Search for strains with gvg clusters

The formyltransferase gene was used as a probe in two general strategies to search for sequenced strains containing the gvg biosynthetic gene cluster. The NCBI database was searched for homologs of the formyltransferases encoded by gvgI from Pfl WH6 (PFWH6_5257; WP_003176394.1) and Pan BRT175 (L585_04235; ERM15483.1) using BLAST+ 2.3.0 (BLASTP and TBLASTN) under default parameters [20]. Locus tags were retrieved from positive hits and used to search the NCBI Nucleotide database to confirm the presence of the other genes in the cluster. The Gene Neighborhood tool in Integrated Microbial Genome (IMG) v4 from the Joint Genome Institute was also used to search for complete gene clusters, again with the gvgI as a probe [21]. Comparisons of the gene content or genetic context were made using the Gene Neighborhood tool or manually based on gene annotations. Sequences were downloaded from NCBI or IMG and imported into CLC Main Workbench v. 6 (Qiagen) for additional analysis. For synteny comparisons, chromosome regions were cropped approximately 25 kb upstream and downstream of the Carbamoyltransferase (CTase) encoding gene and colored based on the synteny of genetic contexts.

Variations of the gvg cluster that may lack homologs of gvgI, termed gvg-like clusters, were identified using the gvgA (PFWH6_5249), gvgC (PFWH6_5251), and gvgF (PFWH6_5254) genes. Additional gvg amino acid sequences and accessions can be found in S2 File. The NCBI database was searched for homologs of the amino acid sequences corresponding to these genes from Pfl WH6 by BLASTP. Additional homologs were retrieved by Genome BLASTP in IMG. The Gene Neighborhood tool in IMG was used to facilitate identification of gene clusters.

Phylogenetic tree construction of the P. fluoresens and P. syringae groups

The multi-locus sequence analysis based phylogenetic tree was generated as previously described [22]. The code for the automated multi-locus sequence analysis pipeline (automlsa2) is available from the python package index (pypi) at https://pypi.org/project/automlsa2/ and builds upon autoMLSA.pl as previously reported [23]. Briefly, TBLASTN+ (v. 2.10.0) was used to identify and extract sequences for 99 of the 100 housekeeping genes as identified in Hesse et al. 2018 [14]; PA4393 was excluded from our analysis as it was unable to be routinely recovered using TBLASTN+ from all selected Pseudomonas spp. Included genome sequences are shown in S1 Table. As P. aeruginosa has previously been shown to be a suitable outgroup for the P. fluorescens and P. syringae groups, three selected P. aeruginosa isolates were used as an outgroup [24]. All genes that passed filter were aligned using MAFFT (v. 7.427) [25, 26].

Phylogenetic tree construction was done using IQ-TREE2 [27–30] with a partitioned nexus file containing references to each aligned sequence as input. Model selection and partition finding was done using the ‘-m MFP—merge rclusterf’ mode in IQ-TREE2, with protein models limited with the ‘—msub nuclear’ flag. Branch supports were determined using 1000 ultra fast bootstraps, and 1000 SH-aLRT test replicates (-B 1000, -alrt 1000). Nodes with > = 95% and > = 80% supports, respectively, were considered as well supported, and are so marked on the trees. Nodes with less support are unlabeled. Trees were visualized using ggtree v2.4.1 in R v4.0.1 [31–34].

In addition, the P. fluorescens and P. gessardii subgroups were trimmed from the larger Pseudomonas phylogeny and annotated using ggtree, as above, and inkscape (https://inkscape.org/). The genes present at a particular locus, between the genes encoding a methyl transferase and single-stranded DNA binding protein (tam and ssb), were identified for these strains. Identification was performed by searching for the sequence encoded by the P. fluorescens tam gene using IMG BLASTP (v4), cross-referencing results with P. fluorescens and P. gessardii subgroups in the phylogeny, and manually examining gene annotations.

A phylogenetic tree including Pseudomonas strains encoding the gvg-gene cluster was constructed generally as above, but with Pan BRT175 as the outgroup.

