Burkholderia cepacia complex strain R-12632 produces ditropolonyl sulfide, an unusual sulfur-containing tropone, via a yet-unknown biosynthetic pathway. Ditropolonyl sulfide purified from a culture of strain R-12632 inhibits the growth of various Gram-positive and Gram-negative resistant bacteria, with MIC values as low as 16 μg/ml. In the present study, we used a transposon mutagenesis approach combined with metabolite analyses to identify the genetic basis for antibacterial activity of strain R-12632 against Gram-negative bacterial pathogens. Fifteen of the 8304 transposon mutants investigated completely lost antibacterial activity against Klebsiella pneumoniae LMG 2095. In these loss-of-activity mutants, nine genes were interrupted. Four of those genes were involved in assimilatory sulfate reduction, two were involved in phenylacetic acid (PAA) catabolism, and one was involved in glutathione metabolism. Via semipreparative fractionation and metabolite identification, it was confirmed that inactivation of the PAA degradation pathway or glutathione metabolism led to loss of ditropolonyl sulfide production. Based on earlier studies on the biosynthesis of tropolone compounds, the requirement for a functional PAA catabolic pathway for antibacterial activity in strain R-12632 indicated that this pathway likely provides the tropolone backbone for ditropolonyl sulfide. Loss of activity observed in mutants defective in assimilatory sulfate reduction and glutathione biosynthesis suggested that cysteine and glutathione are potential sources of the sulfur atom linking the two tropolone moieties. The demonstrated antibacterial activity of the unusual antibacterial compound ditropolonyl sulfide warrants further studies into its biosynthesis and biological role.
IMPORTANCEBurkholderia bacteria are historically known for their biocontrol properties and have been proposed as a promising and underexplored source of bioactive specialized metabolites. Burkholderia cepacia complex strain R-12632 inhibits various Gram-positive and Gram-negative resistant pathogens and produces numerous specialized metabolites, among which is ditropolonyl sulfide. This unusual antimicrobial has been poorly studied and its biosynthetic pathway remains unknown. In the present study, we performed transposon mutagenesis of strain R-12632 and performed genome and metabolite analyses of loss-of-activity mutants to study the genetic basis for antibacterial activity. Our results indicate that phenylacetic acid catabolism, assimilatory sulfate reduction, and glutathione metabolism are necessary for ditropolonyl sulfide production. These findings contribute to understanding of the biosynthesis and biological role of this unusual antimicrobial.
Bacteria within the genus Burkholderia are well known for their diverse lifestyles and metabolism (1). The Burkholderia cepacia complex (Bcc) is a group of closely related species that is notorious for causing infections in immunocompromised individuals, such as persons with cystic fibrosis (CF) (2). Its taxonomy is complex and continuously evolving and at present, the Bcc consists of 23 validly named species (3–5), with two additional species names awaiting validation (6, 7). Besides their association with CF patients, Bcc bacteria are most frequently isolated from rhizosphere soil, where they demonstrate biopesticidal activity toward phytopathogens (8). This ability to protect crops from bacterial and fungal pathogens has been attributed to the production of antibiotic or toxic specialized metabolites (1, 9). Examples of antifungal metabolites include pyrrolnitrin (10), phenazines (11), cepaciamides (12), xylocandins (13), occidiofungins (14), burkholdines (15), and cepacins (16). The production of such bioactive specialized metabolites is often achieved through large multimodular enzymes, such as polyketide synthetases (PKS) and nonribosomal peptide synthases (NRPS).
Several studies have shown that Bcc bacteria also produce specialized metabolites with antibacterial activity against important human pathogens. When grown on a minimal medium with glycerol as the sole carbon source, Burkholderia ambifaria AMMDT produced enacyloxin, an antibiotic with activity against Gram-negative bacteria such as Acinetobacter baumannii and Burkholderia multivorans (17). Enacyloxin is the product of an unusual PKS assembly line and its production is controlled through quorum sensing (QS). Activity against other Burkholderia species was also observed for ubonodin, a novel and unusually long lasso peptide from Burkholderia ubonensis (18). Finally, “Pseudomonas mesoacidophila” ATCC 31433 was recently identified as a member of the Bcc most closely related to B. ubonensis (19) and produces the monobactam antibiotic sulfazecin and the β-lactam potentiating bulgecins (20).
In a previous study (21), we examined the production of antimicrobial compounds by a diverse collection of Burkholderia bacteria and observed several Bcc strains that inhibited multiple Gram-negative bacterial pathogens, including A. baumannii and carbapenem-resistant strains of Klebsiella pneumoniae, Enterobacter spp., and Escherichia coli. Strain R-12632, an unclassified soil isolate from the maize rhizosphere (22) that has been referred to as “Other Bcc group I” and that is closely related to Burkholderia cenocepacia (23), produced a total of 17 specialized metabolites, including pyrrolnitrin, ornibactins (C4, C6, and C8), pyochelin, aerugine, aeruginoic acid, dihydroaeruginoic acid, and ditropolonyl sulfide, and seven potentially novel molecules (21). Through semipreparative fractionation and activity testing, three molecules were found in fractions that inhibited the Gram-negative bacterial pathogens A. baumannii LMG 10520 and Citrobacter freundii R-67508. Whereas pyrrolnitrin showed only weak activity, the fraction containing a mixture of ditropolonyl sulfide and a putative novel molecule with the molecular formula C47H61N3O16 strongly inhibited growth of both pathogens (21). Although ditropolonyl sulfide was previously reported to inhibit Gram-negative bacterial pathogens (24), the pathway for its biosynthesis remains to be elucidated.
The present study aimed to identify genes responsible for the observed antibacterial activity of strain R-12632 through a combination of transposon mutagenesis and genome and specialized metabolite analyses.
Screening a transposon mutant library for loss of antibacterial activity.
