The 'oxygen' in oxygen minimum zones.

Aerobic processes require oxygen, and anaerobic processes are typically hindered by it. In many places in the global ocean, oxygen is completely removed at mid-water depths forming anoxic oxygen minimum zones (A-OMZs). Within the oxygen gradients linking oxygenated waters with A-OMZs, there is a transition from aerobic to anaerobic microbial processes. This transition is not sharp and there is an overlap between processes using oxygen and those using other electron acceptors. This review will focus on the oxygen control of aerobic and anaerobic metabolisms and will explore how this overlap impacts both the carbon and nitrogen cycles in A-OMZ environments. We will discuss new findings on non-phototrophic microbial processes that produce oxygen, and we focus on how oxygen impacts the loss of fixed nitrogen (as N2 ) from A-OMZ waters. There are both physiological and environmental controls on the activities of microbial processes responsible for N2 loss, and the environmental controls are active at extremely low levels of oxygen. Understanding how these controls function will be critical to understanding and predicting how fixed-nitrogen loss in the oceans will respond to future global warming.

Microbe Profile: Nitrosopumilus maritimus.

Nitrosopumilus maritimus is a marine ammonia-oxidizing archaeon with a high affinity for ammonia. It fixes carbon via a modified hydroxypropionate/hydroxybutyrate cycle and shows weak utilization of cyanate as a supplementary energy and nitrogen source. When oxygen is depleted, N. maritimus produces its own oxygen, which may explain its regular occurrence in anoxic waters. Several enzymes of the ammonia oxidation and oxygen production pathways remain to be identified.

Oxygen and nitrogen production by an ammonia-oxidizing archaeon.

Ammonia-oxidizing archaea (AOA) are one of the most abundant groups of microbes in the world's oceans and are key players in the nitrogen cycle. Their energy metabolism­the oxidation of ammonia to nitrite­requires oxygen. Nevertheless, AOA are abundant in environments where oxygen is undetectable. By carrying out incubations for which oxygen concentrations were resolved to the nanomolar range, we show that after oxygen depletion, Nitrosopumilus maritimus produces dinitrogen and oxygen, which is used for ammonia oxidation. The pathway is not completely resolved but likely has nitric oxide and nitrous oxide as key intermediates. N. maritimus joins a handful of organisms known to produce oxygen in the dark. On the basis of this ability, we reevaluate the role of N. maritimus in oxygen-depleted marine environments.

Proteogenomic analysis of Georgfuchsia toluolica revealed unexpected concurrent aerobic and anaerobic toluene degradation.

Denitrifying Betaproteobacteria play a key role in the anaerobic degradation of monoaromatic hydrocarbons. We performed a multi-omics study to better understand the metabolism of the representative organism Georgfuchsia toluolica strain G5G6 known as a strict anaerobe coupling toluene oxidation with dissimilatory nitrate and Fe(III) reduction. Despite the genomic potential for degradation of different carbon sources, we did not find sugar or organic acid transporters, in line with the inability of strain G5G6 to use these substrates. Using a proteomics analysis, we detected proteins of fumarate-dependent toluene activation, membrane-bound nitrate reductase, and key components of the metal-reducing (Mtr) pathway under both nitrate- and Fe(III)-reducing conditions. High abundance of the multiheme cytochrome MtrC implied that a porin-cytochrome complex was used for respiratory Fe(III) reduction. Remarkably, strain G5G6 contains a full set of genes for aerobic toluene degradation, and we detected enzymes of aerobic toluene degradation under both nitrate- and Fe(III)-reducing conditions. We further detected an ATP-dependent benzoyl-CoA reductase, reactive oxygen species detoxification proteins, and cytochrome c oxidase indicating a facultative anaerobic lifestyle of strain G5G6. Correspondingly, we found diffusion through the septa a substantial source of oxygen in the cultures enabling concurrent aerobic and anaerobic toluene degradation by strain G5G6.

The rates and players of denitrification, dissimilatory nitrate reduction to ammonia (DNRA) and anaerobic ammonia oxidation (anammox) in mangrove soils.

Mangroves are ecosystems located in the transition zone between land and sea, characterized by periodic flooding that confer to its unique characteristics. Little is known about the transformation of nutrients that occur during the organic matter degradation in this system. In this study, we monitor the nitrogen transformations in soils from three mangroves with distinct levels of contamination using labeled 15NO3-. We also screened the mangroves metagenomes for the presence of genes that encode enzymes involved in denitrification (nirS, nirK, nosZ, norB and narG), anaerobic oxidation of ammonia (anammox) (hh, hao and hzo) and dissimilatory nitrate reduction to ammonium (DNRA) (nrfA). The transformations of 15NO3- indicated the balance of denitrification over anammox and DNRA in all three mangroves, with lower rates of processes in the mangrove affected by oil contamination. The metagenomic analysis detected 56 sequences related to denitrification, 19 with anammox and 6 with DNRA. Genes related with denitrification were phylogenetically distributed among several groups of bacteria (mainly Gammaproteobacteria). Anammox and DNRA related sequences were affiliated with Planctomycetes and Gammaproteobacteria, respectively. Thus, metagenomic and functional approaches supported the description of denitrification, anammox and DNRA rates in mangrove soils, and identified the major bacterial groups involved in these processes.

