Identification and Biosynthesis of a Novel Xanthomonadin-Dialkylresorcinol-Hybrid from Azoarcus sp. BH72

A novel xanthomonadin-dialkylresorcinol hybrid named arcuflavin was identified in Azoarcus sp. BH72 by a combination of feeding experiments, HPLC-MS and MALDI-MS and gene clusters encoding the biosynthesis of this non-isoprenoid aryl-polyene containing pigment are reported. A chorismate-utilizing enzyme from the XanB2-type producing 3- and 4-hydroxybenzoic acid and an AMP-ligase encoded by these gene clusters were characterized, that might perform the first two steps of the polyene biosynthesis. Furthermore, a detailed analysis of the already known or novel biosynthesis gene clusters involved in the biosynthesis of polyene containing pigments like arcuflavin, flexirubin and xanthomonadin revealed the presence of similar gene clusters in a wide range of bacterial taxa, suggesting that polyene and polyene-dialkylresorcinol pigments are more widespread than previously realized.


Introduction
An underexplored class of natural compounds are the 2,5dialkylresorcinols (DAR), microbial secondary metabolites, which are derived from a condensation of two fatty acid metabolism intermediates [1]. DAR examples with known bioactivities (Figure 1) are the free radical scavengers DB-2073 (1) [2][3][4], resorstatin (2) [4], a multipotent isopropylstilbene (3) [5] or the mammalian cell growth stimulating factor resorcinin (4) [6] which can also be found as part of the flexirubins from Flavobacterium johnsoniae [7]. Flexirubins (5) [8] are orange pigments made of a DAR esterified with a non-isoprenoid arylpolyene carboxylic acid and are used as chemotaxonomic marker for bacteria from the Bacteroidetes phylum. Another class of arylpolyene pigments, the xanthomonadins, has been found in bacteria of the genus Xanthomonas. Besides the dibrominated xanthomonadin I (6) [9], derivatives with different levels of bromination, methylation and chain length are known [10], all lacking a DAR-moiety. The gene cluster for xanthomonadin biosynthesis (Figure 2.B) is conserved in several strains of Xanthomonas [11][12][13] and was recently studied in Lysobacter enzymogenes [14], another bacterium from the family xanthomonadaceae. The gene cluster encodes an acyltransferase, a ketoreductase, a dehydratase, a predicted ketosynthase, an unusual chain-length factor like protein [14] as well as a chorismateutilizing enzyme [15][16][17] suggesting, a type II fatty acid synthase like biosynthesis of xanthomonadin. Whereas the biological role of flexirubin is unknown, it could be shown that xanthomonadins protect Xanthomonas against photooxidative damage and lipidperoxidation [18][19][20]. Similar results were obtained for the xanthomonadin-like pigment from L. enzymogenes, which protects the bacterium from UV/visible-light and H 2 O 2 [14].
Recently we reported that DAR biosynthesis is encoded by a conserved gene cluster which was found in 89 bacterial strains with most of them being associated with other organisms [1]. Among them is Azoarcus sp. BH72, a rod shaped motile bacterium which was isolated from surface-sterilized roots of Kallar grass (Leptochloa fusca) [21] where it lives as diazotroph mutualistic endophyte. Since heterologous expressed DAR-biosynthesis proteins of Azoarcus sp. BH72 led to DAR (7) (Figure 1) production [1] but no DAR was detected in Azoarcus sp. BH72 extracts, we were interested about its fate in the bacterial secondary metabolism. As it is known that Azoarcus sp. BH72 produces a cell-bound yellow pigment [21] we speculated that its DAR might be connected to the yellow compound in a flexirubin-like manner.
Here we report gene clusters responsible for the pigment biosynthesis in Azoarcus sp. BH72 and show that gene clusters containing polyene-biosynthesis associated genes are widespread in bacteria. Furthermore, we show by a combination of labeling experiments, HPLC-MS and MALDI-MS that the pigment from Azoarcus sp. BH72 contains the already reported DAR 7, which is connected to a xanthomonadin-like polyene. Additionally, a benzoate synthase and an AMP-ligase encoded by the Azoarcus pigment biosynthesis gene clusters were characterized that might perform the first two reactions of the polyene assembly.

