Properties of ion channels formed by Staphylococcus aureus delta-toxin

The delta-toxin of Staphylococcus aureus has been investigated in terms of its potential to form ion channels in planar lipid bilayers formed at the tip of patch electrodes. Channel formation has been shown to occur for delta-toxin concentrations in the range 0.1 to 2.0 microM. In 0.5 M KCl, two major classes of channels were seen--'small' with conductances of 70-100 pS, and 'large' with a conductance of approx. 450 pS. Current-voltage relationships for lipid bilayers containing several delta-toxin channels revealed both voltage-dependent and independent components to channel gating. Reversal potential measurements showed the channels to be cation selective. In the presence of 3.0 M KCl, the channel gating kinetics were complex, with multiple open and closed states. The results are interpreted in terms of a model for the channel consisting of a hexameric cluster of alpha-helical delta-toxin molecules.     

Polymyxin B biosynthesis and tricarboxylic acid cycle enzymatic activity]

Activity of pyruvate dehydrogenase (PDG), isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase was found in the extracts of the cells of Bac. polymyxa 153, an organism producing polymyxin B. Dependence of the activity of the above enzymes on the carbon source in the medium, aeration conditions, strain features and culture age was shown. A low level of polymyxin B biosynthesis was observed at high activity of PDG and dehydrogenases of the tricarbonic acid cycle. Increased antibiotic production was recorded against the background of decreases values of the above enzyme activities.     

Detection of Staphylococcus aureus delta-toxin production by whole-cell MALDI-TOF mass spectrometry

The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p?=?0.048; CI 95%: 1.01-10.24; p?=?0.023; CI 95%: 1.20-12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.     

Enhanced post-stationary-phase survival of a clinical thymidine-dependent small-colony variant of Staphylococcus aureus results from lack of a functional tricarboxylic acid cycle

The mechanisms underlying the persistence of the Staphylococcus aureus small-colony variant (SCV) are not fully elucidated. In this study, clinical thymidine-dependent SCVs displayed altered expression of citB, clpC, and arcA genes, reduced acetate catabolization, and enhanced survival. These results implicate the importance of changes in tricarboxylic acid cycle and acetic acid metabolism in SCV survival and persistence.     

Staphylococcus epidermidis polysaccharide intercellular adhesin production significantly increases during tricarboxylic acid cycle stress

Staphylococcal polysaccharide intercellular adhesin (PIA) is important for the development of a mature biofilm. PIA production is increased during growth in a nutrient-replete or iron-limited medium and under conditions of low oxygen availability. Additionally, stress-inducing stimuli such as heat, ethanol, and high concentrations of salt increase the production of PIA. These same environmental conditions are known to repress tricarboxylic acid (TCA) cycle activity, leading us to hypothesize that altering TCA cycle activity would affect PIA production. Culturing Staphylococcus epidermidis with a low concentration of the TCA cycle inhibitor fluorocitrate dramatically increased PIA production without impairing glucose catabolism, the growth rate, or the growth yields. These data lead us to speculate that one mechanism by which staphylococci perceive external environmental change is through alterations in TCA cycle activity leading to changes in the intracellular levels of biosynthetic intermediates, ATP, or the redox status of the cell. These changes in the metabolic status of the bacteria result in the attenuation or augmentation of PIA production.     

Staphylococcus aureus haem biosynthesis and acquisition pathways are linked through haem monooxygenase IsdG

Haem is an essential cofactor in central metabolic pathways in the vast majority of living systems. Prokaryotes acquire haem via haem biosynthesis pathways, and some also utilize haem uptake systems, yet it remains unclear how they balance haem requirements with the paradox that free haem is toxic. Here, using the model pathogen Staphylococcus aureus, we report that IsdG, one of two haem oxygenase enzymes in the haem uptake system, inhibits the formation of haem via the internal haem biosynthesis route. More specifically, we show that IsdG decreases the activity of ferrochelatase and that the two proteins interact both in vitro and in vivo. Further, a bioinformatics analysis reveals that a significant number of haem biosynthesis pathway containing organisms possess an IsdG‐homologue and that those with both biosynthesis and uptake systems have at least two haem oxygenases. We conclude that IsdG‐like proteins control intracellular haem levels by coupling the two pathways. IsdG is thus a target for the treatment of S. aureusinfections.     

Supramolecular organization of tricarboxylic acid cycle enzymes

We propose a spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon). The structure is based on an analysis of data on the interaction between tricarboxylic acid cycle enzymes and the mitochondrial inner membrane, as well as on data on enzyme-enzyme interactions. The alpha-ketoglutarate dehydrogenase complex, adsorbed along one of the 3-fold symmetry axes of the mitochondrial inner membrane, plays a key role in formation of the metabolon. In the interaction with the membrane, two association sites of the alpha-ketoglutarate dehydrogenase complex participate, placed on opposite sides of the complex. 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 nucleoside-diphosphate kinase. Succinate dehydrogenase, which is the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of the metabolon on the membrane. The molecular mass of the complex (without regard to succinate dehydrogenase) is 8 x 10(6) Da. The metabolon symmetry corresponds to the D3 point symmetry group.     

