Construction of a β-galactosidase-gene-based fusion is convenient for screening candidate genes involved in regulation of pyrrolnitrin biosynthesis in <i>Pseudomonas chlororaphis</i> G05

Abstract

In our recent work, we found that pyrrolnitrin, and not phenazines, pyrrolnitrin contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy Fusarium head blight (FHB) disease in cereal crops. However, pyrrolnitrin production of Pseudomonas chlororaphis G05 in King's B medium was very low. Although a few regulatory genes mediating the prnABCD (the prn operon, pyrrolnitrin biosynthetic locus) expression have been identified, it is not enough for us to enhance pyrrolnitrin production by systematically constructing a genetically-engineered strain. To obtain new candidate genes involved in regulation of the prn operon expression, we successfully constructed a fusion mutant G05ΔphzΔprn::lacZ, in which most of the coding regions of the prn operon and the phzABCDEFG (the phz operon, phenazine biosynthetic locus) were deleted, and the promoter region plus the first thirty condons of the prnA was in-frame fused with the truncated lacZ gene on its chromosome. The expression of the fused lacZ reporter gene driven by the promoter of the prn operon made it easy for us to detect the level of the prn expression in terms of the color variation of colonies on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). With this fusion mutant as a recipient strain, mini-Tn5-based random insertional mutagenesis was then conducted. By picking up colonies with color change, it is possible for us to screen and identify new candidate genes involved in regulation of the prn expression. Identification of additional regulatory genes in further work could reasonably be expected to increase pyrrolnitrin production in G05 and to improve its biological control function.

Combining transposon mutagenesis and reporter genes to identify novel regulators of the topA promoter in Streptomyces

“…This technique has been successfully applied for the identification of actinorhodin and landomycin E negative regulators [ 13 , 29 ]. Moreover, transposon mutagenesis combined with a reporter system based on an antibiotic resistance cassette was previously applied to search for repressors for daptomycin production in S. roseosporus [ 40 ]. However, this approach, based on antibiotic resistance genes, limited the screening to negative transcriptional regulators.…”

“…To search for gene expression regulators in vivo, the combination of random transposon mutagenesis with reporter genes (predominantly lacZ or antibiotic resistance cassettes) was successfully developed. This strategy has been applied in a number of bacterial species ( Pseudomonas chlororaphis, Proteus mirabilis, Staphylococcus aureus and Vibrio cholerae ) [ 7 , 40 – 42 , 67 ]. An approach based on the combination of random mutant library construction and the lux reporter gene was used for the identification of regulatory proteins of the lecA in Pseudomonas aeruginosa [ 16 ] and the acs gene in Escherichia coli [ 2 ].…”