The Epipeptide YydF Intrinsically Triggers the Cell Envelope Stress Response of Bacillus subtilis and Causes Severe Membrane Perturbations

Abstract

The Gram-positive model organism and soil bacterium Bacillus subtilis naturally produces a variety of antimicrobial peptides (AMPs), including the ribosomally synthesized and post-translationally modified AMP YydF, which is encoded in the yydFGHIJ locus. The yydF gene encodes the pre-pro-peptide, which is, in a unique manner, initially modified at two amino acid positions by the radical SAM epimerase YydG. Subsequently, the membrane-anchored putative protease YydH is thought to cleave and release the mature AMP, YydF, to the environment. The AMP YydF, with two discreet epimerizations among 17 residues as sole post-translational modification, defines a novel class of ribosomally synthesized and post-translationally modified peptides (RiPPs) called epipeptides, for which the mode-of-action (MOA) is unknown. The predicted ABC transporter encoded by yydIJ was previously postulated as an autoimmunity determinant of B. subtilis against its own AMP. Here, we demonstrate that extrinsically added YydF * kills B. subtilis cells by dissipating membrane potential via membrane permeabilization. This severe membrane perturbation is accompanied by a rapid reduction of membrane fluidity, substantiated by lipid domain formation. The epipeptide triggers a narrow and highly specific cellular response. The strong induction of liaIH expression, a marker for cell envelope stress in B. subtilis, further supports the MOA described above. A subsequent mutational study demonstrates that LiaIH-and not YydIJ-represents the most efficient resistance determinant against YydF * action. Unexpectedly, none of the observed cellular effects upon YydF * treatment alone are able to trigger liaIH expression, indicating that only the unique combination of membrane permeabilization and membrane rigidification caused by the epipetide, leads to the observed cell envelope stress response.

Mini Review: Bacterial Membrane Composition and Its Modulation in Response to Stress

“…B. subtilis PspA is regulated by σ W ( Wiegert et al, 2001 ; Cao et al, 2002a ), whereas the paralog LiaH is regulated by the LiaRS TCS ( Mascher et al, 2004 ; Jordan et al, 2006 ). Both paralogs localize to the membrane in response to stress and protect against membrane-damaging antibiotics ( Wolf et al, 2010 ; Kingston et al, 2013 ; Dominguez-Escobar et al, 2014 ; Popp et al, 2020 ). In the case of LiaH, membrane association is mediated by interaction with the integral membrane protein LiaI ( Dominguez-Escobar et al, 2014 ).…”

“…Subsequent studies revealed that YydF is post-translationally processed to convert two L-amino acids to D-amino acids ( Benjdia et al, 2017 ). The resulting epipeptide, YydF ∗ , induces LiaRS-regulated LiaH and the FloT flotillin ( Popp et al, 2020 ). The modified YydF ∗ peptide kills B. subtilis cells by dissipating the membrane potential via membrane permeabilization.…”

Amphotericin B Specifically Induces the Two-Component System LnrJK: Development of a Novel Whole-Cell Biosensor for the Detection of Amphotericin-Like Polyenes

“…After treatment, cells were transferred to 50 ml falcons and growth was immediately stopped in an ice water bath followed by centrifugation at 8000 rpm at 4 • C for 3 min. The supernatants were discarded, and the resulting pellets were stored at −80 • C. RNA isolation was performed with a phenol-chloroform extraction method as previously described (Popp et al, 2020). The cDNA library was prepared using the NEB Ultra RNA directional prep kit for Illumina and sequencing was performed on an Illumina HiSeq3000 system.…”

Metabolome Analysis of Constituents in Membrane Vesicles for Clostridium thermocellum Growth Stimulation

“…We observed a slight growth inhibition of C. thermocellum in the presence of myo -inositol, however this required a high concentration (1 mg/mL) of myo -inositol ( Figure S2 ). YydF is predicted to be an exported and modified peptide that has antimicrobial and/or signaling properties [ 49 , 50 ]. YxaL, which contains a repeated pyrrolo-quinoline quinone (PQQ) domain that forms a beta-propeller structure, interacts with the DNA helicase PcrA in B. subtilis [ 51 ].…”