Springer-Verlag 1995. David G. Presutti ⢠Hosni M. Hassan. Binding of integration host factor (IHF) to the E$cherichia coil soda gene and its role in the regulation ...
Mol Gen Genet (1995) 246:228-235
© Springer-Verlag 1995
David G. Presutti • Hosni M. Hassan
Binding of integration host factor (IHF) to the E$cherichia coil soda gene and its role in the regulation of a sodA.lacZ fusion gene
Received: 18 March 1994/Accepted: 2 July 1994
Abstract We used the electrophoretic mobility-shift assay to reveal specific DNA-protein interactions between DNA fragments containing the sodA promoter and proteins present in Escherichia coli cell-free extracts. We have shown specific binding of several E. coli proteins to sodA promoter sequences and identified one of these proteins as the integration host factor (IHF). Mobility-shift experiments with cell-free extracts prepared from himA (IHF-negative) mutant strains lacked a specific DNA-protein band relative to shifts made with wild-type extracts. Several potential IHF-binding sites were identified in the sodA promoter region. Purified IHF was found to bind specifically to DNA fragments containing the sodA promoter. Further evidence presented suggests that IHF binds to multiple sites in the sodA promoter. We have also investigated the transcriptional regulation of sodA by monitoring the expression of a sodA-lacZ fusion gene in an IHF-negative E. coli strain under different growth conditions. Under aerobic conditions, a deletion in himA (IHF subunit ~) resulted in a 60% increase in sodA expression, while having no effect on induction by paraquat. The same deletion in himA did not cause derepression of sodAlacZ during anaerobic growth, but resulted in an increased response (about twofold) to the presence of 2,2'-dipyridyl compared to the isogenic wild-type strain.
Communicated by K. Isono H. M. Hassan ( I ~ ) Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA D. G. Presutti. H. M. Hassan Department of Toxicology, North Carolina State University, Raleigh, NC 27695-7633, USA D. G. Presutti - H. M. Hassan Department of Biochemistry, North Carolina State University, Raleigh, NC 27695-7624, USA
Key words Escherichia coli • Superoxide dismutase (SOD) • Integration host factor (IHF) • Mobility-shift
Introduction Superoxide dismutases (SODs; superoxide: superoxide oxidoreductase; EC 1.15.1.1) are ubiquitous enzymes found in nearly all organisms that possess an aerobic lifestyle. SODs catalyze the dismutation of the toxic, partially reduced oxygen intermediate, superoxide radical (O~-) to the less toxic H202 and 02 (Fridovich 1986; Hassan 1989; McCord and Fridovich 1969). Thus, SODs provide a primary line of defense against oxygen toxicity. The facultative anaerobe Escherichia coli possesses two types of homodimeric SODs; one contains iron at its catalytic center (FeSOD; Yost and Fridovich 1973), the other contains manganese (MnSOD; Keele et al. 1970). The FeSOD gene (sodB) is constitutively expressed and its expression is not responsive to changes in oxygen concentration or redox potential (Hassan 1989; Hassan and Fridovich 1977a, b). On the other hand, the MnSOD is not synthesized in anaerobically grown cells but is induced by oxygen and other environmental stresses (Hassan 1989). The soda gene has been cloned (Touati 1983; 1988) and sequenced (Takeda and Avila 1986). The sodA gene is regulated by a complex interconnected regulatory system. Induction of sodA occurs in response to several environmental stimuli. Among these are: oxygen (Gregory et al. 1973; Hassan and Fridovich 1977a), redox cycling compounds, such as paraquat, which increase the intracellular flux of superoxide radical in the presence of air (Hassan and Fridovich 1977b, 1979), ferrous iron chelators (Moody and Hassan 1984; Pugh and Fridovich 1985), nitrate (Smith and Neidhardt 1983; Miyake 1986; Hassan and Moody 1987) or nitrate plus paraquat during anaerobic respiration (Hassan and Moody 1987; Schiavone and
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Hassan 1988; Privalle et al. 1989), strong oxidants (Schiavone and Hassan 1988; Privalle and Fridovich 1988; 1990), and compounds which deplete intracellular glutathione levels (Gardner and Fridovich 1987). Recently sodA expression has been found to be under the influence of three global regulatory systems. Two of these systems, aerobic respiratory control (Arc; Iuchi and Lin 1988; Iuchi et al. 1989; Tardat and Touati 1991; Beaumont and Hassan 1992) and fumarate-nitrate regulation (Fur; Hassan and Sun 1992; Unden and Trageser 1991) are oxygen sensitive regulons (Iuchi and Lin 1988; Iuchi et al. 1989; Unden and Trageser 1991). A third system, Fur, controls sodA (Niederhoffer et al. 1990; Tardat and Touati 1991; Beaumont and Hassan 1992) in response to iron concentration (Bagg and Neilands 1987; Hantke 1981). In addition to these regulators, which repress sodA expression, soda is also positively controlled by the SoxRS regulon (Greenberg et al. 1990; Tsaneva and Weiss 1990; Wu and Weiss 1991). Another genetic locus has also been recently identified (soxQ) which positively regulates soda and several other genes (Greenberg et al. 1991). Additionally, DNA supercoiling appears to play a role in sodA expression (Schrum and Hassan 1992). Takeda and Avila (1986) observed the presence of several potential integration host factor (IHF) binding sites in the sodA promoter region. We have identified four putative IHF-binding sites in the sodA promoter which contain 1-2 mismatches to the consensus sequence (Gamas and Chandler 1987; Goodrich et al. 1990; Yang and Nash 1989) WATCAANNNNTTR (W = A or T, R = A or G, N = any base). IHF has been found to play a role in a variety of cellular processes, including the regulation of gene expression (for reviews see Drlica and Rouviere-Yaniv, 1987; Friedman 1988. The effect of IHF on gene expression can be
Table 1 Bacterial strains and plasmids
Escherichia coIi:
either positive or negative (Friedman 1988). IHF often plays an auxiliary role in these cases, rarely being the primary regulator. Freundlich et al. (1992) have reported that IHF binds to sodA in an area centered around position - 4 5 in the sodA 5' non-transcribed region. In order to understand the role of IHF in sodA expression we have studied the binding of purified IHF to sodA promoter DNA, through use of the electrophoretic mobility-shift assay. We have also investigated the effect of IHF on expression of the sodA gene by monitoring sodA-lacZ expression in a AhimA strain. A preliminary report of the data has been presented elsewhere (Presutti 1992).
