Growth of complete ammonia oxidizers on guanidine.

Guanidine is a chemically stable nitrogen compound that is excreted in human urine and is widely used in manufacturing of plastics, as a flame retardant and as a component of propellants, and is well known as a protein denaturant in biochemistry1-3. Guanidine occurs widely in nature and is used by several microorganisms as a nitrogen source, but microorganisms growing on guanidine as the only substrate have not yet been identified. Here we show that the complete ammonia oxidizer (comammox) Nitrospira inopinata and probably most other comammox microorganisms can grow on guanidine as the sole source of energy, reductant and nitrogen. Proteomics, enzyme kinetics and the crystal structure of a N. inopinata guanidinase homologue demonstrated that it is a bona fide guanidinase. Incubation experiments with comammox-containing agricultural soil and wastewater treatment plant microbiomes suggested that guanidine serves as substrate for nitrification in the environment. The identification of guanidine as a growth substrate for comammox shows an unexpected niche of these globally important nitrifiers and offers opportunities for their isolation.

Cyanate as an energy source for nitrifiers.

Ammonia- and nitrite-oxidizing microorganisms are collectively responsible for the aerobic oxidation of ammonia via nitrite to nitrate and have essential roles in the global biogeochemical nitrogen cycle. The physiology of nitrifiers has been intensively studied, and urea and ammonia are the only recognized energy sources that promote the aerobic growth of ammonia-oxidizing bacteria and archaea. Here we report the aerobic growth of a pure culture of the ammonia-oxidizing thaumarchaeote Nitrososphaera gargensis using cyanate as the sole source of energy and reductant; to our knowledge, the first organism known to do so. Cyanate, a potentially important source of reduced nitrogen in aquatic and terrestrial ecosystems, is converted to ammonium and carbon dioxide in Nitrososphaera gargensis by a cyanase enzyme that is induced upon addition of this compound. Within the cyanase gene family, this cyanase is a member of a distinct clade also containing cyanases of nitrite-oxidizing bacteria of the genus Nitrospira. We demonstrate by co-culture experiments that these nitrite oxidizers supply cyanase-lacking ammonia oxidizers with ammonium from cyanate, which is fully nitrified by this microbial consortium through reciprocal feeding. By screening a comprehensive set of more than 3,000 publically available metagenomes from environmental samples, we reveal that cyanase-encoding genes clustering with the cyanases of these nitrifiers are widespread in the environment. Our results demonstrate an unexpected metabolic versatility of nitrifying microorganisms, and suggest a previously unrecognized importance of cyanate in cycling of nitrogen compounds in the environment.

Complete nitrification by Nitrospira bacteria.

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

Membrane Lipid Composition of the Moderately Thermophilic Ammonia-Oxidizing Archaeon "Candidatus Nitrosotenuis uzonensis" at Different Growth Temperatures.

