A two-component regulatory system controls autoregulated serpin expression in Bifidobacterium breve UCC2003

Abstract

This work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded by serRK, which is believed to control the expression of the ser(2003) locus in Bifidobacterium breve UCC2003. The ser(2003) locus consists of two genes, Bbr_1319 (sagA) and Bbr_1320 (serU), which are predicted to encode a hypothetical membrane-associated protein and a serpin-like protein, respectively. The response regulator SerR was shown to bind to the promoter region of ser(2003), and the probable recognition sequence of SerR was determined by a combinatorial approach of in vitro site-directed mutagenesis coupled to transcriptional fusion and electrophoretic mobility shift assays (EMSAs). The importance of the serRK 2CRS in the response of B. breve to protease-mediated induction was confirmed by generating a B. breve serR insertion mutant, which was shown to exhibit altered ser(2003) transcriptional induction patterns compared to the parent strain, UCC2003. Interestingly, the analysis of a B. breve serU mutant revealed that the SerRK signaling pathway appears to include a SerU-dependent autoregulatory loop.

Fig 1

(a) Schematic representation of the ser₂₀₀₃ gene locus, promoter region, and DNA fragments used in the gel mobility shift assays; the numbers indicate the ends of the fragments relative to the transcriptional start site. Direct repeats (DRs) and inverted repeats (IRs) are indicated; −10 and −35 hexamers, the transcriptional starting site (TSS), and the ribosomal starting site (RBS) are underlined. (b and c) Electrophoretic mobility shift assays carried out using ser₂₀₀₃ promoter fragments and cell crude extracts containing the full SerR protein (b) or its 5′-truncated fragment SerR-T (c). Gradients correspond to 2, 1, 0.5, and 0.25 μg of protein content. Cell extract from E. coli harboring the empty vector pQE30 was used as a negative control.

Fig 2

(a) Schematic representation of the different mutations performed in the ser₂₀₀₃ locus promoter. (b) Gel mobility shift assays carried out with crude cell extracts containing SerR and various mutated versions of the ser₂₀₀₃ promoter region (see panel a); as a positive control, the wild-type ser₂₀₀₃ promoter region was used. (c) GusA assays testing protease-mediated induction of the ser₂₀₀₃ promoter of B. breve UCC2003 harboring plasmid pNZ272-BSMut1, pNZ272-BSMut2, pNZ272-BSMut3, or pNZ272-BSMut4; B. breve UCC2003 harboring pNZ272 with the wild-type ser₂₀₀₃ promoter or without any insert was used as a positive or a negative control, respectively. Data correspond to mean values of three independent experiments, and they were compared by an unpaired t test analysis. Error bars correspond to the standard deviations. **, P ≤ 0.01; ***, P ≤ 0.001.

Fig 3

The relative transcription levels of ser₂₀₀₃ in B. breve UCC2003 and various mutant and complemented strains upon serine protease treatment versus growth in MRS-based medium as analyzed by quantitative reverse transcriptase PCR assays. The histograms indicate the relative amounts of ser₂₀₀₃ mRNAs for the specific samples. The cDNAs were generated from RNA collected following exposure of the bifidobacterial cultures, for 90 min, to kallikrein (KAK), papain (PAP), plasmin (PLA), or trypsin (TRY) or left untreated (MRS; taken as a value of 1).