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Functional analysis of Bifidobacterium genes is essential for understanding bost-Bifidobacterium interactions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover in Bifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-&Dgr;repA, which lacks the plasmid replication gene repA, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harbors repA, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vec-tor from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected in B. longum 105-A using the chromosomal full-length &bgr;-galactosidase gene as a target. Markerless gene deletion was tested using the aga gene, which en-codes &agr;-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion of aga. Carbohydrate assimilation analysis and &agr;-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based aga-comple-mented strain. These functional analyses revealed that aga is the only gene encoding a functional &agr;-galactosidase enzyme in B. longum 105-A. [ABSTRACT FROM AUTHOR]

The infant's gut microbiome is generally rich in the Bifidobacterium genus. The mother's milk contains natural prebiotics, called human milk oligosaccharides (HMOs), as the third most abundant solid component after lactose and lipids, and of the different gut microbes, infant gut-associated bifidobacteria are the most efficient in assimilating HMOs. Indeed, the fecal concentration of HMOs was found to be negatively correlated with the fecal abundance of Bifidobacterium in infants. Given these results, two HMO molecules, 2′-fucosyllactose and lacto-N-neotetraose, have recently been industrialized to fortify formula milk. As of now, however, our knowledge about the HMO consumption pathways in infant gut-associated bifidobacteria is still incomplete. The recent studies indicate that HMO assimilation abilities significantly vary among different Bifidobacterium species and strains. Therefore, to truly maximize the effects of prebiotic and probiotic supplementation in commercialized formula, we need to understand HMO consumption behaviors of bifidobacteria in more detail. In this review, we summarized how different Bifidobacterium species/strains are equipped with varied gene sets required for HMO assimilation. We then examined the correlation between the abundance of the HMO-related genes and bifidobacteria-rich microbiota formation in the infant gut through data mining analysis of a deposited fecal microbiome shotgun sequencing dataset. Finally, we shortly described future perspectives on HMO-related studies. [ABSTRACT FROM AUTHOR]

Indole in the gut is formed from dietary tryptophan by a bacterial tryptophan-indole lyase. Indole not only triggers biofilm formation and antibiotic resistance in gut microbes but also contributes to the progression of kidney dysfunction after absorption by the intestine and sulfation in the liver. As tryptophan is an essential amino acid for humans, these events seem inevitable. Despite this, we show in a proof-of-concept study that exogenous indole can be converted to an immunomodulatory tryptophan metabolite, indole-3-lactic acid (ILA), by a previously unknown microbial metabolic pathway that involves tryptophan synthase β subunit and aromatic lactate dehydrogenase. Selected bifidobacterial strains converted exogenous indole to ILA via tryptophan (Trp), which was demonstrated by incubating the bacterial cells in the presence of (2- 13 C)-labeled indole and l-serine. Disruption of the responsible genes variedly affected the efficiency of indole bioconversion to Trp and ILA, depending on the strains. Database searches against 11,943 bacterial genomes representing 960 human-associated species revealed that the co-occurrence of tryptophan synthase β subunit and aromatic lactate dehydrogenase is a specific feature of human gut-associated Bifidobacterium species, thus unveiling a new facet of bifidobacteria as probiotics. Indole, which has been assumed to be an end-product of tryptophan metabolism, may thus act as a precursor for the synthesis of a host-interacting metabolite with possible beneficial activities in the complex gut microbial ecosystem.

Aim: Bifidobacterium longum subsp. infantis uses a glycoside hydrolase (GH) family 42 β-galactosidase ( Bi Bga42A) for hydrolyzing lacto- N -tetraose (LNT), which is the most abundant core structure of human milk oligosaccharides (HMOs). As such, Bi Bga42A represents one of the pivotal enzymes underpinning the symbiosis between bifidobacteria and breastfed infants. Despite its importance, the structural basis underlying LNT hydrolysis by Bi Bga42A is not understood. Moreover, no substrate-complexed structures are available to date for GH42 family members. Methods: X-ray crystallography was used to determine the structures of Bi Bga42A in the apo- and liganded forms. The roles of the amino acid residues that were presumed to be involved in catalysis and substrate recognition were examined by a mutational study, in which kinetic parameters of each mutant were determined using 4-nitrophenyl-β-D-galactoside, lacto- N -biose I, LNT, and lacto- N -neotetraose (LNnT) as substrates. Conservation of those amino acid residues was examined among structure-determined GH42 β-galactosidases. Results: Crystal structures of the wild-type enzyme complexed with glycerol, the E160A/E318A double mutant complexed with galactose (Gal), and the E318S mutant complexed with LNT were determined at 1.7, 1.9, and 2.2 Å resolutions, respectively. The LNT molecule (excluding the Gal moiety at subsite +2) bound to the E318S mutant is recognized by an extensive hydrogen bond network and several hydrophobic interactions. The non-reducing end Gal moiety of LNT adopts a slightly distorted conformation and does not overlap well with the Gal molecule bound to the E160A/E318A mutant. Twelve of the sixteen amino acid residues responsible for LNT recognition and catalysis in Bi Bga42A are conserved among all homologs including β-1,6-1,3-galactosidase ( Bl Gal42A) from Bifidobacterium animalis subsp. lactis . Conclusion: Bl Gal42A is active on 3-β-galactobiose similarly to Bi Bga42A but is inactive on LNT. Interestingly, we found that the entrance of the catalytic pocket of Bl Gal42A is narrower than that of Bi Bga42A and seems not easily accessible from the solvent side due to the presence of two bulky amino acid side chains. The specificity difference may reflect the structural difference between the two enzymes., Competing Interests: Employment of Sakanaka M at Kyoto University is in part supported by Morinaga Milk Industry Co., Ltd. The other authors declare no conflicts of interest., (© The Author(s) 2023.)