Abstract

Xenorhabdus nematophila produces a wealth of specialized metabolites with promising agricultural and medical applications, among which Xenocoumacin 1 (Xcn1) is a key antifungal secondary metabolite. The transcriptional regulatory mechanisms governing Xcn1 biosynthesis, however, remain incompletely characterized, particularly the direct regulatory links between the two-component system response regulator CpxR and the LysR-type transcriptional regulator LrhA. Here, we combined in vitro protein-DNA binding assays, mutant construction, and transcriptomic analysis to dissect the coordinated regulatory roles of CpxR and LrhA in X. nematophila YL001. Recombinant LrhA and CpxR were heterologously expressed and purified; electrophoretic mobility shift assays demonstrated that LrhA directly binds to the promoters of xcnA, lrhA, leuO, and ompR, whereas CpxR targets the promoters of xcnA, lrhA, and opnP. Notably, the position of His-tag modification critically impacts LrhA’s DNA-binding activity—C-terminal tagging abrogated binding capacity, while N-terminal tagging preserved it. Deletion mutants were constructed via homologous recombination, and RNA sequencing coupled with bioinformatics analysis revealed that LrhA and CpxR exert opposing regulatory effects on overlapping core pathways: LrhA positively regulates flagellar assembly and bacterial chemotaxis, whereas CpxR negatively modulates these processes. GO and KEGG enrichment analyses further uncovered distinct regulatory roles of the two regulators in carbohydrate transport and amino acid metabolism. Collectively, our findings establish that CpxR directly activates lrhA transcription, and LrhA directly represses xcnA expression, forming a regulatory cascade that fine-tunes Xcn1 biosynthesis. This study elucidates the sophisticated transcriptional regulatory network mediated by CpxR and LrhA in X. nematophila, providing a theoretical basis for exploiting this bacterium and its bioactive metabolites for biotechnological app