Background: Characterisation of microbial communities increasingly involves use of high throughput sequencing methods (e.g. MiSeq Illumina) that amplify relatively short sequences of 16S rRNA or functional genes, the latter including ammonia monooxygenase subunit A (amoA), a key functional gene for ammonia oxidising bacteria (AOB) and archaea (AOA). The availability of these techniques, in combination with developments in phylogenetic methodology, provides the potential for better analysis of microbial niche specialisation. This study aimed to develop an approach for sequencing of bacterial and archaeal amoA genes amplified from soil using bioinformatics pipelines developed for general analysis of functional genes and employed sequence data to reassess phylogeny and niche specialisation in terrestrial bacterial ammonia oxidisers. Results: amoA richness and community composition differed with bioinformatics approaches used but analysis of MiSeq sequences was reliable for both archaeal and bacterial amoA genes and was used for subsequent assessment of potential niche specialisation of soil bacteria ammonia oxidisers. Prior to ecological analysis, phylogenetic analysis of Nitrosospira, which dominates soil AOB, was revisited using a phylogenetic analysis of 16S rRNA and amoA genes in available AOB genomes. This analysis supported congruence between phylogenies of the two genes and increased previous phylogenetic resolution, providing support for additional gene clusters of potential ecological significance. Analysis of environmental sequences using these new sequencing, bioinformatics and phylogenetic approaches demonstrated, for the first time, similar niche specialisation in AOB to that in AOA, indicating pH as a key ecological factor controlling the composition of soil ammonia oxidiser communities. Conclusions: This study presents the first bioinformatics pipeline for optimal analysis of Illumina MiSeq sequencing of a functional gene and is adaptable to any amplicon size (even genes larger than 500 bp). The pipeline was used to provide an up-to-date phylogenetic analysis of terrestrial betaproteobacterial amoA genes and to demonstrate the importance of soil pH for their niche specialisation and is broadly applicable to other ecosystems and diverse microbiomes.
Investigation of niche specialization in microbial communities is important in assessing consequences of environmental change for ecosystem processes. Ammonia oxidizing bacteria (AOB) and archaea (AOA) present a convenient model for studying niche specialization. They coexist in most soils and effects of soil characteristics on their relative abundances have been studied extensively. This study integrated published information on the influence of temperature and pH on AOB and AOA into several hypotheses, generating predictions that were tested in soil microcosms. The influence of perturbations in temperature was determined in pH 4.5, 6 and 7.5 soils and perturbations in pH were investigated at 15 C, 25 C and 35 C. AO activities were determined by analysing changes in amoA gene and transcript abundances, stable isotope probing and nitrate production. Experimental data supported major predictions of the effects of temperature and pH, but with several significant discrepancies, some of which may have resulted from experimental limitations. The study also provided evidence for unpredicted activity of AOB in pH 4.5 soil. Other discrepancies highlighted important deficiencies in current knowledge, particularly lack of consideration of niche overlap and the need to consider combinations of factors when assessing the influence of environmental change on microbial communities and their activities.
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