Isolation and Characterization of Three Streptococcus pneumoniae Transformation-Specific Loci by Use of a lacZ Reporter Insertion Vector

Abstract

Although more than a dozen new proteins are produced whenStreptococcus 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. pneumoniae by using the insertional lacZ reporter vector pEVP3. Screening the library for clones with competence-specific β-galactosidase (β-Gal) production yielded three insertion mutants with induced β-Gal levels of about 4, 10, and 40 Miller units. In all three clones, activation of the lacZ reporter correlated with competence and depended on competence-stimulating peptide. Chromosomal loci adjacent to the integrated vector were subcloned from the insertion mutants, and their nucleotide sequences were determined. Genes at two of the loci exhibited strong similarity to parts ofBacillus subtilis com operons. One locus contained open reading frames (ORFs) homologous to the comEA andcomEC genes in B. subtilis but lacked acomEB homolog. A second locus contained four ORFs with homology to the B. subtilis comG gene ORFs 1 to 4, butcomG gene ORFs 5 to 7 were replaced in S. pneumoniae with an ORF encoding a protein homologous to transport ATP-binding proteins. Genes at all three loci were confirmed to be required for transformation by mutagenesis using pEVP3 for insertion duplications or an erm cassette for gene disruptions.

Identification of DNA binding sites for ComE, a key regulator of natural competence in Streptococcus pneumoniae

“…the proteins involved in binding, processing, uptake and integration of DNA. The work of Campbell et al (1998) and Pestova and Morrison (1998) has demonstrated that the late genes share a common regulatory sequence called the competence-induced promoter Q 1999 Blackwell Science Ltd, Molecular Microbiology, 33, 817±827 (CIP) consensus sequence. This sequence is unrelated to the sequence recognized by ComE, showing that ComE activates transcription of the late genes via one or more intermediate factors.…”

ComEA is a DNA receptor for transformation of competent Bacillus subtilis

“…Both of these ComEA orthologues are situated immediately upstream from an open reading frame that encodes an orthologue of ComEC. In the case of S. pneumoniae, this operon is required for transformation (Campbell et al, 1998;Pestova and Morrison, 1998). ComEA is 28.5% identical (60% similar) to MTCY253.05 and 37.8% identical (72.7% similar) to the streptococcal protein.…”

Shuffling genes around in hot environments: the unique DNA transporter ofThermus thermophilus

“…Thermus thermophilus ComEA orthologs are present in different transformable bacteria such as B. subtilis, N. gonorrhoeae, Campylobacter jejuni and S. pneumoniae (Pestova & Morrison, 1998;Provvedi & Dubnau, 1999;Chen & Gotschlich, 2001;Jeon & Zhang, 2007). In contrast to the membrane localization of ComEA in B. subtilis, the neisserial and Campylobacter ortholog, ComE and Cj0011c, respectively, are located in the periplasm.…”