A library of 8304 transposon mutants was generated and the corresponding methanol extracts were screened for loss of inhibition of K. pneumoniae LMG 2095. Mutants showing a loss of activity (n = 658) or reduced activity (n = 139) against this indicator pathogen were retested using an overlay assay (Fig. 1). A total of 15 mutants showed complete loss of antibacterial activity, and an additional 22 mutants showed reduced activity (Table 1). Inhibition zone diameters for wild-type strain R-12632 and the 37 mutants with reduced or no antibacterial activity are presented in Table S2 in the supplemental material.
TABLE 1 Transposon mutants of B. cepacia complex strain R-12632 with loss of or reduced inhibitory activity against K. pneumoniae LMG 2095a
Mutants indicated in bold type were selected for comparative metabolite analysis. KO, KEGG Orthology; APS, adenosine 5′-phosphosulfate; NA, not available; CoA, coenzyme A.
Whole-genome sequencing and identification of the transposon insertion site.
The complete genome sequence of the wild-type strain R-12632 was 8.11 Mbp in size and consisted of three large replicons (3.58 Mbp, 3.05 Mbp, and 1.35 Mbp) and one smaller plasmid (140 kb) with a total of 7,191 protein-coding genes. The R-12632 genome was analyzed using antiSMASH to identify known specialized metabolite gene clusters. A total of 20 biosynthetic gene clusters were predicted, of which 5 were located on the first replicon, 8 on the second replicon, 7 on the third replicon, and none on the plasmid (Table 2). Two NRPS clusters responsible for the production of the siderophores ornibactin and pyochelin were identified, as well as the biosynthetic genes for pyrrolnitrin production. The third replicon included one PKS gene cluster and two large hybrid NRPS-PKS gene clusters, none of which could be linked to the production of a known compound in the database. Finally, the genome of strain R-12632 contained six clusters encoding terpenes, three encoding bacteriocins, two encoding aryl polyenes, and a single cluster for each of the following functional classes: lasso peptide, ectoine, homoserine lactone, and phosphonate (Table 2).
TABLE 2 Genomic specialized metabolite potential of B. cepacia complex strain R-12632a
Most similar known cluster
The genome of strain R-12632 was analyzed using antiSMASH v5.1.0 (57). Similarity refers to the sequence similarity of the detected gene cluster toward a gene cluster present in the antiSMASH database. NRPS, nonribosomal peptide synthase; HSL, homoserine lactone; T1PKS, type 1 polyketide synthetase.
In the 15 mutants that lost antibacterial activity, nine genes were disrupted by a transposon insertion (Table 1). Two mutants with an identical insertion site were identified for BCCR12632_00471, encoding an enoyl coenzyme A (enoyl-CoA) hydratase, and BCCR12632_02547, encoding a 5′-phosphosulfate (APS) reductase. Five mutants with distinct insertion sites were identified for BCCR12632_02955, encoding a glutathione synthetase. Through comparison with KEGG metabolic pathways, BCCR12632_02545, BCCR12632_02546, BCCR12632_02547, and BCCR12632_02549 were identified as part of the assimilatory sulfate reduction pathway, while BCCR12632_00250 and BCCR12632_00471 are involved in phenylacetic acid (PAA) catabolism (Table 1). BCCR12632_00396 is part of a type VI secretion system that is encoded by BCCR12632_03388 through BCCR12632_00404. While BCCR12632_00424 could not be linked to any known metabolic pathway, the gene immediately downstream (BCCR12632_00423) is predicted to encode a glutaredoxin.
In the 22 mutants with reduced antibacterial activity, 10 genes were disrupted (Table 1). Multiple mutants with unique insertion sites were identified for BCCR12632_00209 (n = 2), BCCR12632_00598 (n = 4), BCCR12632_02540 (n = 2), BCCR12632_02888 (n = 2), and BCCR12632_05920 (n = 2). BCCR12632_02540 encodes a cytosol aminopeptidase and is involved in glutathione metabolism. BCCR12632_00598, BCCR12632_02305, BCCR12632_02740, and BCCR12632_05920, encoding a glutamate dehydrogenase, an NADH:ubiquinone oxidoreductase subunit, a succinyl-CoA synthetase subunit, and a malate dehydrogenase, respectively, could be linked to energy metabolism (Table 1). For the five other genes, no link with known metabolic pathways was found.
Semipreparative fractionation of crude extracts to detect ditropolonyl sulfide.
Results from our earlier study (21) suggested that ditropolonyl sulfide was likely responsible for the observed antibacterial activity of strain R-12632. That study also showed that ditropolonyl sulfide could not be detected in crude agar extracts of strain R-12632 and that semipreparative fractionation combined with liquid chromatography–high-resolution mass spectrometry (LC-HRMS) was necessary to detect this compound. Therefore, wild-type strain R-12632 and three loss-of-activity mutants (R-75390, R-75501, and R-75718) were subjected to semipreparative fractionation to detect ditropolonyl sulfide production. Each selected mutant was impaired in a different metabolic pathway: glutathione metabolism (R-73590), PAA catabolism (R-75501), and assimilatory sulfate reduction (R-75718) (Table 1).
Wild-type strain R-12632 produced a characteristic green-brown pigment when grown on BSM-G (basal salts medium supplemented with glycerol) agar, which was not observed in any of the loss-of-activity mutants (Fig. S2). Mutant R-75718 was unable to grow on BSM-G agar, but supplementation of the growth medium with 0.5 mM l-cysteine allowed growth and restored characteristic green-brown pigment production and antibacterial activity similar to those of the wild-type (Table S2).
In wild-type strain R-12632, ditropolonyl sulfide presented as a sharp UV peak with characteristic absorbance at 400 nm and with a retention time between 19 and 20 min (Fig. 2). No characteristic absorbance at 400 nm was observed in any of the fractions of loss-of-activity mutants R-75501 and R-75390, and the ion with exact mass of 275.037 m/z corresponding to ditropolonyl sulfide was not detected (Fig. 2). In mutant R-75718 grown on BSM-G plus cysteine, ditropolonyl sulfide presented as a broad UV peak with characteristic absorbance at 400 nm and with a retention time between 17 and 20 min (Fig. 2).
