The role of mitochondrial aconitate (ACO2) in human sperm motility.

Deficiencies in tricarboxylic acid (TCA) cycle enzymes have been shown to cause a wide spectrum of human diseases, including malignancies and neurological and cardiac diseases. In mammalian spermatozoa mitochondria, the TCA cycle is known to be a crucial metabolic pathway that contributes to produce ATP. There is little known about the role and mechanism of mitochondrial aconitase (ACO2), which is an important regulatory enzyme of the TCA cycle, in asthenozoospermia. In the current study, immunofluorescence staining localized ACO2 to the human sperm mid-piece. By immunoblotting, we demonstrated that the level of ACO2 protein in asthenozoospermic samples was significantly decreased compared with that in normal fertile men. Importantly, we first observed that co-incubation of isocitrate with low motile sperm suspensions significantly improved sperm motility, which might be due to elevated intracellular ATP. The improvement of the sperm motility by isocitrate may have important clinical implications in the treatment of asthenozoospermia and certainly warrants further investigation.

Aconitase regulation of erythropoiesis correlates with a novel licensing function in erythropoietin-induced ERK signaling.

BACKGROUND: Erythroid development requires the action of erythropoietin (EPO) on committed progenitors to match red cell output to demand. In this process, iron acts as a critical cofactor, with iron deficiency blunting EPO-responsiveness of erythroid progenitors. Aconitase enzymes have recently been identified as possible signal integration elements that couple erythropoiesis with iron availability. In the current study, a regulatory role for aconitase during erythropoiesis was ascertained using a direct inhibitory strategy. METHODOLOGY/PRINCIPAL FINDINGS: In C57BL/6 mice, infusion of an aconitase active-site inhibitor caused a hypoplastic anemia and suppressed responsiveness to hemolytic challenge. In a murine model of polycythemia vera, aconitase inhibition rapidly normalized red cell counts, but did not perturb other lineages. In primary erythroid progenitor cultures, aconitase inhibition impaired proliferation and maturation but had no effect on viability or ATP levels. This inhibition correlated with a blockade in EPO signal transmission specifically via ERK, with preservation of JAK2-STAT5 and Akt activation. Correspondingly, a physical interaction between ERK and mitochondrial aconitase was identified and found to be sensitive to aconitase inhibition. CONCLUSIONS/SIGNIFICANCE: Direct aconitase inhibition interferes with erythropoiesis in vivo and in vitro, confirming a lineage-selective regulatory role involving its enzymatic activity. This inhibition spares metabolic function but impedes EPO-induced ERK signaling and disturbs a newly identified ERK-aconitase physical interaction. We propose a model in which aconitase functions as a licensing factor in ERK-dependent proliferation and differentiation, thereby providing a regulatory input for iron in EPO-dependent erythropoiesis. Directly targeting aconitase may provide an alternative to phlebotomy in the treatment of polycythemia vera.

A mycobacterial enzyme essential for cell division synergizes with resuscitation-promoting factor.

The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein-protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall-targeting beta-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein-protein interaction as one way of regulating its activity.

Extracellular fatty acids facilitate flagella-independent translocation by Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is widespread in natural environments such as soil, sewage and plant rhizospheres. Surfactants frequently function in modulating bacterial surface translocation. In this study, rpfB and rpfF orthologues were identified from S. maltophilia strain WR-C, which was isolated from the clogged zone of a septic system. These genes play a role in the biosynthesis of eight extracellular compounds that facilitated flagella-independent translocation by the wild-type or a flagella-defective mutant. This type of surface translocation has not been reported previously for this organism. These eight compounds include cis-delta 2-11-methyl-dodecenoic acid and seven structural derivatives. Two are saturated fatty acids; the others are unsaturated fatty acids with double bonds at position 2. These fatty acids vary in chain length from 12 to 14 carbons and in the position of the branched methyl group. Our results demonstrated that independently cis-delta 2-11-methyl-dodecenoic acid and 11-methyl-dodecanoic acid promoted flagella-independent translocation by S. maltophilia strain WR-C by acting as wetting agents.

Aconitase plays a role in regulating resistance to oxidative stress and cell death in Arabidopsis and Nicotiana benthamiana.

In animals, aconitase is a bifunctional protein. When an iron-sulfur cluster is present in its catalytic center, aconitase displays enzymatic activity; when this cluster is lost, it switches to an RNA-binding protein that regulates the translatability or stability of certain transcripts. To investigate the role of aconitase in plants, we assessed its ability to bind mRNA. Recombinant aconitase failed to bind an iron responsive element (IRE) from the human ferritin gene. However, it bound the 5' UTR of the Arabidopsis chloroplastic CuZn superoxide dismutase 2 (CSD2) mRNA, and this binding was specific. Arabidopsis aconitase knockout (KO) plants were found to have significantly less chlorosis after treatment with the superoxide-generating compound, paraquat. This phenotype correlated with delayed induction of the antioxidant gene GST1, suggesting that these KO lines are more tolerant to oxidative stress. Increased levels of CSD2 mRNAs were observed in the KO lines, although the level of CSD2 protein was not affected. Virus-induced gene silencing (VIGS) of aconitase in Nicotiana benthamiana caused a 90% reduction in aconitase activity, stunting, spontaneous necrotic lesions, and increased resistance to paraquat. The silenced plants also had less cell death after transient co-expression of the AvrPto and Pto proteins or the pro-apoptotic protein Bax. Following inoculation with Pseudomonas syringae pv. tabaci carrying avrPto, aconitase-silenced N. benthamiana plants expressing the Pto transgene displayed a delayed hypersensitive response (HR) and supported higher levels of bacterial growth. Disease-associated cell death in N. benthamiana inoculated with P. s. pv. tabaci was also reduced. Taken together, these results suggest that aconitase plays a role in mediating oxidative stress and regulating cell death.

Bacillus subtilis aconitase is required for efficient late-sporulation gene expression.

Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of sigmaK-dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3' untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.

Characterization of the mitochondrial aconitase promoter and the identification of transcription factor binding.

BACKGROUND: Mitochondrial (m) aconitase plays an important role in the unique pathway of citrate accumulation in prostate epithelial cells through its limited activity. In the current study, we characterized the human m-aconitase gene promoter. METHODS: A 1,411-bp 5'-flanking fragment of the human m-aconitase gene was cloned, followed by 5' serial deletion analysis of promoter activity. Transcriptional start sties and transcription factors bound to the promoter were identified by 5' RACE, DNA pull-down assay and transcription factor array analysis. RESULTS: Two transcriptional start sites were identified. The promoter fragment pulled down 15 transcription factors, some without consensus sequences in the promoter. Deletion of one Sp1 site eliminated all promoter activity. CONCLUSIONS: The m-aconitase promoter is contained in a 153-bp 5' fragment lacking a TATA or CAAT sequence. Sp1 binding to a specific Sp1 site is required for promoter activity while other transcription factors are recruited through protein-protein interactions.

Biofilm formation and dispersal in Xanthomonas campestris.

Xanthomonas campestris pathovar campestris is the causal agent of black rot disease of cruciferous plants. A cell-cell signalling system encoded by genes within the rpf cluster is required for the full virulence of this plant pathogen. This system has recently been implicated in regulation of the formation and dispersal of Xanthomonas biofilms.

Mitochondria: are they the seat of senescence?

The frequently quoted figure for the fractional univalent reduction of oxygen to superoxide in mitochondria is certainly too high by at least one order of magnitude. This is so because the higher number (2%) was derived from mitochondria whose cytochrome c oxidase was blocked with cyanide. Nevertheless, even the more correct number (0.1%) means that the production of O(2)(-) and H(2)O(2) in mitochondria is large and apt to result in damage to macromolecules in spite of such defensive enzymes as superoxide dismutases and glutathione peroxidase. The data available for nematodes and flies provide a compelling case for the view that the accumulation of oxidative damage to specific mitochondrial proteins leads to the progressive dysfunction that we see as senescence. The data available from work with mammals are much weaker and do not yet allow a strong position to be taken.

Staphylococcus aureus aconitase inactivation unexpectedly inhibits post-exponential-phase growth and enhances stationary-phase survival.

Staphylococcus aureus preferentially catabolizes glucose, generating pyruvate, which is subsequently oxidized to acetate under aerobic growth conditions. Catabolite repression of the tricarboxylic acid (TCA) cycle results in the accumulation of acetate. TCA cycle derepression coincides with exit from the exponential growth phase, the onset of acetate catabolism, and the maximal expression of secreted virulence factors. These data suggest that carbon and energy for post-exponential-phase growth and virulence factor production are derived from the catabolism of acetate mediated by the TCA cycle. To test this hypothesis, the aconitase gene was genetically inactivated in a human isolate of S. aureus, and the effects on physiology, morphology, virulence factor production, virulence for mice, and stationary-phase survival were examined. TCA cycle inactivation prevented the post-exponential growth phase catabolism of acetate, resulting in premature entry into the stationary phase. This phenotype was accompanied by a significant reduction in the production of several virulence factors and alteration in host-pathogen interaction. Unexpectedly, aconitase inactivation enhanced stationary-phase survival relative to the wild-type strain. Aconitase is an iron-sulfur cluster-containing enzyme that is highly susceptible to oxidative inactivation. We speculate that reversible loss of the iron-sulfur cluster in wild-type organisms is a survival strategy used to circumvent oxidative stress induced during host-pathogen interactions. Taken together, these data demonstrate the importance of the TCA cycle in the life cycle of this medically important pathogen.

A mitochondrial-like aconitase in the bacterium Bacteroides fragilis: implications for the evolution of the mitochondrial Krebs cycle.

Aconitase and isocitrate dehydrogenase (IDH) enzyme activities were detected in anaerobically prepared cell extracts of the obligate anaerobe Bacteroides fragilis. The aconitase gene was located upstream of the genes encoding the other two components of the oxidative branch of the Krebs cycle, IDH and citrate synthase. Mutational analysis indicates that these genes are cotranscribed. A nonpolar in-frame deletion of the acnA gene that encodes the aconitase prevented growth in glucose minimal medium unless heme or succinate was added to the medium. These results imply that B. fragilis has two pathways for alpha-ketoglutarate biosynthesis-one from isocitrate and the other from succinate. Homology searches indicated that the B. fragilis aconitase is most closely related to aconitases of two other Cytophaga-Flavobacterium-Bacteroides (CFB) group bacteria, Cytophaga hutchinsonii and Fibrobacter succinogenes. Phylogenetic analysis indicates that the CFB group aconitases are most closely related to mitochondrial aconitases. In addition, the IDH of C. hutchinsonii was found to be most closely related to the mitochondrial/cytosolic IDH-2 group of eukaryotic organisms. These data suggest a common origin for these Krebs cycle enzymes in mitochondria and CFB group bacteria.

Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer.

The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha-proteobacterial ancestor of mitochondria should, in phylogenetic trees, branch with homologues that are no longer found in most alpha-proteobacterial genomes, and some should reside on long branches that reveal affinity to eubacterial rather than archaebacterial homologues, but no particular affinity for any specific eubacterial donor.

Direct evidence for mRNA binding and post-transcriptional regulation by Escherichia coli aconitases.

Escherichia coli contains a stationary-phase aconitase (AcnA) that is induced by iron and oxidative stress, and a major but less stable aconitase (AcnB) synthesized during exponential growth. These enzymes were shown to resemble the bifunctional iron-regulatory proteins (IRP1)/cytoplasmic aconitases of vertebrates in having alternative mRNA-binding and catalytic activities. Affinity chromatography and gel retardation analysis showed that the AcnA and AcnB apo-proteins each interact with the 3' untranslated regions (3'UTRs) of acnA and acnB mRNA at physiologically significant protein concentrations. AcnA and AcnB synthesis was enhanced in vitro by the apoaconitases and this enhancement was abolished by 3'UTR deletion from the DNA templates, presumably by loss of acn-mRNA stabilization by bound apoaconitase. In vivo studies showed that although total aconitase activity is lowered during oxidative stress, synthesis of the AcnA and AcnB proteins and the stabilities of acnA and acnB mRNAs both increase, suggesting that inactive aconitase mediates a post-transcriptional positive autoregulatory switch. Evidence for an iron-sulphur-cluster-dependent switch was inferred from the more than threefold higher mRNA-binding affinities of the apo-aconitases relative to the holo-enzymes. Thus by modulating translation via site-specific interactions between apo-enzyme and relevant transcripts, the aconitases provide a new and rapidly reacting component of the bacterial oxidative stress response.

Iron regulatory factor--the conductor of cellular iron regulation.

All cells have to adjust uptake, utilization and storage of iron according to the availability and their requirement for this essential metal. Progress in recent years has led to the elucidation of the molecular control mechanisms that co-ordinate the uptake, utilization and storage of iron in mammalian cells and has highlighted the role of a newly-identified regulatory protein, the iron regulatory factor (IRF). IRF is a cytoplasmic protein that senses the intracellular iron level and responds by adjusting its function. When the iron level is low, it binds to so-called 'iron responsive elements' (IREs) contained in the mRNAs encoding proteins involved in iron metabolism and erythroid haem synthesis. When levels of cellular iron rise, IRF converts into the enzyme aconitase and looses its ability to bind to IREs. We discuss both functions of this Janus face protein and describe how its function is controlled by the status of an iron sulphur cluster in the IRF protein. We also speculate about how an IRF-mediated regulation may relate to certain medical disorders.