Dual role of the PhoP approximately P response regulator: Bacillus amyloliquefaciens FZB45 phytase gene transcription is directed by positive and negative interactions with the phyC promoter.
Several Bacillus strains secrete phytase, an enzyme catalyzing dephosphorylation of myo-inositol hexakisphosphate (phytate). We identified the phyC (phytase) gene from environmental Bacillus amyloliquefaciens FZB45 as a member of the phosphate starvation-inducible PhoPR regulon. In vivo and in vitro assays revealed that PhoP approximately P is essential for phyC transcription. The transcriptional start site was identified downstream of a sigmaA-like promoter region located 27 bp upstream of the probable translation ATG start codon. Inspection of the phyC promoter sequence revealed an unusual structure. The -35 and -10 regions are separated by a window of 21 bp. A pair of tandemly repeated PhoP TT(T/A/C)ACA binding boxes was located within and upstream of the -35 consensus promoter region. A single PhoP box was found within the -10 consensus promoter region. DNase I footprinting experiments performed with isolated PhoP confirmed that PhoP approximately P binds at two sites overlapping with the phyC -35 and -10 consensus promoter region. While binding of dimeric PhoP approximately P at -35 is essential for activation of the phyC promoter, binding of PhoP approximately P at -10 suppresses promoter activity. A sixfold enhancement of phyC gene expression was registered after T:G substitution of nucleotide -13 (mutant MUT13), which eliminates PhoP binding at the single PhoP box without impairing the -10 consensus sequence. Moreover, MUT13 also expressed phyC during phosphate-replete growth, suggesting that the repressing effect due to binding of PhoP approximately P at -10 was abolished. A model is presented in which transcription initiation of phyC is positively and negatively affected by the actual concentration of the PhoP approximately P response regulator.
Makarewicz
Oliwia
O
Institut für Biologie, Humboldt Universität Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.
Dubrac
Sarah
S
Msadek
Tarek
T
Borriss
Rainer
R
D010832:6-Phytase / Q000096:biosynthesis / Q000235:genetics*; D019677:Artificial Gene Fusion; D001407:Bacillus / Q000235:genetics* / Q000502:physiology*; D001426:Bacterial Proteins / Q000502:physiology*; D001665:Binding Sites / Q000235:genetics; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D008954:Models, Biological; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D018085:Regulon; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C061064:PhoP protein, Bacteria; D010832:6-Phytase; D001616:beta-Galactosidase