GAF competence

We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner-transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counter-selection of mutants in 21 different genes during the challenge. Eight of the counter-selected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants in which seven of the eight remaining genes, i.e., spr0459/spr0460, spr0777, spr0838, spr1259/spr1260, and spr1357, were deleted, displayed reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation of chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and DNA damage repair in S. pneumoniae. Keywords: GAF competence Selection for genes essential for transformation. Aliquots of a marinerT7 transposon library generated in S. pneumoniae R6 containing approximately 40,000 independent mutants were transformed with an antibiotic resistance marker and grown without (initial library) or with (challenged library) antibiotics for 25 generation until DNA extraction.