In Bacillus subtilis, NsrR is required for the upregulation of ResDE-dependent genes in the presence of nitric oxide (NO). NsrR was shown to bind to the promoters of these genes and inhibit their transcription in vitro. NO relieves this inhibition by an unknown mechanism. Here we use spectroscopic techniques (UV-vis, resonance Raman, and EPR) to show that anaerobically isolated NsrR from B. subtilis contains a [4Fe-4S] 2+ cluster which reacts with NO to form dinitrosyl iron complexes. This method of NO sensing is analogous to that of the FNR protein of Escherichia coli. The Fe-S cluster of NsrR is also reactive toward other exogenous ligands such as cyanide, dithiothreitol, and O 2 . These results, together with the presence of only three cysteine residues in NsrR, suggest that the 4Fe-4S cluster contains a non-cysteinyl labile ligand to one of the iron atoms, leading to high reactivity. Size exclusion chromatography and crosslinking experiments show that NsrR adopts a dimeric structure in its [4Fe-4S] 2+ holo form as well as in the apo form. These findings provide a first steppingstone to investigate the mechanism of NO sensing in NsrR.The ability to adapt to anaerobic conditions is vital for a great diversity of microorganisms. It is particularly true of pathogenic organisms which may encounter oxygen limitation within their hosts. Such bacteria also commonly face other stresses such as nitric oxide (NO) produced by phagocytic cells as part of the immune response of the host (1). Thus, an understanding of how bacteria sense and respond to these conditions is of considerable importance.An example of the bacterial response to oxygen limitation comes from Bacillus subtilis which, in the absence of oxygen, can grow by nitrate respiration (2). The ResDE two-component regulatory system is required for the induction of genes involved in nitrate respiration (3). These genes include fnr, (the gene encoding anaerobic transcription factor) (4), nasDEF (nitrite reductase genes) (5), and hmp (flavohemoglobin gene) (6). However, anaerobic conditions alone are not sufficient for the full induction of the ResDE-controlled genes, in particular hmp and nasDEF, and the presence of NO is required to attain the full induction of these genes (7). The effect of NO is abrogated in an nsrR mutant strain, indicating a role for NsrR in NOmediated control of the ResDE regulon (8).NsrR belongs to the Rrf2 family of transcription regulators and is widely found in various bacteria (9). Sequence analyses predict that NsrR contains a helix-turn-helix which likely binds to the promoter region of its target genes (9). In vivo studies of nsrR mutants and the effect of * To whom correspondence should be addressed. Tel: 503-748-167, Fax: 503-7481464, Email: ploccoz@ebs.ogi Materials & Methods Purification of N-terminal and C-terminal His 6 -tagged NsrR proteins (NH-NsrR & CH-NsrR)The various strains and plasmids used in this study are listed in Table S1 (see Supporting Information). NH-NsrR expression plasmid, pMMN648, was previously cons...

Arterial cannulation in neonates is usually performed for frequent blood pressure monitoring and blood sampling. The procedure, while easily executed by skilled neonatal staff, can be associated with serious complications such as vasospasm, thrombosis, embolism, hematoma, infection, peripheral nerve damage, ischemia, and tissue necrosis. Several treatment options are available to reverse vascular induced ischemia and tissue damage. Applied interventions depend on the extent of tissue involvement and whether the condition is progressive and deemed life threatening. Standard, noninvasive measures include immediate catheter removal, limb elevation, and warming the contralateral extremity. Topical vasodilators, anticoagulation, thrombolysis, and surgery are considered secondary therapeutic strategies. A comprehensive literature search indicates that topical nitroglycerin has been utilized for the treatment of tissue ischemia in three preterms with umbilical arterial catheters and four with peripheral arterial lines. We report the first successful use of nitroglycerine ointment in a critically ill preterm infant with ischemic hand changes after brachial artery cannulation.

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