Ammonia-oxidizing microorganisms in acidic forest soil and CRISPR-CAS of soil metagenomes

Abstract

Soil acidification is one of the major global issues and affects nitrification adversely due to the limited availability of substrate to ammonia oxidizers and the potential toxicity of dissolved acids. We deciphered the dominant and active ammonia-oxidizing archaea (AOA) and bacteria (AOB) in acidic soils with pH 3.66-4.58 from three natural forests and two re-vegetated forests in Nanling National Nature Reserve of China. Quantitative analysis of the subunit of ammonia monooxygenase (amoA) gene and transcripts revealed AOA was both numerically and functionally dominant over AOB. Three AOA lineages, namely Nitrososphaera, Nitrososphaera sister group and Nitrosotalea were detected in both DNA and RNA, but the relative abundances of them inferred by DNA and RNA differed, implying AOA genera do not contribute to nitrification proportionately. Nitrosotalea was the most abundant but was the second functionally important after Nitrososphaera sister group. Organic matter and exchangeable Al3+ profoundly affect AOA abundance and microbial community structure. Additionally, the effect of forest restoration on ammonia oxidizers in acidic soil was also investigated. AOA community was relatively stable during forest maturation but a more diverse AOB community with a dominance of Nitrosospira briensis and a decreased proportion of Nitrosomonas europaea/mobilis was detected in mature re-vegetated forests. Although soil acidification selects the less efficient ammonia oxidizers, the high abundance and the presence of functionally active AOA genera may help to sustain nitrification in the nitrogen cycling. To unveil the interaction between mobile genetic elements and the hosts in response to soil warming, we first built Hidden Markov Models (HMMs) specific to Cas1, the most conserved and universal CAS in CRISPR-CAS systems, for targeted assembly using Xander. Eight Cas1 HMMs were built and optimized to cover all 17 subtypes of CRISPR-CAS systems with minimum overlap. Cas1 was then assembled from 48 soil metagenomes in Oklahoma, US with 8-year warming treatment. The major soil taxa positively but temporarily responded only to the strongest heating stimuli when soil temperature increased by 40C. After strongest heating treatment, Cas1 prevalence in the major taxa was increased even in the ones with decreased taxa abundances. The enrichment of Cas1 in the corresponding taxa was continuously observed in the next three years. The elevated soil temperature itself has a slow impact on the microbial communities while the increasing preference of CRISPR-CAS system may imply the arising pressure from mobile genetic attacks. We further expand Cas1 detection from temperate environment to more extensive ecosystems including Alaska tundra, Michigan biofuel cropland, U.S. Midwest prairie, Amazon forest and pasture. More abundant Cas1 was found in less diverse soil microbiomes. Type I Cas1 was the most widespread but unevenly distributed in major soil taxa. Cas1 prevalence within the same host varied in different environments. The quantified direct repeats and spacers reveal that Alaska had more redundant CRISPR loci and Midwest grassland rhizospheres may have a higher activity of maintaining spacers. This research provides an overview of CRISPR-CAS in diverse environments and ecosystems.