Genome mining
For the identification of DarAB biosynthesis gene clusters, the primary sequences of the DarAB homologues from C. pinensis DSM 2588 were used for a BLAST-P identification (proteinprotein-BLAST) of homologues in Azoarcus sp. BH72, Variovorax paradoxus S110, Dechloromonas aromatica RCB and Sideroxydans lithotrophicus ES-1. The xanthomonadin gene cluster from Xanthomonas campestris pv. campestris ATCC 33913 was used in a STRING-analysis (version 9.0) [23] for the identification of the putative xanthomonadin polyene cluster in Azoarcus sp. BH72, V. paradoxus S110, D. aromatica RCB and S. lithotropicus ES-1. Gene colors in Figure 2 are based on NCBI-annotation or domain guided annotations from BLAST-P with primary sequences of X. campestris pv. campestris-genes as template (see Table S1, S2, S3, S4). Grey connections between genes highlight BLAST-P results with an identity $40%.
Feeding experiments 400 mL liquid LB-medium was inoculated 1:100 from an overnight culture of Azoarcus sp. BH72. After 3 h L-[methyl-2 H 3 ]methionine or 4-fluoro-3-hydroxybenzoic acid was fed in 1 mM portions to the cultures and feeding was repeated after 12, 24 and 48 h, resulting in a final concentration of 4 mM. As control, a culture without feeding was cultivated at the same time. Due to poor growth compared to the control culture, feeding of Azoarcus sp. BH72 with 4-fluoro-3-hydroxybenzoic acid was stopped after 24 h at a final concentration of 2 mM and cultures were grown for additional 48 h before harvesting. Cultures were harvested 12 h after the last feeding by centrifugation at 10.000 rpm.

Extraction and isolation of compounds
Pelleted cells of liquid cultures were shaken for 30 min with 50 mL acetone in the dark, followed by filtration to remove cell debris. The extracts were dried under reduced pressure. For structure elucidation raw extracts were fractionated using silica gel columns with hexane/ethylacetate (4:1; 1% acetic acid). Yellow fractions were dried under reduced pressure. Samples were stored dry at 220uC in the dark and dissolved in 10-50 mL acetonitrile (ACN) before further analysis.

HPLC-MS
ESI HPLC MS analysis was performed with a Dionex UltiMate 3000 system coupled to a Bruker AmaZon X mass spectrometer and an Acquity UPLC BEH C18 1.7 mm RP column (Waters) as described previously [24].

Molecular-biological methods
Molecular-biological experiments were performed according to standard procedures [27]. The phusion high-fidelity polymerase (Thermo-scientific) was used for polymerase chain reactions (PCR) according to the manufacturer's instructions and with oligonucleotides (Table S6) obtained from Sigma-Aldrich. Plasmid isolation was performed with the GeneJet TM Plasmid Extraction Kit (Fermentas) and DNA extraction from agarose gels with the GeneJet TM Gel Extraction Kit (Fermentas).

Generation of mutants of Azoarcus sp. strain BH72
Genes azo3911 (arcK) and azo0260 (arcT) were disrupted via plasmid integration with plasmid pK18GGST [28]. Briefly, internal gene fragments of the 59 region of the respective genes were amplified and cloned into the expression vector pK18GGST through specific restriction sites, and a single recombination event with the wild type chromosome resulted in plasmid integration and thus a polar mutation. Growth conditions for mutant generation were as previously described [29]. The primers used for amplification were for azo0260 azo0260KOFor (XbaI restriction site) and azo0260KORev (HindIII restriction site), and for azo3911 azo3911KOFor (XbaI restriction site) and azo3911-KORev (HindIII restriction site), respectively. The resulting fragments 0260KOfragment and 3911KOfragment cloned into the XbaI-HindIII restriction sites of pK18GGST spanned nucleotide positions of 7-639 nt of gene azo0260, or of 16-625 nt of gene azo3911, respectively. The correct plasmid sequences were verified by sequencing. Plasmids were transferred to strain BH72 by biparental mating, and the resulting plasmid integration mutants BH0260 and BH3911 were selected by their kanamycin resistance. Integration of the plasmid into the correct target site in the chromosome was verified by Southern blot hybridization.

