Subcellular localization of the magnetosome protein MamC in the marine magnetotactic bacterium Magnetococcus marinus strain MC-1 using immunoelectron microscopy

Magnetotactic bacteria are a diverse group of prokaryotes that biomineralize intracellular magnetosomes, composed of magnetic (Fe₃O₄) crystals each enveloped by a lipid bilayer membrane that contains proteins not found in other parts of the cell. Although partial roles of some of these magnetosome proteins have been determined, the roles of most have not been completely elucidated, particularly in how they regulate the biomineralization process. While studies on the localization of these proteins have been focused solely on Magnetospirillum species, the goal of the present study was to determine, for the first time, the localization of the most abundant putative magnetosome membrane protein, MamC, in Magnetococcus marinus strain MC-1. MamC was expressed in Escherichia coli and purified. Monoclonal antibodies were produced against MamC and immunogold labeling TEM was used to localize MamC in thin sections of cells of M. marinus. Results show that MamC is located only in the magnetosome membrane of Mc. marinus. Based on our findings and the abundance of this protein, it seems likely that it is important in magnetosome biomineralization and might be used in controlling the characteristics of synthetic nanomagnetite.     

Evidence for autotrophic CO2 fixation via the reductive tricarboxylic acid cycle by members of the epsilon subdivision of proteobacteria

Based on 16S rRNA gene surveys, bacteria of the epsilon subdivision of proteobacteria have been identified to be important members of microbial communities in a variety of environments, and quite a few have been demonstrated to grow autotrophically. However, no information exists on what pathway of autotrophic carbon fixation these bacteria might use. In this study, Thiomicrospira denitrificans and Candidatus Arcobacter sulfidicus, two chemolithoautotrophic sulfur oxidizers of the epsilon subdivision of proteobacteria, were examined for activities of the key enzymes of the known autotrophic CO(2) fixation pathways. Both organisms contained activities of the key enzymes of the reductive tricarboxylic acid cycle, ATP citrate lyase, 2-oxoglutarate:ferredoxin oxidoreductase, and pyruvate:ferredoxin oxidoreductase. Furthermore, no activities of key enzymes of other CO(2) fixation pathways, such as the Calvin cycle, the reductive acetyl coenzyme A pathway, and the 3-hydroxypropionate cycle, could be detected. In addition to the key enzymes, the activities of the other enzymes involved in the reductive tricarboxylic acid cycle could be measured. Sections of the genes encoding the alpha- and beta-subunits of ATP citrate lyase could be amplified from both organisms. These findings represent the first direct evidence for the operation of the reductive tricarboxylic acid cycle for autotrophic CO(2) fixation in epsilon-proteobacteria. Since epsilon-proteobacteria closely related to these two organisms are important in many habitats, such as hydrothermal vents, oxic-sulfidic interfaces, or oilfields, these results suggest that autotrophic CO(2) fixation via the reductive tricarboxylic acid cycle might be more important than previously considered.     

Substrate inhibition in the tricarboxylic acid cycle

It has been shown in the experiments on rat liver mitochondria under glucose hexo-kinase load that excess of substrates of (1-20 mM) pyruvate, acetate, propionate, pent-4-enoate and malate may induce oxidation of NAD(P)H and inhibition of mitochondrial respiration (by 20-50% and more) due to a decreased rate of hydrogen production by tricarboxylic acid cycle. It has been concluded from the analysis of mathematical models and metabolite-testings which remove this inhibition that for pyruvate and acetate this inhibition is an autocatalytic one. It is related to a decreased level of CoA and oxaloacetate due to the formation of "traps" such as acetyl-CoA and alpha-kotoglutarate. For propionate and pent-4-enoate in the bicarbonate-free medium suppression of the flux in the cycle is concerned with a decreased level of CoA, acetyl-CoA and succionoyl CoA due to the accumulation of propionyl-CoA. It seems to be also concerned with the inhibition of citrate-synthetase and alpha-ketoglutarate-dehydrogenase by propionyl-CoA. Malate (in the presence of malonate) can inhibit respiration at the expense of direct inhibition of citrate-synthetase.     

Abundance of reverse tricarboxylic acid cycle genes in free-living microorganisms at deep-sea hydrothermal vents

Since the discovery of hydrothermal vents more than 25 years ago, the Calvin-Bassham-Benson (Calvin) cycle has been considered the principal carbon fixation pathway in this microbe-based ecosystem. However, on the basis of recent molecular data of cultured free-living and noncultured episymbiotic members of the epsilon subdivision of Proteobacteria and earlier carbon isotope data of primary consumers, an alternative autotrophic pathway may predominate. Here, genetic and culture-based approaches demonstrated the abundance of reverse tricarboxylic acid cycle genes compared to the abundance of Calvin cycle genes in microbial communities from two geographically distinct deep-sea hydrothermal vents. PCR with degenerate primers for three key genes in the reverse tricarboxylic acid cycle and form I and form II of ribulose 1,5-bisphosphate carboxylase/oxygenase (Calvin cycle marker gene) were utilized to demonstrate the abundance of the reverse tricarboxylic acid cycle genes in diverse vent samples. These genes were also expressed in at least one chimney sample. Diversity, similarity matrix, and phylogenetic analyses of cloned samples and amplified gene products from autotrophic enrichment cultures suggest that the majority of autotrophs that utilize the reverse tricarboxylic acid cycle are members of the epsilon subdivision of Proteobacteria. These results parallel the results of previously published molecular surveys of 16S rRNA genes, demonstrating the dominance of members of the epsilon subdivision of Proteobacteria in free-living hydrothermal vent communities. Members of the epsilon subdivision of Proteobacteria are also ubiquitous in many other microaerophilic to anaerobic sulfidic environments, such as the deep subsurface. Therefore, the reverse tricarboxylic acid cycle may be a major autotrophic pathway in these environments and significantly contribute to global autotrophic processes.     

Evidence for Autotrophy via the Reverse Tricarboxylic Acid Cycle in the Marine Magnetotactic Coccus Strain MC-1

Strain MC-1 is a marine, microaerophilic, magnetite-producing, magnetotactic coccus phylogenetically affiliated with the α-Proteobacteria. Strain MC-1 grew chemolithotrophically with sulfide and thiosulfate as electron donors with HCO₃⁻/CO₂ as the sole carbon source. Experiments with cells grown microaerobically in liquid with thiosulfate and H¹⁴CO₃⁻/¹⁴CO₂ showed that all cell carbon was derived from H¹⁴CO₃⁻/¹⁴CO₂ and therefore that MC-1 is capable of chemolithoautotrophy. Cell extracts did not exhibit ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO) activity, nor were RubisCO genes found in the draft genome of MC-1. Thus, unlike other chemolithoautotrophic, magnetotactic bacteria, strain MC-1 does not appear to utilize the Calvin-Benson-Bassham cycle for autotrophy. Cell extracts did not exhibit carbon monoxide dehydrogenase activity, indicating that the acetyl-coenzyme A pathway also does not function in strain MC-1. The ¹³C content of whole cells of MC-1 relative to the ¹³C content of the inorganic carbon source (Δδ¹³C) was −11.4 . Cellular fatty acids showed enrichment of ¹³C relative to whole cells. Strain MC-1 cell extracts showed activities for several key enzymes of the reverse (reductive) tricarboxylic acid (rTCA) cycle including fumarate reductase, pyruvate:acceptor oxidoreductase and 2-oxoglutarate:acceptor oxidoreductase. Although ATP citrate lyase (another key enzyme of the rTCA cycle) activity was not detected in strain MC-1 using commonly used assays, cell extracts did cleave citrate, and the reaction was dependent upon the presence of ATP and coenzyme A. Thus, we infer the presence of an ATP-dependent citrate-cleaving mechanism. These results are consistent with the operation of the rTCA cycle in MC-1. Strain MC-1 appears to be the first known representative of the α-Proteobacteria to use the rTCA cycle for autotrophy.     

Oxidation of carbon sources via the tricarboxylic acid cycle during calcium-induced conidation of Penicillium notatum

The TCA cycle was examined during Ca²⁺-induced conidiation in Penicillium notatum over the 12-h period after addition of Ca²⁺ to vegetative cultures. Conidiation was independent of Ca²⁺ when certain intermediates and derivatives of the TCA cycle served as sole carbon sources. Arsenite and malonate augmented the effect of Ca²⁺ on conidiation but did not substitute for it. Mitochondria from vegetative cells had low rates of oxidation of TCA cycle intermediates and, with the exception of pyruvate, aconitate and glutamate, these were poorly linked to phosphorylation processes. Calcium ions affected mitochondrial function causing reduced oxidation of oxoglutarate, elimination of pyruvate oxidation and a decline in respiratory control of these substrates with increased oxidation of NADH and NADPH. Radiorespirometric studies and enzyme searches revealed a complete but weakly oxidative TCA cycle in vegetative cells. In Ca²⁺-induced cells oxoglutarate dehydrogenase activity was deleted within 6.5 h of Ca²⁺ addition and this was accompanied by establishment of an incomplete Krebs cycle. Calcium-induced conidiation was associated with increased capacity for acetate and glutamate metabolism involving an activated glyoxylate shunt which may be related to enhanced biosynthetic demand. The metabolic basis of the Ca²⁺ effect on conidiation is discussed in connection with previous findings.     

Supramolecular organization of enzymes of the tricarboxylic acid cycle

In virtue of analysis of data on the interaction of tricarboxylic acid cycle enzymes with the mitochondrial inner membrane and data on the enzyme-enzyme interactions, the spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon) is proposed. The alpha-ketoglutarate dehydrogenase complex, adsorbed on the mitochondrial inner membrane along one of its 3-fold symmetry axes, plays the key role in the formation of metabolon. Two association sites of the alpha-ketoglutarate dehydrogenase complex located on opposite sides of the complex participate in the interaction with the membrane. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleosidediphosphate kinase. Succinate dehydrogenase, the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of metabolon on the membrane. The molecular mass of the complex (ignoring succinate dehydrogenase) is of 8.10(6) daltons. The metabolon symmetry corresponds to the D3 point symmetry group. It is supposed, that the tricarboxylic acid cycle enzyme complex interacts with other multienzyme complexes of the matrix and the electron transfer chain.     

Further characterization of the Krebs tricarboxylic acid cycle metabolon

A preparation of gently disrupted rat liver mitochondria which shows exposed and easily sedimented Krebs tricarboxylic acid cycle enzyme activities has been characterized further. The exposed malate dehydrogenase is inhibited by high molecular weight blue dextran which indicates the availability of the enzyme to the bulk solvent. Further, mitoplasts are not permeable to citrate synthase antibodies ruling out the possibility of vesicularization of high molecular weight substances. The slightly disrupted mitochondria sedimented more slowly than did intact mitochondria on a Ficoll gradient. Electron microscopy, both thin section and scanning, showed slightly swollen mitochondria with some disruption of the membranes. Labeling with ferritin-labeled second antibody to citrate synthase antibodies showed again the accessibility of these disrupted mitochondria to the antibody. When either the oxidation of fumarate or the malate dehydrogenase-citrate synthase coupled system are studied, relative kinetic advantages are observed of the gently disrupted systems over the completely solubilized system. These kinetic advantages are more labile to disruption than is the binding of the enzymes to the particle. These results indicate that the Krebs tricarboxylic acid cycle exists as a sequential complex of enzymes, a metabolon, in situ. This study shows that previous studies which showed interactions between sequential enzymes of this pathway and their binding to the inner surface of the inner membrane actually reflected an in vivo organization of this pathway.