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Agrobacterium may be used as a suitable experimental system for genetic analysis of resistance to (at least Xenorhabdus budapestensis) antimicrobial peptide complexes

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Agrobacterium may be used as a suitable experimental system for genetic analysis of resistance to (at least Xenorhabdus budapestensis) antimicrobial peptide complexes https://t.co/tchDWivH81
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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.

DOI: 10.7287/peerj.preprints.26900v1/supp-1

Additional information about the Agrobacterium strains used in this study

Origin and the most important practical information on them.

DOI: 10.7287/peerj.preprints.26900v1/supp-2

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).

DOI: 10.7287/peerj.preprints.26900v1/supp-3

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).

DOI: 10.7287/peerj.preprints.26900v1/supp-4

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.

DOI: 10.7287/peerj.preprints.26900v1/supp-5

Raw data presented in Fig 1

Raw data of Fig 1 A and B.

DOI: 10.7287/peerj.preprints.26900v1/supp-6

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..

DOI: 10.7287/peerj.preprints.26900v1/supp-7

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).

DOI: 10.7287/peerj.preprints.26900v1/supp-8

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.

DOI: 10.7287/peerj.preprints.26900v1/supp-9

Row data (in excel) presented in Table 3

DOI: 10.7287/peerj.preprints.26900v1/supp-10

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

András Fodor conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

László Makrai performed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft, he provided laboratory for the experiments prior to the work in Madison, WI.

Dávid Vozik performed the experiments, contributed reagents/materials/analysis tools, approved the final draft, he isolated and provided EMA PF.

Ferenc Olasz conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft, he gave Agrobacterium strains HP1836 - HP1843.

János Kiss conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft, he arranged to MALDI Data into 1 informative figure andconstractesód E. coli mutatorstrain.

Michael Gardner Klein conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft, he made the excellent proofreading.

Muhamad-Akbar Bin-Abdul Ghaffar analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft, he was guarding and supervising the statistical analyses.

László Szabados he provided Agrobacterium strains SZL1 - SZL5.

Ahmed Nour El-Deen performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

László Fodor analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft, he provided kaboatrory oprions forthe experments after Madison.

Erzsébet Burgetti Böszörményi performed the experiments, approved the final draft, she made the first observation about the different sensitivities of Arobacterium isolates to EMA CFCMs.

Katalin Bélafi-Bakó contributed reagents/materials/analysis tools, she was the great supervisor of the PhD work of D. Vozik.

Steven A. Forst analyzed the data, authored or reviewed drafts of the paper, approved the final draft, he was our real Xenorhabdus advisor.

Data Deposition

The following information was supplied regarding data availability:

Mendeley

Funding

This project (bench cost for experiments in Madison, WI) was supported by a Fulbright Grant Biological Science Grant (1214102) and funds from Valent Biosciences, both awarded to András Fodor to conduct research in the lab of Heidi Goodrich-Blair at the University of Wisconsin-Madison, USA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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