Unearthing the genomes of plant-beneficial Pseudomonas model strains WCS358, WCS374 and WCS417

General genome characteristics

Sequencing of the genomes of WCS358 from the P. putida group, and WCS374 and WCS417 from the P. fluorescens group was carried out at the Beijing Genome Institute (Beijing, China). A summary
of the general sequence characteristics is given in Table 2. The sizes of the genomes varied from approximately 5.94 Mb for WCS358 to 6.09 Mb
for WCS374 and 6.17 Mb for WCS417 and they were predicted to contain 5188, 5351 and
5506 coding sequences, respectively. Their G?+?C contents ranged from 60 to 63.5 %.
These characteristics are comparable to those described for taxonomically related
and previously sequenced Pseudomonas spp. strains 51], 52].

Table 2. General sequencing and genome characteristics of the WCS Pseudomonas spp. strains

Phylogenetic analysis of WCS358, WCS374, and WCS417

The genus Pseudomonas is very diverse and is still undergoing taxonomic refinement 53]. Based on their nutritional and physiological characteristics, strains WCS374 and
WCS417 were tentatively ascribed to the species Pseudomonas fluorescens, whereas WCS358 was described to belong to Pseudomonas putida54], 55]. To obtain a comprehensive overview of the taxonomic position of the WCS strains,
the sequences of the core housekeeping genes 16S rRNA, gyrB, rpoB and rpoD were compared with the corresponding genes of 107 Pseudomonas species type strains 53] and a selection of additional Pseudomonas spp. strains of which the full genome was available 51], 52], 56]–61]. A phylogenetic tree was generated based on multi-locus sequence analysis (MLSA)
of the concatenated sequences of four core housekeeping genes (Fig. 1). This tree shows that both WCS374 and WCS417 are associated with the P. fluorescens subgroup as defined by Mulet and co-workers 53]. However, they are not most closely related to the P. fluorescens type strain. Similarly, WCS358 is related to the members of the P. putida group, but not most closely to the P. putida type strain. WCS417 has a 100 % nucleotide identity match with the concatenated sequences (NI)
of the type strain of Pseudomonas simiae. WCS374 is most closely related to the type strain of Pseudomonas synxantha (96 % NI), whereas WCS358 shows highest homology with Pseudomonas monteilii (94 % NI). As the species boundary for MLSA was proposed to be 97 % 53], WCS417, which was previously ascribed to the P. fluorescens species, should be regarded as a P. simiae strain, whereas both WCS374 and WCS358 should be considered representatives of yet undescribed
species. WCS417 appears to be closely related to the plant-beneficial P. simiae strain R81 (100 % NI), which was also isolated from wheat roots 60]. Furthermore, WCS374 appears to be closely related to two other plant-beneficial
Pseudomonas spp. strains within its species boundary, i.e. strains A506 (99 % NI; 52]) and SS101 (95 % NI; 52]).

Fig. 1. Phylogenetic tree showing the relationship of WCS358, WCS374, and WCS417 with other
Pseudomonas spp. strains. Phylogenetic tree of the WCS strains (blue; also indicated by arrows)
relative to 107 Pseudomonas sp. type strains (red), 24 selected Pseudomonas sp. strains of which the genomes were already sequenced (green), and Pseudomonas sp. strain CFBP2461 (purple). The tree is based on the alignment of concatenated
sequences of four core housekeeping genes (16S rRNA, gyrB, rpoB, rpoD) of the strains. Bootstrap values from 1000 replicates are indicated at the nodes.
Organization of Pseudomonas groups and subgroups is according to Mulet et al.53]

To further establish the phylogenetic relationship of the WCS strains with other Pseudomonas spp. strains, we compared the whole genomes of our strains to available completely
sequenced strains of the P. fluorescens subgroup and the P. putida group by calculating the Average Nucleotide Identity based on BLAST (ANIb) using
JSpecies 62]. ANIb values of 94 % 63] and 95 % 62] have been proposed for defining the boundaries between species. The phylogeny based
on ANIb values matched with that of MLSA, confirming the phylogenetic separation between
strains of the P. putida group and the P. fluorescens subgroup (Fig. 2). None of the Pseudomonas strains for which the genome was available shared more than 94 % of its total genome
sequence with WCS358, confirming that WCS358 belongs to a thus far undescribed Pseudomonas species within the P. putida group. WCS417 shared 99.8 % nucleotide identity with the draft sequence of Pseudomonas strain R81, indicating that these strains belong to the same species. Furthermore,
Pseudomonas spp. strains A506 and SS101 shared 99.1 and 94.8 % of their respective genomes with
WCS374 indicating that they belong to the same so far undescribed Pseudomonas species within the P. fluorescens subgroup (See also Additional file 1: Figure S1 and Additional file 2: Table S1). Together, our genomic analyses indicate that strain WCS417 belongs to
the species P. simiae of the P. fluorescens subgroup, whereas strains WCS358 of the P. putida group and WCS374 of the P. fluorescens subgroup belong to sofar undescribed species. Here we designate these species as
Pseudomonas capeferrum (type strain WCS358) and Pseudomonas defensor (type strain WCS374; see also Discussion section).

