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“Candidatus Methanoperedens nitroreducens” is an archaeon that couples the anaerobic oxidation of methane to nitrate reduction. In natural and man-made ecosystems, this archaeon is often found at oxic-anoxic interfaces where nitrate, the product of aerobic nitrification, cooccurs with methane produced by methanogens. As such, populations of “Ca. Methanoperedens nitroreducens” could be prone to regular oxygen exposure. Here, we investigated the effect of 5% (vol/vol) oxygen exposure in batch activity assays on a “Ca. Methanoperedens nitroreducens” culture, enriched from an Italian paddy field. Metagenome sequencing of the DNA extracted from the enrichment culture revealed that 83% of 16S rRNA gene reads were assigned to a novel strain, “Candidatus Methanoperedens nitroreducens Verserenetto.” RNA was extracted, and metatranscriptome sequencing upon oxygen exposure revealed that the active community changed, most notably in the appearance of aerobic methanotrophs. The gene expression of “Ca. Methanoperedens nitroreducens” revealed that the key genes encoding enzymes of the methane oxidation and nitrate reduction pathways were downregulated. In contrast to this, we identified upregulation of glutaredoxin, thioredoxin family/like proteins, rubrerythrins, peroxiredoxins, peroxidase, alkyl hydroperoxidase, type A flavoproteins, FeS cluster assembly protein, and cysteine desulfurases, indicating the genomic potential of “Ca. Methanoperedens nitroreducens Verserenetto” to counteract the oxidative damage and adapt in environments where they might be exposed to regular oxygen intrusion.

Understanding how microorganisms respond to environmental disturbance is one of the key focuses in microbial ecology. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are responsible for ammonia oxidation which is a crucial step in the nitrogen cycle. Although the physiology, distribution, and activity of AOA and AOB in soil have been extensively investigated, their recovery from a natural disturbance remains largely unknown. To assess the recovery capacities, including resistance and resilience, of AOA and AOB, soil samples were taken from a reservoir riparian zone which experienced periodically water flooding. The samples were classified into three groups (flooding, recovery, and control) for a high-throughput sequencing and quantitative PCR analysis. We used a relative quantitative index of both the resistance (RS) and resilience (RL) to assess the variation of gene abundance, alpha-diversity, and community composition. The AOA generally demonstrated a better recovery capability after the flooding disturbance compared to AOB. In particular, AOA were more resilient after the flooding disturbance. Taxa within the AOA and AOB showed different RS and RL values, with the most abundant taxa showing in general the highest RS indices. Soil NH4+ and Fe2+/Fe3+ were the main variables controlling the key taxa of AOA and AOB and probably influenced the resistance and resilience properties of AOA and AOB communities. The distinct mechanisms of AOA and AOB in maintaining community stability against the flooding disturbance might be linked to the different life-history strategies: the AOA community was more likely to represent r-strategists in contrast to the AOB community following a K-life strategy. Our results indicated that the AOA may play a vital role in ammonia oxidation in a fluctuating habitat and contribute to the stability of riparian ecosystem.

Contains fulltext : 176765.pdf (publisher's version ) (Open Access), The genomes of the aerobic methanotrophs "Methyloterricola oryzae" strain 73aT and Methylomagnum ishizawai strain 175 were sequenced. Both strains were isolated from rice plants. Methyloterricola oryzae strain 73aT represents the first isolate of rice paddy cluster I, and strain 175 is the second representative of the recently described genus Methylomagnum.

We present the draft genome of anammox bacterium “Candidatus Scalindua brodae,” which at 282 contigs is a major improvement over the highly fragmented genome assembly of related species “Ca. Scalindua profunda” (1,580 contigs) which was previously published., Biotechnology, Applied Sciences

Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides (LCOs). Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accumulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizobium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and isopentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase (CCaMK), a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein2/Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

