Nitrosomonas supralitoralis sp. nov., an ammonia-oxidizing bacterium from beach sand in a supralittoral zone.

A betaproteobacterial chemolithotrophic ammonia-oxidizing bacterium designated APG5T was isolated from supralittoral sand of the Edmonds City Beach, WA, USA. Growth was observed at 10-35 °C (optimum, 30 °C), pH 5-9 (optimum, pH 8) and ammonia concentrations as high as 100 mM (optimum, 1-30 mM NH4Cl). The strain grows optimally in a freshwater medium but tolerates up to 400 mM NaCl. It is most closely related to 'Nitrosomonas ureae' (96.7% 16S rRNA and 92.4% amoA sequence identity). The 3.75-Mbp of AGP5T draft genome contained a single rRNA operon and all necessary tRNA genes and has the lowest G+C content (43.5%) when compared to the previously reported genomes of reference strains in cluster 6 Nitrosomonas. Based on an average nucleotide identity of 82% with its closest relative ('N. ureae' Nm10T) and the suggested species boundary of 95-96%, a new species Nitrosomonas supralitoralis sp. nov. is proposed. The type strain of Nitrosomonas supralitoralis is APG5T (= NCIMB 14870T = ATCC TSD-116T).

A Physiological and Genomic Comparison of Nitrosomonas Cluster 6a and 7 Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) within the genus Nitrosomonas perform the first step in nitrification, ammonia oxidation, and are found in diverse aquatic and terrestrial environments. Nitrosomonas AOB were grouped into six defined clusters, which correlate with physiological characteristics that contribute to adaptations to a variety of abiotic environmental factors. A fundamental physiological trait differentiating Nitrosomonas AOB is the adaptation to either low (cluster 6a) or high (cluster 7) ammonium concentrations. Here, we present physiological growth studies and genome analysis of Nitrosomonas cluster 6a and 7 AOB. Cluster 6a AOB displayed maximum growth rates at ≤ 1 mM ammonium, while cluster 7 AOB had maximum growth rates at ≥ 5 mM ammonium. In addition, cluster 7 AOB were more tolerant of high initial ammonium and nitrite concentrations than cluster 6a AOB. Cluster 6a AOB were completely inhibited by an initial nitrite concentration of 5 mM. Genomic comparisons were used to link genomic traits to observed physiological adaptations. Cluster 7 AOB encode a suite of genes related to nitrogen oxide detoxification and multiple terminal oxidases, which are absent in cluster 6a AOB. Cluster 6a AOB possess two distinct forms of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and select species encode genes for hydrogen or urea utilization. Several, but not all, cluster 6a AOB can utilize urea as a source of ammonium. Hence, although Nitrosomonas cluster 6a and 7 AOB have the capacity to fulfill the same functional role in microbial communities, i.e., ammonia oxidation, differentiating species-specific and cluster-conserved adaptations is crucial in understanding how AOB community succession can affect overall ecosystem function.

Methane oxidation coupled to nitrate reduction under hypoxia by the Gammaproteobacterium Methylomonas denitrificans, sp. nov. type strain FJG1.

Obligate methanotrophs belonging to the Phyla Proteobacteria and Verrucomicrobia require oxygen for respiration and methane oxidation; nevertheless, aerobic methanotrophs are abundant and active in low oxygen environments. While genomes of some aerobic methanotrophs encode putative nitrogen oxide reductases, it is not understood whether these metabolic modules are used for NOx detoxification, denitrification or other purposes. Here we demonstrate using microsensor measurements that a gammaproteobacterial methanotroph Methylomonas denitrificans sp. nov. strain FJG1(T) couples methane oxidation to nitrate reduction under oxygen limitation, releasing nitrous oxide as a terminal product. Illumina RNA-Seq data revealed differential expression of genes encoding a denitrification pathway previously unknown to methanotrophs as well as the pxmABC operon in M. denitrificans sp. nov. strain FJG1(T) in response to hypoxia. Physiological and transcriptome data indicate that genetic inventory encoding the denitrification pathway is upregulated only upon availability of nitrate under oxygen limitation. In addition, quantitation of ATP levels demonstrates that the denitrification pathway employs inventory such as nitrate reductase NarGH serving M. denitrificans sp. nov. strain FJG1(T) to conserve energy during oxygen limitation. This study unravelled an unexpected metabolic flexibility of aerobic methanotrophs, thereby assigning these bacteria a new role at the metabolic intersection of the carbon and nitrogen cycles.