Fig. 2. Average Nucleotide Identity based on BLAST for a selection of bacterial genomes. Black
lines and bold numbers indicate putative species boundaries. Cell colors indicate
similarity scaled from low (red) to high (green). ANIb values were calculated using
Jspecies 62]

The whole-genome comparison unearthed that both WCS374 and WCS417 have a close relationship
with other Pseudomonas spp. strains that were isolated based on their plant-beneficial properties. We aligned
the genomes of WCS374 and WCS417 to their closest relative, i.e. Pseudomonas spp. strain A506 (isolated from pear phyllosphere, California, USA; 64]) and R81 (isolated from wheat rhizosphere in Bhawanipur, India; 60]), respectively, and found that large parts of the genomes were nearly identical,
whereas only small, randomly dispersed genomic regions were missing in the corresponding
relatives (Fig. 3). To further analyze the relationship between the pairs of strains, we used Islandviewer
65] to identify genomic islands in the compared genomes. Genomic islands are genomic
regions that are derived from horizontal transfers 66]. Figure 3 shows a large overlap between genomic islands and the regions missing in the cognate
closest relative, indicating that most of the differences between these closely related
strains result from horizontal gene transfer. Although we did not find a close relative
of WCS358 among the available sequenced Pseudomonas spp. strains, the plant-beneficial Pseudomonas sp. strain CFBP2461 (isolated from bean rhizosphere in Angers, France; 67]) was found to produce exactly the same pyoverdine siderophore as WCS358 68]. Pyoverdines are iron-chelating molecules and their characteristics often closely
reflect the phylogeny of the bacteria that produce them. Hence, in the past this feature
was often used to characterize bacterial identity, a method known as siderotyping
69]. We amplified the housekeeping genes 16S rRNA, gyrB, rpoB and rpoD of CFBP2461 (Genbank accession numbers KM221193- KM221196) and included the concatenated
sequence of this strain in an MLSA as described above (Fig. 1). Pseudomonas sp. CFBP2461 shared 99.5 % NI with WCS358, indicating that these strains are very
similar and should be considered representatives of the same Pseudomonas species. Together, these results indicate that for each of the WCS strains other
representatives of the same species have been found. The fact that these WCS “look-a-likes”
have all been selected in independent searches for plant-beneficial microbes at very
different geographic regions suggests that the WCS genome sequences can serve as representative
genomes for plant-beneficial Pseudomonas spp.

Fig. 3. Whole genome alignment of WCS374 and WCS417 with related Pseudomonas sp. strains. Whole genome alignments of WCS374 with its close relative Pseudomonas sp. strain A506 (a) and WCS417 with its close relative P. simiae R81 (b) were generated with Progressive MAUVE. Colored blocks indicate similar genome regions
between the two strains. White gaps indicate genomic regions that are not shared between
the compared strains. Genomic islands predicted by Islandviewer are indicated in purple
above the black line for each genome. In R81, a large genomic region is represented
indented from the rest of the genome as this region was reversely oriented in comparison
to WCS417

Siderophores

The WCS strains produce siderophores that can inhibit plant pathogens directly via
competition for iron, or indirectly via the onset of ISR. Application of purified
pyoverdines of WCS358, WCS374 or WCS417 to the plant roots can elicit ISR. However,
the pyoverdines of the WCS strains differentially trigger ISR in different plant species
25], 47] suggesting that their structure differs and that these pyoverdines are part of a
specific host-microbe recognition system. Whilst the structures of the pyoverdines
PVD358 and PVD374 of WCS358 and WCS374, respectively, have been elucidated, the structure
of PVD417 of WCS417 is as yet unknown 68], 70]. Pyoverdines consist of a chromophore and a short peptide chain. Both are synthesized
by the sequential action of multiple non-ribosomal peptide synthetases (NRPSs). NRPS
are multi-modular enzymes in which each module is responsible for addition, attachment
or modification of a specific amino acid onto a growing peptide chain 71]. AntiSMASH identified gene clusters that contained NRPSs in all three WCS strains.
In each strain, two NRPS-containing gene clusters could be related to the production
of pyoverdine. One of these gene clusters is likely to be involved in the synthesis
of the chromophore as the only NRPS gene found in this cluster (data not shown) is
an ortholog of NRPS pvdL of P. protegens Pf-5 72]. The other pyoverdine biosynthesis gene cluster contains NRPS genes that are associated
with synthesis of the respective pyoverdine peptide chains (Fig. 4a; indicated in green), pyoverdine transport (Fig. 4a; indicated in blue), or regulation (Fig. 4a; indicated in red). Bioinformatic analysis of the predicted NPRS-mediated peptide
chains confirmed the previously elucidated composition of the pyoverdine peptide chains
of PVD374 and PVD358 (Fig. 4b). In addition, the bioinformatics analysis predicts that the peptide chain of the
WCS417 pyoverdine PVD417 contains the same amino acids as PVD374 in the same order
(Ser-Lys-Gly-Orn-Lys-Orn-Ser; Fig. 4b).

