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1. Regulation

1.1 Transcriptional regulation via one-component systems

DNA-binding transcriptional regulators control the transcription of one or more target genes in response to a specific signal. Besides the two-component systems (see following section) which mostly mediate responses to extracellular or membrane-associated stimuli, C. glutamicum possesses a wide variety of one-component systems which alter transcription of the corresponding target genes in response to intracellular signals. An overview of transcriptional regulators characterised by our group is given in table 1. Together with research groups in Germany, France, Japan and Korea all transcriptional regulators of C. glutamicum should be functionally characterised in the future.

 

Table 1: Transcriptional regulators which have been characterised by our research group.

RegulatorFunctionLiterature
ClgRActivator of the Clp-protease and DNA repair proteins[2-4]
AcnRRepressor of the aconitase[5,6]
RipARepressor of important iron-containing proteins under iron limitation[7]
DtxRGlobal regulator of the iron homeostasis[8]
GntR1/2Regulators of the gluconate catabolism and the glucose uptake[9]
RosRRegulator of the response to oxidative stress[10]
MtrABTwo-component system for osmoregulation and cell wall metabolism[11-13]
PhoRSTwo-component system for regulation of the phosphate starvation response[14,15]
HrrASTwo-component system for the control of the heme  homeostasis[16]
CitABTwo-component system for the control of citrate uptake[17]

 

1.2 Transcriptional regulation via two-component systems

Bacteria are capable of adapting to changing environmental conditions, such as carbon source availability, oxygen concentration, or pH. Therefore, specific extracellular information has to be recognised, transmitted over the membrane and converted into a cellular response. In bacteria, this process is often mediated by two-component signal transduction systems. In the genome of C. glutamicum 13 of such regulatory systems are encoded (Fig. 1). In all cases the output is an altered gene expression. By now four of these two-component systems have been functionally characterised. When citrate is present in the medium the CitAB system activates the expression of two citrate uptake systems which are essential for growth on this carbon source. The PhoRS system plays an important role in the phosphate starvation response and the HrrSA system is involved in heme homeostasis. The MtrAB system is highly conserved in corynebacteria and mycobacteria and regulates genes involved in the response to osmotic stress, in cell wall homeostasis, and cell division.

Picture of genomic regions of C. glutamicumFigure 1: Genomic regions of C. glutamicum comprising the genes for the 13 two-component systems. The different colours of the genes represent different functions of the encoded proteins or different localisations of the gene product. The numbers of the gene loci correspond to those of the GenBank entry BX927147 [18]. put., putative; Me, metal; assoc., associated; biosynth., biosynthesis; mech.-sens., mechano-sensitive; CoA, coenzyme A; transp., transporter.


1.3 Transcriptional regulation of the TCA-cycle

Many industrially relevant metabolites are derived from TCA cycle intermediates (e. g. L-glutamate and L-lysine) or are themselves intermediates of the TCA cycle (e. g. succinate). Therefore, the transcriptional regulation of this central metabolic pathway is of special interest. In principle TCA cycle enzymes are supposed to be present in the cell under all cultivation conditions. However, we could show that the expression of many TCA cycle genes is subjected to a complex transcriptional control thereby allowing the cell to precisely adjust the respective enzyme activities to the specific metabolic requirements. An overview of the current knowledge concerning the transcriptional regulation of the TCA cycle genes is shown in Fig. 2. Our group has studied the regulation of gltA [19], acn [6,7,20] and sdhCAB [21].

Picture of transcriptional regulation of the TCA cycleFigure 2: Transcriptional regulation of the TCA cycle and the glyoxylate shunt in C. glutamicum. The genes encode the following enzymes: gltA, citrate synthase; acn, aconitase; icd, isocitrate dehydrogenase; odhA, aceF, lpdA, 2-oxoglutarate dehydrogenase complex; sucCD, succinyl-CoA synthetase; sdhCAB, succinate dehydrogenase; fum, fumarase; mqo, malate:menaquinone oxidoreductase; aceA, isocitrate lyase; aceB, malate synthase. The currently known and experimentally proven transcriptional regulators of individual genes are indicated. Lines emanating from regulators that end in an arrow indicate transcriptional activation, those ending blunt indicate transcriptional repression. Known and putative effectors are also indicated. Adapted from [22].


1.4 Posttranslational regulation by covalent protein modification: serine/threonine protein kinases

Besides regulation at the transcriptional level, posttranslational regulation via covalent protein modifications has attracted our attention. In the past years we intensively analysed phosphorylation of serine and threonine residues [23,24]. C. glutamicum possesses 4 serine/threonine protein kinases (STPKs: PknA, PknB, PknG und PknL) and one phospho-serine/threonine protein phosphatase (Ppp). Except for PknG all STPKs are membrane-integral proteins (Fig. 3). Our studies concerning PknG have shown that this protein kinase in concert with its target protein OdhI plays an important role in the regulation of the 2-oxoglutarate dehydrogenase complex (ODHC). Unphosphorylated OdhI binds with high affinity (Kd ca. 15 nM) to the OdhA subunit of ODHC, thereby inhibiting the activity of the enzyme complex. As a consequence the oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA in the TCA cycle is reduced whereas the flux from 2-oxoglutarate towards L-glutamate catalysed by glutamate dehydrogenase can be increased. Phosphorylation of OdhI at a conserved threonine residue by PknG inhibits the interaction of OdhI with OdhA, thereby abolishing the inhibition of the ODHC activity (Fig. 3). Deletion of the odhI gene leads to a C. glutamicum strain that has lost the capability of forming high amounts of glutamate under inducing conditions (biotin deprivation, addition of penicillin, addition of tween-20 or addition of ethambutol). Thus OdhI is essential for an efficient glutamate production with C. glutamicum. Besides PknG also the other STPKs PknA, PknB und PknL can phosphorylate OdhI, demonstrating that OdhI can integrate different signals. PknA, PknB, and PknL harbour extracellular domains indicating that these STPKs probably detect extracellular signals which have not been identified, yet.

