Purification and characterization of the Proteus vulgaris BlaA protein, the activator of the beta-lactamase gene.
Induction of the expression of the beta-lactamase gene, blaB, is regulated by the blaA gene located just upstream of blaB in the opposite direction in Proteus vulgaris. The expression of the blaA gene is negatively autoregulated by its own product BlaA, the activator of the blaB gene. The P. vulgaris BlaA protein shares high amino acid homology with the LysR family members, which are prokaryotic transcriptional activators that possess a putative helix-turn-helix DNA binding motif. To characterize its function, we purified the BlaA protein to homogeneity from Escherichia coli carrying the expression plasmid of the blaA gene driven by the tac promoter. The gel shift assay and DNaseI footprinting showed that purified BlaA specifically bound to the blaA promoter region, which resides immediately upstream of that of blaB. The binding region contained an inverted repeat, including a T-N11-T sequence which is similar to the LysR motif (T-N11-A) that is conserved in some LysR family members [Goethals et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1646-1650]. We also showed that the BlaA protein forms a dimer in solution, using glycerol gradient centrifugation and glutaraldehyde crosslinking.
Ishiguro
K
K
Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo.
Sugimoto
K
K
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D003432:Cross-Linking Reagents; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D005798:Genes, Bacterial / Q000235:genetics; D005976:Glutaral; D006706:Homeostasis; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011514:Proteus vulgaris / Q000737:chemistry / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D015533:Transcriptional Activation / Q000502:physiology*; D001618:beta-Lactamases / Q000235:genetics*
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C074160:BlaA protein, Bacteria; D001618:beta-Lactamases; D005976:Glutaral