Fig. 4. Siderophore biosynthesis genes. (a) Siderophore biosynthetic gene clusters in the genomes of WCS358, WCS374, and WCS417
as identified by AntiSMASH. Colors represent different functional gene categories:
biosynthetic genes (green); transport-related genes (blue); regulatory genes (red);
and other genes (grey). (b) Bioinformatic analysis of the NRPSs that synthesize the peptide chain of pyoverdine
in the WCS strains. For each NRPS, the domains recognized in the NRPS to function
in condensation (C), adenylation (A), thiolation (T), epimerization (E), and epimerization
and thioesterase (TE) are shown, as well as the amino acids predicted to be recognized
by the adenylation domains

Recently, it was demonstrated that WCS374 produces exactly the same PVD as Pseudomonas strain A50632], 70]. The peptide chain of the WCS374/A506 PVD was demonstrated to be very similar to
that of Pseudomonas sp. SBW25 as it contains the same amino acid residues in the same order. It was proposed
that the SBW25 PVD differs slightly as its PvdJ contains an epimerization module that
is lacking in the orthologous PvdD of the NRPS of WCS374 and A506 and that changed
the stereo-isomeric configuration of the 6th amino acid in the chain from L-Orn to
D-Orn 32]. Our bioinformatics analysis revealed that the PvdJ ortholog of WCS417 contains a
similar epimerization domain that is lacking in WCS374 (Fig. 4b). This suggests that WCS417 produces the same PVD as SBW25, which is a close relative
of WCS417 in our phylogenetic analysis (Figs. 1 and 2).

Pyoverdine knockout mutants WCS358-PVD
^?
and WCS417-PVD
^?
are completely abolished in siderophore activity in the universal siderophore assay
on CAS agar medium (Fig. 5) 73]–75], indicating that both strains produce only one type of siderophore. However, pyoverdine
knockout mutant WCS374-PVD
^?
still produced a halo on CAS medium (Fig. 5) 75], indicating that this strain produces one or more additional siderophores. Mercado-Blanco
et al.76], identified a second siderophore in WCS374, called pseudomonine (PSM). Pseudomonine
is composed of salicylic acid (SA), cyclothreonine and histamine. Although microbially
produced SA has iron-chelating properties and is implicated to function as a siderophore
itself 77], the latter was questioned by other studies 78], 79]. Mercado-Blanco and co-workers 76] identified the pmsCEAB operon in WCS374 as responsible for production of SA and pseudomonine. Matthijs and
co-workers 80] identified two NRPS genes upstream of pmsCEAB, i.e. basB and basAD, which are thought to function in the adenylation and cyclization of the amino acid
threonine and subsequent assembly of pseudomonine 81]. All the genes required for pseudomonine biosynthesis were also detected in the genome
sequence of WCS374 (Fig. 4a). Interestingly, mutants of WCS374 that do not produce PVD374, PSM or SA, still produce
a halo in the CAS assay and are able to grow on KBA with 800 ?M of the iron chelator
bipyridyl, whereas the pyoverdine-deficient mutants WCS417-PVD
^?
and WCS358-PVD
^?
can only tolerate 400 ?M of 2,2-bipyridyl (Fig. 5). This indicates that WCS374 produces yet another siderophore. Searching the WCS374
genome for additional siderophore biosynthesis genes using antiSMASH revealed a gene
cluster (PD374_19610 – PD374_19665) involved in the production and transport of an
aerobactin-like siderophore (Fig. 4a). This gene cluster is also present in the genomes of the WCS374 relatives SS101
and A506, although the ortholog of PD374_19640, which is related to IucC/IucA and is putatively required for the production of the siderophore, contains a frameshift
mutation in A506.

