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. Consequently, care must always be taken in interpreting the information gleaned from such studies and must be compared with how the same macromolecules function in vivo, otherwise, discrepancies can arise. The importance of combining reductionist approaches with the study of whole-cell microbial physiology is discussed regarding the long-term aim of understanding how a cell functions in its entirety. This can only be achieved by the continued development of high-resolution structural and multi-omic technologies. It is only by studying the whole cell that we can ever hope to understand how living systems function.

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. coli cells in dependence on FocA. Using a wild-type strain synthesizing native FocA, it was shown that using glucose as a source of formate, DCCD-independent proton efflux was ∼2.5 mmol min-1, while a mutant lacking FocA showed only DCCD-inhibited, FOF1-ATPase-dependent proton-efflux. A strain synthesizing a chromosomally-encoded FocAH209N variant that functions exclusively to translocate formic acid out of the cell, showed a further 20 % increase in FocA-dependent proton efflux relative to the parental strain. Cells synthesizing a FocAT91A variant, which is unable to translocate formic acid out of the cell, showed only DCCD-inhibited proton efflux. When exogenous formate was added, formic acid uptake was shown to be both FocA- and proton motive force-dependent. By measuring rates of H2 production, potassium ion flux and ATPase activity, these data support a role for coupling between formate, proton and K+ ion translocation in maintaining pH and ion gradient homeostasis during fermentation. FocA thus plays a key role in maintaining this homeostatic balance in fermenting cells by bidirectionally translocating formic acid.

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

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. Two decades ago, a small 14-kDa protein called YfiD (now called autonomous glycyl radical cofactor [GrcA]) was shown to be capable of restoring activity to O2-inactivated PflB in vitro; however, GrcA has never been shown to have this function in vivo. By constructing a strain with a chromosomally encoded PflB enzyme variant with a G734A residue exchange, we could show that cells retained near-wild type fermentative growth, as well as formate and H2 production; H2 is derived by enzymatic disproportionation of formate. Introducing a grcA deletion mutation into this strain completely prevented formate and H2 generation and reduced anaerobic growth. We could show that the conserved glycine at position 102 on GrcA was necessary for GrcA to restore PflB activity and that this recovered activity depended on the essential cysteine residues 418 and 419 in the active site of PflB. Together, our findings demonstrate that GrcA is capable of restoring in vivo activity to inactive full-length PflB and support a model whereby GrcA displaces the C-terminal glycyl radical domain to rescue the catalytic function of PflB. IMPORTANCE Many facultative anaerobic microorganisms use glycyl radical enzymes (GREs) to catalyze chemically challenging reactions under anaerobic conditions. Pyruvate formate-lyase (PflB) is a GRE that catalyzes cleavage of the carbon-carbon bond of pyruvate during glucose fermentation. The problem is that glycyl radicals are destroyed readily, especially by oxygen. To protect and restore activity to inactivated PflB, bacteria like Escherichia coli have a small autonomous glycyl radical cofactor protein called GrcA, which functions to rescue inactivated PflB. To date, this proposed function of GrcA has only been demonstrated in vitro. Our data reveal that GrcA rescues and restores enzyme activity to an inactive full-length form of PflB in vivo. These results have important implications for the evolution of radical-based enzyme mechanisms.

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. Therefore, this brief review is intended as a form of redress and aims to highlight the role of this remarkable scientist in establishing the Society and in promoting Microbiology as a discipline. Notwithstanding the significance of these achievements, however, it is her overall impact on the field of 'Chemical Microbiology' and what she achieved through her research that are extraordinary, even by today's standards. Marjory Stephenson recognized that in order to understand a biological system, the 'whole' organism must be considered and this can only be achieved by adopting an interdisciplinary approach: inorganic and organic chemistry, biochemistry, genetics, metabolism and ultimately physiology. Her scientific ethos serves today as a beacon for how scientific research should be conducted, and what we as scientists can learn about how to inspire and mentor the next generation. It is impossible to overstate Marjory Stephenson's scientific legacy, or her overall contribution to Microbiology.

