Fig. 3.
(R)-3-HB induction of PA2004-lacZ was RpoN dependent. There is a putative − 24/ − 12 or RpoN promoter located 148 bp upstream of the PA2004 ORF, suggesting that RpoN might be involved in the transcription of the PA2004-bdhA operon in response to (R)-3-HB. Addition of 30 mM (R,S)-3-HB did not induce expression of the PA2004-lacZ reporter in an rpoN mutant (rpoNΩKm) of P. aeruginosa PAO1. Furthermore, substitution of the conserved ‘GG’ nucleotides of the − 24 element with ‘AA’ in the RpoN promoter (–PRpoN) of the PA2004-lacZ reporter made it unresponsive to 30 mM (R,S)-3-HB. Cells were grown in LB supplemented with 5 mM l-Gln to an OD600 of 0.3 and then challenged with 30 mM (R,S)-3-HB. LacZ activity was determined 1, 2, 3 and 4 h post-induction. l-Gln was provided to support the growth of the rpoN mutant (Heurlier et al., 2003). Data points represent mean values ± sd (n = 4). ANOVA was performed using a Dunnett's post-hoc test (α-value of 0.05) to identify significant changes (P < 0.0001).