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Article

The Ever-Expanding Pseudomonas Genus: Description of 43 New Species and Partition of the Pseudomonas putida Group

by
Léa Girard
1,†,
Cédric Lood
1,2,†,
Monica Höfte
3,
Peter Vandamme
4,
Hassan Rokni-Zadeh
5,
Vera van Noort
1,6,
Rob Lavigne
2,* and
René De Mot
1,*
1
Centre of Microbial and Plant Genetics, Faculty of Bioscience Engineering, KU Leuven, Kasteelpark Arenberg 20, 3001 Leuven, Belgium
2
Department of Biosystems, Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21, 3001 Leuven, Belgium
3
Department of Plants and Crops, Laboratory of Phytopathology, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000 Ghent, Belgium
4
Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium
5
Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan 45139-56184, Iran
6
Institute of Biology, Leiden University, Sylviusweg 72, 2333 Leiden, The Netherlands
*
Authors to whom correspondence should be addressed.
†
The authors contributed equally to this work.
Microorganisms 2021, 9(8), 1766; https://doi.org/10.3390/microorganisms9081766
Submission received: 13 July 2021 / Revised: 10 August 2021 / Accepted: 16 August 2021 / Published: 18 August 2021
(This article belongs to the Special Issue Genomics in Bacterial Taxonomy: Impact on the Genus Pseudomonas)
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Versions Notes

Abstract

:
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.

1. Introduction

During the past decade, the landscape of bacterial systematics has changed drastically [1]. Once dominated by a polyphasic approach including phenotypic characterization, DNA–DNA hybridization, and 16S rRNA gene sequencing, the age of microbial genomics and metagenomics has reshaped the foundation of prokaryotic species definition [2,3]. Although 16S rRNA phylogeny remains the most common tool to evaluate the diversity of mixed prokaryotic populations, estimating inter- and intra-species relatedness was traditionally facilitated by DNA-typing methods. For several years, Multi-Locus Sequence Analysis (MLSA) represented the most widely adopted methodology for bacterial systematics, and for the exploration of evolutionary relationships within specific families/genera [4,5,6,7]. The success of high throughput and affordable Whole Genome Sequencing (WGS) technologies has tremendously increased the number of publicly available genomes and, therefore, genome-to-genome comparisons, with the Average Nucleotide Identity (ANI) and digital DNA–DNA Hybridization (dDDH), have become today’s standards for species definition [1,8,9,10,11]. This genome-based elucidation of relatedness at the inter- and intra-species level is now encouraged and, at a larger scale, the creation of a Genome Taxonomy Database (GTDB) has allowed the bacterial taxonomy to be standardized [12,13].
According to GTDB, the Pseudomonadaceae family currently includes seven genera: Azomonas, Azotobacter, Entomomonas, Oblitimonas, Pseudomonas, Thiopseudomonas, and Ventosimonas (https://gtdb.ecogenomic.org/tree?r=f__Pseudomonadaceae, accessed on 10 July 2021). The genus Pseudomonas is the most complex, with 259 validly named species (List of Prokaryotic Names with Standing in Nomenclature (https://lpsn.dsmz.de/genus/pseudomonas, accessed on 10 August 2021), excluding subspecies and synonymous species. However, this number is constantly evolving, with over 30 new Pseudomonas species described between March 2020 and March 2021. Since the first descriptions of Pseudomonas species, which were based on morphological and phenotypical characteristics, several studies updated the taxonomy of Pseudomonas based on 16S rRNA gene sequence analysis [14]. This allowed the differentiation of the genus Pseudomonas from its sister genera, and also the definition of the three main Pseudomonas lineages, P. pertucinogena, P. aeruginosa, and P. fluorescens [6,15]. In a similar fashion, MLSA has guided the redefinition of prokaryotic species and has also impacted the phylogenomics and systematics of the genus Pseudomonas [4,6,16]. Indeed, the analysis based on four housekeeping genes (i.e., 16S rRNA, gyrB, rpoB, and rpoD) enabled the clarification of the Pseudomonas phylogeny by enhancing species delineation. This approach also proved to be a reliable tool for strain identification at the species level [4,6]. We recently demonstrated that the rpoD gene sequence alone provides a strong and low-cost alternative, particularly in the case of taxonomic affiliation of large batches of environmental Pseudomonas isolates [17].
Pseudomonas are motile, non-spore forming, Gram-negative rods belonging to the Gammaproteobacteria. Pseudomonas species are able to colonize and thrive in a wide range of ecological niches (e.g., soil, water, and plants, associated with higher organisms) [18]. In addition to the well-known human pathogen P. aeruginosa, other Pseudomonas species induce diseases in plants, fish, insects, or other animals [19,20,21]. In contrast, a large majority of Pseudomonas species are commensals but can also be used as bioremediation, biostimulation, and biocontrol agents [22,23]. Pseudomonas are ubiquitous bacteria that are often identified as fundamental components of bacterial communities and thus play essential ecological functions in the environment [24,25,26]. Furthermore, Pseudomonas are outstanding producers of bioactive secondary metabolites that often support their eclectic lifestyle (e.g., iron scavenging, swarming motility, biofilm formation, pathogenicity, cooperation, or antagonism) [27,28]. The link between secondary metabolites and Pseudomonas taxonomy has already been made through pyoverdines, a class of pigments used for a long time as a specific marker of classification [18]. Pseudomonas cyclic lipopeptides (CLPs), having a broad antimicrobial activity profile and anti-proliferative properties, have gained the attention of researchers due to their promising application potential [29]. CLP production is widespread within the genus Pseudomonas, and relationships between CLP diversity and Pseudomonas taxonomy were recently highlighted [30,31]. CLP producers tend to be grouped by CLP family and confined to specific groups or subgroups of Pseudomonas. Nonetheless, exceptions occur within the P. putida group, which hosts a large diversity of CLP producers from diverse families (i.e., Xantholysin, Entolysin, Putisolvin, and Viscosin families) [30,31].
In this study, we report 43 new Pseudomonas species and use a combination of Nanopore and Illumina sequencing to provide high quality genomes. Through the genome analysis of these new species, together with type strains of Pseudomonas, we provide an update of the Pseudomonas phylogeny based on a set of 1508 core orthogroup sequences and another based on the rpoD gene. We used nucleotide identities based on the rpoD gene and whole genome comparisons to reassign, respectively, 82 and 41 non-type strains of Pseudomonas to known and newly described Pseudomonas species. A large majority of the new species were affiliated to the P. putida group, increasing species numbers from 35 to 51. We thus explored genetic diversity within the P. putida group in a greater depth and mapped, on an expanded phylogeny of the group, the presence of Biosynthetic Gene Clusters (BGCs) for the production of CLPs.

2. Materials and Methods

2.1. Pseudomonas Strains

In this study we used 273 known type strains of Pseudomonas, including validly published species and recently published species still lacking taxonomic status (https://lpsn.dsmz.de/genus/pseudomonas, accessed on 10 August 2021). Only 270 type strains of Pseudomonas were used for genome analysis because no genome sequences were available for three of these type strains. Eleven type strains of other genera within the Pseudomonadaceae and Cellvibrio japonicus were used for phylogenetic analyses (Figure S5). The list of type strains, including their culture collection codes and accession numbers (i.e., rpoD and whole genome sequences) is provided in Table S1.
We also used 47 strains from our collection of environmental Pseudomonas isolates to describe 43 new Pseudomonas species (the type strains of newly described species are highlighted in bold; Table S2). These 47 isolates were deposited in two culture collections (i.e., Belgian Co-ordinated Collections of Micro-organisms (BCCM/LMG) and Collection Française de Bactéries associées aux Plantes (CFBP)), and their phenotypic profiles were obtained using the Biolog GEN III MicroPlate (BIOLOG, Hayward, CA, USA) according to the manufacturer’s instructions (Table S3). To avoid species description based on single strains, the rpoD sequences of these 43 new type strains of Pseudomonas were then used as a query to search for additional strains using BlastN with default parameters (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch; accessed on 10 August 2021, Tables S4 and S5). We previously defined a cutoff value of 98% nucleotide identity to differentiate strains at the species level but also revealed some inconsistent species affiliations [17]. To avoid any misidentification due to the use of the rpoD gene, we only considered hits with 100% identity, or lower if a genome was available and thus allowed validation by ANIb calculation (> 96.5%, Tables S4 and S5). We thus used a total of 82 non-type strains of Pseudomonas (Table S4), including 29 with whole genome sequences (Table S5).
Finally, a set of 122 strains, including type and non-type strains of Pseudomonas, was specifically used for the P. putida group phylogenetic and genomic analyses (Table S6).