"Candidatus Nitrosotenuis uzonensis" is the only cultured moderately thermophilic member of the thaumarchaeotal order Nitrosopumilales (NP) that contains many mesophilic marine strains. We examined its membrane lipid composition at different growth temperatures (37°C, 46°C, and 50°C). Its lipids were all membrane-spanning glycerol dialkyl glycerol tetraethers (GDGTs), with 0 to 4 cyclopentane moieties. Crenarchaeol (cren), the characteristic thaumarchaeotal GDGT, and its isomer (cren') were present in high abundance (30 to 70%). The GDGT polar headgroups were mono-, di-, and trihexoses and hexose/phosphohexose. The ratio of glycolipid to phospholipid GDGTs was highest in the cultures grown at 50°C. With increasing growth temperatures, the relative contributions of cren and cren' increased, while those of GDGT-0 to GDGT-4 (including isomers) decreased. TEX86 (tetraether index of tetraethers consisting of 86 carbons)-derived temperatures were much lower than the actual growth temperatures, further demonstrating that TEX86 does not accurately reflect the membrane lipid adaptation of thermophilic Thaumarchaeota As the temperature increased, specific GDGTs changed relative to their isomers, possibly representing temperature adaption-induced changes in cyclopentane ring stereochemistry. Comparison of a wide range of thaumarchaeotal core lipid compositions revealed that the "Ca Nitrosotenuis uzonensis" cultures clustered separately from other members of the NP order and the Nitrososphaerales (NS) order. While phylogeny generally seems to have a strong influence on GDGT distribution, our analysis of "Ca Nitrosotenuis uzonensis" demonstrates that its terrestrial, higher-temperature niche has led to a lipid composition that clearly differentiates it from other NP members and that this difference is mostly driven by its high cren' content.IMPORTANCE For Thaumarchaeota, the ratio of their glycerol dialkyl glycerol tetraether (GDGT) lipids depends on growth temperature, a premise that forms the basis of the widely applied TEX86 paleotemperature proxy. A thorough understanding of which GDGTs are produced by which Thaumarchaeota and what the effect of temperature is on their GDGT composition is essential for constraining the TEX86 proxy. "Ca Nitrosotenuis uzonensis" is a moderately thermophilic thaumarchaeote enriched from a thermal spring, setting it apart in its environmental niche from the other marine mesophilic members of its order. Indeed, we found that the GDGT composition of "Ca Nitrosotenuis uzonensis" cultures was distinct from those of other members of its order and was more similar to those of other thermophilic, terrestrial Thaumarchaeota This suggests that while phylogeny has a strong influence on GDGT distribution, the environmental niche that a thaumarchaeote inhabits also shapes its GDGT composition.

Surface-enhanced Raman spectroscopy of microorganisms: limitations and applicability on the single-cell level.

Detection and characterization of microorganisms is essential for both clinical diagnostics and environmental studies. An emerging technique to analyse microbes at single-cell resolution is surface-enhanced Raman spectroscopy (surface-enhanced Raman scattering: SERS). Optimised SERS procedures enable fast analytical read-outs with specific molecular information, providing insight into the chemical composition of microbiological samples. Knowledge about the origin of microbial SERS signals and parameter(s) affecting their occurrence, intensity and/or reproducibility is crucial for reliable SERS-based analyses. In this work, we explore the feasibility and limitations of the SERS approach for characterizing microbial cells and investigate the applicability of SERS for single-cell sorting as well as for three-dimensional visualization of microbial communities. Analyses of six different microbial species (an archaeon, two Gram-positive bacteria, three Gram-negative bacteria) showed that for several of these organisms distinct features in their SERS spectra were lacking. As additional confounding factor, the physiological conditions of the cells (as influenced by e.g., storage conditions or deuterium-labelling) were systematically addressed, for which we conclude that the respective SERS signal at the single-cell level is strongly influenced by the metabolic activity of the analysed cells. While this finding complicates the interpretation of SERS data, it may on the other hand enable probing of the metabolic state of individual cells within microbial populations of interest.

Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells.

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.

Quantification of guanidine in environmental samples using benzoin derivatization and LC-MS analysis.

The recent discovery of guanidine-dependent riboswitches in many microbes raised interest in the biological function and metabolism of this nitrogen-rich compound. However, very little is known about the concentrations of guanidine in the environment. Several methods have been published for quantifying guanidine and guanidino compounds in human urine and blood, often relying on derivatization followed by fluorescence detection. We adapted this analytical approach using benzoin as the derivatization agent to sensitively and selectively quantify guanidine in environmental samples, thereby facilitating future research on the biological and environmental roles of guanidine. This adapted method was applied to human urine, raw wastewater, and biological growth media as relevant matrices. Our liquid chromatography-tandem mass spectrometry analyses of the derivatized solutions identified a different major derivatization product than previously reported. This product was consistently observed across various substrates (guanidine, methylguanidine, and arginine) and derivatization agents (benzoin and anisoin). We observed a constant background signal, restricting our analyses to a lower limit of quantification of 50 nM. Despite this limitation, our method allowed for the quantification of guanidine concentrations significantly lower than those reported in previous derivatization-based studies.•Selective and sensitive detection of guanidine by LC-MS.•Method development and validation for robust detection of guanidine in environmental samples.•Reduction of sample preparation steps and reduced usage of toxic chemicals compared to previous methods.

