Oxygen and nitrate-dependent regulation of dmsABC operon expression in Escherichia coli: sites for Fnr and NarL protein interactions

Abstract

Background: Escherichia coli, can respire anaerobically using dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMAO) as the terminal electron acceptor for anaerobic energy generation. Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present.

Results: The regions of dmsA regulatory DNA required for Fnr and NarL interactions in response to anaerobiosis and nitrate, respectively, were examined. Mutations within the Fnr site that deviated from the wild type sequence, TTGATaccgAACAA, or that removed an entire half-site, either impaired or abolished the anaerobic activation of dmsA-lacZ expression. The region for phosphorylated NarL (NarL-phosphate) binding at the dmsA promoter was identified by DNase I and hydroxyl radical footprinting methods. A large 97 bp region that overlaps the Fnr and RNA polymerase recognition sites was protected by NarL-phosphate but not by the non-phosphorylated form of NarL. Hydroxyl radical footprinting analysis confirmed the NarL-phosphate DNase I protections of both dmsA strands and revealed 8-9 protected sites of 3-5 bp occurring at ten bp intervals that are offset by 3 bp in the 3' direction.

Conclusion: These findings suggest that multiple molecules of phosphorylated NarL bind along one face of the DNA and may interfere with Fnr and/or RNA polymerase interactions at the dmsA regulatory region. The interplay of these transcription factors insures a hierarchical expression of the dmsABC genes when respiration of the preferred electron acceptors, oxygen and nitrate, is not possible.

Figure 1

Nucleotide sequence at the dmsA P1 promoter region and the effects of sequence alterations in the Fnr binding site on dmsA-lacZ expression. The DNA sequence is shown in the middle portion of the figure and is numbered relative to the 5' terminus of the dmsA mRNA (not to scale). The transcriptional start site is located at the guanine residue positioned at 223 bp upstream of the dmsA translational start site and is indicated by the arrow at position +1. The consensus sequence of the RNA polymerase recognition sequences in the -35 and -10 regions are shown below the DNA sequence. The boxed sequences from position -35 to -48 indicate a 14 bp region of dyad symmetry similar to the Fnr consensus recognition sequence (TTGATnnnnATCAA). The location of Fnr-box mutations within the dmsA regulatory region and the corresponding phages carrying the dmsA-lacZ fusions are indicated in the lower portion of the figure. The effect of cis mutations on Fnr-dependent activation of dmsA-lacZ expression is shown in the lower right portion of the figure. β-galactosidase activity was measured from the cells containing the indicated fusion inserted in single copy at the att site. The strains were grown in a buffered LB medium either aerobically or anaerobically. The asterisk represents the fold difference between the fnr^- and fnr⁺ strains.

Figure 2

DNase I and hydroxyl radical footprint analyses of the dmsA coding strand by NarL and NarL-phosphate. The closed boxes denote the hydroxyl radical protected regions whereas the open box indicates the DNase I protected region. The asterisks note positions with increased sensitivity to DNase I cleavage when NarL-phosphate is bound to the DNA. Numbering of the DNA is relative to the start of dmsA transcription. The amount of NarL used in each lane is indicated above each lane. Lane G contains the Maxam-Gilbert sequencing reaction. The NarL protein used in Lanes 2 and 6–9 was phosphorylated with acetyl phosphate prior to incubation with the dmsA fragment.

Figure 3

DNase I and hydroxyl radical footprint analyses of the dmsA non-coding strand by NarL and NarL-phosphate. The closed boxes denote the hydroxyl radical protected regions whereas the open box indicates the DNase I protected region. The asterisks note positions with increased sensitivity to DNase I cleavage when NarL-phosphate is bound to the DNA. Numbering of the DNA is relative to the start of dmsA transcription. The amount of NarL used in each lane is indicated above each lane. Lane G contains the Maxam-Gilbert sequencing reaction. Lanes 1 and 5–8 designate the phosphorylated NarL protein.

Figure 4

Location of the Fnr and NarL binding sites in the dmsA promoter region. The DNA sequence is numbered relative to the start of transcription. The dmsA Fnr recognition sequence is indicated by the open rectangle. The region of DNA protected by NarL-phosphate from DNase I cleavage on each strand is denoted by the brackets whereas the sequences protected from hydroxyl radical cleavage on each strand are represented by the closed boxes. DNase I hypersensitive sites are marked with asterisks. The RNA polymerase recognition sequences in the -35 and -10 regions are in bold italics. The three consensus NarL binding sites are represented by the solid arrows whereas dashed arrows mark NarL consensus sequences with one mismatch and dotted arrows signify two mismatches.

Figure 5

Comparison of the NarL-phosphate protection patterns for the entire dmsA promoter region versus a truncated dmsA promoter fragment. The open box denotes the DNase I protected region for the entire dmsA region (Fragment A, -127 to +62) whereas the closed box indicates the protected region for the truncated dmsA promoter region (Fragment B, -127 to -13). Numbering of the DNA is relative to the start of dmsA transcription. Lane G contains the Maxam-Gilbert sequencing reaction. The noncoding strand of DNA was used in both fragments, and the NarL protein used in Lanes 3–5 and 7–9 was phosphorylated with acetyl phosphate prior to incubation with the dmsA fragment. The amount of NarL used in each lane is indicated above each lane.