Search for genes essential for pneumococcal transformation: the RADA DNA repair protein plays a role in genomic recombination of donor DNA.
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 counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected 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 of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in 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 with 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 in DNA damage repair in S. pneumoniae.
Burghout
Peter
P
Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, P.O. Box 9101 (Route 224), 6500 HB Nijmegen, The Netherlands. P.Hermans@cukz.umcn.nl
Bootsma
Hester J
HJ
Kloosterman
Tomas G
TG
Bijlsma
Jetta J E
JJ
de Jongh
Christa E
CE
Kuipers
Oscar P
OP
Hermans
Peter W M
PW
D001426:Bacterial Proteins / Q000235:genetics*; D018983:DNA Footprinting / Q000379:methods; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D020043:Genes, Essential; D016680:Genome, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D011995:Recombination, Genetic / Q000235:genetics*; D013296:Streptococcus pneumoniae / Q000235:genetics*; D014169:Transformation, Bacterial / Q000235:genetics*; D016335:Zinc Fingers
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C100901:RadA protein, bacteria