Determining the MIC and MBC of ditropolonyl sulfide for 13 resistant bacterial pathogens.
To confirm its antibacterial activity, the MIC and minimum bactericidal concentration (MBC) of purified ditropolonyl sulfide were determined for 13 resistant bacterial pathogens. Ditropolonyl sulfide demonstrated antibacterial activity against K. pneumoniae LMG 2095 and against carbapenem-resistant strains or colistin-resistant strains of A. baumannii, C. freundii, and E. coli (MIC = 16 to 64 μg/ml) (Table 3). Antibacterial activity was also observed against vancomycin intermediate-resistant Staphylococcus aureus (VISA) Mu50 and vancomycin-resistant Enterococcus faecium (VRE) LMG 17188 (Table 3). Bactericidal activity was observed toward K. pneumoniae LMG 2095 (MBC = 32 μg/ml), A. baumannii R-67512 (MBC = 64 μg/ml), and all colistin-resistant strains tested (MBC = 64 μg/ml); all other pathogens had an MBC above 128 μg/ml (Table 3).
TABLE 3 MICs and MBCs for ditropolonyl sulfide against 13 resistant bacterial pathogens
A. baumannii LMG 10520
A. baumannii R-67512
C. freundii R-67508
C. freundii 2200212
Low-level colistin resistant
E. coli R-67506
E. coli 1101443
Low-level colistin resistant
E. coli CD_R02067
High-level colistin resistant
K. pneumoniae LMG 2095
K. pneumoniae AN0127/CP
High-level colistin resistant
K. pneumoniae SA0523
Low-level colistin resistant
P. aeruginosa PAO1
E. faecium LMG 17188
S. aureus Mu50
Methicillin resistant, vancomycin intermediate
Results from a previous study showed that Bcc strain R-12632 inhibited several important Gram-negative bacterial pathogens and suggested that ditropolonyl sulfide might be responsible for the observed activity (21). Ditropolonyl sulfide production was also reported for six additional Burkholderia isolates, including B. cepacia LMG 1222T and B. cenocepacia IIID R-1474 (21). Although antibacterial activity has been reported for this molecule, the genes involved in its biosynthesis have not been identified. The present study aimed to identify the genetic basis for the observed antibacterial activity of strain R-12632 through a transposon mutagenesis approach. Fifteen mutants completely lost antibacterial activity against K. pneumoniae LMG 2095, while 22 additional mutants showed a reduced activity compared to that of wild-type strain R-12632 (Table 1 and Table S2).
Of the 19 transposon-disrupted genes, 13 could be linked to a known metabolic pathway: assimilatory sulfate reduction (n = 4), glutathione metabolism (n = 2), PAA catabolism (n = 2), type VI secretion system (n = 1), or energy metabolism (n = 4) (Table 1). Although the genome of the wild-type strain R-12632 harbors several multimodular biosynthetic gene clusters, including one PKS and two hybrid NRPS-PKS gene clusters representing potentially novel specialized metabolite biosynthetic gene clusters (BGCs) (Table 2), none of these clusters were disrupted in the 37 transposon mutants with reduced or no antibacterial activity. These clusters are therefore unlikely to be responsible for the observed antibacterial activity against K. pneumoniae LMG 2095. It is also possible that many of these specialized metabolite BGCs are not expressed under the growth conditions used in the present study, meaning that disruptions in those BGCs could not be detected using a phenotype-based screen such as antimicrobial activity tests.
Among the 15 loss-of-activity mutants, four transposon-disrupted genes were identified as part of the assimilatory sulfate reduction pathway. In prokaryotes, sulfate reduction can be either dissimilatory or assimilatory. Whereas dissimilatory sulfate reduction occurs in anaerobic organisms, which use sulfate as a terminal electron acceptor for respiration, assimilatory sulfate reduction is carried out by aerobic organisms and is necessary for the production of the amino acid cysteine (25). Although the two reduction pathways are similar in outline, several differences can be observed. The first step in both assimilatory and dissimilatory sulfate reduction is the activation of sulfate by adenylation in a reaction catalyzed by ATP sulfurylase to generate APS (Fig. 3). The latter protein consists of two subunits, CysN and CysD, which are encoded in the genome of R-12632 by the genes BCCR12632_02545 and BCCR12632_02546, respectively. In sulfate dissimilators, APS is directly reduced to sulfite by the enzyme APS reductase. Sulfate assimilators such as E. coli, however, require phosphorylation of APS to 3′-phosphoadenosine 5′-phosphosulfate (PAPS) through an APS kinase (CysC) before the reduction to sulfite can be carried out by PAPS reductase (CysH) (Fig. 3). Although the ability to reduce APS directly was long considered a trait exclusive to dissimilatory sulfate-reducing bacteria and plants, other sulfate-assimilating bacteria were shown to harbor CysH homologs with greater amino acid sequence homology to plant APS reductases than to PAPS reductases (26). That study demonstrated that CysH homologs of both B. cepacia DBO1 and P. aeruginosa PAO1 directly use APS as the substrate for sulfate reduction without the need for the phosphorylated PAPS intermediate. A similar cysH homolog (BCCR12632_02547) was identified in the genome of strain R-12632.
Further steps in assimilatory sulfate reduction involve the reduction of sulfite to sulfide by sulfite reductase (CysI, encoded by BCCR12632_02549) and the incorporation of sulfide into cysteine (Fig. 3). In the present study, mutants with transposon insertions in cysN, cysD, cysH, or cysI, together encoding the first three enzymatic steps in assimilatory sulfate reduction, showed a complete loss of activity against K. pneumoniae LMG 2095 (Table 1). The lack of growth of these mutants on a minimal medium containing inorganic sulfate salts as the sole sulfur source confirms that the assimilatory sulfate reduction pathway is nonfunctional in these mutants. When this minimal medium was supplemented with 0.5 mM l-cysteine, growth and antibacterial activity of these mutants was restored (Table S2). Supplementation of the minimal medium with 0.5 mM l-methionine supported growth, but pigment production and antibacterial activity were not observed (Table S2).
