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A 2.8-kb EcoRI-BglII fragment cloned from the wild-type Haemophilus influenzae Rd chromosome is shown to increase the transformability of the Com-101 mutant through trans complementation. Deletion and sequence analyses indicate that the active region of the clone carries a 687-bp open reading frame. A 0.3-kb insertion in the corresponding EcoRI-BglII fragment of the Com-101 chromosome is shown to be a partial (331-bp) duplication of this open reading frame. The wild-type sequence produces a peptide of a size that is consistent with the sequence data when this sequence is expressed in Escherichia coli with a T7 promoter-based transcription vector. RNA hybridization analysis using a DNA probe derived from the open reading frame suggests that the sequence is transiently expressed during competence development. On the basis of these observations, it is proposed that the open reading frame corresponds to the com101A gene.

A plasmid containing a 13.3-kb insert (pER194) was isolated from an EcoRI genomic library of Haemophilus influenzae on the basis of its ability to increase the transformability of the transformation-deficient mutants Com-78 and Com-101. The plasmid failed to increase the transformability of the Rec-1 and Rec-2 mutants, indicating that the mutations producing the Com-78 and Com-101 phenotypes are distinct from those giving rise to the Rec-1 and Rec-2 phenotypes. The physical mapping of the cloned fragment on the H. influenzae chromosome was found to be consistent with the genetic mapping of the Com-101 trait. A 2.8-kb EcoRI-BglII subfragment, representing one end of the 13.3-kb clone, was found to increase the transformation frequency of the Com-78 and Com-101 mutants when supplied in trans, indicating that the subfragment carries one or more loci required for chromosomal transformation. The corresponding region of the Com-101 chromosome was determined by hybridization analysis to contain a 0.3-kb insertion, suggesting that the Com-101 strain may contain an insertion mutation at this locus. A 3.0-kb EcoRI-MluI subfragment, representing the other end of the 13.3-kb EcoRI fragment, was found to increase the transformation frequency of the Com-101 mutant but not of the Com-78 mutant, suggesting that the Com-101 phenotype results from a complex genotype involving mutations at two or more transformation-related loci. This conclusion is consistent with data indicating that the Com-101 trait can be genetically separated into at least two components.