The extracellular death factor: physiological and genetic factors influencing its production and response in Escherichia coli

Gene pairs specific for a toxin and its antitoxin are called toxin-antitoxin modules and are found on the chromosomes of many bacteria. The most studied of these modules is Escherichia coli mazEF, in which mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. In a previous report from this laboratory, it was shown that mazEF-mediated cell death is a population phenomenon requiring a quorum-sensing peptide called the extracellular death factor (EDF). EDF is the linear pentapeptide NNWNN (32). Here, we further confirm that EDF is a signal molecule in a mixed population. In addition, we characterize some physiological conditions and genes required for EDF production and response. Furthermore, stress response and the gene specifying MazEF, the Zwf (glucose-6-phosphate dehydrogenase) gene, and the protease ClpXP are critical in EDF production. Significant strain differences in EDF production and response explain variations in the induction of mazEF-mediated cell death.     

GLUT4 vesicle recruitment and fusion are differentially regulated by Rac, AS160, and Rab8A in muscle cells

Insulin increases glucose uptake into muscle by enhancing the surface recycling of GLUT4 transporters. In myoblasts, insulin signals bifurcate downstream of phosphatidylinositol 3-kinase into separate Akt and Rac/actin arms. Akt-mediated Rab-GAP AS160 phosphorylation and Rac/actin are required for net insulin gain of GLUT4, but the specific steps (vesicle recruitment, docking or fusion) regulated by Rac, actin dynamics, and AS160 target Rab8A are unknown. In L6 myoblasts expressing GLUT4myc, blocking vesicle fusion by tetanus toxin cleavage of VAMP2 impeded GLUT4myc membrane insertion without diminishing its build-up at the cell periphery. Conversely, actin disruption by dominant negative Rac or Latrunculin B abolished insulin-induced surface and submembrane GLUT4myc accumulation. Expression of non-phosphorylatable AS160 (AS160-4P) abrogated membrane insertion of GLUT4myc and partially reduced its cortical build-up, an effect magnified by selective Rab8A knockdown. We propose that insulin-induced actin dynamics participates in GLUT4myc vesicle retention beneath the membrane, whereas AS160 phosphorylation is essential for GLUT4myc vesicle-membrane docking/fusion and also contributes to GLUT4myc cortical availability through Rab8A.     

A molecular mechanism for mimosine-induced apoptosis involving oxidative stress and mitochondrial activation

Mimosine, a non-protein amino acid, is mainly known for its action as a reversible inhibitor of DNA replication and, therefore, has been widely used as a cell cycle synchronizing agent. Recently, it has been shown that mimosine also induces apoptosis, as mainly reflected in its ability to elicit characteristic nuclear changes. The present study elucidates the mechanism underlying mimosine’s apoptotic effects, using the U-937 leukemia cell line. We now demonstrate that in isolated rat liver mitochondria, mimosine induces mitochondrial swelling that can be inhibited by cyclosporine A, indicative of permeability transition (PT) mega-channel opening. Mimosine-induced apoptosis was accompanied by formation of hydrogen peroxide and a decrease in reduced glutathione levels. The apoptotic process was partially inhibited by cyclosporine A and substantially blocked by the antioxidant N-acetylcysteine, suggesting an essential role for reactive oxygen species formation during the apoptotic processes. The apoptosis induced by mimosine was also accompanied by a decrease in mitochondrial membrane potential, cytochrome c release and caspase 3 and 9 activation. Our results thus imply that mimosine activates apoptosis through mitochondrial activation and formation of H₂O₂, both of which play functional roles in the induction of cell death. Maher Hallak and Liat Vazana have contributed equally to the work.     

The hemerythrin-like diiron protein from Mycobacterium kansasii is a nitric oxide peroxidase

The hemerythrin-like protein from Mycobacterium kansasii (Mka HLP) is a member of a distinct class of oxo-bridged diiron proteins that are found only in mycobacterial species that cause respiratory disorders in humans. Because it had been shown to exhibit weak catalase activity and a change in absorbance on exposure to nitric oxide (NO), the reactivity of Mka HLP toward NO was examined under a variety of conditions. Under anaerobic conditions, we found that NO was converted to nitrite (NO₂⁻) via an intermediate, which absorbed light at 520 nm. Under aerobic conditions NO was converted to nitrate (NO₃⁻). In each of these two cases, the maximum amount of nitrite or nitrate formed was at best stoichiometric with the concentration of Mka HLP. When incubated with NO and H₂O₂, we observed NO peroxidase activity yielding nitrite and water as reaction products. Steady-state kinetic analysis of NO consumption during this reaction yielded a K_m for NO of 0.44 μM and a k_cat/K_m of 2.3 × 10⁵ M⁻¹s⁻¹. This high affinity for NO is consistent with a physiological role for Mka HLP in deterring nitrosative stress. This is the first example of a peroxidase that uses an oxo-bridged diiron center and a rare example of a peroxidase utilizing NO as an electron donor and cosubstrate. This activity provides a mechanism by which the infectious Mycobacterium may combat against the cocktail of NO and superoxide (O₂^•−) generated by macrophages to defend against bacteria, as well as to produce NO₂⁻ to adapt to hypoxic conditions.     

