The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, weshowthatP.acidipropioniciandfreudenreichii,twospecies which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted bythebacteria. Bacterialculturesupernatantsaswell as pure SCFA induced typical signs of apoptosis including a lossofmitochondrialtransmembranepotential,thegeneration ofreactiveoxygenspecies,caspase-3processing,andnuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.

Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, respectively. Traditionally used as Swiss-type cheese starters, propionibacteria also constitute promising human probiotics. Stress tolerance and cross-protection in Propionibacterium freudenreichii were thus examined after exposure to heat, acid, or bile salts stresses. Adapted cells demonstrated acquired homologous tolerance. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, bile salts pretreatment sensitized cells to acid challenge and vice versa. Surprisingly, heat and acid responses did not present significant cross-protection in P. freudenreichii. During adaptations, important changes in cellular protein synthesis were observed using two-dimensional electrophoresis. While global protein synthesis decreased, several proteins were overexpressed during stress adaptations. Thirty-four proteins were induced by acid pretreatment, 34 by bile salts pretreatment, and 26 by heat pretreatment. Six proteins are common to all stresses and represent general stress-response components. Among these polypeptides, general stress chaperones, and proteins involved in energetic metabolism, oxidative stress response, or SOS response were identified. These results bring new insight into the tolerance of P. freudenreichii to heat, acid, and bile salts, and should be taken into consideration in the development of probiotic preparations.

The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, we show that P. acidipropionici and freudenreichii, two species which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted by the bacteria. Bacterial culture supernatants as well as pure SCFA induced typical signs of apoptosis including a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species, caspase-3 processing, and nuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.

In addition to their use in cheese technology, dairy propionibacteria have been identified as potential probiotics. However, to have a probiotic effect, propionibacteria have to survive and to remain metabolically active in the digestive tract. The aim of the present study was to investigate the survival and metabolic activity of Propionibacterium freudenreichii within the gastrointestinal tract of human microbiota-associated rats, and its influence on intestinal microbiota composition and metabolism. Twenty-five dairy Propionibacterium strains were screened for their tolerance towards digestive stresses and their ability to produce propionate in a medium mimicking the content of the human colon. Three strains were selected and a daily dose of 2 £ 10 10 colony-forming units was fed to groups of human microbiota-associated rats for 20 d before microbiological, biochemical and molecular investigations being carried out. These strains all reached 8-log values per g faeces, showing their ability to survive in the gastrointestinal tract. Transcriptional activity within the intestine was demonstrated by the presence of P. freudenreichii-specific transcarboxylase mRNA. The probiotic efficacy of propionibacteria was yet species-and strain-dependent. Indeed, two of the strains, namely TL133 and TL1348, altered the faecal microbiota composition, TL133 also increasing the caecal concentration of acetate, propionate and butyrate, while the third strain, TL3, did not have similar effects. Such alterations may have an impact on gut health and will thus be taken into consideration for further in vivo investigations on probiotic potentialities of P. freudenreichii.

The plasmid pE194 is unable to replicate in Lactococcus lactis subsp. lactis (formerly Streptococcus lactis). When linked to resident bacteriophage sequences, pE194 was able to integrate into the L. lactis subsp. lactis chromosome either by Campbell-like recombination or by double crossing over with deletion. Integration occurred into the DNA of the prophage and prevented its multiplication. When a selective pressure was applied to an integrant in which pE194 was flanked by two direct repeats of prophage fragment, amplification of pE194 and the prophage fragment was observed. The pE194 copy number was assessed at six to nine, and amplification was stable upon growth under nonselective conditions.