Cloning the dapA dapB cluster of the lysine-secreting bacterium Corynebacterium glutamicum

Cremer, Josef; Eggeling, Lothar; Sahm, Hermann

doi:10.1007/bf00391757

Cited by 85 publications

(32 citation statements)

References 7 publications

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“…The resulting 1245-bp PCR product was cloned into the vector pJC1 (12). The resulting plasmid pJC1-ripA and pJC1 were used to transform C. glutamicum wild type and the ⌬ripA strain.…”

Section: Methodsmentioning

confidence: 99%

The AraC-type Regulator RipA Represses Aconitase and Other Iron Proteins from Corynebacterium under Iron Limitation and Is Itself Repressed by DtxR

Wennerhold¹,

Krug²,

Bott

2005

Journal of Biological Chemistry

116

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The mRNA level of the aconitase gene acn of Corynebacterium glutamicum is reduced under iron limitation. Here we show that an AraC-type regulator, termed RipA for "regulator of iron proteins A," is involved in this type of regulation. A C. glutamicum ⌬ripA mutant has a 2-fold higher aconitase activity than the wild type under iron limitation, but not under iron excess. Comparison of the mRNA profiles of the ⌬ripA mutant and the wild type revealed that the acn mRNA level was increased in the ⌬ripA mutant under iron limitation, but not under iron excess, indicating a repressor function of RipA. Besides acn, some other genes showed increased mRNA levels in the ⌬ripA mutant under iron starvation (i.e. those encoding succinate dehydrogenase (sdhCAB), nitrate/nitrite transporter and nitrate reductase (narKGHJI), isopropylmalate dehydratase (leuCD), catechol 1,2-dioxygenase (catA), and phosphotransacetylase (pta)). Most of these proteins contain iron. Purified RipA binds to the upstream regions of all operons mentioned above and in addition to that of the catalase gene (katA). Corynebacterium glutamicum is a nonpathogenic, aerobic Grampositive soil bacterium that is used for large scale industrial production of amino acids, predominantly L-glutamate (1.5 million tons/year) and L-lysine (0.7 million tons/year). In addition, C. glutamicum has gained interest as a suitable model organism for the Corynebacterineae, a suborder of the actinomycetes that includes the genus Mycobacterium. An overview on C. glutamicum biology, genetics, physiology, and biotechnology can be found in a recent monograph (1).The citric acid cycle is of central importance for metabolism in general and for amino acid production in particular, because it provides the biosynthetic precursors of the aspartate and glutamate family of amino acids. Despite its key role, knowledge about the genetic regulation of this pathway in C. glutamicum is scarce. We recently could show that the activity of aconitase (EC 4.2.1.3), which catalyzes the stereospecific and reversible isomerization of citrate to isocitrate via cis-aconitate, varies depending on the carbon source and that this is caused by transcriptional regulation (2). A repressor of the TetR family, called AcnR, was identified, which represses aconitase by binding to an imperfect inverted repeat within the acn promoter region and interfering with the binding of RNA polymerase (2). The factors that control binding of AcnR to its operator are not yet known. DNA microarray experiments revealed that acn expression is not only influenced by the carbon source but also by the iron concentration of the medium (2). Under iron limitation, the acn mRNA level in the wild type was 3-fold lower than under iron excess. In the ⌬acnR mutant, this decrease was even larger (4.8-fold), presumably because the increased expression of aconitase, which contains a 4Fe-4S cluster, leads to an enhanced iron starvation.We now have identified a new transcriptional regulator, designated RipA, which is responsible for iron-dependent re...

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“…The resulting 1245-bp PCR product was cloned into the vector pJC1 (12). The resulting plasmid pJC1-ripA and pJC1 were used to transform C. glutamicum wild type and the ⌬ripA strain.…”

Section: Methodsmentioning

confidence: 99%

The AraC-type Regulator RipA Represses Aconitase and Other Iron Proteins from Corynebacterium under Iron Limitation and Is Itself Repressed by DtxR

Wennerhold¹,

Krug²,

Bott

2005

Journal of Biological Chemistry

116

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“…The resulting PCR fragment was cloned in the vector pET324 (36), resulting in pECmqo for expression in E. coli. For expression in C. glutamicum, the fragment was cloned in pJCBW1, a derivative of pJC1 (8,20) containing the lacZ complementation region and multiple cloning site from pSKϩ (Stratagene), using EcoRV.…”

Section: Methodsmentioning

confidence: 99%

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Escherichia coli

2000

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Oxidation of malate to oxaloacetate in Escherichia coli can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37) and the membrane-associated malate:quinoneoxidoreductase (MQO; EC 1.1.99.16), encoded by the gene mqo (previously called yojH). Expression of the mqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the ␤-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repress mqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genes sfcA and b2463 (coding for EC 1.1.1.38 and EC 1.1.1.40, respectively) and of the phosphoenolpyruvate synthase (EC 2.7.9.2) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of the mdh single mutant to that of the double mutant containing mdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.In Escherichia coli, several enzymes or pathways are able to convert malate to oxaloacetate. The NAD-dependent (cytoplasmic) malate dehydrogenase (MDH; EC 1.1.1.37) has always been considered to be the principal malate-oxidizing enzyme in the citric acid cycle (tricarboxylic acid [TCA] cycle) of this organism (9). Recently, a malate:quinone-oxidoreductase (MQO; EC 1.1.99.16) which is an essential enzyme in the TCA cycle of Corynebacterium glutamicum (reference 23 and accompanying paper) was described. MQO is a flavin adenine dinucleotide (FAD)-and lipid-dependent peripheral membrane protein catalyzing the oxidation of L-malate to oxaloacetate. The electrons are donated to the electron transfer chain at the level of quinones. This reaction is essentially irreversible (17). In C. glutamicum, MQO and the cytoplasmic NAD-dependent malate dehydrogenase, which is capable of reversible oxidation of malate to oxaloacetate, are active at the sam...

