Mutations in each of the tol genes of Pseudomonas putida reveal that they are critical for maintenance of outer membrane stability
Mutations in each of the tol genes of Pseudomonas putida reveal that they are critical for maintenance of outer membrane stability
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DOI：
10.1128/jb.182.17.4764-4772.2000
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发表时间：
2000-09-01
影响因子：
3.2
通讯作者：
Rodríguez-Herva, JJ
中科院分区：
文献类型：
--
作者：
Llamas, MA;Ramos, JL;Rodríguez-Herva, JJ
革兰氏阴性菌的外膜作为一种渗透性屏障，保护细胞免受大量抗菌剂的侵害。恶臭假单胞菌的OprL蛋白已被证明对维持该细胞成分的稳定性至关重要（J. J. Rodriguez-Herva, m . i .）。拉莫斯-冈萨雷斯和j.l.拉莫斯。[j] .中华微生物学杂志，2008,32(2):481 - 481。本研究克隆并诱变了恶臭p.p . putida KT2440中位于oprL基因上游的orf1、role、tolR、toll和tolB基因。利用Omega-Km(r)介子（分别携带两个转录停止信号）和一个无启动子的xylE盒（缺乏任何转录停止信号），在体外产生了恶臭p.p . tale、tolR、tolA和tolB基因的极性和非极性突变。突变结构被用来灭活，通过反向遗传程序，相应的染色体拷贝的基因。对每个突变株的表型进行分析，并与野生型菌株和先前鉴定的恶臭假单胞菌oprL::xylE突变株进行比较。所有突变菌株都表现出相似的表型：细胞形态改变，细胞表面形成气泡，质周和外膜蛋白向细胞外介质释放，对多种化合物（即EDTA，十二烷基硫酸钠，脱氧胆酸钠和某些抗生素）的敏感性增加，纤维形成，细胞运动性严重降低。总之，这些结果证明了Tol-OprL系统对维持恶臭假单胞菌外膜完整性的重要性，并提示了这些蛋白质在组装外膜成分中的可能作用。
The outer membrane of gram-negative bacteria functions as a permeability barrier that protects cells against a large number of antibacterial agents. OprL protein of Pseudomonas putida has been shown to he crucial to maintain the stability of this cell component (J. J. Rodriguez-Herva, M.-I. Ramos-Gonzalez, and J. L. Ramos. J. Bacteriol. 178:1699-1706, 1996). In the present study we cloned and mutagenized the orf1, role, tolR, toll, and tolB genes from P. putida KT2440, which were located upstream of the oprL gene. Polar and nonpolar mutations of the P. putida tale, tolR, tolA, and tolB genes were generated in vitro by using the Omega-Km(r) interposon, which carries two transcriptional stop signals, or a promoterless xylE cassette, lacking any transcriptional stop signal, respectively. The mutant constructs were used to inactivate, by reverse genetics procedures, the corresponding chromosomal copies of the genes. The phenotype of each mutant strain was analyzed and compared with those of the wild-type strain and the previously characterized P. putida oprL::xylE mutant. All mutant strains exhibited a similar phenotype: altered cell morphology, bleb formation at the cell surface, release of periplasmic and outer membrane proteins to the extracellular medium, increased sensitivity to a variety of compounds (i.e., EDTA, sodium dodecyl sulfate, deoxycholate, and some antibiotics), filament formation, and severely reduced cell motility. Altogether, these results demonstrate the importance of the Tol-OprL system for the maintenance of outer membrane integrity in P. putida and suggest a possible role of these proteins in assembling outer membrane components.