Primary amino acid and DNA sequences of gassericin T, a lactacin F-family bacteriocin produced by Lactobacillus gasseri SBT2055
Primary amino acid and DNA sequences of gassericin T, a lactacin F-family bacteriocin produced by Lactobacillus gasseri SBT2055
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DOI：
10.1271/bbb.64.2201
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发表时间：
2000-10-01
影响因子：
1.6
通讯作者：
Itoh, T
中科院分区：
文献类型：
--
作者：
Kawai, Y;Saitoh, B;Itoh, T
通过疏水色谱法从培养物上清液中分离出由加氏乳杆菌 SET 2055（来自人类粪便）产生的广谱细菌素，称为 Gassericin T。通过 SDS-PAGE 和原位活性测定，纯化的 Gassericin T 迁移为单条带，具有细菌素活性，分子大小为 5,400。将染色体DNA的2.9-kbp HindIII-HindIII片段与根据gassericin T的部分N末端氨基酸序列设计的寡核苷酸探针杂交并克隆。通过计算机分析和数据库推导了包含gassericin T结构基因的6个ORF。 Gassericin T（gatA）的结构基因被鉴定为第四个ORF，其编码由75个氨基酸组成的蛋白质，其中包括切割位点的GG基序。对完整氨基酸序列进行化学测序分析表明，gassericin T（57个氨基酸）分子中存在二硫键，且无氨基酸残基修饰，为II类细菌素。 Gassericin T与成熟LafA（57个氨基酸、乳糖素F、约氏乳杆菌VPI11088产生的细菌素）有60%的序列相似性，且加工位点和C端区域周围的序列保存良好。第五个 ORF 被命名为 gatX，编码为由 65 个氨基酸组成的肽，包含假定的裂解位点的 GG 基序，但在培养物上清液中未检测到成熟的 GatX 及其抗菌活性。 GatX 与 LafX 的相似性高于与属于第一个乳糖素 F 家族的乳糖素 A（50 个氨基酸）的相似性。这些结果表明gassericin T属于疏水性II类细菌素和最邻近的乳糖素F家族。
A broad-spectral bacteriocin, named gassericin T, produced by Lactobacillus gasseri SET 2055 (from human feces) was isolated to homogeneity from the culture supernatant by hydrophobic chromatography. By SDS-PAGE and in situ activity assay, the purified gassericin T migrated as a single band with bacteriocin activity and molecular size of 5,400. A 2.9-kbp HindIII-HindIII fragment of chromosome DNA was hybridized with the oligonucleotide probe designed from the partial N-terminal amino acid sequence of gassericin T and was cloned. Six ORFs including the structural gene of gassericin T were deduced by computer analysis and the data bases. The structural gene of gassericin T (gatA) was identified as the fourth ORF, which encoded a protein composed of 75 amino acids that included the GG motif of the cleavage site. Chemical sequencing analysis of the complete amino acid sequence showed that gassericin T (57 amino acids) had a disulfide bond in the molecule and no modified amino acid residues, making it a class II bacteriocin. The gassericin T had 60% sequence similarity to mature LafA (57 amino acids, lactacin F, bacteriocins produced by L. johnsonii VPI11088), and the sequences around the processing site and C-terminal area were well conserved. The fifth ORF was designated as gatX, encoded as a peptide composed of 65 amino acids containing the GG motif of the putative cleavage site, however mature GatX and its antibacterial activity were not detected in the culture supernatant. GatX has higher similarity with LafX than with lactobin A (50 amino acids) belonging to the first lactacin F-family. These results indicated that gassericin T belongs to the hydrophobic class II bacteriocins and the most vicinal lactacin F-family.