Fig. 5. Overview of siderophore production by the WCS strains and their mutant derivatives.
Maximum amount of 2,2-bipyridyl in KBA that still allowed growth of strains after
48 h is indicated. Photographs display production of an orange halo by the bacterial
strains on CAS agar, which is indicative for siderophore production. Abbreviations
stand for Wild type (WT), pyoverdine (PVD), pseudomonine (PSM), pyochelin (PCH) or
salicylic acid (SA). Production of pyochelin by WCS417 has not been demonstrated

Leeman et al.25] investigated the production of SA by the three WCS strains and found that besides
WCS374 also WCS417 is capable of producing SA. To identify the corresponding biosynthesis
gene cluster in WCS417, the WCS374 genes pmsC and pmsB were used as bait in a BLASTp search of the WCS417 genome. This revealed two WCS417
orthologs (PS417_13200 and PS417_13205) in a genomic region that was identified by
antiSMASH as being putatively responsible for the production of a pyochelin-like siderophore
(Fig. 4a). Like pseudomonine, pyochelin is an iron-chelating siderophore found in Pseudomonas spp. that comprises a SA moiety 82]. However, pyoverdine knockout mutant WCS417-PVD
^?
does not show any siderophore activity on CAS agar at 28 °C, nor can it tolerate higher
levels of bipyridyl than the pyoverdine-deficient mutant WCS358-PVD
^?
. Thus, it is unlikely that a pyochelin-like siderophore is indeed produced by WCS417
under the growth conditions tested.

Microbially produced siderophores are re-absorbed after they have complexed ferric
iron from the environment. This is mediated by TonB-dependent proteins (TBDPs) that
specifically recognize and transport siderophore-iron complexes into the periplasm
30]. We identified 40 TBDP-encoding genes in WCS358, 31 in WCS374 and 33 in WCS417. A
minority of these 104 TBDPs also contain a short N-terminal domain, which is typical
for TBDP-mediated transduction of environmental signals to the cytoplasm 83]. Most bacteria contain less than 14 TBDPs in their proteome, although some bacteria
have larger numbers 32]. Recently, Hartney and co-workers 32] demonstrated that P. protegens Pf-5 contains 45 TBDPs. Six of these TBDPs (FpvU to FpvZ) function as ferric-pyoverdine
receptors (FPVs) that facilitate the uptake of specific pyoverdine-iron complexes
and enable this bacterium to use not only its native pyoverdine, but also heterologous
pyoverdines from other strains. Previously it was reported that TBDPs cluster according
to their substrate rather than to phylogeny 72]. Because, the ferric-pyoverdine receptors form a clear clade within the TBDPs 32], 84], we used the corresponding Fpv genes of strain Pf-5 to identify pyoverdine receptor genes in the three WCS strains.
The amino acid sequences of the identified putative pyoverdine receptors were aligned
together with the amino acid sequences of the six previously described pyoverdine
receptors of Pf-5, after which a phylogenetic tree was build (Additional file 3: Figure S2). The six Pf-5 FPV pyoverdine receptors clustered together, confirming
previous findings 72] and this cluster included 10 TBDPs of WCS358, five of WCS417 and four of WCS374.

Using combinations of deletion mutants of the six Fpv genes of Pf-5 it is possible to identify which FPV pyoverdine receptors are required
for the uptake of specific ferric-pyoverdines 32]. Previously, it was demonstrated that Pf-5 can utilize ferric-pyoverdine of WCS374
and A506 through the receptors FpvU and FpvY, while ferric-pyoverdine of SBW25 can
only be utilized through receptor FpvU. This discrepancy was attributed to the different
stereo-isomeric configuration of the ornithine residue on position 6 of the pyoverdine
peptide chain (Fig. 4b). Because from the genome sequence of WCS417 we predicted that PVD417 would be similar
to pyoverdine of SBW25, we set out to functionally validate this using the set of
Pf-5 fpv mutants 32]. To this end, the six fpv deletion mutants of Pf-5 were tested under low-iron conditions for their capacity
to be cross-fed by WCS358, WCS374, or WCS417. In line with the descriptions of the
specific nature of PVD358 33], 68], none of the fpv mutants of Pf-5 could use the pyoverdine produced by WCS358 (Additional file 4: Figure S3). However, all mutants were able to grow in the presence of WCS374, confirming
previous findings by Hartney and co-workers 32]. In the presence of WCS417, all fpv mutants of Pf-5 were able to grow, except mutant fpvU, indicating that pyoverdin receptor FpvU is required for the uptake of ferric-PVD417,
which resembles the findings for SBW25. These results confirm the prediction from
our bioinformatical analysis that WCS417 produces the same pyoverdin as SBW25 and
highlights that slight differences in pyoverdine structure can have implications for
specificity in heterologous uptake of iron-pyoverdin complexes.

