Generalised mutagenesis with transposon Tn5. A laboratory procedure for the identification of genes responsible for a bacterial phenotype and its regulation, illustrated with phenazine production in Pseudomonas chlororaphis

Abstract

ABSTRACT Forward genetics involves the identification of an unknown DNA sequence (genotype) associated with the expression of that sequence (phenotype). This is often done by generating variants (mutants) of that feature. One of the most practical methods is transposon mutagenesis, which facilitates the genotype–phenotype association through a sequence tag. This six-session laboratory practical illustrates the concept of forward genetics in bacteria using the random DNA target selection of the Tn5 mini-transposon to generate a mutant library. The target bacterium is Pseudomonas chlororaphis subsp. aurantiaca strain SMMP3, whose broad-spectrum fungal antagonism is largely due to a diffusible, orange-pigmented antibiotic of the phenazine class. The library is screened on appropriate media to select clones with reduced colony pigmentation. Upon qualitative and quantitative phenotype confirmation, a subset of mutants is subjected to an arbitrary PCR to identify the interrupted genes through an in-silico comparative analysis of the sequencing products in public genomic databases. Students thus identify previously described or novel biosynthetic and regulatory genes involved in phenazine production. Along with the objective of illustrating a forward genetics approach, the exercise emulates a real scientific activity in which supervised students pursue a path to interpret the phenotypic consequences of gene knockouts.