Hydrothermally active sediments are highly productive, chemosynthetic areas which are characterized by the rapid turnover of particulate organic matter under extreme conditions in which ammonia is liberated. These systems might be suitable habitats for anaerobic ammonium oxidizing (anammox) bacteria but this has not been investigated in detail. Here we report the diversity and abundance of anammox bacteria in sediments that seep cold hydrocarbon-rich fluids and hydrothermal vent areas of the Guaymas Basin in the Cortés Sea using the unique functional anammox marker gene, hydrazine synthase (hzsA). All clones retrieved were closely associated to the “Candidatus Scalindua” genus. Phylogenetic analysis revealed two distinct clusters of hzsA sequences (Ca. Scalindua hzsA cluster I and II). Comparison of individual sequences from both clusters showed that several of these sequences had a similarity as low as 76% on nucleotide level. Based on the analysis of this phylomarker, a very high interspecies diversity within the marine anammox group is apparent. Absolute numbers of anammox bacteria in the sediments samples were determined by amplification of a 257 bp fragment of the hszA gene in a qPCR assay. The results indicate that numbers of anammox bacteria are generally higher in cold hydrocarbon-rich sediments compared to the vent areas and the reference zone. Ladderanes, lipids unique to anammox bacteria were also detected in several of the sediment samples corroborating the hzsA analysis. Due to the high concentrations of reduced sulfur compounds and its potential impact on the cycling of nitrogen we aimed to get an indication about the key players in the oxidation of sulfide in the Guaymas Basin sediments using the alpha subunit of the adenosine-5'-phosphosulfate (APS) reductase (aprA). Amplification of the aprA gene revealed a high number of gammaproteobacterial aprA genes covering the two sulfur-oxidizing bacteria aprA lineages as well as sulfate-reducers

Marine microorganisms are important for the global nitrogen cycle, but marine nitrifiers, especially aerobic nitrite oxidizers, remain largely unexplored. To increase the number of cultured representatives of marine nitrite-oxidizing bacteria (NOB), a bioreactor cultivation approach was adopted to first enrich nitrifiers and ultimately nitrite oxidizers from Dutch coastal North Sea water. With solely ammonia as the substrate an active nitrifying community consisting of novel marine Nitrosomonas aerobic ammonia oxidizers (ammonia-oxidizing bacteria) and Nitrospina and Nitrospira NOB was obtained which converted a maximum of 2 mmol of ammonia per liter per day. Switching the feed of the culture to nitrite as a sole substrate resulted in a Nitrospira NOB dominated community (approximately 80% of the total microbial community based on fluorescence in situ hybridization and metagenomic data) converting a maximum of 3 mmol of nitrite per liter per day. Phylogenetic analyses based on the 16S rRNA gene indicated that the Nitrospira enriched from the North Sea is a novel Nitrospira species with Nitrospira marina as the next taxonomically described relative (94% 16S rRNA sequence identity). Transmission electron microscopy analysis revealed a cell plan typical for Nitrospira species. The cytoplasm contained electron light particles that might represent glycogen storage. A large periplasmic space was present which was filled with electron dense particles. Nitrospira-targeted polymerase chain reaction analyses demonstrated the presence of the enriched Nitrospira species in a time series of North Sea genomic DNA samples. The availability of this new Nitrospira species enrichment culture facilitates further in-depth studies such as determination of physiological constraints, and comparison to other NOB species.

Background Sequencing technologies have different biases, in single-genome sequencing and metagenomic sequencing; these can significantly affect ORFs recovery and the population distribution of a metagenome. In this paper we investigate how well different technologies represent information related to a considered organism of interest in a metagenome, and whether it is beneficial to combine information obtained using different technologies. We analyze comparatively three metagenomic datasets acquired from a sample containing the anammox bacterium Candidatus ’Brocadia fulgida’ (B. fulgida). These datasets were obtained using Roche 454 FLX and Sanger sequencing with two different libraries (shotgun and fosmid). Results In each dataset, the abundance of the reads annotated to B. fulgida was much lower than the abundance expected from available cell count information. This was due to the overrepresentation of GC-richer organisms, as shown by GC-content distribution of the reads. Nevertheless, by considering the union of B. fulgida reads over the three datasets, the number of B. fulgida ORFs recovered for at least 80% of their length was twice the amount recovered by the best technology. Indeed, while taxonomic distributions of reads in the three datasets were similar, the respective sets of B. fulgida ORFs recovered for a large part of their length were highly different, and depth of coverage patterns of 454 and Sanger were dissimilar. Conclusions Precautions should be sought in order to prevent the overrepresentation of GC-rich microbes in the datasets. This overrepresentation and the consistency of the taxonomic distributions of reads obtained with different sequencing technologies suggests that, in general, abundance biases might be mainly due to other steps of the sequencing protocols. Results show that biases against organisms of interest could be compensated combining different sequencing technologies, due to the differences of their genome-level sequencing biases, BT/Biotechnology, Applied Sciences

