Turner M S, Timms P, Hafner L M, Giffard P M
Centre for Molecular Biotechnology, School of Life Science, Queensland University of Technology, Brisbane, Australia.
J Bacteriol. 1997 May;179(10):3310-6. doi: 10.1128/jb.179.10.3310-3316.1997.
Extraction of Lactobacillus fermentum BR11 cells with 5 M LiCl yielded a preparation containing a single predominant polypeptide with an apparent molecular mass of 32 kDa. A clone encoding an immunoreactive 32-kDa polypeptide was isolated from a pUC18 library of L. fermentum BR11 DNA by screening with an antiserum raised against whole cells of L. fermentum BR11. Sequence determination of the insert in the clone revealed a complete 795-bp open reading frame (ORF) that defines a 28,625-Da polypeptide (BspA). N-terminal sequencing of the LiCl-extracted polypeptide from L. fermentum BR11 confirmed that it is the same as the cloned BspA. BspA was found to have a sequence similar to those of family III of the bacterial solute-binding proteins. The sequences of two ORFs upstream of bspA are consistent with bspA being located in an operon encoding an ATP-binding cassette-type uptake system. Unusually, BspA contains no lipoprotein cleavage and attachment motif (LXXC), despite its origin in a gram-positive bacterium. Biotin labelling and trypsin digestion of whole cells indicated that this polypeptide is exposed on the cell surface. The isoelectric point as predicted from the putative mature sequence is 10.59. It was consequently hypothesized that the positively charged BspA is anchored by electrostatic interaction with acidic groups on the cell surface. It was shown that BspA could be selectively removed from the surface by extraction with an acidic buffer, thus supporting this hypothesis.
用5 M LiCl提取发酵乳杆菌BR11细胞，得到一种制剂，其中含有一种主要的单一多肽，其表观分子量为32 kDa。通过用针对发酵乳杆菌BR11全细胞产生的抗血清进行筛选，从发酵乳杆菌BR11 DNA的pUC18文库中分离出一个编码免疫反应性32 kDa多肽的克隆。对该克隆中插入片段的序列测定揭示了一个完整的795 bp开放阅读框（ORF），该阅读框定义了一个28,625 Da的多肽（BspA）。对发酵乳杆菌BR11的LiCl提取多肽进行N端测序，证实它与克隆的BspA相同。发现BspA的序列与细菌溶质结合蛋白家族III的序列相似。bspA上游两个ORF的序列与bspA位于编码ATP结合盒式摄取系统的操纵子中一致。不同寻常的是，尽管BspA起源于革兰氏阳性细菌，但它不包含脂蛋白切割和附着基序（LXXC）。对全细胞进行生物素标记和胰蛋白酶消化表明，该多肽暴露在细胞表面。根据推定的成熟序列预测的等电点为10.59。因此推测，带正电荷的BspA通过与细胞表面酸性基团的静电相互作用而锚定。结果表明，用酸性缓冲液提取可以选择性地从表面去除BspA，从而支持了这一假设。
