Agrobacterium may be used as a suitable experimental system for genetic analysis of resistance to (at least Xenorhabdus budapestensis) antimicrobial peptide complexes
Authors: Fodor András , Makrai László , Vozik Dávid , Olasz Ferenc , Kiss János , Klein Michael Gardner , Ghaffar Muhamad-Akbar Bin-Abdul , Szabados László , El-Deen Ahmed Nour , Fodor László , Böszörményi Erzsébet Burgetti , Bélafi-Bakó Katalin , Forst Steven A. 
Published: April 2018

Background Antimicrobial compounds released by the entomopathogenic nematode-symbiont bacterium Xenorhabdus budapestensis (EMA) are oligopeptides and the "trump" is fabclavine. They kill antibiotic multi-resistant Escherichia coli, Salmonella; mastitis-isolate Staphylococcus aureus, E. coli and Klebisella pneumoniae; S. aureus MRSA strain; plant-pathogenic Erwinia amylovora; Xanthomonas, Clavibacter, and Pseudomonas strains. Each tested Phytophthora isolate proved also sensitive. Fabclavine was claimed toxic, however, Proteus, some Pseudomonas and Agrobacterium strains are resistant. Our goal is to establish a suitable system for genetic analysis of antimicrobial peptide (AMP)-resistance by beneficially using the experimental toolkit of Agrobacterium research.

Methods. We tested the anti-Agrobacterium activity of the native cell-free culture media (CFCM) of EMA by agar diffusion assay. EMA_PF2 peptide fraction (of reproducible HPLC and MALDI profile) was then isolated from CFCM of EMA and exerted strong AMP activity on both Gram-negative and positive targets. The sensitive/resistant (S/R) phenotype of Agrobacterium strains of known genotype to EMA_PF2 was determined in liquid culture bio-assays.We tested 1 wild-type (A281) and 3 T-DNA-deleted (AGL1, EHA105, A4T) agropine (L, L,-succinamopine, AGR) catabolizing strains with C58 chromosome and of pTiBo542 plasmid; 5 pTi58-plasmid-cured (HP1836, HP1840, HP1841, HP1842, HP1843) and 1 T-DNA deleted and binary vector harboring (SZL4) nopaline-catabolizing strains of C58 chromosome; and 2 T-DNA deleted octopine-catabolizing (OCT) strains with and without binary vector of Ach5 chromosome (SZL2 and HP 1837, respectively).

Results. Agrobacterium tumefaciens A281, HP1836, HP1840, HP1841, HP1842, HP1843, SZL4 and SZL2 proved resistant; HP1837, AGL1, EHA105 and A4T strains were sensitive to EMA PF2. All but SZL4 showed the same S/R phenotype to CFCM and EMA_PF2.

Discussion. There are both sensitive and resistant strains of C58 and Ach5 chromosome and of different opine type strains. All but one T-DNA(-) strains (SZL2) were sensitive to EMA PF2. All plasmid-cured strains and the wild-type A281 were resistant.

Conclusions. We consider EMA_PF2 as a natural complex of interacting AMP molecules and identified resistant (R) and sensitive (S) Agrobacterium strains to it. The S/R phenotype seems independent on both the chromosome and the opine-type. Each tested T-DNA-Deleted pTiBo542 harboring strain proved sensitive while that of harboring intact plasmid was fully resistant. The availabilities of the T-DNA-Deleted EMA_PF2 (S) and the of the T-DNA-Non-Delated EMA_PF2 (R) pTiBo542 plasmid harboring Agrobacterium strains may provide a suitable system for genetic (complementation) analysis for resistance mechanisms towards EMA_PF2 and maybe towards other AMPs active on Gram-negatives. The main argument is the exceptional unique opportunity for applying the genuine tools binary vector strategy.