Characterization of the serpin-encoding gene of Bifidobacterium breve 210B

Abstract

Members of the serpin (serine protease inhibitor) superfamily have been identified in higher multicellular eukaryotes, as well as in bacteria, although examination of available genome sequences has indicated that homologs of the bacterial serpin-encoding gene (ser) are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least 5, and perhaps up to 9, of the 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria produce serpins that form a separate clade. We characterized the ser(210B) locus of Bifidobacterium breve 210B, which encompasses a number of genes whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, microarray, reverse transcription-PCR (RT-PCR), and quantitative real-time PCR (qRT-PCR) analyses revealed that a 3.5-kb polycistronic mRNA encompassing the ser(210B) operon with a single transcriptional start site is strongly induced following treatment of B. breve 210B cultures with some proteases. Interestingly, transcription of other bifidobacterial ser homologs appears to be triggered by different proteases.

FIG. 1.

Distribution of genes that are upregulated upon protease treatment based on COG functional categories. The y axis indicates the numbers of genes identified for the COG functional categories.

FIG. 2.

Schematic diagram of ser loci of B. breve 210B and other bifidobacterial strains. Each arrow indicates an ORF, and the length of the arrow is proportional to the size of the ORF. The predicted protein functions are indicated above the arrows. The levels of amino acid identity (expressed as percentages) are also indicated.

FIG. 3.

(a) Alignment of the Ser proteins of B. breve 210B, B. breve DSM20213, B. longum subsp. longum DJO10A, B. longum subsp. longum NCC2705, B. longum subsp. infantis ATCC 15697, B. longum subsp. infantis CCUG52486, B. dentium Bd1, and B. dentium ATCC 27678 with the structural template tengpinD31 (2PPE). Shading indicates conservation at a position in at least 50% of the amino acid residues in the alignment (black, identical residues; gray, similar residues). The α-helices and turns are indicated. The RCL region is enclosed in a box. (b) 3D structure of B. breve 210B serpin. The 3D structure of the B. breve 210B serpin (light gray) is superimposed on the predicted serpin model (PDB entry 2PEE) (gray). The potential residues in the various serpins that are predicted to form the RCL regions are indicated.

FIG. 4.

Slot blot hybridization using DNA extracted from different bifidobacterial species and hybridized using a mixed ser probe. The spots contained DNA from the following strains: 1, B. longum subsp. longum ATCC 15707^T; 2, B. bifidum LMG 11041^T; 3, B. pseudolongum subsp. globosum LMG11569^T; 4, B. asteroides LMG 10735^T; 5, B. saeculare LMG 14934^T; 6, B. gallinarum 11586^T; 7, B. angulatum ATCC 27535^T; 8, B. choerinum LMG 10510^T; 9, B. gallicum LMG 11596^T; 10, B. subtile LMG 11597^T; 11, B. dentium LMG 11045^T; 12, B. longum subsp. infantis ATCC 15697^T; 13, B. animalis subsp. lactis DSM 10140^T; 14, B. animalis subsp. animalis ATCC 25527^T; 15, B. ruminatium LMG21811^T; 16, B. thermacidophilum subsp. thermacidophilum LMG 21395^T; 17, B. thermacidophilum subsp. porcinum LMG21689^T; 18, B. longum subsp. suis LMG 21814^T; 19, B. pullorum LMG 21816^T; 20, B. boum LMG 10736^T; 21, B. pseudolongum subsp. pseudolongum LMG 11571^T; 22, B. minimum LMG 11592^T; 23, B. catenulatum LMG 11591^T; 24, B. merycicum LMG 11341^T; 25, B. pseudocatenulatum LMG 10505^T; 26, B. psychraerophilum LMG 21775^T; 27, B. indicum LMG 11587^T; 28, B. coryneforme LMG 18911^T; 29, B. adolescentis ATCC 15703^T; 30, B. magnum LMG 11591^T; 31, B. thermophilum JCM 7027^T; 32, B. scardovii LMG 21589^T; 33, B. animalis subsp. animalis ATCC 27536; 34, B. bifidum 317B; 35, B. pseudocatenulatum 318B; 36, B. cuniculi LMG 10738^T; and 37, B. breve 210B.