Lipopolysaccharides

Lipopolysaccharides (LPS) are molecules in the outermembrane of Gram negative bacteria,
and are recognized as microbe-associated molecular patterns (MAMPs) that are able
to elicit immune responses in plants and animals 85], 86]. Application of purified LPS of WCS417 to the roots of carnation plants was shown
to trigger ISR, resulting in reduced Fusarium wilt disease when Fusarium was inoculated
in the stem 87]. In radish, both WCS374 and WCS417 can trigger ISR, whereas WCS358 cannot. This differential
effectiveness of ISR inducibility could be attributed to strain-specific differences
in LPS, because in radish purified LPS of WCS417 and WCS374 triggered ISR, whereas
that of WCS358 did not 88]. Bacterial LPS usually consists of three domains 86]. The first domain, lipid A, consists of a bisphosphorylated glucosamine-disaccharide
backbone substituted with several fatty acids, which anchors the LPS to the outer
membrane. Attached to lipid A is the LPS core, an oligosaccharide of about 9–10 sugars,
which may be extended with the O-antigen or O-chain. The O-chain is a polysaccharide
comprised of repeating units. The number and nature of sugars in the O-chain units
is highly specific and can differ dramatically even between strains of the same species
86]. Under conditions of high iron availability, mutants of WCS374 and WCS417 that lack
the O-antigen of the LPS (WCS374-?OA and WCS417-?OA) were no longer able to elicit
ISR in radish, demonstrating that under these conditions the LPS of these strains
is the only bacterial determinant implicated in ISR and that the O-antigen is the
active component 88]. The importance of the O-chain of the LPS was also demonstrated in Arabidopsis. In
this plant species, strains WCS417 and WCS358 are able to elicit ISR against Pseudomonas syringae, whereas strains WCS374 cannot 48]. Using LPS-containing cell envelopes of these strains, and mutants lacking the O-antigen
it was shown that the O-chains of WCS417 and WCS358 are ISR-eliciting bacterial determinants,
and that the O-antigen of the LPS of the three WCS strains are differentially recognized
in Arabidopsis and radish.

In order to identify genes of the WCS strains involved in the biosynthesis of the
highly variable O-antigen of LPS, we searched for putative O-antigen biosynthetic
loci (OBL) using different OBL described for P. aeruginosa as bait. In P. aeruginosa, 20 distinct O-antigen serotypes are known for which the biosynthetic loci have been
sequenced 89]. The WCS genomes were mined for orthologs of the predicted proteins in these 20 P. aeruginosa OBL. In each of the WCS strains, only one gene cluster was found with genes coding
for more than four orthologs of the P. aeruginosa OBL proteins (Fig. 6). In P. aeruginosa, an OBL encoding the major enzymes for O-antigen biogenesis is usually found in between
the highly conserved genes himD and wbpM. Orthologs for these two genes were also found in all the OBL of the WCS strains,
although in the WCS strains genes encoding orthologs of the OBL proteins were also
found upstream of himD (Fig. 6). The region upstream of himD is highly conserved between WCS417 and WCS374, but contains an ortholog of rspA that
encodes an essential component of the protein synthesis machinery of Escherichia coli90] and is most likely not involved in LPS biosynthesis.

Fig. 6. LPS O-antigen biosynthetic loci. Putative LPS O-antigen biosynthetic loci (OBL) in
the genomes of WCS358, WCS374, and WCS417 as identified in a BLASTp search using 20
OBL identified in P. aeruginosa as bait. Genes of which the gene tags are designated in red font are orthologs of
genes found in at least one of the 20 P. aeruginosa OBL. Colors of the arrows indicate shared orthologs in the three WCS strains as determined
with reciprocal BLASTp. Graphics under the arrows represent a measure of similarity
as determined with Progressive MAUVE

Previously, the composition of the O-antigens of WCS374 and WCS358 were investigated
biochemically and reported to consist of glucose and fucose (WCS374), and glucose
and quinovosamine (WCS358), respectively 91]. However, the predicted function of the genes in the OBL of WCS374 and WCS358 are
not clearly related to the biosynthesis of such subunits. Furthermore, the complexity
of especially the OBL of WCS358 suggests that the O-antigen composition is not as
clear-cut as described by De Weger and co-workers 91]. In contrast to the O-antigens of WCS358 and WCS374, the composition of the WCS417
O-antigen has not been previously explored. The genome sequence of the WCS417 OBL
gives a number of clues towards the structure of the WCS417 LPS O-antigen. The WCS417
genes with locus tags PS417_08220 – PS417_08245 are homologs of ddhA-ddhD, yerE, and yerF, which are required for the synthesis of the O-antigen subunit yersiniose in Yersinia pseudotuberculosis92], 93] a sugar that has been detected before in a Pseudomonas spp. O-antigen 94]. The presence of the genes with locus tags PS417_08165 – PS417_08175, which encode
orthologs of WbjB, WbjC and WbjD that are required for the synthesis of the O-chain
component N-acetyl-L-fucosamine (L-FucNAc) 95], suggests that L-FucNAc is also part of the WCS417 O-antigen. In addition, an ortholog
of wbpM of P. aeruginosa is also found in the WCS417 OBL. WbpM catalyzes a reaction that ultimately results
in either 2-acetamido-2-deoxy-D-fucose (D-FucNAc) or 2-acetamido-2-deoxy-D-quinovose
(D- QuiNAc) 86]. It is therefore likely that the WCS417 LPS O-antigen contains yersiniose, L-FucNAc
and D-QuiNAc or D-FucNAc. Within the WCS417 OBL, only four genes are shared between
WCS417 and WCS358 (Locus tags PS417_08165, PS417_08175, PS417_08185 and PS417_08190)
and only one of them (PS417_08190) is also present in WCS374 (Fig. 6). This lack of homology between the OBLs of the three WCS strains is likely to result
in variation in O-chain composition, which may be causally related to the observed
host-microbe specificity of LPS in eliciting ISR.

