Goldberg J B, Ohman D E
J Bacteriol. 1984 Jun;158(3):1115-21. doi: 10.1128/jb.158.3.1115-1121.1984.
Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis commonly produce a capsule-like exopolysaccharide called alginate. The alginate-producing (Alg+) phenotype results in a mucoid colony morphology and is an unstable trait. A mutant of P. aeruginosa FRD (a cystic fibrosis isolate) was obtained which was temperature sensitive for alginate production ( Algts ). At elevated growth temperatures (41 degrees C), no alginate was detected in culture supernatants of the Algts mutant, but yields of alginate increased as the temperature of incubation was reduced. The mutation responsible for the Algts phenotype, alg-50(Ts), has been mapped to a region of the FRD chromosome closely linked to trp-2. The alg-50(Ts) marker did not map near the met-l-linked chromosomal mutations responsible for the instability of the Alg+ phenotype. A broad host range cosmid cloning system based upon derivatives of plasmid RK2 was used to construct a P. aeruginosa clone bank. After transfer of the clone bank to the Algts mutant, hybrid plasmids were obtained which complemented the Algts defect. Deletion mapping of the original 20.3 kilobases of P. aeruginosa DNA cloned showed that a 4.7-kilobase fragment would complement the alg-50(Ts) mutation.
从囊性纤维化患者中分离出的铜绿假单胞菌菌株通常会产生一种名为藻酸盐的胶囊样胞外多糖。产生藻酸盐（Alg+）的表型会导致黏液样菌落形态，并且是一种不稳定的性状。获得了铜绿假单胞菌FRD（一种囊性纤维化分离株）的突变体，该突变体对藻酸盐产生具有温度敏感性（Algts）。在较高的生长温度（41摄氏度）下，在Algts突变体的培养上清液中未检测到藻酸盐，但随着孵育温度降低，藻酸盐产量增加。导致Algts表型的突变alg-50（Ts）已被定位到FRD染色体上与trp-2紧密连锁的区域。alg-50（Ts）标记并不位于与Alg+表型不稳定性相关的met-1连锁染色体突变附近。基于质粒RK2衍生物的广泛宿主范围黏粒克隆系统被用于构建铜绿假单胞菌克隆文库。将克隆文库转移到Algts突变体后，获得了能够互补Algts缺陷的杂交质粒。对最初克隆的20.3千碱基铜绿假单胞菌DNA进行缺失定位表明，一个4.7千碱基的片段能够互补alg-50（Ts）突变。
