Development of a Double-Crossover Markerless Gene Deletion System in Bifidobacterium longum: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Functional analysis of<jats:named-content content-type="genus-species">Bifidobacterium</jats:named-content>genes is essential for understanding host-<jats:named-content content-type="genus-species">Bifidobacterium</jats:named-content>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<jats:named-content content-type="genus-species">Bifidobacterium longum</jats:named-content>. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-<jats:italic>ΔrepA</jats:italic>, which lacks the plasmid replication gene<jats:italic>repA</jats:italic>, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harbors<jats:italic>repA</jats:italic>, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected in<jats:named-content content-type="genus-species">B. longum</jats:named-content>105-A using the chromosomal full-length β-galactosidase gene as a target. Markerless gene deletion was tested using the<jats:italic>aga</jats:italic>gene, which encodes α-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<jats:italic>aga</jats:italic>. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based<jats:italic>aga</jats:italic>-complemented strain. These functional analyses revealed that<jats:italic>aga</jats:italic>is the only gene encoding a functional α-galactosidase enzyme in<jats:named-content content-type="genus-species">B. longum</jats:named-content>105-A.</jats:p>

Metabolism of Four α-Glycosidic Linkage-Containing Oligosaccharides by Bifidobacterium breve UCC2003

“…These results show not only that the rafA gene is required for growth on raffinose but also that a mutation in rafB causes this growth-deficient phenotype, implying that the rafBCD genes encode an ABC-type transport system responsible for the internalization of ␣-galactose-containing oligosaccharides. Our results corroborate the findings of a recent study in which a deletion in a homolog of the rafA gene of Bifidobacterium longum 105-A (designated agl) was shown to cause a loss of ␣-galactosidase activity and a growth deficiency on raffinose or melibiose (28).…”

“…To metabolize such ␣-galacto-oligosaccharides, bifidobacteria require ␣-galactosidase enzyme activity, which has been identified and characterized in five bifidobacterial species or strains: Bifidobacterium bifidum JCM 1254 (22), Bifidobacterium adolescentis (23,24), Bifidobacterium bifidum NCIMB 41171 (25), Bifidobacterium breve 203 (26), and Bifidobacterium longum subsp. longum (27,28).…”

A Single Mutation in the Gene Responsible for the Mucoid Phenotype of Bifidobacterium animalis subsp. lactis Confers Surface and Functional Characteristics

“…S1 in the supplemental material). Although a few works have reported gene deletion systems in Bifidobacterium longum (38,39), to our knowledge, this work is the first report in scientific literature of a gene deletion system in B. animalis subsp. lactis, and it paves the way for new opportunities to study the functional characteristics of this commercially relevant bacteria.…”

Developing an efficient and reproducible conjugation-based gene transfer system for bifidobacteria

“…When analysing the sequence, it was evident that aside from the three ORFs previously annotated (mob, rep and orfIV) a new ORF was located overlapping by one nucleotide at the 39 end of the rep gene. This newly identified ORF consisted of 324 base pairs and a BLASTP analysis with a translated sequence revealed homology with a gene predicted to be repB that has been identified in pBS423 from B. longum (Hirayama et al, 2012), pFI2576 from B. longum (Moon et al, 2009), pMB1 from B. longum (Rossi et al, 1996), pBC1 from Bifidobacterium catenulatum (Á lvarez-Martín et al, 2007), pNV18 from Nocardia Conjugation-based gene transfer into bifidobacteria (Chiba et al, 2007), pAL5000 from Mycobacterium fortuitum (Rauzier et al, 1988) and pRGO1 from Propionibacterium acidipropionici (Kiatpapan et al, 2000). In all cases a similar gene organization is shared where this gene is immediately downstream and overlapping a repA gene, as now depicted for pDOJHR-W2 in Fig.…”