Five loss-of-function mutants with distinct insertion sites were identified for BCCR12632_02955, encoding glutathione synthetase (Table 1). Glutathione (γ-l-glutamyl-l-cysteinyl-glycine) is a highly abundant nonprotein thiol found in all eukaryotic cells, most Gram-negative bacteria, and some Gram-positive bacteria. Its main function is to maintain the redox balance within the cell, but it also has an important role in protecting the cell against low pH, chlorine compounds, and oxidative and osmotic stress and it serves as a cysteine reserve (27). The first step in glutathione biosynthesis involves the addition of glutamic acid to cysteine to form γ-glutamylcysteine and is catalyzed by γ-glutamylcysteine synthetase (GshA) (Fig. 3). In the second step, glycine is added by glutathione synthetase (GshB) to form the complete tripeptide. In the present study, five transposon mutants of R-12632 with unique insertion sites in gshB (BCCR12632_02955) completely lost antibacterial activity against K. pneumoniae LMG 2095 (Table 1 and Table S2). Remarkably, none of the mutants with loss of or reduced activity had a disruption of gshA (BCCR12632_02956), although this is considered the rate-limiting step in glutathione biosynthesis (27). Since BCCR12632_02955 (gshB) is located at the 3′ end of its operon, polar effects of this insertion on BCCR12632_02956 (gshA) are unlikely. It is possible that gshA is an essential gene in strain R-12632 or that BCCR12632_03267, a gshA homolog present elsewhere in the genome of R-12632, can act as a functional homolog.
The PAA catabolic pathway represents a third metabolic pathway for which several genes were disrupted in loss-of-activity mutants of strain R-12632 (Table 1). The PAA catabolic pathway is the central route for degradation of many aromatic compounds, such as styrene, phenylalanine, phenylacetyl amide, and phenylalkanoates, which are converted via phenylacetyl-CoA (PAA-CoA) to Krebs cycle intermediates (28). The organization of genes involved in PAA catabolism varies between organisms. In B. cenocepacia K56-2 they are organized into three operons, two of which are located on the first replicon and the third on the second replicon (29). The same three-operon structure was also observed in the genome of strain R-12632 (Fig. 4). Following activation of PAA by PAA-CoA ligase (PaaK), ring hydroxylation is catalyzed by a five-membered monooxygenase complex (PaaABCDE) to yield an unstable epoxide intermediate (30) (Fig. 4). In strain R-12632, this five-membered complex is encoded in the genes BCCR12632_00250 through BCCR12632_00254, of which the paaE gene (BCCR12632_00250) was disrupted in 1 of the 15 loss-of-activity mutants (Table 1). As BCCR12632_00250 (paaE) is located at the 3′ end of its operon, polar effects of the transposon insertion on the expression of the paaABCD genes upstream are unlikely.
The unstable epoxy intermediate is then converted to an oxepin intermediate through an enoyl-CoA isomerase PaaG, encoded by BCCR12632_00471 (Fig. 4). Two transposon mutants with identical insertion sites in the paaG gene also lost inhibitory activity against K. pneumoniae LMG 2095 (Table 1). However, polar effects of a transposon insertion in the paaG gene could also affect the expression of two downstream genes, BCCR12632_00470 (paaI) and BCCR12632_00469 (paaK1) (Fig. 4). The next step in PAA catabolism is ring opening of the oxepin intermediate, followed by β-oxidation-like steps to finally yield acetyl-CoA and succinyl-CoA, which are intermediates of the Krebs cycle (30). When the PAA catabolic pathway was studied in B. cenocepacia K56-2, a paaE mutant exhibited severely reduced growth when PAA was presented as a sole carbon source, indicating that disruption of paaE interferes with PAA catabolism (29). Since paaG acts directly downstream of the paaABCDE complex, it is reasonable to assume that the PAA catabolic pathway of strain R-12632 is nonfunctional in both the paaE and paaG transposon mutants.
The ring opening of the oxepin intermediate, catalyzed by PaaZ, has been proposed as a metabolic branching point in bacteria, serving as the starting material for tropone-like antibiotic compounds (31). Tropones are unusual seven-membered nonbenzenoid aromatic rings and one of the simplest tropones, tropolone, was identified as a virulence factor in the plant pathogen Burkholderia plantarii (32). In our previous study, strain R-12632 was found to produce ditropolonyl sulfide (21), which consists of two tropolone moieties linked together by a sulfur atom (Fig. 4). Although the biosynthesis of tropolone and tropone-related compounds has not been thoroughly investigated in Burkholderia bacteria, several studies have elucidated the molecular basis for production of tropone derivatives in other organisms. Marine bacteria from the Roseobacter clade have been reported to produce tropodithietic acid (TDA), a sulfur-containing tropone compound with antibacterial activity (33).
To identify the genes responsible for TDA biosynthesis, Geng et al. (34) employed transposon mutagenesis of Silicibacter sp. strain TM1040 and found 12 genes critical for the production of this compound. Three of those genes were involved in PAA catabolism, encoding parts of the PaaABCDE monooxygenase complex, and one other gene, cysI (encoding sulfite reductase), was involved in sulfur metabolism (34). These findings are strikingly similar to what was observed in the present study, in which both paaE and cysI transposon mutants lost activity against K. pneumoniae LMG 2095 (Table 1). In addition, loss of TDA production in Silicibacter sp. TM1040 was accompanied by a loss of yellow-brown pigment production, which was also seen during the present study (Fig. S2). Finally, Geng et al. (34) observed that cysteine, but not methionine, could restore TDA production and antimicrobial activity of the cysI mutant. The ability of cysteine, but not methionine, to restore pigment production and antimicrobial activity was also observed in the present study for cysN (R-75718), cysD (R-75500), cysH (R-75588), and cysI (R-75388) mutants (Table S2).
