Broad conservation of milk utilization genes in Bifidobacterium longum subsp. infantis as revealed by comparative genomic hybridization

Abstract

Human milk oligosaccharides (HMOs) are the third-largest solid component of milk. Their structural complexity renders them nondigestible to the host but liable to hydrolytic enzymes of the infant colonic microbiota. Bifidobacteria and, frequently, Bifidobacterium longum strains predominate the colonic microbiota of exclusively breast-fed infants. Among the three recognized subspecies of B. longum, B. longum subsp. infantis achieves high levels of cell growth on HMOs and is associated with early colonization of the infant gut. The B. longum subsp. infantis ATCC 15697 genome features five distinct gene clusters with the predicted capacity to bind, cleave, and import milk oligosaccharides. Comparative genomic hybridizations (CGHs) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations. Multilocus sequence typing provided taxonomic subspecies designations and grouped the strains between B. longum subsp. infantis and B. longum subsp. longum. CGH analysis determined that HMO utilization gene regions are exclusively conserved across all B. longum subsp. infantis strains capable of growth on HMOs and have diverged in B. longum subsp. longum strains that cannot grow on HMOs. These regions contain fucosidases, sialidases, glycosyl hydrolases, ABC transporters, and family 1 solute binding proteins and are likely needed for efficient metabolism of HMOs. Urea metabolism genes and their activity were exclusively conserved in B. longum subsp. infantis. These results imply that the B. longum has at least two distinct subspecies: B. longum subsp. infantis, specialized to utilize milk carbon, and B. longum subsp. longum, specialized for plant-derived carbon metabolism.

FIG. 1.

Evolutionary relationship of B. longum spp. strains used in the study. (a) The MLST-based hierarchical clustering was inferred using the minimum-evolution method (35). The optimal tree with the sum of branch length of 0.12 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (12). The phylogenetic tree was linearized assuming equal evolutionary rates in all lineages (47). The clock calibration to convert distance to time was 2.1 × 10⁸ (time/node height). The tree is drawn to scale, with the branch lengths being in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum-composite-likelihood method (49) and are in units of the number of base substitutions per site. Phylogenetic analyses were conducted in MEGA (version 4) (48). Sequence fragments for the seven MLST loci of B. longum ATCC 55813 and B. longum CCUG 52486 were obtained from NCBI. (b) Hierarchical clustering (Euclidian distance, average linking) based on the entire CGH data set and performed in MeV using the HCL algorithm.

FIG. 2.

CGH analysis of loci related to milk glycans utilization and gut adaptation. Strains are grouped hierarchically on the basis of probe intensities. (a) Urease operon; (b and c) putative fucose utilization regions (H-2 and H-3); (d) putative sialic acid utilization region (H-4); (e) LNB metabolism gene region (H-5); (f) a 43-kb cluster associated with utilization of human milk oligosaccharides (H-1). •, locus absent; *, locus highly divergent in strain JCM 7010.