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

FocA translocates formate/formic acid bi-directionally across the cytoplasmic membrane when Escherichia coli grows by fermentation. It remains unclear, however, what physiological benefit is imparted by FocA, because formic acid (pKa=3.75) can diffuse passively across the membrane, especially at low pH. Here, we monitored changes in intra- and extracellular formate levels during batch-culture fermentation, comparing a parental E. coli K-12 strain with its isogenic focA mutant. Our results show that, regardless of the initial pH in the culture, FocA functions to maintain relatively constant intracellular formate levels during growth. Analysis of a strain synthesizing a FocAT91A variant with an exchange in a conserved threonine residue within the translocation pore revealed the strain accumulated formate intracellularly and imported formate poorly, but in a pH-dependent manner, which was different to uptake by native FocA. We conclude that FocA maintains formate homeostasis, using different mechanisms for efflux and uptake of the anion.

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 Escherichia coli, especially at low pH, formate is then disproportionated to CO2 and H2 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. Formate translocation across the cytoplasmic membrane is controlled by the formate channel, FocA, a member of the formate-nitrite transporter (FNT) family of homopentameric anion channels. This review highlights recent advances in our understanding of how FocA helps to maintain intracellular formate and pH homeostasis during fermentation. Efflux and influx of formate/formic acid are distinct processes performed by FocA and both are controlled through protein interaction between FocA's N-terminal domain with PflB. Formic acid efflux by FocA helps to maintain cytoplasmic pH balance during exponential-phase growth. Uptake of formate against the electrochemical gradient (inside negative) is energetically and mechanistically challenging for a fermenting bacterium unless coupled with proton/cation symport. Translocation of formate/formic acid into the cytoplasm necessitates an active FHL complex, whose synthesis also depends on formate. Thus, FocA, FHL and PflB function together to govern formate homeostasis. We explain how FocA achieves efflux of formic acid and propose mechanisms for pH-dependent uptake of formate both with and without proton symport. We propose that FocA displays both channel- and transporter-like behaviour. Whether this translocation behaviour is shared by other members of the FNT family is also discussed.

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

During mixed-acid fermentation, Escherichia coli 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. It is not understood how the direction of formate/formic acid passage through FocA is controlled by pH. A conserved histidine residue (H209) is located within the translocation pore, suggesting that protonation/deprotonation might be linked to the direction of formate translocation. Using a formate-responsive lacZ-based reporter system we monitored changes in formate levels in vivo when H209 in FocA was exchanged for either of the non-protonatable amino acids asparagine or glutamine, which occur naturally in some FNTs. These FocA variants (with N or Q) functioned as highly efficient formate efflux channels and the bacteria could neither accumulate formate nor produce hydrogen gas. Therefore, the data in this study suggest that this central histidine residue within the FocA pore is required for pH-dependent formate uptake into E. coli cells. We also address why H209 is evolutionarily conserved and provide a physiological rationale for the natural occurrence of N/Q variants of FNT channels.

Setting the Stage: Genes Controlling Mechanosensation and Ca2+ Signaling in Escherichia coli.

Although mechanistic understanding of calcium signaling in bacteria remains inchoate, current evidence clearly links Ca2+ signaling with membrane potential and mechanosensation. Adopting a radically new approach, Luder et al. scanned the Keio collection of Escherichia coli gene knockouts (R. Luder, G. N. Bruni, and J. M. Kralj, J Bacteriol 203:e00509-20, 2021, https://doi.org/10.1128/JB.00509-20) to identify mutations that cause changes in Ca2+ transients. They identify genes associating Ca2+ signaling with outer membrane biogenesis, proton motive force, and, surprisingly, long-term DNA damage. Their work has major implications for electrophysiological communication between bacteria and their environment.

Activity of Spore-Specific Respiratory Nitrate Reductase 1 of Streptomyces coelicolor A3(2) Requires a Functional Cytochrome bcc-aa3 Oxidase Supercomplex.

