Interaction of the regulator proteins RcsA and RcsB with the promoter of the operon for amylovoran biosynthesis in Erwinia amylovora.
The RcsA and RcsB proteins of Erwinia amylovora and Escherichia coli were expressed in E. coli and purified. Their DNA-binding activity was examined using a 1-kb DNA region containing the putative promoter of the ams operon of Ew. amylovora, which is responsible for the biosynthesis of the exopolysaccharide amylovoran. Mobility shift assays indicated specific binding of RcsA and RcsB to a region of 78 bp spanning nucleotide positions -578 to -501 relative to the translational start of the first open reading frame of the operon. This region includes stretches of homology to E. coli sigma 70 promoter consensus sequences and to the E. coli cps promoter region. Binding of the Rcs proteins was not found at a JUMPstart consensus, typical for various promoters of polysaccharide gene clusters. DNA-binding activity was not detected for RcsA alone and only high concentrations of RcsB were able to interact with the ams promoter in our assay. The two proteins bind cooperatively at the indicated region of the ams promoter and further evidence is provided showing that the DNA-protein complex formed involves a heterodimer of RcsA and RcsB. The specific activity of RcsA, but not of RcsB, was enhanced when the protein was expressed in E. coli at 28 degrees C, relative to expression at 37 degrees C. In addition, DNA-protein complex formation is affected by temperature. The E. coli RcsA/RcsB proteins bind to the same region of the ams promoter and are able to interact with the Rcs proteins from Ew. amylovora.
Kelm
O
O
Freie Universität Berlin, Institut für Kristallographie, Germany.
Kiecker
C
C
Geider
K
K
Bernhard
F
F
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004885:Erwinia / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D014157:Transcription Factors
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011135:Polysaccharides, Bacterial; C489568:RcsB protein, E coli; D011994:Recombinant Proteins; D014157:Transcription Factors; C100769:amylovoran; C074889:RcsB protein, Bacteria; C067572:RcsA protein, E coli