Figure 1

Identification of the pyk mutant and the complementation strain. A A schematic of the pyk gene and its flanking genes. The inside and outside fragments amplified using respective primers are indicated. B PCR amplification confirmed deletion of the pyk gene in the pyk mutant. Lane M: DNA marker DL2000 (Takara Bio, Inc., Shiga, Japan). Lanes 1–6: amplification of the inside fragment. Lanes 1–4: four different clones of the pyk mutant, no fragment was amplified. Lane 5: the S2308 strain (positive control), a 493-bp fragment was amplified; Lane 6: sterile water (negative control). Lanes 7 and 8: amplification of the outside fragment. Lane 7: the S2308 strain (positive control), a 1885-bp fragment was amplified; Lane 8: the pyk mutant, a 935-bp fragment was amplified. C Real-time qRT-PCR confirmed the deletion of the pyk gene at the transcriptional level. The pyk deletion did not affect expression of the flanking genes. The gene transcriptional level of the pyk mutant was compared with that of the S2308 strain. ***p ≤ 0.001, ns: no significant difference. D Western blotting confirmed expression of pyk in the complementation strain Δpyk(Pyk-3 × Flag). Lane M: prestained protein ladder (Thermo Fisher Scientific); Lane 1: the S2308 strain (negative control), no flag expression was detected; Lane 2: the complementation strain Δpyk(Pyk-3 × Flag), about 55 kDa of the flag expression product was shown.