Genome-Wide Assessment of Stress-Associated Genes in Bifidobacteria

Abstract

Over the last decade, the genomes of several Bifidobacterium strains have been sequenced, delivering valuable insights into their genetic makeup. However, bifidobacterial genomes have not yet been systematically mined for genes associated with stress response functions and their regulation. In this work, a list of 76 genes related to stress response in bifidobacteria was compiled from previous studies. The prevalence of the genes was evaluated among the genome sequences of 171 Bifidobacterium strains. Although genes of the protein quality control and DNA repair systems appeared to be highly conserved, genome-wide in silico screening for consensus sequences of putative regulators suggested that the regulation of these systems differs among phylogenetic groups. Homologs of multiple oxidative stress-associated genes are shared across species, albeit at low sequence similarity. Bee isolates were confirmed to harbor unique genetic features linked to oxygen tolerance. Moreover, most studied Bifidobacterium adolescentis and all Bifidobacterium angulatum strains lacked a set of reactive oxygen species-detoxifying enzymes, which might explain their high sensitivity to oxygen. Furthermore, the presence of some putative transcriptional regulators of stress responses was found to vary across species and strains, indicating that different regulation strategies of stress-associated gene transcription contribute to the diverse stress tolerance. The presented stress response gene profiles of Bifidobacterium strains provide a valuable knowledge base for guiding future studies by enabling hypothesis generation and the identification of key genes for further analyses. IMPORTANCE Bifidobacteria are Gram-positive bacteria that naturally inhabit diverse ecological niches, including the gastrointestinal tract of humans and animals. Strains of the genus Bifidobacterium are widely used as probiotics, since they have been associated with health benefits. In the course of their production and administration, probiotic bifidobacteria are exposed to several stressors that can challenge their survival. The stress tolerance of probiotic bifidobacteria is, therefore, an important selection criterion for their commercial application, since strains must maintain their viability to exert their beneficial health effects. As the ability to cope with stressors varies among Bifidobacterium strains, comprehensive understanding of the underlying stress physiology is required for enabling knowledge-driven strain selection and optimization of industrial-scale production processes.

Keywords: bifidobacteria; genomics; stress response.

FIG 1

Heat map representing the median sequence identity of the best hit of 76 stress-associated gene products in 22 Bifidobacterium species, including 171 Bifidobacterium strains. The analyzed Bifidobacterium species are members of six previously suggested phylogenetic groups (37, 40). For each stress-associated gene, a query protein sequence was extracted from the genome of a strain in which it was proposed to be involved in stress responses. Homologs of stress-associated gene products across the 22 species were identified using DIAMOND BLASTp (E value, 0.001; sequence identity cutoff, 40%; coverage cutoff, 70%). The maximum likelihood phylogeny tree was constructed using CLC Genomics Workbench. The tree scale gives the average number of substitutions per site. Information on the stress-associated gene products can be found in Table S1.

FIG 2

Distribution of the stress-associated genes in the analyzed Bifidobacterium species. Overall, 171 Bifidobacterium strains were studied, representing 22 species. (A) Proportion of genes found in all strains of all species, in all strains of 95% of the species (21 out of 22 species), in all strains of less than 95% of the species, and in all strains of a single species. (B) Proportion of genes found in all strains of all species, in all strains of 95% of the species, in all strains of less than 95% of the species, and in all strains of a single species in the categories (i) protein quality and DNA repair systems, (ii) oxidative stress response, (iii) acid stress response, and (iv) putative transcriptional regulators of stress responses. The single genes analyzed in the categories organic solvent and bile stress are present in strains of less than 21 species. All species-specific genes were identified in B. subtile KCTC 3272, using genes from B. tibiigranuli TMW 2057 as query genes.

FIG 3

Identification of HemW-specific motifs in HemN homologs of B. animalis BB-12, B. longum NCC2705, and B. asteroides PRL2011. To assess the presence of HemW-specific motifs in the protein sequences of HemN homologs from B. animalis subsp. lactis BB-12 (WP_004217870.1), B. longum subsp. longum NCC2705 (WP_008783712.1), and B. asteroides PRL2011 (WP_033511215.1), their protein sequences were aligned with HemW of L. lactis (WP_003132086.1). The alignment was generated using CLC Genomics Workbench 20.0, default settings, alignment mode set to very accurate. Blue boxes are HemW-specific conserved amino acid residues that distinguish the protein from HemN, identified in L. lactis: H¹³⁴, H¹⁸⁴xxxYxLxxE, Y²³⁴ExS, and especially H²⁴⁸NxxYW (72).

FIG 4

Active-site motif of BCP homologs in Bifidobacterium strains. ★, peroxidatic cysteine; ▾, resolving cysteine, which is absent from BCPs of B. animalis.