Annette Rouault

Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in Propionibacterium freudenreichii.

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.

Survival and metabolic activity of selected strains of Propionibacterium freudenreichii in the gastrointestinal tract of human microbiota-associated rats.

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 x 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.

Lactobacillus helveticus: Strain Typing and Genome Size Estimation by Pulsed Field Gel Electrophoresis

Abstract. Genomic DNAs of 22 strains of Lactobacillus helveticus of various geographical origins were analyzed by pulsed-field gel electrophoresis. Two endonucleases, SmaI and SgrAI, of the 19 tested produced DNA fragments useful for strain comparison. With the endonuclease SmaI, a characteristic restriction pattern was identified for 18 of the 22 strains. The percentage of similarity (Dice coefficient) between the profiles varied between 26% and 100%, and clustering was accomplished by using the unweighted pair group method with arithmetic averages (UPGMA). For the strains showing identical profiles, the high genomic similarity was confirmed when the endonuclease SgrAI was used instead of SmaI. From summation of SmaI and SgrAI fragments from three L. helveticus strains (CNRZ 241, CNRZ 303, and CIP 57.15), the genomic length was estimated at ca. 1.85–2.0 Mb.

Filamentous phage active on the gram-positive bacterium Propionibacterium freudenreichii

We present the first description of a single-stranded DNA filamentous phage able to replicate in a gram-positive bacterium. Phage B5 infects Propionibacterium freudenreichii and has a genome consisting of 5,806 bases coding for 10 putative open reading frames. The organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria. The putative coat protein exhibits homology with the coat proteins of phages PH75 and Pf3 active on Thermus thermophilus and Pseudomonas aeruginosa, respectively. B5 is, therefore, evolutionarily related to the filamentous phages active on gram-negative bacteria.

DNA Fingerprinting of Dairy Propionibacteria Strainsby Pulsed-Field Gel Electrophoresis

Abstract. Restriction endonuclease patterns generated by Pulsed-Field Gel Electrophoresis (PFGE) were used to compare 96 strains of dairy propionibacteria originating from dairy products, international and industrial collections; endonucleases XbaI and SspI gave satisfactory restriction patterns. However, whereas XbaI can be used for Propionibacterium freudenreichii, SspI seems more suitable for the three other species: P. acidipropionici, P. thoenii, and P. jensenii. It is a convenient method to differentiate the dairy propionibacteria from closely related bacteria and from others usually present in dairy products. We observed a considerable restriction fragment length polymorphism among the Propionibacterium chromosomes and especially for P. freudenreichii: among 48 strains we detected 40 different patterns. This species is the most commonly encountered in the Swiss-type cheeses and is the only Propionibacterium species used as a cheese starter. Conversely, the species P. acidipropionici is not very diverse: among nine strains we observed only four different patterns, two of which were closely related. This is probably because this species is not used as a starter in cheese manufacture and consequently is poorly represented in collections. When strains come from geographical different isolates, their patterns are always different with very few common bands. The presence of numerous identical strains was due to the fact that they were present at the same time in the national collections, research laboratory collections, and in the industrial ones.

Electrotransfection of Propionibacterium freudenreichii TL 110

The first highly efficient protocol is described for the electrotransfection of Propionibacterium freudenreichii with DNA phage. The transfection efficiency is 7 times 105 transfectants per μg of DNA under optimal conditions. Optimized parameters included the field strength (12.5 kV, 200 Ohms, 25 μF), phage DNA concentration (1 μg ml‐1) and cell density (1.5 times 1010 cells ml‐1). Growth in the presence of glycine and harvesting of cells during the early exponential growth phase increased the transfection efficiency. This electrotransfection protocol is of importance for the genetic improvement of dairy propionibacteria.