Miller K W, Schamber R, Osmanagaoglu O, Ray B
Departments of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071, USA.
Appl Environ Microbiol. 1998 Jun;64(6):1997-2005. doi: 10.1128/AEM.64.6.1997-2005.1998.
A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to approximately 60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed approximately 2. 8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.
通过对编码细菌素的DNA进行PCR随机诱变，产生了一组植物乳杆菌素AcH氨基酸取代突变体。将诱变后的DNA克隆到一个大肠杆菌malE质粒中，该质粒指导麦芽糖结合蛋白 - 植物乳杆菌素AcH嵌合蛋白的分泌，通过筛选转化菌落对植物乳杆菌NCDO955的杀菌活性来分离突变体（K. W. 米勒、R. 尚伯、Y. 陈和B. 雷，1998年。《应用与环境微生物学》64:14 - 20，1998年）。总共在植物乳杆菌素AcH的44个氨基酸中的14个位置分离出17个取代突变体。七个突变体（N5K、C9R、C14S、C14Y、G37E、G37R和C44W）对植物乳杆菌素AcH敏感菌株植物乳杆菌NCDO955、无害李斯特菌Lin11、粪肠球菌M1、嗜酸乳球菌LB42和肠系膜明串珠菌Ly完全无活性。通过其他方法构建的C24S取代突变体对这些细菌也无活性。当针对无害李斯特菌Lin11进行检测时，其他九个突变体（K1N、W18R、I26T、M31T、A34D、N41K、H42L、K43N和K43E）保留了从不到1%到约60%的野生型活性。一个突变体K11E对该指示菌显示出约2.8倍更高的活性。大约一半的突变定位在细菌素样家族中保守的氨基酸上。发现所有四个半胱氨酸都是活性所必需的，尽管在细菌素样细菌素中只有C9和C14是保守的。还发现几个碱性氨基酸以及位于疏水C末端区域内的非极性氨基酸也很重要。在已提出的细菌素结构模型的背景下讨论了这些突变。