Construction of plasmids for heterologous expression of ArcB and ArcT
An expression cassette encoding the TEV-protease cleavage site and a cherrytag (DelphiGenetics SA) with c-terminal His 6 -tag was Variovorax paradoxus S110 (D) and Sideroxydans lithotrophicus ES-1 (E). Annotation colors follow the Azoarcus gene clusters and genes are connected by grey lines if their identity was $40% in a BLAST-P analysis. All genes are scaled to the depicted size and are listed in Table S1, S2, S3, S4. doi:10.1371/journal.pone.0090922.g002 digested with the restriction endonucleases NcoI and AvrII and ligated into the similar treated pCOLA-DUET1 resulting in pCATI1. The genes arcB (azo3920) and arcT (azo0260) were amplified by PCR from genomic DNA isolated from Azoarcus sp. BH72, resulting in 3920fragment and 0260fragment respectively. For construction of pCATI-arcB, pCATI1 was linearized by PCR giving pCATI1fragment which was fused with azo3920fragment using the Gibson assembly cloning kit (NEB). For construction of pCATI-arcT, 0260fragment was digested with the restriction endonucleases NcoI and XhoI and ligated with the similar treated pCATI1. The inserts of the plasmids pCATI-arcB and pCATI-arcT were sequenced by SeqIT GmbH (Kaiserslautern).

Benzoate synthase activity assays with ArcB
For in vivo activity assays with ArcB, 1 mL auto induction medium was inoculated 1:100 from pre-cultures of E. coli BL21 (DE3) Star containing pCATI-arcB (strain TS3920) or the empty vector (pCATI1). After incubation on a rotary shaker (16 h, 37uC) cultures were extracted with 1 volume ethylacetate, dried and dissolved in 500 mL acetonitrile followed by derivatization with Nmethyl-N-trimethylsilyl-trifluoroacetamid (MSTFA) (55uC, 1 h) and assay products were analyzed by GC-MS. For in vitro assays with ArcB, the tag-less purified protein was buffered in 50 mM Tris (pH 7.4). 100 mL assays (2 mg ArcB, 1 mM chorismic acid (Sigma-Aldrich), 100 mM Tris pH 7.4) were incubated at 37uC and stopped after 10 or 60 minutes by incubation at 100uC for 5 min. Stopped assays were adjusted to pH 4 and extracted 26 with 1 volume of ethylacetate. Dried organic phases were dissolved in 40 mL acetonitrile, derivatized with MSTFA (55uC, 1 h) and analyzed by GC-MS. Assay products were detected on a 7890A gas chromatograph (Agilent) equipped with a DB5ht column (30 m6250 mm60.1 mM, Agilent) coupled with a 5975C mass selective detector (Agilent). The method parameters were the following: Carrier gas: Helium (1 mL/min); injection volume: 1 mL; inlet temperature: 250uC; injection mode splitless; oven starting temperature 5 min 70uC, then 5uC/min to 300uC. Ionization of the analyte molecules were carried out by electron impact ionization at 70 eV. Products were identified with the ''Automated Mass Deconvolution and Identification Software'' (AMDIS) version 2.64 in combination with the NIST-library.

ArcT in vitro assays
To test the adenylation activity of ArcT in vitro, purified tag-less protein was buffered in 20 mM Tris (pH 7.4) and incubated with labeled [c- 18 O 4 ]-ATP, various benzoic acid derivatives (listed in Figure S5), MgCl 2 and pyrophosphate as described previously [26]. Assays were analyzed by MALDI-MS as reported above.

