Cloning and functional analysis by gene disruption of a gene encoding a γ-butyrolactone autoregulator receptor from Kitasatospora setae
Cloning and functional analysis by gene disruption of a gene encoding a γ-butyrolactone autoregulator receptor from Kitasatospora setae
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DOI：
10.1128/jb.186.11.3423-3430.2004
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发表时间：
2004-06-01
影响因子：
3.2
通讯作者：
Nihira, T
中科院分区：
文献类型：
--
作者：
Choi, SU;Lee, CK;Nihira, T
链霉菌属的γ-丁内酯自调节因子受体具有作为DNA结合转录抑制因子的共同活性，控制次级代谢和/或形态分化。首次从非链霉菌属的放线菌中克隆了巴夫洛霉素B产生菌刚毛北里孢菌（Kitasatospora setae）的γ-丁内酯自动调节受体基因，并通过体外和体内分析对其功能进行了评价。该基因片段最初通过PCR克隆，引物设计从两个高度保守的区域链霉菌自动调节受体（BarA，FarA，ScbR，和ArpA），然后通过基因组Southern杂交产生一个7 kb的BamHI片段上的654 bp的受体基因（ksbA）被确定。重组KsbA蛋白与~ 3 H标记的自身调节因子，特别是与[H-3] SCB 1有明显的结合活性，证实了ksbA蛋白编码的是一个真实的K.刚毛为了阐明ksbA在体内的功能，ksbA-破坏的菌株构建后，通过同源重组的方式引入ksbA破坏构建体通过转接合从大肠杆菌。在野生型菌株和ksbA破坏剂之间没有发现形态上的差异。然而，ksbA破坏物开始产生巴弗洛霉素比野生型菌株早18小时，并显示出2.4倍的巴弗洛霉素积累。通过与完整的ksbA互补，表型恢复到原始野生型表型，表明K.刚毛对巴夫洛霉素的生物合成起主要的负调节作用，但对K.刚毛这表明，与灰色链霉菌的A因子受体不同，K.刚毛属于控制次生代谢但在形态分化中不起作用的自动调节受体家族。
gamma-Butyrolactone autoregulator receptors of the genus Streptomyces have a common activity as DNA-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation. A gene encoding a gamma-butyrolactone autoregulator receptor was cloned from a bafilomycin B, producer, Kitasatospora setae, for the first time from a non-Streptomyces genus of actinomycetes, and its function was evaluated by in vitro and in vivo analyses. The gene fragment was initially cloned by PCR with primers designed from two highly conserved regions of Streptomyces autoregulator receptors (BarA, FarA, ScbR, and ArpA), followed by genomic Southern hybridization yielding a 7-kb BamHI fragment on which a 654-bp receptor gene (ksbA) was identified. The recombinant KsbA protein demonstrated clear binding activity toward 3 H-labeled autoregulators, especially toward [H-3]SCB1, confirming that ksbA encodes a real autoregulator receptor of K. setae. To clarify the in vivo function of ksbA, a ksbA-disrupted strain was constructed by means of homologous recombination after introducing a ksbA disruption construct via transconjugation from Escherichia coli. No difference in morphology was found between the wild-type strain and the ksbA disruptants. However, the ksbA disruptants started producing bafilomycin 18 h earlier than the wild-type strain and showed a 2.4-fold-higher accumulation of bafilomycin. The phenotype was restored to the original wild-type phenotype by complementation with intact ksbA, indicating that the autoregulator receptor protein of K. setae acts as a primary negative regulator of the biosynthesis of bafilomycin but plays no role in cytodifferentiation of K. setae. This indicates that, unlike the A-factor receptor of Streptomyces griseus, the autoregulator receptor (ksbA) of K. setae belongs to a family of autoregulator receptors which control secondary metabolism but play no role in morphological differentiation.