Characterization of four plasmids harboured in a Lactobacillus brevis strain encoding a novel bacteriocin, brevicin 925A, and construction of a shuttle vector for lactic acid bacteria and Escherichia coli
Characterization of four plasmids harboured in a Lactobacillus brevis strain encoding a novel bacteriocin, brevicin 925A, and construction of a shuttle vector for lactic acid bacteria and Escherichia coli
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DOI：
10.1099/mic.0.022871-0
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发表时间：
2009-05-01
期刊：
影响因子：
2.8
通讯作者：
Sugiyama, Masanori
中科院分区：
文献类型：
--
作者：
Wada, Takaomi;Noda, Masafumi;Sugiyama, Masanori
在这项研究中，我们从水果，花卉，蔬菜和发酵食品中分离出超过250种乳酸菌（LAB）候选菌，以产生LAB文库。从韩国泡菜（一种由大白菜制成的传统韩国发酵菜肴）中分离的一种菌株，命名为925 A，产生一种新型细菌素，短菌素925 A，其对某些LAB有效，包括乳杆菌属、肠球菌属、链球菌属、芽孢杆菌属和李斯特菌属。菌株925 A，鉴定为短乳杆菌，窝藏至少四个质粒，我们确定了每一个的整个核苷酸序列。这4个质粒命名为pLB 925 A01 -04，分子量分别为1815、3524、8881和65 037 lop。我们通过用新生霉素处理菌株925 A获得了不产生细菌素的衍生物。所有这些对自身抗菌产物敏感的衍生物都丢失了最大的质粒pLB 925 A04，这表明细菌素生物合成（breB和breC）和免疫（breE）的基因位于pLB 925 A04上。对纯化的brevicin 925 A的部分氨基酸序列进行测定，并对pLB 925 A04进行序列分析，结果表明breB是brevicin 925 A的结构基因。我们构建了一个穿梭载体（pLES 003，6134 bp），该载体可以在大肠杆菌和乳酸杆菌如植物乳杆菌、Lb.短乳杆菌、瑞士乳杆菌、希尔加德乳杆菌和海氏肠球菌。为了确定基因breE的功能，将该基因插入pLES 003中，该基因与BLAST搜索数据库中的任何其他序列没有显著的相似性。用pLES 003转化的pLB 925 A04-固化衍生物携带对短蛋白925 A的breE获得性免疫，表明breE编码免疫蛋白。
In this study we isolated over 250 lactic acid bacteria (LAB) candidates from fruit, flowers, vegetables and a fermented food to generate an LAB library. One strain, designated 925A, isolated from kimchi (a traditional Korean fermented dish made from Chinese cabbage) produced a novel type of bacteriocin, brevicin 925A, which is effective against certain LAB, including strains of Lactobacillus, Enterococcus, Streptococcus, Bacillus and Listeria. Strain 925A, identified as Lactobacillus brevis, harboured at least four plasmids and we determined the entire nucleotide sequence of each one. The four plasmids were designated pLB925A01-04, and have molecular sizes of 1815, 3524, 8881 and 65 037 lop, respectively. We obtained bacteriocin non-producing derivatives by treatment of strain 925A with novobiocin. All of these derivatives, which were susceptible to their own antibacterial product, lost the largest plasmid, pLB925A04, suggesting that the genes for bacteriocin biosynthesis (breB and breC) and immunity (breE) are located on pLB925A04. The partial amino acid sequence of purified brevicin 925A and sequence analysis of pLB925A04 showed that breB is the structural gene for brevicin 925A. We constructed a shuttle vector (pLES003, 6134 bp) that can replicate in both Escherichia coli and LAB such as Lactobacillus plantarum, Lb. brevis, Lactobacillus helveticus, Lactobacillus hilgardii and Enterococcus hirae. To determine the function of gene breE, which displays no significant similarity to any other sequences in the BLAST search database, the gene was inserted into pLES003. A pLB925A04-cured derivative transformed with pLES003 carrying breE acquired immunity to brevicin 925A, suggesting that breE encodes an immunity protein.