The respiratory chain of Corynebacterium glutamicum
Introduction
Corynebacterium glutamicum was isolated by Kinoshita and coworkers in the mid 1950s as a biotin-auxotrophic bacterium that excretes large amounts of l-glutamate when growing aerobically under biotin limitation with glucose as carbon source (Kinoshita et al., 1957). It belongs to the Gram-positive bacteria with a high G+C content of the chromosomal DNA (53.8% in the case of C. glutamicum) and, more specifically, to a group of bacteria including the genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus which contain mycolic acids (α-alkyl-β-hydroxy fatty acids) in the cell wall. After the discovery that C. glutamicum is suitable for the conversion of sugars and organic acids to l-glutamate, mutants were isolated that were able to excrete also other amino acids, in particular l-lysine. Nowadays, both l-glutamate, which is mainly used as a flavor enhancer, and l-lysine, mainly used as a feed additive, are produced industrially almost exclusively with C. glutamicum at a scale of 1×106 and 0.5×106 tons per year, respectively (Ikeda, 2003, Kimura, 2003, Pfefferle et al., 2003).
In the past, the common practice to obtain amino acid overproducing strains was by mutagenesis and selection. Within the last decade, however, metabolic engineering, i.e. the improvement of enzymatic, transport and regulatory functions of the cell with the application of recombinant DNA technology (Bailey, 1991), was established as an alternative that allows the rational construction of production strains. Metabolic engineering includes the deletion, overexpression or timed expression of selected genes or the replacement of a wild-type enzyme with one having defined altered properties. In contrast to strain construction by mutagenesis and selection, metabolic engineering requires at least some basic knowledge on the metabolism of the organism investigated and in particular tools for genetic manipulation.
Due to the importance of C. glutamicum for amino acid production, most studies on this organism focused on amino acid biosynthesis, on precursor supply by central metabolic pathways and anaplerotic reactions, on carbon and nitrogen fluxes and on transport processes relevant for amino acid production (for review, see Eikmanns et al., 1993, Sahm et al., 1995, Sahm et al., 2000, Eggeling et al., 1997, Eggeling and Sahm, 2001, Burkovski and Krämer, 2002). In recent years, additional topics received attention, driven (i) by the establishment of the C. glutamicum genome sequence, (ii) by the realization that construction of the “optimal” production strain requires knowledge on the physiology and biochemistry of the organism as a whole rather than only of selected parts, and (iii) by the possibility to use C. glutamicum as a non-pathogenic model organism to study features in common with pathogenic corynebacteria and mycobacteria, e.g. C. diphtheriae and Mycobacterium tuberculosis. Respiration was one of the topics receiving attention, resulting in a continuation of the studies made in the 1970s and 1980s (Kanzaki et al., 1973, Kanzaki et al., 1974, Sugiyama et al., 1973a, Sugiyama et al., 1973b, Trutko et al., 1982, Kawahara et al., 1988). This review summarizes the current knowledge on the C. glutamicum respiratory chain, including relevant information from the genome sequence on aspects that have not yet been studied experimentally.
Section snippets
The early studies on respiration in C. glutamicum
First interest into the respiratory chain of C. glutamicum was probably initiated by the finding that copper ions were necessary for the maximal production of l-glutamate from acetate by an oleic acid-requiring strain of C. glutamicum designated as Brevibacterium thiogenitalis. A similar effect was observed for succinate and fumarate, but not for glucose, lactate, ethanol and n-paraffin. The decreased glutamate formation in the absence of copper ions correlated with an increased oxidation of
Enzymes reducing menaquinol
In this and the following sections, the individual components of the C. glutamicum respiratory chain are described. Fig. 1 gives an overview on these components, whereas Fig. 2 shows more details of their subunit composition, localization, cofactors and involvement in the generation of a proton-motive force.
