Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen-allergic patients. Current pollen monitoring methods are microscope-based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost-effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next-generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring.

Oxygen minimum zones (OMZs) are marine regions where O 2 is undetectable at intermediate depths. Within OMZs, the oxygen-depleted zone (ODZ) induces anaerobic microbial processes that lead to fixed nitrogen loss via denitrification and anammox. Surprisingly, nitrite oxidation is also detected in ODZs, although all known marine nitrite oxidizers (mainly Nitrospina) are aerobes. We used metagenomic binning to construct metagenome-assembled genomes (MAGs) of nitrite oxidizers from OMZs. These MAGs represent two novel Nitrospina-like species, both of which differed from all known Nitrospina species, including cultured species and published MAGs. Relative abundances of different Nitrospina genotypes in OMZ and non-OMZ seawaters were estimated by mapping metagenomic reads to newly constructed MAGs and published high-quality genomes of members from the Nitrospinae phylum. The two novel species were present in all major OMZs and were more abundant inside ODZs, which is consistent with the detection of higher nitrite oxidation rates in ODZs than in oxic seawaters and suggests novel adaptations to anoxic environments. The detection of a large number of unclassified nitrite oxidoreductase genes in the dataset implies that the phylogenetic diversity of nitrite oxidizers is greater than previously thought.

The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Additionally, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named “Candidatus Nitrospira kreftii”, and performed a detailed genomic and physiological characterization. The complete genome of “Ca. N. kreftii” allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (Km(app)_NH3 ≈ 0.040 ± 0.01 µM), but a higher nitrite affinity (Km(app)_NO2- = 12.5 ± 4.0 µM), indicating an adaptation to highly oligotrophic environments. Furthermore, we observed partial inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This inhibition of “Ca. N. kreftii” indicates that differences in ammonium tolerance rather than affinity could potentially be a niche determining factor for different comammox Nitrospira.

Methane is a potent greenhouse gas, which can be converted by microorganism at the expense of oxygen, nitrate, nitrite, metal-oxides or sulfate. The bacterium ‘Candidatus Methylomirabilis oxyfera,’ a member of the NC10 phylum, is capable of nitrite-dependent anaerobic methane oxidation. Prolonged enrichment of ‘Ca. M. oxyfera’ with cerium added as trace element and without nitrate resulted in the shift of the dominant species. Here, we present a high quality draft genome of the new species ‘Candidatus Methylomirabilis lanthanidiphila’ and use comparative genomics to analyze its metabolic potential in both nitrogen and carbon cycling. To distinguish between gene content specific for the ‘Ca. Methylomirabilis’ genus and the NC10 phylum, the genome of a distantly related NC10 phylum member, CSP1-5, an aerobic methylotroph, is included in the analysis. All genes for the conversion of nitrite to N2 identified in ‘Ca. M. oxyfera’ are conserved in ‘Ca. M. lanthanidiphila,’ including the two putative genes for NO dismutase. In addition both species have several heme-copper oxidases potentially involved in NO and O2 respiration. For the oxidation of methane ‘Ca. Methylomirabilis’ species encode a membrane bound methane monooxygenase. CSP1-5 can act as a methylotroph, but lacks the ability to activate methane. In contrast to ‘Ca. M. oxyfera,’ which harbors three methanol dehydrogenases (MDH), both CSP1-5 and ‘Ca. M. lanthanidiphila’ only encode a lanthanide-dependent XoxF-type MDH, once more underlining the importance of rare earth elements for methylotrophic bacteria. The pathways for the subsequent oxidation of formaldehyde to carbon dioxide and for the Calvin–Benson–Bassham cycle are conserved in all species. Furthermore, CSP1-5 can only interconvert nitrate and nitrite, but lacks subsequent nitrite or NO reductases. Thus, it appears that although the conversion of methanol to carbon dioxide is present in several NC10 phylum bacteria, the coupling of nitrite reduction to the oxidation of methane is a trait so far unique to the genus ‘Ca. Methylomirabilis.’

The Bothnian Sea is an oligotrophic brackish basin characterized by low salinity and high concentrations of reactive iron, methane, and ammonium in its sediments, enabling the activity and interactions of many microbial guilds. Here, we studied the microbial network in these sediments by analyzing geochemical and microbial community depth profiles at one offshore and two near coastal sites. Analysis of 16S rRNA gene amplicons revealed a distinct depth stratification of both archaeal and bacterial taxa. The microbial communities at the two near coastal sites were more similar to each other than the offshore site, which is likely due to differences in the quality and rate of organic matter degradation. The abundance of methanotrophic archaea of the ANME-2a clade was shown to be related to the presence of methane and varied with sediment iron content. Metagenomic sequencing of sediment-derived DNA from below the sulfate-methane transition zone revealed a broad potential for respiratory sulfur metabolism via partially reduced sulfur species. The potential for nitrogen cycling was dominated by reductive processes via a truncated denitrification pathway encoded exclusively by bacterial lineages. Gene-centric fermentative metabolism analysis indicated a potential importance for acetate, formate, alcohol, and hydrogen metabolism. Methanogenic/-trophic pathways were dominated by Methanosaetaceae, Methanosarcinaceae, Methanomassiliicoccaceae, Methanoregulaceae, and ANME-2 archaea. Our results indicated flexible metabolic capabilities of core microbial community taxa, which could adapt to changing redox conditions, and with a spatial and depth distribution that is likely governed by the quality and input of available organic substrates and, for ANME-2, of iron oxides.

