Binding to DNA protects Neisseria meningitidis fumarate and nitrate reductase regulator (FNR) from oxygen.

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.

The NfeD protein family and its conserved gene neighbours throughout prokaryotes: functional implications for stomatin-like proteins.

NfeD-like proteins are widely distributed throughout prokaryotes and are frequently associated with genes encoding stomatin-like proteins (slipins). Here, we reveal that the NfeD family is ancient and comprises three major groups: NfeD1a, NfeD1b and truncated NfeD1b. Members of each group are associated with one of four conserved gene partners, three of which have eukaryotic homologues that are membrane raft associated, namely stomatin, paraslipin (previously SLP-2) and flotillin. The first NfeD group (NfeD1b), comprises proteins of approximately 460-aa long that have three functional domains: an N-terminal protease, a middle membrane-spanning region and a soluble C-terminal region rich in beta-strands. The nfeD1b gene is adjacent to eoslipin in prokaryotic genomes except in Firmicutes and Deinococci, where yqfA replaces eoslipin. Proteins in the second major group (NfeD1a) are homologous to the C-terminus of NfeD1b which forms a beta-barrel-like domain, and their genes are associated with paraslipin. Using OrthoMCL clustering, we show that nfeD1b genes have become truncated on many independent occasions giving rise to the third major group. These short NfeD homologues frequently remain associated with their ancestral gene neighbour, resembling NfeD1a in structure, yet are much more related to full-length NfeD1b; we term these "truncated NfeD1b". These conserved associations suggest that NfeD proteins are dependent on gene partners for their function and that the site of interaction may lie within the C-terminal portion that is common to all NfeD homologues. Although NfeD homologues are confined to prokaryotes, this conserved association could represent an excellent system to study slipin and flotillin proteins.

Slipins: ancient origin, duplication and diversification of the stomatin protein family.

BACKGROUND: Stomatin is a membrane protein that was first isolated from human red blood cells. Since then, a number of stomatin-like proteins have been identified in all three domains of life. The conservation among these proteins is remarkable, with bacterial and human homologs sharing 50 % identity. Despite being associated with a variety of diseases such as cancer, kidney failure and anaemia, precise functions of these proteins remain unclear. RESULTS: We have constructed a comprehensive phylogeny of all 'stomatin-like' sequences that share a 150 amino acid domain. We show these proteins comprise an ancient family that arose early in prokaryotic evolution, and we propose a new nomenclature that reflects their phylogeny, based on the name "slipin" (stomatin-like protein). Within prokaryotes there are two distinct subfamilies that account for the two different origins of the eight eukaryotic stomatin subfamilies, one of which gave rise to eukaryotic SLP-2, renamed here "paraslipin". This was apparently acquired through the mitochondrial endosymbiosis and is widely distributed amongst the major kingdoms. The other prokaryotic subfamily gave rise to the ancestor of the remaining seven eukaryotic subfamilies. The highly diverged "alloslipin" subfamily is represented only by fungal, viral and ciliate sequences. The remaining six subfamilies, collectively termed "slipins", are confined to metazoa. Protostome stomatin, as well as a newly reported arthropod subfamily slipin-4, are restricted to invertebrate groups, whilst slipin-1 (previously SLP-1) is present in nematodes and higher metazoa. In vertebrates, the stomatin family expanded considerably, with at least two duplication events giving rise to podocin and slipin-3 subfamilies (previously SLP-3), with the retained ancestral sequence giving rise to vertebrate stomatin. CONCLUSION: Stomatin-like proteins have their origin in an ancient duplication event that occurred early on in the evolution of prokaryotes. By constructing a phylogeny of this family, we have identified and named a number of orthologous groups: these can now be used to infer function of stomatin subfamilies in a meaningful way.

Eukaryotic and prokaryotic stomatins: the proteolytic link.

The 32kD membrane protein stomatin was first studied because it is deficient from the red cell membrane in two forms of the class of haemolytic anaemias known as "hereditary stomatocytosis." The hallmark of these conditions is a plasma membrane leak to the monovalent cations Na+ and K+: the protein is missing only in the most severely leaky of these conditions. No mutation has ever been found in the stomatin gene in these conditions. Stomatin-like proteins have been identified in all three domains of biology, yet their function remains enigmatic. Although the murine knock-out is without phenotype, we have identified a family showing a splicing defect in the stomatin mRNA, in which affected children showed a catastrophic multisystem disease not inconsistent with the now-known wide tissue distribution of stomatin. We report here a study of strongly homologous stomatin-like genes in prokaryotes, which reveals a close connection with a never-studied gene erroneously known as "nfed." This gene codes for a hydrophobic protein with a probable serine protease motif. It is possible that these stomatin-like genes and those which are known as"nfed" form an operon, suggesting that the two protein products are aimed at a common function. The corollary is that stomatin could be a partner protein for a membrane-bound proteolytic process, in both prokaryotes and in eukaryotes generally: this idea is consistent with experimental evidence.