Gene diagram for gvg locus

The gvg locus in each genome was identified using BLAST with the protein sequences from WH6 used as queries against the predicted protein sequences from each of the target genomes as the databases. Subsetted genbank files (accessions listed in the figure) with 10kb regions upstream and downstream the putative gvg loci were extracted for each representative genome (20kb in total). Subset genbank files were used as input to clinker for visualization [35]. Unannotated genes were identified using TBLASTN+ v 2.10.0 using the Gvg protein sequences from WH6 as the query and each individual genome sequence as the target (S2 File; S6 Table).

Comparison of tree topologies

One strain from each clade in the Pseudomonas phylogeny containing the gvg cluster was selected for comparison of the topologies of gvg and species trees with Pan BRT175 as the outgroup. Seven of the Gvg proteins (GvgR, GvgA, GvgC, GvgF, GvgI, and GvgJ) were used in construction of the gvg phylogeny. Tree topology tests, including the approximately unbiased (-au) and Shimodaira-Hasegawa (SH) tests were done using iqtree2 v2.1.2 with 10,000 replicates (-zb 10000) [36, 37].

Phylogenetic tree of carbamoyltransferase proteins

Carbamoyltransferase (CTase) sequences were retrieved using BLASTP (v2.3.0) of the NCBI nt database with gvgF (PFWH6_5254) as the query sequence. Additional sequences were identified by a genome BLASTP search of the IMG database (v4.540). Sequences were confirmed to be present within clusters containing other homologs to gvg cluster genes. CTase sequences from the in-house genomes were added and all sequences were aligned using MAFFT (v. 7.244 [25]) and the percent identities calculated in CLC Main Workbench. A representative sequence of groups of sequences with greater than 95% identity was chosen for further analysis. These sequences were aligned again using MAFFT with the L-INS-i setting and trimmed using Gblocks (v. 0.91b) with half gapped positions allowed (-b5 = h setting). The LG substitution model was selected based on the PROTGAMMAAUTO option in RAxML (v8 [38]). Bootstrap analysis was performed using the autoMRE bootstrapping criterion, resulting in 350 bootstrap replicates. The resulting phylogenetic tree was visualized and annotated in iTOL [39].

Bacterial isolation

Bacterial strains used in this study are listed in Table 1. The strains in the ARS collection of FVG producers were isolated from wheat and grass rhizosphere in the central Willamette Valley of OR, USA as described in [11]. Pseudomonas fluorescens P5A was isolated from the surface of a basidiomycete fungus, Marasmius oreades, at the edge of a Douglas fir plantation in the Willamette Valley. The sample was transferred to sterile water (100 ml) with glass beads and shaken. The resulting suspension was serially diluted, plated on Fluorescent Pseudomonad Media (FPM) agar with cycloheximide (75 mg/L), and incubated at 28°C for 24 h. Colonies were re-streaked on FPM agar and screened for germination arrest and anti-Erwinia activity consistent with FVG production as in [3, 4]. Initial taxonomic identification of P. fluorescens P5A was performed by amplifying and sequencing 16S rDNA using standard protocols.

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Table 1. Strains used in this study.

https://doi.org/10.1371/journal.pone.0247348.t001

The sequencing, assembly and annotation of the genomes is described in Okrent et al. [43]. The contigs were ordered based on whole genome alignment with closely related complete genomes and the presence of the gvg gene cluster was confirmed.

Preparation of culture filtrates and biological assays

Bacteria were inoculated into either 60 mL or 5 mL of Pseudomonas Minimal Salts Medium [3] in a 125 mL Wheaton bottle or 20 mL test tube, respectively. The cultures were grown at 28°C with shaking for either seven (60 mL cultures) or two days (5 mL cultures). Subsequently, cells were removed from the filtrate by centrifugation (3000 x g for 15 min) and filter sterilization (Millipore GP express Steritop, 0.22-μm pore size (EMD Millipore) or 25 mm Pall Acrodisc with 0.22 μm pore size Supor membrane (Pall Corporation), respectively. For initial laser ablation electrospray ionization mass spectrometry (LAESI-MS) experiments, culture filtrate was extracted with 90% ethanol as described previously [1].