Distribution of ether lipids and composition of the archaeal community in terrestrial geothermal springs: impact of environmental variables.

Archaea can respond to changes in the environment by altering the composition of their membrane lipids, for example, by modification of the abundance and composition of glycerol dialkyl glycerol tetraethers (GDGTs). Here, we investigated the abundance and proportions of polar GDGTs (P-GDGTs) and core GDGTs (C-GDGTs) sampled in different seasons from Tengchong hot springs (Yunnan, China), which encompassed a pH range of 2.5-10.1 and a temperature range of 43.7-93.6°C. The phylogenetic composition of the archaeal community (reanalysed from published work) divided the Archaea in spring sediment samples into three major groups that corresponded with spring pH: acidic, circumneutral and alkaline. Cluster analysis showed correlation between spring pH and the composition of P- and C-GDGTs and archaeal 16S rRNA genes, indicating an intimate link between resident Archaea and the distribution of P- and C-GDGTs in Tengchong hot springs. The distribution of GDGTs in Tengchong springs was also significantly affected by temperature; however, the relationship was weaker than with pH. Analysis of published datasets including samples from Tibet, Yellowstone and the US Great Basin hot springs revealed a similar relationship between pH and GDGT content. Specifically, low pH springs had higher concentrations of GDGTs with high numbers of cyclopentyl rings than neutral and alkaline springs, which is consistent with the predominance of high cyclopentyl ring-characterized Sulfolobales and Thermoplasmatales present in some of the low pH springs. Our study suggests that the resident Archaea in these hot springs are acclimated if not adapted to low pH by their genetic capacity to effect the packing density of their membranes by increasing cyclopentyl rings in GDGTs at the rank of community.

Nitrosospira lacus sp. nov., a psychrotolerant, ammonia-oxidizing bacterium from sandy lake sediment.

A Gram-negative, spiral-shaped, chemolithotrophic, ammonia-oxidizing bacterium, designated APG3(T), was isolated into pure culture from sandy lake sediment collected from Green Lake, Seattle, WA, USA. Phylogenetic analyses based on the 16S rRNA gene sequence showed that strain APG3(T) belongs to cluster 0 of the genus Nitrosospira, which is presently not represented by described species, with Nitrosospira multiformis (cluster 3) as the closest species with a validly published name (identity of 98.6 % to the type strain). Strain APG3(T) grew at 4 °C but could not grow at 35 °C, indicating that this bacterium is psychrotolerant. Remarkably, the strain was able to grow over a wide range of pH (pH 5-9), which was greater than the pH range of any studied ammonia-oxidizing bacteria in pure culture. The DNA G+C content of the APG3(T) genome is 53.5 %, which is similar to that of Nitrosospira multiformis ATCC 25196(T) (53.9 %) but higher than that of Nitrosomonas europaea ATCC 19718 (50.7 %) and Nitrosomonas eutropha C71 (48.5 %). The average nucleotide identity (ANI) calculated for the genomes of strain APG3(T) and Nitrosospira multiformis ATCC 25196(T) was 75.45 %, significantly lower than the value of 95 % ANI that corresponds to the 70 % species-level cut-off based on DNA-DNA hybridization. Overall polyphasic taxonomy study indicated that strain APG3(T) represents a novel species in the genus Nitrosospira, for which the name Nitrosospira lacus sp. nov. is proposed (type strain APG3(T) = NCIMB 14869(T) = LMG 27536(T) = ATCC BAA-2542(T)).

Diversity and evolution of bioenergetic systems involved in microbial nitrogen compound transformations.

Nitrogen is an essential element of life that needs to be assimilated in its most reduced form, ammonium. On the other hand, nitrogen exists in a multitude of oxidation states and, consequently, nitrogen compounds (NCs) serve as electron donor and/or acceptors in many catabolic pathways including various forms of microbial respiration that contribute to the global biogeochemical nitrogen cycle. Some of these NCs are also known as reactive nitrogen species able to cause nitrosative stress because of their high redox reactivity. The best understood processes of the nitrogen cycle are denitrification and ammonification (both beginning with nitrate reduction to nitrite), nitrification (aerobic oxidation of ammonium and nitrite) and anaerobic ammonium oxidation (anammox). This review presents examples of the diverse architecture, either elucidated or anticipated, and the high degree of modularity of the corresponding respiratory electron transport processes found in Bacteria and Archaea, and relates these to their respective bioenergetic mechanisms of proton motive force generation. In contrast to the multiplicity of enzymes that catalyze NC transformations, the number of proteins or protein modules involved in connecting electron transport to and from these enzymes with the quinone/quinol pool is comparatively small. These quinone/quinol-reactive protein modules consist of cytochromes b and c and iron-sulfur proteins. Conclusions are drawn towards the evolutionary relationships of bioenergetic systems involved in NC transformation and deduced aspects of the evolution of the biogeochemical nitrogen cycle are presented. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Metabolic environments and genomic features associated with pathogenic and mutualistic interactions between bacteria and plants.