Gene clusters for xanthomonadin and DAR biosynthesis
During our investigations of the DAR biosynthesis we could show that heterologous overexpressed DAR-biosynthesis proteins of Azoarcus sp. BH72 led to DAR 7 production in Escherichia coli [1], but the corresponding DAR was not detectable in Azoarcus extracts. However, Azoarcus sp. BH72 extracts contained a yellow hydrophobic compound that showed no color shift with alkali, indicating that the pigment was not a flexirubin [30]. The gene cluster for xanthomonadin biosynthesis (Figure 2.B, Table S4) is conserved within the gammaproteobacteria in several strains of Xanthomonas [11][12][13] and was also studied in L. enzymogenes [14]. We identified two gene clusters in the genome of the betaproteobacterium Azoarcus sp. BH72 (Rhodocyclales) encoding proteins with high similarity to those of the xanthomonadin biosynthesis ( Figure 2.A, Table 1). Both putative arc loci (azo3910-azo3921 and azo0253-0264) were not physically linked. In addition to their already reported DAR biosynthesis gene clusters [1], the putative polyene biosynthesis gene clusters were also found in other betaproteobacteria like Dechloromonas aromatica RCB (Rhodocyclales) (Figure 2.C, Table S1), Variovorax paradoxus S110 (Burkholderiales) (Figure 2.D, Table S2) and Sideroxydans lithotrophicus ES-1 (Gallionellales) (Figure 2.E, Table S3). Within the identified gene clusters several genes are highly conserved (for example a gene encoding a glycosyl transferase, an outer membrane lipoprotein carrier protein (LolA), a phospholipid/glycerol acyltransferase and an adjacent exporter like protein) and we also found these genes in flexirubin biosynthesis gene clusters [31]. BLAST-P and String analysis with proteins from the gene clusters identified in Azoarcus revealed that gene clusters encoding these polyene biosynthesis associated genes can be found within several different proteobacteria, bacteria from the Bacteroidetes phylum and in one strain from the Spirochaetes (Table S7). In addition, 16 of the 39 reported bacterial genomes in Table S7 contained DAR biosynthesis gene clusters, with nine examples where the DAR biosynthesis genes are located within or adjacent to the polyene biosynthesis associated gene cluster.

Verification of assigned pigment biosynthesis gene clusters
Detailed studies with Xanthomonas strains showed that the biosynthesis of xanthomonadin depends on the reported gene cluster [11][12][13] (Figure 2.B), which was also shown for the biosynthesis of the xanthomonadin-like pigment from L. enzymogenes [14]. Thus we tested whether these gene clusters encoding such homologues are essential for the pigment biosynthesis by insertion of plasmids into gene arcK (azo3911) or arcT (azo0260), respectively. The resulting mutant strains BH3911 and BH0260 appeared colorless on agar plate ( Figure S1), and subsequent acetone extraction and HLPC-UV analysis showed the loss of a UV-signal at 15.7 min in the extracts from the mutants (Figure 3).