2.2. Genome Sequencing, Assembly, and Functional Annotation

We recently highlighted the high discriminative power of the rpoD gene as a reliable tool for the identification of environmental Pseudomonas isolates [17]. In the same study, we released draft genome sequences of 55 environmental Pseudomonas isolates and rpoD gene analysis together with whole genome comparisons allowed us to highlight the presence of 30 new Pseudomonas species (Pseudomonas #5 [17], with strains SWRI59, SWRI68, and SWRI77, was later identified as P. capeferrum). We applied the same methodology to an expanded set of Pseudomonas isolates and identified 17 additional new species. To provide high-quality genomes for the type strains of these 43 new species, we combined Illumina and Nanopore sequencing [32]. An overview of the different sequencing methodologies used for the entire set of strains is shown in Table S2. We controlled the quality of the Illumina reads with FastQC v0.11.9 and used Trimmomatic v0.38 [33] for adapter clipping, quality trimming (LEADING:3 TRAILING:3 SLIDINGWINDOW:4.15), and filtering on length (>50 bp). The quality of the Nanopore reads was assessed with Nanoplot v1.28.2 [34] and we used Porechop v0.2.4 (https://github.com/rrwick/Porechop, accessed on 8 June 2021) for barcode clipping, in addition to NanoFilt v2.6 [34] to filter quality (Q > 8) and length (>500 bp). The genomes were assembled using Unicycler v0.4.8 [35] with default options and the quality of their assemblies was assessed using QUAST v5.1 [36]. The functional annotation was undertaken with the NCBI Prokaryotic Genome Annotation Pipeline [37].

2.3. Taxonomic Affiliations and Phylogenetic Analyses

To define the new species and confirm rpoD-based affiliations, Average Nucleotide Identity (ANI) values were calculated using PYANI v0.2.10 [38] with default parameters, and the ANIb method (Table S7) with a cutoff value of 96.5% [17,18]. In the case of ANIb values considered as ambiguous (i.e., between 95 and 96.5%) we calculated digital DNA–DNA Hybridization (dDDH) using the Genome-to-Genome Distance Calculator (GGDC; https://www.dsmz.de/services/online-tools/genome-to-genome-distance-calculator-ggdc, accessed on 8 June 2021).
The evolutionary relationships between newly described and previously known type strains of Pseudomonas were assessed using rpoD and whole genome phylogenies. The rpoD-based phylogenies were conducted as previously described using MEGA-X (Figure 1, right) [17]. The corresponding similarity matrix, based on a 650 bp fragment of the rpoD gene, including 316 type strains of Pseudomonas (273 known and 43 newly described species), was generated (Table S8). The phylogenetic trees based on whole genomes were inferred with IQ-TREE v1.6.12 [39] with automatic model selection and 1.000 ultrafast bootstraps (UF-Boot) using an alignment of 1508 (genus phylogeny; Figure S1, left) and 2570 (P. putida group phylogeny; Figure S5) core orthogroup sequences that were delineated with the SCARAP pipeline (https://github.com/SWittouck/SCARAP, accessed on 8 June 2021) [40].
Several new phylogenetic groups (G) and subgroups (SG) were delineated based on branch length, grouping, and bootstrap values on both rpoD and whole genome phylogenies (Table 1 and Table 2, Figure 1, Figure 2, and Figure S5). The new groups and subgroups were named after the first species described in a group or subgroup.

2.4. Cyclic Lipopeptide (CLP) NRPS Analysis

The P. putida group was previously highlighted to include CLP producers from the Viscosin (WLIP producers), Putisolvin, Entolysin, and Xantholysin families [30,31]. Among the 16 type strains of the newly described species belonging to the P. putida group, 4 were already described as CLP producers (WLIP and Xantholysin producers) [30,31]. Consequently, all strains belonging to the P. putida group (Table S6), with available genome sequences, were subjected to an antiSMASH analysis (antiSMASH 6.0) [41]. Positive hits were then inspected manually to confirm the typical features of Pseudomonas CLP Non-Ribosomal Peptide Synthetase (NRPS) clusters (i.e., the presence of tandem TE-domains and the absence of epimerization domains) and synteny (i.e., number of modules and their distribution along the encoded NRPSs), all based on previously described CLP NRPS gene cluster annotations [42,43]. All known and newly identified strains carrying CLP BGCs, together with their affiliation to CLP families and the accession numbers of their NRPS genes, are presented in Table S6. The phylogenetic relationship between known and newly identified CLP producers was assessed, by family, based on concatenated NRPS amino acid sequences (Figures S2–S4).

3. Results and Discussion

3.1. Defining New Pseudomonas Species

In a recent study, we performed rpoD-based identifications which allowed us to identify 31 new Pseudomonas species [17]. In the same study, three strains were incorrectly identified as representative strains of a new species (i.e., Pseudomonas #5, SWRI59, SWRI68, and SWRI77) but subsequently identified as P. capeferrum strains. Further rpoD-based identifications enabled us to identify 17 additional Pseudomonas species. Four strains, namely, SWRI22, OE 28.3, SWRI76, and CMR5c, were first assessed as new species but were later assigned to newly published Pseudomonas species (i.e., #29 P. carnis, #30 P. edaphica, #31 P. atacamensis, and #45 P. aestus; Table S7). Finally, a total of 43 new Pseudomonas species could be defined (Appendix A) and the result of their phenotypic profiling, together with assigned culture collection numbers, are presented in Table S3. Hybrid assemblies of the genomes resulted in 22 closed genomes and 18 draft genomes with improved contiguity. Due to technical issues, we have not been able to increase the quality of the draft genomes of strains BW11P2, COW3, and SWRI196. To avoid the proposal of new species based on single strains, the rpoD sequences of the 43 new species were used as queries to search for additional strains. We therefore reassigned 82 Pseudomonas strains, including 29 with whole genome sequences, available through GenBank (Tables S1 and S2). Finally, ANIb values were calculated between a total of 346 Pseudomonas species (270 type strains and 76 (47 + 29) Pseudomonas strains affiliated to new species), and allowed us to confirm these affiliations and the presence of 43 new Pseudomonas species (Table S7). The phylogenetic position of the 43 type strains is shown in Figure 1 and their distribution within the different groups of Pseudomonas is detailed in Table 1. All of the new species are clustering within the P. fluorescens (n = 27) and P. putida (n = 16) groups. We amended the existing subgroups of P. fluorescens as follows: P. asplenii (inclusion of P. vanderleydeniana), P. corrugata (inclusion of P. alvandae, P. marvdashtae, P. tehranensis, P. zanjanensis and P. zarinae), P. fluorescens (inclusion of P. asgharzadehiana, P. azadiae, P. khavaziana, P. salmasensis and P. tritici), P. gessardii (inclusion of P. shahriarae), P. jessenii (inclusion of P. asgharzadehiana and P. azerbaijanoccidens), P. koreensis (inclusion of P. bananamidigenes, P. botevensis, P. ekonensis, P. hamedanensis, P. iranensis, P. khorasanensis, P. monsensis, P. siliginis, P. tensinigenes, P. triticicola and P. zeae), P. mandelii (inclusion of P. farris), P. protegens (inclusion of P. sessiligenes) (Table 1). The remaining sixteen new species allowed the partitioning of the P. putida group into fifteen subgroups, as described in Section 3.3.

3.2. Comparison of Whole Genome and rpoD-based Phylogenies

The phylogenetic relationships between known and newly described type strains of Pseudomonas are presented in Figure 1, respectively, the whole genome, based on 1508 core orthogroups, and the rpoD-based phylogenies. The phylogenies include 273 type strains of Pseudomonas species (270 for the whole genome phylogeny) and 43 type strains of the newly described Pseudomonas species. Three type strains of Pseudomonas were excluded from the analysis: (1) P. hydrolytica, with an abnormally long genome (10.4 Mbp)); and (2) P. hussainii and P. caeni, harboring short genomes (respectively, 3.68 and 3.03 Mbp) and clustering with members of other genera within the Pseudomonadaceae [4,6,17]. We suspect that the latter two are not Pseudomonas species and a dedicated study needs to clarify the taxonomy of other genera within the Pseudomonadaceae family.
Indeed, P. caeni gained the attention of Hesse and colleagues [18] due to its unusual genomic features, and is already displayed as Thiopseudomonas caeni in the GTDB (https://gtdb.ecogenomic.org/tree?r=f__Pseudomonadaceae, accessed on 10 July 2021). A tree of the Pseudomonadaceae family, including P. hussainii and P. caeni (T. caeni), in addition to all type strains of the Azomonas, Azotobacter, Entomomonas, Oblitimonas, Pseudomonas, Thiopseudomonas, and Ventosimonas genera, is shown in Figure S1.
The thirteen groups of Pseudomonas previously identified in several studies (i.e., P. pertucinogena, P. oryzihabitans, P. aeruginosa, P. resinovorans, P. stutzeri, P. linyingensis, P. oleovorans, P. straminea, P. anguilliseptica, P. putida, P. lutea, P. syringae, and P. fluorescens) are all well supported in both trees [4,6,17,18]. In addition to these thirteen groups, three new groups, namely, P. pohangensis, P. massiliensis, and P. rhizosphaerae, were identified based on branch length and the strong bootstrap support values separating them from the neighboring groups (Figure 1). Furthermore, as previously observed, ten species are scattered across the tree and represent orphan groups currently formed by only one species (Figure 1). An overview of all known and newly proposed groups is summarized in Table 1.
Overall, both trees are highly consistent in topology, although the tree inferred by whole genome analysis is supported by stronger bootstrap values. Two main differences can still be highlighted: (1) the position of the P. syringae and P. lutea group, clustering inside the P. fluorescens group in the rpoD-based tree; and (2) the position of P. karstica, P. spelaei, and P. yamanorum, clustering within the P. gessardii subgroup in rpoD and MLSA phylogenies [4,6,17], whereas in phylogenies based on whole genome analysis, they cluster within the P. fluorescens subgroup ([18] and Figure 1).

