A Conserved Nonribosomal Peptide Synthetase in Xenorhabdus bovienii Produces Citrulline-Functionalized Lipopeptides

Abstract

The entomopathogenic bacterium Xenorhabdus bovienii exists in a mutualistic relationship with nematodes of the genus Steinernema. Free-living infective juveniles of Steinernema prey on insect larvae and regurgitate X. bovienii within the hemocoel of a host larva. X. bovienii subsequently produces a complex array of specialized metabolites and effector proteins that kill the insect and regulate various aspects of the trilateral symbiosis. While Xenorhabdus species are rich producers of secondary metabolites, many of their biosynthetic gene clusters remain uncharacterized. Here, we describe a nonribosomal peptide synthetase (NRPS) identified through comparative genomics analysis that is widely conserved in Xenorhabdus species. Heterologous expression of this NRPS gene from X. bovienii in E. coli led to the discovery of a family of lipo-tripeptides that chromatographically appear as pairs, containing either a C-terminal carboxylic acid or carboxamide. Coexpression of the NRPS with the leupeptin protease inhibitor pathway enhanced production, facilitating isolation and characterization efforts. The new lipo-tripeptides were also detected in wild-type X. bovienii cultures. These metabolites, termed bovienimides, share an uncommon C-terminal d-citrulline residue. The NRPS lacked a dedicated chain termination domain, resulting in product diversification and release from the assembly line through reactions with ammonia, water, or exogenous alcohols.

Global analysis of biosynthetic gene clusters reveals conserved and unique natural products in entomopathogenic nematode-symbiotic bacteria

“…20 ). Identical compounds, termed bovienimides, as well as a recognition of the BGC conservativeness, were reported by the Crawford laboratory 65 during revision of this manuscript.…”

Metabolomics and Genomics Enable the Discovery of a New Class of Nonribosomal Peptidic Metallophores from a Marine Micromonospora

“…Although we cannot rule out other enzymatic contributions (details in the SI, Section 8.6), the absence of a thioesterase domain invokes alternative release mechanisms; one of these entails hydrolytic or alcoholysis pathways likely involving methanol and long chain fatty alcohols in this case. This has been observed in the biosynthesis of bovienimides, NRPs from the entomopathogenic bacterium Xenorhabdus bovienii . Perhaps noteworthy is that, despite extensive data set searching, we have never been able to detect C-terminal free acid variants of ecteinamine; we hypothesize that either the observed ecteinamines are extremely stable, their acid forms (if generated) are unstable, or the acid forms are present in vanishingly small quantities that elude detection.…”

Analyses ofXenorhabdus griffiniaegenomes reveal two distinct sub-species that display intra-species variation due to prophages

“…9; Table S3 in Additional File 1). For example, the lpcS and isnAB,GT BGCs in the ID10 genome are predicted to encode the production of group IIA bovienimides [98] and rhabduscin [99], respectively, both of which are insect immunity suppressors, the pxb BGC which encodes the production of the insecticidal photoxenobactins [20], and safA-H, ioc/leu, xvbA-J, bcmA-G, and ape BGCs which respectively encode the production of safracin antibiotics, 3isopropyl-4-oxo-2-oxetanecarboxylic acid (IOC), benzobactins, bicyclomycins, and aryl polyenes [20,[100][101][102]. The gxpS BGC was predicted to encode the synthesis of GameXPeptide C [103], as their predicted peptide sequence was D Val-L Val-D Leu-L Val-L Leu.…”