Biology subjects

Konstantinidis, K. T.

Publications and source records attributed to Konstantinidis, K. T..

44 records · Page 3Linked to original sources

bioRxiv2019

Genome Repository of Oiled Systems (GROS): an interactive and searchable database that expands the catalogued diversity of crude oil-associated microbes

Indigenous microbial communities ultimately control the fate of petroleum hydrocarbons (PHCs) that enters the natural environment through natural seeps or accidental oil spills, but the interactions among microbes and with their chemical environment during oil biodegradation are highly complex and poorly understood. Genome-resolved metagenomics have the potential to help in unraveling these complex interactions. However, the lack of a comprehensive database that integrates existing genomic/metagenomic data from oiled environments with physicochemical parameters known to regulate the fate of PHCs currently limits data analysis and interpretations. Here, we present a curated, comprehensive, and searchable database that documents microbial populations in oiled ecosystems on a global scale, along with underlying physicochemical data, geocoded via GIS to reveal geographic distribution patterns of the populations. Analysis of the ~2,000 metagenome-assembled genomes (MAGs) available in the database revealed strong ecological niche specialization within habitats e.g., specialization to coastal sediments vs. water-column vs. deep-sea sediments. Over 95% of the recovered MAGs represented novel and uncultured species underscoring the limited representation of cultured organisms from oil-contaminated and oil reservoir ecosystems. The majority of MAGs linked to oiled ecosystems are members of the rare biosphere in non-oiled samples, except for the Gulf of Mexico (GoM) which appears to be primed for oil biodegradation. GROS should facilitate future work toward a more predictive understanding of the microbial taxa and their activities that control the fate of oil spills as well as serve as a model approach for building similar resources for additional environmental processes and omic data of interest.

genomics↗

bioRxiv2019

Iterative Subtractive Binning of Freshwater Chronoseries Metagenomes Identifies over Four Hundred Novel Species and their Ecologic Preferences

Recent advances in sequencing technology and accompanying bioinformatic pipelines have allowed unprecedented access to the genomes of yet-uncultivated microorganisms from a wide array of natural and engineered environments. However, the catalogue of available genomes from uncultivated freshwater microbial populations remains limited, and most genome recovery attempts in freshwater ecosystems have only targeted few specific taxa. Here, we present a novel genome recovery pipeline, which incorporates iterative subtractive binning and apply it to a time series of metagenomic datasets from seven connected locations along the Chattahoochee River (Southeastern USA). Our set of Metagenome-Assembled Genomes (MAGs) represents over four hundred genomospecies yet to be named, which substantially increase the number of high-quality MAGs from freshwater lakes and represent about half of the total microbial community sampled. We propose names for two novel species that were represented by high-quality MAGs: \"Candidatus Elulimicrobium humile\" (\"Ca. Elulimicrobiota\" in the \"Patescibacteria\" group) and \"Candidatus Aquidulcis frankliniae\" (\"Chloroflexi\"). To evaluate the prevalence of these species in the chronoseries, we introduce novel approaches to estimate relative abundance and a habitat-preference score that control for uneven quality of the genomes and sample representation. Using these metrics, we demonstrate a high degree of habitat-specialization and endemicity for most genomospecies observed in the Chattahoochee lacustrine ecosystem, as well as wider species ecological ranges associated with smaller genomes and higher coding densities, indicating an overall advantage of smaller, more compact genomes for cosmopolitan distributions.

genomics↗

bioRxiv2019

A Novel Droplet Digital PCR Human mtDNA Assay for Global Fecal Source Tracking

Human mitochondrial DNA (mtDNA) provides a promising target for microbial source tracking because it is unique to humans and universal across human individuals. We developed a droplet digital PCR (ddPCR) assay, hCYTB484, targeting the cytochrome b gene of the human mtDNA and compared the performance of the hCYTB484 assay with a widely used assay targeting human-associated Bacteroides, the HF183/BacR287 assay. We also defined and validated the analytical limit of detection and analytical lower limit of quantification; these analytical limits determine the concentration levels above which samples are declared to be positive and quantifiable for the target, respectively. We found both assays to be highly specific (95%) against cow and pig feces; however, the hCYTB484 was more sensitive when tested against individual human feces from US, Bangladesh, and Mozambique (100% versus a mean of 51% across the 3 countries). To further compare the performance of the two assays, we utilized a receiver operating characteristic curve, showing that the hCYTB484 marker was widely distributed across human feces from the 3 different geographical regions tested and in higher quantities than the HF183/BacR287 marker. The higher performance of the hCYTB484 marker in individual feces is a desirable characteristic in the detection of fecal pollution from sources to which fewer individuals contribute, such as non-sewered types of sanitation that serve most of Earths population and carry the highest risk of exposure to fecal-oral pathogens.\n\nImportanceThe usefulness of an ideal human-specific, culture- and library-independent marker to the microbial source tracking field is reflected by the numerous efforts to develop such markers; however, thus far, microbial-based markers of this type have exhibited variable source-specificity across geographies and variable sensitivity across scales of fecal waste. Most of the worlds population is served by non-sewered forms of sanitation that also carry the highest risk of exposure to fecal-oral pathogens. This reality underscores the need for markers of human fecal contamination that have high sensitivity in fecal pollution sources to which fewer individuals contribute to, such as fecal sludges found in pit latrines. We show that human mtDNA-based methods can be highly source-specific and highly sensitive in smaller scales of fecal pollution, providing a useful addition to the microbial source tracking toolbox by complementing the variable performance of microbial-based markers.

