Expression of a metagenome-derived fumarate reductase from marine microorganisms and its characterization

A potential novel fumarate reductase gene designated frd1A was isolated by screening a marine metagenomic library through a sequence-based strategy. Sequence analyses indicated that Frd1A and other putative fumarate reductases were closely related. The putative fumarate reductase gene was subcloned into a pETBlue-2 vector and expressed in Escherichia coli Tuner(DE3)pLac? cells. The recombinant protein was purified to homogeneity. Functional characterization by high-performance liquid chromatography demonstrated that the recombinant Frd1A protein could catalyze the hydrogenation of fumarate to succinate acid. The Frd1A protein displayed an optimal activity at pH 7.0 and 28 °C, which could be stimulated by adding metal ions such as Zn²⁺ and Mg²⁺. The Frd1A enzyme showed a comparable affinity and catalytic efficiency under optimal reaction conditions: k _m?=0.227 mmol/L, v _max= 29.9 U/mg, and k _cat/k _m=5.44?×?10⁴ per mol/s. The identification of Frd1A protein underscores the potential of marine metagenome screening for novel biomolecules.     

Reduction of fumarate,mesaconate and crotonate by Mfr,a novel oxygen‐regulated periplasmic reductase in Campylobacter jejuni

Methylmenaquinol : fumarate reductase (Mfr) is a newly recognized type of fumarate reductase present in some ε‐proteobacteria, where the active site subunit (MfrA) is localized in the periplasm, but for which a physiological role has not been identified. We show that the Campylobacter jejuni mfrABE operon is transcribed from a single promoter, with the mfrA gene preceded by a small open reading‐frame (mfrX) encoding a C. jejuni‐specific polypeptide of unknown function. The growth characteristics and enzyme activities of mutants in the mfrA and menaquinol : fumarate reductase A (frdA) genes show that the cytoplasmic facing Frd enzyme is the major fumarate reductase under oxygen limitation. The Mfr enzyme is shown to be necessary for maximal rates of growth by fumarate respiration and rates of fumarate reduction in intact cells measured by both viologen assays and ¹H‐NMR were slower in an mfrA mutant. As periplasmic fumarate reduction does not require fumarate/succinate antiport, Mfr may allow more efficient adaptation to fumarate‐dependent growth. However, a further rationale for the periplasmic location of Mfr is suggested by the observation that the enzyme also reduces the fumarate analogues mesaconate and crotonate; fermentation products of anaerobes with which C. jejuni shares its gut environment, that are unable to be transported into the cell. Both MfrA and MfrB subunits were localized in the periplasm by immunoblotting and 2D‐gel electrophoresis, but an mfrE mutant accumulated unprocessed MfrA in the cytoplasm, suggesting a preassembled MfrABE holoenzyme has to be recognized by the TAT system for translocation to occur. Gene expression studies in chemostat cultures following an aerobic‐anaerobic shift showed that mfrA is highly upregulated by oxygen limitation, as would be experienced in vivo. Our results indicate that in addition to a role in fumarate respiration, Mfr allows C. jejuni to reduce analogous substrates specifically present in the host gut environment.     

A conserved lysine residue controls iron–sulfur cluster redox chemistry in Escherichia coli fumarate reductase

The Escherichia coli respiratory complex II paralogs succinate dehydrogenase (SdhCDAB) and fumarate reductase (FrdABCD) catalyze interconversion of succinate and fumarate coupled to quinone reduction or oxidation, respectively. Based on structural comparison of the two enzymes, equivalent residues at the interface between the highly homologous soluble domains and the divergent membrane anchor domains were targeted for study. This included the residue pair SdhB-R205 and FrdB-S203, as well as the conserved SdhB-K230 and FrdB-K228 pair. The close proximity of these residues to the [3Fe–4S] cluster and the quinone binding pocket provided an excellent opportunity to investigate factors controlling the reduction potential of the [3Fe–4S] cluster, the directionality of electron transfer and catalysis, and the architecture and chemistry of the quinone binding sites. Our results indicate that both SdhB-R205 and SdhB-K230 play important roles in fine tuning the reduction potential of both the [3Fe–4S] cluster and the heme. In FrdABCD, mutation of FrdB-S203 did not alter the reduction potential of the [3Fe–4S] cluster, but removal of the basic residue at FrdB-K228 caused a significant downward shift (> 100 mV) in potential. The latter residue is also indispensable for quinone binding and enzyme activity. The differences observed for the FrdB-K228 and Sdh-K230 variants can be attributed to the different locations of the quinone binding site in the two paralogs. Although this residue is absolutely conserved, they have diverged to achieve different functions in Frd and Sdh.     

Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum

Corynebacterium glutamicum, an established industrial amino acid producer, has been genetically modified for efficient succinate production from the renewable carbon source glucose under fully aerobic conditions in minimal medium. The initial deletion of the succinate dehydrogenase genes (sdhCAB) led to an accumulation of 4.7 g l^?1 (40 mM) succinate as well as high amounts of acetate (125 mM) as by‐product. By deleting genes for all known acetate‐producing pathways (pta‐ackA, pqo and cat) acetate production could be strongly reduced by 83% and succinate production increased up to 7.8 g l^?1 (66 mM). Whereas overexpression of the glyoxylate shunt genes (aceA and aceB) or overproduction of the anaplerotic enzyme pyruvate carboxylase (PCx) had only minor effects on succinate production, simultaneous overproduction of pyruvate carboxylase and PEP carboxylase resulted in a strain that produced 9.7 g l^?1 (82 mM) succinate with a specific productivity of 1.60 mmol g (cdw)^?1 h^?1. This value represents the highest productivity among currently described aerobic bacterial succinate producers. Optimization of the production conditions by decoupling succinate production from cell growth using the most advanced producer strain (C. glutamicumΔpqoΔpta‐ackAΔsdhCABΔcat/pAN6‐pyc^P458Sppc) led to an additional increase of the product yield to 0.45 mol succinate mol^?1 glucose and a titre of 10.6 g l^?1 (90 mM) succinate.     

Investigation of the fumarate metabolism of the syntrophic propionate-oxidizing bacterium strain MPOB

The growth of the syntrophic propionate-oxidizing bacterium strain MPOB in pure culture by fumarate disproportionation into carbon dioxide and succinate and by fumarate reduction with propionate, formate or hydrogen as electron donor was studied. The highest growth yield, 12.2 g dry cells/mol fumarate, was observed for growth by fumarate disproportionation. In the presence of hydrogen, formate or propionate, the growth yield was more than twice as low: 4.8, 4.6, and 5.2 g dry cells/mol fumarate, respectively. The location of enzymes that are involved in the electron transport chain during fumarate reduction in strain MPOB was analyzed. Fumarate reductase, succinate dehydrogenase, and ATPase were membrane-bound, while formate dehydrogenase and hydrogenase were loosely attached to the periplasmic side of the membrane. The cells contained cytochrome c, cytochrome b, menaquinone-6 and menaquinone-7 as possible electron carriers. Fumarate reduction with hydrogen in membranes of strain MPOB was inhibited by 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO). This inhibition, together with the activity of fumarate reductase with reduced 2,3-dimethyl-1,4-naphtoquinone (DMNH₂) and the observation that cytochrome b of strain MPOB was oxidized by fumarate, suggested that menequinone and cytochrome b are involved in the electron transport during fumarate reduction in strain MPOB. The growth yields of fumarate reduction with hydrogen or formate as electron donor were similar to the growth yield of Wolinella succinogenes. Therefore, it can be assumed that strain MPOB gains the same amount of ATP from fumarate reduction as W. succinogenes, i.e. 0.7 mol ATP/mol fumarate. This value supports the hypothesis that syntrophic propionate-oxidizing bacteria have to invest two-thirds of an ATP via reversed electron transport in the succinate oxidation step during the oxidation of propionate. The same electron transport chain that is involved in fumarate reduction may operate in the reversed direction to drive the energetically unfavourable oxidation of succinate during syntrophic propionate oxidation since (1) cytochrome b was reduced by succinate and (2) succinate oxidation was similarly inhibited by HOQNO as fumarate reduction. Received: 18 March 1997 / Accepted: 10 November 1997     

The Dual-Functioning Fumarate Reductase Is the Sole Succinate:Quinone Reductase in Campylobacter jejuni and Is Required for Full Host Colonization

