In this study, comparative computational methods were applied to determine the maturation pathway regulating the assembly of functional c-type cytochrome holoforms in four genera of anammox bacteria, using key protein constituents of maturation Systems I-III as biomarkers. Our analysis showed that all anammox genome assemblies contain at least one full set of System II (Ccs) genes. Methods All anammox bacteria belong to the order Brocadiales that branches deeply into the phylum Planctomycetes
and includes five genera (Kuenenia, Scalindua, Brocadia, Jettenia, and Anammoxoglobus). In this study draft genomes representative of four anammox genera were LXH254 cost analyzed. Kuenenia stuttgartiensis [NCBI bioproject: PRJNA16685 ], Scalindua profunda [JGI: 2017108002 and 2022004002 ], and strain KSU-1 (representing Jettenia genus) [NCBI bioprojects: PRJDA163683 and PRJDB68 ] obtained as described elsewhere. Genomic data for Brocadia fulgida were obtained as described here below. Brocadia fulgida genomic data Library preparation and sequencing All kits used in this section were obtained from Life technologies (Life technologies,
Carlsbad, CA, USA). Genomic DNA, isolated using a CTAB phenol/chloroform based method, was sheared for 5 minutes using Lonafarnib the Ion Xpress™ Plus Fragment Quisinostat in vivo Library Kit following the manufacturer’s instructions. Further library preparation was performed using the Ion Plus Fragment Library Kit following manufacturer’s
instructions. Size selection of the library was performed using an E-gel 2% agarose gel. Emulsion PCR was performed using the Onetouch 200 bp kit and sequencing was performed on an IonTorrent PGM using the Ion PGM 200 bp sequencing kit and an Ion 318 chip, resulting in 5.25 million reads with an average length of 179 bp. Assembly and annotation The obtained 5.25 million reads were quality trimmed and all reads below 200 bp were discarded. The remaining 2,22 million reads were assembled using the CLC genomics workbench (v6.5.1, CLCbio, Aarhus, Denmark) with word size 35 and bubble size 5000. Brocadia fulgida accounted for 91% of the assembled reads. Contigs were assigned to Brocadia fulgida based on coverage (>30 fold). The obtained 411 contigs were annotated using Prokka 1.7.2 (Prokka: selleck compound Prokaryotic Genome Annotation System – http://vicbioinformatics.com/). After annotation, a round of manual curation was performed to correct detected frame shifts. Raw reads and assembled data are available under NCBI bioproject PRJEB4876.