Structural studies of Lactococcus lactis bacteriophage proteins

加拿大乳制品行业严重依赖细菌发酵将牛奶转化为奶酪和酸奶等产品。一家奶酪厂平均每天转化约100万升牛奶。发酵剂是乳酸菌的组合，乳酸乳球菌是干酪生产中最重要的菌种。奶酪厂将轮换不同的发酵剂培养物，需要大量的细菌菌株，这些菌株是根据微生物、生化和技术特性精心挑选的。在巴氏杀菌牛奶的非无菌环境中，添加的和首选的细菌细胞将与牛奶中随处可见的有毒噬菌体接触。噬菌体(或噬菌体)是感染细菌并在细菌内复制的病毒。牛奶中的噬菌体浓度通常很低，但在34小时的干酪制作过程中，噬菌体数量会迅速增加；强毒乳球菌噬菌体的周期为30分钟，每个感染细胞释放50-100个病毒粒子。几十年来，乳制品行业一直依赖于一系列控制措施。尽管做出了这些努力，但噬菌体仍在进化，变种不断出现，噬菌体攻击至今仍是奶酪行业制造问题的最常见原因。对噬菌体的深入了解无疑将为这些噬菌体的长期和优化控制带来更好的策略。*我们的主要目标是通过对这些病毒编码的所有蛋白质的表征，显著增加我们对影响加拿大乳制品行业的乳酸乳杆菌噬菌体的了解。尽管噬菌体是地球上最丰富的生物实体(生物圈中约有1032个噬菌体，导致每秒约1023例感染)，但约75%的噬菌体基因具有未知的功能。乳球菌噬菌体分为十个不同的组。936型代表了乳制品生产设施中发现的90%的乳球菌噬菌体，它们是最麻烦的。到目前为止，已有936型噬菌体的56个完整基因组可用。这些dsDNA噬菌体编码的蛋白质很少，其中大部分具有未知的功能。*我们建议使用一种独特的全局方法来处理所有936种类型噬菌体中的所有3000+蛋白质，这些蛋白质具有可用的基因组序列。结构是理解蛋白质功能的基础。此外，蛋白质中的局部动态是无处不在的。这些运动直接影响配基结合和催化等事件；因此，研究蛋白质动力学对于理解功能也是必不可少的。因此，我们还建议深入研究从936型噬菌体中挑选出的高度保守的蛋白质的结构和动力学。在这个项目的前5年，我们将结合现代预测工具、溶液核磁共振和分子动力学模拟来收集936型噬菌体蛋白的结构和动力学数据。

The Canadian dairy industry heavily relies on the bacterial fermentation for the transformation of milk into products such as cheese and yogurt. An average cheese plant transforms ~1,000,000 litres of milk per day. The starter cultures are a combination of lactic acid bacteria, and Lactococcus lactis is the most important species for cheese manufacture. A cheese plant will rotate different starter cultures, requiring numerous bacterial strains that are carefully selected based on microbiological, biochemical, and technical properties.******In the nonsterile environment of pasteurized milk, the added and preferred bacterial cells will come into contact with ubiquitous virulent phages found in milk. Phages (or bacteriophages) are viruses that infect and replicate within a bacterium. Phage concentration is usually low in milk, but phage population can increase rapidly during the 34 hours cheese making process; virulent lactococcal phages have a 30 min cycle and release of 50-100 virions per infected cell. For decades, the dairy industry has relied on an array of control measures. In spite of these efforts, phages are evolving and variants keep emerging, and phage attacks remain today the most common cause of manufacturing problems in the cheese industry. An in depth understanding of phages will, without a doubt, lead to better strategies for the long-term and optimize control of these phages.******Our primary goal is to significantly increase our knowledge of L. lactis phages that affect the Canadian dairy industry through a characterization of all proteins encoded by these viruses. Even though phages are the most abundant biological entities on the planet (~1032 phages in the biosphere resulting in ~1023 infections per second), about 75% of phage genes have unknown functions. Lactococcal phages are classified into ten distinct groups. The 936-type represent 90% of lactococcal phages found in dairy manufacturing facilities and they are the most troublesome. To date, 56 complete genomes of 936-type phages are available. These dsDNA phages encode for few proteins, most of which have unknown functions.******Using a distinct and global approach, we propose to tackle all 3000+ proteins from all 936-type phages with available genome sequences. Structures are essential to understand protein function. In addition, local dynamics in proteins are omnipresent. These movements directly influence events such as ligand binding and catalysis; hence the study of protein dynamics is also essential to understand function. Hence, we also propose in-depth studies of structure and dynamics of highly conserved proteins selected among the 936-type phages. In the first 5 years of this project, we will combine modern prediction tools, solution NMR and molecular dynamics simulations to gather structural and dynamics data on 936-type phage proteins.