Streptococcus pneumoniae is an important human pathogen that is able to take up naked DNA from the environment by a quorum-sensing-regulated process called natural genetic transformation. This property enables members of this bacterial species to efficiently acquire new properties that may increase their ability to survive and multiply in the human host. We have previously reported that induction of the competent state in a liquid culture of Streptococcus pneumoniae triggers lysis of a subfraction of the bacterial population resulting in release of DNA. We have also proposed that such competence-induced DNA release is an integral part of natural genetic transformation that has evolved to increase the efficiency of gene transfer between pneumococci. In the present work, we have further elucidated the mechanism behind competence-induced cell lysis by identifying a putative murein hydrolase, choline-binding protein D (CbpD), as a key component of this process. By using real-time PCR to estimate the amount of extracellular DNA in competent relative to noncompetent cultures, we were able to show that competence-induced cell lysis and DNA release are strongly attenuated in a cbpD mutant. Ectopic expression of CbpD in the presence or absence of other competence proteins revealed that CbpD is essentially unable to cause cell lysis on its own but depends on at least one additional protein expressed during competence.

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The effects of various growth conditions on spontaneous LC3 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 LC3 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 LC3 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 LC3 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.

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.Lactic acid bacteria (LAB) have a long tradition of being used as starter cultures for the fermentation of a variety of food products. In the dairy industry, the species Lactococcus lactis has an important role as a starter for the production of various cheeses. Unfortunately, L. lactis is sensitive to bacteriophage attack, an event that may result in impaired quality of the product because of fermentation failure. Consequently, phages are of great economic significance (6,10,31,36).Studies have shown that lysogeny is common among lactococci (reviewed in references 11, 12, and 22), and recently the presence of six prophages in the genome of L. lactis IL1403 has been reported (7). The impact of lysogenic starter strains on fermentation processes is not fully understood. However, temperate phages and prophage-derived sequences of the P335 species have shown to give rise to new lytic phages (4, 13, 33), and the spontaneously induced temperate phages have the potential to infect and lyse or lysogenize sensitive strains in a mixed starter culture in cheese plants (11,18,23,38). Furthermore, prophages of LAB are found to be involved in the lysis of starter cells during cheese ripening, in which release of desirable enzymes into the surroundings is an important step in flavor development (19,24,35).Despite the increasing knowledge about the molecular basis for the lysis-lysogeny decision of temperate phages, there is still little information available on the influence of the host and environmental conditions (3...

Complete genome sequencing of the P335 temperate Lactococcus lactis bacteriophage phiLC3 (32, 172 bp) revealed fifty-one open reading frames (ORFs). Four ORFs did not show any homology to other proteins in the database and twenty-one ORFs were assigned a putative biological function. phiLC3 contained a unique replication module and orf201 was identified as the putative replication initiator protein-encoding gene. phiLC3 was closely related to the L. lactis r1t phage (73% DNA identity). Similarity was also shared with other lactococcal P335 phages and the Streptococcus pyogenes prophages 370.3, 8232.4 and 315.5 over the non-structural genes and the genes involved in DNA packaging/phage morphogenesis, respectively. phiLC3 contained small homologous regions distributed among lactococcal phages suggesting that these regions might be involved in mediating genetic exchange. Two regions of 30 and 32 bp were conserved among the streptococcal and lactococcal r1t-like phages. These two regions, as well as other homologous regions, were located at mosaic borders and close to putative transcriptional terminators indicating that such regions together might attract recombination. The conserved regions found among lactococcal and streptococcal phages might be used for identification of phages/prophages/prophage remnants in their hosts.