Impacts of chemical gradients on microbial community structure.

Succession of redox processes is sometimes assumed to define a basic microbial community structure for ecosystems with oxygen gradients. In this paradigm, aerobic respiration, denitrification, fermentation and sulfate reduction proceed in a thermodynamically determined order, known as the 'redox tower'. Here, we investigated whether redox sorting of microbial processes explains microbial community structure at low-oxygen concentrations. We subjected a diverse microbial community sampled from a coastal marine sediment to 100 days of tidal cycling in a laboratory chemostat. Oxygen gradients (both in space and time) led to the assembly of a microbial community dominated by populations that each performed aerobic and anaerobic metabolism in parallel. This was shown by metagenomics, transcriptomics, proteomics and stable isotope incubations. Effective oxygen consumption combined with the formation of microaggregates sustained the activity of oxygen-sensitive anaerobic enzymes, leading to braiding of unsorted redox processes, within and between populations. Analyses of available metagenomic data sets indicated that the same ecological strategies might also be successful in some natural ecosystems.

Nitrogen cycling. The environmental controls that govern the end product of bacterial nitrate respiration.

In the biogeochemical nitrogen cycle, microbial respiration processes compete for nitrate as an electron acceptor. Denitrification converts nitrate into nitrogenous gas and thus removes fixed nitrogen from the biosphere, whereas ammonification converts nitrate into ammonium, which is directly reusable by primary producers. We combined multiple parallel long-term incubations of marine microbial nitrate-respiring communities with isotope labeling and metagenomics to unravel how specific environmental conditions select for either process. Microbial generation time, supply of nitrite relative to nitrate, and the carbon/nitrogen ratio were identified as key environmental controls that determine whether nitrite will be reduced to nitrogenous gas or ammonium. Our results define the microbial ecophysiology of a biogeochemical feedback loop that is key to global change, eutrophication, and wastewater treatment.

Rapid succession of uncultured marine bacterial and archaeal populations in a denitrifying continuous culture.

Marine denitrification constitutes an important part of the global nitrogen cycle and the diversity, abundance and process rates of denitrifying microorganisms have been the focus of many studies. Still, there is little insight in the ecophysiology of marine denitrifying communities. In this study, a heterotrophic denitrifying community from sediments of a marine intertidal flat active in nitrogen cycling was selected in a chemostat and monitored over a period of 50 days. The chemostat enabled the maintenance of constant and well-defined experimental conditions over the time-course of the experiment. Analysis of the microbial community composition by automated ribosomal intergenic spacer analysis (ARISA), Illumina sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) revealed strong dynamics in community composition over time, while overall denitrification by the enrichment culture was stable. Members of the genera Arcobacter, Pseudomonas, Pseudovibrio, Rhodobacterales and of the phylum Bacteroidetes were identified as the dominant denitrifiers. Among the fermenting organisms co-enriched with the denitrifiers was a novel archaeon affiliated with the recently proposed DPANN-superphylum. The pan-genome of populations affiliated to Pseudovibrio encoded a NirK as well as a NirS nitrite reductase, indicating the rare co-occurrence of both evolutionary unrelated nitrite reductases within coexisting subpopulations.

Recoding of the stop codon UGA to glycine by a BD1-5/SN-2 bacterium and niche partitioning between Alpha- and Gammaproteobacteria in a tidal sediment microbial community naturally selected in a laboratory chemostat.

Sandy coastal sediments are global hotspots for microbial mineralization of organic matter and denitrification. These sediments are characterized by advective porewater flow, tidal cycling and an active and complex microbial community. Metagenomic sequencing of microbial communities sampled from such sediments showed that potential sulfur oxidizing Gammaproteobacteria and members of the enigmatic BD1-5/SN-2 candidate phylum were abundant in situ (>10% and ~2% respectively). By mimicking the dynamic oxic/anoxic environmental conditions of the sediment in a laboratory chemostat, a simplified microbial community was selected from the more complex inoculum. Metagenomics, proteomics and fluorescence in situ hybridization showed that this simplified community contained both a potential sulfur oxidizing Gammaproteobacteria (at 24 ± 2% abundance) and a member of the BD1-5/SN-2 candidate phylum (at 7 ± 6% abundance). Despite the abundant supply of organic substrates to the chemostat, proteomic analysis suggested that the selected gammaproteobacterium grew partially autotrophically and performed hydrogen/formate oxidation. The enrichment of a member of the BD1-5/SN-2 candidate phylum enabled, for the first time, direct microscopic observation by fluorescent in situ hybridization and the experimental validation of the previously predicted translation of the stop codon UGA into glycine.

Desulfofrigus sp. prevails in sulfate-reducing dilution cultures from sediments of the Benguela upwelling area.

