GAF competence. Streptococcus pneumoniae R6

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 Overall design: 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.我们采用一种名为基因组阵列足迹分析（Genomic Array Footprinting, GAF）的新型负筛选策略，旨在鉴定革兰氏阳性菌肺炎链球菌（Streptococcus pneumoniae）遗传转化所需的基因。以肺炎链球菌R6菌株构建全基因组范围的mariner转座子突变体文库，利用携带抗生素抗性盒的DNA进行转化，并在相应抗生素存在的条件下培养以施加筛选压力。本次GAF筛选发现，hexA与hexB两个基因的突变体出现富集，而在筛选过程中21个不同基因的突变体被反向筛选。其中8个被反向筛选的基因此前已被证实为肺炎链球菌转化所必需。另有4个基因（radA、comGF、parB及spr2011）已被证实与感受态调控子相关，还有一个基因spr2014紧邻必需感受态基因comFA。对剩余8个基因中的7个（即spr0459/spr0460、spr0777、spr0838、spr1259/spr1260及spr1357）构建的定向缺失突变体，其转化速率虽有降低但幅度较小。第8个基因编码响应调节因子RR03，目前尚未发现其与肺炎链球菌转化存在关联。我们进一步证实，编码假定DNA修复蛋白RadA的基因对于染色体标记的高效转化是必需的，而利用复制型质粒DNA进行的转化则未受到显著影响。radA突变体同时对DNA损伤剂甲基甲烷磺酸酯处理表现出更高的敏感性。因此，RadA被认为在肺炎链球菌的供体DNA重组与DNA损伤修复过程中发挥作用。 关键词：GAF 感受态 整体实验设计：筛选转化必需基因。 将肺炎链球菌R6中构建的、包含约40000个独立突变体的marinerT7转座子文库分装，分别利用抗生素抗性标记进行转化：一组不添加抗生素（初始文库），另一组添加抗生素（筛选后文库），培养25代后提取DNA。