Universidad de Guanajuato Campus Irapuato-Salamanca, División Ciencias de la Vida, Posgrado en Biosciencias, México.
Aniversidad Autónoma de Coahuila, Escuela de Ciencias Biológicas, Torreón, Coahuila 27104, México.
Microbiol Res. 2014 Dec;169(12):948-53. doi: 10.1016/j.micres.2014.04.005. Epub 2014 May 10.
Bacteriocins synthesized by entomopathogenic Bacillus thuringiensis are gaining attention owing to their inhibitory effects against a wide variety of pathogenic bacteria. In the present study, we purified and characterized Tolworthcin 524, a bacteriocin synthesized by B. thuringiensis subsp. tolworthi, and compared it with other bacteriocins synthesized by B. thuringiensis. Tolworthcin 524 was separated and purified from the secretome of B. thuringiensis by fast protein liquid chromatography with a gel filtration column to obtain yields of 17% and a specific activity of ∼3600U/mgprotein. The purified product showed two peptides of ∼9 and 6kDa with antimicrobial activity in a gel-screening assay. The purified product was analyzed by two-dimensional electrophoresis and the resolved peptides of ∼9 and 6kDa with isoelectric points of ∼8 were sequenced. Partial sequences (METPVVQPR and DWTCWSCLVCAACS) were obtained suggesting that the ∼9 and 6kDa correspond to the prebacteriocin and mature Tolworthcin 524, respectively. Sequences showed high identity with Thurincin H and Thuricin 17 and had a conserved motif with other bacteriocins of B. thuringiensis. Based on sequence data, Tolworthcin 524 was classified in subclass II.2 (Thuricin-like peptides) of the Bacillus bacteriocin classification scheme. The larger peptide did not harbor a sequence suggestive of a signal peptide neither did it contain the double-glycine (GG) motif characteristic of the secretion leader recognized by the ABC transport system. Implications of these properties in Tolworthcin 524 secretion are discussed.
苏云金芽孢杆菌合成的细菌素由于对各种致病菌具有抑制作用而受到关注。本研究从苏云金芽孢杆菌亚种分离纯化并鉴定了 Tolworthcin 524，比较了其与其他苏云金芽孢杆菌合成的细菌素。Tolworthcin 524 通过快速蛋白液相色谱凝胶过滤柱从苏云金芽孢杆菌的分泌物中分离和纯化，获得了 17%的产率和约 3600U/mgprotein 的比活性。凝胶筛选试验显示，纯化产物具有两种具有抗菌活性的约 9 和 6 kDa 的肽。通过二维电泳分析纯化产物，并对等电点约为 8 的约 9 和 6 kDa 的分辨肽进行测序。获得了部分序列（METPVVQPR 和 DWTCWSCLVCAACS），表明约 9 和 6 kDa 分别对应于前细菌素和成熟的 Tolworthcin 524。序列与 Thurincin H 和 Thuricin 17 具有高度同一性，并且与苏云金芽孢杆菌的其他细菌素有保守的模体。根据序列数据，Tolworthcin 524 被分类为 Bacillus bacteriocin 分类方案的 II.2 亚类（Thuricin 样肽）。较大的肽没有序列提示其含有信号肽，也不包含 ABC 转运系统识别的双甘氨酸 (GG) 模体特征的分泌先导序列。讨论了这些特性对 Tolworthcin 524 分泌的影响。
