Agrobacterium may be used as a suitable experimental system for genetic analysis of resistance to (at least Xenorhabdus budapestensis) antimicrobial peptide complexes
- Published
- Accepted
- Subject Areas
- Genetics, Microbiology
- Keywords
- Agrobacterium, Ti plasmid, intact/wild-type/virulent, cured, T-DNA-Deletd, resistant/sensitive (S/R) to, Antimicrobial peptides (AMPs), Xenorhabdus, X. budapestensis, in vitro liquid bioassay
- Copyright
- © 2018 Fodor et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2018. Agrobacterium may be used as a suitable experimental system for genetic analysis of resistance to (at least Xenorhabdus budapestensis) antimicrobial peptide complexes. PeerJ Preprints 6:e26900v1 https://doi.org/10.7287/peerj.preprints.26900v1
Abstract
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.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
Supplementary material, text, tables and figures
Supplementary material includes supplementary information on Agrobacterium strains; details of preparation and purification of antimicrobial peptides from EMA_CFCM; data and statistic analysis of the data obtained in Agrobacterium strains other than those belonging to opine groups other than the AGR. Tables and Figures from the Supplementary material see also in separate supplementary files.
Additional information about the Agrobacterium strains used in this study
Origin and the most important practical information on them.
Puruficatin of antimicrobial peptide fractions from EMA_CFCM
Table S2: +++ = very strong antimicrobial activity; Abbreviations: EMA= X, budapestensis HGB033; CFCM = Cell-Free Culture Medium; PF = Peptide Rich Fraction; * = Name of HPLC Sample; RPCC = Reverse Phase Column Chromatography; Test organisms; CA =Candida albicans; SA = .S, aureus; EC = E. coli HGB2226; XN = a Xenorhabdus nematophila lab isolate which is extreme sensitive to Xenorhabdus antibiotics. HGB1795 is a transposon-induced insertion mutant of the XNC1_2022 gene (Gene ID: 9430524; Gene Page Link: NCBI UniProtKB; Locus Tag: XNC1_2022 see gene page for GenePage for the XNC1_2022 gene EcoGene-RefSeq) from X. nematophila (strain ATCC 19061 / provided by Prof. Helge Bode via Prof. Heidi Goodrich-Blair. responsible for the biosynthesis of Bicornutin A (Fuchs et al., 2012).
ANOVA PROCEDURE OF OD VALUES DETERMINED IN IN VITRO BIOASSAYS OF EMA_PF2 IN AGROBACTERIUM STRAIN I
Table S3: The data analysis was performed using [SAS/STAT] software, Version [9.4] of the SAS System for [Windows X 64 Based Systems]; (Copyright © [2013 of copyright]; SAS Institute Inc. SAS, Cary, NC, USA. We used ANOVA and GLM Procedures alternatively following the propositions of the SAS 9.4 Software. The design of the experiment was a randomized complete block, design with the number of the respective treatments, concentrations, and replicates. Data have been averaged as to allow the analysis of variance (ANOVA).The significance of differences of the means (α = 0.05) was where determined by using t (LSD) tests or Duncan’s Multiple Range Tests, depending upon the experiment Anova Table S3A summarizes the results of Anova Procedure for all the 180 OD values of (36 untreated control and 144 treated).
ANOVA Analysis: Comparison of OD values the the EMA-PF2 treated Agrobacterium culltures
Table S4: The data analysis was performed using [SAS/STAT] software, Version [9.4] of the SAS System for [Windows X 64 Based Systems]; (Copyright © [2013 of copyright]; SAS Institute Inc. SAS, Cary, NC, USA, see Footnotes to Table S3. The significance of differences of the means (α=0.05) were determined here by using Duncan’s Multiple Range Tests, depending upon the experiment as a part of the Anova Procedure. Duncan’s Multiple Range Test of OD30-75 values measured in Liquid Culture Bioassay of EMA PF on Agrobacterium strains.
Raw data presented in Fig 1
Raw data of Fig 1 A and B.
Raw data: OD values of EMA_PF2 in vitro liquid bioassays (all) for Fig 5, also for Fig 7 andFigS3.(For both review and public information)
This excel file contains the most important data for this Publication. For both review and public information. Data are presented in Fig 5 and are grouped, from different aspects in Fig7 and FigS3. The detailed statistical analyses are given in the Supplementary material, Table S3 andS4..
Raw data presented in Table 2
Raw data on agar-diffusion bioassays (on four sensitive targets) of antimicrobial active peptide fractions isolated from EMA_CFCM by amberlite absorption, (followed by methanol purification and elution, ultrafiltration (EMA_PF1, EMA_PF2), then HPLC fractionation or RPLC purification; and RPLC) EMA30, also followed by HPLC purification (AF103-40,- 43 and 44 fractions).
The OD values of the obtained in in vitro liquid bioassay of EMA_PF2 in NOP strains
Legends /Footnotes to Fig S3: Comparison of OD values obtained in liquid bioassays of EMA PF (antimicrobial active peptide fraction from cell-free media of early stationary phase cultures of Xenorhabdus Xenorhabdus. budapestensis, EMA) on nopaline – catabolizing (NOP) Agrobacterium strains HP1843, HP1842, HP1836, HP1840, HP1841, and SZL4. The tests were carried out in LB liquid cultures of 200 µl final volumes, inoculated with 5 µl O/N culture of the respective test bacterium and incubated at 30 o C for 24h. Note that although the OD values of the PF-treated cultures were significantly lower than those in the respective untreated (control) ones, there was no detectable dose dependence within the range of 30 -75 µg/ml. None of the doses 30, 45, 60 and 75 µg/m exerted a cytotoxic but cytotoxic effect on them. On the basis of their significantly different OD values, these strains could be scored to different Duncan’s Groups (Duncan’s Group A, B, C, D, respectively), which reflects differences in the cytostatic effect of EMA PF on them.