Screening for Candidate Genes Associated with Biocontrol Mechanisms of Bacillus pumilus DX01 Using Tn5 Transposon Mutage
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Screening for Candidate Genes Associated with Biocontrol Mechanisms of Bacillus pumilus DX01 Using Tn5 Transposon Mutagenesis and a 2‑DE‑Based Comparative Proteomic Analysis Yunpeng Chen1 · Tong Liu1 · Qiongjie Li1 · Yifei Ma1 · Jiejie Cheng1 · Lurong Xu1 Received: 20 February 2020 / Accepted: 28 August 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract A total of 1467 mutants of the biocontrol bacterium Bacillus pumilus DX01 were obtained by Tn5 insertional mutagenesis and subjected to the determination of antagonistic capabilities. Compared with the wild-type strain DX01, the mutant M25 was identified to have the most significant reduction in antagonistic capability against the phytopathogen Bipolaris maydis and extracellular proteinase activity. The integration site of the exogenous T-DNA in the genome of mutant M25 was revealed in the coding region of malony CoA-ACP transacylase (MCAT) gene (mcat), which belongs to a polyketide synthase (PKS) gene cluster, DX01pks of B. pumilus DX01. Furthermore, the whole DX01pks gene cluster was cloned using Illumina Solexa sequencing technology, and it has a modular framework different from the other two gene clusters involved in polyketide synthesis in B. amyloliquefaciens FZB42 (pks1) and B. subtilis 168 (pksX). Finally, in order to gain more insights into the molecular mechanisms of biocontrol of B. pumilus DX01, the changes in the relative level of expression of total proteins between the original strain DX01 and the mutant M25 were detected by 2-DE-based proteomic analysis. A total of twenty differentially expressed proteins were identified upon the mcat gene transposition mutagenesis. Of these proteins, seven proteins were up-regulated in expression level and the other proteins were down-regulated. Taken together, the results in this study showed that Tn5 transposon mutagenesis of B. pumilus DX01 can lead to a significant change of antiphytopathogen ability, and the DX01pks gene cluster possibly play a potential role in the biocontrol processes of this bacterium.
Introduction A lot of Bacillus strains are plant growth-promoting bacteria (PGPB) and can be used as biofertilizers [1]. The plantpromoting effect of these microbes is mostly attributed to the release of metabolites directly stimulating growth. Although Nucleotide sequence accession number The nucleotide sequence flanking the integration site in the genome of a mutant M25 generated from Bacillus pumilus DX01 has been deposited in the GenBank database under the accession number JQ513965. Similarly, the DNA sequences of tasA gene encoding the translocation-dependent antimicrobial spore protein and two surfactin synthase genes, surf1 and surf2 have been assigned the GenBank accession numbers, KC692521, KC462182, and KC462183, respectively. * Yunpeng Chen [email protected] 1
Department of Resources and Environment, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
the mechanisms by which Bacillus promote plant growth are not fully
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