Abstract
Anaerobic ammonium-oxidizing (anammox) bacteria are a group of strictly anaerobic chemolithoautotrophic microorganisms. They are capable of oxidizing ammonium to nitrogen gas using nitrite as a terminal electron acceptor, thereby facilitating the release of fixed nitrogen into the atmosphere. The anammox process is thought to exert a profound impact on the global nitrogen cycle and has been harnessed as an environment-friendly method for nitrogen removal from wastewater. In this study, we present the first closed genome sequence of an anammox bacterium, Kuenenia stuttgartiensis MBR1. It was obtained through Single-Molecule Real-Time (SMRT) sequencing of an enrichment culture constituting a mixture of at least two highly similar Kuenenia strains. The genome of the novel MBR1 strain is different from the previously reported Kuenenia KUST reference genome as it contains numerous structural variations and unique genomic regions. We find new proteins, such as a type 3b (sulf)hydrogenase and an additional copy of the hydrazine synthase gene cluster. Moreover, multiple copies of ammonium transporters and proteins regulating nitrogen uptake were identified, suggesting functional differences in metabolism. This assembly, including the genome-wide methylation profile, provides a new foundation for comparative and functional studies aiming to elucidate the biochemical and metabolic processes of these organisms.
Highlights
Anaerobic ammonium-oxidizing bacteria are a group of slow growing, strictly anaerobic chemolithoautotrophic microorganisms affiliated with the order Brocadiales of the phylum Planctomycetes[1,2]
We present the closed genome sequence and the genome-wide methylation profile of Kuenenia stuttgartiensis MBR1, a novel Kuenenia strain growing as suspended planktonic cells in a membrane bioreactor
DNA was extracted from a highly enriched (~95% of total bacterial biomass28) planktonic culture of Kuenenia stuttgartiensis sustained in an anoxic membrane bioreactor and sequenced using the Pacific Biosciences (PacBio) RSII single-molecule real-time (SMRT) sequencing platform
Summary
Inherent to the SMRT sequencing technology, unprocessed reads manifested a relatively low accuracy due to the presence of randomly distributed sequencing errors[26] These errors were corrected using the hierarchical genome-assembly process (HGAP) pipeline[29], yielding 108,054 highly accurate consensus sequences that ranged from 500 bp to over 27 Kb in length with a median length of 2,558 bp (Supplementary Table S1). This revealed deeply covered, fully assembled regions interrupted by numerous unresolved regions, depicted in the graph as bubbles These assembly bubbles indicated the presence of structural variations and consistently showed very distinctive coverage patterns, containing both high and low coverage paths. This observation suggested the presence of at least two highly similar strains at different levels of abundance. Sequencing technology Number of scaffolds Bases in scaffolds Mean sequencing depth GC-content
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