title: Direct interaction of FliX and FlbD is required for their regulatory activity in Caulobacter crescentus.
abstract: BACKGROUND
  The temporal and spatial expression of late flagellar genes in Caulobacter crescentus is activated by the transcription factor FlbD and its partner trans-acting factor FliX. The physical interaction of these two proteins represents an alternative mechanism for regulating the activity of σ54 transcription factors. This study is to characterize the interaction of the two proteins and the consequences of the interaction on their regulatory activity.
  
  
  RESULTS
  FliX and FlbD form stable complexes, which can stand the interference of 2.65 M NaCl. The stability of FliX and FlbD was affected by the co-existence of each other. Five FliX mutants (R71A, L85K, Δ117-118, T130L, and L136K) were created by site-directed mutagenesis in conserved regions of the protein. All mutants were successfully expressed in both wild-type and ΔfliX Caulobacter strains. All but FliXL85K could rescue the motility and cell division defects of a ΔfliX mutant strain. The ability of FliX to regulate the transcription of class II and class III/IV flagellar promoters was fully diminished due to the L85K mutation. Co-immunoprecipitation experiment revealed that FliXL85K was unable to physically interact with FlbD.
  
  
  CONCLUSIONS
  FliX interacts with FlbD and thereby directly regulates the activity of FlbD in response to flagellar assembly. Mutations in highly conserved regions of FliX could severely affect the recognition between FliX and FlbD and hence interrupt the normal progression of flagellar synthesis and other developmental events in Caulobacter.
authors.affiliation: Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, CA 90095-1569, USA. zxu@bgsu.edu
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Xu
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Dutton
authors.forename: James W
authors.initials: JW
authors.lastname: Gober
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016935:Caulobacter crescentus / Q000235:genetics / Q000502:physiology*; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D047468:Immunoprecipitation; D008124:Locomotion; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D051877:RNA Polymerase Sigma 54 / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C089631:FlbD protein, Caulobacter crescentus; C111943:FliX protein, Caulobacter crescentus; D008565:Membrane Proteins; D050505:Mutant Proteins; D051877:RNA Polymerase Sigma 54