Aendekerk Séverine, Ghysels Bart, Cornelis Pierre, Baysse Christine
Laboratory of Microbial Interactions, Department of Immunology, Parasitology and Ultrastructure, Flanders Interuniversity Institute of Biotechnology, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint Genesius Rode, Belgium1.
Microbiology (Reading). 2002 Aug;148(Pt 8):2371-2381. doi: 10.1099/00221287-148-8-2371.
Vanadium has an antibacterial activity against Pseudomonas aeruginosa, especially under conditions of iron limitation. Some degree of resistance to V is inducible by prior exposure to the metal. One mutant (VS1) with a higher sensitivity to V was obtained by transposon mutagenesis of P. aeruginosa PA 59.20, a clinical isolate. This mutant had an insertion in a non-coding region, upstream of a cluster of four genes. Three of them show similarities to genes corresponding to known P. aeruginosa antibiotic efflux systems, including an efflux protein, a membrane fusion protein and an outer-membrane porin. This cluster was named mexGHI-opmD. By allelic exchange, three mutants, ncr (for non-coding region), mexI and opmD were constructed in P. aeruginosa PAO1. Next to V sensitivity, the ncr, mexI and opmD mutants also showed reduced production of elastase, rhamnolipids, pyocyanine, pyoverdine and had reduced swarming motility, phenotypes that are known to be regulated by quorum sensing. All wild-type phenotypes, including growth in the presence of V, were restored by complementation with the complete cluster. The production of N-acyl-homoserine lactones (AHLs) was detected using the Chromobacter violaceum bioassay. Total extracts from the three mutants failed to induce the production of violacein by C. violaceum, although AHLs were detected by TLC and C. violaceum overlay. Violacein production was restored by complementation with mexGHI-opmD. The opmD mutant grew very slowly in LB or CAA medium, indicating that OpmD has an important physiological function for the cell. In conclusion, it is believed that the MexGHI-OpmD pump is probably involved in AHL homeostasis in P. aeruginosa.
钒对铜绿假单胞菌具有抗菌活性，尤其是在铁限制条件下。预先接触该金属可诱导一定程度的钒抗性。通过对临床分离株铜绿假单胞菌PA 59.20进行转座子诱变，获得了一株对钒敏感性更高的突变体（VS1）。该突变体在四个基因簇上游的非编码区域有一个插入。其中三个基因与已知的铜绿假单胞菌抗生素外排系统的对应基因相似，包括一个外排蛋白、一个膜融合蛋白和一个外膜孔蛋白。这个基因簇被命名为mexGHI-opmD。通过等位基因交换，在铜绿假单胞菌PAO1中构建了三个突变体，即ncr（非编码区域）、mexI和opmD。除了对钒敏感外，ncr、mexI和opmD突变体还表现出弹性蛋白酶、鼠李糖脂、绿脓菌素、绿脓菌素的产量降低，以及群体运动性降低，这些表型已知受群体感应调节。通过用完整的基因簇进行互补，所有野生型表型，包括在钒存在下的生长，都得以恢复。使用紫色杆菌生物测定法检测N-酰基高丝氨酸内酯（AHLs）的产生。尽管通过薄层层析和紫色杆菌覆盖检测到了AHLs，但来自这三个突变体的总提取物未能诱导紫色杆菌产生紫菌素。通过用mexGHI-opmD进行互补，紫菌素的产生得以恢复。opmD突变体在LB或CAA培养基中生长非常缓慢，这表明OpmD对细胞具有重要的生理功能。总之，据信MexGHI-OpmD泵可能参与了铜绿假单胞菌中AHL的稳态。
