IDENTIFICATION AND CHARACTERIZATION OF GENES FOR A 2ND ANTHRANILATE SYNTHASE IN PSEUDOMONAS-AERUGINOSA - INTERCHANGEABILITY OF THE 2 ANTHRANILATE SYNTHASES AND EVOLUTIONARY IMPLICATIONS
IDENTIFICATION AND CHARACTERIZATION OF GENES FOR A 2ND ANTHRANILATE SYNTHASE IN PSEUDOMONAS-AERUGINOSA - INTERCHANGEABILITY OF THE 2 ANTHRANILATE SYNTHASES AND EVOLUTIONARY IMPLICATIONS
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DOI：
10.1128/jb.172.2.884-900.1990
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发表时间：
1990-02-01
影响因子：
3.2
通讯作者：
CRAWFORD, IP
中科院分区：
文献类型：
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作者：
ESSAR, DW;EBERLY, L;CRAWFORD, IP
在铜绿假单胞菌中已鉴定出两对邻氨基苯甲酸合酶基因。它们被克隆，测序，通过插入抗生素抗性基因在体外灭活，并返回铜绿假单胞菌，取代野生型基因。一种邻氨基苯甲酸合成酶参与色氨酸合成;其基因被设计为trpE和trpG。另一种邻氨基苯甲酸酯酶，编码phnA和phnB，参与绿脓菌素的合成，绿脓菌素是生物体的特征性吩嗪色素。trpE和trpG是独立转录的;同源基因已从恶臭假单胞菌中克隆。吩嗪途径基因phnA和phnB是共转录的。克隆的phnA phnB基因对互补大肠杆菌的trpE和trpE（G）突变体。恶臭假单胞菌PPG 1（一种非吩嗪产生菌）中未发现同源基因。令人惊讶的是，PhnA和PhnB与E. coliTrpE和TrpG与假单胞菌TrpE和TrpG的亲缘关系较近，而假单胞菌TrpE和TrpG与E. coliPabB和PabA对E. coliTrpE和TrpG。我们将铜绿假单胞菌染色体上的野生型trpE替换为突变形式，该突变形式具有相当大部分的编码序列缺失并被四环素抗性基因盒替换。这导致色氨酸营养缺陷型;然而，自发的色氨酸非依赖性回复突变体以10 - 5至10 - 6的频率出现。这些回复突变体的邻氨基苯甲酸合酶不被色氨酸反馈抑制，表明它来自PhnAB。phnA突变体保持低水平绿脓菌素产生。将失活的trpE基因引入到phnA突变体中废除了残余绿脓菌素的产生，这表明trpE trpG基因产物能够为绿脓菌素合成提供一些邻氨基苯甲酸。
Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designed trpE and trpG. The other anthranilate synthases enzyme, encoded phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Psuedomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10-5 to 10-6. The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutant retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.