Comparative genomics and informational content analysis uncovered internal regions of the core genes rpoD, pepN and gltX for an MLSA with genome-level resolving power within the genus Pseudomonas

[Display omitted] •Current gold standard in bacterial taxonomy is phylogenomics.•Fast, cheap, and taxonomically competent options, like MLSA, are recommendable.•We did an unbiased search of core gene regions with highest taxonomic value for Pseudomonas.•Internal regions of rpoD, pepN, and gltX are the most informative for the Pseudomonas genus.•The rpoD-pepN-gltX MLSA is a robust tool with phylogenetic power comparable to ANI. In the field of prokaryotic taxonomy, there has been a recent transition towards phylogenomics as the gold standard approach. However, genome-based phylogenetics is still restrictive for its cost when managing large amounts of isolates. Fast, cheap, and taxonomically competent alternatives, like multilocus sequence analysis (MLSA) are thus recommendable. Nevertheless, the criteria for selecting the conserved genes for MLSA have not been explicit for different bacterial taxa, including the broadly diverse Pseudomonas genus. Here, we have carried out an unbiased and rational workflow to select internal sequence regions of Pseudomonas core genes (CG) for a MLSA with the best phylogenetic power, and with a resolution comparable to the genome-based ANI approach. A computational workflow was established to inspect 126 complete genomes of representatives from over 60 Pseudomonas species and subspecies, in order to identify the most informative CG internal regions and determine which combinations in sets of three partial CG sequences have comparable phylogenetic resolution to that of the current ANI standard. We found that the rpoD346-1196-pepN1711-2571-gltX86-909 concatenated sequences were the best performing in terms of phylogenetic robustness and resulted highly sensitive and specific when contrasted with ANI. The rpoD-pepN-gltX MLSA was validated in silico and in vitro. Altogether, the results presented here supports the proposal of the rpoD-pepN-gltX MLSA as a fast, affordable, and robust phylogenetic tool for members of the Pseudomonas genus.