Screening for Candidate Genes Associated with Biocontrol Mechanisms ofBacillus pumilusDX01 Using Tn5 Transposon Mutagenesis and a 2-DE-Based Comparative Proteomic Analysis

A total of 1467 mutants of the biocontrol bacteriumBacillus pumilusDX01 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 phytopathogenBipolaris maydisand 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,DX01pksofB.pumilusDX01. Furthermore, the wholeDX01pksgene 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 inB.amyloliquefaciensFZB42 (pks1) andB.subtilis168 (pksX). Finally, in order to gain more insights into the molecular mechanisms of biocontrol ofB.pumilusDX01, 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 themcatgene 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 ofB.pumilusDX01 can lead to a significant change of antiphytopathogen ability, and theDX01pksgene cluster possibly play a potential role in the biocontrol processes of this bacterium.