDNA preparations. In this proof-of-principle study, we demonstrated that, compared with RNase A digestion and two commercial plasmid affinity purification kits, the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps. Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits. We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples. Furthermore, the Mag-bind-based SSMB method costs only 5–10% of most commercial plasmid purification kits on a per sample basis. Thus, the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.
Topography of the respiratory tract bacterial microbiota in cattleChristopher McMullen - 2020
AbstractBackground
Bacterial bronchopneumonia (BP) is the leading cause of morbidity and mortality in cattle. The nasopharynx is generally accepted as the primary source of pathogenic bacteria that cause BP. However, it has recently been shown in humans that the oropharynx may act as the primary reservoir for pathogens that reach the lung. The objective was therefore to describe the bacterial microbiota present along the entire cattle respiratory tract to determine which upper respiratory tract (URT) niches may contribute the most to the composition of the lung microbiota.
Methods
Seventeen upper and lower respiratory tract locations were sampled from 15 healthy feedlot steer calves. Samples were collected using a combination of swabs, protected specimen brushes, and saline washes. DNA was extracted from each sample and the 16S rRNA gene (V3-V4) was sequenced. Community composition, alpha-diversity, and beta-diversity were compared among sampling locations.
Results
Microbiota composition differed across sampling locations, with physiologically and anatomically distinct locations showing different relative abundances of 1137 observed sequence variants (SVs). An analysis of similarities showed that the lung was more similar to the nasopharynx (R-statistic = 0.091) than it was to the oropharynx (R-statistic = 0.709) or any other URT sampling location. Five distinct metacommunities were identified across all samples after clustering at the genus level using Dirichlet multinomial mixtures. This included a metacommunity found primarily in the lung and nasopharynx that was dominated by Mycoplasma. Further clustering at the SV level showed a shared metacommunity between the lung and nasopharynx that was dominated by Mycoplasma dispar. Other metacommunities found in the nostrils, tonsils, and oral microbiotas were dominated by Moraxella, Fusobacterium, and Streptococcus, respectively.
Conclusions
The nasopharyngeal bacterial microbiota is most similar to the lung bacterial microbiota in healthy cattle and therefore may serve as the primary source of bacteria to the lung. This finding indicates that the nasopharynx is likely the most important location that should be targeted when doing bovine respiratory microbiota research.
A rapid, low cost, and highly sensitive SARS-CoV-2 diagnostic based on whole genome sequencingBrian Glenn St Hilaire - 2020
AbstractEarly detection of infection with SARS-CoV-2 is key to managing the current global pandemic, as evidence shows the virus is most contagious on or before symptom onset1,2. Here, we introduce a low-cost, high-throughput method for diagnosis of SARS-CoV-2 infection, dubbed Pathogen-Oriented Low-Cost Assembly & Re-Sequencing (POLAR), that enhances sensitivity by aiming to amplify the entire SARS-CoV-2 genome rather than targeting particular viral loci, as in typical RT-PCR assays. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network (https://artic.network/) with short-read DNA sequencing and de novo genome assembly. We are able to reliably (>95% accuracy) detect SARS-CoV-2 at concentrations of 84 genome equivalents per milliliter, better than the reported limits of detection of almost all diagnostic methods currently approved by the US Food and Drug Administration. At higher concentrations, we are able to reliably assemble the SARS-CoV-2 genome in the sample, often with no gaps and perfect accuracy. Such genome assemblies enable the spread of the disease to be analyzed much more effectively than would be possible with an ordinary yes/no diagnostic, and can help identify vaccine and drug targets. Using POLAR, a single person can process 192 samples over the course of an 8-hour experiment, at a cost of ~$30/patient, enabling a 24-hour turnaround with sequencing and data analysis time included. Further testing and refinement will likely enable greater enhancements in the sensitivity of the above approach.
Influence of introduced arbuscular mycorrhizal fungiand phosphorus sources on plant traits, soil properties,and rhizosphere microbial communities in organiclegume-flax rotationYunliang Li - 2019
AbstractAims We identify P management strategies combiningarbuscular mycorrhizal fungal (AMF) inoculation withrock phosphate or composted manure for intensive or-ganic grain-production systems.Methods We measured the response of plants traits andsoil properties to the factorial combination of three ratesof organic-approved P sources applied in rotation phase-1of legume–flax cropping systems, and of granular AMFinoculant applied in the first, second, or both rotationphases, or not applied. Treatment combinations effectson the rhizosphere communities of AMF, fungi, andbacteria were tested by amplicon sequencing, in twopedoclimates.Results Inoculation had limited effects in both environ-ments. Composted manure decreased lentil yield, butincreased lentil N and P concentrations and soil P fertilityon the Chernozem, while increasing pea productivity onthe Luvisol. Composted manure applied in rotationphase-1 had a residual effect on flax productivity, N andP concentrations, and soil P fertility in both environ-ments. Rock phosphate reduced soil P fertility and flaxproductivity on the Gray Luvisol. The βdiversity of therhizosphere communities was unaffected by treatments,while the αdiversity of bacteria and AMF was altered byAMF inoculation and fertilization only in the GrayLuvisol. Correlations between microbial species andplant traits or soil properties were inconsistent, reflectingthe complex relationships among microbial community,plant identity, and environmental conditions.Conclusion Here, composted manure was more influ-ential than AMF inoculation and rock phosphate. Giventhe influence of environmental conditions, small fieldtrials are recommended before wide-scale adoption oftheir use.
