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.
The paradigm that soil microbial communities, being very diverse, have high functional redundancy levels, so that erosion of microbial diversity is less important for ecosystem functioning than erosion of plant or animal diversity, is often taken for granted. However, this has only been demonstrated for decomposition/ respiration functions, performed by a large proportion of the total microbial community, but not for specialized microbial groups. Here, we determined the impact of a decrease in soil microbial diversity on soil ecosystem processes using a removal approach, in which less abundant species were removed preferentially. This was achieved by inoculation of sterile soil microcosms with serial dilutions of a suspension obtained from the same non-sterile soil and subsequent incubation, to enable recovery of community size. The sensitivity to diversity erosion was evaluated for three microbial functional groups with known contrasting taxonomic diversities (ammonia oxidizers < denitrifiers < heterotrophs). Diversity erosion within each functional group was characterized using molecular fingerprinting techniques: ribosomal intergenic spacer analysis (RISA) for the eubacterial community, denaturing gradient gel electrophoresis (DGGE) analysis of nirK genes for denitrifiers, and DGGE analysis of 16S rRNA genes for betaproteobacterial ammonia oxidizers. In addition, we simulated the impact of the removal approach by dilution on the number of soil bacterial species remaining in the inoculum using values of abundance distribution of bacterial species reported in the literature. The reduction of the diversity of the functional groups observed from genetic fingerprints did not impair the associated functioning of these groups, i.e. carbon mineralization, denitrification and nitrification. This was remarkable, because the amplitude of diversity erosion generated by the dilution approach was huge (level of bacterial species loss was estimated to be around 99.99% for the highest dilution). Our results demonstrate that the vast diversity of the soil microbiota makes soil ecosystem functioning largely insensitive to biodiversity erosion even for functions performed by specialized groups.
SummaryIt is well established that resource quantity and elemental stoichiometry play major roles in shaping below and aboveground plant biodiversity, but their importance for shaping microbial diversity in soil remains unclear. Here, we used statistical modeling on a regional database covering 179 locations and six ecosystem types across Scotland to evaluate the roles of total carbon (C), nitrogen (N) and phosphorus (P) availabilities and ratios, together with land use, climate and biotic and abiotic factors, in determining regional scale patterns of soil bacterial diversity. We found that bacterial diversity and composition were primarily driven by variation in soil resource stoichiometry (total C:N:P ratios), itself linked to different land uses, and secondarily driven by other important biodiversity drivers such as climate, soil spatial heterogeneity, soil pH, root influence (plant-soil microbe interactions) and microbial biomass (soil microbe-microbe interactions). In aggregate, these findings provide evidence that nutrient stoichiometry is a strong predictor of bacterial diversity and composition at a regional scale.
To determine whether the distribution of estuarine ammonia-oxidizing bacteria (AOB) was influenced by salinity, the community structure of betaproteobacterial ammonia oxidizers (AOB) was characterized along a salinity gradient in sediments of the Ythan estuary, on the east coast of Scotland, UK, by denaturant gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rRNA gene fragments. Ammonia-oxidizing bacteria communities at sampling sites with strongest marine influence were dominated by Nitrosospira cluster 1-like sequences and those with strongest freshwater influence were dominated by Nitrosomonas oligotropha-like sequences. Nitrosomonas sp. Nm143 was the prevailing sequence type in communities at intermediate brackish sites. Diversity indices of AOB communities were similar at marine- and freshwater-influenced sites and did not indicate lower species diversity at intermediate brackish sites. The presence of sequences highly similar to the halophilic Nitrosomonas marina and the freshwater strain Nitrosomonas oligotropha at identical sampling sites indicates that AOB communities in the estuary are adapted to a range of salinities, while individual strains may be active at different salinities. Ammonia-oxidizing bacteria communities that were dominated by Nitrosospira cluster 1 sequence types, for which no cultured representative exists, were subjected to stable isotope probing (SIP) with 13C-HCO3-, to label the nucleic acids of active autotrophic nitrifiers. Analysis of 13C-associated 16S rRNA gene fragments, following CsCl density centrifugation, by cloning and DGGE indicated sequences highly similar to the AOB Nitrosomonas sp. Nm143 and Nitrosomonas cryotolerans and to the nitrite oxidizer Nitrospira marina. No sequence with similarity to the Nitrosospira cluster 1 clade was recovered during SIP analysis. The potential role of Nitrosospira cluster 1 in autotrophic ammonia oxidation therefore remains uncertain.