Identification of inulin-responsive bacteria in the gut microbiota via multi-modal activity-based sorting.

Prebiotics are defined as non-digestible dietary components that promote the growth of beneficial gut microorganisms. In many cases, however, this capability is not systematically evaluated. Here, we develop a methodology for determining prebiotic-responsive bacteria using the popular dietary supplement inulin. We first identify microbes with a capacity to bind inulin using mesoporous silica nanoparticles functionalized with inulin. 16S rRNA gene amplicon sequencing of sorted cells revealed that the ability to bind inulin was widespread in the microbiota. We further evaluate which taxa are metabolically stimulated by inulin and find that diverse taxa from the phyla Firmicutes and Actinobacteria respond to inulin, and several isolates of these taxa can degrade inulin. Incubation with another prebiotic, xylooligosaccharides (XOS), in contrast, shows a more robust bifidogenic effect. Interestingly, the Coriobacteriia Eggerthella lenta and Gordonibacter urolithinfaciens are indirectly stimulated by the inulin degradation process, expanding our knowledge of inulin-responsive bacteria.

Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions.

Stable isotope labeling of microbial taxa of interest and their sorting provide an efficient and direct way to answer the question "who does what?" in complex microbial communities when coupled with fluorescence in situ hybridization or downstream 'omics' analyses. We have developed a platform for automated Raman-based sorting in which optical tweezers and microfluidics are used to sort individual cells of interest from microbial communities on the basis of their Raman spectra. This sorting of cells and their downstream DNA analysis, such as by mini-metagenomics or single-cell genomics, or cultivation permits a direct link to be made between the metabolic roles and the genomes of microbial cells within complex microbial communities, as well as targeted isolation of novel microbes with a specific physiology of interest. We describe a protocol from sample preparation through Raman-activated live cell sorting. Subsequent cultivation of sorted cells is described, whereas downstream DNA analysis involves well-established approaches with abundant methods available in the literature. Compared with manual sorting, this technique provides a substantially higher throughput (up to 500 cells per h). Furthermore, the platform has very high sorting accuracy (98.3 ± 1.7%) and is fully automated, thus avoiding user biases that might accompany manual sorting. We anticipate that this protocol will empower in particular environmental and host-associated microbiome research with a versatile tool to elucidate the metabolic contributions of microbial taxa within their complex communities. After a 1-d preparation of cells, sorting takes on the order of 4 h, depending on the number of cells required.

Anaerobic Sulfur Oxidation Underlies Adaptation of a Chemosynthetic Symbiont to Oxic-Anoxic Interfaces.

