Functional analysis of NsrR, a nitric oxide-sensing Rrf2 repressor in Neisseria gonorrhoeae.
Nitric oxide (NO) has been shown to be an important component of the human immune response, and as such, it is important to understand how pathogenic organisms respond to its presence. In Neisseria gonorrhoeae, recent work has revealed that NsrR, an Rrf2-type transcriptional repressor, can sense NO and control the expression of genes responsible for NO metabolism. A highly pure extract of epitope-tagged NsrR was isolated and mass spectroscopic analysis suggested that the protein contained a [2Fe-2S] cluster. NsrR/DNA interactions were thoroughly analysed in vitro. Using EMSA analysis, NsrR::FLAG was shown to interact with predicted operators in the norB, aniA and nsrR upstream regions with a K(d) of 7, 19 and 35 nM respectively. DNase I footprint analysis was performed on the upstream regions of norB and nsrR, where NsrR was shown to protect the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97 or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding, consistent with their presumed role in co-ordination of a NO-sensitive Fe-S centre required for DNA binding.
Isabella
Vincent M
VM
Department of Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue, Rochester, NY, USA.
Lapek
John D
JD
Kennedy
Edward M
EM
Clark
Virginia L
VL
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009344:Neisseria gonorrhoeae / Q000235:genetics* / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D009569:Nitric Oxide