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“…A comparison of the amino acids conserved in the two types of enzymes (N-acetylneuraminate lyases and dihydrodipicolinate synthases) suggests that seven specific domains (a to g in Fig. 4 (5). The enzyme is strongly similar to that of E. coli and shows a domain which is conserved in several dehydrogenases, including the aspartic semialdehyde dehydrogenases (asd) of E. coli, Saccharomyces cerevisiae, and C. glutamicum; N-acetylglutamyl phosphate reductases (argC) of Streptomyces clavuligerus, E. coli, and Bacillus subtilis; glyceraldehyde-3-phosphate dehydrogenases of C. glutamicum, white mustard, and Mesembryanthemum crystallinum; and malate dehydrogenases of E. coli and pig mitochondria.…”

Section: Dap2mentioning

confidence: 99%

“…In addition to the succinylase and acetylase variants common to other procaryotes, a shunt pathway in which A'-piperideine-2,6-dicarboxylate is converted in a single step to diaminopimelate by the enzyme D-diaminopimelate dehydrogenase has been described in corynebacteria (11). Finally, diaminopimelic acid is decarboxylated to form lysine by diaminopimelate decarboxylase (lysA gene).Only preliminary information is available on the nature of the dapA and dapB genes of C. glutamicum (1,5), and no information has been published on that in B. lactofermen-* Corresponding author. tum.…”

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confidence: 99%

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A cluster of three genes (dapA, orf2, and dapB) of Brevibacterium lactofermentum encodes dihydrodipicolinate synthase, dihydrodipicolinate reductase, and a third polypeptide of unknown function

et al. 1993

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The dapA and dapB genes, encoding, respectively, dihydrodipicolinate synthase and dihydrodipicolinate reductase, the two first enzymes of the lysine branch of the aspartic amino acid family, were cloned from the DNA of the amino acid-producing bacterium Brevibacterium lactofermentum. The two genes were clustered in a 3.5-kb Sau3AI-BamHI fragment but were separated by an open reading frame of 750 nucleotides. The protein encoded by this open reading frame had little similarity to any protein in the data banks, and its function remains unknown. The three genes were translated in Escherichia coli, giving the corresponding polypeptides.The lysine biosynthetic pathway in corynebacteria is still poorly understood despite the use of Corynebacterium glutamicum and Brevibacterium lactofermentum for the industrial production of lysine. This pathway shares the first two steps with the threonine and methionine biosynthetic pathways, which involve the activation of aspartic acid to aspartyl phosphate and the reduction of the activated amino acid to aspartyl semialdehyde. In the lysine branch, the aspartyl semialdehyde is converted to dihydrodipicolinate by condensation with pyruvate. This first "branching" reaction, which involves the condensation of a C4 with a C3 fragment to form the seven-carbon dihydrodipicolinate, is carried out by dihydrodipicolinate synthase, encoded in Escherichia coli by the dapA gene. The condensation step is followed by a cyclization reaction which results in the formation of 2,3-dihydrodipicolinate and may involve an enzyme-bound C7 linear intermediate (Fig. 1). Whether this reaction requires more than one protein is uncertain. The dihydrodipicolinate formed is later converted to Al-piperideine-2,6-dicarboxylate by dihydrodipicolinate reductase, which in E. coli is encoded by the dapB gene. In the last steps of the pathway the A'-piperideine-2,6-dicarboxylate is converted to D,L-diaminopimelic acid by at least two alternative pathways (6, 20). In addition to the succinylase and acetylase variants common to other procaryotes, a shunt pathway in which A'-piperideine-2,6-dicarboxylate is converted in a single step to diaminopimelate by the enzyme D-diaminopimelate dehydrogenase has been described in corynebacteria (11). Finally, diaminopimelic acid is decarboxylated to form lysine by diaminopimelate decarboxylase (lysA gene).Only preliminary information is available on the nature of the dapA and dapB genes of C. glutamicum (1,5), and no information has been published on that in B. lactofermen-* Corresponding author. tum. We describe here that the dapA and dapB genes of B.lactofermentum are clustered together with a third open reading frame (ORF) located between them. The three ORFs are translated into polypeptides.Cloning the dapB gene and deletion mapping. Total DNA of B. lactofermentum ATCC 13869 (Table 1) was isolated as described previously (7) and partially digested with Sau3AI or totally digested with HindIII in different experiments. DNA fragments of 4 to 10 kb were ligated with pUC13, and th...

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Cloning the dapA dapB cluster of the lysine-secreting bacterium Corynebacterium glutamicum

Cited by 85 publications

References 7 publications

The AraC-type Regulator RipA Represses Aconitase and Other Iron Proteins from Corynebacterium under Iron Limitation and Is Itself Repressed by DtxR

The AraC-type Regulator RipA Represses Aconitase and Other Iron Proteins from Corynebacterium under Iron Limitation and Is Itself Repressed by DtxR

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Escherichia coli

A cluster of three genes (dapA, orf2, and dapB) of Brevibacterium lactofermentum encodes dihydrodipicolinate synthase, dihydrodipicolinate reductase, and a third polypeptide of unknown function

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