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vfr, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in Pseudomonas chlororaphis G05

  • Wu, Xia (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University) ;
  • Chi, Xiaoyan (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University) ;
  • Wang, Yanhua (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University) ;
  • Zhang, Kailu (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University) ;
  • Kai, Le (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University) ;
  • He, Qiuning (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University) ;
  • Tang, Jinxiu (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University) ;
  • Wang, Kewen (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University) ;
  • Sun, Longshuo (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University) ;
  • Hao, Xiuying (Institute of Applied Microbiology, Xinjiang Academy of Agricultural Sciences) ;
  • Xie, Weihai (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University) ;
  • Ge, Yihe (Department of Applied and Environmental Microbiology, School of Life Sciences, Ludong University)
  • Received : 2019.01.16
  • Accepted : 2019.04.09
  • Published : 2019.08.01

Abstract

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant $G05{\Delta}phz{\Delta}prn::lacZ$ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant $G05{\Delta}vfr$ and $G05{\Delta}phz{\Delta}prn::lacZ{\Delta}vfr$. By quantifying ${\beta}-galactosidase$ activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant $G05{\Delta}vfr$, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

Keywords

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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).

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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).

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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).

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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).

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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).

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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

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Table 2. Oligonucleotide primers used in this study

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