microbiology↗

bioRxiv2019

Metagenomic characterization of creek sediment microbial communities from a major agricultural region in Salinas, California

Little is known about the public health risks associated with natural creek sediments that are affected by runoff and fecal pollution from agricultural and livestock practices. For instance, the persistence of foodborne pathogens originating from agricultural activities such as Shiga Toxin-producing E. coli (STEC) in such sediments remains poorly quantified. Towards closing these knowledge gaps, the water-sediment interface of two creeks in the Salinas River Valley was sampled over a nine-month period using metagenomics and traditional culture-based tests for STEC. Our results revealed that these sediment communities are extremely diverse and comparable to the functional and taxonomic diversity observed in soils. With our sequencing effort (~4 Gbp per library), we were unable to detect any pathogenic Escherichia coli in the metagenomes of 11 samples that had tested positive using culture-based methods, apparently due to relatively low pathogen abundance. Further, no significant differences were detected in the abundance of human- or cow-specific gut microbiome sequences compared to upstream, more pristine (control) sites, indicating natural dilution of anthropogenic inputs. Notably, a high baseline level of metagenomic reads encoding antibiotic resistance genes (ARGs) was found in all samples and was significantly higher compared to ARG reads in metagenomes from other environments, suggesting that these communities may be natural reservoirs of ARGs. Overall, our metagenomic results revealed that creek sediments are not a major sink for anthropogenic runoff and the public health risk associated with these sediment microbial communities may be low.\n\nIMPORTANCECurrent agricultural and livestock practices contribute to fecal contamination in the environment and the spread of food and water-borne disease and antibiotic resistance genes (ARGs). Traditionally, the level of pollution and risk to public health is assessed by culture-based tests for the intestinal bacterium, E. coli. However, the accuracy of these traditional methods (e.g., low quantification, and false positive signal when PCR-based) and their suitability for sediments remains unclear. We collected sediments for a time series metagenomics study from one of the most highly productive agricultural regions in the U.S. in order to assess how agricultural runoff affects the native microbial communities and if the presence of STEC in sediment samples can be detected directly by sequencing. Our study provided important information on the potential for using metagenomics as a tool for assessment of public health risk in natural environments.

microbiology↗

bioRxiv2019

The anaerobic digestion microbiome: a collection of 1600 metagenome-assembled genomes shows high species diversity related to methane production

BackgroundMicroorganisms in biogas reactors are essential for degradation of organic matter and methane production through anaerobic digestion process. However, a comprehensive genome-centric comparison, including relevant metadata for each sample, is still needed to identify the globally distributed biogas community members and serve as a reliable repository.\n\nResultsHere, 134 publicly available datasets derived from different biogas reactors were used to recover 1,635 metagenome-assembled genomes (MAGs) representing different bacterial and archaeal species. All genomes were estimated to be >50% complete and nearly half were [≥]90% complete with [≤]5% contamination. In most samples, specialized microbial communities were established, while only a few taxa were widespread among the different reactor systems. Metabolic reconstruction of the MAGs enabled the prediction of functional traits related to biomass degradation and methane production from waste biomass. An extensive evaluation of the replication index provided an estimation of the growth rate for microbes involved in different steps of the food chain. The recovery of many MAGs belonging to Candidate Phyla Radiation and other underexplored taxa suggests their specific involvement in the anaerobic degradation of organic matter.\n\nConclusionsThe outcome of this study highlights a high flexibility of the biogas microbiome. The dynamic composition and adaptability to the environmental conditions, including temperatures and a wide range of substrates, were demonstrated. Our findings enhance the mechanistic understanding of anaerobic digestion microbiome and substantially extend the existing repository of genomes. The established database represents a relevant resource for future studies related to this engineered ecosystem.

microbiology↗

bioRxiv2018

Comparing DNA, RNA and protein levels for measuring microbial activity in nitrogen-amended soils