Spores have strongly reduced metabolic activity and are produced during the complex developmental cycle of the actinobacterium Streptomyces coelicolor Resting spores can remain viable for decades, yet little is known about how they conserve energy. It is known, however, that they can reduce either oxygen or nitrate using endogenous electron sources. S. coelicolor uses either a cytochrome bd oxidase or a cytochrome bcc-aa3 oxidase supercomplex to reduce oxygen, while nitrate is reduced by Nar-type nitrate reductases, which typically oxidize quinol directly. Here, we show that in resting spores the Nar1 nitrate reductase requires a functional bcc-aa3 supercomplex to reduce nitrate. Mutants lacking the complete qcr-cta genetic locus encoding the bcc-aa3 supercomplex showed no Nar1-dependent nitrate reduction. Recovery of Nar1 activity was achieved by genetic complementation but only when the complete qcr-cta locus was reintroduced to the mutant strain. We could exclude that the dependence on the supercomplex for nitrate reduction was via regulation of nitrate transport. Moreover, the catalytic subunit, NarG1, of Nar1 was synthesized in the qcr-cta mutant, ruling out transcriptional control. Constitutive synthesis of Nar1 in mycelium revealed that the enzyme was poorly active in this compartment, suggesting that the Nar1 enzyme cannot act as a typical quinol oxidase. Notably, nitrate reduction by the Nar2 enzyme, which is active in growing mycelium, was not wholly dependent on the bcc-aa3 supercomplex for activity. Together, our data suggest that Nar1 functions together with the proton-translocating bcc-aa3 supercomplex to increase the efficiency of energy conservation in resting spores.IMPORTANCEStreptomyces coelicolor forms spores that respire with either oxygen or nitrate, using only endogenous electron donors. This helps maintain a membrane potential and, thus, viability. Respiratory nitrate reductase (Nar) usually receives electrons directly from reduced quinone species; however, we show that nitrate respiration in spores requires a respiratory supercomplex comprising cytochrome bcc oxidoreductase and aa3 oxidase. Our findings suggest that the Nar1 enzyme in the S. coelicolor spore functions together with the proton-translocating bcc-aa3 supercomplex to help maintain the membrane potential more efficiently. Dissecting the mechanisms underlying this survival strategy is important for our general understanding of bacterial persistence during infection processes and of how bacteria might deal with nutrient limitation in the natural environment.

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. The osdRK genes (oxygen stress and development) were recently identified to encode a two-component system important for expression of the nar2 operon in mycelium. OsdK is a predicted histidine kinase and we show here that an osdK mutant completely lacks Nar2 enzyme activity in mycelium. Recovery of Nar2 enzyme activity was achieved by re-introduction of the osdRK genes into the mutant on an integrative plasmid. In anoxically incubated spores, however, the osdK mutant retained the ability to synthesize NarG2, the catalytic subunit of Nar2. We could also demonstrate that synthesis of NarG2 in spores occurred only under hypoxic conditions; anoxia, as well as O2 concentrations significantly higher than 1 % in the gas-phase, failed to result in induction of NarG2 synthesis. Together, these findings indicate that, although Nar2 synthesis in both mycelium and spores is induced by oxygen limitation, different mechanisms control these processes and only Nar2 synthesis in mycelium is under the control of the OsdKR two-component system.

Cytochrome bd Oxidase Has an Important Role in Sustaining Growth and Development of Streptomyces coelicolor A3(2) under Oxygen-Limiting Conditions.

Streptomyces coelicolor A3(2) is a filamentously growing, spore-forming, obligately aerobic actinobacterium that uses both a copper aa3 -type cytochrome c oxidase and a cytochrome bd oxidase to respire oxygen. Using defined knockout mutants, we demonstrated that either of these terminal oxidases was capable of allowing the bacterium to grow and complete its developmental cycle. The genes encoding the bcc complex and the aa3 oxidase are clustered at a single locus. Using Western blot analyses, we showed that the bcc-aa3 oxidase branch is more prevalent in spores than the bd oxidase. The level of the catalytic subunit, CydA, of the bd oxidase was low in spore extracts derived from the wild type, but it was upregulated in a mutant lacking the bcc-aa3 supercomplex. This indicates that cytochrome bd oxidase can compensate for the lack of the other respiratory branch. Components of both oxidases were abundant in growing mycelium. Growth studies in liquid medium revealed that a mutant lacking the bcc-aa3 oxidase branch grew approximately half as fast as the wild type, while the oxygen reduction rate of the mutant remained close to that of the wild type, indicating that the bd oxidase was mainly functioning in controlling electron flux. Developmental defects were observed for a mutant lacking the cytochrome bd oxidase during growth on buffered rich medium plates with glucose as the energy substrate. Evidence based on using the redox-cycling dye methylene blue suggested that cytochrome bd oxidase is essential for the bacterium to grow and complete its developmental cycle under oxygen limitation.IMPORTANCE Respiring with oxygen is an efficient means of conserving energy in biological systems. The spore-forming, filamentous actinobacterium Streptomyces coelicolor grows only aerobically, synthesizing two enzyme complexes for O2 reduction, the cytochrome bcc-aa3 cytochrome oxidase supercomplex and the cytochrome bd oxidase. We show in this study that the bacterium can survive with either of these respiratory pathways to oxygen. Immunological studies indicate that the bcc-aa3 oxidase is the main oxidase present in spores, but the bd oxidase compensates if the bcc-aa3 oxidase is inactivated. Both oxidases are active in mycelia. Growth conditions were identified, revealing that cytochrome bd oxidase is essential for aerial hypha formation and sporulation, and this was linked to an important role of the enzyme under oxygen-limiting conditions.

