Catalyzed Reporter Deposition Research Articles

The methane-oxidizing microbial communities of three maar lakes in tropical monsoon Asia.

Methane-oxidizing bacteria (MOB) is a group of planktonic microorganisms that use methane as their primary source of cellular energy. For tropical lakes in monsoon Asia, there is currently a knowledge gap on MOB community diversity and the factors influencing their abundance. Herewith, we present a preliminary assessment of the MOB communities in three maar lakes in tropical monsoon Asia using Catalyzed Reporter Deposition, Fluorescence In-Situ Hybridization (CARD-FISH), 16S rRNA amplicon sequencing, and pmoA gene sequencing. Correlation analysis between MOB abundances and lakes' physicochemical parameters following seasonal monsoon events were performed to explain observed spatial and temporal patterns in MOB diversity. The CARD-FISH analyses detected the three MOB types (I, II, and NC10) which aligned with the results from 16S rRNA amplicons and pmoA gene sequencing. Among community members based on 16S rRNA genes, Proteobacterial Type I MOB (e.g., Methylococcaceae and Methylomonadaceae), Proteobacterial Type II (Methylocystaceae), Verrucomicrobial (Methylacidiphilaceae), Methylomirabilota/NC10 (Methylomirabilaceae), and archaeal ANME-1a were found to be the dominant methane-oxidizers in three maar lakes. Analysis of microbial diversity and distribution revealed that the community compositions in Lake Yambo vary with the seasons and are more distinct during the stratified period. Temperature, DO, and pH were significantly and inversely linked with type I MOB and Methylomirabilota during stratification. Only MOB type I was influenced by monsoon changes. This research sought to establish a baseline for the diversity and ecology of planktonic MOB in tropical monsoon Asia to better comprehend their contribution to the CH4 cycle in tropical freshwater ecosystems.

Growth rates of marine prokaryotes are extremely diverse, even among closely related taxa.

Marine prokaryotes play crucial roles in ocean biogeochemical cycles, being their contribution strongly influenced by their growth rates. Hence, elucidating the variability and phylogenetic imprint of marine prokaryotes' growth rates are crucial for better determining the role of individual taxa in biogeochemical cycles. Here, we estimated prokaryotic growth rates at high phylogenetic resolution in manipulation experiments using water from the northwestern Mediterranean Sea. Experiments were run in the four seasons with different treatments that reduced growth limiting factors: predators, nutrient availability, viruses, and light. Single-amplicon sequence variants (ASVs)-based growth rates were calculated from changes in estimated absolute abundances using total prokaryotic abundance and the proportion of each individual ASV. The trends obtained for growth rates in the different experiments were consistent with other estimates based on total cell-counts, catalyzed reporter deposition fluorescence in situ hybridization subcommunity cell-counts or metagenomic-operational taxonomic units (OTUs). Our calculations unveil a broad range of growth rates (0.3-10 d-1) with significant variability even within closely related ASVs. Likewise, the impact of growth limiting factors changed over the year for individual ASVs. High numbers of responsive ASVs were shared between winter and spring seasons, as well as throughout the year in the treatments with reduced nutrient limitation and viral pressure. The most responsive ASVs were rare in the in situ communities, comprising a large pool of taxa with the potential to rapidly respond to environmental changes. Essentially, our results highlight the lack of phylogenetic coherence in the range of growth rates observed, and differential responses to the various limiting factors, even for closely related taxa.

Translocation of gut microbes to epididymal white adipose tissue drives lipid metabolism disorder under heat stress.

