Effect of heat-shock and bile salts on protein synthesis ofBifidobacterium longumrevealed by [³⁵S]methionine labelling and two-dimensional gel electrophoresis

Abstract

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.

Bile Salt Hydrolase Activity in Probiotics

“…Strain selection has generally been based on in vitro tolerance of physiologically relevant stresses: e.g., low pH, elevated osmolarity, and bile (26,53,77,100). In addition to these physiological assays, molecular investigations are now under way to determine the genetic basis of gastric survival and functionality (11,78,79) and inclusion of molecular markers identified by this approach into screening programs may lead to more well-defined and reliable results.…”

HspR Mutations Are Naturally Selected in Bifidobacterium longum When Successive Heat Shock Treatments Are Applied

“…Our selection of heat shock-tolerant mutants similarly targeted a technological property. However, the genetic response of B. longum to HS is likely to confer a broader stress resistance, as shown by its numerous classical stress-associated genes induced by heat treatment (27,31). The high complexity of the bifidobacterial HS response resembles a genetic stampede for survival.…”

“…Another noticeable convergent upregulation concerned htrA after 7 min of HS. HtrA is a widely distributed serine protease (25), which is strongly induced by HS in B. longum (27,31). Again, its upregulation may be an indirect effect of the dnaK-clpB modulation, which could interfere with the expression of htrA and its proteolytic processing normally undergone during HS in B. longum (31).…”

“…The genetic response of Bifidobacterium longum to heat shock (HS) was recently documented in its complexity (27,31). When doing a global transcriptomic analysis by microarrays, HS altered the transcription of 46% of the genes analyzed.…”

Labeling of Bifidobacterium longum Cells with 13 C-Substituted Leucine for Quantitative Proteomic Analyses

“…Twodimensional electrophoresis and multidimensional chromatography separation techniques coupled to protein identification by mass spectrometry have provided insights into the changes in protein abundance underlying basic biological processes (13,14,19). However, these approaches are not always appropriate for studying specific components of the proteome and subcellular fractions, such as low-abundance proteins, proteins with extreme pI values and M r , and very hydrophobic proteins, including membrane proteins.…”