title: Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans.
abstract: Entry into sporulation in Bacillus subtilis is governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur in trans. To test this hypothesis, we generated a series of B. subtilis strains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in a trans fashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressed in vivo. Taken together, these in vitro and in vivo results reinforce the evidence that KinA autophosphorylation is able to occur in a trans fashion.
  
  
  OBJECTIVE
  Autophosphorylation of histidine kinases is known to occur by either the cis (one subunit of kinase phosphorylating itself within the multimer) or the trans (one subunit of the multimer phosphorylates the other subunit) mechanism. The present study provided direct in vivo and in vitro evidence that autophosphorylation of the major sporulation histidine kinase (KinA) is able to occur in trans within the homotetramer complex. While the physiological and mechanistic significance of the trans autophosphorylation reaction remains obscure, understanding the detailed reaction mechanism of the sporulation kinase is the first step toward gaining insight into the molecular mechanisms of the initiation of sporulation, which is believed to be triggered by unknown factors produced under conditions of nutrient depletion.
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA.
authors.forename: Seram Nganbiton
authors.initials: SN
authors.lastname: Devi
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA.
authors.forename: Brittany
authors.initials: B
authors.lastname: Kiehler
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA.
authors.forename: Lindsey
authors.initials: L
authors.lastname: Haggett
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA mfujita@uh.edu.
authors.forename: Masaya
authors.initials: M
authors.lastname: Fujita
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D000071677:Histidine Kinase; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D013171:Spores, Bacterial / Q000201:enzymology* / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; C080950:Spo0A protein, Bacillus subtilis; D014157:Transcription Factors; C075521:kinA protein, Bacillus subtilis; D011494:Protein Kinases; D000071677:Histidine Kinase