Heat stress induces multi-organ damage and serious physiological dysfunction in mammals, and gut bacteria may translocate to extra-intestinal tissues under heat stress pathology. However, whether gut bacteria translocate to the key metabolic organs and impair function as a result of heat stress remains unknown. Using a heat stress-induced mouse model, heat stress inhibited epididymal white adipose tissue (eWAT) expansion and induced lipid metabolic disorder but did not damage other organs, such as the heart, liver, spleen, or muscle. Microbial profiling analysis revealed that heat stress shifted the bacterial community in the cecum and eWAT but not in the inguinal white adipose tissue, blood, heart, liver, spleen, or muscle. Notably, gut-vascular barrier function was impaired, and the levels of some bacteria, particularly Lactobacillus, were higher in the eWAT, as confirmed by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) staining when mice were under heat stress. Moreover, integrated multi-omics analysis showed that the eWAT microbiota was associated with host lipid metabolism, and the expression of genes involved in the lipid metabolism in eWAT was upregulated under heat stress. A follow-up microbial supplementation study after introducing Lactobacillus plantarum to heat-stressed mice revealed that the probiotic ameliorated heat stress-induced loss of eWAT and dyslipidemia and reduced gut bacterial translocation to the eWAT by improving gut barrier function. Overall, our findings suggest that gut bacteria, particularly Lactobacillus spp., play a crucial role in heat stress-induced lipid metabolism disorder and that there is therapeutic potential for using probiotics, such as Lactobacillus plantarum.

FISHing for ciliates: Catalyzed reporter deposition fluorescence in situ hybridization for the detection of planktonic freshwater ciliates.

Planktonic ciliate species form multiple trophic guilds and are central components of freshwater food webs. Progress in molecular analytical tools has opened new insight into ciliate assemblages. However, high and variable 18S rDNA copy numbers, typical for ciliates, make reliable quantification by amplicon sequencing extremely difficult. For an exact determination of abundances, the classical morphology-based quantitative protargol staining is still the method of choice. Morphotype analyses, however, are time consuming and need specific taxonomic expertise. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) may represent a promising tool for the analysis of planktonic ciliates by combining molecular identification with microscopic quantification. We tested the applicability of CARD-FISH using nine cultured ciliate species. Eight species- and three genus-specific oligonucleotide probes were designed based on their 18S rRNA genes. The CARD-FISH protocol was adapted and the specificity of probes was established. We subsequently examined the precision of quantitation by CARD-FISH on single cultures and mock assemblages. Successful tests on lake water samples proved that planktonic ciliates could be identified and quantified in field samples by CARD-FISH. Double hybridizations allowed studying interspecific predator prey interactions between two ciliate species. In summary, we demonstrate that CARD-FISH with species-specific probes can facilitate studies on the population dynamics of closely related, small sized or cryptic species at high sampling frequencies.

Cryptic and ubiquitous aplastidic cryptophytes are key freshwater flagellated bacterivores

Morphology-based microscopic approaches are insufficient for a taxonomic classification of bacterivorous heterotrophic nanoflagellates (HNF) in aquatic environments since their cells do not display reliably distinguishable morphological features. This leads to a considerable lack of ecological insights into this large and taxonomically diverse functional guild. Here, we present a combination of fluorescence in situ hybridization followed by catalyzed reporter deposition (CARD-FISH) and environmental sequence analyses which revealed that morphologically indistinguishable, so far largely cryptic and uncultured aplastidic cryptophytes are ubiquitous and prominent protistan bacterivores in diverse freshwater ecosystems. Using a general probe for Cryptophyceae and its heterotrophic CRY1 lineage, we analyzed different water layers in 24 freshwater lakes spanning a broad range of trophic states, sizes and geographical locations. We show that bacterivorous aplastidic cryptophytes and the CRY1 lineage accounted for ca. 2/3 and ¼ of total HNF, respectively, in both epilimnetic and hypolimnetic samples. These heterotrophic cryptophytes were generally smaller and more abundant than their chloroplast-bearing counterparts. They had high uptake rates of bacteria, hinting at their important roles in channeling carbon flow from prokaryotes to higher trophic levels. The worldwide ubiquity of Cryptophyceae and its CRY1 lineage was supported by 18S rRNA gene sequence analyses across a diverse set of 297 freshwater metagenomes. While cryptophytes have been considered to be mainly plastidic “algae”, we show that it is the aplastidic counterparts that contribute considerably to bacterial mortality rates. Additionally, our results suggest an undiscovered diversity hidden amongst these abundant and morphologically diverse aplastidic cryptophytes.

