The response regulator Spo0A from Bacillus subtilis is efficiently phosphorylated in Escherichia coli.
The response regulator proteins of two-component systems mediate many adaptations of bacteria to their ever-changing environment. Most response regulators are transcription factors that alter the level of transcription of specific sets of genes. Activation of response regulators requires their phosphorylation on a conserved aspartate residue by a cognate sensor kinase. For this reason, expression of a recombinant response regulator in the absence of the requisite sensor kinase is expected to yield an unphosphorylated product in the inactive state. For Spo0A, the response regulator controlling sporulation in Bacillus subtilis however, we have found that a significant fraction of the purified recombinant protein is phosphorylated. This phosphorylated component is dimeric and binds to Spo0A recognition sequences in DNA. Treatment with the Spo0A-specific phosphatase, Spo0E, leads to dissociation of the dimers and loss of DNA binding. It is therefore necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the fully inactive state of the molecule.
Ladds
Joanne C
JC
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, UK.
Muchová
Katarína
K
Blaskovic
Dusan
D
Lewis
Richard J
RJ
Brannigan
James A
JA
Wilkinson
Anthony J
AJ
Barák
Imrich
I
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D000071677:Histidine Kinase; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013171:Spores, Bacterial / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C080950:Spo0A protein, Bacillus subtilis; D014157:Transcription Factors; D011494:Protein Kinases; D000071677:Histidine Kinase