Identification of 2,4-diacetylphloroglucinol production in the genus Chromobacterium

The compound 2,4-diacetylphloroglucinol (DAPG) is a broad-spectrum antibiotic that is primarily produced by Pseudomonas spp. DAPG plays an important role in the biocontrol disease suppressing activity of Pseudomonas spp. In the current study, we report the discovery of the DAPG biosynthetic cluster in strains of Chromobacterium vaccinii isolated from Brazilian aquatic environments and the distribution of the biosynthetic cluster in the Chromobacterium genus. Phylogenetic analysis of the phlD protein suggests the biosynthetic cluster probably entered the genus of Chromobacterium after a horizontal gene transfer event with a member of the Pseudomonas fluorescens group. We were able to detect trace amounts of DAPG in wild type cultures and confirm the function of the cluster with heterologous expression in Escherichia coli. In addition, we identified and verified the presence of other secondary metabolites in these strains. We also confirmed the ability of C. vaccinii strains to produce bioactive pigment violacein and bioactive cyclic depsipeptide FR900359. Both compounds have been reported to have antimicrobial and insecticidal activities. These compounds suggest strains of C. vaccinii should be further explored for their potential as biocontrol agents.


Materials and methods
Strains.The type strain of C. vaccinii DSM 25150 T was obtained directly from the DSMZ-German Collection of Microorganisms and Cell Cultures.Chromobacterium vaccinii CR1 and CR5 were previously isolated from aquatic environments in Brazil 26 .Both strains were isolated from raw surface water, which was collected directly in sterile flasks from the body of water between 0 and 30 cm water depth, in which the collection and preservation of the samples occurred in accordance to the National Collection Guide and preservation of samples: water, sediment, aquatic communities and liquid effluents 27 .C. vaccinii CR1 was isolated from João Leite stream, Goiânia, Goiás, Brazil-Geographic location 16° 34′ 30.54"S; 49° 13′ 55.02" W. C. vaccinii CR5 was isolated from the Meia Ponte river, Goiânia, Goiás, Brazil-geographic location 16° 54′ 16.3'' S; 49° 07′ 37.8'' W. The isolation details were according to Gomes et al. 28 .
The aquatic environment where strains CR1 and CR5 were isolated from is a river and its tributary.These waters are also impacted by many anthropological pressures, such as industrial, agro-industrial activities (livestock and an intense production of horticultural products), in addition to being used for leisure.The climate of the basin is tropical with two well-defined seasons, dry and rainy, belonging to the Cerrado biome 28 .

Genome mining for secondary metabolites and DAPG cluster distribution. The website version
of the antiSMASH 6.0 software was used to identify putative secondary metabolite clusters in the genomes of C. vaccinii CR1, CR5, and DSM 25150 T29 .Clusters that were not reliably assigned in the antiSMASH 6.0 software were manually searched using BLAST software to determine a potential function.To determine the distribution of the DAPG cluster, the cluster (identified as GenBank LQR3212800..LQR32_12830) was used as a reference.These loci were manually searched using BLAST software against all available genomes (as of June 1, 2022) in the Chromobacterium genus.In addition to the biosynthetic genes of the DAPG cluster (phlACBDE), we searched for the regulatory gene associated with the cluster (phlF, phlG, phlH) commonly found in Pseudomonas strains, accession: CM001558 was used as a reference.

Phylogenetic analysis.
The core genome for the Chromobacterium genus was determined and aligned using the BIGSdb program 30 .Aquitalea magnusonii DSM 23134 T was included as an outgroup.The phylogenetic tree was constructed using MEGA 11 software 31 .The neighbor-joining tree was determined using the Tamura-Nei model (0.40, gamma distributed with invariant sites).Measures of bootstrap support for internal branches were obtained from 1000 pseudoreplicates.For the PhlD phylogeny, the PhlD protein was identified in the genome of C. vaccinii CR1 and searched using BLAST software against all available non-redundant proteins in GenBank.All proteins available (9) in the Chromobacterium genus were included in the alignment.Due to the large number of Pseudomonas proteins available, only a representative sample (11) were included.A Streptomyces sp. protein (PhiD) was used as an outgroup.All alignments were performed using MUSCLE implemented under MEGA 11 31 .The alignment was model tested in MEGA 11 and the Maximum Likelihood method with WAG model was selected.Measures of bootstrap support for internal branches were obtained from 1000 pseudoreplicates.