3.3. Genomic Diversity within the P. putida Group

3.3.1. Identification and Reassignment at the Species Level

Several studies have revealed inconsistencies within public databases in which genomes of Pseudomonas are not identified (Pseudomonas sp.) or incorrectly assigned at the species level [4,44,45]. Within the P. putida group, a huge number of strains are incorrectly assigned to P. putida [4,44]. Here, we propose to update the P. putida group with 16 new Pseudomonas species and tentatively reassign 44 non-type strains of Pseudomonas (Table 3). A total of 25 strains are affiliated to known and newly described species (P. shirazensis (n = 1), P. guariconensis (n = 2), P. wayambapalatensis (n = 2), P. farsensis (n = 1), P. peradeniyensis (n = 1), P. capeferrum (n = 2), P. kermanshahensis (n = 4), P. juntendi (n = 2), P. alloputida (n = 6), and P. kurunegalensis (n = 4)), and the remaining 19 strains represent an additional 13 new species. As previously observed for the genus Pseudomonas, these results confirm the fact that type strains still represent a small fraction of the genomic diversity within the P. putida group.

3.3.2. Distribution of CLP biosynthesis Gene Clusters

CLPs are specialized metabolites that often support important ecological functions including cooperation, phytopathogenicity, or antagonism [29,43,46]. CLPs consist of a fatty acid tail attached to a cyclized oligopeptide and are synthesized by NRPSs [29,42]. The modularity of these enzymes enables Pseudomonas strains to produce a wide diversity of CLPs, resulting in their classification in several families [28,29,42]. The relationship between CLP diversity and Pseudomonas taxonomy was recently highlighted, and it was demonstrated that certain CLP families were exclusive to specific subgroups of P. fluorescens [30,31,43]. In contrast, the P. putida group was demonstrated to host CLP producers from different families [30,31]. CLP production is widespread within the P. putida group and different type strains (i.e., P. capeferrum, P. entomophila, and P. soli) and many non-type strains (e.g., RW10S2, PCL1445, BW11M1, 250J, COR5, COW10, COR19, COR51; Table S7) were formerly characterized as producers of CLPs from the Viscosin (WLIP producers), Putisolvin, Entolysin, and Xantholysin families [30,31,46,47,48,49,50,51,52,53,54,55,56]. Among the 16 type strains of the newly described species, four were previously described as CLP producers: two WLIP producers, P. fakonensis COW40 and P. xanthosomae COR54; and two xantholysin producers, P. maumuensis COW77 and P. muyukensis COW39 (Table S6) [30,31]. We therefore searched for CLP NRPSs in a selection of Pseudomonas genomes, including all type strains belonging to the P. putida group (n = 51) and the 44 genomes of non-type strains presented in Table 3. About 65% of the strains (i.e., 34 of 51 type strains; 28 of 44 non-type strains) did not carry CLP NRPSs in their genomes (Table S6). Our analysis revealed the presence of NRPSs from the Viscosin family (WLIP-like NRPSs) in the genomes of two strains (P. wayambapalatensis RW3S1T and RW3S2); from the Putisolvin family in five type strains (P. fulva, P. kermanshahensis, P. parafulva, P. reidholzensis, and P. vlassakiae) and five non-type strains (P. capeferrum SWRI59 and SWRI68 and P. kermanshahensis SWRI67, SWRI50, E46); and from the Xantholysin family in two type strains (P. peradeniyensis and P. xantholysinigenes) and two non-type strains (P. mosselii BW18S1 and P. peradeniyensis BW16M2) (Figure 2 and Table S6). The poor genome quality of two type strains, namely, P. brassicae and P. juntendi, revealed the presence of NRPS gene fragments coding for tandem thioesterase (TE) domains. Tandem TE domains are specific to Pseudomonas CLP NRPS; therefore, these results indicate P. brassicae and P. juntendi carry CLP NRPS genes and most likely produce CLPs. Further analyses, chemical characterization, and/or a hybrid assembly based on long read sequencing and Illumina sequencing are needed to identify the CLPs. We previously highlighted, in the type strains of P. asplenii and P. fucovaginae, a NRPS system predicted to assemble a lipotridecapeptide (LP-13) but this metabolite still awaits chemical and functional characterization [43]. A putative LP-13 biosynthesis gene cluster is also present in the genome of P. tructae. Oni and colleagues also reported the presence of a new CLP (N8, 17:8, 17 amino acids, of which 8 are in the macrocycle) within the P. putida group [30,31]. Altogether, these results highlight a wide diversity of CLP producers from known, and yet to be described new, CLP families within the P. putida group.

3.3.3. Partitioning of the P. putida Group

To present an integrated approach linking the genetic diversity and the metabolic potential of Pseudomonas species, we mapped the presence of CLP biosynthesis gene clusters on an extended phylogeny of the P. putida group (Figure 2 and Figure S5). As shown in Figure 1, the P. putida group is composed of several subgroups (Figure 1). The extended phylogeny allowed us to define 15 subgroups, P. japonica (n = 6), P. vranovensis (n = 6), P. reidholzensis (n = 3), P. xanthosomae (n = 2), P. mosselii (n = 8), P. vlassakiae (n = 3), P. capeferrum (n = 2), and P. putida (n = 14), including seven orphan subgroups (P. akappagea, P. cremoricolorata, P. guariconensis, P. wayambapalatensis, P. farsensis, P. taiwanensis, and P. plecoglossicida). The distribution of all type strains in the 15 subgroups is detailed in Table 1. Among the 44 non-type strains used in Section 3.3.1, 19 were highlighted to represent 13 new species distributed in seven subgroups: P. japonica (n = 1), P. guariconensis (n = 1), P. wayambapalatensis (n = 1), P. mosselii (n = 1), P. plecoglossicida (n = 3), P. vlassakiae (n = 2), and P. putida (n = 4) (Table 3). These additions to the P. putida phylogenies allowed us to seize a small portion of the genomic diversity among environmental Pseudomonas strains, but also to pinpoint the immediate growing potential of the newly defined subgroups. We observed that, in both rpoD and whole genome phylogenies, the distribution of CLP biosynthesis gene clusters was associated with this phylogenetic subgrouping.
All xantholysin and entolysin producers were grouped within the P. mosselii subgroup, putisolvin producers were clustered in four subgroups (i.e., P. putida, P. reidholzensis, P. vlassakiae, and P. capeferrum), and WLIP producers were distributed over two subgroups (P. xanthosomae and P. wayambapalatensis). Moreover, the phylogenies based on concatenated NRPS amino acid sequences (Xantholysin/Entolysin families, Figure S2; Putisolvin family, Figure S3; and WLIP producers, Figure S4) revealed different clusters that perfectly match the distribution of CLP producers within the different subgroups. These results demonstrate that the rpoD gene allows both the identification of Pseudomonas isolates and the construction of robust phylogenies, providing information about the affiliation of producers to CLP families.
The strong congruence between the phylogenetic tree based on the NRPS sequences and the rpoD- and whole genome-based phylogenies indicates that CLP biosynthesis genes have largely evolved in accordance with the evolutionary history of Pseudomonas species within the P. putida group. However, P. reidholzensis carries a putisolvin biosynthetic gene cluster that is absent from the genome of the closely related species. Furthermore, CLP producers from the Viscosin family, including WLIP producers, are predominantly found within the P. fluorescens group [30,31,57], with the exception of the two subclusters of WLIP producers present in the P. putida group. Altogether, these observations indicate that Pseudomonas CLP NRPS clusters have a complex evolutionary history probably involving both vertical and horizontal gene transfer.

