Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071
Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071
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DOI：
10.1128/aem.66.4.1298-1304.2000
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发表时间：
2000-04-01
影响因子：
4.4
通讯作者：
Holzapfel, WH
中科院分区：
文献类型：
--
作者：
Balla, E;Dicks, LMT;Holzapfel, WH
从哥廷根小型猪粪便中分离得到的faccalis肠球菌BFE 1071的ph中性细胞上清液对肠球菌和其他几种革兰氏阳性细菌的生长有抑制作用。对细胞上清进行硫酸铵沉淀和阳离子交换层析，然后进行质谱分析，得到两种分子质量相似的细菌素样肽：enterocin 1071A （4.285 kDa）和enterocin 1071B （3.899 kDa）。两个肽总是一起分离的。这些肽是耐热的（100摄氏度，60分钟；在121摄氏度，15分钟后仍有50%的活性），在pH 3至12的孵育30分钟后仍有活性，并且对蛋白水解酶的处理敏感。固化实验表明，编码enterocins 1071A和1071B的基因位于一个50 kbp的质粒（pEF1071）上。质粒pEF1071与粪肠球菌FA2-2和OGX1结合后，表达了两个与肠球菌1071A和1071B大小相同的活性肽，对9 ~ 10 kbp的DNA片段进行测序，发现两个开放阅读框ent1071A和ent1071B，分别编码39-氨基酸和m -氨基酸肽。分别。成熟的Ent1071A和Ent1071B肽段的氨基酸序列与乳球菌蛋白G的α肽和β肽的同源性分别为64%和61%。这是代表第四种粪肠杆菌细菌素的两种新抗菌肽的首次报道。
The pH-neutral cell supernatant of Enterococcus faccalis BFE 1071, isolated from the feces of minipigs in Gottingen, inhibited the growth of Enterococcus spp, and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100 degrees C, 60 min; 50% of activity remained after 15 min at 121 degrees C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E.faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B, Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading Frames, ent1071A and ent1071B, which coded for 39- and M-amino-acid peptides? respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the alpha and beta peptides of lactococcin G, respectively. This is the first report of tno nem antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.