Ecogenomics sheds light on diverse lifestyle strategies in freshwater CPR

BackgroundThe increased use of metagenomics and single-cell genomics led to the discovery of organisms from phyla with no cultivated representatives and proposed new microbial lineages such as the candidate phyla radiation (CPR or Patescibacteria). These bacteria have peculiar ribosomal structures, reduced metabolic capacities, small genome, and cell sizes, and a general host-associated lifestyle was proposed for the radiation. So far, most CPR genomes were obtained from groundwaters; however, their diversity, abundance, and role in surface freshwaters is largely unexplored. Here, we attempt to close these knowledge gaps by deep metagenomic sequencing of 119 samples of 17 different freshwater lakes located in Europe and Asia. Moreover, we applied Fluorescence in situ Hybridization followed by Catalyzed Reporter Deposition (CARD-FISH) for a first visualization of distinct CPR lineages in freshwater samples.ResultsA total of 174 dereplicated metagenome-assembled genomes (MAGs) of diverse CPR lineages were recovered from the investigated lakes, with a higher prevalence from hypolimnion samples (162 MAGs). They have reduced genomes (median size 1 Mbp) and were generally found in low abundances (0.02–14.36 coverage/Gb) and with estimated slow replication rates. The analysis of genomic traits and CARD-FISH results showed that the radiation is an eclectic group in terms of metabolic capabilities and potential lifestyles, ranging from what appear to be free-living lineages to host- or particle-associated groups. Although some complexes of the electron transport chain were present in the CPR MAGs, together with ion-pumping rhodopsins and heliorhodopsins, we believe that they most probably adopt a fermentative metabolism. Terminal oxidases might function in O2 scavenging, while heliorhodopsins could be involved in mitigation against oxidative stress.ConclusionsA high diversity of CPR MAGs was recovered, and distinct CPR lineages did not seem to be limited to lakes with specific trophic states. Their reduced metabolic capacities resemble the ones described for genomes in groundwater and animal-associated samples, apart from Gracilibacteria that possesses more complete metabolic pathways. Even though this radiation is mostly host-associated, we also observed organisms from different clades (ABY1, Paceibacteria, Saccharimonadia) that appear to be unattached to any other organisms or were associated with ‘lake snow’ particles (ABY1, Gracilibacteria), suggesting a broad range of potential life-strategies in this phylum.-F5-UcTDsZ5zHRoTaQByA1Video

Dissimilatory microbial sulfur and methane metabolism in the water column of a shallow meromictic lake

Lake Harutori is a brackish meromictic lake with a steep physicochemical gradient in shallow water. Anoxic water below the chemocline has been characterized by high concentrations of sulfide (>10 mM) and methane (>1.5 mM). Previously, we reported that uncultured bacteria in the SEEP-SRB1 group were major sulfate reducers in the lake [21], but knowledge of sulfur oxidation and methane metabolism was scarce. In this current study, the Lake Harutori microbial community structure in the mixolimnion (at depths of 1.5 m and 3.0 m), upper chemocline (3.5 m), and monimolimnion (4.5 m) was further investigated by 16S rRNA gene amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Reads of type I and II methanotrophs were retrieved mainly from 3.5 m and above. Methanotrophic bacteria detected by CARD-FISH accounted for 3.1% of DAPI-stained cells at 3.5 m. Detection frequencies of reads affiliated with the genera Sulfurimonas and Thiomicrorhabdus, which are known to comprise sulfur oxidizers, were relatively high at 3.5 m. Methanogenic archaeal reads were retrieved from the monimolimnion and they affiliated with the genus Methanosaeta. CARD-FISH counts indicated that the cells of Methanosaeta/Methanosarcina/Methanomicrobiales accounted for up to 0.8% of the DAPI-stained cells in the monimolimnion. On the other hand, many of the reads retrieved primarily from the monimolimnion were affiliated with phylogenetically novel uncultured groups.

