Identification and Characterization of the Anti-Methicillin-Resistant Staphylococcus aureus WAP-8294A2 Biosynthetic Gene Cluster from Lysobacter enzymogenes OH11

Abstract

Lysobactor enzymogenes strain OH11 is an emerging biological control agent of fungal and bacterial diseases. We recently completed its genome sequence and found it contains a large number of gene clusters putatively responsible for the biosynthesis of nonribosomal peptides and polyketides, including the previously identified antifungal dihydromaltophilin (HSAF). One of the gene clusters contains two huge open reading frames, together encoding 12 modules of nonribosomal peptide synthetases (NRPS). Gene disruption of one of the NRPS led to the disappearance of a metabolite produced in the wild type and the elimination of its antibacterial activity. The metabolite and antibacterial activity were also affected by the disruption of some of the flanking genes. We subsequently isolated this metabolite and subjected it to spectroscopic analysis. The mass spectrometry and nuclear magnetic resonance data showed that its chemical structure is identical to WAP-8294A2, a cyclic lipodepsipeptide with potent antimethicillin-resistant Staphylococcus aureus (MRSA) activity and currently in phase I/II clinical trials. The WAP-8294A2 biosynthetic genes had not been described previously. So far, the Gram-positive Streptomyces have been the primary source of anti-infectives. Lysobacter are Gram-negative soil/water bacteria that are genetically amendable and have not been well exploited. The WAP-8294A2 synthetase represents one of the largest NRPS complexes, consisting of 45 functional domains. The identification of these genes sets the foundation for the study of the WAP-8294A2 biosynthetic mechanism and opens the door for producing new anti-MRSA antibiotics through biosynthetic engineering in this new source of Lysobacter.

Antibiotics from neglected bacterial sources

“…Although the precise mode of action has not been published, previous studies have hinted at an interaction with bacterial membrane phospholipids as the mechanism for the bactericidal activity of WAP-8294A2 (Kato et al, 1998). The biosynthetic pathway for these cyclic lipodepsipeptides was recently identified in Lysobacter enzymogenes OH11 and confirmed what had previously been assumed, that WAP-8294A2 is the product of a 12 module NRPS (Zhang et al, 2011b). As with the enacyloxins (see above), it is conceivable that engineering of the WAP-8294A pathway may lead to further derivatives with altered or better anti-infective properties (Xie et al, 2012).…”

“…Many species within this order are plant pathogens and others, such as several Lysobacter species have been used as biocontrol agents for fungal plant infections (Zhang et al, 2011b). Although research into antibiotic compounds produced by xanthomonads is an emerging area compared to many other bacteria, Lysobacter spp.…”

Transcriptomic analysis reveals new regulatory roles of Clp signaling in secondary metabolite biosynthesis and surface motility in Lysobacter enzymogenes OH11

“…In this genus, L. enzymogenes , first proposed by Christensen and Cook in 1978, is most extensively studied and is emerging as a potentially novel biocontrol agent against pathogens of crop plants (Giesler and Yuen 1998; Qian et al 2009). The antagonistic nature of this bacterium is thought to be due at least in part to the production of a large number of lytic enzymes capable for degrading the cell walls of pathogens (Christensen and Cook 1978; Kobayashi et al 2005) and to the antimicrobial metabolites HSAF (dihydromaltophilin; heat-stable and broad-spectrum antifungal compound with a novel mode of action) (Li et al 2006; Yu et al 2007) and the cyclic lipodepsipeptide WAP-8294A2, which also exhibits anti-MRSA (methicillin-resistant Staphylococcus aureus ) activity (Zhang et al 2011). In addition to its antagonism, L. enzymogenes directly attaches itself to the hypha of fungal pathogens and subsequently causes a pathogenic infection to the fungal host (Mathioni et al 2013; Patel et al 2011).…”

Lysobacter enzymogenes Uses Two Distinct Cell-Cell Signaling Systems for Differential Regulation of Secondary-Metabolite Biosynthesis and Colony Morphology

“…The production of lytic enzymes, such as chitinases, proteases, and glucanases, is tightly linked to L. enzymogenes biocontrol activity. Recently, two antimicrobial secondary metabolites, including heat-stable antifungal factor (HSAF) and WAP-8294A2, have also been shown to be important in L. enzymogenes for growth inhibition of filamentous fungi and Gram-positive bacteria, respectively (31)(32)(33)(34).…”