Genomic characteristics discriminating parasitic and mutualistic relationship of bacterial symbionts with plants are poorly understood. This study comparatively analyzed the genomes of 54 mutualists and pathogens to discover genomic markers associated with the different phenotypes. Using metabolic network models, we predict external environments associated with free-living and symbiotic lifestyles and quantify dependences of symbionts on the host in terms of the consumed metabolites. We show that specific differences between the phenotypes are pronounced at the levels of metabolic enzymes, especially carbohydrate active, and protein functions. Overall, biosynthetic functions are enriched and more diverse in plant mutualists whereas processes and functions involved in degradation and host invasion are enriched and more diverse in pathogens. A distinctive characteristic of plant pathogens is a putative novel secretion system with a circadian rhythm regulator. A specific marker of plant mutualists is the co-residence of genes encoding nitrogenase and ribulose bisphosphate carboxylase/oxygenase (RuBisCO). We predict that RuBisCO is likely used in a putative metabolic pathway to supplement carbon obtained heterotrophically with low-cost assimilation of carbon from CO2. We validate results of the comparative analysis by predicting correct phenotype, pathogenic or mutualistic, for 20 symbionts in an independent set of 30 pathogens, mutualists, and commensals.

Thaumarchaeotal signature gene distribution in sediments of the northern South China Sea: an indicator of the metabolic intersection of the marine carbon, nitrogen, and phosphorus cycles?

Thaumarchaeota are abundant and active in marine waters, where they contribute to aerobic ammonia oxidation and light-independent carbon fixation. The ecological function of thaumarchaeota in marine sediments, however, has rarely been investigated, even though marine sediments constitute the majority of the Earth's surface. Thaumarchaeota in the upper layer of sediments may contribute significantly to the reservoir of nitrogen oxides in ocean waters and thus to productivity, including the assimilation of carbon. We tested this hypothesis in the northern South China Sea (nSCS), a section of a large oligotrophic marginal sea with limited influx of nutrients, including nitrogen, by investigating the diversity, abundance, community structure, and spatial distribution of thaumarchaeotal signatures in surface sediments. Quantitative real-time PCR using primers designed to detect 16S rRNA and amoA genes in sediment community DNA revealed a significantly higher abundance of pertinent thaumarchaeotal than betaproteobacterial genes. This finding correlates with high levels of hcd genes, a signature of thaumarchaeotal autotrophic carbon fixation. Thaumarchaeol, a signature lipid biomarker for thaumarchaeota, constituted the majority of archaeal lipids in marine sediments. Sediment temperature and organic P and silt contents were identified as key environmental factors shaping the community structure and distribution of the monitored thaumarchaeotal amoA genes. When the pore water PO4(3-) concentration was controlled for via partial-correlation analysis, thaumarchaeotal amoA gene abundance significantly correlated with the sediment pore water NO2(-) concentration, suggesting that the amoA-bearing thaumarchaeota contribute to nitrite production. Statistical analyses also suggest that thaumarchaeotal metabolism could serve as a pivotal intersection of the carbon, nitrogen, and phosphorus cycles in marine sediments.

Environment-dependent distribution of the sediment nifH-harboring microbiota in the Northern South China Sea.

