Broad Conservation of Milk Utilization Genes in
            <i>Bifidobacterium longum</i>
            subsp.
            <i>infantis</i>
            as Revealed by Comparative Genomic Hybridization

Human milk oligosaccharides (HMOs) are considered to play a key role in establishing and maintaining the infant gut microbiota. Lacto-N-triose forms part of both type 1 and type 2 HMOs and also of the glycan moieties of glycoproteins. Upstream of the previously characterized gene cluster involved in lacto-N-biose and galacto-N-biose metabolism from Lactobacillus casei BL23, there are two genes, bnaG and manA, encoding a ␤-N-acetylglucosaminidase precursor and a mannose-6-phosphate isomerase, respectively. In this work, we show that L. casei is able to grow in the presence of lacto-N-triose as a carbon source. Inactivation of bnaG abolished the growth of L. casei on this oligosaccharide, demonstrating that BnaG is involved in its metabolism. Interestingly, whole cells of a bnaG mutant were totally devoid of ␤-N-acetylglucosaminidase activity, suggesting that BnaG is an extracellular wall-attached enzyme. In addition to hydrolyzing lacto-N-triose into N-acetylglucosamine and lactose, the purified BnaG enzyme also catalyzed the hydrolysis of 3=-N-acetylglucosaminyl-mannose and 3=-N-acetylgalactosaminyl-galactose. L. casei can be cultured in the presence of 3=-N-acetylglucosaminyl-mannose as a carbon source, but, curiously, the bnaG mutant strain was not impaired in its utilization. These results indicate that the assimilation of 3=-N-acetylglucosaminyl-mannose is independent of BnaG. Enzyme activity and growth analysis with a manA-knockout mutant showed that ManA is involved in the utilization of the mannose moiety of 3=-N-acetylglucosaminyl-mannose. Here we describe the physiological role of a ␤-N-acetylglucosaminidase in lactobacilli, and it supports the metabolic adaptation of L. casei to the N-acetylglucosaminide-rich gut niche. G lycans in human milk are present as free oligosaccharides or conjugated to proteins and lipids (1, 2), and they have been proposed to directly influence the composition of the infant gut microbiota (3, 4). Furthermore, the free human milk oligosaccharides (HMOs), the third largest solid component in milk, act as prebiotics to promote colonization by beneficial bacterial species (5, 6). HMOs contain a lactose moiety (Gal␤1-4Glc) at their reducing end, which is elongated by ␤1,3-linked lacto-N-biose units (Gal␤1-3GlcNAc) to give the type 1 HMOs, including lacto-Ntetraose (Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc), or by ␤1,3/6-linked N-acetyllactosamine units (Gal␤1-4GlcNAc) to give the type 2 HMOs, such as lacto-N-neotetraose (Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc). Further elongation of these core structures is made by the addition of fucose and sialic acid residues (1). Both types of HMOs contain a lacto-N-triose unit (GlcNAc␤1-3Gal␤1-4Glc), highlighting the importance of this trisaccharide in the total pool of HMOs. In addition, lacto-N-triose and other N-acetylhexosaminyl-oligosaccharides also form part of the structure of glycans conjugated to proteins and lipids present in human milk. The carbohydrate moieties of these molecules also have a prebiotic role, and besides the monosaccharides described abov...

supporting

confidence: 50%

The Extracellular Wall-Bound β- N -Acetylglucosaminidase from Lactobacillus casei Is Involved in the Metabolism of the Human Milk Oligosaccharide Lacto- N -Triose

Bidart

Rodrı́guez-Dı́az

Yebra

2016

“…The usefulness of such a polyphasic approach has previously been demonstrated for many microbial groups such as lactobacilli (21,33,34), as well as for various bifidobacterial species, such as Bifidobacterium breve (9), Bifidobacterium longum subsp. infantis (35), Bifidobacterium dentium (14), and Bifidobacterium bifidum (13). The current study represents an example of the application of this polyphasic approach allowing insights into the genetic diversity of the B. adolescentis phylogenetic group, which includes key components of the gut microbiota of adults (B. adolescentis, B. pseudocatenulatum, and B. catenulatum) (4).…”

