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

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, gfpmut3a. 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 concentrationdependent 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.Plants play host to a wide variety of microorganisms, including bacteria. The relationships between the bacteria and their host plants are diverse and include pathogenicity, symbiotic root nodule formation and nitrogen fixation, plant growth promotion, disease suppression, and probably other, as-yet-undiscovered, interactions. Two of the best-studied interactions between plant hosts and bacteria include the root nodule-inhabiting Rhizobium spp. and gall-forming Agrobacterium tumefaciens. Study of these systems led to the discovery that plants and bacteria communicate by using chemical signals that determine the outcome of the relationship between the organisms (32,44,45,52). Since these pioneering studies, research has revealed that many relationships between plants and gram-negative bacteria are mediated by compounds that influence bacterial gene expression (3,11,29,35,50). In contrast, little attention has focused on the gram-positive bacteria, which are common in soil and on plant tissue (8, 10). To address this knowledge gap, we are investigating chemical communication between grampositive bacteria and their plant hosts.For more than two decades, genetic analysis of mutants has been the archetypal approach to understanding plant-microbe interactions. Nonnodulating mutants of Rhizobium spp. (24), avirulent mutants of A. tumefaciens (9), Ralstonia solanacearum (4, 18), and P...

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