Rodríguez-Herva J J, Ramos-Gonzalez M I, Ramos J L
Consejo Superior de Investigaciones Cientificas, Department of Biochemistry and Molecular and Cellular Biology of Plants, Granada, Spain.
J Bacteriol. 1996 Mar;178(6):1699-706. doi: 10.1128/jb.178.6.1699-1706.1996.
Pseudomonas putida 14G-3, a derivative of the natural soil inhabitant P. putida KT2440, exhibited a chromosomal insertion of a mini-Tn5/'phoA transposon that resulted in reduced ability to colonize soil. In vitro characterization of P. putida 14G-3 revealed that it exhibited an altered cell morphology and envelope, as revealed by electron microscopy. The derived strain was sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA, produced clumps when it reached high cell densities in the late logarithmic growth phase, and did not grow on low-osmolarity medium. The P. putida DNA surrounding the mini-Tn5/'phoA insertion was cloned and used as a probe to rescue the wild-type gene, which was sequenced. Comparison of the deduced peptide sequence with sequences in the Swiss-Prot database allowed the knocked-out gene to be identified as that encoding the peptidoglycan-associated lipoprotein (Pal or OprL) of P. putida. The protein was identified in coupled transcription and translation assays in vitro.
恶臭假单胞菌14G-3是天然土壤栖居菌恶臭假单胞菌KT2440的衍生物,其染色体插入了一个mini-Tn5/'phoA转座子,导致其在土壤中定殖的能力下降。对恶臭假单胞菌14G-3的体外特性分析表明,通过电子显微镜观察,它呈现出改变的细胞形态和包膜。衍生菌株对十二烷基硫酸钠、脱氧胆酸盐和乙二胺四乙酸敏感,在对数生长后期细胞密度较高时会形成菌团,并且在低渗培养基上无法生长。围绕mini-Tn5/'phoA插入位点的恶臭假单胞菌DNA被克隆,并用作探针来拯救野生型基因,该基因随后被测序。将推导的肽序列与瑞士蛋白质数据库中的序列进行比较,使得被敲除的基因被鉴定为编码恶臭假单胞菌肽聚糖相关脂蛋白(Pal或OprL)的基因。该蛋白质在体外的偶联转录和翻译试验中得到了鉴定。