James I. Prosser

Archaea predominate among ammonia-oxidizing prokaryotes in soils.

Ammonia oxidation is the first step in nitrification, a key process in the global nitrogen cycle that results in the formation of nitrate through microbial activity. The increase in nitrate availability in soils is important for plant nutrition, but it also has considerable impact on groundwater pollution owing to leaching. Here we show that archaeal ammonia oxidizers are more abundant in soils than their well-known bacterial counterparts. We investigated the abundance of the gene encoding a subunit of the key enzyme ammonia monooxygenase (amoA) in 12 pristine and agricultural soils of three climatic zones. amoA gene copies of Crenarchaeota (Archaea) were up to 3,000-fold more abundant than bacterial amoA genes. High amounts of crenarchaeota-specific lipids, including crenarchaeol, correlated with the abundance of archaeal amoA gene copies. Furthermore, reverse transcription quantitative PCR studies and complementary DNA analysis using novel cloning-independent pyrosequencing technology demonstrated the activity of the archaea in situ and supported the numerical dominance of archaeal over bacterial ammonia oxidizers. Our results indicate that crenarchaeota may be the most abundant ammonia-oxidizing organisms in soil ecosystems on Earth.

The influence of soil pH on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria.

Autotrophic ammonia oxidation occurs in acid soils, even though laboratory cultures of isolated ammonia oxidizing bacteria fail to grow below neutral pH. To investigate whether archaea possessing ammonia monooxygenase genes were responsible for autotrophic nitrification in acid soils, the community structure and phylogeny of ammonia oxidizing bacteria and archaea were determined across a soil pH gradient (4.9-7.5) by amplifying 16S rRNA and amoA genes followed by denaturing gradient gel electrophoresis (DGGE) and sequence analysis. The structure of both communities changed with soil pH, with distinct populations in acid and neutral soils. Phylogenetic reconstructions of crenarchaeal 16S rRNA and amoA genes confirmed selection of distinct lineages within the pH gradient and high similarity in phylogenies indicated a high level of congruence between 16S rRNA and amoA genes. The abundance of archaeal and bacterial amoA gene copies and mRNA transcripts contrasted across the pH gradient. Archaeal amoA gene and transcript abundance decreased with increasing soil pH, while bacterial amoA gene abundance was generally lower and transcripts increased with increasing pH. Short-term activity was investigated by DGGE analysis of gene transcripts in microcosms containing acidic or neutral soil or mixed soil with pH readjusted to that of native soils. Although mixed soil microcosms contained identical archaeal ammonia oxidizer communities, those adapted to acidic or neutral pH ranges showed greater relative activity at their native soil pH. Findings indicate that different bacterial and archaeal ammonia oxidizer phylotypes are selected in soils of different pH and that these differences in community structure and abundances are reflected in different contributions to ammonia oxidizer activity. They also suggest that both groups of ammonia oxidizers have distinct physiological characteristics and ecological niches, with consequences for nitrification in acid soils.

Autotrophic Nitrification in Bacteria

Publisher Summary This chapter highlights the physiology of autotrophic ammonia- and nitrite-oxidizing bacteria. Several aspects of the physiology of nitrifiers of relevance to their growth and activity in natural environments are considered. The chapter describes the basic features of the biochemistry of ammonia and nitrite oxidation and discusses the growth limiting factors and activity of these organisms. The influence of oxygen concentration, pH value, and inhibitors on their physiology is also described. Nitrification plays a central role in the nitrogen cycle of terrestrial and aquatic ecosystems, converting the most reduced form of nitrogen, NH3, to the most oxidized form, NO3. Nitrifying bacteria occupy niches in many ecosystems and compete successfully with faster and more efficiently growing organisms for oxygen, ammonia, and carbon dioxide. Nitrifiers are capable of reversing the nitrification process, carrying out denitrification and producing nitrite, ammonia, nitrous and nitric oxides, and gaseous nitrogen. Ammonia oxidizers can metabolize urea and can assimilate carbon from methane while nitrite oxidizers can grow anaerobically in the presence of organic compounds and nitrate.

The role of ecological theory in microbial ecology

Microbial ecology is currently undergoing a revolution, with repercussions spreading throughout microbiology, ecology and ecosystem science. The rapid accumulation of molecular data is uncovering vast diversity, abundant uncultivated microbial groups and novel microbial functions. This accumulation of data requires the application of theory to provide organization, structure, mechanistic insight and, ultimately, predictive power that is of practical value, but the application of theory in microbial ecology is currently very limited. Here we argue that the full potential of the ongoing revolution will not be realized if research is not directed and driven by theory, and that the generality of established ecological theory must be tested using microbial systems.

Growth, activity and temperature responses of ammonia-oxidizing archaea and bacteria in soil microcosms

Ammonia oxidation, as the first step in the nitrification process, plays a central role in the global cycling of nitrogen. Although bacteria are traditionally considered to be responsible for ammonia oxidation, a role for archaea has been suggested by data from metagenomic studies and by the isolation of a marine, autotrophic, ammonia-oxidizing, non-thermophilic crenarchaeon. Evidence for ammonia oxidation by non-thermophilic crenarchaea in marine and terrestrial environments is largely based on abundance of bacterial and archaeal ammonia monooxygenase (amo) genes, rather than activity. In this study, we have determined the influence of temperature on the response of ammonia-oxidizing bacteria and archaea in nitrifying soil microcosms using two approaches, involving analysis of transcriptional activity of 16S rRNA genes and of a key functional gene, amoA, which encodes ammonia monooxygenase subunit A. There was little evidence of changes in relative abundance or transcriptional activity of ammonia-oxidizing bacteria during nitrification. In contrast, denaturing gradient gel electrophoresis analysis of crenarchaeal 16S rRNA and crenarchaeal amoA genes provided strong evidence of changes in community structure of active archaeal ammonia oxidizers. Community structure changes were similar during incubation at different temperatures and much of the activity was due to a group of non-thermophilic crenarchaea associated with subsurface and marine environments, rather than soil. The findings suggest a role for crenarchaea in soil nitrification and that further information is required on their biogeography.