The agar diffusion bioassays for antimicrobial activity against Erwinia amylovora were performed generally as described in [4], however smaller volumes were used than reported previously. Aliquots of E. amylovora cultures grown overnight and diluted to an OD600 of 0.2 (300 μL) were spread on a 100x100x15 mm square plate containing 35 mL of 925 agar media [4]. Four wells per plate were cut using a #2 cork borer and the plugs removed. Samples of filtrate (40 μL) were added per well and plates incubated at 28°C for 2 days. Plates were scanned and the areas of the zones of inhibition were measured on triplicate wells for each sample using ImageJ from the National Institutes of Health [51].

Laser ablation electrospray ionization mass spectrometry analysis

LAESI-MS analysis was performed by Protea Biosciences, Inc. Samples of crude culture filtrate, non-inoculated filtrate, or extracted culture filtrate (20 μl) were aliquoted into individual wells of a 96-well plate and analyzed with a LAESI DP-1000 (Protea Biosciences) coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) according to the methods described in [52].

Conditions were slightly altered for analysis of FVG in crude culture filtrates. Settings for the LAESI DP-1000 were electrospray (+ 4000 V, 1.0 μl/min flow, 50% methanol, 0.1% acetic acid), 100 pulses per well at 20 Hz laser repetition rate, and laser energy ~600 μJ. The samples were analyzed in selected-ion monitoring mode at a resolution of 140,000 with scans centered on the sodium adduct of FVG, [M+Na]⁺ m/z 183.037. The average scan signal intensities were extracted on each well using LAESI Bridge software. Samples were analyzed by direct analysis using the LAESI-MS interface to the Q Exactive mass spectrometer without any sample preparation. Samples were analyzed in SIM mode at resolution of 140,000 for greatest sensitivity, specificity, and accuracy. Mass accuracy was maintained through analyses at < 0.5 ppm error with the sodium adduct detected. After optimization on positive and negative controls, all remaining samples were run using the same ablation and scanning conditions. Measurements were also performed on a second set of biological replicates for comparison.

Identification of potential FVG-producers

The gvg gene cluster in Pfl WH6 contains 12 genes (gvgR, gvgA-gvgK), encoding putative regulatory, transferase or transport activity, and two small ORFs encoding peptides of less than 50 amino acids (Fig 1B) [16, 17]. Three genes, gvgD, gvgE and gvgK, are dispensable for production of FVG [17]. In preliminary analyses, the gvgI gene, encoding a formyltransferase, was proposed as a specific marker for FVG biosynthesis [13]. Based on previous studies, we hypothesized that the gvgI gene would be found in a limited number of isolates within the three previously reported groups of Pseudomonas (i.e. WH6-like strains, P. chlororaphis, and few P. syringae isolates), in addition to Pantoea ananatis isolate known to encode the gvg locus.

Our strategy for prediction of additional FVG-producing strains was to mine genome databases for the gvgI gene and confirm its presence within a gvg cluster. This strategy identified over 30 additional sequenced strains containing a gvg cluster in NCBI and IMG databases as of December 2020. Counter to our expected result, the strains originated from a broad spectrum of geographic locations and varied taxonomic units (S2 Table). In general, strains encoding the gvg cluster were collected from rhizosphere or soil samples and belong to the genus Pseudomonas. However, the gvg cluster was also found in other genera including Thiomonas, Pantoea and Burkholderia, and strains from environmental or clinical samples (S2 Table).

Distribution of gvg-cluster containing strains

The distribution of strains containing the gvg cluster within the genus Pseudomonas is not readily apparent by species name alone. The Pseudomonas genus contains over 230 recognized species names (www.bacterio.net/pseudomonas.html). The P. fluorescens and P. syringae groups or species complexes are diverse and heterogeneous, and are each further divided into subgroups. Recent phylogenies have grouped P. fluorescens into 9 subgroups [24, 53] and P. syringae into at least 9 genomospecies or 11 phylogroups with multiple subgroups [54]. As a further complication, several of the Pseudomonas strains containing a gvg cluster have not been conclusively assigned to species (S2 Table). Additionally, researchers have suggested that some species designations in Pseudomonas may be incorrect based on newer tools, namely comparisons to type strains using multilocus or whole genome-based methods [14, 24, 55].

