An interactive regulatory network controls stress response in Bifidobacterium breve UCC2003

Abstract

Members of the genus Bifidobacterium are gram-positive bacteria that commonly are found in the gastrointestinal tract (GIT) of mammals, including humans. Because of their perceived probiotic properties, they frequently are incorporated as functional ingredients in food products. From probiotic production to storage and GIT delivery, bifidobacteria encounter a plethora of stresses. To cope with these environmental challenges, they need to protect themselves through stress-induced adaptive responses. We have determined the response of B. breve UCC2003 to various stresses (heat, osmotic, and solvent) using transcriptome analysis, DNA-protein interactions, and GusA reporter fusions, and we combined these with results from an in silico analysis. The integration of these results allowed the formulation of a model for an interacting regulatory network for stress response in B. breve UCC2003 where HspR controls the SOS response and the ClgR regulon, which in turn regulates and is regulated by HrcA. This model of an interacting regulatory network is believed to represent the paradigm for stress adaptation in bifidobacteria.

FIG. 1.

Deduced stress regulatory binding sites described in Table 3 depicted in WebLOGO format based on the comparative sequence analysis of actual target sequences in the B. breve UCC2003 genome. (A) HAIR; (B) CIRCE; (C) SOS box; and (D) ClgR binding site.

FIG. 2.

(A to E) EMSAs of H6-HspR interaction with DNA fragments encompassing the promoter regions of dnaK (A), clpB (B), clgR (C), nfo (D), and hrdB (E). DNA fragments were obtained by PCR using IRD-800-labeled primers. Lane X contained the probe without added protein. The remaining lanes contained probe samples incubated with increasing concentrations of H6-HspR (concentrations ranged from 50 to 400 nM). For each successive lane from left to right, the concentration of H6-HspR was doubled. (F to I) EMSAs of H6-HrcA interactions with promoter regions of hrcA (F), groEL (G), groES (H), and hspR (I) (negative control). DNA fragments were obtained by PCR using IRD-800-labeled primers. Lane X contained the probe without added protein. The remaining lanes contained probe samples incubated with increasing concentrations of H6-HrcA (concentrations ranged from 50 to 400 nM). For each successive lane from left to right, the concentration of H6-HrcA was doubled. (J to N) EMSAs of H6-ClgR-T interactions with promoter regions of clpC (J), clpP (K), clgR (L), and hrcA (M). Panel N represents an EMSA in which full-length ClgR was used in combination with the labeled promoter region of clpC. DNA fragments were obtained by PCR using IRD-800-labeled primers. Lane X contained the probe without added protein. The remaining lanes contained probe samples incubated with increasing concentrations of H6-ClgR-T or H6-ClgR (concentrations ranged from 15 to 120 nM). For each successive lane from left to right, the concentration of H6-ClgR-T or H6-ClgR was doubled. (O) EMSAs of H6-ClgR-T with a concentration of 60 nM on mutated promoter regions of the ClgR motif of PclpC. Original sequences and mutations are shown above the image. +, original promoter sequence; −, promoter region of hsp20. (P) EMSAs of H6-HspR with a concentration of 175 nM on mutated promoter regions of the HAIR motif of PclpB. N, mutation of the AAAA 3′ extension to GGGG; WT, original promoter sequence; and C, mutation of the TTTT 5′ extension to CCCC.

FIG. 3.

Schematic representation of the stress gene regulatory network of B. breve UCC 2003. Dotted lines indicate the predicted interaction, and closed lines indicate a proven interaction. A dash at the end of a line indicates repression, while a triangle at the end of a line indicates activation.