A spontaneous translational fusion of Bacillus cereus PlcR and PapR activates transcription of PlcR-dependent genes in Bacillus anthracis via binding with a specific palindromic sequence.
Transformation of Bacillus anthracis with plasmid pUTE29-plcR-papR carrying the native Bacillus cereus plcR-papR gene cluster did not activate expression of B. anthracis hemolysin genes, even though these are expected to be responsive to activation by the global regulator PlcR. To further characterize the action of PlcR, we examined approximately 3,000 B. anthracis transformants containing pUTE29-plcR-papR and found a single hemolytic colony. The hemolytic strain contained a plasmid having a spontaneous plcR-papR intergenic region deletion. Transformation of the resulting plasmid pFP12, encoding a fused PlcR-PapR protein, into the nonhemolytic B. anthracis parental strain produced strong activation of B. anthracis hemolysins, including phosphatidylcholine-specific phospholipase C and sphingomyelinase. The fused PlcR-PapR protein present in a lysate of B. anthracis containing pFP12 bound strongly and specifically to the double-stranded palindrome 5'-TATGCATTATTTCATA-3' that matches the consensus PlcR-binding site. In contrast, native PlcR protein in a lysate from a B. anthracis strain expressing large amounts of this protein did not demonstrate binding with the palindrome. The results suggest that the activation of PlcR by binding of a PapR pentapeptide as normally occurs in Bacillus thuringiensis and B. cereus can be mimicked by tethering the peptide to PlcR in a translational fusion, thereby obviating the need for PapR secretion, extracellular processing, retrieval into the bacterium, and binding with PlcR.
Pomerantsev
Andrei P
AP
Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-4350, USA.
Pomerantseva
Olga M
OM
Leppla
Stephen H
SH
D000595:Amino Acid Sequence; D001408:Bacillus anthracis / Q000166:cytology / Q000235:genetics* / Q000502:physiology; D001409:Bacillus cereus / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D006461:Hemolysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D012641:Selection, Genetic; D017421:Sequence Analysis; D013379:Substrate Specificity; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D010738:Type C Phospholipases / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046911:Macromolecular Substances; C466079:PapR protein, Bacillus cereus; C099480:PlcR protein, Bacillus; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C