Sediments of coastal upwelling areas are generally characterized by a high content of organic carbon that is mainly degraded via anaerobic microbial processes including sulfate reduction as a major terminal oxidation step. Despite the high importance of sulfate reduction in these sediments, the identity of sulfate-reducing bacteria (SRB) has remained almost unknown. Here, we applied a cultivation-based approach using selective enrichment conditions to study the diversity and distribution of active SRB in sediments along a transect perpendicular to the continental slope off the coast of Namibia (Meteor-cruise M76/1). To promote growth of the most abundant SRB, dilution series were prepared and amended with hydrogen, acetate, or a mixture of monomers representing typical substrates for SRB. Growth of SRB could be detected in the presence of all electron donors and from sediment down to 4 m depth. 16S rRNA gene-based DGGE analysis and sequencing revealed the predominance of SRB related to psychrophiles in particular to the genus Desulfofrigus, which made up 1 % of the total microbial community, accounting for an absolute abundance of up to 4.8 × 10(7)  cells mL(-1) . In general, the abundance of cultured SRB changed with depth and between the different sampling sites and correlated with the content of organic carbon as previously reported. Growth of chemolithotrophic SRB in relatively high dilution steps and the enrichment of methanogens as well as acetogens from deeper sediment point to a competition between hydrogen-utilizing microbial processes and their biogeochemical significance in deep sediment layers of the Benguela upwelling area.

The binning of metagenomic contigs for microbial physiology of mixed cultures.

So far, microbial physiology has dedicated itself mainly to pure cultures. In nature, cross feeding and competition are important aspects of microbial physiology and these can only be addressed by studying complete communities such as enrichment cultures. Metagenomic sequencing is a powerful tool to characterize such mixed cultures. In the analysis of metagenomic data, well established algorithms exist for the assembly of short reads into contigs and for the annotation of predicted genes. However, the binning of the assembled contigs or unassembled reads is still a major bottleneck and required to understand how the overall metabolism is partitioned over different community members. Binning consists of the clustering of contigs or reads that apparently originate from the same source population. In the present study eight metagenomic samples from the same habitat, a laboratory enrichment culture, were sequenced. Each sample contained 13-23 Mb of assembled contigs and up to eight abundant populations. Binning was attempted with existing methods but they were found to produce poor results, were slow, dependent on non-standard platforms or produced errors. A new binning procedure was developed based on multivariate statistics of tetranucleotide frequencies combined with the use of interpolated Markov models. Its performance was evaluated by comparison of the results between samples with BLAST and in comparison to existing algorithms for four publicly available metagenomes and one previously published artificial metagenome. The accuracy of the new approach was comparable or higher than existing methods. Further, it was up to a 100 times faster. It was implemented in Java Swing as a complete open source graphical binning application available for download and further development (http://sourceforge.net/projects/metawatt).

Influence of different biomaterials on the viability of Aggregatibacter actinomycetemcomitans.

OBJECTIVES: The aim of the present in vitro study was to evaluate the effects of different biomaterials used for regenerative periodontal surgery on the growth of the periodontopathogen Aggregatibacter actinomycetemcomitans. METHODS: Three commercially available biomaterials of synthetic origin (hydroxyapatite/beta-tricalcium phosphate, nanostructured hydroxyapatite paste, oily calcium hydroxide suspension), a bovine-derived xenograft as well as an enamel matrix derivative (EMD) were added in different concentrations to calibrated suspensions of A. actinomycetemcomitans ATCC 43718/33384 (serotype b/c). Equal aliquots (0.1 ml) for the viability assay were taken after 5 min, 1h, 3h, 8h and 24h, plated on blood agar and incubated in an anaerobic environment for 48 h at 37°C. Viable cell counts were expressed as colony forming units (cfu)/0.1 ml. RESULTS: The results demonstrated that none of the investigated biomaterials could inhibit the growth of A. actinomycetemcomitans serotype b. A marked growth reduction of A. actinomycetemcomitans serotype c was observed in the presence of oily calcium hydroxide suspension and nanostructured hydroxyapatite. In contrast, no significant growth inhibition could be observed in the presence of hydroxyapatite/beta-tricalcium phosphate, enamel matrix derivative and bovine-derived xenograft. CONCLUSIONS: The results of the present study suggest that none of the investigated biomaterials possesses antimicrobial properties against A. actinomycetemcomitans serotype b. Therefore, the use of these biomaterials for regenerative procedures should be weighted critically in the presence of A. actinomycetemcomitans serotype b.

Microbial nitrate respiration--genes, enzymes and environmental distribution.

Nitrate is a key node in the network of the assimilatory and respiratory nitrogen pathways. As one of the 'fixed' forms of nitrogen, nitrate plays an essential role in both nature and industry. For bacteria, it is both a nitrogen source and an electron acceptor. In agriculture and wastewater treatment, nitrate respiration by microorganisms is an important issue with respect to economics, greenhouse gas emission and public health. Several microbial processes compete for nitrate: denitrification, dissimilatory nitrate reduction to ammonium and anaerobic ammonium oxidation. In this review we provide an up to date overview of the organisms, genes and enzymes involved in nitrate respiration. We also address the molecular detection of these processes in nature. We show that despite rapid progress in the experimental and genomic analyses of pure cultures, knowledge on the mechanism of nitrate reduction in natural ecosystems is still largely lacking.