Effects of diverse environmental conditions on {phi}LC3 prophage stability in Lactococcus lactis

Abstract

The effects of various growth conditions on spontaneous phiLC3 prophage induction in Lactococcus lactis subsp. cremoris IMN-C1814 was analyzed with a half fraction of a 4(4) factorial experimental design. The four factors included in the study were nutrient availability, acidity, osmolarity, and temperature, each applied at four levels. These environmental factors are related to the fermentation processes in the dairy industry, in which bacteriophage attacks on sensitive starter strains are a constant threat to successful fermentation processes. The frequency of spontaneous phiLC3 induction was determined by quantitative analyses of restored DNA attachment sites (attB) on the bacterial chromosomes in a population of lysogenic cells. Statistical analysis revealed that all four environmental factors tested affected phiLC3 prophage stability and that the environmental factors were involved in interactions (interactions exist when the effect of one factor depends on the level of another factor). The spontaneous phiLC3 induction frequency varied from 0.08 to 1.76%. In general, the induction frequency remained at the same rate or decreased when level 1 to 3 of the four environmental factors was applied. At level 4, which generally gave the least favorable growth conditions, the induction frequency was either unchanged, decreased, or increased, depending on the type of stress. It appeared that the spontaneous induction frequency was independent of the growth behavior of the host. It was the environmental growth conditions that were the decisive factor in induction frequency.

FIG. 1.

Main effect plots of nutrient availability (M17) (A), acidity (pH) (B), osmolarity (NaCl) (C), and temperature (D) on spontaneous prophage induction frequency. Error bars show the standard error of the means.

FIG. 2.

Interaction effect plots of nutrient availability and osmolarity (M17×NaCl) (A), acidity and temperature (pH×Temp) (B), and osmolarity and temperature (NaCl×Temp) (C) on spontaneous prophage induction frequency (standard error = 0.08).

FIG. 3.

Growth curves of Lactococcus lactis subsp. cremoris IMN-C1814. The strain was grown in different combinations of four environmental factors according to a factorial design. The curves represent growth behavior in the presence of different combinations of nutrient availability, acidity, osmolarity, and temperature as follows; 1-fold M17, pH 6.6, 0.0% NaCl, and 30°C (A), 0.4-fold M17, pH 6.6, 0.0% NaCl, and 30°C (B), 1-fold M17, pH 6.2, 1.5% NaCl, and 33°C (C) and 0.4-fold M17, pH 6.2, 1.5% NaCl, and 33°C (D).