Antimicrobial compounds

Meziane and co-workers 44] investigated the redundancy of ISR-eliciting bacterial determinants in different
plant species. They found that WCS358 could elicit ISR against Botrytis cinerea in tomato. This ISR could also be triggered by applying purified LPS or pyoverdine to the roots
but not by applying flagella of WCS358. Nonetheless, a double knockout derivative
of WCS358 that did not produce the LPS O-antigen or PVD358 still elicited ISR. This indicated that at least a third WCS358 determinant is able to elicit ISR in
tomato 44]. In addition to siderophores, LPS and flagella, antimicrobial compounds are bacterial
determinants that can induce systemic resistance 45], 96]–98]. However, to date no antimicrobial metabolites have been described for the three
WCS strains under investigation. Tran and co-workers 96] demonstrated that the cyclic lipopeptide massetolide A, produced by Pseudomonas strain SS101, can induce ISR. Like siderophores, cyclic lipopeptides are non-ribosomally
produced by NRPSs. All NRPS-containing gene clusters identified by antiSMASH in the
genomes of the three WCS strains could be related to the production of the siderophores
described above, except for one gene cluster in WCS358 (PC358_04000 – PC358_4180).
This gene cluster putatively encodes three NRPS that share 100 % identity in a BLASTp
search against PsoA, PsoB and PsoC. PsoA-C are responsible for the biosynthesis of
the cyclo lipopeptides putisolvin I and II in P. putida PCL1445 99]. In PCL1445, three additional genes have been shown to be important for putisolvin
production. The LuxR-type regulator gene psoR is located upstream of psoA and is required for expression of the pso cluster, whereas macA and macB, located downstream of psoC, are involved in putisolvin production or export 99], 100]. Interestingly, also the entire psoR-macB cluster of PCL1445 shares 100 % nucleotide identity with WCS358. Tot test if WCS358
is indeed capable of producing these cyclic lipopeptides, a drop collapse assay was
performed. In this assay, WCS358 was able to reduce surface tension, whereas WCS374
and WCS417 were not, demonstrating that WCS358 indeed produces a surfactant (Additional
file 5: Figure S4).

Besides the putisolvin biosynthesis cluster, other genes that are potentially involved
in the synthesis of compounds with broad-spectrum antibiotic activity could not be
identified in the genomes of WCS358, WCS374 and WCS417. For instance, biosynthesis
genes for the antimicrobial compounds 2,4-diacetylphloroglucinol, phenazines, hydrogen
cyanide, and pyrrolnitrin, which are abundantly present in root-associated Pseudomonas strains 52], 82], are not present in the genomes of the sequenced WCS strains. This corroborates with
early observations that the in vitro antagonistic activity of these strains was only apparent at low-iron conditions,
which suggested that the observed antagonistic activity of the WCS strains is predominantly
based on siderophore-mediated competition for iron 20], 21]. However, some genes were detected that encode putative bacteriocins. Bacteriocins
are bacteriocidal proteins that are generally effective against a narrow taxonomic
range of bacteria closely related to the producer 101]. We mined the WCS genomes with BAGEL3 102] for orthologs of bacteriocin-encoding genes that were previously identified in taxonomically
related strains 52]. Four putative bacteriocins were identified in WCS417, one related to S-type pyocins
of P. aeruginosa (PS417_07930, pyocin AP41-like) and three to R-type pyocins (PS417_05796, PS417_10225
and PS417_05705). In WCS374, three bacteriocins were identified: again one related
to S-type pyocins (PD374_22800, pyocin S6-like) and two to R-type pyocins (PD374_05705
and PD374_06430). In WCS358, no bacteriocins could be identified. Together, the genomes
of WCS358, WCS374, and WCS417 indicate that these Pseudomonas strains produce a relatively small pallet of known antibiotics, confirming that their
plant protective capacity is rather based on other mechanisms (e.g. siderophore-mediated
competition for iron and ISR).

Protein secretion systems

In order to deal with different environments, competing microbes and accommodating
hosts, bacteria need to secrete enzymes and other proteins into the extracellular
environment 103]. Five different protein secretion systems (type I, II, III, V, and VI) are typically
found within the Pseudomonas genus 104]. Differences in these protein secretion systems are likely to be important for the
excretion of proteins involved in traits that influence rhizosphere competence of
the WCS strains and for delivery of putative ISR-eliciting determinants.

