Role of Temperate Bacteriophage ϕ20617 on Streptococcus thermophilus DSM 20617T Autolysis and Biology

Abstract

Streptococcus thermophilus DSM 20167^T showed autolytic behavior when cultured in lactose- and sucrose-limited conditions. The amount of cell lysis induced was inversely related to the energetic status of the cells, as demonstrated by exposing cells to membrane-uncoupling and glycolysis inhibitors. Genome sequence analysis of strain DSM 20617^T revealed the presence of a pac-type temperate bacteriophage, designated Φ20617, whose genomic organization and structure resemble those of temperate streptococcal bacteriophages. The prophage integrated at the 3'-end of the gene encoding the glycolytic enzyme enolase (eno), between eno and the lipoteichoic acid synthase-encoding gene ltaS, affecting their transcription. Comparative experiments conducted on the wild-type strain and a phage-cured derivative strain revealed that the cell-wall integrity of the lysogenic strain was compromised even in the absence of detectable cell lysis. More importantly, adhesion to solid surfaces and heat resistance were significantly higher in the lysogenic strain than in the phage-cured derivative. The characterization of the phenotype of a lysogenic S. thermophilus and its phage-cured derivative is relevant to understanding the ecological constraints that drive the stable association between a temperate phage and its bacterial host.

Keywords: Streptococcus thermophilus; bacteriophage; bioenergetics; biofilm; heat-resistance.

FIGURE 1

Growth of Streptococcus thermophilus DSM 20617^T (squares) and its derivative A33 (circles) strain in M17 broth in the presence of sucrose, and lactose. (A) Growth of S. thermophilus strains in the presence of sucrose at a final concentration of 0.2% (white symbols) and 1% (black symbols). Strain DSM 20617^T (autolytic) (white and black squares), strain A33 (non-autolytic) (white and black circles). Flow cytometry of SYBR Green I stained cells of S. thermophilus DSM 20617^T grown in M17 (0.2 % wt/v sucrose). (B) Cells after 4 h of growth at 37°C (blue gate). (C) Cells and cell debris (red gate) after 8 h of growth at 37°C. (D) Cells and cell debris (red gate) after 8 h of growth at 37°C treated with DNAse. (E) M17 broth SYBR Green I stained.

FIGURE 2

Streptococcus thermophilus DSM 20617^T-induced lysis (A) and cell morphology (B) after 10 h of incubation at 37°C, in the presence of gramicidin (black triangles), membrane uncoupling reagent, or sodium oxamate (black squares), which is a lactate dehydrogenase inhibitor. Cells grown in M17 (2% wt/vol lactose) at 37°C were collected at O.D. 0.55, and washed and suspended in fresh M17 at 37°C, without lactose (white squares) in the absence/presence of gramicidin or sodium oxamate. Error bars represent the standard deviations based on three replicates. (C) S. thermophilus DSM 20617^T was grown in microtiter plates using increasing concentrations of sucrose (0.05–3%) in presence of sodium oxamate. The growth curves were extrapolated as example for a total of 384 different culture conditions as reported in Supplementary Figure S3.

FIGURE 3

Correlation between the level of cell lysis and the total amount of sucrose in M17 broth, in presence or absence of sodium oxamate. For each condition analyzed here, the cell lysis was calculated as follow: [(maximum cell density reached – cell density at the end of the incubation time)/maximum cell density reached] × 100. The inoculum concentration was indicated. Linear regression equations and the R²-values are indicated in each graph. Shaded graphs represent the data obtained when M17 was supplemented with 10 mM sodium oxamate.

FIGURE 4

(A) Schematic representation of integration (INT) and excision (EXC) structure in the S. thermophilus DSM 20617^T genome. The 43 bp sequence recombination site is indicated. eno, enolase coding gene; ltaS, lipoteichoic acid synthase coding gene; int, phage integrase coding genes. The location of PCR primers designed for the specific amplification and identification of the integration and excision structure are indicated. (B) PCR amplification for the identification of integration and excision structures on genomic DNA extracted from S. thermophilus DSM 20617T and A33 phage-cured strains. Line 1, 3, 4, and 5 PCR product obtained using as template DNA from S. thermophilus DSM 20617^T grown in M17 lactose 1%, 0.5%, sucrose 1%, and sucrose 0.2%, respectively. Line 2, PCR product using as template DNA from S. thermophilus A33 phage-cured. M, molecular weight marker. cn, PCR negative control. Primer sets and the relative expected dimension of PCR fragment are reported. Dynamics of excision events (C) and BODIPY-FL vancomycin fluorescence (D) during the growth of S. thermophilus DSM 20617^T in M17 containing 0.2% (white symbols) and 1% (wt/vol) (black symbols) sucrose. S. thermophilus wild-type (squares), and A33 phage-cured (triangles). Error bars represent the standard deviation based on three replicates.

FIGURE 5

Cell-wall integrity in S. thermophilus wild-type and A33 phage-cured derivative. (A) Lisozyme/SDS sensitivity was reported as reduction (%) of the cell density (O.D. _595nm) after the enzymatic treatment. (B) ß-galactosidase activity was reported as mO.D. _415nm per min. exp = cells collected during the exponential growth phase (O.D. _{600 nm} 0.5). st = cells collected during the stationary phase (O.D. _{600 nm} 1.5). Error bars represent the standard deviation based on three replicates.

FIGURE 6

Survival to heat-treatment of cell suspensions of S. thermophilus DSM 20617^T (black circle) and A33 (white circle) phage-cured derivative. S. thermophilus cells have been collected in exponential (A) or stationary (B) phase of growth, washed and suspended in saline solution at a final concentration of 2 10⁹ event/ml. The data are represented as the average of three independent heat-treatments. The error bars represent the standard deviation. The temperature which affected the survival of each tested strains is indicated.