Metagenome Analysis of a Complex Community Reveals the Metabolic Blueprint of Anammox Bacterium “Candidatus Jettenia asiatica”

Ziye Hu,Zhe-Xue Quan,Kees-Jan Francoijs,M S M Jetten,D R Speth

doi:10.3389/fmicb.2012.00366

Ziye Hu, Zhe-Xue Quan + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2012.00366

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Abstract

Anaerobic ammonium-oxidizing (anammox) bacteria are key players in the global nitrogen cycle and responsible for significant global nitrogen loss. Moreover, the anammox process is widely implemented for nitrogen removal from wastewaters as a cost-effective and environment-friendly alternative to conventional nitrification-denitrification systems. Currently, five genera of anammox bacteria have been identified, together forming a deep-branching order in the Planctomycetes-Verrucomicrobium-Chlamydiae superphylum. Members of all genera have been detected in wastewater treatment plants and have been enriched in lab-scale bioreactors, but genome information is not yet available for all genera. Here we report the metagenomic analysis of a granular sludge anammox reactor dominated (∼50%) by “Candidatus Jettenia asiatica.” The metagenome was sequenced using both Illumina and 454 pyrosequencing. After de novo assembly 37,432 contigs with an average length of 571 nt were obtained. The contigs were then analyzed by BLASTx searches against the protein sequences of “Candidatus Kuenenia stuttgartiensis” and a set of 25 genes essential in anammox metabolism were detected. Additionally all reads were mapped to the genome of an anammox strain KSU-1 and de novo assembly was performed again using the reads that could be mapped on KSU-1. Using this approach, a gene encoding copper-containing nitrite reductase NirK was identified in the genome, instead of cytochrome cd1-type nitrite reductase (NirS, present in “Ca. Kuenenia stuttgartiensis” and “Ca. Scalindua profunda”). Finally, the community composition was investigated through MetaCluster analysis, 16S rRNA gene analysis and read mapping, which showed the presence of other important community members such as aerobic ammonia-oxidizing bacteria, methanogens, and the denitrifying methanotroph “Ca. Methylomirabilis oxyfera”, indicating a possible active methane and nitrogen cycle in the bioreactor under the prevailing operational conditions.

Highlights

Two microbial processes are responsible for the release of fixed nitrogen: denitrification and anaerobic ammonium oxidation
Using environmental shotgun sequencing, which is used to retrieve microbial genomes or genomic fragments from complicated environmental samples, the first metagenome of an anammox bacterium was sequenced from an enrichment culture (∼75%) of “Candidatus Kuenenia stuttgartiensis”
Other hao-like proteins retrieved from Kuenenia genome could be partially mapped by assembled Jettenia contigs, some of them, such as kustc0458, were nearly fully mapped (Tables 1 and 2). These hao-like proteins might be involved in some other reactions in the anammox metabolism, such as electron transfer, detoxification of potentially hazardous nitrogen compounds, dissimilatory nitrate/nitrite reduction to ammonia (DNRA) or ammonia formation (Campbell et al, 2009; Kartal et al, 2011; Van de Vossenberg et al, 2012)

Summary

INTRODUCTION

Two microbial processes are responsible for the release of fixed nitrogen: denitrification and anaerobic ammonium oxidation (anammox). Using environmental shotgun sequencing, which is used to retrieve microbial genomes or genomic fragments from complicated environmental samples, the first metagenome of an anammox bacterium was sequenced from an enrichment culture (∼75%) of “Candidatus Kuenenia stuttgartiensis” (hereafter: Kuenenia; Strous et al, 2006) Based on this metagenome, the central metabolic pathways of anammox bacteria were predicted and resolved (Strous et al, 2006; de Almeida et al, 2011; Kartal et al., 2011). The genome sequences of three more anammox species were reported; a marine species “Candidatus Scalindua profunda” (hereafter: Scalindua), a waste water species KSU-1, and a freshwater species “Candidatus Brocadia fulgida” (Hereafter: Brocadia) and all of them showed differences to Kuenenia (Gori et al, 2011; Hira et al, 2012; Van de Vossenberg et al, 2012) This indicated that genomic analyses of other anammox bacteria could contribute to a more comprehensive understanding of the metabolic features of these extraordinary microorganisms. Important anammox genes in the dataset were identified and compared to those in the genomes of Kuenenia and the anammox strain KSU-1 (Hira et al, 2012)

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUDING REMARKS