Section: Resultsmentioning

confidence: 99%

Exploration of the Genomic Diversity and Core Genome of the Bifidobacterium adolescentis Phylogenetic Group by Means of a Polyphasic Approach

Duranti

Turroni

Milani

et al. 2013

In the current work, we describe genome diversity and core genome sequences among representatives of three bifidobacterial species, i.e., Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Bifidobacterium pseudocatenulatum, by employing a polyphasic approach involving analysis of 16S rRNA gene and 16S-23S internal transcribed spacer (ITS) sequences, pulsed-field gel electrophoresis (PFGE), and comparative genomic hybridization (CGH) assays.

“…It can be speculated that the metabolic activity of the microbiota might also be different in the absence or presence of B. longum subsp. infantis, because this species is particularly well adapted among the bifidobacteria to the utilization of HMO (33).…”

Section: Discussionmentioning

confidence: 99%

“…B. longum subsp. infantis is probably the most intensively studied of the bifidobacteria with respect to biochemistry (33), but it may be an endangered species in western countries. Further international prevalence studies using a single validated identification method are desirable.…”

Section: Discussionmentioning

confidence: 99%

Comparison of the Compositions of the Stool Microbiotas of Infants Fed Goat Milk Formula, Cow Milk-Based Formula, or Breast Milk

Tannock

Lawley

Munro

et al. 2013

181

174

The aim of the study was to compare the compositions of the fecal microbiotas of infants fed goat milk formula to those of infants fed cow milk formula or breast milk as the gold standard. Pyrosequencing of 16S rRNA gene sequences was used in the analysis of the microbiotas in stool samples collected from 90 Australian babies (30 in each group) at 2 months of age. Beta-diversity analysis of total microbiota sequences and Lachnospiraceae sequences revealed that they were more similar in breast milk/goat milk comparisons than in breast milk/cow milk comparisons. The Lachnospiraceae were mostly restricted to a single species (Ruminococcus gnavus) in breast milk-fed and goat milk-fed babies compared to a more diverse collection in cow milkfed babies. Bifidobacteriaceae were abundant in the microbiotas of infants in all three groups. Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium bifidum were the most commonly detected bifidobacterial species. A semiquantitative PCR method was devised to differentiate between B. longum subsp. longum and B. longum subsp. infantis and was used to test stool samples. B. longum subsp. infantis was seldom present in stools, even of breast milk-fed babies. The presence of B. bifidum in the stools of breast milk-fed infants at abundances greater than 10% of the total microbiota was associated with the highest total abundances of Bifidobacteriaceae. When Bifidobacteriaceae abundance was low, Lachnospiraceae abundances were greater. New information about the composition of the fecal microbiota when goat milk formula is used in infant nutrition was thus obtained. Natural microbial communities such as those that inhabit the human bowel carry out diverse and complex biochemical processes (1, 2). Investigations of factors involved in community structure and function require an understanding of the trophic requirements of the microbial members. Optimally, this requires laboratory experiments with cultured bacteria. However, the first step in ecological research is to determine the phylogenetic composition of the microbial community of interest.Most infant formulas are manufactured using cow milk as a base. Goat milk provides an alternative basis for the production of infant formula. Like cow milk, goat milk needs to be fortified to provide optimal nutrition for infants (3). The amount of lactose in cow and goat milk is about the same, but there are other compositional differences (4). Alpha-s1 casein is present in ruminant milk but not in breast milk. Compared to cow milk, goat milk contains much lower concentrations of alpha-s1 casein and higher concentrations of nucleotides and polyamines as well as some of the essential amino acids. Breast milk differs from ruminant milks in that sialylated and fucosylated oligosaccharides (human milk oligosaccharides [HMO]) are the third largest component (5). The HMO are utilized for growth by bifidobacteria, and their presence in breast milk is the likely explanation as to why there is generally a greater abundance of these bacteria in th...