4. Conclusions

Our update of the genus with 43 new species together with our analysis of 313 genomes of type strains allowed us to propose a robust revised phylogeny of the Pseudomonas spp. This study aimed to fill the gap between the currently named species and the real genomic diversity within the genus Pseudomonas. Additional work is needed to complete this task and genome-based standards for species definition should be favored over highly variable phenotypic tests for publication. Our study validated the use of the rpoD gene for species identification, and for the study of the evolutionary relationships within the genus Pseudomonas. Furthermore, rpoD-based phylogenies can also be highly useful to specifically prospect for CLP biosynthesis gene clusters and affiliation of producers to known CLP families. Finally, the use of genomic sequences appears to be essential to reveal the ecological and metabolic potential of Pseudomonas spp.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/microorganisms9081766/s1, Figure S1: Genome-based phylogeny of the Pseudomonadaceae. Figure S2: Phylogenetic tree based on concatenated NRPS proteins from the Xantholysin family. Figure S3: Phylogenetic tree based on concatenated NRPS proteins from the Putisolvin family. Figure S4: Phylogenetic tree based on concatenated NRPS proteins of WLIP producers from the Viscosin family. Figure S5: Genome-based phylogeny of the P. putida group. Table S1: List of type strains used in this study. Table S2: List of environmental Pseudomonas isolates used to describe 43 new Pseudomonas species. Table S3: Phenotypic profiles of the 43 new Pseudomonas species. Table S4: rpoD-based affiliation of strains to newly described Pseudomonas species. Table S5: Whole genome-based affiliation of strains to newly described Pseudomonas species. Table S6: Prospection of CLP biosynthesis gene clusters within the P. putida group. Table S7: ANIb matrix including 313 type and 33 non-type strains of Pseudomonas. Table S8: rpoD similarity matrix based on 316 type strains of Pseudomonas.

Author Contributions

Conceptualization, methodology, investigation, writing original draft and editing, L.G. and C.L.; methodology, investigation and writing-review and editing, M.H. and P.V.; resources and formal analysis, H.R.-Z.; writing- review and editing and funding acquisition, V.v.N., R.L. and R.D.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research and the APC were funded by the EOS grant 30650620 (RHIZOCLIP). C.L. is supported by an SB PhD fellowship from FWO Vlaanderen (1S64720N).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article or Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A. Descriptions of the 43 New Pseudomonas Species

