Molecular characterization of LhpR in control of hydroxyproline catabolism and transport in Pseudomonas aeruginosa PAO1

Utilization of hydroxy-L-proline (L-Hyp) in Pseudomonas aeruginosa requires conversion of L-Hyp to D-Hyp followed by the D-Hyp dehydrogenase pathway; however, the molecular mechanism in control of L-Hyp catabolism and transport was not clear. DNA microarray analysis revealed twelve genes in two adjacent loci that were induced by exogenous L-Hyp and D-Hyp. The first locus includes lhpABFE encoding a Hyp epimerase (LhpA) and D-Hyp dehydrogenase (LhpBEF), while the second locus codes for a putative ABC transporter (LhpPMNO), a protein of unknown function (LhpH), Hyp/Pro racemase (LhpK) and two enzymes in L-Hyp catabolism (LhpC and LhpG). Proximal to these two loci, lhpR encodes a transcriptional regulator of the AraC family. The importance of these genes on L-Hyp catabolism was supported by growth phenotype analysis on knockout mutants. Induction of the lhpA and lhpP promoters by exogenous L-Hyp and D-Hyp was demonstrated by the measurement of beta-galactosidase activities from promoter-lacZ fusions in PAO1, and no induction could be detected in the Delta lhpR mutant. Induction of the lhpA promoter by D-Hyp was completely abolished in the lhpA lhpK double mutant devoid of two epimerases, while the induction effect of L-Hyp remained unchanged. The purified His-tagged LhpR binds specifically to the lhp promoter regions, and formation of nucleoprotein complexes is affected by the presence of L-Hyp but not D-Hyp. Putative LhpR binding sites were deduced from serial deletions and comparative genomic sequence analysis. In summary, expression of lhp genes for Hyp catabolism and uptake requires the transcriptional activator LhpR and L-Hyp as the signalling compound.