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。一个普通的奶酪厂每天可生产约1,000,000升牛奶。发酵剂是乳酸菌的组合，乳酸乳球菌是奶酪生产中最重要的菌种。奶酪厂将轮换不同的发酵剂，需要根据微生物学、生化学和技术特性仔细选择许多菌株。******在巴氏消毒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无处不在的毒性噬菌体接触。噬菌体（或噬菌体）是在细菌中感染和复制的病毒。牛奶中的噬菌体浓度通常较低，但在奶酪制作过程的34小时内，噬菌体数量会迅速增加；毒力强的乳球菌噬菌体周期为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，噬菌体仍在不断进化，变种不断出现，噬菌体攻击仍然是今天奶酪行业生产问题的最常见原因。毫无疑问，对噬菌体的深入了解将为噬菌体的长期和优化控制提供更好的策略。******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，每秒约有1023次感染），但约75%的噬菌体基因功能未知。乳球菌噬菌体被分为十个不同的类群。936型占乳制品生产设施中发现的乳球菌噬菌体的90%，它们是最麻烦的。迄今为止，已有936种噬菌体的56个完整基因组。这些dsDNA噬菌体编码很少的蛋白质，其中大多数具有未知的功能。******采用独特的全局方法，我们建议使用可用的基因组序列来处理所有936型噬菌体的所有3000+蛋白质。结构对于理解蛋白质的功能至关重要。此外，蛋白质中的局部动力学是无处不在的。这些运动直接影响配体结合和催化等事件；因此，蛋白质动力学的研究对于理解蛋白质的功能也是至关重要的。因此，我们还建议对936型噬菌体中选择的高度保守蛋白的结构和动力学进行深入研究。在项目的前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，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.