Organization of Krebs tricarboxylic acid cycle enzymes

Binding of enzymes of the Krebs TCA cycle to biological membranes was characterized with respect to intracellular location, susceptibility to various chemical and physical treatments, and extractability as a macromolecular component of the mitochondrial inner membrane. It was shown that citrate synthase and malate dehydrogenase bind to the inner membrane in an ionic strength-sensitive, saturable, and specific manner to a relatively thermostabile component manifested on the inner (matrix) surface of the inner membrane of the mitochondrion. From these data several arguments in support of the physiological applicability of these processes were deduced, and the question of whether these two enzymes bind to the same or different membrane components was considered. Also, experiments preliminary to purification of the citrate synthase binding component were presented.     

Impaired tricarboxylic acid cycle activity in mouse livers lacking cytosolic phosphoenolpyruvate carboxykinase

Liver-specific phosphoenolpyruvate carboxykinase (PEPCK) null mice, when fasted, maintain normal whole body glucose kinetics but develop dramatic hepatic steatosis. To identify the abnormalities of hepatic energy generation that lead to steatosis during fasting, we studied metabolic fluxes in livers lacking hepatic cytosolic PEPCK by NMR using 2H and 13C tracers. After a 4-h fast, glucose production from glycogenolysis and conversion of glycerol to glucose remains normal, whereas gluconeogenesis from tricarboxylic acid (TCA) cycle intermediates was nearly absent. Upon an extended 24-h fast, livers that lack PEPCK exhibit both 2-fold lower glucose production and oxygen consumption, compared with the controls, with all glucose production being derived only from glycerol. The mitochondrial reduction-oxidation (red-ox) state, as indicated by the NADH/NAD+ ratio, is 5-fold higher, and hepatic TCA cycle intermediate concentrations are dramatically increased in the PEPCK null livers. Consistent with this, flux through the TCA cycle and pyruvate cycling pathways is 10- and 40-fold lower, respectively. Disruption of hepatic cataplerosis due to loss of PEPCK leads to the accumulation of TCA cycle intermediates and a nearly complete blockage of gluconeogenesis from amino acids and lactate (an energy demanding process) but intact gluconeogenesis from glycerol (which contributes to net NADH production). Inhibition of the TCA cycle and fatty acid oxidation due to increased TCA cycle intermediate concentrations and reduced mitochondrial red-ox state lead to the development of steatosis.     

The tricarboxylic acid cycle of Helicobacter pylori.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.     

Physiology and metabolism of pathogenic neisseria: tricarboxylic acid cycle activity in Neisseria gonorrhoeae.

Tricarboyxlic acid cycle activity was examined in Neisseria gonorrhoeae CS-7. The catabolism of glucose in N. gonorrheae by a combination of the Entner-Doudoroff and pentose phosphate pathways resulted in the accumulation of acetate, which was not further catabolized until the glucose was depleted or growth became limiting. Radiorespirometric studies revealed that the label in the 1 position of acetate was converted to CO2 at twice the rate of the label in the 2 position, indicating the presence of a tricarboxylic acid cycle. Growth on glucose markedly reduced the levels of all tricarboxylic acid cycle enzymes except citrate synthase (EC 4.1.3.7). Extracts of glucose-grown cells contained detectable levels of all tricarboxylic acid cycle enzymes except aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), and a pyridine nucleotide-dependent malate dehydrogenase (EC 1.1.1.37). Extracts of cells capable of oxidizing acetate lacked only the pyridine nucleotide-dependent malate dehydrogenase. In lieu of this enzyem, a particulate pyridine nucleotide-independent malate oxidase (EC 1.1.3.3) was present. This enzyme required flavin adenine dinucleotide for activity and appeared to be associated with the electron transport chain. Radiorespirometric studies utilizing labeled glutamate demonstrated that a portion of the tricarboxylic acid cycle functioned during glucose catabolism. In spite of the presence of all tricarboxylic acid cycle enzymes, N. gonorrhoeae CS-7 was unable to grow in medium supplemented with cycle intermediates.     

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.     

Relationship between sugar consumption and tricarboxylic acid cycle enzyme activity in a high bialaphos-producing strain

The biosynthesis of bialaphos [a herbicide, 2-amino-4(hydroxy)(methyl)-phosphinoylbutyryl-alanylalanine] produced by Streptomyces hygroscopicus, proceeds through the degradation of glucose to phosphoenol pyruvate, the formation of a CP bond, acetic addition by acetyl-CoA and alanine addition. Therefore, bialaphos formation is considered to be closely related to glucose metabolism. Based on this hypothesis, sugar consumption and the activities of the tricarboxylic acid cycle enzymes were examined using a highly productive strain and a strain of the lower productivity. It was clear that the highly productive strain has a lower sugar consumption rate and lower yield of cells, compared with the lower productivity strain. The activities of the tricarboxylic acid cycle enzymes of the highly productive strain were lower than those of the lower productivity strain, while the activities of the glyoxylic acid cycle enzymes of the highly productive strain were higher. From these findings, it is suggested that the highly productive strain suppresses the flow from acetyl-CoA and pyruvate (as substrates of bialaphos) to the tricarboxylic acid cycle, and efficiently directs these substrates to the secondary metabolism by activation of the glyoxylic acid cycle, resulting in a high rate of bialaphos production in this strain.