Bacteriophages and plasmids in Streptococcus thermophilus

奶制品行业是我们遗产和经济的重要组成部分。它是加拿大农业食品经济的第三大部门。2012年，奶制品养殖业创造了59亿美元的收入，乳制品加工商的销售额达到147亿美元。总体而言，乳制品行业占我国食品饮料行业加工销售额的15.1%。加拿大奶牛场经营者超过22,000人，全国有480家奶牛场，雇佣了超过24,000名工人。这使乳制品行业成为食品行业的第二大雇主，生产了1050多个奶酪品种。2012年，加拿大奶酪生产商经历了第二好的一年，生产了408,197吨奶酪，比2011年增长了1.0%。408,647吨的纪录是在2010年创造的。2012年，酸奶产量也位居第二，为312,633吨(2011年为315,413吨)。没有迹象表明这种生产趋势会改变。然而，我国乳制品行业面临着国内和国际市场的激烈竞争，必须提供高质量的产品。奶酪和酸奶的生产需要在牛奶中接种精心挑选的细菌细胞(发酵剂培养)，以适当控制发酵并获得高质量的产品。发酵剂是产乳酸菌的组合，嗜热链球菌(St)是其中最重要的一种。众所周知，现有生产设施内生产率的提高将导致牛奶发酵失败，因为细菌病毒(噬菌体)会破坏发酵剂培养。这一自然现象导致产品质量低下，造成经济损失。我们的酸奶和奶酪工厂正面临着ST菌株的噬菌体感染问题。这项研究计划将鉴定这些噬菌体，以帮助开发抗噬菌体的ST菌株。我们将集中在三个一般领域1-噬菌体生物学。我们之前的基因组学研究对两个模型噬菌体进行了深入的分析，代表了ST噬菌体的两个主要组。我们现在计划对我们收集的另外50个ST噬菌体的基因组进行测序，以阐明它们的进化过程。我们还将提炼这两个噬菌体组的核心和旁观组。此外，我们此前已经在这两个噬菌体群中鉴定了几个结构蛋白。我们现在打算研究非结构噬菌体蛋白，其中许多功能尚不清楚。2-噬菌体抗性。自2005年以来，我们在ST对CRISPR-CAS系统进行了广泛的研究，取得了几项里程碑式的发现。CRISPR-CAS是目前微生物学领域最活跃、最具竞争力的研究领域之一。我们将继续研究这一噬菌体抵抗系统，特别是间隔区获取步骤，并将调查参与噬菌体复制的其他宿主因素。3.质粒生物学。我们之前已经证明，在ST中，一些质粒出人意料地稳定。我们将使用测序和转换策略来研究这种现象。作为操纵DNA的工具，质粒在我们的ST研究中也发挥着核心作用。由于没有遗传工具可用于修改强毒ST噬菌体的基因组，我们计划构建一个用于噬菌体基因组编辑的质粒系统来研究每个噬菌体基因/蛋白。这一雄心勃勃和最先进的计划将导致对革兰氏阳性细菌中噬菌体-宿主相互作用的最全面分析。这项研究将为我们的加拿大乳制品行业提供基础知识，使其具有竞争优势。这一知识也将适用于工业发酵中其他易受噬菌体攻击的细菌菌株。

The dairy industry is a significant part of our heritage and our economy. It is the third largest sector of the Canadian agri-food economy. In 2012, dairy farming generated $5.9 billion and sales from dairy processors equaled $14.7 billion. Overall, the dairy industry accounted for 15.1% of all processing sales in our nation’s food and beverage sector. Canadian dairy farm operators number over 22,000 and 480 dairy plants, nation-wide, employ over 24,000 additional workers. This makes the dairy sector the second largest employer in the food industry, producing more than 1050 cheese varieties. In 2012, Canadian cheese producers had their second best year, producing 408,197 tons of cheeses, an increase of 1.0% from 2011. The record of 408,647 tons was established in 2010. In 2012, yogurt production also had its second best year, with 312,633 tons (vs. 315,413 tons in 2011). There is no indication that this production trend will change. However, our dairy industry faces intense competition in national and international markets and must deliver high-quality products. Cheese and yogurt manufacture requires the inoculation of milk with carefully selected bacterial cells (starter culture) to properly control the fermentation and to obtain high-quality products. The starter culture is a combination of lactic acid-producing bacteria, from which Streptococcus thermophilus (ST) is one of the most important species. It is well documented that increased productivity within existing manufacturing facilities will lead to milk fermentation failures due to bacterial viruses (phages) that destroy the starter cultures. This natural phenomenon leads to low quality products and economic losses. Our yogurt and cheese factories are facing phage infection problems with their ST strains. This research program will characterize these phages to aid the development of phage-resistant ST strains. We will focus on three general areas.1- PHAGE BIOLOGY. Our previous genomics research produced an in-depth analysis of two model phages, representing the two main groups of ST phages. We now plan to sequence the genomes of 50 additional ST phages from our collection to elucidate their evolution processes. We will also refine the core and pangenome of both phage groups. In addition, we have previously identified several structural proteins in these two phage groups. We intend now to investigate non-structural phage proteins, many of which have no known function. 2- PHAGE RESISTANCE. Since 2005, we have extensively studied CRISPR-Cas systems in ST, making several landmark discoveries. CRISPR-Cas is now one of the most active and competitive research areas in microbiology. We will continue to study this phage resistance system, especially the spacer acquisition step, and we will investigate other host factors involved in phage replication. 3- PLASMID BIOLOGY. We previously showed that some plasmids are surprisingly stable in ST. We will investigate this phenomenon using sequencing and transformation strategies. Plasmids also play a central role in our ST studies as tools for manipulating DNA. Because no genetic tool is available to modify the genome of virulent ST phages, we plan to construct a plasmid system for phage genome editing to study each phage gene/protein.This ambitious and state-of-the-art program will lead to the most comprehensive analysis of phage-host interactions in a Gram-positive bacterium. This research will provide fundamental knowledge to our Canadian dairy industry to give them a competitive edge. This knowledge will also be applicable to other bacterial strains susceptible to phage attack in industrial fermentations.