Initiation of the arcuflavin polyene biosynthesis
Previously it was shown that xanB2 (Xcc4014) from X. campestris is responsible for production of the diffusible factor (DF) 3hydroxybenzoic acid (3-HBA), which is essential for xanthomonadin biosynthesis [12,15]. Recent research revealed that xanB2 encodes a structurally novel and bifunctional chorismatase converting chorismic acid into 3-HBA, the probable biosynthetic precursor of the xanthomonadin ring moiety, and 4-hydroxybenzoic acid (4-HBA) which is needed for coenzyme Q8 biosynthesis [16,17]. A homologue of XanB2 in Azoarcus is encoded by arcB within the postulated pigment biosynthesis gene cluster (Figure 2.A). Like XanB2, ArcB contains a YjgF-like C-terminal domain, which is also present in the chorismate utilizing enzymes Hyg5, FkbO and RapK from Streptomyces [32]. Based on their reaction products, these enzymes were sorted in three distinct types of chorismatases [33]. The FkbO type produces 3,4dihydroxycyclohexa-1,5-dienoic acid, the Hyg5-type forms 3-HBA whereas the XanB2-type converts chorismate to 3-HBA and 4-HBA [33]. Since from sequence alignment alone it is actually not possible to predict accurately the reaction catalyzed by these chorismate-converting enzymes and to get experimental evidence for the catalyzed reaction, arcB was overexpressed in E. coli and the culture supernatants were extracted. GC-MS analysis of these extracts ( Figure 5) revealed the formation of compounds with similar retention times and mass spectra ( Figure S3.A) as 3-HBA and 4-HBA standards. Subsequent purification of ArcB ( Figure S4) allowed its incubation with chorismic acid. In all assays 4-HBA was formed non-enzymatically ( Figure S3.BIV) as reported before [16,34], whereas 3-HBA was only detectable in assays containing ArcB ( Figure S3.III). Since it was postulated that xanthomonadin biosynthesis is performed by a type II fatty acid synthase like biosynthesis using 3-HBA as polyene precursor [15], 3-HBA should be activated for the polyketide synthase (PKS) machinery. Such a reaction might be performed by the putative AMP-ligase encoded by Xcc4015 in X. campestris pv. campestris, which is essential for the pigment biosynthesis in this organism [15]. The homologue ArcT, encoded in the pigment gene cluster from Azoarcus sp. BH72, was overexpressed in E. coli and purified ( Figure S4). The adenylation activity of ArcT was tested with a method measuring the isotopic back exchange of unlabelled pyrophosphate into [c- 18 O 4 ]-ATP if the enzyme adenylates a substrate [26]. MALDI-MS analysis showed ATP-label exchange in assays with 3-HBA and various other benzoic acids with modifications in 4-position which was not detected if no substrate, 3-methoxybenzoic acid or 2-substituted benzoic acids were added ( Figure S5).

