Bacteriophages and plasmids in Streptococcus thermophilus

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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.