FIG. 5.

Genetic evidence implicating folX in tetrahydromonapterin synthesis in P. aeruginosa and supporting the tetrahydromonapterin requirement of P. aeruginosa PhhA in situ. (A) Fluorometric HPLC analysis of pterins extracted from wild-type (PAO1) and ΔfolX strains grown to an A₆₀₀ of 0.6 in liquid M9 medium supplemented with 0.4% glucose. Note the loss of the monapterin peak in the ΔfolX strain. Abbreviations are the same as those in the legend to Fig. 3A. (B) Quantitation of intra- and extracellular monapterin (per mg cellular protein) in wild-type and ΔfolX strains grown to an A₆₀₀ of 0.6 in the medium described for panel A. Data are means and standard errors from three replicates. (C) Abolition of the tyrosine prototrophy of a ΔtyrA strain by the deletion of folX. Wild-type P. aeruginosa was included as a control. Plates contained M9 medium supplemented with 0.4% glucose, 0.1% arabinose, 50 μg/ml l-tryptophan, 10 μM p-aminobenzoate, 10 μM 4-hydroxybenzoate, and 10 μM 2,3-dihydroxybenzoate, with or without 50 μg/ml l-phenylalanine and 54 μg/ml l-tyrosine. Note that the ΔtyrA strain requires l-phenylalanine as well as l-tryptophan and p-aminobenzoate, because P. aeruginosa TyrA is a bifunctional cyclohexadienyl dehydrogenase/5-enolpyruvylshikimate-3-phosphate synthase whose ablation eliminates the synthesis of chorismate as well as that of tyrosine.