Enzymes oxidizing menaquinol
Reduced-minus-oxidized difference spectra obtained with intact cells, cell extracts and membranes of C. glutamicum revealed peaks at 600, 560 and 550 nm, indicating the presence of a-, b- and c-type cytochromes, respectively (Sugiyama et al., 1973a, Sugiyama et al., 1973b, Trutko et al., 1982). In the CO-reduced-minus-reduced difference spectrum a peak at 427 nm and a trough at 443 nm were found, typical for a cytochrome aa3-type terminal oxidase (Trutko et al., 1982). Isolated membranes
Evidences for an additional terminal oxidase activity
Genome analysis revealed that cytochrome aa3 and cytochrome bd are the only terminal oxidases present in C. glutamicum. However, some experimental results indicated the presence of an additional terminal oxidase activity whose molecular nature has not yet been elucidated: (i) The cyanide-resistant respiration of C. glutamicum measured by Trutko et al., 1982, Matsushita et al., 1998 was not accompanied by the appearance of cytochrome d in redox difference spectra. (ii) Kusumoto et al. (2000)
F1F0-ATP synthase
F1F0-ATP synthase is essential for ATP generation by oxidative phosphorylation. The genes encoding the eight subunits of this enzyme are organized as in most other bacteria in the atpIBEFHAGDC operon (Cgl1205–1213), which includes the atpI gene of unknown function. The promoter-proximal genes atpB, atpE and atpF encode the three components of the membrane-integral F0 part, i.e. the a-, c- and b-subunit, respectively. The promoter-distal genes atpH, atpA, atpG, atpD and atpC encode the five
Bioenergetic considerations
According to our present knowledge, the C. glutamicum respiratory chain using oxygen as terminal electron acceptor involves three enzymes which couple electron transfer to the generation of an electrochemical proton gradient across the cytoplasmic membrane, i.e. the cytochrome bc1 complex, cytochrome aa3 oxidase and cytochrome bd oxidase. According to generally accepted values (Nicholls and Ferguson, 1992), the number of protons formally transported across the membrane per electron (H+/e−) is
MK biosynthesis
MK was identified as the only isoprenoid quinone present in C. glutamicum, with dihydromenaquinone-8 (i.e. MK containing eight isoprene units with one double bond hydrogenated) and MK-9 as minor components and dihydromenaquinone-9 as major component (Kanzaki et al., 1974, Collins et al., 1977, Collins et al., 1979). Genome analysis led to the identification of all genes required for the synthesis of MK from chorismate, an intermediate in the biosynthesis of aromatic amino acids (Fig. 6). These
Heme biosynthesis and cytochrome c maturation
Heme biosynthesis in C. glutamicum (Fig. 7) occurs via the C5 pathway that uses glutamate as the substrate for the synthesis of δ-aminolevulinic acid. The genes for heme biosynthesis (reviewed in O'Brian and Thöny-Meyer, 2002) in C. glutamicum are arranged in one large cluster (Cgl0429–0442, Fig. 8) except for the hemA, hemC, hemN and hemH genes encoding glutamyl-tRNA reductase (EC 1.2.1.-, Cgl0417), porphobilinogen deaminase (EC 4.3.1.8, Cgl0418), coproporphyrinogen III dehydrogenase (EC
Biotechnological aspects
C. glutamicum is an aerobic respiratory organism and thus synthesizes most of its ATP by oxidative phosphorylation. As shown by the effects of copper (Kanzaki et al., 1973, Sugiyama et al., 1973b), cyanide (Trutko et al., 1982) or a defective F1F0-ATP synthase (Sekine et al., 2001), interferences with the system of oxidative phosphorylation can have a strong influence on metabolism in general and on amino acid production in particular. Further support for this finding comes from a number of
Concluding remarks
The recent experimental studies in several laboratories and the information obtained by the genome sequence have established a good picture of the aerobic respiratory chain of C. glutamicum. It involves several dehydrogenases, MK, a cytochrome bc1–aa3 supercomplex and cytochrome bd menaquinol oxidase. Cytochrome c1 represents the only c-type cytochrome of C. glutamicum and is unusual in that it contains two covalently bound heme groups, both of which are essential for assembly, stability and
Acknowledgements
Support of the studies on Corynebacterium glutamicum in the laboratory of M.B. by the EU project “VALPAN”, the BMBF “Genomik” program (Genome research on bacteria relevant for agriculture, environment and biotechnology; cluster IV: Corynebacteria), the BMBF “Proteomics” program (New methods for proteome analysis: application and combination with metabolome analysis using Corynebacterium glutamicum as an example), the Degussa AG, and the DFG-Graduiertenkolleg “Molekulare Physiologie: Stoff- und
References (94)
- et al.
