CbpA: a polarly localized novel cyclic AMP-binding protein in Pseudomonas aeruginosa.
In Pseudomonas aeruginosa, cyclic AMP (cAMP) signaling regulates the transcription of hundreds of genes encoding diverse virulence factors, including the type II secretion system (T2SS) and type III secretion system (T3SS) and their associated toxins, type IV pili (TFP), and flagella. Vfr, a cAMP-dependent transcriptional regulator that is homologous to the Escherichia coli catabolite repressor protein, is thought to be the major cAMP-binding protein that regulates these important virulence determinants. Using a bioinformatic approach, we have identified a gene (PA4704) encoding an additional putative cAMP-binding protein in P. aeruginosa PAO1, which we herein refer to as CbpA, for cAMP-binding protein A. Structural modeling predicts that CbpA is composed of a C-terminal cAMP-binding (CAP) domain and an N-terminal degenerate CAP domain and is structurally similar to eukaryotic protein kinase A regulatory subunits. We show that CbpA binds to cAMP-conjugated agarose via its C-terminal CAP domain. Using in vitro trypsin protection assays, we demonstrate that CbpA undergoes a conformational change upon cAMP binding. Reporter gene assays and electrophoresis mobility shift assays defined the cbpA promoter and a Vfr-binding site that are necessary for Vfr-dependent transcription. Although CbpA is highly regulated by Vfr, deletion of cbpA did not affect known Vfr-dependent functions, including the T2SS, the T3SS, flagellum- or TFP-dependent motility, virulence in a mouse model of acute pneumonia, or protein expression profiles. Unexpectedly, CbpA-green fluorescent protein was found to be localized to the flagellated old cell pole in a cAMP-dependent manner. These results suggest that polar localization of CbpA may be important for its function.
Department of Medicine, University of California, San Francisco, California 941431, USA.
Takayuki
T
Endoh
Joanne N
JN
Engel
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms; D000242:Cyclic AMP / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D000242:Cyclic AMP