Molecular basis of allicin resistance in Pseudomonas fluorescens Allicin Resistant-1

Borlinghaus, Jan; Slusarenko, Alan (Thesis advisor); Blank, Lars Mathias (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019

Abstract

Allicin is a sulfur containing compound from garlic (Allium sativum) and has a strong broad range of antimicrobial activity against bacteria and fungi. To date, three modes of action are known to be responsible for its activity: (1) reactivity with exposed thiol groups from proteins, (2) the cause of oxidative stress by depletion of cellular redox buffers like glutathione, and (3) pore formation in biological membranes. Microbial resistance against allicin is rarely found but offers the chance to investigate the specific action and cellular targets of allicin in more detail. In this work, the genetic basis of allicin resistance from a bacterial isolate from garlic, named Pseudomonas fluorescens Allicin Resistant-1 (PfAR-1) was investigated. A genomic DNA library of PfAR-1 was constructed and eight genomic clones were identified that conferred allicin resistance to E. coli DH10B and Pseudomonas syringae 4612 (Ps4612). Seven clones have a core of ten genes in common (annotated as OsmC, SDR, TetR, DsbA, Trx, KefC, KefF, 4-OT, OYE, and AhpD) while one clone is missing two of these (OYE and AhpD) and confers less resistance. A bioinformatic analysis revealed that the genomic fragments of these clones originated from three genomic repeats in PfAR-1. These are part of three larger genomic regions with lowered GC content (approx. 28.3 kbp, 26.5 kbp, and 55.3 kbp in size). These genomic regions can be considered as genomic islands since a codon usage analysis and a phylogenetic tree construction for the core genome and these genomic regions highly indicate that these were obtained by horizontal gene transfer from a so far unknown organism. Syntenic regions in other pathogenic and non-pathogenic Pseudomonas were very likely obtained by horizontal gene transfer as well. The cluster of these ten genes is highly conserved in these syntenic regions, indicating that these genes are organized as at least two operons. The presence of syntenic regions in pathogenic Pseudomonas like in the garlic pathogen P. salomonii or in the well studied P. syringae pv. tomato DC3000 might indicate a function in host pathogen interactions. A transposon mutagenesis of one genomic clone indicated Trx, DsbA, KefC, OYE, and AhpD as allicin resistance factors. Single gene overexpression of AhpD and DsbA in Ps4612 conferred a high amount of allicin resistance. Single gene expression of KefC and OsmC seemed to be lethal, and the remaining genes showed no gain of allicin resistance when expressed alone in Ps4612. The genomic clones conferred specific resistance against allicin but not against other oxidants like cumen hydroperoxide, N-ethyl maleimide or hydrogen peroxide in E. coli. However, single gene expression of AhpD showed only a weak gain of resistance while the expression of DsbA conferred no resistance to E. coli which is why the specificity of allicin resistance needs to be further investigated in another genetic background. An additional feature of PfAR-1 is the presence of three copies of glutathione reductase (glr), one in the core genome and two on genomic islands. Since allicin is known to be buffered by glutathione inside of cells, these additional copies of glr are most likely very significant for allicin resistance as well.

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