Enzymatic resistance to the lipopeptide surfactin as identified through imaging mass spectrometry of bacterial competition

Abstract

Many species of bacteria secrete natural products that inhibit the growth or development of competing species. In turn, competitors may develop or acquire resistance to antagonistic molecules. Few studies have investigated the interplay of these countervailing forces in direct competition between two species. We have used an imaging mass spectrometry (IMS) approach to track metabolites exchanged between Bacillus subtilis and Streptomyces sp. Mg1 cultured together. Surfactin is a cyclic lipopeptide produced by B. subtilis that inhibits the formation of aerial hyphae by streptomycetes. IMS analysis exposed an addition of 18 mass units to surfactin in the agar proximal to Streptomyces sp. Mg1 but not other streptomycetes tested. The spatially resolved change in the mass of surfactin indicated hydrolysis of the molecule. We observed that the aerial growth of Streptomyces sp. Mg1 was resistant to inhibition by surfactin, which suggests that hydrolysis was a mechanism of resistance. To identify possible enzymes from Streptomyces sp. Mg1 with surfactin hydrolase activity, we isolated secreted proteins and identified candidates by mass spectrometry. We purified one candidate enzyme that hydrolyzed surfactin in vitro. We tested the role of this enzyme in surfactin resistance by deleting the corresponding gene from the S . Mg1 genome. We observed that aerial growth by the Δ sfhA mutant strain was now sensitive to surfactin. Our results identify an enzyme that hydrolyzes surfactin and confers resistance to aerial growth inhibition, which demonstrates the effective use of an IMS approach to track natural product modifications during interspecies competition.

More than Motility: Salmonella Flagella Contribute to Overriding Friction and Facilitating Colony Hydration during Swarming

“…In this model, overexpression of FlhDC may increase osmolyte production through alternate metabolic pathways, given that flagellar master regulators control the expression of other regulators (16,42), which in turn control a large number of genes, including those for sugar transport and general metabolism in different bacteria (46,58,59). The model can be tested by using new mass spectrometry methodologies to directly detect secreted metabolites within bacterial colonies (60,61), correlating their presence to a stalled, nonswitching, or rotating motor. In both models, the dry colony morphologies (absence of the "Swiss cheese" appearance) of fliL and motA mutants are explained by a role for flagellar rotation in hydration (Fig.…”

Multifaceted Interfaces of Bacterial Competition

“…Bacillus subtilis requires surfactin for biofilm development and some types of motility (27,37,38). Intriguingly, surfactin also antagonizes aerial development of many Streptomyces species (39,40). Insight into the mechanism arose from S. coelicolor, which when treated with surfactin was unable to process and secrete SapB to support aerial growth (41).…”

“…strain Mg1 hydrolyzes surfactin ( Fig. 1A and B) (40). The enzyme, surfactin hydrolase, was shown to specifically inactivate surfactin and plipastatin, another lipopeptide produced by B. subtilis (40).…”

“…In addition to SMs, bacteria secrete enzymes that participate in competition. Secreted enzymes that confer antibiotic resistance have a clear competitive benefit (40,42). Additionally, bacteria benefit by interfering with the development of their competitors, e.g., using enzymes to degrade signaling mol- ecules, like acyl homoserine lactones (48)(49)(50)(51).…”

“…1A and B) (40). The enzyme, surfactin hydrolase, was shown to specifically inactivate surfactin and plipastatin, another lipopeptide produced by B. subtilis (40). Hydrolytic inactivation is a common resistance mechanism for many antibiotics (42).…”