Chemosynthetic symbioses occur worldwide in marine habitats, but comprehensive physiological studies of chemoautotrophic bacteria thriving on animals are scarce. Stilbonematinae are coated by thiotrophic Gammaproteobacteria. As these nematodes migrate through the redox zone, their ectosymbionts experience varying oxygen concentrations. However, nothing is known about how these variations affect their physiology. Here, by applying omics, Raman microspectroscopy, and stable isotope labeling, we investigated the effect of oxygen on "Candidatus Thiosymbion oneisti." Unexpectedly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions, but carbon fixation genes and incorporation of 13C-labeled bicarbonate were not. Instead, several genes involved in carbon fixation were upregulated under oxic conditions, together with genes involved in organic carbon assimilation, polyhydroxyalkanoate (PHA) biosynthesis, nitrogen fixation, and urea utilization. Furthermore, in the presence of oxygen, stress-related genes were upregulated together with vitamin biosynthesis genes likely necessary to withstand oxidative stress, and the symbiont appeared to proliferate less. Based on its physiological response to oxygen, we propose that "Ca. T. oneisti" may exploit anaerobic sulfur oxidation coupled to denitrification to proliferate in anoxic sand. However, the ectosymbiont would still profit from the oxygen available in superficial sand, as the energy-efficient aerobic respiration would facilitate carbon and nitrogen assimilation. IMPORTANCE Chemoautotrophic endosymbionts are famous for exploiting sulfur oxidization to feed marine organisms with fixed carbon. However, the physiology of thiotrophic bacteria thriving on the surface of animals (ectosymbionts) is less understood. One longstanding hypothesis posits that attachment to animals that migrate between reduced and oxic environments would boost sulfur oxidation, as the ectosymbionts would alternatively access sulfide and oxygen, the most favorable electron acceptor. Here, we investigated the effect of oxygen on the physiology of "Candidatus Thiosymbion oneisti," a gammaproteobacterium which lives attached to marine nematodes inhabiting shallow-water sand. Surprisingly, sulfur oxidation genes were upregulated under anoxic relative to oxic conditions. Furthermore, under anoxia, the ectosymbiont appeared to be less stressed and to proliferate more. We propose that animal-mediated access to oxygen, rather than enhancing sulfur oxidation, would facilitate assimilation of carbon and nitrogen by the ectosymbiont.

Transparent soil microcosms for live-cell imaging and non-destructive stable isotope probing of soil microorganisms.

Microscale processes are critically important to soil ecology and biogeochemistry yet are difficult to study due to soil's opacity and complexity. To advance the study of soil processes, we constructed transparent soil microcosms that enable the visualization of microbes via fluorescence microscopy and the non-destructive measurement of microbial activity and carbon uptake in situ via Raman microspectroscopy. We assessed the polymer Nafion and the crystal cryolite as optically transparent soil substrates. We demonstrated that both substrates enable the growth, maintenance, and visualization of microbial cells in three dimensions over time, and are compatible with stable isotope probing using Raman. We applied this system to ascertain that after a dry-down/rewetting cycle, bacteria on and near dead fungal hyphae were more metabolically active than those far from hyphae. These data underscore the impact fungi have facilitating bacterial survival in fluctuating conditions and how these microcosms can yield insights into microscale microbial activities.

An automated Raman-based platform for the sorting of live cells by functional properties.

Stable-isotope probing is widely used to study the function of microbial taxa in their natural environment, but sorting of isotopically labelled microbial cells from complex samples for subsequent genomic analysis or cultivation is still in its early infancy. Here, we introduce an optofluidic platform for automated sorting of stable-isotope-probing-labelled microbial cells, combining microfluidics, optical tweezing and Raman microspectroscopy, which yields live cells suitable for subsequent single-cell genomics, mini-metagenomics or cultivation. We describe the design and optimization of this Raman-activated cell-sorting approach, illustrate its operation with four model bacteria (two intestinal, one soil and one marine) and demonstrate its high sorting accuracy (98.3 ± 1.7%), throughput (200-500 cells h-1; 3.3-8.3 cells min-1) and compatibility with cultivation. Application of this sorting approach for the metagenomic characterization of bacteria involved in mucin degradation in the mouse colon revealed a diverse consortium of bacteria, including several members of the underexplored family Muribaculaceae, highlighting both the complexity of this niche and the potential of Raman-activated cell sorting for identifying key players in targeted processes.

Publisher Correction: An automated Raman-based platform for the sorting of live cells by functional properties.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-nucleotide primer extension assay for detection and sequence typing of "Dehalococcoides" spp.

A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis was developed for the detection and typing of populations of the genus "Dehalococcoides". The specificity of the assay was evaluated with 16S rRNA gene sequences obtained from an isolate and an environmental sample representing two Dehalococcoides subgroups, i.e., the Cornell and the Pinellas subgroups. Only one sequence type, belonging to the Pinellas subgroup, was detected in a Bitterfeld-Wolfen region aquifer containing chlorinated ethenes as the main contaminants. The three-primer hybridization assay thus provided a fast and easy-to-implement method for confirming the specificity of taxon-specific PCR and allowed rapid additional taxonomic classification into subgroups. This study demonstrates the great potential of SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples.

