Ulrich R L, Hughes T A
Department of Microbiology and Molecular Medicine, Clemson University, SC 29634, USA.
J Appl Microbiol. 2001 Dec;91(6):1067-73. doi: 10.1046/j.1365-2672.2001.01480.x.
A cell wall-associated lactacin B inducer protein (IP) was purified from Lactobacillus delbrueckii subsp. lactis ATCC 4797 (Lact. lactis) by chromatofocusing and gel filtration HPLC (Barefoot et al. 1994).
N-terminal sequence of the purified IP was used to design an oligonucleotide (24-mer) for gene identification by Southern and colony hybridizations. Southern hybridization on Lact. lactis chromosomal DNA digested with EcoRI and PstI produced a single 4-5 kbp DNA fragment. Colony hybridizations with 6250 clones produced four positive recombinants for the proposed IP. Sequence of the DNA isolated from RU43e9 revealed a 4623 bp DNA fragment containing three open reading frames (ORF) potentially encoding enzymes that function in glycolysis. One ORF, coding for an active triosephosphate isomerase (Tpi), showed 98% homology to the N-terminal domain of the HPLC purified IP. PCR primers were designed to amplify the ORF encoding the proposed IP for subcloning, protein expression, purification and bacteriocin enhancing assays on pure cultures of Lactobacillus acidophilus N2.
The regions flanking the Tpi gene (data not shown) were also sequenced and it is concluded that the proposed IP reported by Barefoot et al. (1994) is located on an operon containing several glycolytic enzymes that function in glycolysis.
The findings of this study do not support previously published research (Barefoot et al. 1994) hypothesizing that a purified IP from Lact. lactis, homologous to a Bacillus stearothermophilus Tpi, is capable of enhancing bacteriocin synthesis in Lact. acidophilus N2.
通过层析聚焦和凝胶过滤高效液相色谱法从德氏乳杆菌乳酸亚种ATCC 4797（乳酸乳杆菌）中纯化出一种细胞壁相关的乳酸链球菌素B诱导蛋白（IP）（Barefoot等人，1994年）。
利用纯化的IP的N端序列设计了一个寡核苷酸（24聚体），通过Southern杂交和菌落杂交进行基因鉴定。用EcoRI和PstI消化的乳酸乳杆菌染色体DNA进行Southern杂交产生了一个单一的4 - 5 kb DNA片段。与6250个克隆进行菌落杂交产生了4个针对所提出的IP的阳性重组体。从RU43e9分离的DNA序列显示一个4623 bp的DNA片段，包含三个开放阅读框（ORF），可能编码参与糖酵解的酶。其中一个编码活性磷酸丙糖异构酶（Tpi）的ORF与高效液相色谱纯化的IP的N端结构域显示出98%的同源性。设计PCR引物扩增编码所提出的IP的ORF，用于亚克隆、蛋白质表达、纯化以及对嗜酸乳杆菌N2纯培养物的细菌素增强测定。
对Tpi基因两侧的区域也进行了测序（数据未显示），得出结论，Barefoot等人（1994年）报道的所提出的IP位于一个包含几种参与糖酵解的糖酵解酶的操纵子上。
本研究的结果不支持先前发表的研究（Barefoot等人，1994年）所假设的，即从乳酸乳杆菌纯化的与嗜热脂肪芽孢杆菌Tpi同源的IP能够增强嗜酸乳杆菌N2中细菌素的合成。