As the species labels on these isolates do not allow us to make conclusions about phylogenetic relationships between them, and in order to investigate the distribution of strains containing the gvg cluster within the genus Pseudomonas, a maximum likelihood phylogenetic tree was constructed from the amino acid sequences of 99 housekeeping genes [14, 41], focusing on groups containing strains with gvg-like clusters. The resulting phylogenetic tree, shown in Fig 2A, is largely consistent with the phylogenetic tree published in Hesse et al. 2018 [14, 24, 53, 56] and clustering of average amino acid identities from whole genomes [14, 57]. Strains encoding the gvg cluster are shown using blue or green circles at the tips for the P. fluorescens and P. syringae groups, respectively.

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Fig 2. The gvg locus has a patchy distribution in genomes within the Pseudomonas genus.

(A) Full maximum likelihood phylogenetic tree based on 99 housekeeping genes. The fluorescens and syringae groups are highlighted in blue and green, respectively. Red nodes correspond to the expanded clades in B and C. (B) Expanded P. fluorescens clade. (C) Expanded P. syringae clade. The tree was rooted on P. aeruginosa. Strain names in bold contain a gvg cluster. Nodes indicated with a gray circle are well supported (>95% UF-bootstraps & >80% SH-aLRT replicates). Genome accessions are listed in S1 Table.

https://doi.org/10.1371/journal.pone.0247348.g002

Despite the known limitations of accurate taxonomic designations within the Pseudomonas genus, analysis of the Pseudomonas phylogeny revealed that strains with homologous gvg clusters are present in the P. fluorescens and P. syringae groups only. Further, and contrary to our original hypothesis, the gene clusters appear in multiple subgroups within the P. fluorescens group, i.e. the P. fluorescens and P. chlororaphis subgroups, as well as Pfl FW300-N2E3, not yet placed in a subgroup. Strains containing the cluster within the P. fluorescens subgroup are not monophyletic, but instead are dispersed throughout the subgroup (Fig 2B; bold). The strains from the original Willamette Valley collection of FVG producers are found in multiple clades within the P. fluorescens subgroup (Fig 2B; bold). In contrast, gvg clusters are restricted to two paraphyletic groups in a small subclade of P. syringae (Fig 2C; bold). These strains are variously annotated as P. cannabina and P. syringae and include the model Arabidopsis pathogen Psy ES4326. The full phylogenetic tree is included as S1 File.

The gvg cluster was also found in several non-pseudomonads, including one strain of Pantoea ananatis, Thiomonas sp. and several strains of Burkholderia. In each case, the presence of a gvg-like cluster is not typical of the species. For example, a PCR-based survey of 117 strains of Pantoea failed to detect the cluster in any strain other than Pan BRT175 [19]. Similarly, of the 22 sequenced strains of Pantoea ananatis in the IMG database as of December 2020, Pan BRT175 and the closely related Pan PANS 99–36 are the only strains with the cluster. The gvg cluster is also unique to Thiomonas sp. (Tsp) CB2 of the 11 strains of Thiomonas that have been sequenced. Likewise, although there were approximately 40 sequenced genomes in the Burkholderia cepacia complex, only three contain complete gvg clusters (S2 Table).

Gene content of gvg clusters

As gvg gene clusters are present in a variety of bacterial strains, we were interested in determining whether there are variations in the gene content. To this end, the clusters were examined for gene loss, gain or rearrangement. Rearrangement was not evident in these clusters, as genes consistently appear in the same order. However, gene loss and gain were both evident and consistent with the phylogeny, as shown in Fig 3. The three genes not essential for FVG production, gvgD, gvgE and gvgK, are absent in some clusters. While most strains within the P. fluorescens subgroup have the same set of genes as Pfl WH6, the two strains in the E24 clade, Pfl E24 and Pfl ATCC 13525, as well as Pfl FW300-N2E3, lack the partially-redundant LysE transporter gene, gvgK. The gene cluster in Pfl FW300-N2E3 lacks segments of the non-essential amidinotransferase encoded by gvgD, indicative of gene decay. The strains within the P. syringae consistently lack gvgK but contain all other genes (Fig 3).