Analysis of HypD Disulfide Redox Chemistry via Optimization of Fourier Transformed ac Voltammetric Data.

Rapid disulfide bond formation and cleavage is an essential mechanism of life. Using large amplitude Fourier transformed alternating current voltammetry (FTacV) we have measured previously uncharacterized disulfide bond redox chemistry in Escherichia coli HypD. This protein is representative of a class of assembly proteins that play an essential role in the biosynthesis of the active site of [NiFe]-hydrogenases, a family of H2-activating enzymes. Compared to conventional electrochemical methods, the advantages of the FTacV technique are the high resolution of the faradaic signal in the higher order harmonics and the fact that a single electrochemical experiment contains all the data needed to estimate the (very fast) electron transfer rates (both rate constants ≥ 4000 s-1) and quantify the energetics of the cysteine disulfide redox-reaction (reversible potentials for both processes approximately -0.21 ± 0.01 V vs SHE at pH 6). Previously, deriving such data depended on an inefficient manual trial-and-error approach to simulation. As a highly advantageous alternative, we describe herein an automated multiparameter data optimization analysis strategy where the simulated and experimental faradaic current data are compared for both the real and imaginary components in each of the 4th to 12th harmonics after quantifying the charging current data using the time-domain response.

Of mothballs and old yellow enzymes.

Polycyclic aromatic hydrocarbons (PAHs) are persistent and toxic environmental pollutants that accumulate in anoxic habitats. With the exception of naphthalene, nothing is known about the microbial degradation of PAH in these environments. The challenge that must be met in anaerobic PAH degradation is the destabilization of the resonance energy of the aromatic ring system, which requires electrons with very negative redox potentials. Estelmann et al. (2014) identify two enzymes from sulphate-reducing bacteria that perform successive 2-electron reductions of a coenzyme A thioester derivative of naphthalene. The first reduces 2-naphthoyl-CoA to 5,6-dihydro-2-naphthoyl-CoA and the second generates 5,6,7,8-tetrahydro-2-naphthoyl-CoA. Surprisingly, both enzymes are members of the 'old yellow enzyme' (OYE) family of flavoproteins. Neither uses adenosine triphosphate to achieve reduction of the aromatic ring. Typically, OYEs have flavin mononucleotide as cofactor and use nicotinamide adenine dinucleotide (phosphate) as reductant. Both ring reductases have flavin adenine dinucleotide and an iron-sulphur cluster as additional cofactors. Evidence also suggests that in the sulphate-reducing bacteria, these enzymes form a complex, allowing substrate channeling. The findings of this superb study represent unprecedented biochemistry. This work sheds light on how microbes meet the thermodynamic challenges of life at the redox limit.

Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement.

Dehalococcoides mccartyi strain CBDB1 is an obligate organohalide-respiring bacterium using only hydrogen as electron donor and halogenated organics as electron acceptor. Here, we studied proteins involved in the respiratory chain under non-denaturing conditions. Using blue native gel electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250-270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster containing subunit (CbdbA131) of the hydrogen uptake hydrogenase (Hup). No colocalisation between the catalytically active subunits of hydrogenase and reductive dehalogenase was found. By two-dimensional BN/SDS-PAGE the stability of the complex towards detergents was assessed, demonstrating stepwise disintegration with increasing detergent concentrations. Chemical cross-linking confirmed the presence of a higher molecular mass reductive dehalogenase protein complex composed of RdhA, CISM I and Hup hydrogenase and proved to be a potential tool for stabilising protein-protein interactions of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi.

Phosphate and oxygen limitation induce respiratory nitrate reductase 3 synthesis in stationary-phase mycelium of Streptomyces coelicolor A3(2).

