Dormancy: Metabolite pools prime the cyanobacterial dormancy-resuscitation switch.

Non-N-fixing cyanobacteria enter a state of dormancy when fixed nitrogen becomes limiting. Resuscitation from this state involves a complex program of events. A new study reveals how the dormancy-resuscitation switch is governed by metabolite-level control of glucose-6-phosphate dehydrogenase activity.

The formate-hydrogen axis and its impact on the physiology of enterobacterial fermentation.

Formic acid (HCOOH) and dihydrogen (H) are characteristic products of enterobacterial mixed-acid fermentation, with H generation increasing in conjunction with a decrease in extracellular pH. Formate and acetyl-CoA are generated by radical-based and coenzyme A-dependent cleavage of pyruvate catalysed by pyruvate formate-lyase (PflB). Formate is also the source of H, which is generated along with carbon dioxide through the action of the membrane-associated, cytoplasmically-oriented formate hydrogenlyase (FHL-1) complex.

Evidence the Isc iron-sulfur cluster biogenesis machinery is the source of iron for [NiFe]-cofactor biosynthesis in Escherichia coli.

[NiFe]-hydrogenases have a bimetallic NiFe(CN)CO cofactor in their large, catalytic subunit. The 136 Da Fe(CN)CO group of this cofactor is preassembled on a distinct HypC-HypD scaffold complex, but the intracellular source of the iron ion is unresolved. Native mass spectrometric analysis of HypCD complexes defined the [4Fe-4S] cluster associated with HypD and identified + 26 to 28 Da and + 136 Da modifications specifically associated with HypC.

Evidence for bidirectional formic acid translocation in vivo via the Escherichia coli formate channel FocA.

Pentameric FocA permeates either formate or formic acid bidirectionally across the cytoplasmic membrane of anaerobically growing Escherichia coli. Each protomer of FocA has its own hydrophobic pore, but it is unclear whether formate or neutral formic acid is translocated in vivo. Here, we measured total and dicyclohexylcarbodiimide (DCCD)-inhibited proton flux out of resting, fermentatively grown, stationary-phase E.

Perspective elucidating the physiology of a microbial cell: Neidhardt's Holy Grail.

A living microbial cell represents a system of high complexity, integration, and extreme order. All processes within that cell interconvert free energy through a multitude of interconnected metabolic reactions that help to maintain the cell in a state of low entropy, which is a characteristic of all living systems. The study of macromolecular interactions outside this cellular environment yields valuable information about the molecular function of macromolecules but represents a system in comparative disorder.

A redox-active HybG-HypD scaffold complex is required for optimal ATPase activity during [NiFe]-hydrogenase maturation in Escherichia coli.

Four Hyp proteins build a scaffold complex upon which the Fe(CN) CO group of the [NiFe]-cofactor of hydrogenases (Hyd) is made. Two of these Hyp proteins, the redox-active, [4Fe-4S]-containing HypD protein and the HypC chaperone, form the basis of this scaffold complex. Two different scaffold complexes exist in Escherichia coli, HypCD, and the paralogous HybG-HypD complex, both of which exhibit ATPase activity.

FocA and its central role in fine-tuning pH homeostasis of enterobacterial formate metabolism.

During enterobacterial mixed-acid fermentation, formate is generated from pyruvate by the glycyl-radical enzyme pyruvate formate-lyase (PflB). In , especially at low pH, formate is then disproportionated to CO and H by the cytoplasmically oriented, membrane-associated formate hydrogenlyase (FHL) complex. If electron acceptors are available, however, formate is oxidized by periplasmically oriented, respiratory formate dehydrogenases.

Distinguishing functional from structural roles of conserved pore residues during formate translocation by the FocA anion channel.

The formate-specific anion channel FocA of Escherichia coli belongs to the superfamily of homopentameric formate-nitrite transporters (FNT). Minimally nine amino acid residues are conserved in the formate translocation pore of each protomer of the pentamer, including a histidine (H209) and a threonine (T91), both of which are crucial for bidirectional formate translocation through the pore. Information regarding in vivo functional or structural roles for the other seven conserved residues is limited, or nonexistent.

Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development.

Access to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi status are unknown.