Construction of phlACB expression vector.
Genomic DNA was extracted from 1 ml of an overnight culture of C. vaccini DSM 25150 T as previously described by 32 .The putative phlACB cluster (~ 2.8 KB) was amplified from C. vaccini DSM 25150 T genomic DNA using the primers cluster F (ATG AAG AAG GCA GGC ATA GTG AGC TAT GGC AG) and cluster R (ATC TTC CAG CAC GAA CTT GTA GGC GTA TTG CCA) using Invitrogen Platinum SuperFi II DNA polymerase (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions.The gel purified PCR product was reamplified with new primers containing the restriction sites Nco I (5ʹ end) and Hind III (3ʹ end) for cloning into the pET28b expression vector.Following ligation, three independent expression clones with the phlACB cluster were identified from plasmids that had been transformed into E. coli DH 5α.The correct sequence of the amplified phlACB cluster in each clone was verified by comparison to the genome sequence of C. vaccini CR1 following sequencing of each clone using a MiSeq DNA sequencer.The plasmid clones phlACB.1, phlACB.3 and phlACB.5, as well as empty vector pET28b, were individually transformed into BL21 E. coli.

Metabolism of DAPG.
Each E. coli BL21 phlACB cell line, as well as the control BL21 cells with pET28b, was grown overnight in LB broth with kanamycin (50 µg/ml).The overnight culture was then used to inoculate M9 minimal medium (200 µl of inoculum into 10 ml media) with kanamycin (50 µg/ml) and grown at 37 °C with shaking (180 RPM) until the OD 600 reached 0.6.Afterwards, each culture received 1 mg of DAPG (dissolved in ethanol, Cayman Chemical, Ann Arbor, Michigan) and Isopropyl-β-d-1-thiogalactopyranoside (IPTG) for a final concentration of 0.2 mM, and incubated at 30 °C Aliquots of the cultures were removed at various time points and stored at 4 °C.

LC-MS analysis of Chromobacterium metabolites.
For each time point (1, 2, 3, 4, and 7 days) and each media condition (LB or KMB), 5 μL of Chromobacterium culture media was analyzed by LC-MS using a Vanquish HPLC equipped with an ODS-18 column (3 mm × 150 mm, 3 μm particle size, Inertsil, GL Sciences, Inc., Torrance, CA) elution of analytes was accomplished by a linear gradient form 5:95-95:5% 18 MΩ water + 0.1% formic acid: Optima grade methanol + 0.1%formic acid at a flow rate of 0.550 ml/min over 30 min at 50 °C.Column effluent was monitored by UV at wavelengths 254 nm, 280 nm, and 270 nm prior to introduction into an Orbitrap ID-X-tribrid mass spectrometer (ThermoElectron, West Palm Beach, CA) under Xcalibur 4.4 control.Mass spectral data from 200 to 2000 Da m/z were collected in the orbitrap at 120,000 resolution.
www.nature.com/scientificreports/Tandem mass spectral data of target metabolites was collected using higher-energy collision dissociation (HCD, CE = 25 and 35) in the Orbitrap analyzer.

Analysis of E. coli cultures expressing phlACB.
Aliquots of E. coli media at times 8, 20.5, 28.5, 48, and 68 h from three transformants (pET28-phlACB-1, -3, -5) and one plasmid-only control (pET28) were centrifuged at 10,000 RPM for 10 min.A sample of the supernatant (250 μL) was removed and diluted with 250 μL 18 MΩ water.A portion of this dilution (5 μL) was analyzed by LC-MS under the same LC conditions listed above, with the only modification to the mass spectrometric analyses was to lower the mass of detection to 100-1000 Da m/z to allow for the detection of phloroglucinol and MAPG.Authentic standards for DAPG, and phloroglucinol (Acros Organics, Geel, Belgium) were analyzed at 100 μg/ml to establish retention times and MS 2 fragmentation patterns of expected products.Based on the manufacturer's specifications the level of detection of these analytes is approximately 100 pg/ml under these conditions.