The saprophytic actinobacterium Streptomyces coelicolor A3(2) requires oxygen for filamentous growth. Surprisingly, the bacterium also synthesizes three active respiratory nitrate reductases (Nar), which are believed to contribute to survival, or general fitness, of the bacterium in soil when oxygen becomes limiting. In this study, we analysed Nar3 and showed that activity of the enzyme is restricted to stationary-phase mycelium of S. coelicolor. Phosphate limitation was shown to be necessary for induction of enzyme synthesis. Nar3 synthesis was inhibited by inclusion of 20 mM phosphate in a defined 'switch assay' in which highly dispersed mycelium from exponentially growing cultures was shifted to neutral MOPS-glucose buffer to induce Nar3 synthesis and activity. Quantitative assessment of nar3 transcripts revealed a 30-fold induction of gene expression in stationary-phase mycelium. Transcript levels in stationary-phase mycelium incubated with phosphate were reduced by a little more than twofold, suggesting that the negative influence of phosphate on Nar3 synthesis was mainly at the post-transcriptional level. Furthermore, it was demonstrated that oxygen limitation was necessary to induce high levels of Nar3 activity. However, an abrupt shift from aerobic to anaerobic conditions prevented appearance of Nar3 activity. This suggests that the bacterium regulates Nar3 synthesis in response to the energy status of the mycelium. Nitrate had little impact on regulation of the Nar3 level. Together, these data identify Nar3 as a stationary-phase nitrate reductase in S. coelicolor and demonstrate that enzyme synthesis is induced in response to both phosphate limitation and hypoxia.

Coordination of Synthesis and Assembly of a Modular Membrane-Associated [NiFe]-Hydrogenase Is Determined by Cleavage of the C-Terminal Peptide.

UNLABELLED: During biosynthesis of [NiFe]-hydrogenase 2 (Hyd-2) of Escherichia coli, a 15-amino-acid C-terminal peptide is cleaved from the catalytic large subunit precursor, pro-HybC. This peptide is removed only after NiFe(CN)2CO cofactor insertion by the Hyp accessory protein machinery has been completed, suggesting that it has a regulatory function during enzyme maturation. We show here that in hyp mutants that fail to synthesize and insert the NiFe cofactor, and therefore retain the peptide, the Tat (twin-arginine translocon) signal peptide on the small subunit HybO is not removed and the subunit is degraded. In a mutant lacking the large subunit, the Tat signal peptide was also not removed from pre-HybO, indicating that the mature large subunit must actively engage the small subunit to elicit Tat transport. We validated the proposed regulatory role of the C-terminal peptide in controlling enzyme assembly by genetically removing it from the precursor of HybC, which allowed assembly and Tat-dependent membrane association of a HybC-HybO heterodimer lacking the NiFe(CN)2CO cofactor. Finally, genetic transfer of the C-terminal peptide from pro-HyaB, the large subunit of Hyd-1, onto HybC did not influence its dependence on the accessory protein HybG, a HypC paralog, or the specific protease HybD. This indicates that the C-terminal peptide per se is not required for interaction with the Hyp machinery but rather suggests a role of the peptide in maintaining a conformation of the protein suitable for cofactor insertion. Together, our results demonstrate that the C-terminal peptide on the catalytic subunit controls biosynthesis, assembly, and membrane association of Hyd-2. IMPORTANCE: [NiFe]-hydrogenases are multisubunit enzymes with a catalytic subunit containing a NiFe(CN)2CO cofactor. Results of previous studies suggested that after synthesis and insertion of the cofactor by the Hyp accessory proteins, this large subunit changes conformation upon proteolytic removal of a short peptide from its C terminus. We show that removal of this peptide is necessary to allow the cleavage of the Tat signal peptide from the small subunit with concomitant membrane association of the heterodimer to occur. Genetic removal of the C-terminal peptide from the large subunit allowed productive interaction with the small subunit and Tat-dependent membrane insertion of a NiFe cofactor-free enzyme. Results based on swapping of C-terminal peptides between hydrogenases suggest that this peptide governs enzyme assembly via a conformational switch.

Physiology and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2-producing reactions in Escherichia coli.

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H2 to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H2 evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H2 evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H2-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H2 oxidation or H2 evolution in whole cells, reversible H2-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H2 evolution mode. Our findings suggest that Hyd-2 switches between H2-consuming and H2-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H2 evolution from glycerol requires reverse electron transport.

Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family.

