Random transposon mutagenesis of Lactobacillus plantarum by using the pGh9:IS S1 vector to clone genes involved in the regulation of phenolic acid metabolism.

The lactic acid bacterium Lactobacillus plantarum possesses a promising inducible padA promoter that controls the expression of the padA gene encoding a phenolic acid decarboxylase, and which is transcriptionally regulated by phenolic acids. A strategy was followed in order to clone genes involved in the transcriptional regulation of the padA gene. The pGh9:IS S1 plasmid was used to perfect the mutagenesis of L. plantarum by transposition. This plasmid transposed randomly in the L. plantarum NC8 chromosome, with a frequency of 0.03% at a non-permissive replicating temperature of 42 degrees C. A L. plantarum mutant strain harbouring the transcriptional fusion padA:cat that displayed a chloramphenicol acetyl transferase (CAT) activity induced by p-coumaric acid was constructed. This strain was mutated by transposition with pGh9:IS S1 in order to select mutants with a constitutive CAT activity therefore able to grow on medium containing chloramphenicol without the phenolic acid inducer. Four mutants were identified and three of them harboured an inserted single copy of the pGh9:IS S1 vector. Analysis of IS S1 target sites allowed the identification of two genes: one encoding a putative protein that displays similarity with histidine-binding protein, the other belongs to the ATP-dependent RNA helicase family, protein which are usually involved in gene expression.