The Streptococcus thermophilus Autolytic Phenotype Results from a Leaky Prophage

Autolysis of lactic acid bacteria used as starters appears to be a crucial step in the flavor development of fermented dairy products (12, 15). It indeed causes the opening of cells and the subsequent release into the curd of intracellular enzymes that are involved in the flavor compound formation. The activity of these enzymes can therefore be enhanced by better accessibility to their substrates, proteins, lipids, or carbohydrates from the milk. It has been shown that the main consequence of cell autolysis in cheese is peptidolysis acceleration, leading to an increased rate of free amino acid production and to a decrease in bitter taste (2, 3, 13, 16, 25, 32, 34, 35, 55). Bacterial autolysis results from the degradation of the peptidoglycan, which is the major structural component of the bacterial cell wall, by enzymes called peptidoglycan hydrolases. Bacteria synthesize their own peptidoglycan hydrolases, named autolysins (45). These cell wall-associated enzymes are potentially lethal for the cell and thus require stringent regulation. It has been proposed that autolysins are involved in different cellular processes including cell wall expansion and turnover, cell division, and transformation (47). Autolysis would result from an uncontrolled action of the bacterial autolysins after inhibition of peptidoglycan synthesis (47). For lysogenic strains, lysis can be caused by induction of the resident prophage. The prophage lysis system is then responsible for the host lysis. It contains in most cases two effectors, a peptidoglycan hydrolase, named endolysin, and a second protein, a so-called holin. Holins are small proteins causing nonspecific lesions in the membrane. They thus allow the endolysin, usually devoid of a signal peptide, access to the peptidoglycan to cause subsequent host envelope disruption (56). Hitherto, most of the studies dealing with lactic acid bacteria autolysis concern the genus Lactococcus and to a lesser extent Lactobacillus. Autolysis was investigated by physiological studies and also by analysis of the peptidoglycan hydrolase content. The autolytic character of Lactococcus and Lactobacillus strains was studied in liquid medium (2, 3, 28, 33, 41, 54) and for some of them during cheese ripening (5, 13, 16, 32, 35, 53, 55). It appears to vary from strain to strain. Various factors could account for the different autolytic behaviors: the cellular content in peptidoglycan hydrolases, the regulation of either the expression or the activity of these enzymes, or the cell wall composition. Another alternative is the involvement of a prophage in the cell lysis, as demonstrated for the lactococcal strain AM2 (32). The bacteriolytic enzymes of Lactococcus lactis (31, 38, 41) and Lactobacillus spp. (10, 52) have been studied by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which allows detection of peptidoglycan hydrolase activities after renaturation in a substrate-containing gel. In L. lactis, an endogenous autolysin (9) and a prophage-encoded enzyme (31) were identified by this technique. Streptococcus thermophilus received little attention regarding autolysis despite its industrial significance as starter used in the manufacture of yogurts and Italian and Swiss type cheeses. For S. thermophilus, spontaneously autolytic strains have been previously identified (43, 50, 58). Lysis occurs at the end of the exponential growth phase, resulting in a typical bell-shaped growth curve. Independent studies of S. thermophilus temperate bacteriophages led furthermore to the observation that lysogens exhibit an identical autolytic phenotype (19). The aim of our study was to investigate the cellular mechanisms involved in the triggering of lysis of S. thermophilus and to specify the link between the autolytic phenotype and lysogeny in this species. For this purpose, 6 different S. thermophilus strains identified as autolytic out of 146 S. thermophilus strains screened were further characterized in this work. All of them were found to be lysogenic. Different environmental factors, such as lactose depletion and organic solvents, were identified as triggers of premature lysis. A bacteriolytic enzyme of 31 kDa was detected by renaturing SDS-PAGE exclusively in the autolytic strains. It was shown to be prophage encoded and homologous to the endolysin Lyt51 of the streptococcal temperate bacteriophage φ01205 (44). From all these results, we propose a mechanism of lysis triggering according to which S. thermophilus lysis is triggered and achieved under unfavorable environmental conditions via the lysis proteins of a leaky prophage.