Type I secretion systems consist of three components that together span the bacterial
cell envelop and transport their products from the cytosol directly to the extracellular
environment 104]. To identify gene clusters in the WCS genomes that encode the Type I secretion system
(T1SS), a BLASTP search was performed with protein sequences encoded by the P. aeruginosa aprDEF gene cluster as bait. Two complete T1SS loci were found in the WCS417 genome (Additional
file 6: Table S2). One T1SS gene cluster contained genes encoding a lipase and an ortholog
of the alkaline protease AprA 105], whereas the second T1SS gene cluster contained a gene encoding an ortholog of the
hemophore HasA 106]. It is likely that the T1SSs are dedicated to the secretion of these proteins that
are encoded in their gene cluster. Also in WCS374, the genes for two complete T1SSs
were found of which one also shared an operon with aprA and a lipase. For the second T1SS in WCS374 and for both T1SSs found in WCS358, the
substrate was not so obvious, as the corresponding gene clusters did not provide strong
clues (Additional file 6: Table S2). For instance, no ortholog of AprA could be found in the WCS358 genome.
To experimentally confirm this, we used a milk powder assay to detect AprA protease
activity. WCS374 and WCS417, which possess an aprA gene in one of their T1SS gene clusters, both showed a clearing zone around their
colonies when grown on KBA supplemented with milk powder. For P. syringae pv. tomato DC3000, such a clearing zone could be attributed to AprA activity as its aprA mutant did not show a halo (Additional file 7: Figure S5). Also WCS358 lacked such extracellular protease activity, confirming
previous observations 107].

Unlike the T1SS, proteins secreted by type II secretion systems (T2SS) do not directly
transfer the secreted proteins over the entire envelop, but make a stopover in the
periplasmic space 104]. To identify gene clusters that encode the Type II secretion system (T2SS) that transports
proteins across the outermembrane, a BLASTp search was performed using protein sequences
encoded by the P. aeruginosa xcpP-xcpZ and hxcP-hxcZ gene clusters. Clusters containing four or more genes with significant similarity
to the bait genes (e-value 10
^?5
) were considered T2SS loci. In this way, two T2SS loci were found in the WCS417 genome,
whereas the WCS374 genome seemed to contain only one T2SS locus (Additional file 6: Table S2). Also in WCS358, only a single T2SS locus was found using the P. aeruginosa T2SSs as baits. This WCS358 T2SS locus resembled the Xcp T2SS of P. aeruginosa PA01, which was recently found to secrete the PhoX-type phosphatase UxpB under phosphate-limited
conditions, thereby stimulating the phosphate uptake machinery of the bacterium 108]. A second T2SS of WCS358 was detected using the Xcm cluster described in P. putida GB-1 and KT2440 as bait (88 % nucleotide identity shared with the cluster of GB-1;
109]). Activity of this T2SS has not been confirmed in WCS358.

There is a strong resemblance between T2SS and the machinery required for type IV
pili biogenesis 110]. In WCS358, type IV pilus genes were previously investigated, because type IV pili
might have a role in attachment of bacteria to the root surface 111]. Even though type IV pili could not be detected on the surface of WCS358, a cluster
containing orthologs of the pilA, pilC and pilD genes of P. aeruginosa PAO1 could be identified. However, a gene for an ortholog of the traffic ATPase PilB
was lacking in this gene cluster. In the complete genome of WCS358, we could detect
five genes encoding proteins with significant homology to P. aeruginosa PAO1 PilB. However, the PilB ortholog with the highest similarity (PC358-2543; 43 %
AA identity) was found to be XcpR encoded in the WCS358 Type II secretion xcp gene cluster. A PilB ortholog is apparently not present in WCS358, which explains
the lack of type IV pili in WCS358.

The type III secretion system (T3SS), which consists more than 20 proteins, is the
most complex of all known protein secretion systems found in bacteria 104]. In the genomes of WCS417 and WCS374, ORFs encoding structural and regulatory components
of the T3SS are organized in 26-kb and 18-kb clusters, respectively, and several ORFs
within each cluster display a significant degree of similarity to the hrp/hrc cluster of pathogenic bacteria. Following the nomenclature initially proposed by
Preston and co-workers 112] for the type three secretion (tts) gene cluster of P. fluorescens SBW25, we named these genes rsp (rhizosphere-expressed secretion protein) or rsc (rsp conserved). No tts gene cluster or individual ORFs encoding T3SS components could be identified in the
WCS358 genome, suggesting that this strain lacks a typical T3SS. Immediately adjacent
to the rspL regulatory gene of the WCS417 tts cluster, a large ORF is present that encodes RopE of the AvrE family of effectors.
Effectors of the AvrE family can suppress the plant’s basal immune responses and promote
cell death of the host 113]. To identify additional type III effectors in the WCS417 and WCS374 genomes, we employed
bioinformatical analyses searching for conserved Hrp (Rsp) “box motifs” in the promoter
regions of putative effectors and exploring the N-terminal protein sequence of candidate
effectors for features typical of type III secreted effectors (i.e. abundance of Ser
and polar residues, acidic residues in the first 12 positions, and an aliphatic amino
acid in position 3 or 4) 114], 115]. Based on these criteria, we identified 11 putative effectors for WCS417 and 15 putative
effectors for WCS374 (Additional file 8: Table S3), whereas not a single putative effector could be identified in WCS358.
Interestingly, the majority of WCS417 and WCS374 effectors show no homology to known
effector families (data not shown), suggesting that these proteins represent novel
effectors.

