Biosynthesis of Ditropolonyl Sulfide, an Antibacterial Compound Produced by Burkholderia cepacia Complex Strain R-12632

Abstract

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 multidrug resistant bacteria, with minimum inhibitory concentration (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 in phenylacetic acid (PAA) catabolism and one 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. Importance Burkholderia 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 ditropolonyl sulfide. This unusual antimicrobial has been poorly studied and its biosynthetic pathway remained 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 the phenylacetic acid catabolism, assimilatory sulfate reduction and glutathione metabolism are necessary for ditropolonyl sulfide production. These findings contribute to understanding the biosynthesis and biological role of this unusual antimicrobial.