Double-Wing Motif Protein is a Novel Biofilm Regulatory Factor of the Plant Disease Biocontrol Agent, <i>Bacillus subtilis</i>

Transposon mutagenesis screening of <i>Bacillus subtilis</i> YB-1471, a novel rhizosphere biocontrol agent of Fusarium crown rot (FCR) of wheat, resulted in the identification of <i>orf04391</i>, linked to reduced biofilm formation. The gene encodes a protein possessing a putative tertiary structure of a "double-wing" DNA-binding domain. Expression of <i>orf04391</i> increased during biofilm development in stationary cultures and during rapid growth in shaking cultures. An <i>orf04391</i> deletion strain showed reduced biofilm production related to lower levels of the extracellular matrix, and the mutant also had reduced sporulation, adhesion, root colonization, and FCR biocontrol efficiency. Transcriptome analysis of YB-1471 and Δorf04391 in stationary culture showed that the loss of <i>orf04391</i> resulted in altered expression of numerous genes, including <i>sinI</i>, an initiator of biofilm formation. DNA binding was shown with his-tagged Orf04391 binding to the <i>sinIR</i> operon in vivo and in vitro. Orf04391 appears to be a transcriptional regulator of biofilm formation in <i>B. subtilis</i> through the Spo0A-SinI/SinR pathway.