The Operon, Structure and Biological Activities of the Lipopeptide Bacillomycin L Produced by Bacillus subtilis Bs916
The Operon, Structure and Biological Activities of the Lipopeptide Bacillomycin L Produced by Bacillus subtilis Bs916
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发表时间：
2010
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通讯作者：
C. Luo;Xiaoyu Wang;Chen Zhiyi;Yongfeng Liu;Zhang Jie;Youzhou Liu;Y. Nie;Jun-jie Yu;Xiaole Yin
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作者：
C. Luo;Xiaoyu Wang;Chen Zhiyi;Yongfeng Liu;Zhang Jie;Youzhou Liu;Y. Nie;Jun-jie Yu;Xiaole Yin
[目的]从枯草芽孢杆菌Bs916中克隆并测序与杆菌霉素合成有关的Bac操纵子，并对其结构和生物活性进行鉴定。[方法]采用LA-PCR法和基因步行法克隆芽孢杆菌L合成Bac操纵子，并利用生物信息学对其遗传结构进行分析。采用基质辅助激光解吸电离飞行时间质谱仪(MS)测定杆菌素L同系物的相对分子质量。用电喷雾电喷雾串联质谱仪测定了杆菌素L多肽的一级结构。[结果]从枯草芽孢杆菌BS-916中鉴定并克隆了与杆菌素L合成有关的39.0kbBac操纵子。该操纵子含有一个启动子和四个开放阅读框，四个开放阅读框分别命名为BacD、BacA、Bacb和Bacc。虽然Bac操纵子编码的氨基酸序列与ITU、myc和bam操纵子编码的相应氨基酸序列有很高的相似性，但也发现了一个低相似性区域，推测它是一个新的Ser激活区。根据BAc序列，推测该脂肽属于杆菌素L，其分子量为1 008、1 022、1 036和1 050 Da。推测它们属于不同于-CH2结构的同系物。杆菌素L的多肽部分一级结构为[环-(天冬氨酸-酪氨酸-天冬氨酸-丝氨酸-谷氨酸-丝氨酸-苏氨酸-β-氨基酸)]，与以前报道的杆菌素L的多肽序列相同。对病原真菌的体外试验表明，芽孢菌素L具有广泛的抗真菌活性和溶血活性。[结论]本文报道了杆菌素L合成操纵子Bac的克隆、测序和鉴定，通过生物信息学和化学分析，进一步证实了以前报道的杆菌素L的一级结构。由Bac合成的杆菌霉素L具有广泛的抗真菌活性，在BS-916生防活性中起着至关重要的作用。
【Objective】The aim of this study is to clone and sequence the Bac operon responsible for synthesis of the bacillomycin L from Bacillus subtilis Bs916 and determine the structure and biological activities of the bacillomycin L. 【Method】 LA-PCR and gene walking were performed to clone the Bac operon responsible for synthesis of bacillomycin L. Analysis of the Bac operon genetic structure was made using bioinformatics. The molecular weight of the bacillomycin L homologues was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS). ESI/MS-CID was used to determine the bacillomycin L peptide moiety primary structure. 【Result】 The 39.0 kb Bac operon responsible for synthesis of bacillomycin L was identified and cloned from Bacillus subtilis Bs-916. The operon contained a promoter and four ORFs and the four ORFs designatedBacD, BacA, BacB, and BacC, respectively. Although the amino acid sequences encoded by the Bac operon share high similarity with the countpart amino acid sequences encoded by itu, myc and bam operons, a low similarity region was also found and it presumed to be a novel Ser activation domain. According to the Bac sequence, the lipoepeptide presumed to belong to bacillomycin L. The molecular weights of the bacillomycin L were 1 008,1 022,1 036 and 1 050 Da. They were presumed to belong to homologues differed by a structure of -CH2. The bacillomycin L peptide moiety primary structure was [cyclo- (Asn-Tyr-Asn-Ser-Glu-Ser-Thr-β-amino fatty acid)] and it is the same as the peptide sequence of bacillomycin L reported previously. In vitro tests to pathogenic fungi indicated that bacillomycin L have a broad antifungal activities and hemolytic activities. 【Conclusion】This paper reported the cloning, sequencing and characterization of a whole operon Bac which is responsible for synthesis of bacillomycin L. Through bio-information and chemical analysis, the authors also further confirmed the primary structure of bacillomycin L which has paradox reported before. The bacillomycin L synthesized by Bac has a broad antifungal activities and plays crucial part in Bs-916 bio-control activities.