Campylobacter jejuni encodes all the enzymes necessary for a complete oxidative tricarboxylic acid (TCA) cycle. Because of its inability to utilize glucose, C. jejuni relies exclusively on amino acids as the source of reduced carbon, and they are incorporated into central carbon metabolism. The oxidation of succinate to fumarate is a key step in the oxidative TCA cycle. C. jejuni encodes enzymes annotated as a fumarate reductase (Cj0408 to Cj0410) and a succinate dehydrogenase (Cj0437 to Cj0439). Null alleles in the genes encoding each enzyme were constructed. Both enzymes contributed to the total fumarate reductase activity in vitro. The frdA::cat⁺ strain was completely deficient in succinate dehydrogenase activity in vitro and was unable to perform whole-cell succinate-dependent respiration. The sdhA::cat⁺ strain exhibited wild-type levels of succinate dehydrogenase activity both in vivo and in vitro. These data indicate that Frd is the only succinate dehydrogenase in C. jejuni and that the protein annotated as a succinate dehydrogenase has been misannotated. The frdA::cat⁺ strain was also unable to grow with the characteristic wild-type biphasic growth pattern and exhibited only the first growth phase, which is marked by the consumption of aspartate, serine, and associated organic acids. Substrates consumed in the second growth phase (glutamate, proline, and associated organic acids) were not catabolized by the the frdA::cat⁺ strain, indicating that the oxidation of succinate is a crucial step in metabolism of these substrates. Chicken colonization trials confirmed the in vivo importance of succinate oxidation, as the frdA::cat⁺ strain colonized chickens at significantly lower levels than the wild type, while the sdhA::cat⁺ strain colonized chickens at wild-type levels.Campylobacter jejuni causes approximately two million cases of bacterial gastroenteritis in the United States annually (34). Humans are most often infected due to cross-contamination resulting from improper handling of poultry (27), which is the natural habitat of C. jejuni (28). The eradication of C. jejuni from poultry flocks is an important goal in reducing the number of campylobacteriosis cases.C. jejuni can rely solely on catabolism of small organic acids and amino acids as a carbon and energy source, and the products of this catabolism are used for glycolysis and the tricarboxylic acid (TCA) cycle (15, 29). Fumarate and succinate are key intermediates in the TCA cycle, and the interconversion of these compounds is a vital process in organisms that use the TCA cycle for central carbon metabolism. C. jejuni encodes a complete oxidative TCA cycle, which converts TCA intermediates (carboxylic acids) to CO₂, ATP, and reducing equivalents. One of the conversion steps, oxidation of succinate to fumarate, forms a reducing equivalent and is required for a complete cycle. Reduction of fumarate to succinate also occurs as part of the reductive TCA cycle, and this carbon fixation pathway has been proposed to be utilized by ɛ-proteobacteria found in deep-sea hydrothermal vents (3). C. jejuni encodes many of the reversible enzymes necessary for the reductive TCA cycle, including 2-oxoglutarate ferredoxin oxidoreductase (encoded by oorDABC) and pyruvate carboxylase (encoded by pycA and pycB) (29); however, C. jejuni does not encode an ATP citrate lyase, which is required for full cyclic reductive carboxylation (3). The fumarate-succinate interconversion is also involved in respiration (11), and fumarate has specifically been implicated as an electron acceptor that is an alternative to oxygen in other ɛ-proteobacteria (5, 17).C. jejuni encodes an enzyme which is annotated as a fumarate reductase (Cj0408 to Cj0410) and an enzyme which is annotated as a succinate dehydrogenase (Cj0437 to Cj0439) (29). Both of these enzymes are part of a large family of proteins called the succinate:quinone oxidoreductases (SQRs). These compounds are membrane-bound enzymes that either catalyze the two-electron oxidation of succinate to the two-electron reduction of quinone/quinol or, in the reverse direction, couple the oxidation of quinol/quinone to the reduction of fumarate to succinate. The amino acid sequence, however, does not dictate the in vivo function (18), and in characterized organisms like Escherichia coli both enzymes are able to reduce fumarate and oxidize succinate, albeit with a preference for one substrate (6, 21).The SQRs can be divided into three distinct classes based on function, all of which have similar subunit compositions and primary amino acid sequences. Class 1 SQRs couple the oxidation of succinate to the reduction of a high-redox-potential quinone like ubiquinone in vivo. Class 2 SQRs are the quinol:fumarate reductases, which couple the oxidation of menaquinol to the reduction of fumarate. And class 3 SQRs couple the oxidation of succinate to the reduction of a low-potential quinone, such as menaquinone, in vivo (11). Although each class has shared motifs, the in vivo function of an SQR enzyme cannot be resolved based on the primary sequence and must be determined experimentally. Fumarate reductase (Frd) activity has been reported to occur in the particulate fraction of C. jejuni cell lysates, and addition of formate to whole cells increased Frd activity (38), which implies that there is an active electron transport pathway. However, C. jejuni is unable to utilize fumarate as an alternative electron acceptor under anaerobic conditions (37, 41). C. jejuni can also use succinate as an electron donor to a respiratory quinone (12), which has been identified as either a menaquinone-6 or methylmenaquinone-6 (4). Yet succinate oxidation via menaquinone is an endergonic reaction; succinate has a redox midpoint potential (E_m) of 30 mV, and menaquinone is more electronegative (E_m = −80 mV). Although succinate oxidation coupled to menaquinone reduction would be an “uphill” reaction, class 3 SQRs can catalyze this reaction. Studies of gram-positive bacteria belonging to the genus Bacillus, as well as studies of sulfate-reducing bacteria, have shown that oxidation of succinate through menaquinone is driven by reverse transmembrane electron transport (18, 36, 45), and it is hypothesized that C. jejuni behaves similarly. The C. jejuni Frd enzyme contains three subunits, FrdC, FrdA, and FrdB, and the gene order in the operon is similar to that in Wolinella succinogenes (16, 19) and Helicobacter pylori (1, 9, 40). Based on Frd enzymes of other bacteria, FrdC (Cj0408) is the membrane anchor and diheme cytochrome b, FrdA (Cj0409) is a flavoprotein where the reduction of fumarate to succinate occurs, and FrdB (Cj0410) is an Fe-S protein (29). The succinate dehydrogenase of C. jejuni is also composed of three subunits, SdhABC encoded by Cj0437 to Cj0439 (29). SdhA is annotated as a succinate dehydrogenase flavoprotein subunit, SdhB is a putative succinate dehydrogenase Fe-S protein, and SdhC is a putative succinate dehydrogenase subunit C. According to ClustalW pairwise alignment, FrdA and SdhA of C. jejuni share 29% identity, FrdB and SdhB share 18% identity, and FrdC and SdhC share 13% identity.A better understanding of the C. jejuni TCA cycle may help identify metabolic pathways that are crucial to C. jejuni''s ability to thrive in poultry. The roles of the C. jejuni fumarate reductase and succinate dehydrogenase in the TCA cycle and respiration were investigated. Both enzymes contribute to the total fumarate reductase activity. We determined that the protein annotated as the fumarate reductase functions as the sole succinate dehydrogenase and that this enzyme is required for full colonization of chickens by C. jejuni. The sdh operon has been misannotated as the enzyme that it encodes exhibits no succinate dehydrogenase activity, as has recently been reported to be the case for the annotated succinate dehydrogenase of W. succinogenes (14).     

