Mutation of the putative nucleotide binding site of the Bacillus subtilis membrane protein ComFA abolishes the uptake of DNA during transformation.
ComFA is a membrane protein required for the uptake of transforming DNA following its binding to the Bacillus subtilis competent-cell surface. ComFA, which resembles members of the DEAD family of ATP-driven helicases, contains sequences similar to those found in many ATP-binding proteins and thought to represent the ATP-binding sites of these proteins. We have suggested that ComFA may function as a DNA translocase and/or helicase, using the energy of ATP hydrolysis to mediate the uptake of DNA. As a partial test of this hypothesis, we have introduced mutations into highly conserved glycyl and lysyl residues of the putative ATP-binding site, located, respectively, at positions 151 and 152, and determined the effects of these alterations on in vivo function. A substitution of the conserved lysyl by a glutamyl residue (K152E) and a double G151R-K152N mutation each resulted in a nearly 1,000-fold decrease in transformability, equivalent to that observed in a ComFA null mutant. A K152N mutation caused a partial loss-of-function phenotype. These effects were manifested at the level of DNA uptake; no marked effects on the final levels of DNA binding were noted. When either the K152E mutant allele or the G151R-K152N double mutant allele was combined in single copy with wild-type comFA, a dominant negative phenotype expressed on the level of DNA uptake was observed, suggesting that ComFA acts in a complex with other proteins, with additional molecules of ComFA, or with both.
Londoño-Vallejo
J A
JA
Public Health Research Institute, New York, New York 10016.
Dubnau
D
D
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D017354:Point Mutation; D014170:Transformation, Genetic / Q000235:genetics*
D001426:Bacterial Proteins; C061262:ComA protein, Bacteria; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009711:Nucleotides