Results
Genome mining environmental strains of Chromobacterium vaccinii.Chromobacterium vaccinii strains CR1 and CR5 were originally isolated from aquatic environments in the state of Goiás, Brazil 26 .The type strain of C. vaccini DSM 25150 T was also included in the analysis 33 .Table 1 shows five clusters were found across all three strains.The antiSMASH software was only able to assign the function of the violacein and the potential DAPG clusters.A large 68.4 kb trans-AT-PKS cluster was identified using BLAST software to be the biosynthetic cluster of cyclic depsipeptide FR900359 34 .The final two small clusters (6.9 and 8.6 kb) possess domains that suggests they are probable siderophores.

Distribution of biosynthetic clusters in the Chromobacterium genus.
The initial discovery of the potential DAPG biosynthetic cluster in genomes of select C. vaccinii strains prompted the exploration of the distribution of this cluster in the Chromobacterium genus, given the historical importance of this molecule that is normally found in Pseudomonas spp.strains 12 .After a broader investigation using all available Chromobacterium genomes, the cluster was identified in 11 genomes in the genus that appeared in three main clades (Fig. 1).The results show the cluster was found in all C. vaccinii genomes and a closely related Chromobacterium piscinae strain.A second clade containing the cluster was identified in Chromobacterium sphagni.The third clade was consists of Chromobacterium amazonense and a closely related, recently described new species of Chromobacterium sinusclupearum 35 .The cluster had a broad geographical distribution with seven of the strains from the United States [36][37][38] , two from Brazil 26 , one from China (CP022344), and one from Malayasia 39 .
All the biosynthetic genes of the DAPG cluster (phlACBDE) were found in all of the Chromobacterium strains reported to contain the cluster.Interestingly, the Chromobacterium strains do not contain all the regulatory genes associated with this cluster in Pseudomonas strains.Typically, the DAPG cluster in Pseudomonas strains contain the phlF, phlG and phlH genes, which are known to regulate the expression of the cluster 12 .The Chromobacterium strains only contain the phlF gene and lack the phlG and phlH genes, which suggests they utilize a different regulatory mechanism than in Pseudomonas strains.
Additionally, the biosynthetic cluster for the depsipeptide FR900359 was also confined to the C. vaccinii genomes and one very closely related strain, C. piscinae XC0014.The violacein biosynthetic cluster was found in all genomes in Fig. 1, except for the two Chromobacterium phragmitis genomes.

Phylogeny of DAPG cluster.
A phylogeny of the polyketide synthase protein PhlD, which is fundamental to the DAPG cluster, was created to understand the possible origin on the cluster.Figure 2 shows the cluster was closely related to strains in the Pseudomonas fluorescens group 40 .The average amino acid identity between the proteomes of C. vaccinii CR1 and the closest Pseudomonas proteome was 47% while the average amino acid identity between two DAPG clusters was 80%.In all Chromobacterium genomes containing the cluster, the cluster was in the same physical region of the genome and shared the same flanking regions.A survey of Chromobacterium genomes lacking the cluster did not find any alternative genes or clusters at this insertion site.

Confirmation of biosynthetic cluster assignment and activity.
To confirm the assignment and activity of the biosynthetic clusters LC-MS/MS was used to confirm the presence of the metabolites in culture.The three assigned biosynthetic clusters were violacein, depsipeptide FR900359, and DAPG. Figure 3 shows confirmation for the assignment of violacein and depsipeptide FR900359 using high accuracy mass spectrometry.For all three strains the violacein signal was strong and the depsipeptide FR900359 was much weaker.A www.nature.com/scientificreports/very weak signal for DAPG was observed in the strains, which was consistent with the retention time and mass spectrometry of a DAPG reference standard.Based on the results, it was assumed the DAPG cluster was active but not highly expressed under these culture conditions.We evaluated DAPG production under a variety of

