Identification of a solo acylhomoserine lactone synthase from the myxobacterium Archangium gephyra

Abstract

Considered a key taxon in soil and marine microbial communities, myxobacteria exist as coordinated swarms that utilize a combination of lytic enzymes and specialized metabolites to facilitate predation of microbes. This capacity to produce specialized metabolites and the associated abundance of biosynthetic pathways contained within their genomes have motivated continued drug discovery efforts from myxobacteria. Of all myxobacterial biosynthetic gene clusters deposited in the antiSMASH database, only one putative acylhomoserine lactone (AHL) synthase, agpI, was observed, in genome data from Archangium gephyra. Without an AHL receptor also apparent in the genome of A. gephyra, we sought to determine if AgpI was an uncommon example of an orphaned AHL synthase. Herein we report the bioinformatic assessment of AgpI and discovery of a second AHL synthase from Vitiosangium sp. During axenic cultivation conditions, no detectible AHL metabolites were observed in A. gephyra extracts. However, heterologous expression of each synthase in Escherichia coli provided detectible quantities of 3 AHL signals including 2 known AHLs, C8-AHL and C9-AHL. These results suggest that A. gephyra AHL production is dormant during axenic cultivation. The functional, orphaned AHL synthase, AgpI, is unique to A. gephyra, and its utility to the predatory myxobacterium remains unknown.

Figure 1

(A) Cluster 32 from A. gephyra deposited in the antiSMASH database which includes the putative AHL synthase, agpI. (B) Genomic context for vitI from Vitiosangium sp. All annotated features within NCBI are labelled and all hypothetical features are in grey. Percentage GC content for each gene within the cluster provided for comparison and depicted in parentheses.

Figure 2

(A) Alignment of LuxI synthases including AgpI and VitI with conserved residues boxed in black. (B) Alignment of myxobacterial features homologous to AgpI with asterisks (*) indicating conserved residues associated with the LasI autoinducer domain (COG3916) and red lines (-) indicating residues from the LasI domain that are not conserved. (C) Minimum Evolution tree including AgpI and VitI rendered in MEGA7 using ClustalW aligned with AHL synthases experimentally confirmed to produce AHLs (68). Branch lengths ≤ 0.2 not depicted.

Figure 3

Molecular family from the molecular network of LC–MS/MS datasets from extracts of heterologous E. coli expressing AgpI rendered by GNPS (50). Detected m/z values from raw data positioned over each node with node diameter depicting associated intensities for each AHL.

Figure 4

MS/MS fragmentation spectra with diagnostic fragments indicated for each AHL detected in extracts from heterologous E. coli expressing AgpI.