The Xenorhabdus nematophila LrhA transcriptional regulator modulates production of γ-keto- N -acyl amides with inhibitory activity against mutualistic host nematode egg hatching

is a symbiotic Gammaproteobacterium that produces diverse natural products that facilitate mutualistic and pathogenic interactions in their nematode and insect hosts, respectively. The interplay between secondary metabolism and symbiosis stage is tuned by various global regulators. An example of such a regulator is the LysR-type protein transcription factor LrhA, which regulates amino acid metabolism and is necessary for virulence in insects and normal nematode progeny production. Here, we utilized comparative metabolomics and molecular networking to identify small molecule factors regulated by LrhA and characterized a rare γ-ketoacid (GKA) and two new -acyl amides, GKA-Arg ( ) and GKA-Pro ( ) which harbor a γ-keto acyl appendage. A null mutant produced elevated levels of compound and reduced levels of compound relative to wild type. -acyl amides and were shown to be selective agonists for the human G-protein-coupled receptors (GPCRs) C3AR1 and CHRM2, respectively. The CHRM2 agonist deleteriously affected the hatch rate and length of nematodes. This work further highlights the utility of exploiting regulators of host-bacteria interactions for the identification of the bioactive small molecule signals that they control. bacteria are of interest due to their symbiotic relationship with nematodes and their ability to produce a variety of natural bioactive compounds. Despite their importance, the regulatory hierarchy connecting specific natural products and their regulators is poorly understood. In this study, comparative metabolomic profiling was utilized to identify the secondary metabolites modulated by the global regulator LrhA. This analysis led to the discovery of three metabolites, including an -acyl amide that inhibited the egg hatching rate and length of nematodes. These findings support the notion that LrhA influences the symbiosis between and through -acyl amide signaling. A deeper understanding of the regulatory hierarchy of these natural products could contribute to a better comprehension of the symbiotic relationship between and .