Discussion
DAPG has been an important molecule in the bioactivities of rhizosphere colonizing Pseudomonas species and the subject of extensive investigations for 40 years 12 .It is for this reason we found it interesting to discover strains of C. vaccinii isolated from aquatic environments have the DAPG cluster 26 .The type strain of C. vaccinii and other representative isolates of C. vaccinii were originally isolated from the rhizosphere and water environments associated with native and cultivated cranberries (Vaccinium macrocarpon Ait.) 33 .This coincidence suggests that DAPG could provide a similar ecological function in Chromobacterium as seen in Pseudomonas species.
In Pseudomonas species, DAPG has long been known to suppress phytopathogens and other microbes in the rhizosphere 41 .In addition, it was shown that DAPG containing Pseudomonas strains were a key component of disease suppressive soils in controlling the wheat root disease "take-all decline" 5 .It is possible that DAPG in Chromobacterium may play a similar ecological role for plants in bogs or similar aquatic environments.It is also noteworthy that these genera are only distantly related, and the closest shared clade is at the phylum level (Proteobacteria) with Chromobacterium belonging to the class Betaproteobacteria and Pseudomonas belonging to the class Gammaproteobacteria.The distribution of the DAPG biosynthetic cluster in the Chromobacterium genus is confined to a few different clades.It is interesting to find all members of C. vaccinii, C. sphagni and C. sinusclupearum have this cluster (Fig. 1).However, these species have limited genome representation with 5, 2, and 2 whole genomes sequenced, respectively.This suggests there is a selection pressure to maintain the cluster in the species, notably in C. vaccinii where all strains are from diverse environments.The analysis also shows the DAPG cluster has broad geographical distribution with genomes from North America, South America, and Asia.In addition to the DAPG cluster, The depsipeptide FR900359 was found to be a powerful and selective inhibitor of G protein-coupled receptors (GPCR) in humans and other mammals 42,43 .GPCRs are important elements for intracellular signal transduction which respond to external ligand binding and initial response cascades through guanosine triphosphate and guanosine diphosphate exchange 44 .Now depsipeptide FR900359 and closely related compounds, collectively known as chromodepsins, are important pharmacological tools for use as therapeutics and tools to study GPCR function 45 .Interestingly, chromodespins were initially isolated from a plant (Ardisia crenata) used in traditional medicine 46 .Later, it was discovered the compound was actually produced by an endosymbiotic bacteria belonging to the genus Burkholderia 47 .The ecological role of this compound is not well understood.Chromodespins demonstrate a lack of inhibition toward plant GPCRs 43 .It has been suggested that they may provide a benefit to their plant host by acting as a herbivore deterrent 47 .Interestingly, chromodespins have been shown to have www.nature.com/scientificreports/insecticidal activity against bean bug (Riptortus pedestris), as well as inhibit the GPCRs of the pest insects white fly (Bemisia tabaci) and silk moth (Bombyx mori) 45 .This suggests strains of C. vacinnii may also be useful in the control of insect pests.All Chromobacterium genomes except the two C. phragmitis genomes contained the purple pigmented compound violacein.Violacein has been reported to have a variety of bioactive properties.It has been shown to have antifungal properties against a large variety of phytopathogenic and human fungal pathogens 48 .It has been reported to have antibacterial activities 49 and antinematicidal activities 50 .The compound showed antifeedant, larvicidal and pupicidal activities against Asian armyworm (Spodoptera litura Fab.) 51 .These broad-based bioactivities suggest that violacein may also contribute to the biocontrol potential of these Chromobacterium strains.
The phylogeny of the PhlD protein (Fig. 2) suggests all DAPG clusters in Chromobacterium share a common ancestor and the closest non-Chromobacterium ancestor likely originated in the Pseudomonas flourescens group.From the phylogenetic tree it is difficult to definitively infer if the cluster had one transfer event into Chromobacterium and some descendent lineages lost the biosynthetic cluster or if other horizontal gene transfer (HGT) events occurred within the Chromobacterium genus (e.g. a HGT from C. vaccinii to C. sinusclupearum).