Anaerobic ammonium-oxidizing (anammox) bacteria primarily grow by the oxidation of ammonium coupled to nitrite reduction, using CO2 as the sole carbon source. Although they were neglected for a long time, anammox bacteria are encountered in an enormous species (micro)diversity in virtually any anoxic environment that contains fixed nitrogen. It has even been estimated that about 50% of all nitrogen gas released into the atmosphere is made by these ‘impossible’ bacteria. Anammox catabolism most likely resides in a special cell organelle, the anammoxosome, which is surrounded by highly unusual ladder-like (ladderane) lipids. Ammonium oxidation and nitrite reduction proceed in a cyclic electron flow through two intermediates, hydrazine and nitric oxide, resulting in the generation of proton-motive force for ATP synthesis. Reduction reactions associated with CO2 fixation drain lectrons from this cycle, and they are replenished by the oxidation of nitrite to nitrate. Besides ammonium or nitrite, anammox bacteria use a broad range of organic and inorganic compounds as electron donors. An analysis of the metabolic opportunities even suggests alternative chemolithotrophic lifestyles that are independent of these compounds. We note that current concepts are still largely hypothetical and put forward the most intriguing questions that need experimental answers., BT/Biotechnology, Applied Sciences

In marine oxygen minimum zones (OMZs), ammonia-oxidizing archaea (AOA) rather than marine ammonia-oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium-oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory-scale model system under oxygen limitation. A bioreactor containing Candidatus Scalindua profunda marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. Ca. Scalindua profunda and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C18 anammox [3]-ladderane fatty acid was highly abundant in the oxygen-limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 mu M finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.

Bacteriohopanepolyols (BHPs) are bacterial biomarkers with a likely potential to identify present and past methanotrophic communities. To unravel the methanotrophic community in peat bogs, we report the BHP signatures of type I and type II methanotrophs isolated from Sphagnum mosses and of an extreme acidophilic verrucomicrobial methanotroph. A type I Methylovulum-like strain (M200) contains a remarkable combination of BHPs, including a complete suite of mono-unsaturated aminobacteriohopanepentol, -tetrol and -triol. The Methylomonas-like strain (M5) mainly produces aminobacteriohopanepentol, characteristic for type I methanotrophs, and the Methylosinus-like strain (29) contains both aminobacteriohopanetetrol and aminobacteriohopanetriol, typical for a type II methanotroph. The type II methanotroph Methylocella palustris and the verrucomicrobial Methylacidiphilum fumariolicum strain SolV primarily produce aminotriol, which is also produced by many other bacteria. In Sphagnum mosses and underlying peat from a peat bog from Moorhouse, UK, the only detectable BHPs indicative of methanotrophs are aminobacteriohopanepentol (aminopentol) and aminobacteriohopanetetrol (aminotetrol), although both are relatively low in abundance compared to other BHPs. Aminopentol serves as a marker for type I methanotrophs, while aminotetrol may reflect the presence of both type I and type II methanotrophs. The similar quantities of aminotetrol and aminopentol indicate that the methanotrophic community in Sphagnum peat probably consist of a combination of both type I and type II methanotrophs, which is in line with previously published pmoA-based micro-array results.

