Gain-of-function mutations in FlbD can by-pass the transcriptional requirement for FliX, suggesting that FliX is a trans-acting factor rather than a structural component of the flagellum [36]. Additionally, FliX enhances FlbD-activated transcription in vitro by

stimulating purified FlbD to form higher-order oligomers [35]. Interestingly, overexpression of FliX suppresses FlbD-activated transcription in vivo, and a mutant allele of fliX, fliX 1, has been isolated that can by-pass the early selleck compound flagellar assembly requirement for class III and IV transcription [38]. These observations suggest that upon click here the complete assembly of an early class II flagellar basal body structure, FliX switches from a negative to a positive regulator of FlbD. The physical interaction of FliX and FlbD represents a novel mechanism for regulating the activity of a σ54 transcription factor [35]. Here, we describe a genetic and biochemical analysis dissecting the role of FliX in regulating FlbD activities. We present evidence that FliX and FlbD are in stable complexes under physiological conditions. Furthermore, we show that highly-conserved regions of FliX are critical for its productive interaction with FlbD and for proper

regulation of flagellar gene expression in response to the progression of flagellar assembly. TEW-7197 Methods Bacterial strains and plasmids Bacterial strains and plasmids involved in this work are summarized in Table 1. Caulobacter crescentus strains were grown in peptone-yeast extract (PYE) [39] at 31°C. Antibiotics were supplemented when necessary to a final concentration of 2.5 μg/ml of chloramphenicol, 2 μg/ml of tetracycline, or 20 μg/ml of nalidixic acid. PYE motility plates contained 0.3% (w/v) agar. E. coli strains were grown at 37°C in Luria-Bertani broth supplemented with one or more of the following antibiotics: chloramphenicol (30 μg/ml), tetracycline (12.5 μg/ml), or ampicillin (50 μg/ml). DNA manipulations were carried out

according to standard procedures. Plasmids were introduced into C. crescentus by conjugation with E. coli S17-1. Table 1 Bacterial strains and plasmids used in this study Strains or plasmids Genotypes or descriptions Sources C. Megestrol Acetate crescentus LS107 syn-1000, bla-6, amps derivative of NA1000 Stephens et al. [45] JG1172 syn-1000 bla-6 ΔfliX Muir et al. [38] SC1032 flbD198::Tn5 Ohta et al. [41] E. coli S17-1 Rp4-2, Tc::Mu, Km::Tn7 Simon et al. [46] BL21(DE3) F- ompT gal [dcm] [lon] hsdS B (rB – mB – ; an E. coli B strain) with DE3, a λ prophage carrying the T7 RNA polymerase gene Novagen Plasmids pX21b derivative of pET-21b carrying histidine-tagged FliX under the control of T7 promoter, Apr Muir & Gober [36] pBBR1MCS broad host range cloning vector, multicopy, Cmr Kovach et al.