The phenotypic descriptions are presented in Table S3. RpoD and whole genome-based assignment of additional Pseudomonas strains to the newly described species are shown, respectively, in Tables S1 and S2.
(#1) Description of Pseudomonas anuradhapurensis sp. nov.
Pseudomonas anuradhapurensis (a.nu.ra.dha.pur.en’sis. n.L. fem. adj. anuradhapurensis, from Anuradhapura, a city in Sri Lanka).
The type strain is RD8MR3T (LMG 32021T = CFBP 8837T) and was isolated from the endorhizosphere of rice, Anuradhapura, Sri Lanka in 1990. Its G + C content is 63.43 mol% (calculated based on its genome sequence). The 16S rRNA gene, rpoD, and whole-genome sequence of RD8MR3T are publicly available through the accession numbers AM911640, MT621460, and CP077097, respectively.
(#2) Description of Pseudomonas oryzicola sp. nov.
Pseudomonas oryzicola (o.ry.zi’co.la. L. fem n. Oryza rice, L. suff. -cola (from L. n. incola) inhabitant dweller; N.L. n. oryzicola, rice dweller).
The type strain is RD9SR1T (LMG 32022T = CFBP 8838T) and was isolated from the exorhizosphere of rice, Anuradhapura, Sri Lanka in 1990. Its G + C content is 62.91 mol% (calculated based on its genome sequence). The 16S rRNA gene, rpoD, and whole-genome sequence of RD9SR1T are publicly available through the accession numbers AM911646, MT621461, and JABWRZ000000000, respectively.
(#3) Description of Pseudomonas kurunegalensis sp. nov.
Pseudomonas kurunegalensis (ku.ru.ne.gal.en’sis. N.L. fem. adj. kurunegalensis, from Kurunegala, a city in Sri Lanka).
The type strain is RW1P2T (LMG 32023T = CFBP 8839T) and was isolated from the rhizoplane of rice, Kurunegala, Sri Lanka in 1990. Its G + C content is 62.09 mol% (calculated based on its genome sequence). The 16S rRNA gene, rpoD, and whole-genome sequence of RW1P2T are publicly available through the accession numbers AM911650, MT621449, and JABWSB000000000, respectively.
(#4) Description of Pseudomonas kermanshahensis sp. nov.
Pseudomonas kermanshahensis (ker.man.shah.en’sis. N.L. fem. adj. kermanshahensis, from Kermanshah, a city in Iran).
The type strain is SWRI100T (LMG 32035T = CFBP 8840T) and was isolated from the rhizosphere of wheat (cultivar Marvdasht), Kermanshah, Iran in 2004. Its G + C content is 62.22 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI100T are publicly available through the accession numbers MT621423 and JABWRY000000000, respectively.
(#5) Description of Pseudomonas wayambapalatensis sp. nov.
Pseudomonas wayambapalatensis (wa.yam.ba.pa.lat.en’sis. N.L. fem. adj. wayambapalatensis, from Wayamba Palata, the name of the north-western province in Sri Lanka).
The type strain is RW3S1T (LMG 32024T = CFBP 8841T) and was isolated from the exorhizosphere of rice, Kurunegala, Sri Lanka in 1990. Its G + C content is 63.24 mol% (calculated based on its genome sequence). The 16S rRNA gene, rpoD, and whole-genome sequence of RW3S1T are publicly available through the accession numbers AM911665, MT621434, and CP077096, respectively.
(#6) Description of Pseudomonas xantholysinigenes sp. nov.
Pseudomonas xantholysinigenes (xan.tho.ly.si.ni’ge.nes. N.L. neut. n. xantholysinum, xantholysin; Gr. v. gennao to produce; N.L. part. adj. xantholysinigenes, xantholysin producing).
The type strain is RW9S1AT (LMG 32025T = CFBP 8842T) and was isolated from the exorhizosphere of rice, Kurunegala, Sri Lanka in 1990. Its G + C content is 64.16 mol% (calculated based on its genome sequence). The 16S rRNA gene, rpoD, and whole-genome sequence of RW9S1AT are publicly available through the accession numbers AM911667, MT621442, and CP077095, respectively.
(#7) Description of Pseudomonas peradeniyensis sp. nov.
Pseudomonas peradeniyensis (pe.ra.de.niy.en’sis. N.L. fem. adj. peradeniyensis, from Peradeniya, a city in Sri Lanka).
The type strain is BW13M1T (LMG 32026T = CFBP 8887T) and was isolated from banana plant endorhizosphere, Peradeniya, Sri Lanka in 1990. Its G + C content is 64.62 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of BW13M1T are publicly available through the accession numbers MT621446 and JABWRJ000000000, respectively.
(#8) Description of Pseudomonas vlassakiae sp. nov.
Pseudomonas vlassakiae (vlas.sak.i.a’e. N.L. gen. n. vlassakiae, from Katrien Vlassak, a Belgian microbiologist who isolated the strain RW4S2, in addition to RD3MR3, RD9SR1, RW1P2, RW3S1, RW9S1A, BW13M1, RW10S1, RW8P3, and BW11P2, which represent 10 new Pseudomonas species).
The type strain is RW4S2T (LMG 32027T = CFBP 8843T) and was isolated from the exorhizosphere of rice, Kurunegala, Sri Lanka in 1990. Its G + C content is 62.98 mol% (calculated based on its genome sequence). The 16S rRNA gene, rpoD, and whole-genome sequence of RW4S2T are publicly available through the accession numbers AM911658, MT621428, and JABWRP000000000, respectively.
(#9) Description of Pseudomonas promysalinigenes sp. nov.
Pseudomonas promysalinigenes (pro.my.sa.li.ni’ge.nes. N.L. neut. n. promysalinum, promysalin; Gr. v. gennao to produce; N.L. part. adj. promysalinigenes, promysalin producing).
The type strain is RW10S1T (LMG 32028T = CFBP 8844T) and was isolated from the exorhizosphere of rice, Kurunegala, Sri Lanka in 1990. Its G + C content is 60.62 mol% (calculated based on its genome sequence). The 16S rRNA gene, rpoD, and whole-genome sequence of RW10S1T are publicly available through the accession numbers AM911668, MT621430, and CP077094, respectively.
(#10) Description of Pseudomonas urmiensis sp. nov.
Pseudomonas urmiensis (ur.mi.en’sis. N.L. fem. adj. urmiensis, from Urmia, a city in Iran).
The type strain is SWRI10T (LMG 32036T = CFBP 8845T) and was isolated from the rhizosphere of wheat (cultivar Marvdasht), West Azerbaijan, Iran in 2004. Its G + C content is 61.81 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI10T are publicly available through the accession numbers MT621419 and JABWRE000000000, respectively.
(#11) Description of Pseudomonas shirazensis sp. nov.
Pseudomonas shirazensis (shi.raz.en’sis. N.L. fem. adj. shirazensis, from Shiraz, a city in Iran).
The type strain is SWRI56T (LMG 32037T = CFBP 8846T) and was isolated from the rhizosphere of wheat (cultivar Shiraz), Shiraz, Iran in 2004. Its G + C content is 61.85 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI56T are publicly available through the accession numbers MT621418 and JABWRD000000000, respectively.
(#12) Description of Pseudomonas farsensis sp. nov.
Pseudomonas farsensis (fars.en’sis. N.L. fem. adj. farsensis, from Fars, a province in Iran).
The type strain is SWRI107T (LMG 32038T = CFBP 8847T) and was isolated from the rhizosphere of wheat (cultivar Azadi), Shiraz, Iran in 2004. Its G + C content is 62.58 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI107T are publicly available through the accession numbers MT621411 and JABWRF000000000, respectively.
(#13) Description of Pseudomonas vanderleydeniana sp. nov.
Pseudomonas vanderleydeniana (van.der.ley.den.i.a’na. N.L. fem. adj. vanderleydeniana, from Jos Vanderleyden, a Belgian microbiologist who studied plant growth-promoting properties of root-associated alpha- and gammaproteobacteria, including nitrogen-fixing and fluorescent Pseudomonas isolates.
The type strain is RW8P3T (LMG 32029T = CFBP 8848T) and was isolated from the rhizoplane of rice, Kurunegala, Sri Lanka in 1990. Its G + C content is 62.97 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of RW8P3T are publicly available through the accession numbers MT621472 and CP077093, respectively.
(#14) Description of Pseudomonas bananamidigenes sp. nov.
Pseudomonas bananamidigenes (ba.na.na.mi.di’ge.nes. N.L. neut. n. bananamidum, bananamide; Gr. v. gennao to produce; N.L. part. adj. bananamidigenes, bananamide producing).
The type strain is BW11P2T (LMG 32030T = CFBP 8849T) and was isolated from banana plant exorhizosphere, Galagedara, Sri Lanka in 1990. Its G + C content is 60.62 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of BW11P2T are publicly available through the accession numbers MT621496 and LRUN00000000, respectively.
(#15) Description of Pseudomonas iranensis sp. nov.
Pseudomonas iranensis (i.ran.en’sis. N.L. fem. adj. iranensis, from Iran).
The type strain is SWRI54T (LMG 32039T = CFBP 8850T) and was isolated from the rhizosphere of wheat (cultivar Shiraz), Shiraz, Iran in 2004. Its G + C content is 59.89 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI54T are publicly available through the accession numbers MT621504 and CP077092, respectively.
(#16) Description of Pseudomonas khorasanensis sp. nov.
Pseudomonas khorasanensis (kho.ra.san.en’sis. N.L. fem. adj. khorasanensis, from Khorasan, a province in Iran).
The type strain is SWRI153T (LMG 32040T = CFBP 8851T) and was isolated from the rhizosphere of wheat (cultivar Kaasparoo), Khorasan, Iran in 2004. Its G + C content is 59.71 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI153T are publicly available through the accession numbers MT621508 and JABWQP000000000, respectively.
(#17) Description of Pseudomonas hamedanensis sp. nov.
Pseudomonas hamedanensis (ha.me.dan.en’sis. N.L. fem. adj. hamedanensis, from Hamedan, a city in Iran).
The type strain is SWRI65T (LMG 32041T = CFBP 8852T) and was isolated from the rhizosphere of wheat, Hamedan, Iran in 2004. Its G + C content is 59.99 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI65T are publicly available through the accession numbers MT621514 and CP077091, respectively.
(#18) Description of Pseudomonas zeae sp. nov.
Pseudomonas zeae (ze’ae. L. gen. n. zeae, from Zea mays, corn).
The type strain is OE 48.2T (LMG 32031T = CFBP 8853T) and was isolated from the rhizosphere of maize, in Belgium, ~1984–1985. Its G + C content is 58.99 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of OE 48.2T are publicly available through the accession numbers MT621498 and CP077090, respectively.
(#19) Description of Pseudomonas tensinigenes sp. nov.
Pseudomonas tensinigenes (ten.si.ni’ge.nes. N.L. neut. n. tensinum, tensin; Gr. v. gennao to produce; N.L. part. adj. tensinigenes, tensin producing).
The type strain is ZA 5.3T (LMG 32032T = CFBP 8882T) and was isolated from the rhizosphere of wheat, in Belgium, ~1984–1985. Its G + C content is 59.17 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of ZA 5.3T are publicly available through the accession numbers MT621501 and CP077089, respectively.
(#20) Description of Pseudomonas monsensis sp. nov.
Pseudomonas monsensis (mons.en’sis. N.L. fem. adj. monsensis, from Mons, a city in Belgium).
The type strain is PGSB 8459T (LMG 32033T = CFBP 8854T) and was isolated from the rhizosphere of maize, Mons, Belgium, ~1984–1985. Its G + C content is 60.05 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of PGSB 8459T are publicly available through the accession numbers MT621495 and CP077087, respectively.
(#21) Description of Pseudomonas zanjanensis sp. nov.
Pseudomonas zanjanensis (zan.jan.en’sis. N.L. fem. adj. zanjanensis, from Zanjan, a city in Iran).
The type strain is SWRI12T (LMG 32042T = CFBP 8855T) and was isolated from the rhizosphere of wheat (cultivar Alvand), Zanjan, Iran in 2004. Its G + C content is 61.21 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI12T are publicly available through the accession numbers MT621484 and JABWRB000000000, respectively.
(#22) Description of Pseudomonas zarinae sp. nov.
Pseudomonas zarinae (za.ri’nae. N.L. gen. n. zarinae, from Zarin, a wheat cultivar).
The type strain is SWRI108T (LMG 32043T = CFBP 8856T) and was isolated from the rhizosphere of wheat (cultivar Zarin), Kermanshah, Iran in 2004. Its G + C content is 60.86 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI108T are publicly available through the accession numbers MT621493 and CP077086, respectively.
