Effects of Diverse Environmental Conditions on φLC3 Prophage Stability in Lactococcus lactis

Lactic acid bacteria are widely used to ferment milk for production of cheese and other dairy products. Virulent bacteriophages are a constant threat to such fermentation processes, as they are occur naturally in milk (17) and can survive pasteurization (9). In addition, many of the starter strains used are lysogenic, carrying one ore more potentially inducible prophages, suggesting that they represent a source of phages (10). In fact, recent research has revealed that prophage-related sequences in a number of bacterial genomes take up more than 10% of the DNA content (reviewed in references 7 and 8). The three lactococcal phage species most frequently found in dairy environments are the 936, c2, and P335 phage species (1, 19, 36). The 936 and c2 species contain only virulent phages, while the P335 species encompasses both virulent and temperate phages. Temperate phages and prophage-derived sequences within the P335 species have been shown to give rise to new virulent derivatives (5, 11, 33). For several years it has been discussed whether spontaneously induced P335 phages are a matter of significant concern to the dairy industry. However, there is still little information available on how and when the bacterial host and the environmental conditions may influence the induction of a prophage. Consequently, improved knowledge about conditions that would prevent or permit spontaneous prophage induction from lysogenic lactic acid bacteria is of importance in starter culture technology as well as a question of fundamental interest. During milk fermentation processes, lactic acid bacteria are exposed to various environmental stress conditions, such as temperature fluctuations, acid pH, high osmotic pressure, and absence of available nutrients. Many of these conditions will often coincide. Like other bacteria, lactic acid bacteria have evolved intricate stress response systems enabling them to adapt to adverse conditions in order to survive. The stress responses of the industrially important species Lactococcus lactis have gained increased interest in recent years, and reports include studies of responses to heat and cold shock, low pH, UV light, salts, starvation, oxidation, DNA damage, and chloride (reviewed in references 38, 42, and 46). Environmental conditions affect the switch between the lytic and lysogenic life styles of the well-studied temperate Escherichia coli phage λ (13, 16). The molecular basis for the lysis-lysogeny switch is well understood for λ, but the exact mechanisms for sensing environmental conditions and how the signaling routes are coupled to the molecular regulation mechanisms are not well defined. Recently, regulatory mechanisms involved in the lysis-lysogeny decision have been reported for several lactic acid bacterial phages (3, 4, 6, 15, 18, 20, 23, 24, 30, 35, 45), and similarities exist between the regulatory mechanisms of lysogenic lactic acid bacterial phages and λ. Only a limited number of studies have been performed regarding the effect of environmental factors on the lysis-lysogeny decision of temperate lactic acid bacterial phages. Feirtag and McKay (14) demonstrated that a shift in growth temperature (from 30 to 40°C) led to the induction of temperate bacteriophages into the lytic life cycle, followed by host lysis. For Lactococcus lactis subsp. cremoris SK110, it was shown that this thermoinducible lysis was strongly dependent on the cell growth rate and the pH of the medium, in which high growth rates and neutral pHs gave the highest thermolytic response (31). We have previously reported that the spontaneous induction frequency of the temperate lactococcal bacteriophage φLC3 in five lysogenic L. lactis strains was affected by growth temperature and varied between 0.32 and 9.1% among the lysogenic strains (29). The temperate phage φLC3, isolated from Lactococcus lactis subsp. cremoris IMN-C3 (27), belongs to the P335 species of small isometric-headed lactococcal phages. The genome sequence of phage φLC3 was recently determined (2). In this work, phage φLC3 was used as a model to study prophage induction in lactic acid bacteria under diverse environmental conditions related to milk fermentation processes. A statistically designed factorial experiment, in which the environmental factors were varied together, was used instead of the more time-consuming one-factor-at-a-time experimental approach (34). Factorial experiments can reveal relationships that are otherwise difficult to observe by analyzing both the main effect of a factor and the interaction effects between the factors. The spontaneous φLC3 prophage induction frequency was monitored with a recently developed method based on quantitative analysis of specific DNA sites involved in φLC3 phage life cycles (28, 29). This study will provide a better understanding of how dairy-related environmental growth conditions affect prophage maintenance in lactic acid bacteria.