Transcriptional organization and dynamic expression of the hbpCAD genes, which encode the first three enzymes for 2-hydroxybiphenyl degradation in Pseudomonas azelaica HBP1

Pseudomonas azelaica HBP1 degrades the toxic substance 2-hydroxybiphenyl (2 -HBP) by means of three enzymes that are encoded by structural genes hbpC, hbpA, and hbpD). These three genes form a small noncontiguous cluster. Thei r expression is activated by the product of regulatory gene hbpR, which is located directly upstream of the hbpCAD genes. The HbpR protein is a transc ription activator and belongs to the so-called XylR/DmpR subclass within th e NtrC family of transcriptional activators. Transcriptional fusions betwee n the different hbp intergenic regions and the luxAB genes of Vibrio harvey i in P. azelaica and in Escherichia coli revealed the existence of two HbpR -regulated promoters; one is located in front of hbpC, and the other one is located in front of hbpD. Northern analysis confirmed that the hbpC and hb pA genes are cotranscribed, whereas the hbpD gene is transcribed separately . No transcripts comprising the entire hbpCAD cluster were detected, indica ting that transcription from P-hbpC is terminated after the hbpA gene. E. c oli mutant strains lacking the structural genes for the RNA polymerase sigm a (54) subunit or for the integration host factor failed to express biolumi nescence from P-hbpC- and P-hbpD-luxAB fusions when a functional hbpR gene was provided in trans. This pointed to the active role of sigma (54) and in tegration host factor in transcriptional activation from these promoters. P rimer extension analysis revealed that both P-hbpC and P-hbpD contain the t ypical motifs at position -24 (GG) and -12 (GC) found in sigma (54)-depende nt promoters. Analysis of changes in the synthesis of the hbp mRNAs, in act ivities of the 2-HBP pathway enzymes, and in concentrations of 2-HBP interm ediates during the first 4 h after induction of continuously grown P. azela ica cells with 2-HBP demonstrated that the specific transcriptional organiz ation of the hbp genes ensured smooth pathway expression.