Use of a Promoter Trap To Identify Bacillus cereus Genes Regulated by Tomato Seed Exudate and a Rhizosphere Resident, Pseudomonas aureofaciens

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Abstract
The goal of this study was to identify genes in Bacillus cereus , a bacterium commonly associated with plant seeds and roots, that are affected by compounds originating from a host plant, tomato, or another rhizosphere resident, Pseudomonas aureofaciens . We constructed a B. cereus chromosomal DNA library in a promoter-trap plasmid, pAD123, which contains a promoterless version of the green fluorescent protein (GFP) gene, gfp mut3a. The library was screened by using fluorescence-activated cell sorting for clones showing a change in GFP expression in response to either tomato seed exudate or culture supernatant of P. aureofaciens strain 30-84. We identified two clones carrying genes that were induced by the presence of tomato seed exudate and nine clones carrying genes that were repressed by P. aureofaciens culture supernatant. A clone chosen for further study contained an open reading frame, designated lipA , that encodes a deduced protein with a lipoprotein signal peptide sequence similar to lipoproteins in B. subtilis. Expression of gusA under control of the lipA promoter increased twofold when cells were exposed to tomato seed exudate and in a concentration-dependent manner when exposed to a mixture of amino acids. When the wild type and a 10-fold excess of a lipA mutant were applied together to tomato seeds, 2 days after planting, the wild type displayed medium-dependent culturability, whereas the lipA mutant was unaffected. This study demonstrates the power of a promoter trap to identify genes in a gram-positive bacterium that are regulated by the biotic environment and resulted in the discovery of lipA , a plant-regulated gene in B. cereus .