Although recently the genes for a Type IV secretion system (T4SS) were found in a
Pseudomonas genome 61], T4SSs are uncommon in Pseudomonas spp. and were not detected in the WCS strains.

The type V secretion system (T5SS) is the most simple of all Gram-negative bacterial
secretion systems as the secreted proteins are transported across the outer membrane
with the aid of a covalently connected translocator domain (autotransporters) or via
a single dedicated outer membrane protein (called two-partner secretion or TPS) 104]. The C-terminal translocator domain of T5SS (PFAM03797) is conserved among different
classical monomeric autotransporters (T5aSS). In total, we found 2, 10, and 5 T5aSS
autotransporters with this domain in WCS358, WCS374, and WCS417, respectively (Additional
file 6: Table S2). Although the functions of these autotransporters are unclear, some are
homologous to autotransporters of P. aeruginosa PAO1 or other mammalian pathogens with functions in host immune activation 116], 117], peptidase activity 118], or biofilm formation and motility 119], 120].

In addition to the T5aSS, the WCS genomes were mined for orthologs of ORFs PA0692,
PA4540 and PA4624, which code for the outer membrane components of the T5bSS two-partner
secretion systems in P. aeruginosa PAO1. Nine orthologs were found in the WCS genomes: 2 in WCS358, 4 in WCS374, and
3 in WCS417. The orthologs found in this way all contained a periplasmic polypeptide
transport-associated (POTRA) domain required for recognition of the substrate protein
TpsA that is transported by the T5bSS. Although significant orthologs for P. aeruginosa PAO1 tpsA genes could not be found in the WCS genomes, six putative tpsA genes could be identified that contained the “haemagglutinin activity domain” (pfam05860),
which specifically interacts with the POTRA domains in the TpsB component 104]. The tpsA genes were found in the same operon and adjacent to a tpsB gene that is likely dedicated to its secretion. Many TpsAs are toxins that play a
role in contact-dependent growth inhibition of neighboring bacteria 121]. A clear example is PD374_00815, which carries a DUF637 and a pre-toxin Hint (PT-Hint)
domain, which are characteristic for such toxic TpsAs. It is therefore likely that
TpsAs enable the WCS strains to inhibit their competitors and successfully colonize
the plant roots. For three of the putative TpsBs, no TpsA component could be detected.
To our knowledge, such stand-alone TpsB proteins have not been previously described.

Using YadA and invasin of Yersinia enterocolitica as bait in BLAST searches, we did not detect members of the trimeric autotransporters
(T5cSS) or intimin/invasin family (T5eSS) in the WCS strains. However, in each of
the WCS genomes, a single ortholog of Patatin-like protein PlpD of P. aeruginosa PAO1 was found, which was recently suggested to represent a novel T5SS (T5dSS) 121]. Patatins form a group of glycoproteins with lipolytic activity found in potato tubers
and have proposed functions against plant pathogens 104], 122], 123].

Type VI secretion systems (T6SS) are functionally similar to T3SS in that they deliver
effector-like proteins into other organisms. Therefore, like T3SS, T6SS have been
implied to play a role in manipulation of host immunity. They also play a prominent
role in bacterial warfare by delivering toxic proteins into competing micro-organisms
124]. The machinery structurally resembles the contractile tails of bacteriophages and
is used, in this case, for injecting toxins into target cells. Protein sequences corresponding
to the genes encoded in the T6SS loci of P. aeruginosa PAO1 were used as bait in BLASTp searches of the three WCS genomes. Clusters containing
five or more genes with significant similarity to the bait genes (e-value??10
^?5
) were considered a T6SS locus 125]. The genomes of WCS417 and WCS374 each contained two T6SS loci, while WCS358 contained
one.

Overall, these data display the diversity of the protein secretion systems in WCS358,
WCS374, and WCS417. Their presence is not surprising, but highlight the sophisticated
mechanisms that these plant-beneficial rhizosphere bacteria have evolved in order
to successfully compete with other soil microbiota and sustain a long-term mutualistic
relationship with their host plants.