Development and characterization of a fusion mutant with the truncated lacZ to screen regulatory genes for phenazine biosynthesis in Pseudomonas chlororaphis G05

Abstract

Pseudomonas chlororaphis G05 can produce biologically active agent phenazine-1-carbosylic acid and its derivatives, which contribute to suppression of mycelial growth of some pathogenic fungi and protection of crops in modern agriculture. A seven-gene operon phz (phzABCDEFG) in its genome was identified to be responsible for expression of enzymes involving phenazine biosynthesis. Although a few regulators mediating the phz expression have been identified, it is not enough for us to generate clear regulatory pathways or networks governing phenazine biosynthesis in detail. To identify novel genes involved in regulation of the phz expression, we successfully constructed a translational phzC-lacZ fusion mutant ΔG05Z, in which most of the coding region of the phzCDEFG operon was deleted and the phzC was in frame fused with the truncated lacZ in chromosome. The bacterial colony of this mutant turned blue on LB medium plates supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). This is a convenient and simple method to check the level of the phz expression by measuring β-galactosidase activity because dark blue colony indicates that the phz operon promoter is up-regulated and overexpressed. On the contrary, light blue or white colony means that the phz promoter is down-regulated and expressed weakly. Therefore, employing this fusion mutant as a recipient makes it easy and possible for us to screen new regulatory genes involved in the phz expression by random insertional mutagenesis mediated with transposon mini-Tn5Kan later.