(#23) Description of Pseudomonas tehranensis sp. nov.
Pseudomonas tehranensis (teh.ran.en’sis. N.L. fem. adj. tehranensis, from Tehran, a city in Iran).
The type strain is SWRI196T (LMG 32044T = CFBP 8857T) and was isolated from the rhizosphere of wheat, Tehran, Iran in 2004. Its G + C content is 60.46 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI196T are publicly available through the accession numbers MT621473 and JABWQV000000000, respectively.
(#24) Description of Pseudomonas marvdashtae sp. nov.
Pseudomonas marvdashtae (marv.dash’tae. N.L. gen. n. marvdashtae, from Marvdasht, a wheat cultivar).
The type strain is SWRI102T (LMG 32045T = CFBP 8858T) and was isolated from the rhizosphere of wheat (cultivar Marvdasht), Kermanshah, Iran in 2004. Its G + C content is 60.64 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI102T are publicly available through the accession numbers MT621490 and JABWQX000000000, respectively.
(#25) Description of Pseudomonas shahriarae sp. nov.
Pseudomonas shahriarae (shah.ri.a’rae. N.L. gen. n. shahriarae, from Shahriar, a wheat cultivar).
The type strain is SWRI52T (LMG 32046T = CFBP 8859T) and was isolated from the rhizosphere of wheat (cultivar Shahriar), Zanjan, Iran in 2004. Its G + C content is 60.59 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI52T are publicly available through the accession numbers MT621521 and CP077085, respectively.
(#26) Description of Pseudomonas azadiae sp. nov.
Pseudomonas azadiae (a.za’di.ae. N.L. gen. n. azadiae, from Azadi, a wheat cultivar).
The type strain is SWRI103T (LMG 32047T = CFBP 8860T) and was isolated from the rhizosphere of wheat (cultivar Azadi), Shiraz, Iran in 2004. Its G + C content is 60.69 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI103T are publicly available through the accession numbers MT621536 and JAHSTY000000000, respectively.
(#27) Description of Pseudomonas tritici sp. nov.
Pseudomonas tritici (tri’ti.ci. L. gen. n. tritici, of Triticum, wheat).
The type strain is SWRI145T (LMG 32048T = CFBP 8883T) and was isolated from the rhizosphere of wheat, Zanjan, Iran in 2004. Its G + C content is 59.87 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI145T are publicly available through the accession numbers MT621537 and CP077084, respectively.
(#28) Description of Pseudomonas salmasensis sp. nov.
Pseudomonas salmasensis (sal.mas.en’sis. N.L. fem. adj. salmasensis, from Salmas, a city in Iran).
The type strain is SWRI126T (LMG 32049T = CFBP 8861T) and was isolated from the rhizosphere of wheat (cultivar Zarin), Salmas, Iran in 2004. Its G + C content is 60.16 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of SWRI126T are publicly available through the accession numbers MT621526 and CP077083, respectively.
(#29) SWRI22 (LMG 32050 = CFBP 8862): Pseudomonas carnis
(#30) OE 28.3 (LMG 32034 = CFBP 8863): Pseudomonas edaphica
(#31) SWRI76 (LMG 32051 = CFBP 8864): Pseudomonas atacamensis
(#32) Description of Pseudomonas triticicola sp. nov.
Pseudomonas triticicola (tri.ti.ci.co’la. L. fem. n. Triticum, wheat; L. suff. –cola (from L. n. incola), inhabitant dweller; N.L. n. triticicola, wheat dweller).
The type strain is SWRI88T (LMG 32052T = CFBP 8865T) and was isolated from the rhizosphere of wheat (cultivar Marvdasht), Kermanshah, Iran in 2004. Its G + C content is 59.99 mol% (calculated based on its genome sequence). The whole-genome sequence of SWRI88T is publicly available through the accession JAHSTX000000000.
(#33) Description of Pseudomonas siliginis sp. nov.
Pseudomonas siliginis (si.li’gi.nis. L. gen. n. siliginis, of siligo, winter wheat).
The type strain is SWRI31T (LMG 32053T = CFBP 8866T) and was isolated from the rhizosphere of wheat (cultivar Zarin), Kermanshah, Iran in 2004. Its G + C content is 59.97 mol% (calculated based on its genome sequence). The whole-genome sequence of SWRI31T is publicly available through the accession JAHSTW000000000.
(#34) Description of Pseudomonas farris sp. nov.
Pseudomonas farris (far’ris. L. gen. n. farris, of husked wheat, of a grain).
The type strain is SWRI79T (LMG 32054T = CFBP 8867T) and was isolated from the rhizosphere of wheat (cultivar Local), Zanjan, Iran in 2004. Its G + C content is 58.74 mol% (calculated based on its genome sequence). The whole-genome sequence of SWRI79T is publicly available through the accession JAHSTV000000000.
(#35) Description of Pseudomonas azerbaijanoccidens sp. nov.
Pseudomonas azerbaijanoccidens (a.zer.bai.jan.oc’ci.dens. Azerbaijan, geographic name; L. fem. adj. occidens, western; N.L. fem. adj. azerbaijanoccidens, from West Azerbaijan, a province in Iran).
The type strain is SWRI74T (LMG 32055T = CFBP 8868T) and was isolated from the rhizosphere of wheat (cultivar Zarin), Salmas, Iran in 2004. Its G + C content is 59.30 mol% (calculated based on its genome sequence). The whole-genome sequence of SWRI74T is publicly available through the accession JAHSTU000000000.
(#36) Description of Pseudomonas alvandae sp. nov.
Pseudomonas alvandae (al.van’dae. N.L. gen. n. alvandae, from Alvand, a wheat cultivar).
The type strain is SWRI17T (LMG 32056T = CFBP 8869T) and was isolated from the rhizosphere of wheat (cultivar Alvand), Zanjan, Iran in 2004. Its G + C content is 60.86 mol% (calculated based on its genome sequence). The whole-genome sequence of SWRI17T is publicly available through the accession CP077080.
(#37) Description of Pseudomonas asgharzadehiana sp. nov.
Pseudomonas asgharzadehiana (as.ghar.za.deh.i.a’na. N.L. fem. adj. asgharzadehiana, from Ahmad Asgharzadeh, an Iranian microbiologist who, together with Kazem Khavazi, isolated the strain SWRI132, in addition to SWRI10, SWRI12, SWRI17, SWRI31, SWRI52, SWRI54, SWRI56, SWRI65, SWRI74, SWRI79, SWRI88, SWRI100, SWRI102, SWRI103, SWRI107, SWRI108, SWRI123, SWRI124, SWRI126, SWRI145, SWRI153, and SWRI196, which represent 23 new Pseudomonas species).
The type strain is SWRI132T (LMG 32057T = CFBP 8870T) and was isolated from the rhizosphere of wheat (cultivar Marvdasht), Kermanshah, Iran in 2004. Its G + C content is 60.59 mol% (calculated based on its genome sequence). The whole-genome sequence of SWRI132T is publicly available through the accession CP077079.
(#38) Description of Pseudomonas azerbaijanoriens sp. nov.
Pseudomonas azerbaijanoriens (a.zer.bai.jan.o’ri.ens. Azerbaijan, geographic name; L. fem. adj. oriens, eastern; N.L. fem. adj. azerbaijanoriens, from East Azerbaijan, a province in Iran).
The type strain is SWRI123T (LMG 32058T = CFBP 8871T) and was isolated from the rhizosphere of wheat (cultivar Zarin), East Azerbaijan, Iran in 2004. Its G + C content is 60.11 mol% (calculated based on its genome sequence). The whole-genome sequence of SWRI123T is publicly available through the accession CP077078.
(#39) Description of Pseudomonas khavaziana sp. nov.
Pseudomonas khavaziana (kha.va.zi.a’na. N.L. fem. adj. khavaziana, from Kazem Khavazi, an Iranian microbiologist who, together with Ahmad Asgharzadeh, isolated the strain SWRI124, in addition to SWRI10, SWRI12, SWRI17, SWRI31, SWRI52, SWRI54, SWRI56, SWRI65, SWRI74, SWRI79, SWRI88, SWRI100, SWRI102, SWRI103, SWRI107, SWRI108, SWRI123, SWRI126, SWRI132, SWRI145, SWRI153, and SWRI196, which represent 23 new Pseudomonas species).
The type strain is SWRI124T (LMG 32059T = CFBP 8872T) and was isolated from the rhizosphere of wheat (cultivar Zarin), East Azerbaijan, Iran in 2004. Its G + C content is 59.61 mol% (calculated based on its genome sequence). The whole-genome sequence of SWRI124T is publicly available through the accession JAHSTT000000000.
(#40) Description of Pseudomonas botevensis sp. nov.
Pseudomonas botevensis (bo.tev.en’sis. N.L. fem. adj. botevensis, from Boteva, a city in Cameroon).
The type strain is COW3T (LMG 32176T = CFBP 8873T) and was isolated from the roots of white cocoyam (Xanthosoma sagittifolium), Boteva, Cameroon in 2008. Its G + C content is 61.21 mol% (calculated based on its genome sequence). The 16S rRNA gene, rpoB, gyrB, rpoD, and whole-genome sequence of COW3T are publicly available through the accession numbers MT507065, MT506178, MT506955, MT506158, and JAHTKI000000000 respectively.
(#41) Description of Pseudomonas ekonensis sp. nov.
Pseudomonas ekonensis (e.kon.en’sis. N.L. fem. adj. ekonensis, from Ekona, a city in Cameroon).
The type strain is COR58T (LMG 32175T = CFBP 8874T) and was isolated from the roots of red cocoyam (Xanthosoma sagittifolium), Ekona, Cameroon in 2008. Its G + C content is 64.87 mol% (calculated based on its genome sequence). The 16S rRNA gene, rpoB, gyrB, rpoD, and whole-genome sequence of COR58T are publicly available through the accession numbers MT507072, MT506185, MT506962, MT506165, and JAHSTS000000000, respectively.
(#42) Description of Pseudomonas maumuensis sp. nov.
Pseudomonas maumuensis (mau.mu.en’sis. N.L. fem. adj. maumuensis, from Maumu, a city in Cameroon).
The type strain is COW77T (LMG 32179T = CFBP 8888T) and was isolated from the roots of white cocoyam (Xanthosoma sagittifolium), Maumu, Cameroon in 2008. Its G + C content is 64.12 mol% (calculated based on its genome sequence). The rpoB, rpoD, and whole-genome sequence of COW77T are publicly available through the accession numbers MH594167, MK251918, and CP077077, respectively.
(#43) Description of Pseudomonas fakonensis sp. nov.
Pseudomonas fakonensis (fa.kon.en’sis. N.L. fem. adj. fakonensis, from Fako, a county in Cameroon).
The type strain is COW40T (LMG 32178T = CFBP 8875T) and was isolated from the roots of white cocoyam (Xanthosoma sagittifolium), Ekona (Fako county), Cameroon in 2008. Its G + C content is 64.27 mol% (calculated based on its genome sequence). The rpoB, rpoD, and whole-genome sequence of COW40T are publicly available through the accession numbers MH594146, MK251899, and CP077076, respectively.
(#44) Description of Pseudomonas xanthosomae sp. nov.
Pseudomonas xanthosomae (xan.tho.so’mae. L. gen. n. xanthosomae, of Xanthosoma, cocoyam).
The type strain is COR54T (LMG 32174T = CFBP 8876T) and was isolated from the roots of red cocoyam (Xanthosoma sagittifolium), Ekona, Cameroon in 2008. Its G + C content is 64.19 mol% (calculated based on its genome sequence). The rpoB, rpoD, and whole-genome sequence of COR54T are publicly available through the accession numbers MH594196, MK251872, and CP077075, respectively.
(#45) CMR5c (LMG 32172 = CFBP 8889): Pseudomonas aestus
(#46) Description of Pseudomonas sessilinigenes sp. nov.
Pseudomonas sessilinigenes (ses.si.li.ni’ge.nes. N.L. neut. n. sessilinum, sessilin; Gr. v. gennao to produce; N.L. part. adj. sessilinigenes, sessilin producing).
The type strain is CMR12aT (LMG 32173T = CFBP 8877T) and was isolated from the roots of red cocoyam (Xanthosoma sagittifolium), Bokwai, Cameroon in 2001. Its G + C content is 62.80 mol% (calculated based on its genome sequence). The 16S rRNA gene, rpoB, gyrB, and whole-genome sequence of CMR12aT are publicly available through the accession numbers FJ652622, FJ652703, FJ652730, and CP077074, respectively.
(#47) Description of Pseudomonas muyukensis sp. nov.
Pseudomonas muyukensis (mu.yuk.en’sis. N.L. fem. adj. muyukensis, from Muyuka, a district in Cameroon).
The type strain is COW39T (LMG 32177T = CFBP 8890T) and was isolated from the roots of white cocoyam (Xanthosoma sagittifolium), Ekona, Cameroon in 2008. Its G + C content is 65.11 mol% (calculated based on its genome sequence). The rpoD and whole-genome sequence of COW39T are publicly available through the accession numbers MK329212 and CP077073, respectively.

