Transcription Factor NsrR from Bacillus subtilis Senses Nitric Oxide with a 4Fe-4S Cluster (†).

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 fact that there are only three cysteine residues in NsrR, suggest that the 4Fe-4S cluster contains a noncysteinyl labile ligand to one of the iron atoms, leading to high reactivity. Size exclusion chromatography and cross-linking 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 stepping stone to investigate the mechanism of NO sensing in NsrR.

Purification and characterization of extracellular Pseudomonas aeruginosa urate oxidase enzyme.

Urate oxidase (uricase) was isolated and purified from Pseudomonas aeruginosa to apparent homogeneity using ammonium sulphate precipitation followed by ion exchange and gel filtration chromatography. The specific activity of the purified uricase enzyme was found to be 636.36 with the use of uric acid as a substrate. The purified uricase enzyme is a monomeric protein with molecular weight of 64 kilodaltons. The optimal pH and temperature of the purified enzyme is 9.0 and 30 degrees C, respectively. The effect of some metal ions was studied. Sulphate forms of Fe+2, Zn+2 and Co+2 inhibit the uricolytic activity whereas; NaCl and CaCl2 enhance the enzyme activity. Moreover, the purified enzyme is inhibited by EDTA and KCN.

Identification, cloning and expression of Pseudomonas aeruginosa Ps-x putative urate oxidase gene in Escherichia coli.

In a previous study we reported for the first time the isolation and characterization ofurate oxidase enzyme from Pseudomonas aeruginosa. In this work we isolated and cloned a 1.350 kilobase DNA fragment that encode a putative urate oxidase gene from the genomic library of P. aeruginosa Ps-x. The nucleotide sequence of the cloned DNA insert revealed an open reading frame that encodes a protein of a molecular weight of 54.0 kDa. The cloned DNA fragment showed an uricolytic activity when expressed in E. coli DH5alpha. Surprisingly, the nucleotide sequence of the cloned gene showed more than 99% identity to the gene encoding hypothetical protein of P. aeruginosa PAO1. Moreover, the sequence of the cloned gene was closely similar to the corresponding uricase gene of Cellulomonas flavigena (44% similarity), but showed lower similarity values to that of Bacillus sp. BT-90 (24% similarity), Candida utilis (24% similarity). Interestingly, the isolated uricase gene showed closer similarity to uricase from yeast-like symbiotic fungi Beauveria bassiana (35%), Tolypocladium inflatum (29%), Paecilomyces tenuipes (27%) and Cerataphis fransseni (24%).