Exchange of a Single Amino Acid Residue in the HybG Chaperone Allows Maturation of All H-Activating [NiFe]-Hydrogenases in .

The biosynthesis of the NiFe(CN)CO organometallic cofactor of [NiFe]-hydrogenase (Hyd) involves several discreet steps, including the synthesis of the Fe(CN)CO group on a HypD-HypC scaffold complex. HypC has an additional function in transferring the Fe(CN)CO group to the apo-precursor of the Hyd catalytic subunit. Bacteria that synthesize more than one Hyd enzyme often have additional HypC-type chaperones specific for each precursor.

The FocA channel functions to maintain intracellular formate homeostasis during fermentation.

FocA translocates formate/formic acid bi-directionally across the cytoplasmic membrane when grows by fermentation. It remains unclear, however, what physiological benefit is imparted by FocA, because formic acid (p=3.75) can diffuse passively across the membrane, especially at low pH.

The Autonomous Glycyl Radical Protein GrcA Restores Activity to Inactive Full-Length Pyruvate Formate-Lyase .

During glucose fermentation, Escherichia coli and many other microorganisms employ the glycyl radical enzyme (GRE) pyruvate formate-lyase (PflB) to catalyze the coenzyme A-dependent cleavage of pyruvate to formate and acetyl-coenzyme A (CoA). Due to its extreme reactivity, the radical in PflB must be controlled carefully and, once generated, is particularly susceptible to dioxygen. Exposure to oxygen of the radical on glycine residue 734 of PflB results in cleavage of the polypeptide chain and consequent inactivation of the enzyme.

A paean to the ineffable Marjory Stephenson.

It is now 75 years since Marjory Stephenson became the second President of the Society for General Microbiology (SGM). Around the time of her death at the end of 1948 many articles appeared extolling Marjory Stephenson's contribution to the fields of Biochemistry and Microbiology. Not that much has been written about her since that time, which is unfortunate.

A single amino acid exchange converts FocA into a unidirectional efflux channel for formate.

During mixed-acid fermentation, initially translocates formate out of the cell, but re-imports it at lower pH. This is performed by FocA, the archetype of the formate-nitrite transporter (FNT) family of pentameric anion channels. Each protomer of FocA has a hydrophobic pore through which formate/formic acid is bidirectionally translocated.

Native mass spectrometry identifies the HybG chaperone as carrier of the Fe(CN)CO group during maturation of E. coli [NiFe]-hydrogenase 2.

[NiFe]-hydrogenases activate dihydrogen. Like all [NiFe]-hydrogenases, hydrogenase 2 of Escherichia coli has a bimetallic NiFe(CN)CO cofactor in its catalytic subunit. Biosynthesis of the Fe(CN)CO group of the [NiFe]-cofactor occurs on a distinct scaffold complex comprising the HybG and HypD accessory proteins.

Changes of the Proteome and Acetylome during Transition into the Stationary Phase in the Organohalide-Respiring Strain CBDB1.

The strictly anaerobic bactGIerium obligatorily depends on organohalide respiration for energy conservation and growth. The bacterium also plays an important role in bioremediation. Since there is no guarantee of a continuous supply of halogenated substrates in its natural environment, the question arises of how maintains the synthesis and activity of dehalogenating enzymes under these conditions.

Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa oxidase supercomplex from spores of Streptomyces coelicolor A3(2).

In order to reduce nitrate in vivo, the spore-specific respiratory nitrate reductase, Nar1, of Streptomyces coelicolor relies on an active cytochrome bcc-aa oxidase supercomplex (bcc-aa supercomplex). This suggests that membrane-associated Nar1, comprising NarG1, NarH1, and NarI1 subunits, might not act as a classical menaquinol oxidase but could either receive electrons from the bcc-aa supercomplex, or require the supercomplex to stabilize the reductase in the membrane to allow it to function. To address the biochemical basis for this dependence on the bcc-aa supercomplex, we purified two different Strep-tagged variants of Nar1 and enriched the native enzyme complex from spore extracts using different chromatographic and electrophoretic procedures.

Setting the Stage: Genes Controlling Mechanosensation and Ca Signaling in Escherichia coli.