Materials and methods Materials Ampicillin, kanamycin, tetracycline, chtoramphenicol, RNase A, 4 [2-hydroxyethyl]-1-piperazine-ethanesulfonic acid (HEPES), methyl viologen, (paraquat, pQ2+), 2,2'-dipyridyl, and o-nitrophenyl fl-D-galactoside were purchased from Sigma. Restriction enzymes and Klenow fragment of DNA polymerase were purchased from Promega. [c~-3ep]dCTP was from DuPont/NEN. Purified IHF protein was a gift from H. Nash (National Institutes of Mental Health) and a partially purified protein isolated during the current study was also used. Other reagents used were of molecular biology grade or of the highest available purity. MacTargsearch program was generously provided by W. R. McClure (Goodrich et al. 1990).
Bacterial strains and plasmids The E. coli K12 strains and plasmids used in this study are listed in Table 1. The chromosomal (sodA-lacZ)49 fusion (Touati 1988) is a protein fusion which faithfully mimics transcriptional induction of
Relevant genotype
Source
GC4468 QC772 QC774
F - , AlacU169, rpsL F - , AlacU169, rpsL, ~(sodA-lacZ)49 same as QC772, but ~(sodB-kan) l-A2 (sodA, sodB double mutant)
D. Touati D. Touati D. Touati
MH9100 MH4295 MH9110 NC297
9alK2, rpsL 9alK2, rpsL, himAA81 9aIK2, rpsL, himA42
M. Howe M. Howe M. Howe M. Beaumont (This laboratory) M. Beaumont (This laboratory) M. Beaumont (This laboratory)
strain or plasmid
NC298 NC299 Plasmids: pSN(-)4
Same as MH9100 but ~(sodA-lacZ)49 cmr MH9100 x P1 (QC772) Same as MH9110 but 4)(sodA-lacZ)49cmr MH9110 x P1 (QC772) Same as MH4295 but ~(sodA-IacZ)49 cmr MH4295 x P1 (QC772) Vector pBluescript SK (-), plus 1.05 kb sodA gene (1.05 Aval fragment, containing sodA, sequenced by Takeda and Avila 1986)
S. Naik (This laboratory)
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sodA. A chloramphenicol-resistance (cm r) marker is linked to (sodA-lacZ)49. The sodA-lacZ fusion was transduced to the different E. coli strains (Table 1) by P1 transduction.
Media and growth conditions Strains used for plasmid isolations were grown in LB medium (Miller 1973). Plasmid purifications were performed by the methods of Birnboim and Doly (1979) or Holmes and Quigley (1981). General molecular biological techniques were as described by Sambrook et al. (1989) or Ausubel et al. (1989). Trypticase-soy yeast extract (TSY) medium [containing per liter: 30 g tryptic soy broth (Difco) and 5 g yeast extract (Difco)] was used for growing cells to be utilized in the preparation of cell-free extracts. All anaerobic cultures were prepared in media which had been equilibrated in an anaerobic environment in a Coy anaerobic chamber (Coy Laboratory Products, Ann Arbor, Mich., USA). Inocula used for anaerobic growth were from cultures subcultured twice inside the anaerobic chamber. Anaerobic and aerobic cultures were grown essentially as described by Moody and Hassan (1984). Cultures used for fl-galactosidase assays were grown in LBG (LB supplemented with 1% glucose). Aerobic cultures treated with pQ2+ were inoculated to an initial optical density at 600 nm (ODroo) of 0.05 and 0.1raM pQ2+ was added after 1 h of growth. Anaerobic cultures treated with 0.5 mM 2,2'-dipyridyl were inoculated to an initial OD6oo of 0.02, and the iron chelator was added after 1 h of growth.
Preparation of cell-free extracts Cells were harvested by centrifugation at 4 ° C for 10 min at 10 000 X g. Cell pellets were washed once with 10 mM HEPES, pH 7.8, and centrifuged. The pellets were then frozen at - 8 0 ° C, thawed, and resuspended in 10 mM HEPES, pH 7.8, at 1/50 of the original volume of culture used. Sonication of the cells was as described by Moody and Hassan (1984). The lysates were cleared by centrifugation at 20 000 X g and frozen in aliquots at - 80 ° C. All cell-free extracts were prepared from anaerobically grown cultures unless otherwise stated.
Assays 3-Galactosidase was assayed by the method of Miller (1973). flGalactosidase activity was monitored during the entire logarithmic growth phase of the cells, and the data were plotted in the form of differential plots (i.e., changes in Units/ml versus changes in ODroo). The slope of the differential plot represents the activity of fl-galactosidase per unit cell density (Units/OD6oo) determined over the entire logarithmic growth phase of the culture. Variations within the same experiment were
