Computational protocol: Assessment of the relevance of the antibiotic 2 amino 3 (oxirane 2,3 dicarboxamido) propanoyl valine from Pantoea agglomerans biological control strains against bacterial plant pathogens

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Protocol publication

[…] APV-negative mutants were constructed by transposon mutagenesis with the mini-Tn5 donor plasmid pRL27 (). Kanamycin-resistant mutants were screened on agar diffusion assays for their growth inhibition activity. Transposon insertions were analyzed as described previously (). Transposon insertion sites were subcloned by plasposon rescue cloning and the flanking regions were sequenced by GATC-Biotech (Konstanz, Germany). Self-formed adaptor PCR (SEFA-PCR) was used to close gaps in the sequence of the biosynthesis cluster, and to analyze sequences upstream and downstream of the gene cluster (). For alignments, Vector NTI 9.0 AlignX (Invitrogen, Life Technologies GmbH, Darmstadt, Germany) was used.Southern blots were performed as described previously () using a digoxigenin (DIG) DNA labeling and luminescent detection kit (Boehringer-Mannheim, Mannheim, Germany). A DIG-labeled DNA probe was prepared from a PCR amplification product carrying a 0.5-kb fragment of apvD gene from Pa48b. Hybridizations were carried out using a hybridization temperature of 60°C and two 10-min washes with 0.1× SSC (1× SSC consists of 0.15 mol/L NaCl plus 0.015 mol/L sodium citrate) and 0.1% sodium dodecyl sulfate at 60°C. Megaplasmids were prepared as described previously (). [...] Data management and computation were performed using Microsoft Excel software (Microsoft Corporation, Redmond, WA). The mean values and the standard deviations were calculated and statistically compared by t-test using SigmaPlot 9.0 (Systat Software, Inc.). […]

Pipeline specifications

Software tools Vector NTI Advance, SigmaPlot
Applications Miscellaneous, Protein sequence analysis
Organisms Erwinia amylovora, Pantoea agglomerans, Malus domestica, Glycine max, Pseudomonas syringae
Diseases Bacterial Infections