Although mechanistic understanding of calcium signaling in bacteria remains inchoate, current evidence clearly links Ca signaling with membrane potential and mechanosensation. Adopting a radically new approach, Luder et al. scanned the Keio collection of gene knockouts (R.

Solid phase-based cross-matching for solid organ transplantation: Currently out-of-stock but urgently required for improved allograft outcome.

Transplant recipients who have undergone sensitizing events, such as pregnancy, blood transfusion or previous transplants, frequently develop antibodies directed against the highly polymorphous human leukocyte antigen (HLA)-molecules. These pre-formed, donor-specific antibodies (DSA) present a high risk of causing organ failure or even complete loss of the grafted organ as a consequence of antibody-mediated, hyper-acute or acute allograft rejection. In order to detect DSA, the so-called functional complement-dependent lymphocytotoxicity assay (CDC-XM) was established about 50 years ago.

Influence of C -Dcu transporters on hydrogenase and formate dehydrogenase activities in stationary phase-grown fermenting Escherichia coli.

During mixed-acid fermentation, Escherichia coli transports succinate mainly via transporters of the Dcu family. Here, we analyze the influence of Dcu transporters on hydrogenase (Hyd) and fermentative formate dehydrogenase (FDH-H) activities and how this is affected by external pH and carbon source. Using selected dcu mutations, it was shown that Dcu carriers mainly affect Hyd and FDH-H activities during glycerol but not glucose fermentation at acidic pH.

Molecular Hydrogen Metabolism: a Widespread Trait of Pathogenic Bacteria and Protists.

Pathogenic microorganisms use various mechanisms to conserve energy in host tissues and environmental reservoirs. One widespread but often overlooked means of energy conservation is through the consumption or production of molecular hydrogen (H). Here, we comprehensively review the distribution, biochemistry, and physiology of H metabolism in pathogens.

The iron-sulfur-containing HypC-HypD scaffold complex of the [NiFe]-hydrogenase maturation machinery is an ATPase.

HypD and HypC, or its paralogue HybG in Escherichia coli, form the core of the scaffold complex that synthesizes the Fe(CN) CO component of the bimetallic NiFe-cofactor of [NiFe]-hydrogenase. We show here that purified HypC-HypD and HybG-HypD complexes catalyse hydrolysis of ATP to ADP (k  ≅ 0.85·s ); the ATPase activity of the individual proteins was between 5- and 10-fold lower than that of the complex.

Delimiting the Function of the C-Terminal Extension of the [NiFe]-Hydrogenase 2 Large Subunit Precursor.

The active site of all [NiFe]-hydrogenases (Hyd) has a bimetallic NiFe(CN)CO cofactor that requires the combined action of several maturation proteins for its biosynthesis and insertion into the precursor form of the large subunit of the enzyme. Cofactor insertion is an intricately controlled process, and the large subunit of almost all Hyd enzymes has a C-terminal oligopeptide extension that is endoproteolytically removed as the final maturation step. This extension might serve either as one of the recognition motifs for the endoprotease, as well as an interaction platform for the maturation proteins, or it could have a structural role to ensure the active site cavity remains open until the cofactor is inserted.

Anaerobic nitrate respiration in the aerobe Streptomyces coelicolor A3(2): helping maintain a proton gradient during dormancy.

Respiratory nitrate reductases (Nar) catalyse the reduction of nitrate to nitrite, coupling this process to energy conservation. The obligate aerobic actinobacterium Streptomyces coelicolor synthesizes three Nar enzymes that contribute to maintenance of a membrane potential when either the mycelium or the spores become hypoxic or anoxic. No growth occurs under such conditions but the bacterium survives the lack of O by remaining metabolically active; reducing nitrate is one means whereby this process is aided.

Hypoxia-induced synthesis of respiratory nitrate reductase 2 of Streptomyces coelicolor A3(2) depends on the histidine kinase OsdK in mycelium but not in spores.

The three nitrate reductases (Nar) of the saprophytic aerobic actinobacterium Streptomyces coelicolor A3(2) contribute to survival when oxygen becomes limiting. In the current study, we focused on synthesis of the Nar2 enzyme, which is the main Nar enzyme present and active in exponentially growing mycelium. Synthesis of Nar2 can, however, also be induced in spores after extended periods of anoxic incubation.