The biosynthesis of tropodithietic acid was also studied in Phaeobacter inhibens, in which the tdaB gene in the plasmid-borne tdaA-tdaF gene cluster was found to encode a glutathione S-transferase predicted to catalyze the addition of sulfur to the tropone backbone in the form of S-thiocysteine (35). This indicated that sulfur metabolism, in particular synthesis of cysteine and glutathione, is essential for tropodithietic acid production in P. inhibens, and the same appears to be true for the production of some antibacterial metabolites in strain R-12632. The additional requirement of a functional PAA catabolic pathway for antibacterial activity led us to hypothesize that ditropolonyl sulfide is indeed responsible for the observed inhibitory activity of strain R-12632.
To confirm this hypothesis, crude agar extracts of wild-type strain R-12632 and three loss-of-activity mutants cultivated on BSM-G (or BSM-G plus cysteine in the case of R-75718) were subjected to semipreparative fractionation and the resulting fractions were analyzed via LC-HRMS to determine the presence or absence of ditropolonyl sulfide. The observed characteristic absorbance at 400 nm and the detection of the ion with exact mass of 275.037 m/z confirmed the presence of ditropolonyl sulfide in semipreparative fractions of wild-type strain R-12632 (Fig. 2). Ditropolonyl sulfide was not detected in the two loss-of-activity mutants R-75390 and R-75501 (Fig. 2). For mutant R-75718 cultured on BSM-G plus cysteine, ditropolonyl sulfide was detected in several fractions (Fig. 2), highlighting the importance of the sulfur metabolism, in particular the amino acid cysteine, for ditropolonyl sulfide production.
In light of studies on the biosynthesis of other sulfur-containing tropone compounds, the lack of antibacterial activity and ditropolonyl sulfide production (Fig. 2) in transposon mutants defective in PAA catabolism and sulfur metabolism (Table 1) suggests that both pathways are necessary for the biosynthesis of this compound by strain R-12632. Although the PAA catabolism could provide the tropone backbone, none of the genes affected in loss-of-activity mutants can provide a clue as to how the two tropolone moieties become linked by a sulfur atom. Bacterial sulfur-containing specialized metabolites are rather uncommon, and the incorporation of sulfur is known to proceed through only a few mechanisms (36). One possibility is the incorporation of a sulfur-containing amino acid such as cysteine or methionine by an NRPS assembly line. Although these amino acids are underrepresented as NRPS monomers (37), cysteine is an important building block of antimicrobials such as penicillins and cephalosporins (38), bacitracin (39), and siderophores such as pyochelin (40), in which the highly reactive thiol group is involved in cyclization with another monomer.
Another way to incorporate sulfur into natural products is found in sulfated molecules such as the mycobacterial sulfolipids (41), the monobactam antibiotic sulfazecin (42), and the coproduced bulgecins from “Pseudomonas mesoacidophila” ATCC 31433 (20). In these cases, sulfotransferase enzymes catalyze the addition of sulfate, and PAPS, an intermediate in the assimilatory sulfate reduction, acts as the activated source of sulfur. Although Burkholderia bacteria possess a CysH homolog that can directly use APS as a substrate without the need for the phosphorylated intermediate PAPS (26), an APS kinase gene (cysC) was identified in the genome of strain R-12632 (BCCR12632_00700), suggesting that this strain is capable of PAPS production as well (Fig. 3). While this exemplifies the metabolic diversity of Burkholderia bacteria, and strain R-12632 in particular, the presence of a sulfide rather than a sulfite group in ditropolonyl sulfide suggests that PAPS is unlikely to be the source of sulfur in this molecule.
Finally, glutathione is known to act as a sulfur donor in specialized metabolism, as is the case for allicin and gliotoxin biosynthesis (36), although known examples seem to be restricted to eukaryotes. However, in the case of TDA biosynthesis, the protein TdaB, with homology to glutathione S-transferases, was proposed to catalyze the addition of S-thiocysteine, rather than glutathione, to the tropone backbone (35). The fact that no tdaB homologs were identified in the genome of strain R-12632 is not surprising, since the tdaA-tdaF gene cluster shows poor sequence conservation even among members of the Roseobacter clade (34). Nevertheless, the latter mechanism of sulfur addition to a troponoid 7-membered ring is perhaps the most plausible biosynthetic route for the production of ditropolonyl sulfide.
The simultaneous lack of ditropolonyl sulfide production and loss of antibacterial activity in transposon mutants R-75390 and R-75501 strongly suggests that this molecule is responsible for the antibacterial activity of strain R-12632 against the Gram-negative pathogen K. pneumoniae. To confirm this, ditropolonyl sulfide was purified from wild-type strain R-12632 and MIC and MBC values toward 13 resistant bacterial pathogens were determined. Antibacterial activity was observed against both Gram-positive (S. aureus and E. faecium) and Gram-negative (K. pneumoniae, A. baumannii, C. freundii and E. coli) pathogens. Ditropolonyl sulfide showed bactericidal activity toward K. pneumoniae, a carbapenem-resistant A. baumannii strain, and all colistin-resistant strains tested (Table 3).
In the present study, we identified ditropolonyl sulfide as the main molecule responsible for the broad antibacterial activity in Bcc strain R-12632. Results obtained with transposon mutants lacking antibacterial activity and comparison with data from earlier studies indicated that the PAA catabolic pathway likely provides the tropolone backbone in ditropolonyl sulfide. Loss of activity observed in mutants defective in assimilatory sulfate reduction and glutathione biosynthesis suggested that cysteine and glutathione are potential sources of the sulfur atom linking the two tropolone moieties through a yet-uncharacterized mechanism. Its inhibition of multiple problematic Gram-negative bacterial pathogens warrants further investigation into the biosynthesis and the biological role of this unusual antimicrobial compound.
MATERIALS AND METHODS
Strains, media, and growth conditions.
