Reliable Identification of Environmental Pseudomonas Isolates Using the rpoD Gene.

Léa Girard,Cédric Lood,Vera Van Noort,Hassan Rokni-Zadeh,Rob Lavigne,René De Mot

doi:10.3390/microorganisms8081166

Léa Girard, Cédric Lood + Show 4 more

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https://doi.org/10.3390/microorganisms8081166

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The taxonomic affiliation of Pseudomonas isolates is currently assessed by using the 16S rRNA gene, MultiLocus Sequence Analysis (MLSA), or whole genome sequencing. Therefore, microbiologists are facing an arduous choice, either using the universal marker, knowing that these affiliations could be inaccurate, or engaging in more laborious and costly approaches. The rpoD gene, like the 16S rRNA gene, is included in most MLSA procedures and has already been suggested for the rapid identification of certain groups of Pseudomonas. However, a comprehensive overview of the rpoD-based phylogenetic relationships within the Pseudomonas genus is lacking. In this study, we present the rpoD-based phylogeny of 217 type strains of Pseudomonas and defined a cutoff value of 98% nucleotide identity to differentiate strains at the species level. To validate this approach, we sequenced the rpoD of 145 environmental isolates and complemented this analysis with whole genome sequencing. The rpoD sequence allowed us to accurately assign Pseudomonas isolates to 20 known species and represents an excellent first diagnostic tool to identify new Pseudomonas species. Finally, rpoD amplicon sequencing appears as a reliable and low-cost alternative, particularly in the case of large environmental studies with hundreds or thousands of isolates.

Highlights

Pseudomonas species are ubiquitous bacteria present in terrestrial, aquatic, and marine environments [1,2]
The P. aeruginosa lineage is divided in eight phylogenetic groups (P. oryzihabitans, P. stutzeri, P. oleovorans, P. aeruginosa, P. resinovorans, P. linyingensis, P. anguilliseptica, and P. straminea)
The P. fluorescens lineage is divided in five phylogenetic groups (P. putida, P. asplenii, P. lutea, P. syringae, and P. fluorescens), and overall these groups are supported by relatively high bootstrap values (Figures 1 and 2)

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Introduction

Pseudomonas species are ubiquitous bacteria present in terrestrial, aquatic, and marine environments [1,2]. The Pseudomonas phylogeny based on the 16S rRNA gene and the concatenation of 4, 100, and 120 genes was compared to whole genome analyses [12] As a result, it seems that the MLSA based on four housekeeping genes has the best price–performance ratio, the use of only two housekeeping genes (rpoB and rpoD) can be found in the literature [19,20]. It seems that the MLSA based on four housekeeping genes has the best price–performance ratio, the use of only two housekeeping genes (rpoB and rpoD) can be found in the literature [19,20] These methodologies are time-consuming, demanding, and/or expensive, in the case of large environmental studies with hundreds or thousands of isolates. The use of the 16S rRNA gene to identify Pseudomonas isolates remains relevant despite being prone to misidentifications, as observed frequently in public databases [21,22]

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