FIG. 5.

Phylogenetic tree obtained using serpin homologs from various bacteria. The scale bar indicates phylogenetic distance. The serpin protein sequences of bacteria are enclosed in a box, while the serpin proteins of bifidobacteria are indicated by dark shading.

FIG. 6.

Plots of relative levels of transcription of B. breve 210B, B. longum subsp. infantis ATCC 15697, B. longum subsp. longum ATCC 15707, and B. dentium Bd1 serpin-encoding genes after serine protease treatment versus growth in MRS-based media as analyzed by quantitative real-time PCR assays (a) and by slot blot hybridization (b). The bars indicate the relative amounts of ser mRNAs for the specific samples. The cDNAs used were synthesized using RNA collected from bifidobacterial cultures exposed for 150 min to α-chymotrypsin (αCH), chymotrypsin (CHY), neutrophil elastase (HNE), kallikrein (KAK), pancreatic elastase (PPE), papain (PAP), plasmin (PLA), trypsin (TRY), or thrombin (TRO). For panel b, induction of the ser_210B gene was evaluated by slot blot hybridization. Total RNA (25 μg per slot) was isolated from B. breve 210B cells exposed for up to 150 min to different serine proteases (see above) and probed with biotin-labeled ser. The resulting hybridization signals obtained in the autoradiograms were quantified. The amount of mRNA synthesized under the conditions described above was normalized to the amount present in cultures grown in MRS medium.

FIG. 7.

Transcriptional organization of the B. breve 210B ser operon. (a) Schematic diagram of the B. breve 210B ser locus and Northern blot analysis of B. breve 210B ser transcription. Hairpin symbols indicate predicted secondary structures. The transcript identified is indicated by a solid arrow, which points toward the 3′ end of the mRNA. RNA was isolated from a culture before and after exposure to pancreatic elastase (PPE). The molecular weights, calculated from the hybridization signal and position of 16S and 23S rRNA, are indicated. 0, 90, and 180, incubation for 0, 90, and 180 min, respectively. (b) Products generated by RT-PCR. PCR products were obtained with primers spanning the intergenic regions between ORF1502 and ser_210B (primers ser1-uni and ser1-rev) (lanes 1 to 4), between ser_210B and lacI (primers lacI-uni and lacI-rev) (lanes 5 to 8), and between lacI and ORF1505 (primers lac2-uni and lac2-rev) (lanes 9 to 12). The positions of the primer pairs used in RT-PCR experiments are shown in panel a. PCR products were obtained under the following conditions: lanes 1, 5, and 9, cDNA prepared from B. breve 210B RNA; lanes 3, 7, and 11, negative control in which B. breve RNA was used but reverse transcriptase was omitted; lanes 4, 8, and 12, no-template control; lanes 2, 6, and 10, positive control (B. breve DNA). Lanes MK contained a 1-kb DNA molecular marker.

FIG. 8.

Determination of the B. breve 210B ser transcription initiation site by primer extension analysis. (a) Primer extension results obtained using oligonucleotide SER-prom1 and mRNA isolated from exposition of pancreatic elastase at the times indicated. The deduced −10 hexamer and −35 hexamers are indicated by bold type and underlined; bold type with asterisks indicates the transcription start points; and the start codon is underlined. RBS, ribosome binding site. (b) Comparison of the promoter regions of ser genes from different bifidobacteria, including B. breve 210B, B. breve UCC2003, B. longum subsp. longum NCC2705, B. longum subsp. longum DJO10A, B. longum subsp. infantis CCUG52486, B. longum subsp. infantis ATCC 55813, B. longum subsp. infantis ATCC 15697^T, and B. dentium Bd1.