Abstract
Here, we report the overexpression, purification, and characterization of the transcriptional activator fumarate and nitrate reductase regulator from the pathogenic bacterium Neisseria meningitidis (NmFNR). Like its homologue from Escherichia coli (EcFNR), NmFNR binds a 4Fe-4S cluster, which breaks down in the presence of oxygen to a 2Fe-2S cluster and subsequently to apo-FNR. The kinetics of NmFNR cluster disassembly in the presence of oxygen are 2-3x slower than those previously reported for wild-type EcFNR, but similar to constitutively active EcFNR* mutants, consistent with earlier work in which we reported that the activity of FNR-dependent promoters in N. meningitidis is only weakly inhibited by the presence of oxygen (Rock, J. D., Thomson, M. J., Read, R. C., and Moir, J. W. (2007) J. Bacteriol. 189, 1138-1144). NmFNR binds to DNA containing a consensus FNR box sequence, and this binding stabilizes the iron-sulfur cluster in the presence of oxygen. Partial degradation of the 4Fe-4S cluster to a 3Fe-4S occurs, and this form remains bound to the DNA. The 3Fe-4S cluster is converted spontaneously back to a 4Fe-4S cluster under subsequent anaerobic reducing conditions in the presence of ferrous iron. The finding that binding to DNA stabilizes FNR in the presence of oxygen such that it has a half-life of approximately 30 min on the DNA has implications for our appreciation of how oxygen switches off FNR activatable genes in vivo.
Highlights
Our results indicate that NmFNR—FNR from N. meningitidis (NmFNR) is less sensitive to oxygen than E. coli fumarate and nitrate reduction regulator (FNR) (EcFNR)
A spectral scan of purified NmFNR (Fig. 1 and supplemental Fig. 2) displays features typical of an iron-sulfur cluster, with a prominent feature ϳ405 nm and a shoulder ϳ310 nm, very similar to the spectra of the 4Fe-4S form of EcFNR [11]
The overexpression, purification, and characterization of FNR from N. meningitidis reported here indicates that this regulator has similar overall features to its homologue from E. coli
Summary
A stable base line for the oxygen content of PBS and 2 mM DTT was measured, and, subsequently, solutions containing NmFNR (ϮDNA) were added, and the change in oxygen concentration was followed over time. Spectra were measured under anaerobic conditions for NmFNR in the presence of a 1.2-fold molar excess of duplex DNA containing the FNR consensus binding site.
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