Microorganisms are main drivers of the sulfur, nitrogen and carbon biogeochemical cycles. These elemental cycles are interconnected by the activity of different guilds in sediments or wastewater treatment systems. Here, we investigated a nitrate-reducing microbial community in a laboratory-scale bioreactor model that closely mimicked estuary or brackish sediment conditions. The bioreactor simultaneously consumed sulfide, methane and ammonium at the expense of nitrate. Ammonium oxidation occurred solely by the activity of anammox bacteria identified as Candidatus Scalindua brodae and Ca. Kuenenia stuttgartiensis. Fifty-three percent of methane oxidation was catalyzed by archaea affiliated to Ca. Methanoperedens and 47% by Ca. Methylomirabilis bacteria. Sulfide oxidation was mainly shared between two proteobacterial groups. Interestingly, competition for nitrate did not lead to exclusion of one particular group. Metagenomic analysis showed that the most abundant taxonomic group was distantly related to Thermodesulfovibrio sp. (87-89% 16S rRNA gene identity, 52-54% average amino acid identity), representing a new family within the Nitrospirae phylum. A high quality draft genome of the new species was recovered, and analysis showed high metabolic versatility. Related microbial groups are found in diverse environments with sulfur, nitrogen and methane cycling, indicating that these novel Nitrospirae bacteria might contribute to biogeochemical cycling in natural habitats.

BackgroundClostridium difficile strain 630Δerm is a spontaneous erythromycin sensitive derivative of the reference strain 630 obtained by serial passaging in antibiotic-free media. It is widely used as a defined and tractable C. difficile strain. Though largely similar to the ancestral strain, it demonstrates phenotypic differences that might be the result of underlying genetic changes. Here, we performed a de novo assembly based on single-molecule real-time sequencing and an analysis of major methylation patterns.ResultsIn addition to single nucleotide polymorphisms and various indels, we found that the mobile element CTn5 is present in the gene encoding the methyltransferase rumA rather than adhesin CD1844 where it is located in the reference strain.ConclusionsTogether, the genetic features identified in this study may help to explain at least part of the phenotypic differences. The annotated genome sequence of this lab strain, including the first analysis of major methylation patterns, will be a valuable resource for genetic research on C. difficile.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1252-7) contains supplementary material, which is available to authorized users.

BackgroundMicroorganisms drive high rates of methanogenesis and carbon mineralization in wetland ecosystems. These signals are especially pronounced in the Prairie Pothole Region of North America, the tenth largest wetland ecosystem in the world. Sulfate reduction rates up to 22 μmol cm−3 day−1 have been measured in these wetland sediments, as well as methane fluxes up to 160 mg m−2 h−1—some of the highest emissions ever measured in North American wetlands. While pore waters from PPR wetlands are characterized by high concentrations of sulfur species and dissolved organic carbon, the constraints on microbial activity are poorly understood. Here, we utilized metagenomics to investigate candidate sulfate reducers and methanogens in this ecosystem and identify metabolic and viral controls on microbial activity.ResultsWe recovered 162 dsrA and 206 dsrD sequences from 18 sediment metagenomes and reconstructed 24 candidate sulfate reducer genomes assigned to seven phyla. These genomes encoded the potential for utilizing a wide variety of electron donors, such as methanol and other alcohols, methylamines, and glycine betaine. We also identified 37 mcrA sequences spanning five orders and recovered two putative methanogen genomes representing the most abundant taxa—Methanosaeta and Methanoregulaceae. However, given the abundance of Methanofollis-affiliated mcrA sequences, the detection of F420-dependent alcohol dehydrogenases, and millimolar concentrations of ethanol and 2-propanol in sediment pore fluids, we hypothesize that these alcohols may drive a significant fraction of methanogenesis in this ecosystem. Finally, extensive viral novelty was detected, with approximately 80% of viral populations being unclassified at any known taxonomic levels and absent from publicly available databases. Many of these viral populations were predicted to target dominant sulfate reducers and methanogens.ConclusionsOur results indicate that diversity is likely key to extremely high rates of methanogenesis and sulfate reduction observed in these wetlands. The inferred genomic diversity and metabolic versatility could result from dynamic environmental conditions, viral infections, and niche differentiation in the heterogeneous sediment matrix. These processes likely play an important role in modulating carbon and sulfur cycling in this ecosystem.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0522-4) contains supplementary material, which is available to authorized users.