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Fig 3. The gvg biosynthetic locus exhibits co-linearity but also gene gain and/or loss within Pseudomonas spp.

The gvg clusters are color coded according to legend, shared with Fig 1. Syntenic sets of neighboring genes are additionally shown in the same color (not listed in legend). Sequences shared between strains are connected by bands that are shaded according to percent identity (black = 100% identity, white = 0% identity). Strains are ordered by dendrogram on the left, which follows the species tree. (*) Annotated gvgB have been removed from NCBI genome annotations, but are found in all strains. (†) Genomes encoding unannotated gvgG (S6 Table). (‡) ATCC 13525 has a) an assembly error or b) frameshift mutation causing a lack of gvgA. Paralogs gvgE, gvgJ, and gvgK intentionally share the arrow color and have different border colors.

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Outside of Pseudomonas, the gvg-like clusters are more variable. Pan BRT175 lacks orthologs of gvgD, gvgE and gvgK (Fig 3 and S1 Fig). Tsp CB2 lacks gvgK only and Burkholderia strains lack both extra LysE genes, gvgD and gvgK (S1 Fig). Uniquely, Thiomonas and Burkholderia clusters have gene sequences inserted between the gvgR and gvgA orthologs and between the gvgH and gvgI orthologs (S1 Fig). These genes encode putative amino acid transporters, a metallohydrolase, and proteins of unknown function. What effect the presence of these genes may have on the metabolites produced by these strains is unknown; they may produce a variant of FVG rather than the compound itself.

Formylaminooxyvinylglycine chemical analysis

The routine determination of an FVG phenotype typically requires an ethanol-based extraction of crude culture filtrate followed by thin-layer chromatography [1] as well as two biological assays [3, 4]. However, this protocol is not amenable to high-throughput analysis and is sometimes ambiguous. We recently developed a LAESI-MS-based method to directly detect FVG by mass in crude culture filtrates without extraction [51]. This protocol was used here to analyze a diverse selection of strains to determine if the presence of a gvg-like cluster corresponds to the ability to produce FVG. The culture filtrates were analyzed by LAESI-MS and, using Erwinia amylovora as an indicator strain, inhibition using the agar diffusion assay. All tested strains within the P. fluorescens group produced FVG, including strains in the P. fluorescens and P. chlororaphis subgroups (Table 2). As previously reported, the non-pseudomonad Pan BRT175 also produced FVG (Table 2) [51]. However, neither biological activity consistent with FVG production nor the mass associated with FVG were detected from the three strains within the P. syringae group (Table 2). The absence of FVG production from these strains is consistent with previous analyses of Psy ES4326 [16, 19, 51]. We investigated multiple strains from the P. syringae group in the current study, due to the possibility that a mutation in the laboratory strain Psy ES4326 was responsible for the lack of FVG production previously observed. This was not the case, as the closely related isolates of Pca also did not produce FVG.

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Table 2. Zone of inhibition of bacterial filtrates against Erwinia amylovora and detection of FVG (m/z 183.037) by LAESI-MS for various strains.

https://doi.org/10.1371/journal.pone.0247348.t002

Although we tested several possible hypotheses for the lack of FVG production in P. syringae strains, we were not able to conclusively demonstrate why these strains do not produce FVG under standard conditions. However, the most likely explanation is related to differences in gene regulation. P. syringae lack the sigma factor/anti-sigma factor pair involved in regulation of FVG production in WH6 encoded by the prtIR locus [58]. This suggests that regulation of FVG production differs in these strains relative to P. fluorescens. In P. fluorescens WH6, transcription of the gvg cluster occurs from multiple promoters, with one located in the intergenic region between gvgR and gvgA as well as additional promoters internal to the cluster [17]. We thus examined the promoter regions in P. syringae and compared them to those of several strains in the P. fluorescens group. The predicted promoter sequences located between gvgR and gvgA are conserved within P. fluorescens group strains (including P. chlororaphis) but substantially different in P. syringae strains (S2 Fig). Therefore, P. syringae may require different conditions for induction of the gvg cluster, which may reflect the different ecological niches or nutrient acquisition strategies of the two species.

