Menges R, Muth G, Wohlleben W, Stegmann E
Lehrstuhl für Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Universität Tübingen, Tübingen, Germany.
Appl Microbiol Biotechnol. 2007 Nov;77(1):125-34. doi: 10.1007/s00253-007-1139-x. Epub 2007 Sep 6.
All known gene clusters for glycopeptide antibiotic biosynthesis contain a conserved gene supposed to encode an ABC-transporter. In the balhimycin-producer Amycolatopsis balhimycina this gene (tba) is localised between the prephenate dehydrogenase gene pdh and the peptide synthetase gene bpsA. Inactivation of tba in A. balhimycina by gene replacement did not interfere with growth and did not affect balhimycin resistance. However, in the supernatant of the tba mutant RM43 less balhimycin was accumulated compared to the wild type; and the intra-cellular balhimycin concentration was ten times higher in the tba mutant RM43 than in the wild type. These data suggest that the ABC transporter encoded in the balhimycin biosynthesis gene cluster is not involved in resistance but is required for the efficient export of the antibiotic. To elucidate the activity of Tba it was heterologously expressed in Escherichia coli with an N-terminal His-tag and purified by nickel chromatography. A photometric assay revealed that His(6)-Tba solubilised in dodecylmaltoside possesses ATPase activity, characteristic for ABC-transporters.
所有已知的糖肽类抗生素生物合成基因簇都包含一个保守基因，推测其编码一种ABC转运蛋白。在巴龙霉素产生菌拜莱霉素拟无枝酸菌（Amycolatopsis balhimycina）中，该基因（tba）位于预苯酸脱氢酶基因pdh和肽合成酶基因bpsA之间。通过基因置换使拜莱霉素拟无枝酸菌中的tba失活，既不影响生长，也不影响对巴龙霉素的抗性。然而，与野生型相比，tba突变体RM43的上清液中积累的巴龙霉素较少；并且tba突变体RM43中的细胞内巴龙霉素浓度比野生型高10倍。这些数据表明，巴龙霉素生物合成基因簇中编码的ABC转运蛋白不参与抗性，但对抗生素的有效输出是必需的。为了阐明Tba的活性，将其与N端His标签在大肠杆菌中进行异源表达，并通过镍层析进行纯化。一项光度测定显示，溶解在十二烷基麦芽糖苷中的His(6)-Tba具有ABC转运蛋白特有的ATP酶活性。
