Tn4001 mutagenesis identified a new competence gene in Streptococcus gordonii Challis designated comYA. A comYA mutant was completely deficient in transformation and exhibited decreased levels of DNA binding and hydrolysis. The deduced 319-amino-acid ComYA protein exhibited 57% similarity and 33% identity to the ComGA transporter protein of Bacillus subtilis and contained the Walker A-box motif conserved in ATP-binding proteins as well as aspartic acid boxes Asp-1 and Asp-2 present in some components of the general secretory pathway of gram-negative bacteria. comYA appeared to be part of a putative operon encompassing a comGB homolog, designated comYB, together with sequences that could encode ComGC- and ComGD-like peptides designated ComYC and ComYD, respectively, as well as other components. The putative ComYC and ComYD peptides had leader sequences similar to the type IV N-methylphenylalanine pilins of gram-negative bacteria, but unlike other examples in this class, including B. subtilis, they contained an alanine at position -1 of the leader instead of the usual glycine residue. Northern analysis identified a single 6.0-kb comYA-containing transcript strictly dependent on exogenous competence factor for expression in ComA1 cells. An identical pattern of expression was seen in wild-type Challis cells grown under conditions of maximal competence but not in cells that were noncompetent.