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. 2012 Jul;78(14):4984-94.
doi: 10.1128/AEM.00588-12. Epub 2012 May 11.

Development of a double-crossover markerless gene deletion system in Bifidobacterium longum: functional analysis of the α-galactosidase gene for raffinose assimilation

Yosuke Hirayama  1 , Mikiyasu Sakanaka, Hidenori Fukuma, Hiroki Murayama, Yasunobu Kano, Satoru Fukiya, Atsushi Yokota
Affiliations

Development of a double-crossover markerless gene deletion system in Bifidobacterium longum: functional analysis of the α-galactosidase gene for raffinose assimilation

Yosuke Hirayama et al. Appl Environ Microbiol. 2012 Jul.

Abstract

Functional analysis of Bifidobacterium genes is essential for understanding host-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-ΔrepA, 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 vector 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 β-galactosidase gene as a target. Markerless gene deletion was tested using the aga 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 aga. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based aga-complemented strain. These functional analyses revealed that aga is the only gene encoding a functional α-galactosidase enzyme in B. longum 105-A.

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Figures

Fig 1
Fig 1
Outline of markerless gene deletion system in Bifidobacterium longum 105-A. GeneA represents a model target gene for markerless gene deletion. ΔGeneA lacks the internal region of GeneA and is cloned in the conditional replication vector (first plasmid), which lacks the repA gene encoding the plasmid replication initiation protein RepA (indicated as ΔrepA). The region absent from ΔGeneA is identified by a cross. Theoretically, two types of GeneA alleles can be generated by the first crossover, and ΔGeneA deletion mutants and wild-type revertants can arise in the second-crossover recombinants. Here, only the simplified scheme for the generation of ΔGeneA deletion mutants was described to represent the concept. In the first crossover, the first plasmid harboring ΔGeneA was introduced into the host strain B. longum 105-A. Here, the 5′ region of ΔGeneA is used as a homologous region for the first crossover. The first-crossover integrant harbors both the GeneA and ΔGeneA alleles in its chromosome. The region derived from the first plasmid harboring ΔGeneA in the chromosome of the first-crossover integrants is identified by a dashed line. For transformation of the RepA+ plasmid, the introduction of the repA-harboring second plasmid (RepA+ plasmid) into the first-crossover integrant results in the supply of the plasmid replication protein RepA to the cells. During the second crossover and excision, the integrated first plasmid is expected to be excised with GeneA from the chromosome as a result of the second crossover event facilitated by RepA. In exclusion, the excised first plasmid with GeneA has been excluded due to the incompatibility between two plasmids. Objective ΔGeneA deletion mutants can be found among the second-crossover recombinants. AntAr, resistance gene for antibiotic A; AntBr, resistance gene for antibiotic B.
Fig 2
Fig 2
Structures of the plasmids used in markerless gene deletion. Genes are identified by open arrows, except for the repA gene, which is identified by black arrows. Representative restriction sites in the plasmids are shown. DNA regions in plasmids pBS423 (A), pTBR101 (C), and pTBR101-CM (D) are shown in the surrounding circle (dashed lines) of each plasmid and are divided according to the source of the DNA region. Names of derived genetic materials are shown on the surrounding circle. (A) The E. coli-Bifidobacterium shuttle vector pBS423 was constructed by cloning the cryptic plasmid pTB4 from B. longum BK25 into the BamHI site of pTANS19. (B) The conditional replication vector pBS423-ΔrepA was constructed by the self-circularization of the 4.4-kbp HincII fragment of pBS423. Because pBS423-ΔrepA lacks the repA gene, which encodes the replication initiation protein RepA, it cannot replicate in B. longum 105-A. (D) To construct pTBR101-CM, which is incompatible with pBS423-ΔrepA, a chloramphenicol resistance gene (Cmr) from pC194 (cryptic plasmid of Staphylococcus aureus) was cloned into the HindIII site of pTBR101 (C), which consists of pTB4 from B. longum BK25 and pBR322. Spr, spectinomycin resistance gene; Apr, ampicillin resistance gene.
Fig 3
Fig 3
(A) Schematic of the markerless gene deletion procedures conducted in B. longum 105-A. The first crossover event is shown in the upper panel, and the second crossover event is in the lower panel. The first crossover between the target gene (aga) and the conditional replication vector pBS423-ΔrepAΔaga, harboring the Δaga allele, which lacks the internal region of aga (C in the chromosome structure), can take place with homologous region A or B, yielding two types of first-crossover integrants, designated a and b. Primers used to check the genotypes of first-crossover integrants are shown as open arrows, with names above the predicted structure of the target locus of each first-crossover integrant. The expected lengths of the amplicons generated using these primers are indicated by dashed lines below the predicted structures. The second crossover event, facilitated by the supply of RepA by the plasmid pTBR101-CM, can take place with homologous region A or B in the first-crossover integrants, designated a and b. The second crossover event yielded two types of strains, aga deletion mutants and wild-type revertants. Predicted restriction sites for BamHI and the predicted lengths of the restriction fragments are indicated in the structure of the aga locus for each strain. (B to D) Genotype analyses of the pBS423-ΔrepAΔaga transformants to confirm integration of pBS423-ΔrepAΔaga into the aga locus. Amplified fragments produced using the aga11f/aga8r primer pair (B), the aga11f/TB4-1 primer pair (C), and the SpR4/aga8r primer pair (D) were subjected to agarose gel electrophoresis. A 1-kb DNA ladder (New England BioLabs, Inc., Ipswich, MA) was used as a molecular mass marker (lane M). The sizes of representative marker fragments are shown to the left of each panel. Each lane shows amplified DNA generated from a DNA template: lanes 1 to 8, candidate first-crossover integrants; lane P, pBS423-ΔrepAΔaga (positive control for amplification); lane W, B. longum 105-A (wild-type); and lane N, no template DNA (negative control for amplification). The sizes of the amplified fragments are shown to the right of each panel. (E) Southern hybridization analysis of markerless aga deletion. Samples of BamHI-digested chromosomal DNA (4 μg) were electrophoresed in a 0.7% agarose gel. Each lane corresponds to a DNA sample from a Bifidobacterium strain: lane 1, B. longum 105-A; lane 2, 105-A Δaga; lane 3, wild-type revertant (after the second crossover). Electrophoresed total DNA was transferred to a nylon membrane. The 3′ region of aga (region B in panel A) was used as a probe. Hybridization signals were detected after exposure for 30 min. The sizes of the hybridized fragments are indicated to the left of the panel.
Fig 4
Fig 4
Growth profiles of B. longum strains 105-A, 105-A Δaga, 105-A Δaga/pAga135, and 105-A Δaga/pKKT427. Bacteria were cultured anaerobically at 37°C for 24 h in semisynthetic medium containing glucose (filled bars), raffinose (gray bars), or melibiose (open bars) as the sole carbohydrate source. Striped bars indicate cultures without the addition of a carbohydrate source. Data are shown as means ± standard errors of three independent experiments.
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