B. cepacia complex strain R-12632 (also known as strain MVP-C1-16) was isolated from maize rhizosphere in Italy in 1996 (22). This strain and all derived mutants were routinely grown on tryptone soya agar (TSA; Oxoid, USA) at 37°C and maintained at −80°C in 15% glycerol. Super optimal broth (SOB) medium was used for preparation of electrocompetent cells and for electrotransformation of R-12632 (43). For the preparation of selective plates, a 50-μg/ml kanamycin (Sigma-Aldrich, USA) stock was freshly prepared in distilled water and filter sterilized using 0.22-μm cellulose acetate filters (Whatman, Germany). Both TSA and a basal salts medium supplemented with glycerol (BSM-G) (44) were used as the media for antimicrobial production during the specialized metabolite analyses. BSM-G had a pH of 7 and contained the following per liter: 3.24 g K2HPO4, 1.13 g NaH2PO4·2H2O, 2 g NH4Cl, 0.2 g MgSO4·7H2O, 0.1 g nitrilotriacetic acid, 4.7 g glycerol, 3 mg MnSO4·H2O, 3 mg ZnSO4·7H2O, 1 mg CoSO4·7H2O, 7.7 mg FeSO4·nH2O (n = 1.5), and 15 g agar. After autoclaving and cooling to 55°C, the BSM-G medium was supplemented with a filter-sterilized l-cysteine hydrochloride solution to a final concentration of 0.5 mM when necessary.
K. pneumoniae LMG 2095 was used for activity testing of the mutant library and was cultivated in cation-adjusted Mueller-Hinton II broth (MHB; BD, USA) for the liquid inhibition assay or Iso-Sensitest broth (ISB; Oxoid, UK) for the overlay assay, both at 37°C. Strains for MIC and minimum bactericidal concentration (MBC) determinations were cultivated in MHB at 37°C. These included A. baumannii LMG 10520, A. baumannii R-67512 (carbapenem resistant), C. freundii R-67508 (carbapenem resistant), C. freundii 2200212 (low-level colistin resistant), E. coli R-67506 (carbapenem resistant), E. coli 1101443 (low-level colistin resistant), E. coli CD_R02067 (high-level colistin resistant), K. pneumoniae LMG 2095, K. pneumoniae AN0127/CP (high-level colistin resistant), K. pneumoniae SA0523 (low-level colistin resistant), Enterococcus faecium LMG 17188 (vancomycin resistant), and Staphylococcus aureus Mu50 (vancomycin-intermediate methicillin resistant).
Generation of a transposon mutant library of R-12632.
(i) Preparation of electrocompetent cells. Electrocompetent cells of strain R-12632 were generated using a modified version of the protocol previously optimized for electrotransformation of B. cenocepacia J2315T (45). Two well-isolated colonies from a 24-h culture on TSA were inoculated in 5 ml SOB and incubated at 37°C and 200 rpm for 18 h. This overnight culture was diluted to an optical density at 590 nm (OD590) of 0.01 in SOB in an Erlenmeyer flask. The culture was incubated at 37°C and 200 rpm for 4 h to an OD590 of approximately 0.2. Next, the culture was cooled on ice for 5 min and centrifuged at 4°C and 5,000 × g for 8 min. The resulting cell pellet was carefully washed twice using ice-cold 0.5 M sucrose, followed by centrifugation as described above. The cells were resuspended in 500 μl ice-cold 0.5 M sucrose with 10% glycerol, aliquoted, and frozen at −80°C.
The EZ-Tn5 <R6Kγori/KAN-2> Tnp Transposome kit (Lucigen, USA) was used to generate transposon mutants of strain R-12632. An aliquot of 55 μl electrocompetent cells was mixed with 1 μl EZ-Tn5 <R6Kγori/KAN-2> Tnp Transposome in a 0.1-cm-gap electroporation cuvette (Sigma-Aldrich, USA) and subjected to a pulse of 1,800 V using the preset “Ec1” protocol on the MicroPulser electroporator (Bio-Rad, USA). Nuclease-free water was used as a negative control. The electroporated cells were immediately resuspended in 950 μl SOB supplemented with 20 mM glucose and incubated while standing at 37°C for 4 h to allow the expression of the kanamycin resistance gene. Cells were diluted 1:1 in SOB broth, plated onto selective SOB agar supplemented with 50 μg/ml kanamycin, and incubated at 37°C for 24 h. Colonies were transferred with sterile toothpicks to 96-well microtiter plates (MTPs; Greiner Bio-One, Austria) containing SOB and were incubated at 37°C for another 24 h. The resulting liquid cultures were preserved at −80°C in 15% glycerol in 96-well MTPs using a Viaflo 96-well pipetting robot (Integra Biosciences, Switzerland).
Screening of a transposon mutant library for loss of antibacterial activity.
(i) Preparation of agar extracts. Each step of the screening for loss of antibacterial activity was performed in 96-well MTPs. Frozen glycerol stocks of mutant cultures were thawed and 10-μl stock cell suspensions were inoculated into 190 μl SOB using the Viaflo 96-well pipetting robot and incubated at 37°C for 24 h. A 25-μl volume of this preculture was inoculated onto a 50-μl TSA agar plug using the Viaflo 96-well pipetting robot and incubated at 28°C for 4 days to allow for specialized metabolite production. Uninoculated SOB was used as a negative control (no growth and no antibacterial activity) and a liquid culture of strain R-12632 was used as a positive control for growth and strong antibacterial activity. Specialized metabolites were extracted from the 4-day cultures by adding 200 μl methanol per well. After 2 h of extraction, methanol extracts were transferred to new 96-well MTPs and left to air dry overnight. Dried extracts were resuspended in 40 μl sterile ultrapure water and immediately used in a liquid inhibition assay to determine the presence or absence of antibacterial activity.
(ii) Liquid inhibition assay.
