A membrane protein with similarity to N-methylphenylalanine pilins is essential for DNA binding by competent Bacillus subtilis.
In a cloned copy of comG open reading frame 3 (ORF3), an in-frame deletion was generated by site-directed in vitro mutagenesis, removing the coding sequence for 15 amino acids from the central portion of this pilin-related protein. The mutagenized ORF3 was incorporated into the Bacillus subtilis chromosome, replacing the wild-type ORF3. The presence of the deleted ORF3 in the chromosome, as confirmed by Southern analysis, was associated with the complete loss of competence by the mutant strain. The ability of the mutant cells to bind exogenous radiolabeled DNA was reduced to the level of nonspecific binding of DNA by noncompetent cells. The chromosomal ORF3 mutation was partially complemented in trans by a plasmid-encoded wild-type ORF3 copy under PSPAC control upon induction of the PSPAC promoter. Using antiserum raised against a synthetic 14-mer oligopeptide deduced from the ORF3 sequence, an immunoreactive band of approximately the expected molecular size was obtained in Western blot (immunoblot) experiments with extracts of cells containing the plasmid-encoded inducible gene. A signal was also detected when cells harboring the chromosomal wild-type or mutant ORF3 in single copy were grown in competence medium. This signal was detected only in the light-buoyant-density (competent) cell fraction and only after the transition from the exponential to the stationary growth phase. In cell fractionation experiments with competent cell extracts, the immunoreactive protein was found in both the NaOH-insoluble and -soluble membrane fractions and was sensitive to proteinase K treatment of either protoplasts or whole cells.
Breitling
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Department of Microbiology, Public Health Research Institute, New York, New York 10016.
Dubnau
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D
D000483:Alleles; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002872:Chromosome Deletion; D002876:Chromosomes, Bacterial; D004268:DNA-Binding Proteins / Q000378:metabolism*; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000378:metabolism; D015870:Gene Expression; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids
D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D036781:Fimbriae Proteins