Abstract：
In this study, an in silico screening approach was employed to mine potential bacteriocin clusters in genome-sequenced isolates of Lacticaseibacillus zeae UD 2202 and Lacticaseibacillus casei UD 1001. Two putative undescribed bacteriocin gene clusters (Cas1 and Cas2) closely related to genes encoding class IIa bacteriocins were identified. No bacteriocin activity was recorded when cell-free supernatants of strains UD 2202 and UD 1001 were tested against Listeria monocytogenes. Genes encoding caseicin A1 (casA1) and caseicin A2 (casA2) were heterologously expressed in Escherichia coli BL21 (DE3) using the nisin leader peptide cloned in-frame to the C-terminal of the green fluorescent gene (mgfp5). Nisin protease (NisP) was used to cleave caseicin A1 (casA1) and caseicin A2 (casA2) from GFP-Nisin leader fusion proteins. Both heterologously expressed peptides (casA1 and casA2) inhibited the growth of L. monocytogenes, suggesting that casA1 and casA2 are either silent in the wild-type strains or are not secreted in an active form. The minimum inhibitory concentration (MIC) of casA1 and casA2, determined using HPLC-purified peptides, ranged from < 0.2 µg/mL to 12.5 µg/mL when tested against Listeria ivanovii, Listeria monocytogenes, and Listeria innocua, respectively. A higher MIC value (25 µg/mL) was recorded for casA1 and casA2 when Enterococcus faecium HKLHS was used as the target. The molecular weight of heterologously expressed casA1 and casA2 is 5.1 and 5.2 kDa, respectively, as determined with tricine-SDS-PAGE. Further research is required to determine if genes within Cas1 and Cas2 render immunity to other class IIa bacteriocins.
摘要：
在这项研究中,采用计算机筛选方法在玉米乳杆菌UD2202和干酪乳杆菌UD1001的基因组测序分离株中挖掘潜在的细菌素簇。鉴定了与编码IIa类细菌素的基因密切相关的两个推定的未描述的细菌素基因簇(Cas1和Cas2)。当针对单核细胞增生李斯特菌测试菌株UD2202和UD1001的无细胞上清液时,没有记录到细菌素活性。编码酪蛋白A1（casA1）和酪蛋白A2（casA2）的基因在大肠杆菌BL21（DE3）中使用在框架内克隆到绿色荧光基因（mgfp5）的C末端的乳链菌肽前导肽异源表达。使用NisP蛋白酶(NisP)从GFP-Nisin前导融合蛋白切割酪蛋白A1(casA1)和酪蛋白A2(casA2)。两种异源表达的肽（casA1和casA2）都抑制了单核细胞增生李斯特菌的生长，表明casA1和casA2在野生型菌株中是沉默的或不以活性形式分泌。casA1和casA2的最小抑制浓度（MIC），使用HPLC纯化的肽，当针对伊万诺维李斯特菌进行测试时，范围为<0.2µg/mL至12.5µg/mL，单核细胞增生李斯特菌,和无害李斯特菌,分别。当使用屎肠球菌HKLHS作为靶标时，casA1和casA2的MIC值（25µg/mL）更高。异源表达的casA1和casA2的分子量为5.1和5.2kDa，分别,如用tricine-SDS-PAGE测定。需要进一步的研究来确定Cas1和Cas2中的基因是否对其他IIa类细菌素具有免疫力。
