Biological activities associated with the volatile compound 2,5-bis(1-methylethyl)-pyrazine.

Thierry K S Janssens,Paolina Garbeva,Wietse De Boer,Olaf Tyc,Harrie Besselink

doi:10.1093/femsle/fnz023

Abstract

Pyrazines are 1,4-diazabenzene-based volatile organic compounds and known for their broad-spectrum antimicrobial activity. In the present study, we assessed the antimicrobial activity of 2,5-bis(1-methylethyl)-pyrazine, produced by Paenibacillus sp. AD87 during co-culture with Burkholderia sp. AD24. In addition, we were using transcriptional reporter assays in E. coli and mammalian cells to decipher the possible mode of action. Bacterial and mammalian luciferase reporter strains were deployed to elucidate antimicrobial and toxicological effects of 2,5-bis(1-methylethyl)-pyrazine. At high levels of exposure, 2,5-bis(1-methylethyl)-pyrazine exerted strong DNA damage response. At lower concentrations, cell-wall damage response was observed. The activity was corroborated by a general toxicity reporter assay in E. coli ΔampD, defective in peptidoglycan turnover. The maximum E. coli cell-wall stress activity was measured at a concentration close to the onset of the mammalian cytotoxicity, while other adverse outcome pathways, such as the activation of aryl hydrocarbon and estrogenic receptor, the p53 tumour suppressor and the oxidative stress-related Nrf2 transcription factor, were induced at elevated concentrations compared to the response of mammalian cells. Because of its broad-spectrum antimicrobial activity at lower concentrations and the relatively low mammalian toxicity, 2,5-bis(1-methylethyl)-pyrazine is a potential bio-based fumigant with possible applications in food industry, agriculture or logistics.

Highlights

Many microorganisms are able to produce volatile organic compounds that can modulate the growth, differentiation and behaviour of other organisms in their surroundings (Kai et al, 2009, Schmidt et al, 2015, Schulz-Bohm et al, 2018)
Bacterial luciferase reporter assays for antimicrobial mode of action of pure 2,5-bis(1methylethyl)-pyrazine The glycerol batches of the bacterial reporter strains were diluted 20-fold in MOPS-buffered minimal medium (8.5 mM NaCl, 18.7 mM NH4Cl, 47 mM MOPS, 0.3 mM Na2HPO4.2H2O, 0.3 mM KH2PO4, 2 mM of MgCl2; 0.1 mM of CaCl2; 0.2 % glucose, pH=7) and exposed at 1% v/v DMSO in a final volume of 150 μl in Greiner Bio-One white polystyrene 96 wells plates (Greiner bio-one, Cat# 655075)
The results show that the produced pyrazine compound (RT=19.77, m/z=164.135) is 2,5-bis(1-methylethyl)-pyrazine as indicated by a molecular-ion peak at m/z=164 and a base peak at m/z=149 (Fig. 1)

Summary

Introduction

Many microorganisms are able to produce volatile organic compounds that can modulate the growth, differentiation and behaviour of other organisms in their surroundings (e.g. bacteria, fungi and nematodes) (Kai et al, 2009, Schmidt et al, 2015, Schulz-Bohm et al, 2018). Bacterial and mammalian luciferase reporter strains were deployed to elucidate antimicrobial and toxicological effects of 2,5-bis(1methylethyl)-pyrazine. In a previous study we observed that the volatile organic compound 2,5-bis(1-methylethyl)-pyrazine was produced during co-cultivation of two phylogenetically different soil bacterial isolates, namely, Burkholderia sp.

Results

Conclusion