Adaptation to and tolerance of bile stress are among the main limiting factors to ensure survival of bifidobacteria in the intestinal environment of humans. The effect of bile salts on protein expression patterns of Bifidobacterium longum was examined. Protein pattern comparison of strains grown with or without bile extract allowed us to identify 34 different proteins whose expression was regulated. The majority of these proteins were induced after both a minor (0.6 g liter ؊1 ) and a major (1.2 g liter ؊1 ) exposure to bile. These include general stress response chaperones, proteins involved in transcription and translation and in the metabolism of amino acids and nucleotides, and several enzymes of glycolysis and pyruvate catabolism. Remarkably, xylulose 5-phosphate/fructose 6-phosphate phosphoketolase, the key enzyme of the so-called bifidobacterial shunt, was found to be upregulated, and the activity on fructose 6-phosphate was significantly higher for protein extracts of cells grown in the presence of bile. Changes in the levels of metabolic end products (acetate and lactate) were also detected. These results suggest that bile salts, to which bifidobacteria are naturally exposed, induce a complex physiological response rather than a single event in which proteins from many different functional categories take part. This study has extended our understanding of the molecular mechanism underlying the capacity of intestinal bifidobacteria to tolerate bile.
Bifidobacteria are one of the main microbial inhabitants of the human colon. Usually administered in fermented dairy products as beneficial microorganisms, they have to overcome the acidic pH found in the stomach during the gastrointestinal transit to be able to colonize the lower parts of the intestine. The mechanisms underlying acid response and adaptation in Bifidobacterium longum biotype longum NCIMB 8809 and its acid-pH-resistant mutant B. longum biotype longum 8809dpH were studied. Comparison of protein maps, and protein identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, allowed us to identify nine different proteins whose production largely changed in the mutant strain. Furthermore, the production of 47 proteins was modulated by pH in one or both strains. These included general stress response chaperones and proteins involved in transcription and translation as well as in carbohydrate and nitrogen metabolism, among others. Significant differences in the levels of metabolic end products and in the redox status of the cells were also detected between the wild-type strain and its acid-pHresistant mutant in response to, or as a result of, adaptation to acid. Remarkably, the results of this work indicated that adaptation and response to low pH in B. longum biotype longum involve changes in the glycolytic flux and in the ability to regulate the internal pH. These changes were accompanied by a higher content of ammonium in the cytoplasm, likely coming from amino acid deamination, and a decrease of the bile salt hydrolase activity.
Lactococcus lactis is a Gram-positive bacteria, which belongs to the group of lactic acid bacteria among which several genera play an essential role in the manufacture of food products. Cytosolic proteins of L. lactis IL1403 cultivated in M17 broth have been resolved by two-dimensional gel electrophoresis using two pH gradients (pH 4-7, 4.5-5.5). More than 230 spots were identified by peptide mass fingerprints, corresponding to 25% of the predicted acid proteome. The present study made it possible to describe at the proteome level a significant number of cellular pathways (glycolysis, fermentation, nucleotide metabolism, proteolysis, fatty acid and peptidoglycan synthesis) related to important physiological processes and technological properties. It also indicated that the fermentative metabolism, which characterizes L. lactis is associated with a high expression of glycolytic enzymes. Thirty-four proteins were matched to open reading frames for which there is no assigned function. The comparison at the proteome level of two strains of L. lactis showed an important protein polymorphism. The comparison of the proteomes of glucose- and lactose-grown cells revealed an unexpected link between the nature of the carbon source and the metabolism of pyrimidine nucleotides.
Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cell's physiology.
When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to R. gnavus. This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.
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