Novel approach using substrate-mediated radiolabelling of RNA to link metabolic function with the structure of microbial communities.

A novel concept was developed applying radioisotope-labelled substrate incorporation into the biomass. The resulting radiolabelled RNA was used both as an indicator of activity and as a template for gaining structural and functional information about a substrate-utilizing microbial community. Sequences of PCR products are separated via cloning or using molecular fingerprinting techniques. Nucleic acids from predominant clones or the whole molecular fingerprinting pattern are transferred to a membrane and hybridized with the radiolabelled sample RNA. Scanning of the hybridized blots for radioactivity indicates the members involved in the utilization of the substrate. This novel 'random walk' approach using radioisotope probing was evaluated in a model community experiment.

Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis.

In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using 'universal' bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61 degrees C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.

The effect of easily degradable substrate feeding on the community structure of laboratory-scale wastewater sludge digesters.

The effect of several easily degradable substrates, such as protein, starch and sunflower oil was investigated on the bacterial community of a laboratory-scale biogas model system. Besides measuring gas yield, Denaturing Gradient Gel Electrophoresis (DGGE), Phospholipids Fatty Acid Analysis (PLFA) for Bacteria and T-RFLP analysis of the mcrA gene for Archaea were used. The community of the examined biogas reactors adapted to the new substrates through a robust physiological reaction followed by moderate community abundance shifts. Gas yield data clearly demonstrated the physiological adaptation to substrate shifts. Statistical analysis of DNA and chemotaxonomic biomarkers revealed community abundance changes. Sequences gained from DGGE bands showed the dominance of the phyla Bacteroidetes and the presence of Firmicutes (Clostridia) and Thermotogae. This was supported by the detection of large amounts of branched 15-carbon non-hydroxy fatty acids in PLFA profiles, as common PLFA markers of the Bacteroidetes group. Minor abundance ratios changes were observed in the case of Archaea in accordance with changes of the fed substrates.

Spatial and temporal changes of bacterial communities inhabiting the well waters of Harkány Spa.

In this study, changes in the bacterial community composition of the well waters of Harkány Spa were examined. Physical and chemical properties of mixing subsurface cold and thermal karst waters were correlated to shifts in bacterial community structures analyzed by denaturing gradient gel electrophoresis (DGGE) and principal component analysis (PCA). In addition, mineral components of the pellets were investigated by scanning electron microscopy. Samples from the effluent waters of Büdöstapolca I and II, Matty and Thermal VI wells were taken seasonally in 2007 and 2008. The comparison of the results of DGGE and PCA analyses showed that bacterial communities from the Büdöstapolca wells were distinct from those of Matty and Thermal VI, but seasonal changes were not detected. According to the phylogenetic analysis of the excised DGGE bands, presence of chemolithotrophic Proteobacteria (Thiobacillus, Thiothrix, and distant relatives of Sulfurospirillum) were typical in the Büdöstapolca wells, while members of Actinobacteria (Plantibacter, Actinobacterium, Microbacterium) and Firmicutes (Planococcus) were characteristic to the Matty and Thermal VI wells. In the pellets pyrite framboid crystals were observed by electron microscopy, which are minerals known to be biologically induced by dissimilatory iron- and sulfur (sulfate)-reducing bacteria.

Enrichment and genome sequence of the group I.1a ammonia-oxidizing Archaeon "Ca. Nitrosotenuis uzonensis" representing a clade globally distributed in thermal habitats.

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called "marine" group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as "Candidatus Nitrosotenuis uzonensis", is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, "Candidatus N. uzonensis" also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in "Ca. N. uzonensis", which potentially include genetic determinants of ecological niche differentiation.