Jacob Bælum

Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill

The Deepwater Horizon (DWH) oil spill in the spring of 2010 resulted in an input of ∼4.1 million barrels of oil to the Gulf of Mexico; >22% of this oil is unaccounted for, with unknown environmental consequences. Here we investigated the impact of oil deposition on microbial communities in surface sediments collected at 64 sites by targeted sequencing of 16S rRNA genes, shotgun metagenomic sequencing of 14 of these samples and mineralization experiments using 14C-labeled model substrates. The 16S rRNA gene data indicated that the most heavily oil-impacted sediments were enriched in an uncultured Gammaproteobacterium and a Colwellia species, both of which were highly similar to sequences in the DWH deep-sea hydrocarbon plume. The primary drivers in structuring the microbial community were nitrogen and hydrocarbons. Annotation of unassembled metagenomic data revealed the most abundant hydrocarbon degradation pathway encoded genes involved in degrading aliphatic and simple aromatics via butane monooxygenase. The activity of key hydrocarbon degradation pathways by sediment microbes was confirmed by determining the mineralization of 14C-labeled model substrates in the following order: propylene glycol, dodecane, toluene and phenanthrene. Further, analysis of metagenomic sequence data revealed an increase in abundance of genes involved in denitrification pathways in samples that exceeded the Environmental Protection Agency (EPA)’s benchmarks for polycyclic aromatic hydrocarbons (PAHs) compared with those that did not. Importantly, these data demonstrate that the indigenous sediment microbiota contributed an important ecosystem service for remediation of oil in the Gulf. However, PAHs were more recalcitrant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem.

The microbiome of New World vultures

Vultures are scavengers that fill a key ecosystem niche, in which they have evolved a remarkable tolerance to bacterial toxins in decaying meat. Here we report the first deep metagenomic analysis of the vulture microbiome. Through face and gut comparisons of 50 vultures representing two species, we demonstrate a remarkably conserved low diversity of gut microbial flora. The gut samples contained an average of 76 operational taxonomic units (OTUs) per specimen, compared with 528 OTUs on the facial skin. Clostridia and Fusobacteria, widely pathogenic to other vertebrates, dominate the vultures gut microbiota. We reveal a likely faecal-oral-gut route for their origin. DNA of prey species detectable on facial swabs was completely degraded in the gut samples from most vultures, suggesting that the gastrointestinal tracts of vultures are extremely selective. Our findings show a strong adaption of vultures and their bacteria to their food source, exemplifying a specialized host-microbial alliance.

Direct analysis of tfdA gene expression by indigenous bacteria in phenoxy acid amended agricultural soil.

Expression of the functional gene tfdA involved in degradation of phenoxyacetic acids such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) was investigated during degradation scenarios in natural unseeded soil samples. The results illustrate how messenger RNA (mRNA)-based analysis is well suited to quantitatively study the activity of specific microbial populations in soil using phenoxyacetic acid biodegradation as a model system. Via quantitative real-time PCR, a clear response to the presence of phenoxy acids was shown during degradation in soil amended with 20 mg 2,4-D or MCPA per kg soil. Further, we found a relatively high degree of correlation between expression of the functional gene and the rates of mineralization. Melting curve analyses of real-time PCR products, supported by tfdA-denaturing gradient gel electrophoresis analysis showed that, although only class I tfdA genes were apparent in the indigenous microbial population, class III tfdA genes became predominant during incubation, and were the only genes expressed during degradation of MCPA in soil. In contrast, both classes were expressed during degradation of the structurally similar compound 2,4-D. The ability to quantify microbial transcripts directly in environmental samples will have a profound impact on our understanding of microbial processes in the environment in future studies.