The South China Sea (SCS), the largest marginal sea in the Western Pacific Ocean, is a huge oligotrophic water body with very limited influx of nitrogenous nutrients. This suggests that sediment microbial N(2) fixation plays an important role in the production of bioavailable nitrogen. To test the molecular underpinning of this hypothesis, the diversity, abundance, biogeographical distribution, and community structure of the sediment diazotrophic microbiota were investigated at 12 sampling sites, including estuarine, coastal, offshore, deep-sea, and methane hydrate reservoirs or their prospective areas by targeting nifH and some other functional biomarker genes. Diverse and novel nifH sequences were obtained, significantly extending the evolutionary complexity of extant nifH genes. Statistical analyses indicate that sediment in situ temperature is the most significant environmental factor influencing the abundance, community structure, and spatial distribution of the sediment nifH-harboring microbial assemblages in the northern SCS (nSCS). The significantly positive correlation of the sediment pore water NH(4)(+) concentration with the nifH gene abundance suggests that the nSCS sediment nifH-harboring microbiota is active in N(2) fixation and NH(4)(+) production. Several other environmental factors, including sediment pore water PO(4)(3-) concentration, sediment organic carbon, nitrogen and phosphorus levels, etc., are also important in influencing the community structure, spatial distribution, or abundance of the nifH-harboring microbial assemblages. We also confirmed that the nifH genes encoded by archaeal diazotrophs in the ANME-2c subgroup occur exclusively in the deep-sea methane seep areas, providing for the possibility to develop ANME-2c nifH genes as a diagnostic tool for deep-sea methane hydrate reservoir discovery.

Genome sequence of the haloalkaliphilic methanotrophic bacterium Methylomicrobium alcaliphilum 20Z.

Methylomicrobium strains are widespread in saline environments. Here, we report the complete genome sequence of Methylomicrobium alcaliphilum 20Z, a haloalkaliphilic methanotrophic bacterium, which will provide the basis for detailed characterization of the core pathways of both single-carbon metabolism and responses to osmotic and high-pH stresses. Final assembly of the genome sequence revealed that this bacterium contains a 128-kb plasmid, making M. alcaliphilum 20Z the first methanotrophic bacterium of known genome sequence for which a plasmid has been reported.

Draft genome sequence of the volcano-inhabiting thermoacidophilic methanotroph Methylacidiphilum fumariolicum strain SolV.

The draft genome of Methylacidiphilum fumariolicum SolV, a thermoacidophilic methanotroph of the phylum Verrucomicrobia, is presented. Annotation revealed pathways for one-carbon, nitrogen, and hydrogen catabolism and respiration together with central metabolic pathways. The genome encodes three orthologues of particulate methane monooxygenases. Sequencing of this genome will help in the understanding of methane cycling in volcanic environments.

The Wolinella succinogenes mcc gene cluster encodes an unconventional respiratory sulphite reduction system.

Assimilatory and dissimilatory sulphite reductions are key reactions in the biogeochemical sulphur cycle and several distinct sirohaem-containing sulphite reductases have been characterized. Here, we describe that the Epsilonproteobacterium Wolinella succinogenes is able to grow by sulphite respiration (yielding sulphide) with formate as electron donor. Sulphite is reduced by MccA, a prototypical member of an emerging new class of periplasmic cytochrome c sulphite reductases that, phylogenetically, belongs to a multihaem cytochrome c superfamily whose members play crucial roles in the global sulphur and nitrogen cycles. Within this family, MccA represents an unconventional octahaem cytochrome c containing a special haem c group that is bound via two cysteine residues arranged in a unique CX(15)CH haem c binding motif. The phenotypes of numerous W.succinogenes mutants producing MccA variants underlined the structural importance of this motif. Several open reading frames of the mcc gene cluster were individually inactivated and characterization of the corresponding mutants indicated that the predicted iron-sulphur protein MccC, the putative quinol dehydrogenase MccD (a member of the NrfD/PsrC family) as well as a peptidyl-prolyl cis-trans isomerase, MccB, are essential for sulphite respiration. MccA synthesis in W. succinogenes was found to be induced by sulphite (but not by thiosulphate or sulphide) and repressed in the presence of fumarate or nitrate. Based on the results, a sophisticated model of respiratory sulphite reduction by the Mcc system is presented.

Effects of nitrogen dioxide and anoxia on global gene and protein expression in long-term continuous cultures of Nitrosomonas eutropha C91.

Nitrosomonas eutropha is an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO(2) on gene and protein expression under oxic and anoxic conditions were determined by maintaining N. eutropha strain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO(2), and anoxic-plus-NO(2) culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure of N. eutropha C91 to NO(2) under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO(2) resulted in increased representation of proteins and transcripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO(2) and anoxic-plus-NO(2) conditions. The results indicate that exposure to NO(2) results in an energy-deprived state of N. eutropha C91 and that anaerobic growth could not be supported with NO(2) as an oxidant.

Adaptations to submarine hydrothermal environments exemplified by the genome of Nautilia profundicola.

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment--some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20 degrees C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere--anaerobic, sulfur, H2- and CO2-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles.

Genome sequence of Nitrosomonas sp. strain AL212, an ammonia-oxidizing bacterium sensitive to high levels of ammonia.