Fumarate metabolism and the microaerophily of Campylobacter species.

(1) The role of fumarate metabolism in the microaerophily of the Campylobacter genus and the effects of therapeutic agents against it were investigated. (2) NMR spectroscopy was employed to determine the properties of Campylobacter fumarase (Fum) and fumarate reductase (Frd). Radiotracer analysis was used to determine the production of carbon dioxide by Campylobacter cells. Standard microbiological techniques were used to measure the effects of environmental conditions and inhibitors on bacterial growth. (3) All Campylobacter species tested showed both Fum and Frd activities. Frd activity was observed with or without the addition of an exogenous electron donor in the particulate fractions obtained from lysates. Fumarate was oxidized to carbon dioxide via the acetyl-CoA cleavage pathway. The genes encoding proteins involved in fumarate metabolism were identified in the Campylobacter jejuni genome. Cells grew better in atmospheres with 5 and 10% oxygen levels. Fum activity was the same in cultures grown under different oxygen tensions and did not vary with the age of cultures. Frd activity was higher in cultures which grew at faster rates and decreased with the age of cultures. Four Frd inhibitors showed bactericidal effects against Campylobacter spp. with different potencies. The relative strengths of inhibition of the compounds followed the same order as the bactericidal effects. (4) The results suggested that Frd and Fum are constitutive and play a fundamental role in these microaerophiles which show characteristics of anaerobic metabolism, and that the Frd inhibitors tested would not be of therapeutic use.     

