Summary: The growth phase during which probiotic bacteria are harvested and consumed can strongly influence their performance as health‐promoting agents. In this study, global transcriptomic and proteomic changes were studied in the widely used probiotic Lactobacillus rhamnosus GG during growth in industrial‐type whey medium under strictly defined bioreactor conditions. The expression of 636 genes ( P ≤ 0.01) and 116 proteins ( P < 0.05) changed significantly over time. Of the significantly differentially produced proteins, 61 were associated with alterations at the transcript level. The most remarkable growth phase‐dependent changes occurred during the transition from the exponential to the stationary growth phase and were associated with the shift from glucose fermentation to galactose utilization and the transition from homolactic to mixed acid fermentation. Furthermore, several genes encoding proteins proposed to promote the survival and persistence of L. rhamnosus GG in the host and proteins that directly contribute to human health showed temporal changes in expression. Our results suggest that L. rhamnosus GG has a highly flexible and adaptable metabolism and that the growth stage during which bacterial cells are harvested and consumed should be taken into consideration to gain the maximal benefit from probiotic bacteria.

Medical device-associated staphylococcal infections are a common and challenging problem. However, detailed knowledge of staphylococcal biofilm dynamics on clinically relevant surfaces is still limited. In the present study, biofilm formation of the Staphylococcus aureus ATCC 25923 strain was studied on clinically relevant materials—borosilicate glass, plexiglass, hydroxyapatite, titanium and polystyrene—at 18, 42 and 66 h. Materials with the highest surface roughness and porosity (hydroxyapatite and plexiglass) did not promote biofilm formation as efficiently as some other selected materials. Matrix-associated poly-N-acetyl-β-(1-6)-glucosamine (PNAG) was considered important in young (18 h) biofilms, whereas proteins appeared to play a more important role at later stages of biofilm development. A total of 460 proteins were identified from biofilm matrices formed on the indicated materials and time points—from which, 66 proteins were proposed to form the core surfaceome. At 18 h, the appearance of several r-proteins and glycolytic adhesive moonlighters, possibly via an autolysin (AtlA)-mediated release, was demonstrated in all materials, whereas classical surface adhesins, resistance- and virulence-associated proteins displayed greater variation in their abundances depending on the used material. Hydroxyapatite-associated biofilms were more susceptible to antibiotics than biofilms formed on titanium, but no clear correlation between the tolerance and biofilm age was observed. Thus, other factors, possibly the adhesive moonlighters, could have contributed to the observed chemotolerant phenotype. In addition, a protein-dependent matrix network was observed to be already well-established at the 18 h time point. To the best of our knowledge, this is among the first studies shedding light into matrix-associated surfaceomes of S. aureus biofilms grown on different clinically relevant materials and at different time points.

Propionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B12. Production of active form of vitamin is a prerequisite for attempts to naturally fortify foods with B12 by microbial fermentation. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Genomic data indicate that P. freudenreichii possesses a fusion gene, bluB/cobT2, coding for a predicted phosphoribosyltransferase/nitroreductase, which is presumably involved in production of vitamin B12. Understanding the mechanisms affecting the synthesis of different vitamin forms is useful for rational strain selection and essential for engineering of strains with improved B12 production properties. Here, we investigated the activity of heterologously expressed and purified fusion enzyme BluB/CobT2. Our results show that BluB/CoBT2 is responsible for the biosynthesis of the DMBI base and its activation into α-ribazole phosphate, preparing it for attachment as the lower ligand of cobalamin. The fusion enzyme was found to be efficient in metabolite channeling and the enzymes’ inability to react with adenine, a lower ligand present in the pseudovitamin, revealed a mechanism favoring the production of the active form of the vitamin. P. freudenreichii did not produce cobalamin under strictly anaerobic conditions, confirming the requirement of oxygen for DMBI synthesis. In vivo experiments also revealed a clear preference for incorporating DMBI over adenine into cobamide under both microaerobic and anaerobic conditions. The herein described BluB/CobT2 is responsible for the production and activation of DMBI. Fusing those two activities results in high pressure towards production of the true vitamin B12 by efficiently activating DMBI formed within the same enzymatic complex. This indicates that BluB/CobT2 is the crucial enzyme in the B12 biosynthetic pathway of P. freudenreichii. The GRAS organism status and the preference for synthesizing active vitamin form make P. freudenreichii a unique candidate for the in situ production of vitamin B12 within food products.

Experimental conditions for efficient protein radiolabelling and two-dimensional gel electrophoresis were developed for Bifidobacterium longum. Using these tools, protein synthesis in cells before and after heat-shock and bile salts treatment was investigated. Following heat-stress, 13 proteins were upregulated, of which HtrA, DnaK and GroEL were also moderately induced by bile salts, indicating close relationship between the heat and bile salts responses in bifidobacteria. Our work indicated that, as a consequence of prolonged heat-stress, HtrA undergoes sequential modification and proteolysis, and that this mechanism could be employed by bifidobacteria to respond to heat-stress.