Based on earlier results (21) (Table S1), K. pneumoniae LMG 2095 was chosen as an indicator organism to screen for loss of antibacterial activity in the transposon mutant library. The frozen glycerol stock of K. pneumoniae LMG 2095 was streaked on TSA for checking purity and incubated at 37°C for 24 h. Two well-separated colonies were used to inoculate 10 ml MHB which was incubated at 37°C overnight. This overnight culture was diluted in MHB to an OD590 of 0.1 and then again diluted 1:100 in MHB to obtain a suspension of approximately 5 × 105 cells/ml. In a 96-well MTP, a 90-μl volume of this cell suspension was mixed with 10 μl resuspended methanol extract and incubated at 37°C for 24 h. Noninoculated MHB served as a sterility control, an extract of the wild-type strain R-12632 was used as a positive control for antibacterial activity, and an extract of noninoculated growth medium was used as a negative control (no antibacterial activity).
Due to the inherent turbidity of the resuspended extracts, it was not possible to use turbidity alone to measure growth of the indicator pathogen K. pneumoniae LMG 2095. Therefore, a 10-μl volume of a freshly prepared filter-sterilized 0.125-mg/ml resazurin (Sigma-Aldrich, USA) stock was added to each well and plates were shaken at 200 rpm for 1 min and incubated at 37°C in the dark for 1 h. Plates were then examined visually and any color changes were noted. Resazurin is blue in its original oxidized state, but it is irreversibly converted to the pink compound resorufin through metabolic activity. Blue wells indicated an absence of pathogen growth and were interpreted as presence of antibacterial activity in the corresponding methanol extract. Pink or purple wells indicated pathogen growth and were interpreted as a reduction or absence of antibacterial activity in the corresponding extract.
(iii) Overlay assay.
Transposon mutants for which the methanol extract did not inhibit pathogen growth (i.e., resazurin was converted from blue to purple or pink) were tested for antibacterial activity using an overlay method as described earlier (21) to confirm and quantify the results. In short, transposon mutants were grown on TSA at 28°C for 48 h and were subcultured twice prior to performing the overlay assay. For each strain, a dense suspension (approximately 1 × 108 CFU/ml) was prepared and 3 μl was inoculated onto the center of a TSA plate. Plates were incubated at 28°C for 4 days to allow the production of antimicrobials and were then exposed to chloroform vapors for 5 min. A diluted overnight culture of the indicator strain K. pneumoniae LMG 2095 was added to 100 ml soft agar (ISB plus 1% agar) to obtain approximately 1 × 105 CFU/ml. Chloroform-exposed plates were overlaid with this inoculated soft agar and incubated at 37°C for 24 h. Antibacterial activity was scored by measuring the diameter of the inhibition zone, where no pathogen growth was observed. Loss of antibacterial activity was defined as the absence of an inhibition zone, whereas reduced activity was defined as an inhibition zone diameter that was at least 25% smaller than the inhibition zone produced by the wild-type strain R-12632.
Whole-genome sequencing and identification of the transposon insertion site.
Genomic DNA of strain R-12632 and 37 selected mutants (Table 1) was prepared from a 48-h culture grown at 28°C on TSA. Cells were harvested using a 10-μl inoculation loop and suspended in 300 μl 4 M guanidine isothiocyanate solution (Fisher Scientific, USA). DNA extraction was performed using the Maxwell 16 automated nucleic acid purification system using the Maxwell 16 tissue DNA purification kit (Promega, USA) according to the manufacturer’s instructions. DNA was quantified using the QuantiFluor ONE double-stranded DNA (dsDNA) quantification system (Promega). A total of 3 μg genomic DNA of wild-type strain R-12632 was subjected to polyethylene glycol precipitation (final concentration: 5.5% polyethylene glycol 8000 [PEG 8000] and 0.8 M NaCl) to remove fragments shorter than 1 kb. Library preparation was performed using the ligation sequencing kit 1D (SQK-LSK109) according to the manufacturer’s instructions (Oxford Nanopore Technologies, UK). The library was sequenced for 3 h on a MinION R9.4 flow cell (Oxford Nanopore Technologies) to obtain approximately 700 MB of raw data. Reads were base called using Guppy 3.0.3 (Oxford Nanopore Technologies), retaining only reads with a qscore of >7, and quality reports were generated using NanoPlot (46). Short reads were generated for strain R-12632 and the 37 selected mutants by sequencing 150-bp paired-end libraries on an Illumina NovaSeq 6000 sequencer (Oxford Genomics Centre—Wellcome Centre for Human Genetics, Oxford, UK). Short reads were trimmed using fastp with –qualified_quality_phred 30 –length_required 50 –correction options, and adapters were automatically trimmed (47). Quality reports were generated using FastQC (48).
A hybrid assembly of Illumina and Nanopore long reads was performed using Unicycler (49). In the first step, Unicycler was used to build an initial assembly graph from Illumina short reads by using the de novo assembler SPAdes with a wide range of k-mer sizes. Long Nanopore reads were then aligned to the short-read assembly graph to bridge gaps between individual contigs and resolve repeats. The resulting assembly was subjected to several rounds of polishing by Pilon (50) using the high-quality short reads to reduce errors. Annotation was performed using Prokka v1.12 (51) with a genus-specific database based on reference genomes from the Burkholderia Genome Database v8.1 (52). Further functional annotation of protein-coding genes was performed using the KEGG Automatic Annotation Server (53), and KEGG pathways were visually reconstructed using KEGG Mapper (54). To confirm the correct annotation and functionality of genes in KEGG pathways, amino acid sequences were checked for the presence of conserved functional domains using the HMMER Web server (55). Gene orientation and operon organization were inspected visually using the Integrated Genome Viewer (56). antiSMASH 5.1.0 was used to mine genomes for biosynthetic gene clusters responsible for the production of specialized metabolites, including PKS, NRPS, siderophores, and bacteriocins (57).
The trimmed Illumina reads for the 37 selected mutants (Table 1) were assembled using shovill (58) with subsampling to 80× coverage depth and retaining only contigs longer than 500 bp. Ragout v2.2 (59) was used to scaffold the obtained contigs using the complete genome of strain R-12632 as the reference genome. Annotation was performed as described above. The insertion site of the transposon was determined via BLAST (60) and visually examined using Integrated Genome Viewer (56).
Semipreparative fractionation of crude extracts to detect ditropolonyl sulfide.