Isolation and Properties of Fumarate Reductase Mutants of Escherichia coli

Escherichia coli produces two enzymes which interconvert succinate and fumarate: succinate dehydrogenase, which is adapted to an oxidative role in the tricarboxylic acid cycle, and fumarate reductase, which catalyzes the reductive reaction more effectively and allows fumarate to function as an electron acceptor in anaerobic growth. A glycerol plus fumarate medium was devised for the selection of mutants (frd) lacking a functional fumarate reductase by virtue of their inability to use fumarate as an anaerobic electron acceptor. Most of the mutants isolated contained less than 1% of the parental fumarate reduction activity. Measurements of the fumarate reduction and succinate oxidation activities of parental strains and frd mutants after aerobic and anaerobic growth indicated that succinate dehydrogenase was completely repressed under anaerobic conditions, the assayable succinate oxidation activity being due to fumarate reductase acting reversibly. Fumarate reductase was almost completely repressed under aerobic conditions, although glucose relieved this repression to some extent. The mutations, presumably in the structural gene (frd) for fumarate reductase, were located at approximately 82 min on the E. coli chromosome by conjugation and transduction with phage P1. frd is very close to the ampA locus, and the order of markers in this region was established as ampA-frd-purA.     

RNA synthesis during morphogenesis of the fungusMucor racemosus

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO₂. Active CO₂ exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.     

Flavinylation and Assembly of Succinate Dehydrogenase Are Dependent on the C-terminal Tail of the Flavoprotein Subunit

The enzymatic function of succinate dehydrogenase (SDH) is dependent on covalent attachment of FAD on the ∼70-kDa flavoprotein subunit Sdh1. We show presently that flavinylation of the Sdh1 subunit of succinate dehydrogenase is dependent on a set of two spatially close C-terminal arginine residues that are distant from the FAD binding site. Mutation of Arg⁵⁸² in yeast Sdh1 precludes flavinylation as well as assembly of the tetrameric enzyme complex. Mutation of Arg⁶³⁸ compromises SDH function only when present in combination with a Cys⁶³⁰ substitution. Mutations of either Arg⁵⁸² or Arg⁶³⁸/Cys⁶³⁰ do not markedly destabilize the Sdh1 polypeptide; however, the steady-state level of Sdh5 is markedly attenuated in the Sdh1 mutant cells. With each mutant Sdh1, second-site Sdh1 suppressor mutations were recovered in Sdh1 permitting flavinylation, stabilization of Sdh5 and SDH tetramer assembly. SDH assembly appears to require FAD binding but not necessarily covalent FAD attachment. The Arg residues may be important not only for Sdh5 association but also in the recruitment and/or guidance of FAD and or succinate to the substrate site for the flavinylation reaction. The impaired assembly of SDH with the C-terminal Sdh1 mutants suggests that FAD binding is important to stabilize the Sdh1 conformation enabling association with Sdh2 and the membrane anchor subunits.     

The quaternary structure of the Saccharomyces cerevisiae succinate dehydrogenase. Homology modeling, cofactor docking, and molecular dynamics simulation studies

Succinate dehydrogenases and fumarate reductases are complex mitochondrial or bacterial respiratory chain proteins with remarkably similar structures and functions. Succinate dehydrogenase oxidizes succinate and reduces ubiquinone using a flavin adenine dinucleotide cofactor and iron-sulfur clusters to transport electrons. A model of the quaternary structure of the tetrameric Saccharomyces cerevisiae succinate dehydrogenase was constructed based on the crystal structures of the Escherichia coli succinate dehydrogenase, the E. coli fumarate reductase, and the Wolinella succinogenes fumarate reductase. One FAD and three iron-sulfur clusters were docked into the Sdh1p and Sdh2p catalytic dimer. One b-type heme and two ubiquinone or inhibitor analog molecules were docked into the Sdh3p and Sdh4p membrane dimer. The model is consistent with numerous experimental observations. The calculated free energies of inhibitor binding are in excellent agreement with the experimentally determined inhibitory constants. Functionally important residues identified by mutagenesis of the SDH3 and SDH4 genes are located near the two proposed quinone-binding sites, which are separated by the heme. The proximal quinone-binding site, located nearest the catalytic dimer, has a considerably more polar environment than the distal site. Alternative low energy conformations of the membrane subunits were explored in a molecular dynamics simulation of the dimer embedded in a phospholipid bilayer. The simulation offers insight into why Sdh4p Cys-78 may be serving as the second axial ligand for the heme instead of a histidine residue. We discuss the possible roles of heme and of the two quinone-binding sites in electron transport.     

Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z

Actinobacillus sp. 130Z fermented glucose to the major products succinate, acetate, and formate. Ethanol was formed as a minor fermentation product. Under CO₂-limiting conditions, less succinate and more ethanol were formed. The fermentation product ratio remained constant at pH values from 6.0 to 7.4. More succinate was produced when hydrogen was present in the gas phase. Actinobacillus sp. 130Z grew at the expense of fumarate and l-malate reduction, with hydrogen as an electron donor. Other substrates such as more-reduced carbohydrates (e.g., d-sorbitol) resulted in higher succinate and/or ethanol production. Actinobacillus sp. 130Z contained the key enzymes involved in the Embden-Meyerhof-Parnas and the pentose-phosphate pathways and contained high levels of phosphoenolpyruvate (PEP) carboxykinase, malate dehydrogenase, fumarase, fumarate reductase, pyruvate kinase, pyruvate formate-lyase, phosphotransacetylase, acetate kinase, malic enzyme, and oxaloacetate decarboxylase. The levels of PEP carboxykinase, malate dehydrogenase, and fumarase were significantly higher in Actinobacillus sp. 130Z than in Escherichia coli K-12 and accounted for the differences in succinate production. Key enzymes in end product formation in Actinobacillus sp. 130Z were regulated by the energy substrates. Received: 2 September 1996 / Accepted: 10 January 1997     

Succinate Dehydrogenase is the Regulator of Respiration in Mycobacterium tuberculosis

In chronic infection, Mycobacterium tuberculosis bacilli are thought to enter a metabolic program that provides sufficient energy for maintenance of the protonmotive force, but is insufficient to meet the demands of cellular growth. We sought to understand this metabolic downshift genetically by targeting succinate dehydrogenase, the enzyme which couples the growth processes controlled by the TCA cycle with the energy production resulting from the electron transport chain. M. tuberculosis contains two operons which are predicted to encode succinate dehydrogenase enzymes (sdh-1 and sdh-2); we found that deletion of Sdh1 contributes to an inability to survive long term stationary phase. Stable isotope labeling and mass spectrometry revealed that Sdh1 functions as a succinate dehydrogenase during aerobic growth, and that Sdh2 is dispensable for this catalysis, but partially overlapping activities ensure that the loss of one enzyme can incompletely compensate for loss of the other. Deletion of Sdh1 disturbs the rate of respiration via the mycobacterial electron transport chain, resulting in an increased proportion of reduced electron carrier (menaquinol) which leads to increased oxygen consumption. The loss of respiratory control leads to an inability to recover from stationary phase. We propose a model in which succinate dehydrogenase is a governor of cellular respiration in the adaptation to low oxygen environments.     

Anaerobic growth of Escherichia coli on d-tartrate depends on the fumarate carrier DcuB and fumarase, rather than the l-tartrate carrier TtdT and l-tartrate dehydratase

Escherichia coli is able to grow under anaerobic conditions on D: -tartrate when glycerol is supplied as an electron donor (D-tartrate fermentation). D-Tartrate was converted to succinate. Growth was lost in strains deficient for DcuB, the fumarate/succinate antiporter of fumarate respiration. The L-tartrate/succinate antiporter TtdT of L-tartrate fermentation, or the C4-dicarboxylate carriers DcuA and DcuC, were not able to support D-tartrate transport and fermentation. Deletion of fumB demonstrated, that fumarase B is required for growth on D-tartrate. The mutant lost most (about 79%) of D-tartrate dehydratase activity. L-Tartrate dehydratase (TtdAB), and fumarase A or C, showed no or only a small contribution to D-tartrate dehydratase activity. Therefore D-tartrate is metabolised by a sequence of reactions analogous to that from L-tartrate fermentation, including dehydration to oxaloacetate, which is then converted to malate, fumarate and succinate. The stereoisomer specific carrier TtdT and dehydratase TtdAB of L-tartrate fermentation are substituted by enzymes from general anaerobic fumarate metabolism, the antiporter DcuB and fumarase B, which have a broader substrate specificity. No D-tartrate specific carriers and enzymes are involved in the pathway.     