Detection of hydrocarbon-degrading bacteria on deepwater corals of the northeast Atlantic using CARD-FISH

Recently, studies have begun to identify oil-degrading bacteria and host-taxon specific bacterial assemblages associated with the coral holobiont, including deep-sea cold-water corals, which are thought to provide metabolic functions and additional carbon sources to their coral hosts. Here, we describe the identification of Marinobacter on the soft tissue of Lophelia pertusa coral polyps by Catalyzed Reporter Deposition Fluorescence in situ Hybridization (CARD-FISH). L. pertusa samples from three reef sites in the northeast Atlantic (Logachev, Mingulay and Pisces) were collected at depth by vacuum seal to eliminate contamination issues. After decalcification, histological processing and sagittal sectioning of the soft coral polyp tissues, the 16S rRNA-targeted oligonucleotide HRP-labelled probe Mrb-0625-a, and Cyanine 3 (Cy3)-labelled tyramides, were used to identify members of the hydrocarbon-degrading genus Marinobacter. Mrb-0625-a-hybridized bacterial cell signals were detected in different anatomical sites of all polyps collected from each of the three reef sites, suggesting a close, possibly intimate, association between them, but the purpose of which remains unknown. We posit that Marinobacter, and possibly other hydrocarbon-degrading bacteria associated with Lophelia, may confer the coral with the ability to cope with toxic levels of hydrocarbons in regions of natural oil seepage and where there is an active oil and gas industry presence.

Circulating bacterial signature is linked to metabolic disease and shifts with metabolic alleviation after bariatric surgery

BackgroundThe microbiome has emerged as an environmental factor contributing to obesity and type 2 diabetes (T2D). Increasing evidence suggests links between circulating bacterial components (i.e., bacterial DNA), cardiometabolic disease, and blunted response to metabolic interventions. In this aspect, thorough next-generation sequencing-based and contaminant-aware approaches are lacking. To address this, we tested whether bacterial DNA could be amplified in the blood of subjects with obesity and high metabolic risk under strict experimental and analytical control and whether a putative bacterial signature is related to metabolic improvement after bariatric surgery.MethodsSubjects undergoing bariatric surgery were recruited into sex- and BMI-matched subgroups with (n = 24) or without T2D (n = 24). Bacterial DNA in the blood was quantified and prokaryotic 16S rRNA gene amplicons were sequenced. A contaminant-aware approach was applied to derive a compositional microbial signature from bacterial sequences in all subjects at baseline and at 3 and 12 months after surgery. We modeled associations between bacterial load and composition with host metabolic and anthropometric markers. We further tested whether compositional shifts were related to weight loss response and T2D remission. Lastly, bacteria were visualized in blood samples using catalyzed reporter deposition (CARD)-fluorescence in situ hybridization (FISH).ResultsThe contaminant-aware blood bacterial signature was associated with metabolic health. Based on bacterial phyla and genera detected in the blood samples, a metabolic syndrome classification index score was derived and shown to robustly classify subjects along their actual clinical group. T2D was characterized by decreased bacterial richness and loss of genera associated with improved metabolic health. Weight loss and metabolic improvement following bariatric surgery were associated with an early and stable increase of these genera in parallel with improvements in key cardiometabolic risk parameters. CARD-FISH allowed the detection of living bacteria in blood samples in obesity.ConclusionsWe show that the circulating bacterial signature reflects metabolic disease and its improvement after bariatric surgery. Our work provides contaminant-aware evidence for the presence of living bacteria in the blood and suggests a putative crosstalk between components of the blood and metabolism in metabolic health regulation.

