The genus Pseudomonas hosts an extensive genetic diversity and is one of the largest genera among Gram-negative bacteria. Type strains of Pseudomonas are well known to represent only a small fraction of this diversity and the number of available Pseudomonas genome sequences is increasing rapidly. Consequently, new Pseudomonas species are regularly reported and the number of species within the genus is constantly evolving. In this study, whole genome sequencing enabled us to define 43 new Pseudomonas species and provide an update of the Pseudomonas evolutionary and taxonomic relationships. Phylogenies based on the rpoD gene and whole genome sequences, including, respectively, 316 and 313 type strains of Pseudomonas, revealed sixteen groups of Pseudomonas and, together with the distribution of cyclic lipopeptide biosynthesis gene clusters, enabled the partitioning of the P. putida group into fifteen subgroups. Pairwise average nucleotide identities were calculated between type strains and a selection of 60 genomes of non-type strains of Pseudomonas. Forty-one strains were incorrectly assigned at the species level and among these, 19 strains were shown to represent an additional 13 new Pseudomonas species that remain to be formally classified. This work pinpoints the importance of correct taxonomic assignment and phylogenetic classification in order to perform integrative studies linking genetic diversity, lifestyle, and metabolic potential of Pseudomonas spp.

Lipopeptides (LPs) are a prominent class of molecules among the steadily growing spectrum of specialized metabolites retrieved from Pseudomonas, in particular soil-dwelling and plant-associated isolates. Among the multiple LP families, pioneering research focussed on phytotoxic and antimicrobial cyclic lipopeptides (CLPs) of the ubiquitous plant pathogen Pseudomonas syringae (syringomycin and syringopeptin). Their non-ribosomal peptide synthetases (NRPSs) are embedded in biosynthetic gene clusters (BGCs) that are tightly co-clustered on a pathogenicity island. Other members of the P. syringae group (Pseudomonas cichorii) and some species of the Pseudomonas asplenii group and Pseudomonas fluorescens complex have adopted these biosynthetic strategies to co-produce their own mycin and peptin variants, in some strains supplemented with an analogue of the P. syringae linear LP (LLP), syringafactin. This capacity is not confined to phytopathogens but also occurs in some biocontrol strains, which indicates that these LP families not solely function as general virulence factors. We address this issue by scrutinizing the structural diversity and bioactivities of LPs from the mycin, peptin, and factin families in a phylogenetic and evolutionary perspective. BGC functional organization (including associated regulatory and transport genes) and NRPS modular architectures in known and candidate LP producers were assessed by genome mining.

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.

Pseudomonas lipopeptides (LPs) are involved in diverse ecological functions and have biotechnological potential associated with their antimicrobial and/or anti-proliferative activities. They are synthesized by multi-modular non-ribosomal peptide synthetases which, together with transport and regulatory proteins, are encoded by large biosynthetic gene clusters (BGCs). These secondary metabolites are classified in distinct families based on sequence and length of the oligopeptide, and size of the macrocycle, if present. Phylogeny of PleB, the MacB-like transporter that is part of a dedicated ATP-dependent tripartite efflux system driving export of Pseudomonas LPs, revealed a strong correlation with LP chemical diversity. As each LP BGC carries its cognate pleB , PleB is suitable as a diagnostic sequence for genome mining, allowing assignment of the putative metabolite to a particular LP family. In addition, pleB proved a suitable target gene for an alternative PCR method to detect LP-producing Pseudomonas , not relying on amplification of catalytic domains of the biosynthetic enzymes. Combined with amplicon sequencing, this approach enabled typing of Pseudomonas strains as potential producers of a LP belonging to one of ten different families, underscoring its value for strain prioritization. This was validated by chemical characterization of known LPs from three different families secreted by novel producers isolated from the rice or maize rhizosphere, namely the type strains of Pseudomonas fulva (putisolvin), Pseudomonas zeae (tensin) and Pseudomonas xantholysinigenes (xantholysin). In addition, a new member of the Bananamide family, prosekin, was discovered in the type strain of Pseudomonas prosekii , an Antarctic isolate. Importance Pseudomonas are ubiquitous bacteria able to thrive in a wide range of ecological niches and lipopeptides often support their lifestyle but also their interaction with other micro- and macro-organisms. Therefore, the production of lipopeptides is widespread among Pseudomonas strains. Consequently, Pseudomonas lipopeptide research affects not only chemists and microbiologists but touches a much broader audience, including biochemists, ecologists and plant biologists. In this study we present a reliable transporter gene-guided approach for the detection and/or typing of Pseudomonas lipopeptide producers. Indeed, it allows to readily assess the lipopeptide diversity among sets of Pseudomonas isolates and differentiate strains likely to produce known lipopeptides from producers of potentially novel lipopeptides. This work provides a valuable tool that can also be integrated in a genome mining strategy and adapted for the typing of other specialized metabolites.

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Pseudomonas species are prominent producers of lipopeptides that support proliferation in a multitude of environments and foster varied lifestyles. By genome mining of biosynthetic gene clusters (BGCs) with lipopeptide-specific organization, we mapped the global Pseudomonas lipopeptidome and linked its staggering diversity to taxonomy of the producers, belonging to different groups within the major Pseudomonas fluorescens lineage.

Since the discovery of quorum sensing (QS) in the 1970s, many studies have demonstrated that Vibrio species coordinate activities such as biofilm formation, virulence, pathogenesis, and bioluminescence, through a large group of molecules called N-acyl homoserine lactones (AHLs). However, despite the extensive knowledge on the involved molecules and the biological processes controlled by QS in a few selected Vibrio strains, less is known about the overall diversity of AHLs produced by a broader range of environmental strains. To investigate the prevalence of QS capability of Vibrio environmental strains we analyzed 87 Vibrio spp. strains from the Banyuls Bacterial Culture Collection (WDCM911) for their ability to produce AHLs. This screening was based on three biosensors, which cover a large spectrum of AHLs, and revealed that only 9% of the screened isolates produced AHLs in the defined experimental conditions. Among these AHL-producing strains, Vibrio tasmaniensis LGP32 is a well-known pathogen of bivalves. We further analyzed the diversity of AHLs produced by this strain using a sensitive bioguided UHPLC-HRMS/MS approach (Ultra-High-Performance Liquid Chromatography followed by High-Resolution tandem Mass Spectrometry) and we identified C10-HSL, OH-C12-HSL, oxo-C12-HSL and C14:1-HSL as QS molecules. This is the first report that documents the production of AHL by Vibrio tasmaniensis LGP32.

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