Characterization of a Prophage-Free Derivative Strain of Lactococcus lactis ssp. lactis IL1403 Reveals the Importance of Prophages for Phenotypic Plasticity of the Host

Abstract

Lactococcus lactis is a lactic acid bacterium of major importance for the dairy industry and for human health. Recent sequencing surveys of this species have provided evidence that all lactococcal genomes contain prophages and prophage-like elements. The prophage-related sequences encompass up to 10% of the bacterial chromosomes and thus contribute significantly to the genetic diversity of lactococci. However, the impact of these resident prophages on the physiology of L. lactis is presently unknown. The genome of the first sequenced prototype strain, L. lactis ssp. lactis IL1403, contains six prophage-like elements which together represent 6.7% of the IL1403 chromosome. Diverse prophage genes other than those encoding phage repressors have been shown to be expressed in lysogenic conditions, suggesting that prophage genes are indeed able to modulate the physiology of their host. To elucidate the effect of resident prophages on the behavior of L. lactis in different growth conditions, we constructed and characterized, for the first time, a derivative strain of IL1403 that is prophage-free. This strain provides unique experimental opportunities for the study of different aspects of lactococcal physiology using the well-defined genetic background of IL1403. Here, we show that resident prophages modify the growth and survival of the host strain to a considerable extent in different conditions, including in the gastrointestinal environment. They also may affect cellular autolytic properties and the host cells' susceptibility to virulent bacteriophages and antimicrobial agents. It thus appears that prophages contribute significantly to lactococcal cell physiology and might play an important role in the adaptation of L. lactis to cultivation and environmental conditions.

Keywords: Lactococcus lactis IL1403; physiology of Lactococcus lactis; prophage impact; prophage-cured strain; prophages.

FIGURE 1

L. lactis IL1403 prophages. (A) Location of prophages on the chromosome of L. lactis IL1403. The prophage positions are marked by red rectangles; the position of dnaA within the oriC region is designated by a triangle. (B) Genome organization of the L. lactis IL1403 prophages. Genome boundaries correspond to attB sites. Genes are represented by arrows oriented in the direction of transcription. Functions of selected early- and late-gene products are indicated.

FIGURE 2

Morphology of the lysis plaques formed by (A) the 936-like small isometric-headed phage bIL14 and (B) the prolate-headed phage c2 in the parental strain IL1403 and its derivative strains: IL6351 (bIL285-, bIL286-free), IL6345 (bIL286-free), IL6260 (bIL285-, bIL286-, bIL309-, bIL310-free), IL6328 (bIL285-, bIL286-, bIL309-, bIL310-, bIL312-free), and IL6288 (prophage-free). The experiment was reproduced at least three times. The results shown are from a representative experiment.

FIGURE 3

Sensitivity of L. lactis IL1403 and its derivative strains IL1946 (bIL285-free), IL6353 (bIL286-, bIL309-free), and IL6288 to (A) lysozyme and (B) nisin. Serial dilutions of overnight cultures (from 10^-3 to 10^-5) were spotted on M17glu agar containing either lysozyme (from 0 to 0.5 mg/ml; A) or nisin (from 0 to 300 ng/ml; B). Colonies were counted after 36 h of incubation at 30°C. The bars represent average values from three independent experiments, including two biological replicas for each strain.

FIGURE 4

Autolytic properties of IL1403 and its prophage-free derivative strain IL6288. (A) Lytic activity of growing L. lactis IL1403 and IL6288 cells was examined by plating serial dilutions of overnight cultures on M17glu agar plates that contained 0.2% (wt/vol) autoclaved, lyophilized cells of M. luteus ATCC 4698. Images were acquired with a ChemiDoc MP System after 48 h of incubation at 30°C or 37°C. Autolysis of L. lactis IL1403 and its derivative strains IL6351 (bIL285-, bIL286-free) and IL6288 (prophage-free) at 30°C (B) and 37°C (C). Exponentially grown cells were washed and re-suspended in 50 mM potassium phosphate buffer in the absence or presence of 0.05% Triton X-100 (+TX100). Lysis was monitored by measuring the OD₆₀₀ of the cell suspensions at 15-min intervals with an automated multimode plate reader (Synergy 2, BioTek Instruments, Inc). For each strain, the mean values of three independent suspensions, analyzed simultaneously, are plotted and expressed as the percentage decrease in OD₆₀₀, the SDs were ≤ 10%. Similar autolytic profiles were obtained in three independent experiments. The results presented are of a representative experiment.

FIGURE 5

Growth and survival of L. lactis IL1403 and its derivative strains at 30°C and 37°C. (A) For growth experiments, non-aerated cultures of strains IL1403 and IL6288 were incubated in M17glu medium at the given temperature in a multimode microplate reader (Synergy 2, BioTek Instruments, Inc). OD₆₀₀ was measured at 15-min intervals for the indicated time; cultures were gently agitated prior to sampling. Each experiment included four independent cultures of each strain. For each strain, the mean values of three independent cultures, analyzed simultaneously, are plotted, and the SDs were ≤ 10%. Growth curves of each strain were established at least four times. Survival experiments were performed in static growth conditions at 30°C (B) and 37°C (C). Cultures of IL1403, IL6250 (bIL285-, bIL286-, bIL309-free), and prophage-free IL6288 were incubated in M17glu medium at the indicated temperature. Cell survival was monitored by counting CFUs after plating bacterial cultures on M17glu agar. Colonies were counted after 36 h of incubation at 30°C. Each data point (CFUs/ml) is the mean of three counts.

FIGURE 6

Effect of heme on the growth of L. lactis IL1403 and its prophage-free derivative strain IL6288. For the assay of heme-induced toxicity (A), autoclaved heme was added to static cultures of L. lactis IL1403 and IL6288 at an OD₆₀₀ of 0.6 at the indicated concentrations. Cultures were incubated at 30°C under non-aerated conditions. After 1 h, viable cell numbers were determined by spotting indicated serial dilutions of the bacterial cultures onto M17glu agar. Images were acquired with a ChemiDoc MP system after 36 h of incubation at 30°C. This experiment was performed twice; the results of a representative experiment are shown. For the characterization of growth under aerated conditions (B–E), cultures of IL1403 and its derivative strains were incubated in M17glu medium with (+H) or without heme in a multimode microplate reader (Synergy 2, BioTek Instruments, Inc) with constant shaking at 30°C (B,D) or 37°C (C,E). Each experiment included four independent cultures of each strain. For each strain, the mean values of three independent cultures, analyzed simultaneously, are plotted. Growth curves of each strain were established three times. The results presented are of a representative experiment.

FIGURE 7

Survival of L. lactis IL1403 and its prophage-free derivative strain, IL6288 after passage through the gastrointestinal tract of germ-free mice. Over 3 consecutive days, mice were orally gavaged with 16% glycerol solution in PBS buffer containing either 10⁹ CFUs of strain IL1403, or 10⁹ CFUs of strain IL6288, or the mixture of 10⁹ CFUs of strain IL1403:Em^R plus 10⁹ CFU of strain IL6288:Cm^R. Each strain was recovered from mouse feces at the indicated time points; cell survival was determined by counting CFUs on M17glu agar plates after 36 h of incubation at 30°C. Each data point (CFUs/ml) is the mean of at least three measurements.