title: An alternate route to phosphorylating DegU of Bacillus subtilis using acetyl phosphate.
abstract: BACKGROUND
  Two-component signal transduction pathways allow bacteria to sense and respond to the environment. Typically such pathways comprise a sensor histidine kinase and a response regulator. Phosphorylation of the response regulator commonly results in its activation, allowing the protein to bind to target promoter elements to regulate transcription. Several mechanisms are used to prevent inappropriate phosphorylation of the response regulator, thereby ensuring a specific response. In Bacillus subtilis, the DegS-DegU two-component system controls transcription of target genes in a manner dependent on the level of the phosphorylated response regulator, DegU. Previous work has tentatively indicated that DegU, and DegU H(12)L, a DegU variant which displays enhanced stability of the phosphoryl moiety, can be phosphorylated in the absence of the kinase, DegS.
  
  
  RESULTS
  The data presented here reveal that DegU H(12)L requires aspartic acid 56 (D(56)), the identified DegU phosphorylation site, for its activity. By indirectly measuring the level of DegU ~ P in the cell by assessment of several well recognised DegU regulated processes it was shown that DegU H(12)L retains its activity in the absence of DegS, and that mutation of D(56) produced an inactive protein. Further experiments designed to raise the level of acetyl phosphate within the cell suggest that DegU can be phosphorylated by acetyl phosphate in the absence of degS. Additionally, the phenotypic and biochemical experiments presented indicate that DegU H(12)L can reliably mimic high levels of phosphorylated DegU.
  
  
  CONCLUSIONS
  The ability of acetyl phosphate to modify DegU, and indeed DegU H(12)L, reveal an additional layer of regulation for DegU phosphorylation that will be relevant when the level of DegS is low or in the absence of degS. Given the number of processes that DegU can activate or inhibit, extensive regulation at a number of levels is required to ensure that the system is not inappropriately stimulated. DegS has both kinase and phosphatase activity and our findings demonstrate that the phosphatase activity of DegS is essential to control the level of DegU phosphate. Overall we contribute to our understanding of how the intricate signalling pathway DegS-DegU is regulated in B. subtilis.
authors.affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. lynne.cairns@tufts.edu.|Current address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, 02111, USA. lynne.cairns@tufts.edu.
authors.forename: Lynne S
authors.initials: LS
authors.lastname: Cairns
authors.affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. jessica.martyn@path.ox.ac.uk.|Current address: Sir William Dunn School of Pathology, South Parks Road Oxford, Oxford University, Oxford, OX1 3RE, UK. jessica.martyn@path.ox.ac.uk.
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Martyn
authors.affiliation: James Clerk Maxwell Building, School of Physics, University of Edinburgh, Edinburgh, EH9 3JZ, UK. kbromley@staffmail.ed.ac.uk.
authors.forename: Keith
authors.initials: K
authors.lastname: Bromley
authors.affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. n.r.stanleywall@dundee.ac.uk.
authors.forename: Nicola R
authors.initials: NR
authors.lastname: Stanley-Wall
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D053858:Metabolic Networks and Pathways; D010755:Organophosphates / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; C066497:DegU protein, Bacteria; D010755:Organophosphates; C011632:acetyl phosphate