Discussion
In previous studies the xanthomonadin biosynthesis gene cluster was found in genomes of other gamma-and betaproteobacteria and homologues of the chorismate-utilizing enzyme XanB2 (Xcc4014) were also found in genomes outside of the proteobacteria [16]. However, only the gene cluster of the closely related bacterium L. enzymogenes (family xanthomonadaceae) was studied and a non-brominated pigment was found in this bacterium, which is most likely xanthomonadin-like [14]. Here we identified for the first time similar polyene biosynthesis gene clusters in the genome of Azoarcus sp. BH72 which additionally contains a DARbiosynthesis gene cluster. Our results with plasmid insertion mutants show that both xanthomonadin-like biosynthesis gene clusters, although they are not physically linked, are essential for pigment 8 biosynthesis in Azoarcus sp. BH72, indicating that its product is indeed a xanthomonadin-like compound.
Results from MS 2 experiments with pigment 8 show that its fragmentation is similar to those of flexirubin where the basepeak was shown to result from the cleavage of the polyene-DAR ester bond [31]. Therefore we predict that DAR 7 and the xanthomonadin-like polyene are connected in a flexirubin-like manner by an ester bond between the polyene carboxylic acid and a phenolic hydroxyl group. Similar to flexirubin, the formation of a radical cation was observed that might be explained by direct absorption of the laser light by the polyene of pigment 8 instead of matrix mediated ionization mechanisms. Since the MS 3 of the assigned DAR-fragment m/z 293.3 is identical to those from heterologous produced DAR 7 [1], we conclude that the Azoarcus pigment 8 has a DAR moiety identical to 7. It was postulated that 3-HBA is a biosynthetic intermediate of the xanthomonadin biosynthesis [15]. Our labeling result with 4F-3HBA now confirms that this is indeed the case in the biosynthesis of pigment 8 from Azoarcus which additionally contains a methionine derived methylgroup. Furthermore the MS 3 of the assigned polyene fragment m/z 317.2 shows mass shifts of D28 Da (CO), D78 Da (C 6 H 6 ) or D26 Da (C 2 H 2 ) ( Figure S2.C), already known from mass spectrometric fragmentation of flexirubin-like polyenes [31,35]. These results suggest the structure of a nonbrominated arylheptaene for the polyene moiety of 8, reflecting xanthomonadin group-11 previously found in Xanthomonas [36,37]. Based on these findings we conclude that a new xanthomonadin-DAR hybrid has been characterized, which we named arcuflavin (from Azoarcus and latin for yellow) and propose it to have the structure of 8. Since arcuflavin is only produced in low amounts and becomes insoluble in various organic solvents after enrichment, similar to the L. enzymogenes xanthomonadin-like pigment [14], further structure elucidation by means of NMR was not possible.
Combinations of xanthomonadin-like polyene gene cluster and DAR-gene cluster were also found in D. aromatica RCB, three strains of V. paradoxus and S. lithotrophicus ES-1. Whereas in previous genome screenings only the xanthomonadin-like gene clusters from D. aromatica RCB and V. paradoxus EPS and S110 were found [14,16], we could now identify their DAR gene clusters and it will be interesting to see in future studies if the combination of both gene cluster types leads to the identification of polyene-DAR hybrids also in these organisms. In addition we found that genes associated with known polyene biosynthesis gene clusters with and without DAR biosynthesis encoding genes can be found within a wide range of bacterial genomes including medically important taxa like Burkholderia, Vibrio, Escherichia and Pseudomonas (Table S7). We therefore predict that more pigments like flexirubin, xanthomonadin or arcuflavin will be found in these organisms and that these compounds might also be involved in the pathophysiology of these organisms. Moreover, our findings suggest that these pigments cannot be used as chemotaxonomic markers, especially in the case of flexirubin for the Bacteroidetes phylum, as they seem to be much more widespread than originally thought.
We investigated the first steps of the arcuflavin-polyene biosynthesis and could show that ArcB has in vitro the assumed catalytic activity of a chorismate-utilizing enzyme that produces 3-HBA and 4-HBA. This is similar to the reaction performed by its homologue in the xanthomonadin biosynthesis pathway [16,17] and makes it the third biochemically characterized example of the XanB2-type of chorismate-utilizing enzymes. Furthermore the activity of the AMP-ligase ArcT with 3-HBA could be shown in vitro. The result that 3-methoxybenzoic acid was not a good substrate for ArcT suggests that the methylation of pigment 8 takes place after the activation of the 3-HBA starter. Although ArcT showed in vitro a low substrate specificity, no other derivatives of arcuflavin 8 were found in the extracts of Azoarcus, showing that in vivo the arcuflavin-biosynthesis machinery is more selective in the precursor or intermediate selection.

Conclusions
Due to their structural similarity one might conclude a functional similarity for arcuflavin with xanthomonadins and DARs. Xanthomonadins from Xanthomonas oryzae pv. oryzae and X. campestris pv. campestris are located in the bacterial outer membrane [13,38]and have been shown to protect their producers from photooxidative damage and lipid peroxidation [18][19][20]. On the other hand, the DARs DB-2073 (1) and resorstatin (2) were reported to act as free radical scavengers to protect against lipid peroxidation [4] and show striking structural similarity to the DAR from arcuflavin. Therefore DAR or polyene-DAR hybrids like arcuflavin might protect the cell against oxidative damage, suggesting the fusion of two protective moieties in polyene-DAR hybrids. Bacteria from the genus Xanthomonas are well known plant pathogens, Azoarcus sp. BH72 is a diazotroph endophyte of rice and other grasses [39], whereas V. paradoxus S110 is a growthpromoting endophyte of various plants [40]. As transient production of reactive oxygen species is found in most plantmicroorganism interactions [41], arcuflavin and similar polyene pigments might play a role in establishing or maintaining the symbiotic life of the bacterium with its host.      acid I: 2,3-dihydroxybenzoic acid J: 3,5-dihydroxybenzoic acid K: benzoic acid L: p-coumaric acid M: E-cinnamic acid. Colours indicate if no (red), ,50% (orange) or .50% label exchange (green) was detected. (TIF)