Anaerobic ammonium-oxidizing (anammox) bacteria are responsible for a significant portion of the loss of fixed nitrogen from the oceans, making them important players in the global nitrogen cycle. To date, marine anammox bacteria found in marine water columns and sediments worldwide belong almost exclusively to the ‘Candidatus Scalindua’ species, but the molecular basis of their metabolism and competitive fitness is presently unknown. We applied community sequencing of a marine anammox enrichment culture dominated by ‘Candidatus Scalindua profunda’ to construct a genome assembly, which was subsequently used to analyse the most abundant gene transcripts and proteins. In the S. profunda assembly, 4756 genes were annotated, and only about half of them showed the highest identity to the only other anammox bacterium of which a metagenome assembly had been constructed so far, the freshwater ‘Candidatus Kuenenia stuttgartiensis’. In total, 2016 genes of S. profunda could not be matched to the K. stuttgartiensis metagenome assembly at all, and a similar number of genes in K. stuttgartiensis could not be found in S. profunda. Most of these genes did not have a known function but 98 expressed genes could be attributed to oligopeptide transport, amino acid metabolism, use of organic acids and electron transport. On the basis of the S. profunda metagenome, and environmental metagenome data, we observed pronounced differences in the gene organization and expression of important anammox enzymes, such as hydrazine synthase (HzsAB), nitrite reductase (NirS) and inorganic nitrogen transport proteins. Adaptations of Scalindua to the substrate limitation of the ocean may include highly expressed ammonium, nitrite and oligopeptide transport systems and pathways for the transport, oxidation, and assimilation of small organic compounds that may allow a more versatile lifestyle contributing to the competitive fitness of Scalindua in the marine realm., BT/Biotechnology, Applied Sciences

Sphagnum peatlands are important ecosystems in the methane cycle. Methane-oxidizing bacteria in these ecosystems serve as a methane filter and limit methane emissions. Yet little is known about the diversity and identity of the methanotrophs present in and on Sphagnum mosses of peatlands, and only a few isolates are known. The methanotrophic community in Sphagnum mosses, originating from a Dutch peat bog, was investigated using a pmoA microarray. A high biodiversity of both gamma- and alphaproteobacterial methanotrophs was found. With Sphagnum mosses as the inoculum, alpha-and gammaproteobacterial acidophilic methanotrophs were isolated using established and newly designed media. The 16S rRNA, pmoA, pxmA, and mmoX gene sequences showed that the alphaproteobacterial isolates belonged to the Methylocystis and Methylosinus genera. The Methylosinus species isolated are the first acid-tolerant members of this genus. Of the acidophilic gammaproteobacterial strains isolated, strain M5 was affiliated with the Methylomonas genus, and the other strain, M200, may represent a novel genus, most closely related to the genera Methylosoma and Methylovulum. So far, no acidophilic or acid-tolerant methanotrophs in the Gammaproteobacteria class are known. All strains showed the typical features of either type I or II methanotrophs and are, to the best of our knowledge, the first isolated (acidophilic or acid-tolerant) methanotrophs from Sphagnum mosses.

The role of the microbial processes governing methane emissions from tundra ecosystems is receiving increasing attention. Recently, cooperation between methanotrophic bacteria and submerged Sphagnum was shown to reduce methane emissions but also to supply CO

Recently discovered microorganisms affiliated to the bacterial phylum NC10, named “Candidatus Methylomirabilis oxyfera”, perform nitrite-dependent anaerobic methane oxidation. These microorganisms could be important players in a novel way of anaerobic wastewater treatment where ammonium and residual dissolved methane might be removed at the expense of nitrate or nitrite. To find suitable inocula for reactor startup, ten selected wastewater treatment plants (WWTPs) located in The Netherlands were screened for the endogenous presence of M. oxyfera using molecular diagnostic methods. We could identify NC10 bacteria with 98% similarity to M. oxyfera in nine out of ten WWTPs tested. Sludge from one selected WWTP was used to start a new enrichment culture of NC10 bacteria. This enrichment was monitored using specific pmoA primers and M. oxyfera cells were visualized with fluorescence oligonucleotide probes. After 112 days, the enrichment consumed up to 0.4 mM NO2- per day. The results of this study show that appropriate sources of biomass, enrichment strategies, and diagnostic tools existed to start and monitor pilot scale tests for the implementation of nitrite-dependent methane oxidation in wastewater treatment at ambient temperature

The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes exc