Large-scale comparative analysis of cytogenetic markers across Lepidoptera

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.

Assigning Function to Phylogeny: FISH-nanoSIMS.

Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) is an imaging method used to identify microorganisms in environmental samples based on their phylogeny. CARD-FISH can be combined with nano-scale secondary ion mass spectrometry (nanoSIMS) to directly link the cell identity to their activity, measured as the incorporation of stable isotopes into hybridized cells after stable isotope probing. In environmental microbiology, a combination of these methods has been used to determine the identity and growth of uncultured microorganisms, and to explore the factors controlling their activity. Additionally, FISH-nanoSIMS has been widely used to directly visualize microbial interactions in situ. Here, we describe a step-by-step protocol for a combination of CARD-FISH, laser marking, and nanoSIMS analysis on samples from aquatic environments.

Crystalline iron oxides stimulate methanogenic benzoate degradation in marine sediment-derived enrichment cultures

Elevated dissolved iron concentrations in the methanic zone are typical geochemical signatures of rapidly accumulating marine sediments. These sediments are often characterized by co-burial of iron oxides with recalcitrant aromatic organic matter of terrigenous origin. Thus far, iron oxides are predicted to either impede organic matter degradation, aiding its preservation, or identified to enhance organic carbon oxidation via direct electron transfer. Here, we investigated the effect of various iron oxide phases with differing crystallinity (magnetite, hematite, and lepidocrocite) during microbial degradation of the aromatic model compound benzoate in methanic sediments. In slurry incubations with magnetite or hematite, concurrent iron reduction, and methanogenesis were stimulated during accelerated benzoate degradation with methanogenesis as the dominant electron sink. In contrast, with lepidocrocite, benzoate degradation, and methanogenesis were inhibited. These observations were reproducible in sediment-free enrichments, even after five successive transfers. Genes involved in the complete degradation of benzoate were identified in multiple metagenome assembled genomes. Four previously unknown benzoate degraders of the genera Thermincola (Peptococcaceae, Firmicutes), Dethiobacter (Syntrophomonadaceae, Firmicutes), Deltaproteobacteria bacteria SG8_13 (Desulfosarcinaceae, Deltaproteobacteria), and Melioribacter (Melioribacteraceae, Chlorobi) were identified from the marine sediment-derived enrichments. Scanning electron microscopy (SEM) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) images showed the ability of microorganisms to colonize and concurrently reduce magnetite likely stimulated by the observed methanogenic benzoate degradation. These findings explain the possible contribution of organoclastic reduction of iron oxides to the elevated dissolved Fe2+ pool typically observed in methanic zones of rapidly accumulating coastal and continental margin sediments.

NanoSIMS sample preparation decreases isotope enrichment: magnitude, variability and implications for single-cell rates of microbial activity.

The activity of individual microorganisms can be measured within environmental samples by detecting uptake of isotope-labelled substrates using nano-scale secondary ion mass spectrometry (nanoSIMS). Recent studies have demonstrated that sample preparation can decrease 13 C and 15 N enrichment in bacterial cells, resulting in underestimates of activity. Here, we explore this effect with a variety of preparation types, microbial lineages and isotope labels to determine its consistency and therefore potential for correction. Specifically, we investigated the impact of different protocols for fixation, nucleic acid staining and catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) on >14 500 archaeal and bacterial cells (Methanosarcina acetivorans, Sulfolobus acidocaldarius and Pseudomonas putida) enriched in 13 C, 15 N, 18 O, 2 H and/or 34 S. We found these methods decrease isotope enrichments by up to 80% - much more than previously reported - and that the effect varies by taxa, growth phase, isotope label and applied protocol. We make recommendations for how to account for this effect experimentally and analytically. We also re-evaluate published nanoSIMS datasets and revise estimated microbial turnover times in the marine subsurface and nitrogen fixation rates in pelagic unicellular cyanobacteria. When sample preparation is accounted for, cell-specific rates increase and are more consistent with modelled and bulk rates.