The potential for oxidation of ammonia in anoxic marine sediments exists through anaerobic oxidation by Nitrosomonas-like organisms, utilizing nitrogen dioxide, coupling of nitrification, manganese reduction, and anaerobic oxidation of ammonium by planctomycetes (the Anammox process). Here we describe the presence of microbial communities with the potential to carry out these processes in a natural marine sediment system (Loch Duich, Scotland). Natural microbial communities of Planctomycetales-Verrucomicrobia and -and ␥-proteobacterial ammonia-oxidizing bacteria were characterized by analysis of 16S rRNA genes amplified using group-specific primers by PCR-and reverse transcription-PCR amplification of 16S rDNA and RNA, respectively. Amplification products were analyzed by sequencing of clones and by denaturant gradient gel electrophoresis (DGGE). Amplification of primers specific for Planctomycetales-Verrucomicrobia and -proteobacterial ammonia-oxidizing bacteria generated products at all sampling sites and depths, but no product was generated using primers specific for ␥-proteobacterial ammonia-oxidizing bacteria. 16S rDNA DGGE banding patterns indicated complex communities of -proteobacterial ammonia-oxidizing bacteria in anoxic marine sediments. Phylogenetic analysis of sequences from clones and those excised from DGGE gels suggests dominance of Nitrosospira cluster 1-like organisms and of strains belonging to a novel cluster represented in dominant bands in 16S rRNA DGGE banding patterns. Their presence indicates a group of organisms closely related to recognized -proteobacterial ammonia-oxidizing bacteria that may be selected in anoxic environments and may be capable of anoxic ammonia oxidation. Sequence analysis of planctomycete clone libraries and sequences excised from DGGE gels also demonstrated a diverse microbial community and suggested the presence of new subdivisions, but no sequence related to recognized Anammox organisms was detected.
The transcription dynamics of subunit A of the key gene in methanogenesis (methyl coenzyme M reductase; mcrA) was studied to evaluate the relationship between process rate (methanogenesis) and gene transcription dynamics in a peat soil ecosystem. Soil methanogen process rates were determined during incubation of peat slurries at temperatures from 4 to 37°C, and real-time quantitative PCR was applied to quantify the abundances of mcrA genes and transcripts; corresponding transcriptional dynamics were calculated from mcrA transcript/gene ratios. Internal standards suggested unbiased recovery of mRNA abundances in comparison to DNA levels. In comparison to those in pure-culture studies, mcrA transcript/gene ratios indicated underestimation by 1 order of magnitude, possibly due to high proportions of inactive or dead methanogens. Methane production rates were temperature dependent, with maxima at 25°C, but changes in abundance and transcription of the mcrA gene showed no correlation with temperature. However, mcrA transcript/gene ratios correlated weakly (regression coefficient ؍ 0.76) with rates of methanogenesis. Methanogen process rates increased over 3 orders of magnitude, while the corresponding maximum transcript/gene ratio increase was only 18-fold. mcrA transcript dynamics suggested steady-state expression in peat soil after incubation for 24 and 48 h, similar to that in stationary-phase cultures. mcrA transcript/gene ratios are therefore potential in situ indicators of methanogen process rate changes in complex soil systems.
Molecular approaches have revealed considerable diversity and uncultured novelty in natural prokaryotic populations, but not direct links between the new genotypes detected and ecosystem processes. Here we describe the influence of the structure of communities of ammonia-oxidizing bacteria on nitrogen cycling in microcosms containing natural and managed grasslands and amended with artificial sheep urine, a major factor determining local ammonia concentrations in these environments. Nitrification kinetics were assessed by analysis of changes in urea, ammonia, nitrite and nitrate concentrations and ammonia oxidizer communities were characterized by analysis of 16S rRNA genes amplified from extracted DNA using ammonia oxidizer-specific primers. In natural soils, ammonia oxidizer community structure determined the delay preceding nitrification, which depended on the relative abundance of two Nitrosospira clusters, termed 3a and 3b. In batch cultures, pure culture and enrichment culture representatives of Nitrosospira 3a were sensitive to high ammonia concentration, while Nitrosospira cluster 3b representatives and Nitrosomonas europaea were tolerant. Delays in nitrification occurred in natural soils dominated by Nitrosospira cluster 3a and resulted from the time required for growth of low concentrations of Nitrosospira cluster 3b. In microcosms dominated by Nitrosospira cluster 3b and Nitrosomonas, no substantial delays were observed. In managed soils, no delays in nitrification were detected, regardless of initial ammonia oxidizer community structure, most probably resulting from higher ammonia oxidizer cell concentrations. The data therefore demonstrate a direct link between bacterial community structure, physiological diversity and ecosystem function.
The factors driving the abundance and community composition of soil microbial communities provide fundamental knowledge on the maintenance of biodiversity and the ecosystem services they underpin. Several studies have suggested that microbial communities are spatially organized, including functional groups and much of the observed variation is explained by geographical location or soil pH. Soil ammonia-oxidizing archaea (AOA) and bacteria (AOB) are excellent models for such study due to their functional, agronomic and environmental importance and their relative ease of characterization. To identify the dominant drivers of different ammonia oxidizers, we used samples (n = 713) from the National Soil Inventory of Scotland (NSIS). Our results indicate that 40-45% of the variance in community compositions can be explained by 71 environmental variables. Soil pH and substrate, which have been regarded as the two main drivers, only explained 13-16% of the total variance. We provide strong evidence of multi-factorial drivers (land use, soil type, climate and N deposition) of ammonia-oxidizing communities, all of which play a significant role in the creation of specific niches that are occupied by unique phylotypes. For example, one AOA phylotype was strongly linked to woodland/semi-natural grassland, rainfall and N deposition. Some soil typologies, namely regosols, have a novel AOA community composition indicating typology as one of the factors which defines this ecological niche. AOA abundance was high and strongly linked the rate of potential nitrification in the highly acidic soils supporting the argument that AOA are main ammonia oxidizers in acidic soils. However, for AOB, soil pH and substrate (ammonia) were the main drivers for abundance and community composition. These results highlight the importance of multiple drivers of microbial niche formation and their impact on microbial biogeography that have significant consequences for ecosystem functioning.
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