Fur positive regulation of iron superoxide dismutase in Escherichia coli: functional analysis of the sodB promoter

Abstract

In Escherichia coli, the expression of sodB, which encodes iron superoxide dismutase, has been suggested to be activated by Fur, the iron-responsive global regulator initially characterized as a transcriptional repressor. We investigated sodB regulation by functional analysis of the sodB promoter using sodB-lac fusions with various truncated and mutated promoters. Several cis- and trans-acting elements involved in sodB regulation have been identified. The beta-galactosidase activity of sodB-lacZ reporter fusions and RNA analysis showed sevenfold iron-dependent, Fur-mediated activation of expression. A region just downstream from -10, including a large palindromic sequence encompassing the +1 position followed by a 14-bp AT-rich motif, is the site of Fur positive regulation, and the integrity of both sequences was required for full Fur-mediated activation. The life span of sodB mRNA was three times longer in a fur(+) strain, indicating that Fur-mediated activation proceeds, at least in part, at the posttranscriptional level. The H-NS and IHF histone-like factors also affected sodB expression. IHF slightly repressed sodB expression independently of Fur regulation. In contrast, H-NS negative regulation operated only in the absence of Fur. Remarkably, psodB behaved like a "pure extended -10" promoter. Deletion of the -35 region did not affect expression, whereas expression was totally abolished by a TG-to-CC mutation in the extended -10 sequence TGcTACCCT.

FIG. 1

Nucleotide sequence of the sodB promoter region. Position +1 is the start site for transcription. The −35 and −10 regions are boxed. The ATG start site of translation is in bold. Inverted repeats are shown by arrows. The AT-rich region is underlined by a hatched bar. The putative IHF box is indicated by a thick line. Fragments of the sodB promoter indicated below the sequence were fused to lacZ to create the corresponding fusions.

FIG. 2

Effect of a Δfur mutation on expression of Φ(sodB-lacZ) transcriptional and translational fusions. Strains QC2597, QC2598 [Φ(sodB-lacZ)₂], QC2599, and QC2600 [Φ(sodB′-′lacZ)_2-0] were grown in LB medium and assayed for β-galactosidase (β-gal) activity as described in Materials and Methods. β-Galactosidase activity, expressed in units per milliliter, is plotted against units of OD₆₀₀. The values shown are means of three experiments, and individual values did not differ by more than 15% from the means. Symbols: ○, QC2597 (wild type); □, QC2598 (Δfur mutant); ●, QC2599 (wild type); ■, QC2600 (Δfur mutant).

FIG. 3

Analysis by primer extension of the sodB transcript in wild-type and Δfur strains. RNAs isolated from E. coli strains QC2461 and QC2558 were hybridized with the ³²P-labeled 28-mer sodB-BamHI oligonucleotide and used as a template for avian myeloblastosis virus reverse transcriptase. Lanes: 1, primer extension product of RNA from QC2461; 2, primer extension product of RNA from QC2558. GATC is the sequence obtained with the same primer. Position +1 is the start site for transcription.

FIG. 4

Effects of deletions in the sodB promoter on sensitivity to Fur regulation. Conditions were as described in the legend to Fig. 2. Symbols: circles, wild type; squares, Δfur strains. Panels: A, Φ(sodB-lacZ)₃ (QC2700 and QC2704); B, Φ(sodB-lacZ)₉ (QC2682 and QC2683); C, Φ(sodB-lacZ)₁₆ (QC2920 and QC2921). Open symbols correspond to expression from the wild-type promoter [Φ(sodB-lacZ)₂ strains QC2597 and QC2598].

FIG. 5

Effect of Fur on sodB mRNA stability. Strains containing plasmid pHS1-8 were grown at 37°C to an OD₆₀₀ of about 1. Rifampin was added to a final concentration of 150 μg/ml, and samples were taken following incubation at 37°C for Northern blot analysis. Panels: A and B, Northern blots with RNAs from fur⁺ and Δfur strains, respectively; C, quantitative Northern blot analysis of fur⁺ (circles) and Δfur (squares) strains. Half-lives were calculated from the slope of each plot, and half-life errors were estimated from the standard deviation of the slopes. The measured half-lives were 14 ± 2 min in the fur⁺ strain and 4.75 ± 1 min in the Δfur strain.

FIG. 6

sodB expression from the extended −10 promoter. sodB expression was measured, as described in the legend to Fig. 2, from the sodB promoter truncated at position −25 and the sodB promoter mutated from TGN to CCN (as shown in Fig. 1). Symbols: ○, Φ(sodB-lacZ)₂; ●, Φ(sodB-lacZ)₁₁; ▴, Φ(sodB-lacZ)₁₂. β-gal, β-galactosidase.