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Pipeline publication

[…] teins across different bacterial phyla, proteomes (.faa files) of representative genomes were downloaded from refseq NCBI: https://www.ncbi.nlm.nih.gov/refseq/about/prokaryotes/., A total of 4,771 genomes (date: 01.05.2017) were retrieved, and the proteomes were queried based on regular NCBI annotations for the presence of FliO, FliP, FliQ, FliR, and flagellin proteins. For the de novo prediction of homologs of FliO, FliP, FliQ, FliR, and flagellin proteins, HMMs were generated using the curated Pfam database models for FliO (PF04347), FliP (PF00813), FliQ (PF01313), FliR (PF01311), and flagellin (PF00669+PF00700), and all representative genomes were screened using the hmmsearch function in HMMER3 [] with the default Pfam database gathering cutoff (GA) per domain. This HMM approach identified a large number of FliO proteins for genomes in which FliP and the other flagella components have been predicted. The majority of novel FliO proteins corresponded to hypothetical genes. Results from the annotated FliO, FliP, FliQ, FliR, and flagellin proteins and the de novo prediction were integrated using custom Python scripts. For the phylogenetic representation, the NCBI taxonomy IDs were extracted using ETEtools [], and the tree topology was obtained for a given list of TaxIDs. For tree visualization and rendering, iTOL [] was used, and set comparison was visualized using the UpSet tool []., Equal amounts of an overnight culture were inoculated in 0.3% swim agar plates supplemented with appropriate additives. Plates were incubated at 37°C and scanned after 4 h to 7 h. The sizes of the motility halos were measured using ImageJ., Cells were grown in LB at 37°C until OD600 reached 0.8−0.9, and 1.9 ml of culture was harvested and cooled down on ice. Cells and supernatant were separated by 3 × 3 min centrifugation at 18,000 × g at 4°C. The pellet and 1.5-ml supernatant of each strain were precipitated with 10% TCA. After normalization to the OD600, samples were loaded on a 15% SDS-PAGE and analyzed by Western blot., Overnight cultures of chromosomally encoded FliPQ223 […]

Pipeline specifications

Software tools HMMER, iTOL, UpSet