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首页> 外文期刊>Journal of bacteriology >Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.
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Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

机译：铜绿假单胞菌中鼠李糖脂生物表面活性剂合成的调控基因的分离与鉴定。

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A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.
机译：通过使用铜绿假单胞菌PG201野生型基因文库，对不能产生鼠李糖脂生物表面活性剂并且缺乏鼠李糖基转移酶活性的铜绿假单胞菌的突变株（65E12）进行了遗传互补。通过使用同胞选择策略，在转导结合子的继代培养物的表面张力测量的基础上，分离了单个互补粘粒。互补粘粒克隆的亚克隆产生了一个2-kb的片段，该片段能够恢复鼠李糖脂的生物合成，鼠李糖基转移酶的活性，以及​​利用十六烷作为突变体65E12中的C源。确定了互补的2-kb片段的核苷酸序列，并且鉴定了723bp的单个开放阅读框（rhlR），其指定了推定的28kDa蛋白（RhlR）。 RhlR蛋白与某些调节蛋白（如铜绿假单胞菌的LasR，费氏弧菌弧菌，豆科根瘤菌的RhiR和推定的大肠杆菌28-kDa UvrC）之间的序列同源性表明RhlR蛋白是转录激活因子。已经鉴定了由RhlR蛋白调节的推定的靶启动子，其在rhR基因的上游2.5kb处。多个基于质粒的rhlR基因拷贝对含十六烷的基本培养基中的铜绿假单胞菌野生型菌株的生长，鼠李糖脂的产生以及绿脓素生色团的产生具有刺激作用。铜绿假单胞菌野生型rhlR基因座的破坏导致鼠李糖脂缺乏突变株，从而直接证实该基因是鼠李糖脂生物合成所必需的。另外，此类PG201 :::'rhlR'突变株缺乏弹性蛋白酶活性，表明RhlR蛋白是多效性调节剂。

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