Crude extracts of the wild-type strain R-12632 and three loss-of-activity mutants grown on BSM-G (R-75390 and R-75501) or BSM-G supplemented with l-cysteine (R-75718) were prepared as described previously (21). Extracts were dissolved in 100% dimethyl sulfoxide (DMSO) and filtered (0.45-μm pore size) prior to semipreparative fractionation using a Gilson GX-281 322H2 instrument (Gilson Technologies, USA). Each extract was subjected to semipreparative reversed-phase high-performance liquid chromatography (HPLC) (Zorbax SB-C18 column, 9.4 by 250 mm, 5-μm particle size, 3.6 ml/min, UV detection at 210 and 400 nm) eluting with acetonitrile/water, in a linear gradient from 5% to 100% acetonitrile in 45 min and yielding 1.8 ml per fraction every 0.5 min to generate 80 central fractions. Based on the retention time and the obtained UV absorbance at 210 and 400 nm, four to six fractions per sample were subjected to LC-HRMS as described above to determine the presence of ditropolonyl sulfide. The extracted-ion chromatogram at m/z 275 was examined for the ion with exact mass of 275.037 ± 0.005 m/z, corresponding to ditropolonyl sulfide.
Determining the MICs and MBCs of ditropolonyl sulfide for 13 resistant bacterial pathogens.
(i) Purification of ditropolonyl sulfide. For the extraction and purification of ditropolonyl sulfide, a dense suspension of wild-type strain R-12632 was prepared as described above. A total of 1.3 liters BSM-G agar, contained within 13 100-ml agar plates, was inoculated with this suspension and incubated at 28°C for 4 days. For each plate, the agar was ground, transferred to 500-ml glass flasks, and extracted by adding an equal volume of acetone (100 ml) and shaking at 220 rpm for 2 h. After centrifugation at 7,500 rpm for 10 min and filtration over paper in a Büchner funnel, the acetone extract was concentrated under a heated nitrogen stream to a final volume of 1.3 liters (100% water). The aqueous residue was loaded onto an SP207ss resin column (65 g, 100 by 22 mm) for reversed-phase flash fractionation with a 10% step gradient of acetone in water starting from 10% acetone to 100% acetone in 25 min at 10 ml/min. Aliquots of every flash fraction were analyzed and the presence of ditropolonyl sulfide was determined by LC-HRMS and the characteristic UV spectrum. Those fractions that presented the compound were dried, redissolved in 100% DMSO, and filtered (0.2-μm pore size) prior to HPLC fractionation using a Gilson GX-281 322H2 instrument (Gilson Technologies, USA). Enriched flash fractions were subjected to two preparative reversed-phase HPLC batches (Zorbax SB-C8 column, 21.2 by 250 mm, 7-μm particle size, 20 ml/min, UV detection at 210 and 280 nm) eluting with acetonitrile/water, in a linear gradient from 5 to 100% acetonitrile in 45 min, yielding 10 ml per fraction every 0.5 min to generate 80 central fractions. Enriched HPLC preparative fractions, as determined by LC-HRMS, were subjected to six semipreparative reversed-phase HPLC fractionation batches (Zorbax RX-C8 column, 9.4 by 250 mm, 5 μm particle size, 3.6 ml/min, UV detection at 210 and 280 nm) eluting with a linear gradient from 20 to 25% of acetonitrile in water for 35 min, yielding 10 ml per fraction every 0.5 min to generate 80 central fractions. Fractions were analyzed by LC-HRMS for the presence of ditropolonyl sulfide, which eluted in a sharp peak at 15 min with a broad long tail of 8 min. 1H nuclear magnetic resonance (NMR) was used to confirm the presence of ditropolonyl sulfide (Fig. S1) (61). The purification yielded, in total, 2 mg of the pure compound (98% purity).
Determination of MICs and MBCs.
MICs and MBCs of ditropolonyl sulfide against A. baumannii LMG 10520, A. baumannii R-67512, C. freundii R-67508, C. freundii 2200212, E. coli R-67506, E. coli 1101443, E. coli CD_R02067, K. pneumoniae LMG 2095, K. pneumoniae AN0127/CP, K. pneumoniae SA0523, Pseudomonas aeruginosa PAO1, E. faecium LMG 17188, and S. aureus Mu50 were determined using flat-bottom 96-well MTPs (TPP, Switzerland) as described previously (62). Ditropolonyl sulfide was dissolved in ultrapure Milli-Q water (Millipore, USA) and was filter sterilized using 0.22-μm cellulose acetate filters (Whatman, Germany). Ditropolonyl sulfide concentrations tested ranged from 0.25 to 128 μg/ml. Blank wells with corresponding concentrations of ditropolonyl sulfide were used to correct for absorbance of the compound. The lowest concentration of antibiotic for which a similar optical density was observed in the inoculated and the blank wells was recorded as the MIC. A 100-μl aliquot from the inoculated wells for which an optical density similar to that of the blank wells was observed was used to determine the presence of surviving cells by plating on MH agar. The MBC was defined as the lowest concentration for which no CFU were recovered after 48 h of incubation at 37°C.
The data sets generated for this study can be found in the European Nucleotide Archive under accession number PRJEB40633. The whole-genome sequence of strain R-12632 was deposited in GenBank under the accession numbers FR989675 (replicon 1), FR989676 (replicon 2), FR989677 (replicon 3), and FR989678 (plasmid).
E.D. was supported by fellowships from the Fund for Scientific Research Flanders (FWO; 1119517N and 1119519N). This work was carried out with the support of the Industrial Research Fund (IOF) of Ghent University (F2015_IOF_ConcepTT_142 and F2017_IOF_StarTT_208). The research leading to results presented in this publication was carried out with infrastructure funded by EMBRC Belgium—FWO project GOH3817N.
We thank Aurélien Carlier and Andrea Sass for helpful discussions and Petra Rigole for technical assistance. We thank Surbhi Malhotra and Herman Goossens (University of Antwerp, Belgium) for providing the colistin- and carbapenem-resistant strains.
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