Tomb J F, Barcak G J, Chandler M S, Redfield R J, Smith H O
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Bacteriol. 1989 Jul;171(7):3796-802. doi: 10.1128/jb.171.7.3796-3802.1989.
A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.
用mini-Tn10kan在大肠杆菌中对流感嗜血杆菌Rd基因组DNA的PstI片段质粒文库进行诱变。诱变后的PstI片段通过转化导入流感嗜血杆菌染色体，通过环磷酸腺苷- DNA平板法筛选卡那霉素抗性转化体的转化缺陷表型。分离出54个突变菌株，其中包含24个独特的插入片段，这些插入片段定位于10个不同的PstI片段。对携带独特插入片段的菌株分别进行DNA摄取、转化效率、紫外线敏感性和生长速率测试。这些突变体的转化频率降低了10^(-2)到10^(-6)倍。其中5个突变体具有正常的感受态诱导DNA摄取，其余的在感受态发育方面存在不同程度的缺陷。3个菌株对紫外线中度敏感。除一个菌株外，所有菌株的倍增时间都在野生型的50%以内。将突变基因克隆到流感嗜血杆菌-大肠杆菌穿梭载体中，并通过体内重组交换回收野生型基因座。这些克隆与脉冲场凝胶中分离的SmaI基因组片段的杂交表明，这些插入片段在染色体的特定区域没有聚集。
