title: The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels.
abstract: The Obg subfamily of bacterial GTP-binding proteins are biochemically distinct from Ras-like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene (cgtAC) which, in Ras-like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP-binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtAC, we generated a temperature-sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtAC is important for 50S assembly and/or stability. Surprisingly, at the non-permissive temperature, G80E cells rapidly lose viability and yet do not display an additional ribosome defect. Thus, the essential nature of the cgtAC gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1-S arrest. Therefore, CgtAC is necessary for DNA replication and progression through the cell cycle.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Kaustuv
authors.initials: K
authors.lastname: Datta
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Skidmore
authors.forename: Kun
authors.initials: K
authors.lastname: Pu
authors.forename: Janine R
authors.initials: JR
authors.lastname: Maddock
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016935:Caulobacter crescentus / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D002453:Cell Cycle; D004261:DNA Replication; D005798:Genes, Bacterial; D020043:Genes, Essential; D006160:Guanosine Triphosphate / Q000378:metabolism; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009154:Mutation; D011485:Protein Binding; D012270:Ribosomes / Q000378:metabolism*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D006160:Guanosine Triphosphate; C108621:CgtA protein, bacteria; D020559:Monomeric GTP-Binding Proteins