Simplified Digital Enzyme-Linked Immunosorbent Assay Using Tyramide Signal Amplification and Fibrin Hydrogels.

Many protein biomarkers occur at very low concentrations in biofluids like blood and saliva, and ultrasensitive detection methods are required in order to measure them. Approaches such as digital enzyme-linked immunosorbent assays (ELISA) and single molecule arrays (Simoa) have been developed to accurately quantitate protein concentrations as low as attomolar levels. Although these techniques are being implemented in research and clinical laboratories to develop ultrasensitive clinical diagnostic assays, the size and cost of the instruments required to run these digital assays have precluded them from being implemented into point-of-care diagnostic formats. Here, we report the development of a simplified digital ELISA format that is more amenable to point-of-care technologies, referred to as catalyzed reporter deposition digital ELISA (CARD-dELISA). On-bead signal generation using the CARD tyramide signal amplification technique is combined with bead immobilization in fibrin hydrogels for single molecule counting in a simplified workflow format. CARD-dELISA allows for ultrasensitive protein detection (IL-6: ∼1 fM) with a dynamic range similar to the conventional Simoa assay. We use CARD-dELISA to measure IL-6 in saliva samples and show good agreement with conventional Simoa.

A Differential Metabarcoding Approach to Describe Taxonomy Profiles of Bacteria and Archaea in the Saltern of Margherita di Savoia (Italy).

Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9–36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of Bacteria and Archaea, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of Archaea in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for Bacteria and at 24.1% salinity for Archaea. While Bacteria showed the greatest number of genera in the first ponds (salinity range 4.9–14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1–36.0%). Among prokaryotes, Salinibacter was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal Haloquadratum (~43% at 36.0% salinity) and Natronomonas (~18% at 13.1% salinity) and the bacterial “Candidatus Aquiluna” (~19% at 14.5% salinity). Interestingly, “Candidatus Aquiluna” had not been identified before in thalassohaline waters.

Unusual marine cyanobacteria/haptophyte symbiosis relies on N2 fixation even in N-rich environments

The microbial fixation of N2 is the largest source of biologically available nitrogen (N) to the oceans. However, it is the most energetically expensive N-acquisition process and is believed inhibited when less energetically expensive forms, like dissolved inorganic N (DIN), are available. Curiously, the cosmopolitan N2-fixing UCYN-A/haptophyte symbiosis grows in DIN-replete waters, but the sensitivity of their N2 fixation to DIN is unknown. We used stable isotope incubations, catalyzed reporter deposition fluorescence in-situ hybridization (CARD-FISH), and nanoscale secondary ion mass spectrometry (nanoSIMS), to investigate the N source used by the haptophyte host and sensitivity of UCYN-A N2 fixation in DIN-replete waters. We demonstrate that under our experimental conditions, the haptophyte hosts of two UCYN-A sublineages do not assimilate nitrate (NO3−) and meet little of their N demands via ammonium (NH4+) uptake. Instead the UCYN-A/haptophyte symbiosis relies on UCYN-A N2 fixation to supply large portions of the haptophyte’s N requirements, even under DIN-replete conditions. Furthermore, UCYN-A N2 fixation rates, and haptophyte host carbon fixation rates, were at times stimulated by NO3− additions in N-limited waters suggesting a link between the activities of the bulk phytoplankton assemblage and the UCYN-A/haptophyte symbiosis. The results suggest N2 fixation may be an evolutionarily viable strategy for diazotroph–eukaryote symbioses, even in N-rich coastal or high latitude waters.

Adipose tissue derived bacteria are associated with inflammation in obesity and type 2 diabetes

ObjectiveBacterial translocation to various organs including human adipose tissue (AT) due to increased intestinal permeability remains poorly understood. We hypothesised that: (1) bacterial presence is highly tissue specific and (2)...

Laccase-Mediated Catalyzed Fluorescent Reporter Deposition for Live-Cell Imaging.