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Microorganisms 09 01766 g001a 550Microorganisms 09 01766 g001b 550
Figure 1. Phylogenetic tree based on 1508 core orthogroups using IQ-TREE with automatic model selection and 1000 ultrafast bootstraps (left) and the rpoD gene (right); maximum likelihood tree, GTR + G+I model (MEGA-X)) including, respectively, 313 and 316, type strains of Pseudomonas. Bootstrap values were calculated based on 1000 replications. Type strains of newly described species are highlighted in bold. Cellvibrio japonicus is used as the outgroup.
Figure 1. Phylogenetic tree based on 1508 core orthogroups using IQ-TREE with automatic model selection and 1000 ultrafast bootstraps (left) and the rpoD gene (right); maximum likelihood tree, GTR + G+I model (MEGA-X)) including, respectively, 313 and 316, type strains of Pseudomonas. Bootstrap values were calculated based on 1000 replications. Type strains of newly described species are highlighted in bold. Cellvibrio japonicus is used as the outgroup.
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Figure 2. Phylogenetic tree of the P. putida group based on the rpoD gene of 122 Pseudomonas strains (Table S6). All the strains included in this analysis, together with their accession numbers and the output of the prospection for CLP BGCs, are detailed in Table S6. The maximum likelihood phylogenetic tree was constructed using the GTR + G+I model (MEGA-X). Bootstrap values were calculated based on 1000 replications and only bootstrap values higher than 50% are indicated. Type strains of newly described species are highlighted in bold. The P. rhizosphaerae group is used as the outgroup. The corresponding tree based on whole genome sequences is shown in Figure S5.
Figure 2. Phylogenetic tree of the P. putida group based on the rpoD gene of 122 Pseudomonas strains (Table S6). All the strains included in this analysis, together with their accession numbers and the output of the prospection for CLP BGCs, are detailed in Table S6. The maximum likelihood phylogenetic tree was constructed using the GTR + G+I model (MEGA-X). Bootstrap values were calculated based on 1000 replications and only bootstrap values higher than 50% are indicated. Type strains of newly described species are highlighted in bold. The P. rhizosphaerae group is used as the outgroup. The corresponding tree based on whole genome sequences is shown in Figure S5.
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Table
Table 1. Newly proposed and emended groups of Pseudomonas. Newly described Pseudomonas species are highlighted in bold. ANIb and rpoD identity ranges were extracted from Tables S7 and S8).
Table 1. Newly proposed and emended groups of Pseudomonas. Newly described Pseudomonas species are highlighted in bold. ANIb and rpoD identity ranges were extracted from Tables S7 and S8).
Groups/SubgroupsSpecies/SubspeciesTotal No. of Species/ SubspeciesrpoD Identity (%) 1ANIb (%) 1
Existing Groups
P. aeruginosa GP. aeruginosa, P. citronellolis, P. delhiensis, P. humi, P. jinjuensis, P. knackmussii, P. nicosulfuronedens, P. nitritireducens, P. nitroreducens, P. panipatensis1077.85–95.9880.57–94.48
P. anguilliseptica GP. anguilliseptica, P. benzenivorans, P. borbori, P. cuatrocienegasensis, P. guineae, P. leptonychotis, P. marincola, P. peli, P. segetis, P. taeanensis1072.33–91.5876.68–89.45
P. fluorescens G 134––
P. asplenii SGP. agarici, P. asplenii, P. batumici, P. fuscovaginae, P. vanderleydeniana584.41–89.6684.10–88.35
P. chlororaphis SGP. chlororaphis subsp. aurantiaca, P. chlororaphis subsp. aureofaciens, P. chlororaphis subsp. chlororaphis, P. chlororaphis subsp. piscium497.83–98.4594.73–96.95
P. corrugata SGP. alvandae, P. beijieensis, P. brassicacearum, P. corrugata, P. kilonensis, P. marvdashtae, P. mediterranea, P. tehranensis, P. thivervalensis, P. viciae, P. zanjanensis, P. zarinae1289.75–97.3685.55–95.75
P. fluorescens SGP. allii, P. antartica, P. asgharzadehiana, P. aylmerense, P. azadiae, P. azotoformans, P. canadensis, P. carnis, P. cedrina subsp. cedrina, P. cedrina subsp. fulgida, P. costantinii, P. cremoris, P. cyclaminis, P. edaphica, P. extremaustralis, P. extremorientalis, P. fildesensis, P. fluorescens, P. grimontii, P. haemolytica, P. kairouanesis, P. karstica, P. khavaziana, P. kitaguniensis, P. lactis, P. libanensis, P. lurida, P. marginalis, P. nabeulensis, P. orientalis, P. palleroniana, P. panacis, P. paracarnis, P. paralactis, P. pisciculturae, P. poae, P. rhodesiae, P. salmasensis, P. salomonii, P. simiae, P. sivasensis, P. spelaei, P. synxantha, P. tolaasii, P. tritici, P. trivialis, P. veronii, P. yamanorum4885.16–98.9183.52–95.68
P. fragi SGP. bubulae, P. deceptionensis, P. endophytica, P. fragi, P. helleri, P. lundensis, P. psychrophila, P. saxonica, P. taetrolens, P. versuta, P. weihenstephanensis1183.00–97.6780.50–90.49
P. gessardii SGP. brennerii, P. gessardii, P. mucidolens, P. proteolytica, P. shahriarae590.57–97.5385.57–92.54
P. jessenii SGP. asgharzadehiana, P. azerbaijanoccidens, P. izuensis, P. jessenii, P. laurylsulfatiphila, P. laurylsulfatovorans, P. mohnii, P. moorei, P. reinekei, P. umsongensis, P. vancouverensis1190.37–10084.91–95.51
P. koreensis SGP. atacamensis, P. atagosis, P. baetica, P. bananamidigenes, P. botevensis, P. crudilactis, P. ekonensis, P. glycinae, P. granadensis, P. hamedanensis, P. helmanticensis, P. iranensis, P. khorasanensis, P. koreensis, P. kribbensis, P. monsensis, P. moraviensis, P. neuropathica, P. siliginis, P. tensinigenes, P. triticicola, P. zeae2285.40–99.5382.48–96.09
P. mandelii SGP. arsenicoxydans, P. farris, P. frederiksbergensis, P. gregormendelii, P. lini, P. mandelii, P. migulae, P. mucoides, P. piscium, P. prosekii, P. silesiensis1191.04–96.8984.68–94.29
P. protegens SGP. aestus, P. protegens, P. saponiphila, P. sessilinigenes489.52–95.5786.41–91.86
P. kielensis SGP. kielensis1––
P. linyingensis GP. guangdongensis, P. linyingensis, P. oryzae, P. sagittaria479.94–93.8585.19–92.01
P. lutea GP. abietaniphila, P. bohemica, P. graminis, P. lutea483.31–88.3081.89–85.81
P. oleovorans GP. alcaliphila, P. chaetoceroseae, P. chengduensis, P. composti, P. guguanensis, P. hydrolytica, P. indoloxydans, P. khazarica, P. mendocina, P. oleovorans, P. pseudoalcaligenes, P. sediminis, P. sihuisensis, P. toyotomiensis1488.51–98.7686.06–95.79
P. oryzihabitans GP. asuensis, P. duriflava, P. luteola, P. oryzihabitans, P. psychrotolerans, P. rhizoryzae, P. zeshuii766.46–94.1873.64–88.62
P. pertucinogena GP. abyssi, P. aestusnigri, P. bauzanensis, P. formosensis, P. gallaeciencis, P. jilinensis, P. litoralis, P. oceani, P. pachastrellae, P. pelagia, P. pertucinogena, P. phragmitis, P. populi, P. profundi, P. sabulinigri, P. salegens, P. salina, P. saliphila, P. saudimassiliensis, P. xiamenensis, P. xinjiangensis, P. yangmingensis2264.53–92.9874.65–89.65
P. putida G 51
P. akappagea SGP. akappagea1––
P. japonica SGP. brassicae, P. defluvii, P. huaxiensis, P. japonica, P. laurentiana, P. qingdaonensis682.69–95.0580.96–91.58
P. vranovensis SGP. alkylphenolica, P. donghuensis, P. hutmensis, P. tructae, P. vranovensis, P. wadenswillerensis684.23–94.1084.52–93.03
P. cremoricolorata SGP. cremoricolorata1––