Degradation of 4-Chloro-2-Methylphenoxyacetic Acid in Top- and Subsoil Is Quantitatively Linked to the Class III tfdA Gene

ABSTRACT The tfdA gene is known to be involved in the first step of the degradation of the phenoxy acid herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in several soil bacteria, but bacteria containing other tfdA-like genes have been isolated as well. A quantitative real-time PCR method was used to monitor the increase in the concentration of tfdA genes during degradation of MCPA in sandy topsoil and subsoil over a period of 115 days. Quantitative PCR revealed growth in the tfdA-containing bacterial community, from 500 genes g−1 soil to approximately 3 × 104 genes g−1 soil and to 7 × 105 genes g−1 soil for topsoil initially added to 2.3 mg MCPA kg−1 (dry weight) soil and 20 mg MCPA kg−1 (dry weight) soil, respectively. We analyzed the diversity of the tfdA gene during the degradation experiment. Analyses of melting curves of real-time PCR amplification products showed that a shift in the dominant tfdA population structure occurred during the degradation period. Further denaturing gradient gel electrophoresis and sequence analysis revealed that the tfdA genes responsible for the degradation of MCPA belonged to the class III tfdA genes, while the tfdA genes present in the soil before the occurrence of degradation belonged to the class I tfdA genes. The implications of these results is that the initial assessment of functional genes in soils does not necessarily reflect the organisms or genes that would carry out the degradation of the compounds in question.

Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses.

The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below −10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.

Meta-genomic analysis of toilet waste from long distance flights; a step towards global surveillance of infectious diseases and antimicrobial resistance.

Human populations worldwide are increasingly confronted with infectious diseases and antimicrobial resistance spreading faster and appearing more frequently. Knowledge regarding their occurrence and worldwide transmission is important to control outbreaks and prevent epidemics. Here, we performed shotgun sequencing of toilet waste from 18 international airplanes arriving in Copenhagen, Denmark, from nine cities in three world regions. An average of 18.6 Gb (14.8 to 25.7 Gb) of raw Illumina paired end sequence data was generated, cleaned, trimmed and mapped against reference sequence databases for bacteria and antimicrobial resistance genes. An average of 106,839 (0.06%) reads were assigned to resistance genes with genes encoding resistance to tetracycline, macrolide and beta-lactam resistance genes as the most abundant in all samples. We found significantly higher abundance and diversity of genes encoding antimicrobial resistance, including critical important resistance (e.g. blaCTX-M) carried on airplanes from South Asia compared to North America. Presence of Salmonella enterica and norovirus were also detected in higher amounts from South Asia, whereas Clostridium difficile was most abundant in samples from North America. Our study provides a first step towards a potential novel strategy for global surveillance enabling simultaneous detection of multiple human health threatening genetic elements, infectious agents and resistance genes.

TaqMan probe-based real-time PCR assay for detection and discrimination of class I, II, and III tfdA genes in soils treated with phenoxy acid herbicides.

ABSTRACT Separate quantification of three classes of tfdA genes was performed using TaqMan quantitative real-time PCR for 13 different soils subsequent to mineralization of three phenoxy acids. Class III tfdA genes were found to be involved in mineralization more often than class I and II tfdA genes.

The impact of bioaugmentation on dechlorination kinetics and on microbial dechlorinating communities in subsurface clay till

A molecular study on how the abundance of the dechlorinating culture KB-1 affects dechlorination rates in clay till is presented. DNA extracts showed changes in abundance of specific dechlorinators as well as their functional genes. Independently of the KB-1 added, the microbial dechlorinator abundance increased to the same level in all treatments. In the non-bioaugmented microcosms the reductive dehalogenase gene bvcA increased in abundance, but when KB-1 was added the related vcrA gene increased while bvcA genes did not increase. Modeling showed higher vinyl-chloride dechlorination rates and shorter time for complete dechlorination to ethene with higher initial concentration of KB-1 culture, while cis-dichloroethene dechlorination rates were not affected by KB-1 concentrations. This study provides high resolution abundance profiles of Dehalococcoides spp. (DHC) and functional genes, highlights the ecological behavior of KB-1 in clay till, and reinforces the importance of using multiple functional genes as biomarkers for reductive dechlorination.

Corrigendum: The microbiome of New World vultures

Nature Communications 5: Article number: 5498 (2014); Published: 25 November 2014; Updated: 21 October 2015. The original version of this Article contained an error in the spelling of the author Thomas Sicheritz-Ponten, which was incorrectly given as Thomas Sicheritz Ponten. This has now been corrected in both the PDF and HTML versions of the Article.