Catalyzed reporter deposition (CARD) is a widely established method for labeling biological samples analyzed using microscopy. Horseradish peroxidase, commonly used in CARD to amplify reporter signals, requires the addition of hydrogen peroxide, which may perturb samples used in live-cell microscopy. Herein we describe an alternative method of performing CARD using a laccase enzyme, which does not require exogenous hydrogen peroxide. Laccase is an oxidative enzyme which can carry out single-electron oxidations of phenols and related compounds by reducing molecular oxygen. We demonstrate proof-of-concept for this technique through the nontargeted covalent labeling of bovine serum albumin using a fluorescently labeled ferulic acid derivative as the laccase reporter substrate. We further demonstrate the viability of this approach by performing live-cell CARD with an antibody-conjugated laccase against a surface-bound target. CARD using laccase produces an amplified fluorescence signal by labeling cells without the need for exogenous hydrogen peroxide.

Distinct relationships between fluorescence in situ hybridization and 16S rRNA gene- and amplicon-based sequencing data of bacterioplankton lineages

Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) and amplicon sequencing of the total (16S rRNA gene) and potentially active (16S rRNA transcripts), community are the major state of the art approaches for assessing the composition of bacterial communities in marine pelagic and other ecosystems. However, CARD-FISH and amplicon sequencing methods have not yet been directly compared to assess the composition of bacterioplankton communities. Therefore, these approaches were used to study the composition of bacterial communities in two North Sea seawater mesocosm experiments supplemented with diatom-derived organic matter (OM). All approaches revealed Proteobacteria and Bacteroidetes as major components of the bacterioplankton communities. The Roseobacter group and its RCA cluster, as well as Bacteroidetes and Gammaproteobacteria, responded most strongly to OM addition, whereas the SAR11 clade responded in only one of the two mesocosms. A correlation analysis showed that CARD-FISH and amplicon sequencing data of the SAR11 clade and the Roseobacter group, together with its RCA cluster, were highly significantly correlated, whereas Bacteroidetes did not yield any significant correlation and Gammaproteobacteria was only correlated with the potentially active fraction. However, subgroups of these phylogenetic groups, the SAR92 clade, the genera Pseudoalteromonas and Polaribacter, exhibited significant correlations in one of the two mesocosms. Correlations of CARD-FISH with amplicon sequencing data from the total and potentially active fractions of these lineages exhibited distinct differences. The study showed that CARD-FISH and amplicon sequencing data of distinct bacterioplankton groups and especially the phylogenetic lineages at a higher taxonomic level were correlated but reflected different aspects of their growth dynamics.

Anaerobic methane oxidation coupled to sulfate reduction in a biotrickling filter: Reactor performance and microbial community analysis

The aim of this work was to evaluate the performance of a biotrickling filter (BTF) packed with polyurethane foam and pall rings for the enrichment of microorganisms mediating anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) by activity tests and microbial community analysis. A BTF was inoculated with microorganisms from a known AOM active deep sea sediment collected at a depth of 528 m below the sea level (Alpha Mound, Gulf of Cadiz). The microbial community analysis was performed by catalyzed reporter deposition - fluorescence in situ hybridization (CARD-FISH) and 16S rRNA sequence analysis. The AOM occurrence and rates in the BTF were assessed by performing batch activity assays using 13C-labelled methane (13CH4). After an estimated start-up time of ∼20 days, AOM rates of ∼0.3 mmol l−1 day−1 were observed in the BTF, values almost 20 times higher than previously reported in a polyurethane foam packed BTF. The microbial community consisted mainly of anaerobic methanotrophs (ANME-2, 22% of the total number of cells) and sulfate reducing bacteria (SRB, 47% of the total number of cells). This study showed that the BTF is a suitable reactor configuration for the enrichment of microbial communities involved in AOM coupled to SR at ambient pressure and temperature with a relatively short start-up time.