Evidence of horizontal gene transfer

The observed pattern of gvg clusters, present in a given phylogenetic group but not closely related strains, is consistent with horizontal gene transfer (HGT) and/or the inclusion of clusters in genomic islands. Horizontally transferred genes typically demonstrate a lack of correspondence between phylogenetic trees representing gene evolution and those representing species evolution [59]. We therefore compared the phylogeny of the gvg genes and the housekeeping genes for strains within Pseudomonas. One representative from each clade that contains a gvg cluster was chosen for construction of maximum likelihood phylogenetic trees for amino acid sequences encoded by gvg and housekeeping genes. A comparison of the species tree and a partitioned Gvg protein maximum likelihood tree is shown in Fig 4. The most striking finding is that the placement of Psy isolates in the Gvg phylogeny is inconsistent with the species tree. Additionally, using the approximately unbiased (AU) and Shimodaira-Hasegawa (SH) tests with 10,000 resamplings, we determined that these two phylogenies had significantly different topologies (p < 0.01 for both tests). While we are unable to determine the origin of these gene loci in the Pseudomonas spp., this pattern is indicative of two or more HGT events within and/or between gvg-encoding Pseudomonas spp.

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Fig 4. The Pseudomonas species and Gvg trees are incongruent.

Trees were generated using maximum likelihood analysis. The species tree was inferred from the amino acid sequences of 99 housekeeping genes, while the Gvg tree was generated using a partitioned analysis of seven of the Gvg protein sequences. The trees were rooted to Pantoea ananatis BRT175. Nodes indicated with a gray circle are well supported (>95% UF-bootstraps & >80% SH-aLRT replicates). Pan, Pantoea ananatis; Psy, Pseudomonas syringae; Pch, Pseudomonas chlororaphis; Pfl, Pseudomonas fluorescens; Pfi, Pseudomonas fildensis; Psn, Pseudomonas synxantha.

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Genomic context of the gvg locus

The gvg cluster appears in several distinct genetic contexts (Fig 3). These different genetic contexts correspond to both phylogeny and gene content of clusters, i.e. clusters in closely related strains have the same gene content and are located in the same genomic context (Fig 3). The genetic context is the same for all strains in the P. syringae group, while there are six observed in the P. fluorescens group (Fig 3). Strains that are closely related to gvg-cluster-containing strains but that do not contain gvg clusters maintain synteny of the neighboring genes. This suggests specific insertion of gvg clusters rather than larger genetic rearrangements. We propose that each example of a cluster located in a distinct genetic context represents an independent insertion of the gvg cluster in that lineage.

There is one example where gvg clusters are present in the same genetic context even though the strains are in multiple clades of the phylogeny. These strains consist of members of the Pfl WH6 clade, the Psn A342 clade and Pfl 1112. In all of these strains, the gvg cluster is located in the same chromosomal location, between tam and ssb genes. The tam gene encodes a methyl transferase and ssb encodes a single stranded DNA-binding protein. Remarkably, this common location of the gvg cluster is true despite the many related strains lacking the cluster (S3 Fig). Two possible explanations for this observation are that 1) the gene cluster has been inserted in at least three lineages in the same location or 2) the common ancestor to these three clades contained the gvg cluster at this location but that it has been lost in multiple lineages. In strains without the gvg cluster at this location, alternative genes present between tam and ssb encode a LysR transcription factor, chloroperoxidase, chemotaxis protein, and various hypothetical proteins. In some strains, an integrase, transposase or restriction enzyme is also apparent. Like the presence of the gvg cluster, these variations are only partially consistent with phylogeny (S3 Fig). For example, chloroperoxidase and lysR regulatory proteins are present in this location in several disparate clades (S3 Fig). This analysis reveals that the position between the tam and ssb genes is highly variable in the P. fluorescens subgroups, perhaps indicating a hotspot for recombination or insertion.