Multi-omic techniques can offer a comprehensive overview of microbial communities at the gene, transcript and protein levels. However, to what extent these levels reflect in situ process rates is less clear, especially in highly complex habitats such as soils. Here we performed microcosm incubations using soil from a site with a history of agricultural management. Microcosms, amended with isotopically labelled ammonium and urea to simulate a fertilization event, showed nitrification (up to 4.1 {+/-} 0.87 {micro}g N-NO3- g-1 dry soil d-1) and accumulation of N2O after 192 hours of incubation. Nitrification activity (NH4+[->]NH2OH[->]NO2-[->]NO3-) was accompanied by a 6-fold increase in relative expression of the 16S rRNA gene (RNA/DNA) between 10 and 192 hours of incubation for ammonia-oxidizing bacteria (AOB) Nitrosomonas and Nitrosospira. In contrast, ammonia-oxidizing archaea (AOA) and complete ammonia oxidizer (comammox) nitrifiers showed stable gene expression during incubations but were generally more abundant (DNA level) than their Betaproteobacteria AOB counterparts. A strong relationship between nitrification activity and (mostly) betaproteobacterial ammonia monooxygenase (amoA; NH4+[->]NH2OH) and nitrite oxidoreductase (nxrA; NO2-[->]NO3-) transcript abundances revealed that mRNA levels quantitatively reflected measured activity and were generally more sensitive than the DNA level in the microcosm incubations. Although peptides related to housekeeping proteins from nitrite-oxidizing microorganisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. Altogether, these findings underscore the strengths and limitations of multi-omic approaches for assessing complex microbial communities and provide the molecular means to assess nitrification processes in soils.\n\nIMPORTANCEEven though the use of omic approaches has expanded our knowledge of the diversity of microbial communities in natural and engineered systems, it is less clear how well the use of whole community DNA-, RNA- or protein-based approaches reflect microbial activities. To this end, we directly compared the different levels of molecular information (i.e., DNA, RNA or proteins) in order to assess which level best correlated with isotope-based measurements of nitrification activity in agricultural soils after fertilization. This work reveals the strengths as well as the associated limitations of metagenomic, metatranscriptomic, and metaproteomic approaches in serving as reliable proxies for examining microbial activities in highly diverse environments like soils.

microbiology↗

bioRxiv2018

Microbial Diversity in a Military Impacted Lagoon (Vieques, Puerto Rico) as Revealed by Metagenomics

The Anones Lagoon, located in the island municipality of Vieques, Puerto Rico (PR), received extensive bombing during military practices by the US Navy for decades. After military activities ceased in 2003, the bombing range was designated as part of a larger Superfund site by US EPA. Here, we employed shotgun metagenomic sequencing to investigate how microbial communities responded to pollution by heavy metals and explosives at this lagoon. Sediment samples (0-5 cm) from Anones were collected in 2005 and 2014 and compared to samples from two reference lagoons, i.e., Guaniquilla, Cabo Rojo (a natural reserve) and Condado, San Juan (PRs capital city). Consistent with selection under low anthropogenic impacts, Guaniquilla exhibited the highest degree of diversity with lower frequency of genes related to xenobiotics metabolism among the three lagoons. Notably, a clear shift was observed in Anones, with Euryarchaeota becoming enriched (9% of total) and a concomitant increase in community diversity, by about one order of magnitude, after almost 10 years without bombing activities. In contrast, genes associated with explosives biodegradation and heavy metal transformation significantly decreased in abundance in Anones 2014 (by 91.5%). Five unique population genomes were recovered from the Anones 2005 sample that encoded genetic determinants implicated in biodegradation of contaminants. Collectively, these results provided new insights into the natural attenuation of explosive contaminants by the benthic microbial communities of the Anones lagoon and could serve as reference points to enhance bioremediation actions at this site and for assessing other similarly impacted sites.\n\nImportanceThis study represents the first assessment of the benthic microbial community in the Anones Lagoon in Vieques, Puerto Rico after the impact of intense pollution by bombs and unconventional weapons during military training exercises. Evaluating the microbial diversity of Anones, represents an opportunity to assess the microbial succession patterns during the active process of natural attenuation of pollutants. The culture-independent techniques employed to study these environmental samples allowed the recovery of almost complete genomes of several abundant species that were likely involved in the biodegradation of pollutants and thus, represented species responding to the strong selection pressure posed by military activities. Further, our results showed that natural attenuation has proceeded to a great extend ten years after the cease of military activities.

microbiology↗

bioRxiv2017

High-throughput ANI Analysis of 90K Prokaryotic Genomes Reveals Clear Species Boundaries

A fundamental question in microbiology is whether there is a continuum of genetic diversity among genomes or clear species boundaries prevail instead. Answering this question requires robust measurement of whole-genome relatedness among thousands of genomes and from diverge phylogenetic lineages. Whole-genome similarity metrics such as Average Nucleotide Identity (ANI) can provide the resolution needed for this task, overcoming several limitations of traditional techniques used for the same purposes. Although the number of genomes currently available may be adequate, the associated bioinformatics tools for analysis are lagging behind these developments and cannot scale to large datasets. Here, we present a new method, FastANI, to compute ANI using alignment-free approximate sequence mapping. Our analyses demonstrate that FastANI produces an accurate ANI estimate and is up to three orders of magnitude faster when compared to an alignment (e.g., BLAST)-based approach. We leverage FastANI to compute pairwise ANI values among all prokaryotic genomes available in the NCBI database. Our results reveal a clear genetic discontinuity among the database genomes, with 99.8% of the total 8 billion genome pairs analyzed showing either >95% intra-species ANI or <83% inter-species ANI values. We further show that this discontinuity is recovered with or without the most frequently represented species in the database and is robust to historic additions in the public genome databases. Therefore, 95% ANI represents an accurate threshold for demarcating almost all currently named prokaryotic species, and wide species boundaries may exist for prokaryotes.

bioinformatics↗