Fig. 1. Characterizations of the conjugant G05W02 and its derivatives. (A) Color of colonies shown in the LB medium supplemented with X-gal. Arabic numbers from 1 to 4 stand for the wild-type strain G05, the fusion mutant G05ΔphzΔprn::lacZ, the transposon mutant G05W02, and the transformant G05W02/pME10V, respectively. (B) β-Galactosidase activities were quantified when they were grown in GA medium at 30℃ for 72 h. The values from three independent experiments were presented as the average ± standard deviation. Superscript of asterisk followed the strains indicated no significant differences (P > 0.05).
Fig. 2. Characterizations of the site-directed knockout mutant G05ΔphzΔprn::lacZΔvfr and its derivatives. (A) Color of colonies shown in the LB medium plate supplemented with X-gal. Arabic numbers from 2 to 7 stand for the fusion mutant G05ΔphzΔprn::lacZ, the vfr-knockout mutant G05ΔphzΔprn::lacZΔvfr, the transformant G05ΔphzΔprn::lacZΔvfr/pME10V, and the transformant G05ΔphzΔprn::lacZΔvfr/pME6010, respectively. (B) β-Galactosidase activities were quantified when they grown in GA medium at 30℃ for 72 h. The values from three independent experiments were presented as the average ± standard deviation. Different superscript lowercase letters followed strains indicate significant difference (P < 0.05) according to duncan’s multiple range test, and different superscript uppercase letters indicate extremely significant difference (P < 0.01).
Fig. 3. Regulatory effects of deletion of the vfr on fungal metabolites production in P. chlororaphis G05. All experiments were performed in triplicate, and each value was presented as the means ± standard deviation. (A) Pyrrolnitrin produced by the wild-type strain G05 and its derivatives in GA broth. According to duncan’s multiple range test, different superscript lowercase letters followed the strains indicated significant difference (P < 0.05), and different superscript uppercase letters followed the strains indicated extremely significant difference (P < 0.01). (B) Phenazine-1-carboxylic acid produced by the wild-type strain G05 and its derivatives in GA broth. Asterisks at top of columns mean no significant difference (P > 0.05).
Fig. 4. Translational lacZ fusion vectors pME15Z and pME15N were employed to examine Vfr regulation in P. chlororaphis G05. (A) β-Galactosidase activities produced by pME15N in the wild-type strain G05 and the mutant G05Δvfr were quantified. The transformants G05/pME6015 and G05Δvfr/pME6015 were used as negative controls. (B) β-Galactosidase activities produced by pME15Z in the wildtype strain G05 and the mutant G05Δvfr were quantified. The transformants G05/pME6015 and G05Δvfr/pME6015 were used as negative controls. All experiments were performed in triplicate, and each value was presented as the means ± standard deviation. Asterisks at top of columns mean no significant difference (P > 0.05).
Fig. 5. Translational lacZ fusion vectors pME22Z and pME22N were employed to examine Vfr regulation in P. chlororaphis G05. (A) β-Galactosidase activities produced by pME22Z in the wild-type strain G05 and the mutant G05Δvfr were quantified. The transformant G05/pME6522 and G05Δvfr/pME6522 were used as negative controls. (B) β-Galactosidase activities produced by pME22N in the wildtype strain G05 and the mutant G05Δvfr were quantified. The transformant G05/pME6522 and G05Δvfr/pME6522 were used as negative controls. All experiments were performed in triplicate, and each value was presented as the means ± standard deviation. Asterisks at top of columns mean no significant difference (P > 0.05).
Fig. 6. Gene expression of prnA by RT-qPCR assay in P. chlororaphis G05 and its derivative mutant G05Δvfr. Expression level of the tested prnA in the wild-type strain G05 was considered 1. Relative expressions of prnA in the mutant G05Δvfr compared to the wild-type strain G05 grown in GA medium for 24 h, 48 h, and 72 h were determined by the 2−ΔΔCT method. Asterisks at top of columns mean no significant difference (P > 0.05).
Table 1. Bacterial strains and plasmids used in this study
Table 2. Oligonucleotide primers used in this study
참고문헌
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