The application of high-throughput sequencing technology to analysis of amoA phylogeny and environmental niche specialisation of terrestrial bacterial ammonia-oxidisers

Abstract

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.

The pH-based ecological coherence of active canonical methanotrophs in paddy soils

“…3) when compared to the other two high-pH soils (dominated by OTU_17). Our study therefore implied that the contradicting effects of N fertilization on methane oxidation (inhibition or stimulation) frequently reported in different soils (Alam and Jia, 2012;Bodelier et al, 2000;Cai and Yan, 1999;Zheng et al, 2014) might be determined by the dominant methanotrophic phylotypes, but a larger-scale sampling with activity-based molecular analysis (e.g., RNAor SIP-based tools) is required to test this hypothesis in the future.…”

“…Recent studies have provided compelling evidence for niche specialization of biogeochemically important guilds associated with pH variation and consequent distinct patterns of soil resource utilization. For example, the biogeographical distribution of ammonia oxidizers is more strongly associated with soil pH than other parameters tested in soils (Aigle et al, 2019;Gubry-Rangin et al, 2011), as is that of denitrifiers (Liu et al, 2010). It is implied that type I and II methanotrophs might also be selectively favored under different pH conditions in natural wetland system, despite no systematic comparison has yet been made.…”

Cold Adapted Nitrosospira sp.: A Potential Crucial Contributor of Ammonia Oxidation in Cryosols of Permafrost-Affected Landscapes in Northeast Siberia

“…The pH could be the crucial parameter for the differentiation. Aigle et al [48] addressed the question if the pH was a key factor of ecological distribution. Some AOA are adapted to lower pH than AOB [34,82].…”

pH-based ecological coherence of active canonical methanotrophs in paddy soils

“…For example, the biogeographical distribution of ammonia-oxidizing oxidizers is more strongly associated with soil pH than other parameters tested in soils (Gubry-Rangin et al, 2011;Aigle et al, 2019), as is that of denitrifiers (Liu et al, 2010). It is implied that type I and II methanotrophs might also be selectively favoured under different pH conditions in natural wetland system, despite no systematic comparison has yet been made.…”