Redesigned purification yields a fully functional PutA protein dimer from Escherichia coli
J. Biol. Chem.
(1992) - et al.
The relationship between structure and function for the sulfite reductases
Curr. Opin. Struct. Biol.
(1996) - et al.
The cell wall barrier of Corynebacterium glutamicum and amino acid efflux
J. Biosci. Bioeng.
(2001) - et al.
The fruits of molecular physiology: engineering the l-isoleucine biosynthesis pathway in Corynebacterium glutamicum
J. Biotechnol.
(1997) - et al.
The respiratory complex I (NDH I) from Klebsiella pneumoniae, a sodium pump
J. Biol. Chem.
(2002) Succinate: quinone oxidoreductases. Variations on a conserved theme
Biochim. Biophys. Acta
(1997)- et al.
A structural model for the membrane-integral domain of succinate:quinone oxidoreductases
FEBS Lett.
(1996) - et al.
Essential histidine and tryptophan residues in CcsA, a system II polytopic cytochrome c biogenesis protein
J. Biol. Chem.
(2003) Succinate:quinone oxidoreductase in the bacteria Paracoccus denitrificans and Bacillus subtilis
Biochim. Biophys. Acta
(2002)- et al.
Quinones of Brevibacterium
Biochim. Biophys. Acta
(1974)
Fumarate respiration of Wolinella succinogenes: enzymology, energetics and coupling mechanism
Biochim. Biophys. Acta
Succinate: quinone oxidoreductases: new insights from X-ray crystal structures
Biochim. Biophys. Acta
Sequence analysis identifies the proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase domains of the multifunctional Escherichia coli PutA protein
J. Mol. Biol.
Escherichia coli pyruvate oxidase: interaction of a peripheral membrane protein with lipids
Biophys. J.
NADH dehydrogenase of Corynebacterium glutamicum. Purification of an NADH dehydrogenase II homolog able to oxidize NADPH
FEMS Microbiol. Lett.
Mechanism of superoxide and hydrogen peroxide formation by fumarate reductase, succinate dehydrogenase, and aspartate oxidase
J. Biol. Chem.
Purification of a cytochrome bc1–aa3 supercomplex with quinol oxidase activity from Corynebacterium glutamicum. Identification of a fourth subunit of cytochrome aa3 oxidase and mutational analysis of diheme cytochrome c1
J. Biol. Chem.
Cysteine is exported from the Escherichia coli cytoplasm by CydCD, an ABC-type transporter required for cytochrome assembly
J. Biol. Chem.
Physical interaction of CcmC with heme and the heme chaperone CcmE during cytochrome c maturation
J. Biol. Chem.
A new iron–sulfur flavoprotein of the respiratory chain: a component of the fatty acid beta oxidation pathway
J. Biol. Chem.
Metabolic design in amino acid producing Corynebacterium glutamicum
FEMS Microbiol. Rev.
Heme O biosynthesis in Escherichia coli: the cyoE gene in the cytochrome bo operon encodes a protoheme IX farnesyltransferase
Biochem. Biophys. Res. Commun.
