OmpR Indirectly Regulates Biosynthesis of Xenocoumacin 1 in Xenorhabdus nematophila

Abstract

Xenorhabdus nematophila has excellent potential for application in both medicine and agriculture due to its various active secondary metabolites. The transcriptional regulator OmpR negatively regulates Xenocoumacin 1 (Xcn1), which has wide antimicrobial activity. Here, we expressed and purified OmpR and verified its binding activities to promoters via an electrophoretic mobility shift assay. RNA sequencing was used to analyze the relevance and difference of differentially expressed genes between X. nematophila and its mutant ΔompR. Compared with the WT, 1127 differentially expressed genes were found in ΔompR, while 4150 co-expressed genes were detected. RT-qPCR data validated the RNA-seq results with 20 randomly selected genes. OmpR positively regulates the process of porphyrin metabolism, quorum sensing, β-Lactam resistance and glyoxylate and dicarboxylate metabolism, while negatively regulating the phosphotransferase system, two-component system and bacterial chemotaxis. OmpR indirectly regulates the biosynthesis of Xcn1 by positively regulating the process of glyoxylate metabolism, which consumes energy and precursors, and negatively regulates biomacromolecules biosynthesis, which provides energy and precursors. Overall, this work revealed the indirect effects of OmpR on the biosynthesis of Xcn1, serving as a foundation for future research into the intricate regulatory network of X. nematophila.

Keywords: EMSA; OmpR; Xenorhabdus nematophila; transcriptome; xenocoumacin 1.

Figure 1

Heterologous expression of OmpR of X. nematophila YL001. (a) Vector containing ompR for OmpR labeled with 6 histidine. (b) Verification of recombinant vectors for protein expression by PCR using primers 28a-F/R. (c) Purified protein OmpR-N_6His (29.52 kDa) pointed by purple arrows.

Figure 2

Verification of OmpR binding to promoter of various genes. LPAP, lithium potassium acetyl phosphate. The numbers in brackets represent the position of DNA in the genome of X. nematophila YL001. (a) OmpR bound to the promoter of opnP. (b) OmpR did not bind to the promoter of xcnA. (c) OmpR did not bind to the promoter of lrhA. (d) OmpR did not bind to the promoter of leuO. (e) OmpR did not bind to the promoter of cpxR. (f) OmpR did not bind to the promoter of ompR.

Figure 3

Construction and verification of strains ΔompR. (a) Graphical overview of gene knockout, including primers (yellow arrow) and location of ompR (cyan box) and kanR (red box). (b) Fragments for construction of ΔompR. Up: upstream of ompR (1038 bp); KanR: kanamycin-resistant cassette (941 bp); Down: downstream of ompR (990 bp). (c) Identification of the ΔompR with internal and external primers: 454 bp PCR products were generated by ompR-in-F/R with wild type (wt) as template, while no products were generated with ΔompR as template; 1118 bp PCR products were generated by primers ompR-out-F/R with wt as template; 1424 bp PCR products were generated with ΔompR as template.

Figure 4

Correlation and differentially expressed genes analysis based on RNA-Seq. (a) Correlation of gene expression levels between X. nematophila YL001 (WT) and ΔompR. (b) Principal component analysis (PCA) of gene expression levels between WT and ΔompR. (c) Venn diagram of co-expressed genes between WT and ΔompR. (d) Volcano map of differentially expressed genes between ΔompR and WT. (e) Differentially expressed genes of RNA-Seq in ΔompR were validated by RT-qPCR.

Figure 5

Gene Ontology (GO) enrichment analysis of differentially expressed genes. Abscissa represents the GO term that was significantly enriched, and ordinate represents the significance level of the GO term enriched. A higher value of ordinate indicates that the term was enriched more significantly. Different colors represent the three subclasses: biological process (BP, red), cellular component (CC, green) and molecular function (MF, blue). (a) Enriched terms of decreased genes in the ΔompR. (b) Enriched terms of increased genes in the ΔompR.

Figure 6

KEGG pathway enrichment analysis of differentially expressed genes. The abscissa is the ratio of the number of DEGs annotated to the KEGG pathway and the total number of DEGs, and the ordinate is the KEGG pathway. The number of DEGs was displayed by the size of the dots. The color of the dots gradually changes from purple to red, indicating that the pathway was enriched more significantly. (a) KEGG pathway enrichment analysis of down-regulated genes in ΔompR. (b) KEGG pathway enrichment analysis of up-regulated genes in ΔompR.

Figure 7

OmpR regulates biosynthesis of Xcn1 indirectly in X. nematophila. Green arrows represent positive regulation, and red T-shaped arrows represent negative regulation.