Laboratory for Molecular Genetics of Industrial Microorganisms, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444/a, P.O. Box 23, 11010 Belgrade, Serbia.
Int J Food Microbiol. 2010 Jun 15;140(2-3):117-24. doi: 10.1016/j.ijfoodmicro.2010.04.010. Epub 2010 Apr 14.
A new shuttle-cloning vector, pA13, was constructed and successfully introduced into Escherichia coli, Lactobacillus and Lactococcus strains. It showed high segregational and structural stability in all three hosts. The natural plasmid pSJ2-8 from L. paracasei subsp. paracasei BGSJ2-8 was cloned into pA13 using BamHI to obtain the construct, pB5. Sequencing and in silico analysis of pB5 revealed fifteen open reading frames (ORF). Plasmid pSJ2-8 harbours genes encoding the production of two bacteriocins, BacSJ and acidocin 8912. Combined N-terminal amino acid sequencing of BacSJ in combination with DNA sequencing of the bacSJ2-8 gene enabled determination of the primary structure of bacteriocin BacSJ. The bacSJ2-8 gene encodes 68-amino-acid peptide with a double-glycine leader peptide consisting of 18 amino acids, followed by the orf2 (bacSJ2-8i) which encodes the immunity protein of BacSJ. The production and functional expression of BacSJ in homologous and heterologous hosts suggest that bacSJ2-8 and bacSJ2-8i together with the genes encoding the ABC transporter and accessory protein are the minimal requirements for production of BacSJ. Biochemical and genetic analyses showed that BacSJ belongs to class II bacteriocins.
构建了一种新型穿梭克隆载体 pA13，并成功将其导入大肠杆菌、乳杆菌和乳球菌菌株。它在三种宿主中均表现出高度的分离和结构稳定性。从副干酪乳杆菌亚种副干酪乳杆菌 BGSJ2-8 中分离出的天然质粒 pSJ2-8 用 BamHI 克隆到 pA13 中，得到构建体 pB5。对 pB5 的测序和计算机分析揭示了 15 个开放阅读框（ORF）。质粒 pSJ2-8 编码两种细菌素的产生，即 BacSJ 和酸菌素 8912。BacSJ 的 N 端氨基酸测序与 bacSJ2-8 基因的 DNA 测序相结合，确定了细菌素 BacSJ 的一级结构。bacSJ2-8 基因编码 68 个氨基酸的肽，带有由 18 个氨基酸组成的双甘氨酸前导肽，其后是 orf2（bacSJ2-8i），它编码 BacSJ 的免疫蛋白。BacSJ 在同源和异源宿主中的生产和功能表达表明，bacSJ2-8 和 bacSJ2-8i 以及编码 ABC 转运蛋白和辅助蛋白的基因是产生 BacSJ 的最小要求。生化和遗传分析表明，BacSJ 属于 II 类细菌素。
