ISOLATION AND CHARACTERIZATION OF 3 STREPTOCOCCUS-PNEUMONIAE TRANSFORMATION SPECIFIC LOCI BY USE OF A LACZ REPORTER INSERTION VECTOR

Although more than a dozen new proteins are produced when Streptococcu s pneumoniae cells become competent for genetic transformation, only a few of the corresponding genes have been identified to date. To find genes responsible for the production of competence-specific proteins, a random lacZ transcriptional fusion library was constructed in S. pne umoniae by using the insertional lacZ reporter vector pEVP3, Screening the library for clones with competence-specific beta-galactosidase (b eta-Gal) production yielded three insertion mutants with induced beta- Gal levels of about 4, 10, and 40 Miller units. Tn all three clones, a ctivation of the lacZ reporter correlated with competence and depended on competence-stimulating peptide. Chromosomal loci adjacent to the i ntegrated vector were subcloned from the insertion mutants, and their nucleotide sequences were determined. Genes at two of the loci exhibit ed strong similarity to parts of Bacillus subtilis com operons, One lo cus contained open reading frames (ORFs) homologous to the comEA and c omEC genes in B. subtilis but lacked a comEB homolog, A second locus c ontained four ORFs with homology to the B. subtilis comG gene ORFs 1 t o 4, but comG gene ORFs 5 to 7 were replaced in S. pneumoniae with an ORF encoding a protein homologous to transport ATP-binding proteins. G enes at all three loci were confirmed to be required for transformatio n by mutagenesis using pEVP3 for insertion duplications or an erm cass ette for gene disruptions.