A self-produced trigger for biofilm disassembly that targets exopolysaccharide

Biofilms are structured communities of bacteria that are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often have a limited lifespan, disassembling as nutrients become exhausted and waste products accumulate. D-amino acids were previously identified as a self-produced factor that mediates biofilm disassembly by causing the release of the protein component of?the matrix in Bacillus subtilis. Here we report that?B.?subtilis produces an additional biofilm-disassembly factor, norspermidine. Dynamic light scattering and scanning electron microscopy experiments indicated that norspermidine interacts directly and specifically with exopolysaccharide. D-amino acids and norspermidine acted together to break down existing biofilms and mutants blocked in the production of both factors formed long-lived biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B.?subtilis, Escherichia coli, and Staphylococcus aureus.     

Astrocyte Leucine Metabolism: Significance of Branched-Chain Amino Acid Transamination

Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a V _max of 53.6 ± 3.2 nmol/mg of protein/min and a K _m of 449.2 ± 94.9 µ M . Virtually all leucine transport was found to be Na⁺ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α-aminooxyacetic acid (5 m M ), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α-ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent K _m for leucine conversion to extracellular KIC was a medium [leucine] of 58 µ M with a V _max of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α-ketoglutarate ↮ KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the "buffering" of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.     

Diazotrophs are overlooked contributors to carbon and nitrogen export to the deep ocean

Diazotrophs are widespread microorganisms that alleviate nitrogen limitation in 60% of our oceans, thereby regulating marine productivity. Yet, the group-specific contribution of diazotrophs to organic matter export has not been quantified, which so far has impeded an accurate assessment of their impact on the biological carbon pump. Here, we examine the fate of five groups of globally-distributed diazotrophs by using an original combination of mesopelagic particle sampling devices across the subtropical South Pacific Ocean. We demonstrate that cyanobacterial and non-cyanobacterial diazotrophs are exported down to 1000 m depth. Surprisingly, group-specific export turnover rates point to a more efficient export of small unicellular cyanobacterial diazotrophs (UCYN) relative to the larger and filamentous Trichodesmium. Phycoerythrin-containing UCYN-B and UCYN-C-like cells were recurrently found embedded in large (>50 µm) organic aggregates or organized into clusters of tens to hundreds of cells linked by an extracellular matrix, presumably facilitating their export. Beyond the South Pacific, our data are supported by analysis of the Tara Oceans metagenomes collected in other ocean basins, extending the scope of our results globally. We show that, when diazotrophs are found in the euphotic zone, they are also systematically present in mesopelagic waters, suggesting their transport to the deep ocean. We thus conclude that diazotrophs are a significant part of the carbon sequestered in the deep ocean and, therefore, they need to be accounted in regional and global estimates of export.Subject terms: Biogeochemistry, Microbiology     

Enzyme-enzyme interaction in the chloroplast: Glyceraldehyde-3-phosphate dehydrogenase,triose phosphate isomerase and aldolase

Apparent physical interaction between pea chloroplast (Pisum sativum L.) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase (EC 4.1.2.13) is seen in phase-partitioning, fluorescent-anisotropy and isoelectric-focusing experiments. Similarly, results obtained in phase-partitioning and isoelectric-focusing experiments indicate physical interaction between aldolase and triose-phosphate isomerase (EC 5.3.1.1). Kinetic experiments suggest that both aldolase-bound glyceraldehyde-3-phosphate and triose-phosphate isomerase bound glyceraldehyde-3-phosphate can act as substrate for glyceraldehyde-3-phosphate dehydrogenase. These results are consistent with the notion that there is interaction between these three enzymes both during photosynthetic CO₂ fixation and during glycolysis in the chloroplast.Abbreviations FITC fluorescein isothiocyanate - glyceraldehyde3-P glyceraldehyde-3-phosphate - K partition coefficient - K _m (ALD) apparent K _m value obtained when aldolase levels are varied - K _m (GAP) K _m value obtained when glyceraldehyde-3-P concentrations are varied - K _m (PGK) apparent K _m value obtained when phosphoglycerate kinase levels are varied - K _m (TPI) apparent K _m value obtained when triose-P isomerase levels are varied - PEG polyethyleneglycol - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - triose-P triose phosphate We thank Fred J. Stevens, Argonne National Laboratory, for help in analysis of the tertiary structures, Göte Johansson, University of Lund, for hosting two of us in his laboratory where we did the initial phase-partitioning experiments, Chang-hou Li, Shanghai Research Centre of Biotechnology, for the use of the fluorimeter, Lawrence Sykora and the University of Illinois greenhouse staff for growing the pea plants, Jack T. Gibbons for electron microscopy, and Christie Aljets, Xua Ming Da, Xiang He, Arif Ali Khan, Fang Luo, Martha Pacold, Michael Pacold, Lei Shi, Hyun Moon Shin and Qi Zhao for their assistance with these experiments. Support came from the University of Illinois-Chicago Research Board, the US National Science Foundation (Grants DCB 9018265, INT 91-15490 and INT 91-13311) and the Chinese National Science Foundation (Grant 39230050).