Novel Modifications of Nonribosomal Peptides from Brevibacillus laterosporus MG64 and Investigation of Their Mode of Action

Abstract

Nonribosomal peptides (NRPs) are a class of secondary metabolites usually produced by microorganisms. They are of paramount importance in different applications, including biocontrol and pharmacy. Brevibacillus spp. are a rich source of NRPs yet have received little attention. In this study, we characterize four novel bogorol variants (bogorols I to L, cationic linear lipopeptides) and four succilins (succilins I to L, containing a succinyl group that is attached to the Orn₃/Lys₃ in bogorols I to L) from the biocontrol strain Brevibacillus laterosporus MG64. Further investigation revealed that the bogorol family of peptides employs an adenylation pathway for lipoinitiation, different from the usual pattern, which is based on an external ligase and coenzyme A. Moreover, the formation of valinol was proven to be mediated by a terminal reductase domain and a reductase encoded by the bogI gene. Furthermore, succinylation, which is a novel type of modification in the family of bogorols, was discovered. Its occurrence requires a high concentration of the substrate (bogorols), but its responsible enzyme remains unknown. Bogorols display potent activity against both Gram-positive and Gram-negative bacteria. Investigation of their mode of action reveals that bogorols form pores in the cell membrane of both Gram-positive and Gram-negative bacteria. The combination of bogorols and relacidines, another class of NRPs produced by B. laterosporus MG64, displays a synergistic effect on different pathogens, suggesting the great potential of both peptides as well as their producer B. laterosporus MG64 for broad applications. Our study provides a further understanding of the bogorol family of peptides as well as their applications.IMPORTANCE NRPs form a class of secondary metabolites with biocontrol and pharmaceutical potential. This work describes the identification of novel bogorol variants and succinylated bogorols (namely, succilins) and further investigates their biosynthetic pathway and mode of action. Adenylation domain-mediated lipoinitiation of bogorols represents a novel pathway by which NRPs incorporate fatty acid tails. This pathway provides the possibility to engineer the lipid tail of NRPs without identifying a fatty acid coenzyme ligase, which is usually not present in the biosynthetic gene cluster. The terminal reductase domain (TD) and BogI-mediated valinol formation and their effect on the biological activity of bogorols are revealed. Succinylation, which is rarely reported in NRPs, was discovered in the bogorol family of peptides. We demonstrate that bogorols combat bacterial pathogens by forming pores in the cell membrane. We also report the synergistic effect of two natural products (relacidine B and bogorol K) produced by the same strain, which is relevant for competition for a niche.

Keywords: Brevibacillus laterosporus; biosynthesis; bogorol variants; lipoinitiation; mode of action; reduction; succilins; succinylation; synergy.

FIG 1

Bogorols and succilins produced by B. laterosporus MG64. (A) Gene cluster of bogorols harbored by B. laterosporus MG64. Green, core biosynthetic gene; blue, transport-related gene; gray, unknown gene; red, additional biosynthetic gene. (B) Structures of bogorols and succilins characterized by LC-MS/MS and NMR. Amino acid residues at positions 3 to 5 (red shaded) of different peptides are shown in the table below the structures. (C) Two-dimensional (2D) NMR correlations (red, ¹H-¹H TOCSY; blue, ¹H-¹H NOESY) confirming succinylation at the third amino acid residue (Lys) of succilin K (see also Fig. S3 in the supplemental material).

FIG 2

Adenylation domain-mediated lipoinitiation of bogorol peptides. (A) Production of bogorols by the wild-type (WT) and ΔbogJ mutant strains of B. laterosporus LMG15441. Strains were grown in MEM broth for 48 h and extracted with a C₁₈ column filled with silica gel. The extracted products were subsequently subjected to HPLC. (B) Phylogenetic tree of A domains from the bogorol gene cluster harbored by B. laterosporus MG64 and reference A domains that incorporate hydroxy acid. Auriporcine was retrieved from B. laterosporus PE36, while cereulide and valinomycin were retrieved from the miBIG database. The A domains activating alpha-hydroxy acids are indicated in red. (C) Usual pattern of fatty acid incorporation in NRPs. CoA, coenzyme A; FACL, fatty acid coenzyme ligase. (D) Proposed pattern of bogorols incorporating fatty acid.

FIG 3

Formation of valinol in bogorol peptides. (A and B) Bogorols and their intermediates (aldehyde form) produced by wild-type (A) and ΔbogI mutant (B) strains of B. laterosporus LMG15441. The masses (daltons, based on MALDI-TOF analysis) of the three major compounds produced by each strain are indicated. (C) Two-step formation of alcohol-form amino acids in the bogorol family of peptides. (D) Spot-on-lawn assay of aldehyde-form and alcohol-form bogorols. X. campestris pv. campestris NCCB92058 was used as an indicator. Each compound was added at the same amount, namely, 10 μl of a 200-μg/ml stock.

FIG 4

Production of bogorols and succilins by B. laterosporus MG64 at different time points. Fresh cells (10-μl culture with an OD₆₀₀ of 1.0) were inoculated in 50 ml MEM broth and grown at 28°C for 14 h, 16 h, 18 h, and 20 h. The compounds were extracted from the supernatant at each time point and analyzed by HPLC and MALDI-TOF mass spectrometry.

FIG 5

Mode of action employed by the bogorol family of peptides. (A) Growth curves of S. aureus in the presence of bogorol K and LTA. (B) Permeation of bogorol K into the cell membrane of S. aureus. (C) Growth curve of X. campestris in the presence of bogorol K and LPS. (D) Permeation of bogorol K into the cell membrane of X. campestris. (E and F) Permeation of bogorol K and succilin K into the outer membrane (E) and inner membrane (F) of X. campestris. The permeabilization of the outer membrane is indicated by the staining of NPN in the hydrophobic part, while PI staining indicates inner membrane permeabilization. The MIC values of bogorol K and succilin K are around 2.5 μM and 20 μM, respectively. Con, control; BogK, bogorol K; PlyB, polymyxin B; SucK, succilin K.