PMC

Open complex formation at the glnK promoter. The presence (+) or absence (−) of NtrC(D55E,S161F) and/or IHF in each reaction mixture is indicated. ATP (final concentration, 4 mM) was present in all reaction mixtures to promote catalysis of open complex formation by NtrC. Where indicated above each lane, an additional nucleotide (final concentration, 0.5 mM) was added to provide the potential initiating nucleotide.

Expression of a nifLA-lacZ translational fusion in E. coli ntrC mutant strain ET8556 complemented with P. putida ntrC. Black bars, empty vector control; gray bars, wild-type P. putida ntrC; white bars, P. putida constitutive ntrC(D55E,S161F) mutant. Values are averages from at least three independent measurements. Error bars indicate standard deviations of the means.

IHF DNase I footprint of the glnK promoter region. The coordinates are relative to the glnK transcriptional start site. IHF concentrations were 0.5, 1, and 2 μM of dimer. The protected regions (black bars) and hypersensitive positions (dots) are marked in the footprint and on the promoter sequence beside it. The σ^N promoter and the putative IHF binding site identified by sequencing are also marked on the sequence, as described in the legend for Fig. .

Primer extension analysis of dppA transcript. A primer extension reaction mixture with total RNA was prepared from cultures of P. putida KT2442 (wild type [wt]) and MPO201 (ΔntrC) grown on minimal medium under nitrogen-sufficient (NS, ammonium plus serine) or nitrogen-limiting (S, serine) conditions. GATC lanes indicate the sequencing ladder (noncoding strand). A 13-nucleotide sequence around the transcriptional start (coding strand) is shown. Arrows indicate the mapped 5′ ends of the transcripts.

NtrC DNase I footprint of the glnK promoter region. (A) Top- and bottom-strand footprint patterns. Predicted NtrC binding sites (open boxes), protected regions (black bars), and hypersensitive positions (dots) are marked. NtrC(D55E,S161F) concentrations were 0, 50, 100, 200, and 500 nM for each strand. The coordinates are relative to the glnK transcriptional start site. (B) Schematic representation of the protected regions on the sequence of the glnK promoter, with the same indications as for the footprint pattern.

Primer extension analysis of the glnK transcript. (A) Primer extension reaction mixture with total RNA prepared from cultures of P. putida KT2442 grown under nitrogen-sufficient (NS, ammonium plus serine) or nitrogen-limiting (S, serine) conditions. GATC lanes show the sequencing ladder (noncoding strand). (B) Sequence of the glnK promoter region, showing the putative NtrC binding sites (open boxes), the σ^N promoter (sequence underlined with black boxes), and the IHF binding site (dotted box). The transcription initiation site (G in bold with an arrow) and the translation initiation site (underlined ATG) are also highlighted for comparison.

In vivo expression from the ureD, dppA, and codB promoters of P. putida. Expression was measured as β-galactosidase activity of lacZ transcriptional fusions under nitrogen excess or nitrogen-limiting conditions. Values are averages from at least three independent assays. Error bars indicate standard deviations of the means. (A) ureD-lacZ fusion expression in KT2442 (wild-type [wt]) and MPO201 (ΔntrC) strains. Black bars, control empty vector (pMPO234); white bars, ureD-lacZ fusion (pMPO346). (B) codB-lacZ fusion expression under the same conditions as for ureD-lacZ fusion. Black bars, control empty vector (pMPO234); white bars, codB-lacZ complete fusion (pMPO342); gray bars, codB-lacZ fusion lacking the putative NtrC binding site (pMPO343). (C) dppA-lacZ fusion activity under the same conditions as for ureD-lacZ fusion. Black bars, control empty vector (pMPO234); white bars, dppA-lacZ complete fusion (pMPO340); gray bars, dppA-lacZ fusion lacking the putative NtrC binding site (pMPO341).

DNase I footprints of dppA, codB, and ureD promoter regions. The strand with the most informative footprint pattern is shown in each case. Predicted NtrC binding sites (open boxes), protected regions (black bars), and hypersensitive positions (dots) are marked. Dotted boxes represent palindromic sequences within the protected regions that may serve as NtrC binding sites, which were not initially identified. Coordinates are relative to the first G of the −24 box of the promoter. (A) DNase I footprint pattern of dppA. NtrC(D55E,S161F) concentrations were 0, 0.2, 0.5, 1, 2, and 4 μM. (B) DNase I footprint pattern of codB. NtrC concentrations were 0, 0.5, 1, 1.5, and 2 μM. (C) DNase I footprint pattern of ureD. NtrC concentrations were 0, 0.2, 0.35, 0.5, and 1 μM. (D) Sequences of the promoter regions covering the putative σ^N promoter (marked as a sequence underlined with black boxes) and the regions bound by NtrC (marked as boxes).