Dupont Kitt, Janzen Thomas, Vogensen Finn Kvist, Josephsen Jytte, Stuer-Lauridsen Birgitte
Department of Food Science, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
Appl Environ Microbiol. 2004 Oct;70(10):5825-32. doi: 10.1128/AEM.70.10.5825-5832.2004.
The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that phi645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that phi 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.
本研究旨在鉴定乳酸乳球菌乳酸亚种IL1403和乳酸乳球菌乳脂亚种Wg2中分别对936型噬菌体bIL170和phi 645吸附至关重要的基因。利用载体pGh9:ISS1对这两株乳酸乳球菌进行随机插入诱变，并筛选出对噬菌体感染具有抗性且噬菌体吸附减少的整合子。在乳酸乳球菌IL1403中，ycaG和rgpE基因发生了整合；而在乳酸乳球菌Wg2中，与乳酸乳球菌IL1403的ycbC和ycbB同源的两个基因发生了整合。rgpE和ycbB编码推定的糖基转移酶，而ycaG和ycbC编码功能未知的推定跨膜蛋白。有趣的是，ycaG、rgpE、ycbC和ycbB在乳酸乳球菌IL1403中均属于同一个操纵子。该操纵子可能参与细胞壁多糖（WPS）的生物合成和转运。结合与感染研究表明，phi645能结合并感染乳酸乳球菌Wg2、乳酸乳球菌IL1403以及ycaG和rgpE中带有pGh9:ISS1整合的乳酸乳球菌IL1403菌株，而bIL170仅能结合并感染乳酸乳球菌IL1403，无法感染Wg2。这些结果表明，phi 645结合到乳酸乳球菌IL1403和乳酸乳球菌Wg2中均存在的一种WPS结构上，而bIL170结合到乳酸乳球菌Wg2中不存在的另一种WPS结构上。bIL170和phi 645与不同WPS结构的结合得到了bIL170和phi 645受体结合蛋白比对结果的支持，该比对结果显示其C端部分不存在同源性。
