Construction and analyses of mutant ftsH alleles of Bacillus subtilis involving the ATPase- and Zn-binding domains.
The ftsH gene, present in all eubacterial species, is anchored in the cytoplasmic membrane and contains an ATP- and a Zn-binding domain that are both part of a metalloprotease activity. The Bacillus subtilis ftsH is not essential, but null mutants exhibit a pleiotropic phenotype including filamentous growth; hypersensitivity towards heat and salt stress and a failure to sporulate. To find out whether one or the other functional domain is involved in these different phenotypes, point mutations were introduced into the coding region for both domains leading to a replacement of conserved amino acid residues. The mutant alleles were fused to a xylose-inducible promoter and integrated ectopically into two different strains, one expressing the wild-type ftsH allele and the other carrying a ftsH knockout. While none of the strains exhibited a growth defect in rich medium at 37 degrees C, those strains expressing only the mutant alleles did not resume growth after heat or salt stress challenge. Furthermore, none of the mutant alleles promoted sporulation. While only those purified mutant FtsH proteins with an intact Walker A box exhibited ATPase activity, all of them failed to degrade beta-casein.
Kotschwar
Matthias
M
Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany.
Harfst
Evita
E
Ohanjan
Tanja
T
Schumann
Wolfgang
W
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002364:Caseins / Q000378:metabolism; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020860:Subtilisin / Q000096:biosynthesis; D015032:Zinc / Q000378:metabolism
D001426:Bacterial Proteins; D002364:Caseins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D020860:Subtilisin; D000251:Adenosine Triphosphatases; D015032:Zinc