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. 2013;32(2):59-68.
doi: 10.12938/bmfh.32.59. Epub 2013 Apr 27.

The pyrE Gene as a Bidirectional Selection Marker in Bifidobacterium Longum 105-A

Kouta Sakaguchi  1 , Nobutaka Funaoka  2 , Saori Tani  2 , Aya Hobo  2 , Tohru Mitsunaga  2 , Yasunobu Kano  3 , Tohru Suzuki  1
Affiliations

The pyrE Gene as a Bidirectional Selection Marker in Bifidobacterium Longum 105-A

Kouta Sakaguchi et al. Biosci Microbiota Food Health. 2013.

Abstract

We constructed a deletion mutant of the pyrE gene in Bifidobacterium longum 105-A. A pyrE knockout cassette was cloned into pKKT427, a Bifidobacterium-Escherichia coli shuttle vector, and then introduced into B. longum 105-A by electroporation. The transformants were propagated and spread onto MRS plates containing 5-fluoroorotic acid (5-FOA) and uracil. 5-FOA-resistant mutants were obtained at a frequency of 4.7 × 10(-5) integrations per cell. To perform pyrE gene complementation, the pyrE gene was amplified by PCR and used to construct a complementation plasmid (pKKT427-pyrE (+)). B. longum 105-A ∆pyrE harboring this plasmid could not grow on MRS plates containing 5-FOA, uracil and spectinomycin. We also developed a chemically defined medium (bifidobacterial minimal medium; BMM) containing inorganic salts, glucose, vitamins, isoleucine and tyrosine for positive selection of pyrE transformants. B. longum 105-A ∆pyrE could not grow on BMM agar, but the same strain harboring pKKT427-pyrE (+) could. Thus, pyrE can be used as a counterselection marker in B. longum 105-A and potentially other Bifidobacterium species as well. We demonstrated the effectiveness of this system by constructing a knockout mutant of the xynF gene in B. longum 105-A by using the pyrE gene as a counterselection marker. This pyrE-based selection system will contribute to genetic studies of bifidobacteria.

Keywords: 5-FOA; B. longum 105-A; bifidobacterial minimal medium (BMM); gene inactivation; gene knockout; homologous recombination; pyrimidine metabolism.

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Figures

Fig. 1.
Fig. 1.
Pyrimidine metabolism in B. longum. (A) Orotic acid and 5-FOA are metabolized by the same enzymes. pyrE encodes orotate phosphoribosyl transferase (OPRTase, EC:2.4.2.10), and pyrF encodes orotidine 5′-monophosphate decarboxylase (OMPase, EC:4.1.1.23). 5-FOA, an analog of orotic acid, is sequentially converted to 5-fluoroorotidine monophosphate (5-FOMP) by pyrE and then 5-fluorouridine monophosphate (5-FUMP) by pyrF. 5-FUMP is converted to pyrimidine-containing nucleotides in further steps and is toxic to cells. The following abbreviations of genes were used: carA, carbamoyl phosphate synthase small subunit (EC:6.3.5.5); pyrB, aspartate carbamoyltransferase catalytic subunit (EC:2.1.3.2); pyrC, dihydroorotase (EC:3.5.2.3); pyrD, dihydroorotate dehydrogenase; pyrG, CTP synthetase; pyrH, uridylate kinase (EC:2.7.4.22); pyrI, aspartate carbamoyltransferase regulatory subunit (EC:2.1.3.2); pyrK, dihydroorotate dehydrogenase electron transfer subunit; upp, uracil phosphoribosyl transferase (EC:2.4.2.9). (B) Genomic organization of pyrimidine metabolic genes. pyrB-F and pyrI are clustered in the B. longum NCC2705 genome. pyrG (BL0874) and pyrH (BL1505) are located upstream and downstream of these genes, respectively.
Fig. 2.
Fig. 2.
Construction of B. longum 105-A pyrE knockout mutants. (A) Schematic diagram of the construction of pyrE knockout mutants. The plasmid pKKT427-∆pyrE was introduced into B. longum 105-A. Transformants were cultured on MRS plates containing 5-FOA and uracil at 37°C. The cells in which DCO homologous recombination had occurred between the plasmid and chromosome showed a 5-FOAr and Sps phenotype. The arrows indicate primers, pyrE 1200-Fw, pyrE 1200-Rv and theoretical products of PCR confirmation for the pyrE deletion in Panel B. Ori indicates the origin of replication of the plasmid pTB6. (B) PCR analysis of B. longum 105-A wild-type and B. longum 105-A pyrE knockout mutants. The region flanking pyrD, BL0787, was amplified by PCR (Table 2, No.4). Lane M indicates the DNA marker, λ-EcoT14I, with the following size order from top to bottom: 19,329 bp, 7,743 bp, 6,223 bp, 4,254 bp, 3,472 bp, 2,690 bp, 1,882 bp, 1,489 bp and 925 bp. Lane 1, B. longum 105-A wild-type; lane 2, B. longum 105-A ∆pyrE1; and lane 3, B. longum 105-A ∆pyrE2.
Fig. 3.
Fig. 3.
Growth of transformants on the selection media. (A) Growth of pyrE knockout mutants, B. longum 105-A ∆pyrE1 and B. longum 105-A ∆pyrE2, as compared with B. longum 105-A wild type. Each strain was spread on an MRS plate in the presence or absence of uracil (200 µg/ml) or with uracil and 5-FOA (500 µg/ml). (B) The pyrE deletion was complemented by pKKT427-pyrE+. B. longum 105-A ∆pyrE1 and B. longum 105-A ∆pyrE2 were transformed with pKKT427-pyrE+. Spr colonies were selected and streaked on an MRS plate containing Sp (75 µg/ml) or uracil and 5-FOA and on a BMM plate containing Sp. Sp is the selection marker for pKKT427-pyrE+.
Fig. 4.
Fig. 4.
Purification of growth-promoting compounds from yeast extract. (A) Yeast extract powder was sequentially extracted with ethyl acetate, acetone and methanol. The methanol extract was separated by reverse-phase (RP) column chromatography using an ODS column as described in Materials and Methods. (B) Growth assays were performed in the basal medium supplemented with reverse-phase column chromatography fractions (panel A). The turbidity was measured using a 96-well plate reader at 490 nm. YE, yeast extract.
Fig. 5.
Fig. 5.
Construction of B. longum 105-A xynF knockout mutants. (A) Schematic diagram of the construction of xynF knockout mutants. The plasmid pKEC58-∆xynF was introduced into B. longum 105-A ∆pyrE. Transformants were cultured on MRS plates containing 5-FOA, uracil and Sp at 37°C. The cells in which DCO homologous recombination had occurred between the plasmid and chromosome showed a 5-FOAr and Spr phenotype. The arrows indicate primers, xynF 1200-Fw, xynF 1200-Rv and theoretical products of PCR. Ori indicates the origin of replication of the plasmid pTB6. (B) PCR analysis of B. longum 105-A ∆pyrE and B. longum 105-A xynF knockout mutants (Table 2, No. 10). Lane M: DNA size marker as described in Fig. 2. Lane 1, B. longum 105-A ∆pyrE; lane 2, B. longum 105-A ∆xynF1; and lane 3, B. longum 105-A ∆xynF2.
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