Investigation of the mechanism of xenoautoxin induced autolysis in bacteria

Abstract

Natural products (NPs) are chemical compounds isolated from sources like plants, bacteria, and fungi, often produced by secondary metabolic pathways. These compounds, particularly from bacteria, play essential roles in drug discovery due to their complex structures and interactions with biological targets, leading to various therapeutic activities such as anticancer and antibiotic effects. In this project, we worked with a symbiotically associated entomopathogenic bacteria, Xenorhabdus doucetiae that produces several natural products essential for the infection and death of a wide range of insects. We focused on two NPs produced by X. doucetiae, xenoautoxin and xenoamicin, of which xenoautoxin has interesting autolysis properties that we wanted to primarily investigate. To study the NPs, we generated various genomic mutants (promoter exchanges and fluorescent fusions) of X. doucetiae and established a standardized protocol for genomic editing. We confirmed mutant functionality through mass spectrometry and fluorescence microscopy. We were able to observe specific localization of the xenoautoxin NRPS (non-ribosomal peptide synthetase) in the bacterial cell and could contrast it with a homogenous distribution for the xenoamicin NRPS. We also observed homogenous localization of the xenoautoxin NRPS when the inducible promoter was moved in front of the xaxB gene suggesting that localisation of xenoautoxin NRPS is dependent on either the presence of XaxA or XaxABC complex. To explore the autolysis mechanism, we aimed to identify the conditions for xenoautoxin or xenoamicin production by developing reporter strains linked to protein or mRNA production. Plate reader experiments with the common soil microbe Bacillus subtilis showed no significant activation, indicating that further analysis of other conditions is needed to understand the expression of xenoautoxin or xenoamicin.