Abstract: To elucidate the regulatory mechanism of the acid tolerance response (ATR) in Bifidobacterium longum BBMN68, this study investigated the role of response regulator BBMN68_47 in mediating ATR through genomic and proteomic approaches. An overexpression strain of BBMN68_47 with a 3 × FLAG tag was constructed via molecular cloning, and the recombinant plasmids (pDP152-BBMN68_47, pDP152-3 × FLAG, and pDP152-3 × FLAG-BBMN68_47) were validated through polymerase chain reaction (PCR), sequencing, and Western blot. Under weak acid induction (pH 4.5), chromatin immunoprecipitation sequencing (ChIP-seq) identified 118 high-confidence DNA binding peaks, over 80% of which were localized at transcription start sites (TSS). Bioinformatics analysis predicted seven candidate DNA binding motifs. Electrophoretic mobility shift assay (EMSA) analysis further confirmed specific binding of BBMN68_47 to two conserved motifs: CTGGGCGTTT (Probe 0235) and CTGGACGAATCCTG (Probe 0075), regulating the expression of its own gene BBMN68_47 (autoregulatory) and the S-adenosylmethionine synthetase (metK) gene, respectively. These findings establish BBMN68_47 as a central transcriptional regulator in B. longum ATR, orchestrating key acid-resistant gene networks through conserved and novel DNA motifs. This work offers a theoretical basis for elucidating the molecular mechanism of the ATR of B. longum and engineering stress resistance in probiotics.

Key words: Bifidobacterium longum BBMN68; acid tolerance response; DNA-protein interaction; chromatin immunoprecipitation sequencing; electrophoretic mobility shift assay