ELUCIDATING THE RESISTANCE MECHANISM OF MUROBACTIN ANTIBIOTICS

Abstract

Biosynthetic gene clusters (BGCs) encode secondary metabolites, such as antibiotics, which can be produced through bacterial interspecies competition. Cognate resistance genes to the produced antibiotic compounds are typically found within BGCs. Type V glycopeptides – now called murobactins – are a newly characterized sub-class of GPAs that exhibit an unprecedented mechanism of action through the inhibition of cell wall remodelling rather than biosynthesis. However, its cognate resistance cassette has not yet been identified. A putative cassette is present in several murobactin-producing strains; this is composed of a two-component regulatory system (TCS) homologous to vanRS, homologues to the FtsEX ATP-binding cassette family, one protein containing a peptidoglycan-binding domain, and one protein of unknown function. The resistance core lacks the predicted TCS. To confirm that the putative resistance cassette confers resistance to murobactin antibiotics, susceptible hosts were chosen, and constructs containing the resistance core of two murobactin producers and the cassette from one producer were synthesized. The minimum inhibitory concentration (MIC) of the murobactin complestatin was assessed in Streptomyces and Bacillus species, and there was no change in MIC with the introduction of the putative resistance cassette. Phenotypic analysis of Streptomyces species when treated with complestatin did not reveal a distinctive phenotype. When the complestatin resistance core was introduced into B. subtilis, there was slight restoration to a healthy phenotype in the presence of complestatin. MICs in murobactin producers, non-producers, and non-producers containing the putative resistance cassette also showed no resistance. As these genes are present across many species, they may contribute to cell division. Developing deletion mutants of these genes within the murobactin biosynthetic gene cluster could aid in understanding the purpose of this gene cluster. This research provides insight into the underlying resistance mechanism utilized by murobactin-producing bacteria and guides engineering approaches that use Streptomyces strains for future GPA development and discovery.