Nitrosomonas sp. strain AL212 is an obligate chemolithotrophic ammonia-oxidizing bacterium (AOB) that was originally isolated in 1997 by Yuichi Suwa and colleagues. This organism belongs to Nitrosomonas cluster 6A, which is characterized by sensitivity to high ammonia concentrations, higher substrate affinity (lower K(m)), and lower maximum growth rates than strains in Nitrosomonas cluster 7, which includes Nitrosomonas europaea and Nitrosomonas eutropha. Genome-informed studies of this ammonia-sensitive cohort of AOB are needed, as these bacteria are found in freshwater environments, drinking water supplies, wastewater treatment systems, and soils worldwide.

Genome sequence of the Arctic methanotroph Methylobacter tundripaludum SV96.

Methylobacter tundripaludum SV96(T) (ATCC BAA-1195) is a psychrotolerant aerobic methane-oxidizing gammaproteobacterium (Methylococcales, Methylococcaceae) living in High Arctic wetland soil. The strain was isolated from soil harvested in July 1996 close to the settlement Ny-Ålesund, Svalbard, Norway (78°56'N, 11°53'E), and described as a novel species in 2006. The genome includes pmo and pxm operons encoding copper membrane monooxygenases (Cu-MMOs), genes required for nitrogen fixation, and the nirS gene implicated in dissimilatory nitrite reduction to NO but no identifiable inventory for further processing of nitrogen oxides. These genome data provide the basis to investigate M. tundripaludum SV96, identified as a major player in the biogeochemistry of Arctic environments.

Complete genome sequence of the aerobic marine methanotroph Methylomonas methanica MC09.

Methylomonas methanica MC09 is a mesophilic, halotolerant, aerobic, methanotrophic member of the Gammaproteobacteria, isolated from coastal seawater. Here we present the complete genome sequence of this strain, the first available from an aerobic marine methanotroph.

Genome sequence of the methanotrophic alphaproteobacterium Methylocystis sp. strain Rockwell (ATCC 49242).

Methylocystis sp. strain Rockwell (ATCC 49242) is an aerobic methane-oxidizing alphaproteobacterium isolated from an aquifer in southern California. Unlike most methanotrophs in the Methylocystaceae family, this strain has a single pmo operon encoding particulate methane monooxygenase but no evidence of the genes encoding soluble methane monooxygenase. This is the first reported genome sequence of a member of the Methylocystis species of the Methylocystaceae family in the order Rhizobiales.

Molecular characterization of putative biocorroding microbiota with a novel niche detection of Epsilon- and Zetaproteobacteria in Pacific Ocean coastal seawaters.

Submerged metal surfaces in marine waters undergo rapid microbial colonization and biocorrosion, causing huge damage to marine engineering facilities and significant financial losses. In coastal areas, an accelerated and particularly severe form of biocorrosion termed accelerated low water corrosion (ALWC) is widespread globally. While identification of biocorroding microorganisms and the dynamics of their community structures is the key for understanding the processes and mechanisms leading to ALWC, neither one is presently understood. In this study, analysis of constructed clone libraries and qPCR assays targeting group-specific 16S rRNA or functional marker genes were used to determine the identity and abundance of putative early carbon steel surface-colonizing and biocorroding microbes in coastal seawater. Diverse microbial groups including 10 bacterial phyla, archaea and algae were found to putatively participate in the surface-colonizing process. Analysis of the community structure of carbon steel surface microbiota revealed a temporal succession leading to ALWC. By extending the current state of knowledge, our work demonstrates the global importance of Alphaproteobacteria (mainly Rhodobacterales), Gammaproteobacteria (mainly Alteromonadales and Oceanospirillales), Bacteroidetes (mainly Flavobacteriales) and microalgae as the pioneer and sustaining surface colonizers that contribute to initial formation and development of surface biofilms. We also discovered Epsilonproteobacteria and the recently described Zetaproteobacteria as putative corrosion-causing microorganisms during early steps of the ALWC process. Hence, our study reports that Zetaproteobacteria may be ubiquitous also in non-hydrothermal coastal seawaters and that ALWC of submerged carbon steel surfaces in coastal waters may involve a highly diverse, complex and dynamic microbial consortium. Our finding that Epsilon- and Zetaproteobacteria may play pivotal roles in ALWC provides a new starting point for future investigation of the ALWC process and mechanism in marine environments. Further studies of Epsilon- and Zetaproteobacteria in particular may thus help with the design of effective corrosion prevention and control strategies.