Isolation of a flagellar operon in Azospirillum brasilense and functional analysis of FlbD

A 10 kb fragment containing fliF, fliH, fliN, motA, flbD, flhA, flhF and fleN genes was cloned from the genomic DNA of Azospirillum brasilense Yu62. These eight genes appear to be structurally organized as an operon. FlbD, encoded by flbD, has a HTH DNA binding domain and shows homology to σ54-dependent transcriptional activators such as NtrC, NifA and DctD. An in-frame deletion of flbD in A. brasilense abolishes biosynthesis of lateral flagella and swarming ability when grown on semi-solid surfaces. An intact copy of flbD on a plasmid complemented the ΔflbD mutant by restoring lateral flagellation and swarming ability. Transcriptional analysis demonstrated that FlbD is involved in the genetic regulation of flagella biosynthesis and acts as both an activator and a repressor of flagellum gene expression in A. brasilense. DNA binding assays indicated direct interaction between FlbD and the promoter regions of laf1, fliF and flgB genes. We propose that A. brasilense has a genetic regulation profile for flagella biosynthesis similar to that observed in Caulobacter crescentus.