Fig. 5.

ΔtrpE tryptophan auxotrophy is dependent upon phnAB expression. (a) The final growth yields of ΔtrpE and ΔtrpE ΔphnAB on increasing concentrations of tryptophan were determined from OD₆₀₀. ΔtrpE growth became tryptophan-independent upon reaching OD₆₀₀ ~ 0.5. Growth of ΔtrpE ΔphnAB was tryptophan-dependent. (b) Exogenous addition of 20 µM PQS rescued ΔtrpE tryptophan auxotrophy, while ΔtrpE ΔphnAB remained auxotrophic for tryptophan in the presence of PQS. Growth yields were also tryptophan-dependent in ΔtrpE ΔpqsA, which is unable to generate PQS. (c) RT-PCR confirms that phnA expression levels correspond to the loss of ΔtrpE tryptophan auxotrophy. Expression of phnA was analysed at three stages of growth (OD₆₀₀ 0.07, 0.2 and 1.0) by PCR amplification from 25 ng cDNA, and levels were compared with constitutive expression of rplU, amplified from 5 ng cDNA. Genomic DNA (gDNA) and RNA served as positive and negative controls, respectively. Gel images are inverted for clarity. (d) RT-PCR confirms that trpE expression levels decrease late in growth when phnAB expression levels are increasing. Expression of trpE was analysed at OD₆₀₀ 0.15, 0.3 and 1.8 by PCR amplification from 20 ng cDNA, and levels are compared with constitutive expression of rplU, amplified from 5 ng cDNA. gDNA and RNA serve as positive and negative controls, respectively. Gel images are inverted for clarity.