P. reidholzensis SGP. reidholzensis, P. shirazensis, P. urmiensis385.78–92.8984.27–86.77
P. guariconensis SGP. guariconensis1––
P. wayambapalatensis SGP. wayambapalatensis1––
P. farsensis SGP. farsensis1––
P. xanthosomae SGP. fakonensis, P. xanthosomae297.8495.06
P. mosselii SGP. entomophila, P. maumuensis, P. mosselii, P. muyukensis, P. peradeniyensis, P. sichuanensis, P. soli, P. xantholysinigenes887.48–95.3587.35–94.87
P. taiwanensis SGP. taiwanensis1––
P. plecoglossicida SGP. plecoglossicida1––
P. vlassakiae SGP. hunanensis, P. promysalinigenes, P. vlassakiae389.34–93.0486.58
P. capeferrum SGP. capeferrum, P. kermanshahensis293.0190.26
P. putida SGP. alloputida, P. anuradhapurensis, P. asiatica, P. fulva, P. inefficax, P. juntendi, P. kurunegalensis, P. monteilii, P. oryzicola, P. parafulva, P. putida, P. pyomelaminifaciens, P. persica, P. shirazica1485.32–97.9982.66–95.79
P. resinovorans GP. furukawaii, P. lalkuanensis, P. mangiferae, P. otitidis, P. resinovorans581.47–90.4679.25–87.84
P. straminea GP. argentinensis, P. daroniae, P. dryadis, P. flavescens, P. punonensis, P. seleniipraecipitans, P. straminea786.02–93.6382.83–88.54
P. stutzeri GP. azotofigens, P. balearica, P. chloritidismutans, P. kirkiae, P. kunmingensis, P. nitritititolerans, P. nosocomialis, P. perfectomarina, P. saudiphocaensis, P. songnenensis, P. stutzeri, P. urumqiensis, P. xanthomarina, P. zhaodongensis1473.60–89.6976.39–88.15
P. syringae GP. amygdali, P. asturieensis, P. avellanae, P. cannabina, P. caricapapayae, P. caspiana, P. cerasi, P. cichorii, P. congelans, P. coronafaciens, P. ficuserectae, P. floridensis, P. meliae, P. ovata, P. savastanoi, P. syringae, P. tremae, P. viridiflava1878.36–99.5478.20–94.57
Newly described groups
P. pohangensis GP. mangrovi, P. pohangensis267.0277.07
P. massiliensis GP. massiliensis, P. typographi280.5376.86
P. rhizosphaerae GP. baltica, P. coleopterorum, P. rhizosphaerae391.89–94.7088.28–90.55
Orphan groups
P. indica GP. indica1––
P. kuykendallii GP. kuykendallii1––
P. thermotolerans GP. thermotolerans1––
P. flexibilis GP. flexibilis, P. tuomuerensis2––
P. fluvialis GP. fluvialis, P. pharmacofabricae2––
P. alcaligenes GP. alcaligenes1––
P. matsuisoli GP. matsuisoli1––
P. turukhanskensis GP. turukhanskensis1––
1 Excluding synonymous species (Table 2).
Table
Table 2. Synonymous species of Pseudomonas (Table S7). Species are considered synonymous when ANIb values are greater than or equal to 96.5% [18].
Table 2. Synonymous species of Pseudomonas (Table S7). Species are considered synonymous when ANIb values are greater than or equal to 96.5% [18].
Groups/SubgroupsPseudomonas SpeciesANIbEarlier Synonyms
P. aeruginosa groupP. citronellolisP. humi96.70P. citronellolis
P. nitroreducensP. nitritireducens98.85P. nitroreducens
P. oleovorans groupP. oleovoransP. pseudoalcaligenes97.17P. oleovorans
P. chengduensisP. sihuiensis96.25P. chengduensis
P. oryzihabitans groupP. oryzihabitansP. psychrotolerans98.22P. oryzihabitans
P. luteolaP. zeshuii97.87P. luteola
P. pertucinogena groupP. phragmitisP. jilinensis98.70P. phragmitis
P. gallaeciensisP. abyssi97.56P. gallaeciensis
P. putida groupP. asiaticaP. pyomelaninifaciens99.03P. asiatica
P. shirazica99.17
P. stutzeri groupP. chloritidismutansP. kunmingensis96.49P. chloritidismutans
P. syringae groupP. tremaeP. coronafaciens98.74P. tremae
P. amygdaliP. ficuserectae97.42P. amygdali
P. meliae98.27
P. savastanoi98.75
P. fluorescens groupP. aspleniiP. fuscovaginae98.23P. asplenii
P. veroniiP. panacis99.95P. veronii
Orphan groupsP. flexibilisP. tuomuerensis98.69P. flexibilis
P. fluvialisP. pharmacofabricae98.61P. fluvialis
Table
Table 3. Phylogenetic affiliation based on ANIb values for the 44 whole genome sequenced strains belonging to the P. putida group, previously not assigned, or incorrectly assigned at the species level. Accession numbers are shown in Table S6.
Table 3. Phylogenetic affiliation based on ANIb values for the 44 whole genome sequenced strains belonging to the P. putida group, previously not assigned, or incorrectly assigned at the species level. Accession numbers are shown in Table S6.
SubgroupsStrainClosest Type StrainANIb %Re-identified Species
P. japonicaP. putida CSV86P. japonica86.94Pseudomonas sp. #1
P. reidholzensisP. putida 02C-26P. shirazensis97.25P. shirazensis
P. guariconensisP. putida IEC33019P. guariconensis91.47Pseudomonas sp. #2
P. putida WP4-W18-CRE-03P. guariconensis99.38P. guariconensis
P. putida WP8-W18-CRE-01 99.47
P. wayambapalatensisP. putida NX-1P. wayambapalatensis94.69Pseudomonas sp. #3
P. putida PC2 94.78
Pseudomonas sp. RW3S2P. wayambapalatensis99.21P. wayambapalatensis
Pseudomonas sp. RW10S2 99.26
P. farsensisPseudomonas sp. SWRI51P. farsensis98.65P. farsensis
P. mosseliiPseudomonas sp. 250JP. peradeniyensis96.15*Pseudomonas sp. #4
Pseudomonas sp. BW16M2P. peradeniyensis96.59P. peradeniyensis
P. plecoglossicidaP. putida GM84P. plecoglossicida91.13Pseudomonas sp. #5
P. putida GIMC5401-PPKH-115P. plecoglossicida87.01Pseudomonas sp. #6
P. putida BR-PH17P. plecoglossicida86.75Pseudomonas sp. #7
P. putida W619 85.75
P. vlassakiaeP. putida AA7P. vlassakiae90.88Pseudomonas sp. #8
P. putida W5P. vlassakiae91.64Pseudomonas sp. #9
P. capeferrumPseudomonas sp. SWRI68P. capeferrum98.66P. capeferrum
Pseudomonas sp. SWRI59 98.65
P. putida E41P. kermanshahensis97.48P. kermanshahensis
P. putida E46 97.55
Pseudomonas sp. SWRI50 99.39
Pseudomonas sp. SWRI67 99.99
P. putidaP. putida SY153P. jutendi98.15P. jutendi
P. putida TIJ-51 97.77
P. putida GB-1P. alloputida90.49Pseudomonas sp. #10
P. putida PP112420 90.54
P. putida S13-1-2P. putida94.55Pseudomonas sp. #11
P. putida KF715 93.73Pseudomonas sp. #12
P. putida ZXPA-20 93.40
P. putida H8234 93.33
P. putida B1 93.40
P. putida R51P. alloputida95.00*Pseudomonas sp. #13
P. putida BS3701P. alloputida96.67P. alloputida
P. putida MX-2 96.49
P. putida LS46 96.44
P. putida 15420352 96.42
P. putida YC-AE1 96.40
P. putida T25-27 96.51
P. monteilii 170620603REP. kurunegalensis99.45P. kurunegalensis
P. monteilii 170918607 99.44
P. monteilii STW0522-72 99.64
P. monteilii FDAARGOS171 99.77
* dDDH < 70%.
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Girard, L.; Lood, C.; Höfte, M.; Vandamme, P.; Rokni-Zadeh, H.; van Noort, V.; Lavigne, R.; De Mot, R. The Ever-Expanding Pseudomonas Genus: Description of 43 New Species and Partition of the Pseudomonas putida Group. Microorganisms 2021, 9, 1766. https://doi.org/10.3390/microorganisms9081766

AMA Style

Girard L, Lood C, Höfte M, Vandamme P, Rokni-Zadeh H, van Noort V, Lavigne R, De Mot R. The Ever-Expanding Pseudomonas Genus: Description of 43 New Species and Partition of the Pseudomonas putida Group. Microorganisms. 2021; 9(8):1766. https://doi.org/10.3390/microorganisms9081766

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Girard, Léa, Cédric Lood, Monica Höfte, Peter Vandamme, Hassan Rokni-Zadeh, Vera van Noort, Rob Lavigne, and René De Mot. 2021. "The Ever-Expanding Pseudomonas Genus: Description of 43 New Species and Partition of the Pseudomonas putida Group" Microorganisms 9, no. 8: 1766. https://doi.org/10.3390/microorganisms9081766

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