In vitro heme O synthesis by the cyoE gene product from Escherichia coli
J. Biol. Chem.
Expression and characterization of two pathogenic mutations in human electron transfer flavoprotein
J. Biol. Chem.
Chemical and functional properties of the native and reconstituted forms of the membrane-bound, aerobic glycerol-3-phosphate dehydrogenase of Escherichia coli
J. Biol. Chem.
Direct correlationship between proton translocation and growth yield: an analysis of the respiratory chain of Bacillus stearothermophilus
J. Biosci. Bioeng.
A novel hydrophobic diheme c-type cytochrome. Purification from Corynebacterium glutamicum and analysis of the qcrCBA operon encoding three subunit proteins of a putative cytochrome reductase complex
Biochim. Biophys. Acta
Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement
Biochim. Biophys. Acta
Nitrate reduction as a marker for hypoxic shiftdown of Mycobacterium tuberculosis
Tuber. Lung Dis.
Isolation, analysis, and deletion of the gene coding for subunit IV of cytochrome c oxidase in Paracoccus denitrificans
J. Biol. Chem.
Molecular structure of flavocytochrome b2 at 2.4 Å resolution
J. Mol. Biol.
Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli
Microbiology
Toward a science of metabolic engineering
Science
Four genes are required for the system II cytochrome c biogenesis pathway in Bordetella pertussis, a unique bacterial model
Mol. Microbiol.
Electron-transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties
Biochemistry
The coordination and function of the redox centres of the membrane-bound nitrate reductases
Cell. Mol. Life Sci.
Identification of novel hemes generated by heme A synthase: evidence for two successive monooxygenase reactions
Biochemistry
Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications
Appl. Microbiol. Biotechnol.
Unusual redox properties of electron-transfer flavoprotein from Methylophilus methylotrophus
Biochemistry
Energetic efficiency of Escherichia coli: effects of mutations in components of the aerobic respiratory chain
J. Bacteriol.
The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K-12: two nitrate and three nitrite transporters
Mol. Microbiol.
Distribution of menaquinones in actinomycetes and corynebacteria
J. Gen. Microbiol.
Isoprenoid quinones in the classification of coryneform and related bacteria
J. Gen. Microbiol.
PutA protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator
Proc. Natl. Acad. Sci. USA
Functional analysis of a divergent system II protein, Ccs1, involved in c-type cytochrome biogenesis
J. Biol. Chem.
The crystal structure of d-lactate dehydrogenase, a peripheral membrane respiratory enzyme
Proc. Natl. Acad. Sci. USA
Molecular aspects of lysine, threonine, and isoleucine biosynthesis in Corynebacterium glutamicum
Antonie Van Leeuwenhoek
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2021, Journal of BiotechnologyCitation Excerpt :Among these complexes, cytochrome aa3-bc1 oxidase (encoded by the qcrCAB and ctaDCFE genes) and cytochrome bd oxidase (encoded by the cydAB operon) are terminal oxidation branches under aerobic respiration, which requires oxygen as a terminal electron acceptor to form a functional electron transport chain to produce ATP (Bott and Niebisch, 2003; Matsushita, 2013). Under aerobic conditions, the energy-coupling efficiencies of NADH dehydrogenase, the cytochrome bc1-aa3 supercomplex and cytochrome bd oxidase in C. glutamicum correspond to H+/e− ratios of 0, 3 and 1, respectively (Fig. 1) (Bott and Niebisch, 2003; Matsushita, 2013). Duwat et al. reconstituted a functional cytochrome by supplying heme to the medium and inducing the gene cydA expression in L.lactis, drastically shifting the redox balance (Duwat et al., 2001).
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2021, Metabolic EngineeringCitation Excerpt :Furthermore, C. glutamicum serves as a model organism for the order Corynebacteriales, which includes a number of important pathogens, e.g. Mycobacterium tuberculosis. As many bacteria, C. glutamicum possesses a branched respiratory chain, with at least six different dehydrogenases that transfer electrons to menaquinone (without translocating protons), and can use either oxygen or nitrate as terminal electron acceptor (Bott and Niebisch, 2003) (Fig. 1). It is important to note that the composition of the respiratory chain differs from that in mitochondria in that the proton-translocating complex I (NADH dehydrogenase) is missing.