In wild type C. vaccinii isolates, the production of DAPG could only be detected in trace amounts.Efforts to increase the production of DAPG through screening a variety of media conditions and carbon sources were unsuccessful.It may be possible that molecules in aquatic environments induce the biosynthesis of DAPG in Chromobacterium.Nevertheless, we confirmed that the biosynthetic genes in Chromobacterium for DAPG production were intact and functional.Our results also suggest the regulatory mechanism of Chromobacterium strains is significantly different than Pseudomonas strains since they lack the known regulatory genes phlG and phlH 12 .In addition, they lack the Pseudomonas post translational regulatory genes rsmA and rsmE (data not shown) 12 .More experiments will need to be performed in the future to discover the regulatory mechanism of DAPG production in Chromobacterium.We assumed the lack of activity was due to the lack of expression of these genes.We transferred the phlACB cluster into E. coli to have direct control over the expression of these genes.Initial testing of the transformant failed to produce DAPG from phloroglucinol.This is consistent with previous observations that expression of the phlACB cluster in E. coli produced no or trace amounts of DAPG 2,52 .However, in past studies the reverse reaction, conversion of DAPG to MAPG and phloroglucinol was active 2,52 .Monitoring of the reverse reaction showed a time-dependent decrease in the amount of DAPG present in the media when phlACB was expressed.There was an increase in the UV 270nm at retention times corresponding to the exact mass of MAPG and phloroglucinol (Fig. 4E) demonstrating the cluster could convert DAPG to MAPG and phloroglucinol.It should be noted in experiments where E. coli expressing phlACB transformants were incubated with phloroglucinol in an attempt to produce DAPG, the phloroglucinol used contained a small DAPG (< 1%) impurity.When incubated with the E. coli expressing phlACB transformants, this DAPG impurity was converted to phloroglucinol and no reaction was observed in the direction of DAPG formation.It is unknown why phlACB transformants favored the production of phloroglucinol and the biotransformation to DAPG did not occur.However, Liu et al. 52 recently demonstrated enhanced production of DAPG in E. coli expressing phlACB transformants through co-expression of acc, marA and phlE genes.
A survey of literature identified a few examples of strains of Chromobacterium being evaluated as potential biocontrol agents to control plant pathogens [22][23][24][25] .Two of the studies use Chromobacterium haemolyticum C-61, which displayed strong antifungal activities against several plant pathogens 23,24 .These studies identified another novel cyclic lipopeptide which they named chromobactomycin, which was responsible for the observed activity 24 .The genome is available for this strain, and it does not contain the DAPG cluster.Han et al. identified Chromobacterium sp.JH7 during a screening assay to identify antagonists of Cylindrocarpon destructans, a root rot pathogen of ginseng [22][23][24][25] .The authors were not able to identify the strain to the species level.It is possible that DAPG or depsipeptide FR900359 may be associated with this activity, but there is currently no data to support this speculation.In a recent study, C. vaccinii was shown to inhibit several fungal plant pathogens through emission of bioactive volatiles [22][23][24][25] .We could not find any reported DAPG activity in Chromobacterium sp. in the literature.
Our results confirm the presence and activity of the DAPG biosynthetic cluster in the Chromobacterium genus.These findings and the limited investigations of the biocontrol activity against plant pathogens by the Chromobacterium genus suggests additional research in this area is warranted.

Figure 1 .
Figure 1.Distribution of DAPG and depsipeptide FR900359 biosynthetic clusters in Chromobacterium genus.The phylogeny is based on the core genome of the strains listed in the figure.Measures of bootstrap support for internal branches were obtained from 1000 pseudoreplicates.Aquitalea magnusonii DSM 23134 T was used as an outgroup and not shown.

Figure 2 .
Figure 2. Maximum likelihood phylogeny of PhlD protein from the DAPG biosynthetic cluster.Measures of bootstrap support for internal branches were obtained from 1000 pseudoreplicates.