Transcriptional Organization and Dynamic Expression of the hbpCAD Genes, Which Encode the First Three Enzymes for 2-Hydroxybiphenyl Degradation in Pseudomonas azelaica HBP1

Abstract

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. Their expression is activated by the product of regulatory gene hbpR, which is located directly upstream of the hbpCAD genes. The HbpR protein is a transcription activator and belongs to the so-called XylR/DmpR subclass within the NtrC family of transcriptional activators. Transcriptional fusions between the different hbp intergenic regions and the luxAB genes of Vibrio harveyi 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 hbpA genes are cotranscribed, whereas the hbpD gene is transcribed separately. No transcripts comprising the entire hbpCAD cluster were detected, indicating that transcription from P hbpC is terminated after the hbpA gene. E. coli mutant strains lacking the structural genes for the RNA polymerase 54 subunit or for the integration host factor failed to express bioluminescence from P hbpC -and P hbpD -luxAB fusions when a functional hbpR gene was provided in trans. This pointed to the active role of 54 and integration host factor in transcriptional activation from these promoters. Primer extension analysis revealed that both P hbpC and P hbpD contain the typical motifs at position ؊24 (GG) and ؊12 (GC) found in 54 -dependent promoters. Analysis of changes in the synthesis of the hbp mRNAs, in activities of the 2-HBP pathway enzymes, and in concentrations of 2-HBP intermediates during the first 4 h after induction of continuously grown P. azelaica cells with 2-HBP demonstrated that the specific transcriptional organization of the hbp genes ensured smooth pathway expression.

Identification and Physical Characterization of the HbpR Binding Sites of the hbpC and hbpD Promoters

“…In a previous study, it was shown that HbpR activates transcription from two promoters, P hbpC and P hbpD , localized in the hbpRC and hbpAD intergenic regions, respectively (11). When tested in GMSAs, the CBP-HbpR purified fraction indeed bound both the hbpRC and the hbpAD intergenic regions but not the hbpCA region, which has no demonstrated HbpR-dependent promoter (data not shown).…”

“…To determine the in vivo activity of the CBP-HbpR fusion protein, we used HbpR-mediated activation of the luxAB genes transcriptionally fused to the hbpC promoter as described before (11). The CBP-HbpR fusion of pHB151 was completed with the 2.75-kb BamHI fragment of plasmid pHYBP109 (containing the native hbpRC intergenic region fused to the luxAB genes).…”

“…Two promoters, designated P hbpC and P hbpD (13), control the expression of the hbpCA and hbpD genes, respectively. In the presence of 2-HBP, transcriptional activation from both promoters is mediated by the HbpR regulatory protein (11). On the basis of sequence similarities, HbpR has been identified as a member of the XylR/DmpR subclass of the NtrC family of prokaryotic enhancer binding proteins.…”

“…Second, HbpR is the only member of the XylR/DmpR subclass described so far which is activated by biaromatic compounds, such as 2-HBP and 2,2Ј-dihydroxybiphenyl. Third, HbpR activates transcription from two promoters within a rather small cluster of only three genes (hbpCA and hbpD), an unusual scenario (11). Finally, the proposed binding sites for HbpR have slightly larger spacing between the UASs (13).…”

“…Previous promoter fusion studies and the GMSAs identified coarsely those regions on the DNA with which HbpR interacted. Sequence comparisons had suggested the presence within those regions of pairs of palindromic sequences which were similar to the so-called UASs of transcription activators of the XylR/DmpR type (11). To identify whether HbpR was indeed binding to these putative UASs, we performed DNase I footprinting analyses with CBP-HbpR and with labeled fragments containing UASs C-1 and C-2 (fragment hbpCCC6, within the hbpC promoter) or UASs D-1 and D-2 (fragment hbpD3D4, within the hbpD promoter).…”