Turbellaria Phagocata sibirica: Some Enzymes of Carbohydrate and Energy Metabolisms

Activities and properties of some enzymes of carbohydrate and energy metabolisms in free-living turbellaria Phagocata sibirica are studied. The enzymes are studied in various subcellular fractions. A high activity of hexokinase is accompanied by high activity of glucose-6-phosphate dehydrogenase (G6PDG). The level of pyruvate kinase activity is sufficient to provide dissimilation of phosphoenolpyruvate with formation of pyruvate. P. sibirica has highly-active lactate dehydrogenase (LDH) and malate dehydrogenase (MDH); a predominance of MDH activity over LDH and a low activity of phosphoenolpyruvate carboxykinase is revealed. NADP-dependent isocitrate dehydrogenase is found, which is activated by Mn²⁺ and Mg²⁺ and inhibited by salts of heavy metals and p-chloromercuribenzoate. Activities and properties of -ketoglutarate dehydrogenase, succinate dehydrogenase (SDH), and fumarate reductase are studied, and it is concluded that in P. sibirica there is the system of succinate oxidation, whereas the system of fumarate reduction into succinate is absent. Mitochondrial and microsomal fractions from P. sibirica had Mg²⁺- and Ca²⁺-dependent adenosine triphosphatases.     

Sodium-dependent succinate decarboxylation by a new anaerobic bacterium belonging to the genus Peptostreptococcus

An anaerobic bacterium was isolated from a polluted sediment, with succinate and yeast extract as carbon and energy sources. The new strain was Gram-positive, the cells were coccal shaped, the mol% G+C content of the genomic DNA was 29, and the peptidoglycan was of the L-ornithine-D-glutamic acid type. Comparative sequence analysis of the 16S rRNA gene showed the new strain to belong to the genus Peptostreptococcus. Succinate, fumarate, pyruvate, 3-hydroxybutyrate and lysine supported growth. Succinate was degraded to propionate and presumably CO₂, with a stoichiometric cell yield. Key enzymes of the methylmalonyl-CoA decarboxylase pathway were present. The methylmalonyl-CoA decarboxylase activity was avidin-sensitive and sodium dependent, and about 5 mM Na⁺ was required for maximal activity. Whole cells, however, required at least 50 mM sodium for maximal succinate decarboxylation activity and to support the maximum growth rate. Sodium-dependent energy conservation coupled to succinate decarboxylation is shown for the first time to occur in a bacterium belonging to the group of Gram-positive bacteria containing the peptostreptococci and their relatives.     

Cloning and expression of the genes of two fumarate reductase subunits from Wolinella succinogenes

The fumarate reductase complex of the anaerobic bacterium Wolinella succinogenes catalyzes the electron transfer from menaquinol to fumarate. Two structural genes coding for subunits of the enzyme have been cloned in Escherichia coli. The genes were isolated from a lambda EMBL3 phage gene bank by immunological screening and subcloned in an expression vector. The genes frdA and frdB, which encode the FAD protein (Frd A, Mr 79,000) and the iron-sulfur protein (Frd B, Mr 31,000) of the fumarate reductase complex, were cloned together with a W. succinogenes promoter. The gene order was promoter-frdA-frdB. The FAD protein and the iron-sulfur protein were expressed in the correct molar mass in E. coli from the clones. The identity of the frdA gene and the suggested polarity were confirmed by comparing the amino-terminal sequence of the Frd A protein with that predicted from the 5'-terminal nucleotide sequence of frdA. The frdA and frdB genes are present only once in the genome. A region downstream of frdB, possibly a gene encoding cytochrome b of the fumarate reductase complex, hybridizes with a second site in the genome.     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa