Sara Hallin

Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE

We re-evaluated PCR primers targeting nirS, nirK and nosZ genes for denaturing gradient gel electrophoresis as a tool to survey denitrifying community composition in environmental samples. New primers for both nirS and nosZ were combined with existing primers, while for nirK the previously published F1aCu:R3Cu set was chosen for denaturing electrophoresis. All three sets yielded amplicons smaller than 500 bp and amplified the correct fragment in all environmental samples. The denaturing gradient gel electrophoresis worked satisfactorily for nirK and nosZ, but not for nirS. This was probably due to the multiple melting domains in this particular nirS fragment. From the excised and sequenced bands, only sequences related to the target genes were detected and tree analysis showed that the selected primers acted as broad range primers for each of the three genes. By use of the new nirS primers it was demonstrated that agricultural soil harbours a substantial diversity of nirS denitrifiers.

Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment

The relative importance of size and composition of microbial communities in ecosystem functioning is poorly understood. Here, we investigated how community composition and size of selected functional guilds in the nitrogen cycle correlated with agroecosystem functioning, which was defined as microbial process rates, total crop yield and nitrogen content in the crop. Soil was sampled from a 50-year fertilizer trial and the treatments comprised unfertilized bare fallow, unfertilized with crop, and plots with crop fertilized with calcium nitrate, ammonium sulfate, solid cattle manure or sewage sludge. The size of the functional guilds and the total bacterial community were greatly affected by the fertilization regimes, especially by the sewage sludge and ammonium sulfate treatments. The community size results were combined with previously published data on the composition of the corresponding communities, potential ammonia oxidation, denitrification, basal and substrate-induced respiration rates, in addition to crop yield for an integrated analysis. It was found that differences in size, rather than composition, correlated with differences in process rates for the denitrifier and ammonia-oxidizing archaeal and total bacterial communities, whereas neither differences in size nor composition was correlated with differences in process rates for the ammonia-oxidizing bacterial community. In contrast, the composition of nitrate-reducing, denitrifying and total bacterial communities co-varied with primary production and both were strongly linked to soil properties.

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.

Activity and Composition of the Denitrifying Bacterial Community Respond Differently to Long-Term Fertilization

ABSTRACT The objective of this study was to explore the long-term effects of different organic and inorganic fertilizers on activity and composition of the denitrifying and total bacterial communities in arable soil. Soil from the following six treatments was analyzed in an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO3)2, (NH4)2SO4, and unfertilized and unfertilized bare fallow. All plots but the fallow were planted with corn. The activity was measured in terms of potential denitrification rate and basal soil respiration. The nosZ and narG genes were used as functional markers of the denitrifying community, and the composition was analyzed using denaturing gradient gel electrophoresis of nosZ and restriction fragment length polymorphism of narG, together with cloning and sequencing. A fingerprint of the total bacterial community was assessed by ribosomal intergenic spacer region analysis (RISA). The potential denitrification rates were higher in plots treated with organic fertilizer than in those with only mineral fertilizer. The basal soil respiration rates were positively correlated to soil carbon content, and the highest rates were found in the plots with the addition of sewage sludge. Fingerprints of the nosZ and narG genes, as well as the RISA, showed significant differences in the corresponding communities in the plots treated with (NH4)2SO4 and sewage sludge, which exhibited the lowest pH. In contrast, similar patterns were observed among the other four treatments, unfertilized plots with and without crops and the plots treated with Ca(NO3)2 or with manure. This study shows that the addition of different fertilizers affects both the activity and the composition of the denitrifying communities in arable soil on a long-term basis. However, the treatments in which the denitrifying and bacterial community composition differed the most did not correspond to treatments with the most different activities, showing that potential activity was uncoupled to community composition.

Phylogenetic Analysis of Nitrite, Nitric Oxide, and Nitrous Oxide Respiratory Enzymes Reveal a Complex Evolutionary History for Denitrification

Denitrification is a facultative respiratory pathway in which nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) are successively reduced to nitrogen gas (N(2)), effectively closing the nitrogen cycle. The ability to denitrify is widely dispersed among prokaryotes, and this polyphyletic distribution has raised the possibility of horizontal gene transfer (HGT) having a substantial role in the evolution of denitrification. Comparisons of 16S rRNA and denitrification gene phylogenies in recent studies support this possibility; however, these results remain speculative as they are based on visual comparisons of phylogenies from partial sequences. We reanalyzed publicly available nirS, nirK, norB, and nosZ partial sequences using Bayesian and maximum likelihood phylogenetic inference. Concomitant analysis of denitrification genes with 16S rRNA sequences from the same organisms showed substantial differences between the trees, which were supported by examining the posterior probability of monophyletic constraints at different taxonomic levels. Although these differences suggest HGT of denitrification genes, the presence of structural variants for nirK, norB, and nosZ makes it difficult to determine HGT from other evolutionary events. Additional analysis using phylogenetic networks and likelihood ratio tests of phylogenies based on full-length sequences retrieved from genomes also revealed significant differences in tree topologies among denitrification and 16S rRNA gene phylogenies, with the exception of the nosZ gene phylogeny within the data set of the nirK-harboring genomes. However, inspection of codon usage and G + C content plots from complete genomes gave no evidence for recent HGT. Instead, the close proximity of denitrification gene copies in the genomes of several denitrifying bacteria suggests duplication. Although HGT cannot be ruled out as a factor in the evolution of denitrification genes, our analysis suggests that other phenomena, such gene duplication/divergence and lineage sorting, may have differently influenced the evolution of each denitrification gene.

Ecology of Denitrifying Prokaryotes in Agricultural Soil

Denitrification is a microbial respiratory process during which soluble nitrogen oxides are used as an alternative electron acceptor when oxygen is limiting. It results in considerable loss of nitrogen, which is the most limiting nutrient for crop production in agriculture. Denitrification is also of environmental concern, since it is the main biological process responsible for emissions of nitrous oxide, one of the six greenhouse gases considered by the Kyoto protocol. In addition to natural variations, agroecosystems are characterized by the use of numerous practices, such as fertilization and pesticide application, which can influence denitrification rates. This has been widely documented in the literature, illustrating the complexity of the underlying mechanisms regulating this process. In the last decade, however, application of molecular biology approaches has given the opportunity to look behind denitrification rates and to describe genes, transcripts, and enzymes responsible for the process. In order to reduce denitrification in arable soil, it is important to understand how different factors influence denitrification and how the denitrifier community structure is related to in situ activity. This chapter focuses on the impact of natural events as well as agricultural practices on denitrifying microorganisms.

The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink

Nitrous oxide (N2O) is a major radiative forcing and stratospheric ozone-depleting gas emitted from terrestrial and aquatic ecosystems. It can be transformed to nitrogen gas (N2) by bacteria and archaea harboring the N2O reductase (N2OR), which is the only known N2O sink in the biosphere. Despite its crucial role in mitigating N2O emissions, knowledge of the N2OR in the environment remains limited. Here, we report a comprehensive phylogenetic analysis of the nosZ gene coding the N2OR in genomes retrieved from public databases. The resulting phylogeny revealed two distinct clades of nosZ, with one unaccounted for in studies investigating N2O-reducing communities. Examination of N2OR structural elements not considered in the phylogeny revealed that the two clades differ in their signal peptides, indicating differences in the translocation pathway of the N2OR across the membrane. Sequencing of environmental clones of the previously undetected nosZ lineage in various environments showed that it is widespread and diverse. Using quantitative PCR, we demonstrate that this clade was most often at least as abundant as the other, thereby more than doubling the known extent of the overall N2O-reducing community in the environment. Furthermore, we observed that the relative abundance of nosZ from either clade varied among habitat types and environmental conditions. Our results indicate a physiological dichotomy in the diversity of N2O-reducing microorganisms, which might be of importance for understanding the relationship between the diversity of N2O-reducing microorganisms and N2O reduction in different ecosystems.

Soil Resources Influence Spatial Patterns of Denitrifying Communities at Scales Compatible with Land Management

ABSTRACT Knowing spatial patterns of functional microbial guilds can increase our understanding of the relationships between microbial community ecology and ecosystem functions. Using geostatistical modeling to map spatial patterns, we explored the distribution of the community structure, size, and activity of one functional group in N cycling, the denitrifiers, in relation to 23 soil parameters over a 44-ha farm divided into one organic and one integrated crop production system. The denitrifiers were targeted by the nirS and nirK genes that encode the two mutually exclusive types of nitrite reductases, the cd1 heme-type and copper reductases, respectively. The spatial pattern of the denitrification activity genes was reflected by the maps of the abundances of nir genes. For the community structure, only the maps of the nirS community were related to the activity. The activity was correlated with nitrate and dissolved organic nitrogen and carbon, whereas the gene pools for denitrification, in terms of size and composition, were influenced by the soil structure. For the nirS community, pH and soil nutrients were also important in shaping the community. The only unique parameter related to the nirK community was the soil Cu content. However, the spatial pattern of the nirK denitrifiers corresponded to the division of the farm into the two cropping systems. The different community patterns, together with the spatial distribution of the nirS/nirK abundance ratio, suggest habitat selection on the nirS- and nirK-type denitrifiers. Our findings constitute a first step in identifying niches for denitrifiers at scales relevant to land management.

Biochemical cycling in the rhizosphere having an impact on global change

Changes in chemical properties in soil around plant roots influence many microbial processes, including those having an impact on greenhouse gas emissions. To potentially mitigate these emissions according to the Kyoto protocol, knowledge about how and where these gases are produced and consumed in soils is required. In this review, we focus on the greenhouse gases nitrous oxide and methane, which are produced by nitrifying and denitrifying prokaryotes and methanogenic archaea, respectively. After describing the microbial processes involved in production and consumption of nitrous oxide and methane and how they can be affected in the rhizosphere, we give an overview of nitrous oxide and methane emissions from the rhizosphere and soils and sediments with plants. We also discuss strategies to mitigate emissions from the rhizosphere and consider possibilities for carbon sequestration.

Ecological and evolutionary factors underlying global and local assembly of denitrifier communities

The conversion of nitrite to nitric oxide in the denitrification pathway is catalyzed by at least two structurally dissimilar nitrite reductases, NirS and NirK. Although they are functionally equivalent, a genome with genes encoding both reductases has yet to be found. This exclusivity raises questions about the ecological equivalency of denitrifiers with either nirS or nirK, and how different ecological and evolutionary factors influence community assembly of nirS and nirK denitrifiers. Using phylogeny-based methods for analyzing community structure, we analyzed nirS and nirK data sets compiled from sequence repositories. Global patterns of phylogenetic community structure were determined using Unifrac, whereas community assembly processes were inferred using different community relatedness metrics. Similarities between globally distributed communities for both genes corresponded to similarities in habitat salinity. The majority of communities for both genes were phylogenetically clustered; however, nirK marine communities were more phylogenetically overdispersed than nirK soil communities or nirS communities. A more in-depth analysis was performed using three case studies in which a comparison of nirS and nirK community relatedness within the sites could be examined along environmental gradients. From these studies we observed that nirS communities respond differently to environmental gradients than nirK communities. Although it is difficult to attribute nonrandom patterns of phylogenetic diversity to specific niche-based or neutral assembly processes, our results indicate that coexisting nirS and nirK denitrifier communities are not under the same community assembly rules in different environments.