Taxonomic classification of a lipopeptide-producing Pseudomonas

The rpoD-based phylogeny is an efficient diagnostic tool for the reliable identification of environmental Pseudomonas isolates close to the species level (Girard, 2020, Girard, 2021, Lauritsen, 2021). It enables to quickly assign the taxonomic affiliation of an unidentified (potential) CLiP producer when nucleotide identity is ≧ 98%. Ideally, the complete rpoD gene sequence of a producer strain is extracted from its genome (identified by annotation or by Blast search with a known Pseudomonas rpoD) and compared with those of type strains of Pseudomonas species.

Phylogenetic analysis of rpoD

Alignment of rpoD sequences is performed with tools such as MUSCLE (Edgar, 2004). Alignments are trimmed to remove short unaligned ends, if any, to avoid phylogenetic noise (Adamek, 2019). Then, a phylogenetic tree can be inferred using IQ-TREE (Minh, 2020) or other equivalent software. An advantage of using IQ-TREE is the possibility of using ultrafast (UF) bootstraps (Hoang, 2018) which significantly improves the computation time. Online tools such as iTOL (Letunic, 2021) support the visualization or annotation of the phylogenetic trees.

We make a list of rpoD sequences (archive of .FASTA formatted files) available, from either known CLiP producers (including some type strains) [download ‘rpoD producers’] or type strains from (sub)groups with known or predicted CLiP producers [download ‘rpoD type strains non-producers’] (versions: October 2021).

Parameter	Value
Statistical method	Maximum Likelihood
Substitution model	General Time Reversible (GTR)
Distribution among sites	Gamma Distributed with Invariant Sites (G+I)
Ultrafast bootstrap replicates	1000

Useful links

IQ-TREE	http://www.iqtree.org	Minh, 2020
iTOL	https://itol.embl.de	Letunic, 2021
MUSCLE	https://drive5.com/muscle5/manual/	Edgar, 2004

References

Adamek, et al. “Applied evolution: phylogeny-based approaches in natural products research.” Natural Product Reports 36, 9 (2019): https://dx.doi.org/10.1039/C9NP00027E.

Edgar. “MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Res 32, 5 (2004): https://dx.doi.org/10.1093/nar/gkh340.

Girard, et al. “The ever-expanding Pseudomonas genus: Description of 43 new species and partition of the Pseudomonas putida group.” Microorganisms 9, 8 (2021): https://dx.doi.org/10.3390/microorganisms9081766.

Girard, et al. “Reliable Identification of Environmental Pseudomonas Isolates Using the rpoD Gene.” Microorganisms 8, 8 (2020): https://dx.doi.org/10.3390/microorganisms8081166.

Hoang, et al. “UFBoot2: Improving the Ultrafast Bootstrap Approximation.” Mol Biol Evol 35, 2 (2018): https://dx.doi.org/10.1093/molbev/msx281.

Lauritsen, et al. “Identification and Differentiation of Pseudomonas Species in Field Samples Using an rpoD Amplicon Sequencing Methodology.” mSystems 6, 4 (2021): https://dx.doi.org/10.1128/mSystems.00704-21.

Letunic, et al. “Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation.” Nucleic Acids Research 49, W1 (2021): https://dx.doi.org/10.1093/nar/gkab301.

Minh, et al. “IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era.” Mol Biol Evol 37, 5 (2020): https://dx.doi.org/10.1093/molbev/msaa015.