These observations regarding HGT of the FVG biosynthetic cluster are consistent with the evidence of HGT for other specialized metabolites produced by plant-associated bacteria, including several metabolites commonly associated with Pseudomonas (reviewed in [60, 61]). For example, the gene clusters responsible for biosynthesis of 2,4-diacetylphloroglucinol (DAPG), phenazines and pyochelin are observed in Pseudomonas as well as disparate, unrelated taxa [62–65]. As we observed with the gvg cluster, biosynthetic gene clusters frequently are present in both Pseudomonas and Burkholderia in particular, which may be explained by the presence of these bacteria in the same habitats [60].

Similarly, the vinylglycine rhizobitoxine is produced by bacteria from multiple genera, including the rhizobial Bradyrhizobium elkanii and the plant pathogen Burkholderia andropogonsis. Rhizobitoxine biosynthesis (rtx) genes flanked by insertion sequences have been reported in Xanthomonas oryzae pv. oryzae KACC10331 [66], though rhizobitoxine production has not been reported in this strain. AMB production has only been reported in Pseudomonas aeruginosa and AVG in one strain of Streptomyces sp., though systematic analyses have not been performed for other vinylglycines.

Characteristics of gvg-like gene clusters

In addition to the strains containing gvg gene clusters, there are many strains with only some of the genes of the cluster, sometimes in combination with different genes. These variations are present in a broader range of bacteria than is the complete gvg cluster (S4 Table). Genera with strains containing these gvg-like clusters include the Alphaproteobacterium Salinarimonas; Betaproteobacteria Burkholderia, Ralstonia and Thiomonas; Gammaproteobacteria Pseudomonas, Pantoea, Acinetobacter and Serratia; and the Actinobacteria Streptomyces and Saccharopolyspora (S4 Table). These clusters contain genes in various arrangements and do not contain all of the genes shown to be necessary for FVG production [17], and thus would not be predicted to produce FVG. The core genes of these cluster variants are gvgA, gvgB, gvgC, and gvgF. The vast majority of these gene clusters are uncharacterized.

The evolutionary relationship of the gvg and gvg-like clusters was examined by constructing a phylogeny of the carbamoyltransferase (CTase) encoded by gvgF, which is common to the majority of the gene clusters, including the complete gvg clusters discussed above. The CTases from gene clusters with the same gene content tend to be in the same clades, regardless of taxonomy (Fig 5). Additionally, the CTases from the complete gvg clusters form a separate clade from those in gvg-like clusters, irrespective of taxonomy. (The exception is CTases encoded by truncated clusters from the Burkholderia mimosarum strains, which are also found in this clade, suggesting a relatively recent origin from a complete gvg cluster.) (Fig 5).

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Fig 5. The structure of the gvg gene cluster is correlated with the inferred gvg phylogeny.

This is a maximum likelihood phylogenetic tree of the carbamoyltransferase encoded by gvg clusters and other gene clusters. Clades are colored and labeled based on the genes present in the gene cluster. The red A clade corresponds to a complete gvg cluster with all of the genes present for production of FVG. In the key, the genes present in each cluster are listed, with parentheses around genes that are optional and curly braces indicating insertions. Sequence accessions are listed in S5 Table.

https://doi.org/10.1371/journal.pone.0247348.g005

At least eight cluster variations are present (Fig 5), leading to the hypothesis that each contributes to the production of a unique compound, perhaps an amino acid or similar product. One variant is located within a known cluster for biosynthesis of the macrolide antibiotics aldgamycin and chalcomycin in Streptomyces sp. HK2006-1 [67]. This cluster variation contains homologs of gvgA, gvgC, gvgF and gvgG, annotated as almUII, almUIII, almDIV and almUIV, respectively. However, exactly how these genes may function in the synthesis of the cluster products is unknown. These gvg-like loci are promising candidates for future molecular characterization. An important question is whether these gene clusters lead to the production of as yet undiscovered compounds or to previously identified compounds for which the biosynthetic genes are not yet known.

The presence of subsets of core biosynthetic genes in unrelated strains has also been observed for the phenazine phz and DAPG phl clusters encoded in other Pseudomonas spp. [49, 50]. This recycling of biosynthetic sub-clusters into new clusters may be a key method for evolution of biosynthetic diversity [68, 69].