Six Hyp maturation proteins (HypABCDEF) are conserved in micro-organisms that synthesize [NiFe]-hydrogenases (Hyd). Of these, the HypC chaperones interact directly with the apo-form of the catalytically active large subunit of Hyd enzymes and are believed to transfer the Fe(CN)2CO moiety of the bimetallic cofactor from the Hyp machinery to this large subunit. In E. coli, HypC is specifically required for maturation of Hyd-3 while its paralogue, HybG, is specifically required for Hyd-2 maturation; either HypC or HybG can mature Hyd-1. In this study, we demonstrate that the products of the hypABFCDE operon from the deeply branching hydrogen-dependent and obligate organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 were capable of maturing and assembling active Hyd-1, Hyd-2 and Hyd-3 in an E. coli hyp mutant. Maturation of Hyd-1 was less efficient, presumably because HypB of E. coli was necessary to restore optimal enzyme activity. In a reciprocal maturation study, the highly O2-sensitive H2-uptake HupLS [NiFe]-hydrogenase from D. mccartyi CBDB1 was also synthesized in an active form in E. coli. Together, these findings indicated that HypC from D. mccartyi CBDB1 exhibits promiscuity in its large subunit interaction in E. coli. Based on these findings, we generated amino acid variants of E. coli HybG capable of partial recovery of Hyd-3-dependent H2 production in a hypC hybG double null mutant. Together, these findings identify amino acid regions in HypC accessory proteins that specify interaction with the large subunits of hydrogenase and demonstrate functional compatibility of Hyp accessory protein machineries.

Oxygen-dependent control of respiratory nitrate reduction in mycelium of Streptomyces coelicolor A3(2).

Several members of the obligately aerobic genus Streptomyces are able to reduce nitrate, catalyzed by Nar-type respiratory nitrate reductases. A unique feature of Streptomyces coelicolor A3(2) compared with other streptomycetes is that it synthesizes three nonredundant Nar enzymes. In this study, we show that Nar2 is the main Nar enzyme active in mycelium and could characterize the conditions governing its synthesis. Nar2 was present at low levels in aerobically cultivated mycelium, but synthesis was induced when cultures were grown under oxygen limitation. Growth in the presence of high oxygen concentrations prevented the induction of Nar2 synthesis. Equally, an abrupt shift from aerobiosis to anaerobiosis did not result in the immediate induction of Nar2 synthesis. This suggests that the synthesis of Nar2 is induced during a hypoxic downshift, probably to allow maintenance of a proton gradient during the transition to anaerobiosis. Although no Nar2 could be detected in freshly harvested mature spores, synthesis of the enzyme could be induced after long-term (several days) incubation of these resting spores under anaerobic conditions. Induction of Nar2 synthesis in spores was linked to transcriptional control. Nar2 activity in whole mycelium was strictly dependent on the presence of a putative nitrate transporter, NarK2. The oxygen-dependent inhibition of nitrate reduction by Nar2 was mediated by NarK2-dependent nitrate:nitrite antiport. This antiport mechanism likely prevents the accumulation of toxic nitrite in the cytoplasm. A deletion of the narK2 gene had no effect on Nar1-dependent nitrate reduction in resting spores. Together, our results indicate redox-dependent transcriptional and posttranslational control of nitrate reduction by Nar2.

Mapping cell envelope and periplasm protein interactions of Escherichia coli respiratory formate dehydrogenases by chemical cross-linking and mass spectrometry.

During anaerobic growth Escherichia coli synthesizes two large, highly homologous respiratory formate dehydrogenases (Fdh's), Fdh-N and Fdh-O, which are associated with the inner membrane but have their respective active site located within the periplasm. The Fdh-N enzyme extends 90 Å into the periplasmic compartment, which in E. coli ranges between 100 and 150 Å from the inner to the outer membrane leaflet. To date, little is known about the interaction partners of Fdh-N and Fdh-O in the periplasmic space that might be involved in stabilizing these enzymes after maturation and translocation across the cytoplasmic membrane has occurred. To address this question, we performed chemical cross-linking in combination with mass spectrometry. We present for the first time the identification of cell envelope interaction partners of Fdh-N and -O from anaerobically grown E. coli using a heterobifunctional amine/photo-reactive cross-linker followed by mass spectrometric analysis of the cross-linked products. We additionally mapped the interface regions within the Fdh/protein complexes for four selected Fdh-binding partners, the chaperone Skp, the l,d-transpeptidase ErfK, OppA, and TolB. Our work yields first structural and functional insights into the mechanisms that support the postmaturation of the multisubunit enzymes Fdh-N and Fdh-O in the periplasm of E. coli.