impA, a gene coding for an inner membrane protein, influences colonial morphology of Actinobacillus actinomycetemcomitans

Directed mutagenesis of a gene coding for a membrane protein of the periodo ntopathogen Actinobacillus actinomycetemcomitans was achieved by conjugatio n. The gene was disrupted by insertion of an antibiotic cassette into a uni que endonuclease restriction sequence engineered by inverse PCR. The disrup ted gene was cloned into a conjugative plasmid and transferred from Escheri chia coli to A. actinomycetemcomitans. The allelic replacement mutation res ulted in the loss of a 22-kDa inner membrane protein. The loss of this prot ein (ImpA) resulted in changes in the outer membrane protein composition of the bacterium. Concurrent with the mutation in impA was a change in the pa ttern of growth of the mutant bacteria in broth cultures. The progenitor ba cteria grew as a homogeneous suspension of cells compared to a granular, au toaggregating adherent cell population described for the mutant bacteria. T hese data suggest that ImpA may play a regulatory role or be directly invol ved in protein(s) that are exported and associated with colony variations i n A. actinomycetemcomitans.