Relative contributions of archaea and bacteria to aerobic ammonia oxidation in the environment.

Traditionally, organisms responsible for major biogeochemical cycling processes have been determined by physiological characterization of environmental isolates in laboratory culture. Molecular techniques have, however, confirmed the widespread occurrence of abundant bacterial and archaeal groups with no cultivated representative, making it difficult to determine their ecosystem function. Until recently, ammonia oxidation, the first step in the globally important process of nitrification, was thought to be performed almost exclusively by bacteria. Metagenome studies, followed by laboratory isolation, then demonstrated the potential for significant ammonia oxidation by mesophilic crenarchaea, whose ecosystem function was previously unknown. Re-assessment of the role of bacteria in ammonia oxidation is now required and this article reviews the current evidence for the relative importance of bacteria and archaea. Much of this evidence is based on metagenomic analysis and molecular techniques for estimation of gene and gene transcript abundance, changes in ammonia oxidizer community structure during active nitrification and phylogeny of natural communities. These studies have been complemented by physiological characterization of a laboratory isolate and by incorporation of labelled substrates. Data from these studies provide increasingly convincing evidence for the importance of archaeal ammonia oxidizers in the global nitrogen cycle. They also highlight the need to re-assess the importance of ammonia-oxidizing bacteria, the requirement and limitations of molecular techniques in linking specific microbial groups to ecosystem function and the limitations of reliance on laboratory cultures.

Plant host habitat and root exudates shape soil bacterial community structure

The rhizosphere is active and dynamic in which newly generated carbon, derived from root exudates, and ancient carbon, in soil organic matter (SOM), are available for microbial growth. Stable isotope probing (SIP) was used to determine bacterial communities assimilating each carbon source in the rhizosphere of four plant species. Wheat, maize, rape and barrel clover (Medicago truncatula) were grown separately in the same soil under 13CO2 (99% of atom 13C) and DNA extracted from rhizosphere soil was fractionated by isopycnic centrifugation. Bacteria-assimilating root exudates were characterized by denaturing gradient gel electrophoresis (DGGE) analysis of 13C-DNA and root DNA, whereas those assimilating SOM were identified from 12C-DNA. Plant species root exudates significantly shaped rhizosphere bacterial community structure. Bacteria related to Sphingobacteriales and Myxococcus assimilated root exudates in colonizing roots of all four plants, whwereas bacteria related to Sphingomonadales utilized both carbon sources, and were identified in light, heavy and root compartment DNA. Sphingomonadales were specific to monocotyledons, whereas bacteria related to Enterobacter and Rhizobiales colonized all compartments of all four plants, used both fresh and ancient carbon and were considered as generalists. There was also evidence for an indirect important impact of root exudates, through stimulation of SOM assimilation by a diverse bacterial community.

Archaeal and bacterial ammonia- oxidisers in soil: the quest for niche specialisation and differentiation

Autotrophic archaeal and bacterial ammonia-oxidisers (AOA and AOB) drive soil nitrification. Ammonia limitation, mixotrophy, and pH have been suggested as factors providing niche specialisation and differentiation between soil AOA and AOB. However, current data from genomes, cultures, field studies, and microcosms suggest that no single factor discriminates between AOA and AOB. In addition, there appears to be sufficient physiological diversity within each group for growth and activity in all soils investigated, with the exception of acidic soils (pH <5.5), which are dominated by AOA. Future investigation of niche specialisation in ammonia-oxidisers, and other microbial communities, requires characterisation of a wider range of environmentally representative cultures, emphasis on experimental studies rather than surveys, and greater consideration of small-scale soil heterogeneity.

The ecological coherence of high bacterial taxonomic ranks

The species is a fundamental unit of biological organization, but its relevance for Bacteria and Archaea is still hotly debated. Even more controversial is whether the deeper branches of the ribosomal RNA-derived phylogenetic tree, such as the phyla, have ecological importance. Here, we discuss the ecological coherence of high bacterial taxa in the light of genome analyses and present examples of niche differentiation between deeply diverging groups in terrestrial and aquatic systems. The ecological relevance of high bacterial taxa has implications for bacterial taxonomy, evolution and ecology.

Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms

The first step of nitrification, oxidation of ammonia to nitrite, is performed by both ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soil, but their relative contributions to ammonia oxidation and existence in distinct ecological niches remain to be determined. To determine whether available ammonia concentration has a differential effect on AOA and AOB growth, soil microcosms were incubated for 28 days with ammonium at three concentrations: native (control), intermediate (20 μg NH4+-N per gram of soil) and high (200 μg NH4+-N per gram of soil). Quantitative PCR demonstrated growth of AOA at all concentrations, whereas AOB growth was prominent only at the highest concentration. Similarly, denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in AOA communities at all ammonium concentrations, whereas AOB communities changed significantly only at the highest ammonium concentration. These results provide evidence that ammonia concentration contributes to the definition of distinct ecological niches of AOA and AOB in soil.