Posttranslational regulation of OdhlFigure 3: Posttranslational regulation of OdhI by the serine/threonine protein kinases PknG, PknA, PknB, and PknL and by the phospho-serine/threonine protein phosphatase Ppp and influence of these proteins on glutamate production. OdhA, AceF, and Lpd, subunits of the 2-oxoglutarate dehydrogenase complex; Gdh, glutamate dehydrogenase; MscCG, glutamate exporter.


2. Respiratory chain and bioenergetics

C. glutamicum is a facultative anaerobic organism that can use oxygen and nitrate as terminal electron acceptor. However, growth under anoxic condition with nitrate is limited because the formed nitrite is toxic and cannot be metabolised further by C. glutamicum itself. In contrast, aerobic growth enables the formation of high biomass concentrations. Fig. 4 represents a schematic overview of the corynebacterial respiratory chain [25]. Reduction equivalents formed in the course of substrate oxidation are transferred to menaquinone by various dehydrogenases. Under aerobic conditions the reoxidation of menaquinol is mainly mediated by the cytochrome bc1-aa3 supercomplex or alternatively by the cytochrome bd oxidase. The existence of a cytochrome bc1-aa3 supercomplex (Fig. 5) was shown by co-purification experiments in our group [26]. A specific characteristic is the existence of only one c-type cytochrome in C. glutamicum, i. e.  cytochrome c1. It carries two covalently bound heme groups one of which seems to transfer the electrons to the aa3 oxidase [27]. Whereas the supercomplex presumably transports 6 H+/2 e- out of the cell, the cytochrome bd oxidase translocates only 2 H+/2 e-. By deleting the cytochrome bd oxidase genes cydAB, the efficiency of the respiratory chain and thereby also product formation can presumably be improved, as shown e.g. for L-lysine production (see below) [28].


Picture of an overview on major components of the respiratory chain of C. glutamicumFigure 4: Overview on major components of the respiratory chain of C. glutamicum.

Schematic representation of cytochrome bc1-aa3 supercomplexFigure 5: Schematic representation of subunit composition, cofactors, topology, electron flow, and involvement in the generation of proton-motive force for the cytochrome bc1-aa3 supercomplex. Black squares, heme groups; black circles, copper ions.


3. Strain development by metabolic engineering

The knowledge of metabolism and its regulation gained by the studies described above is applied for the development of new or optimised C. glutamicum production strains by metabolic engineering. Three examples are outlined below.

 

3.1 Application of “regulatory” knowledge

Elucidation of the new type of regulation of the oxoglutarate dehydrogenase complex (ODHC) by the inhibitory protein OdhI and serine/threonine protein kinase PknG provided the basis for the construction of a C. glutamicum strain that exhibits an increased L-glutamate production [29]. Therefore, the pknG gene was deleted in the genome of C. glutamicum. This strain offers a higher level of unphosphorylated OdhI which binds to the OdhA subunit of ODHC, thereby reducing its activity. Consequently, under specific glutamate-inducing conditions, as e. g. biotin limitation, the glutamate production rate and the glutamate titre were highly increased in the ΔpknG mutant.

 

3.2 Application of “bioenergetic” knowledge

Deletion of the cydAB genes coding for the cytochrome bd oxidase in the L-lysine producer C. glutamicum MH20-22B had almost no influence on growth rate or biomass formation from glucose, but the L-lysine synthesis rate and the final L-lysine titre increased about 12% [28]. This positive effect seems to rely on the fact that the electrons from substrate oxidation are exclusively transferred to oxygen via the cytochrome bc1-aa3 supercomplex and thus allows a more efficient energy conservation compared to the simultaneous utilisation of both the supercomplex and the energetically less efficient cytochrome bd oxidase. As a consequence, more glucose is available for L-lysine production.


3.3. Application of “metabolic” knowledge:

A critical parameter for L-lysine production is the NADPH supply [30]. Besides the NADPH-generating reactions of the central metabolism many bacteria, but not C. glutamicum, possess a membrane-integral transhydrogenase which can reduce NADP+ to NADPH with concomitant oxidation of NADH to NAD+. The required energy is provided by the electrochemical proton gradient. To make this type of NADPH formation available for C. glutamicum, the heterologous transhydrogenase PntAB from Escherichia coli was overproduced in the L-lysine production strain C. glutamicum DM1730. In the recombinant strain, the L-lysine titre increased between 10 to 300% depending on the carbon source (glucose, fructose, glucose + fructose or saccharose). Thus, the presence of a proton-motive force-coupled transhydrogenase is an efficient strategy to enhance L-lysine production.

 

 

4. References

1. Eggeling L, Bott M (2005) Handbook of Corynebacterium glutamicum. Boca Raton, FL.: CRC Press.

2. Engels S, Ludwig C, Schweitzer J-E, Mack C, Bott M, et al. (2005) The transcriptional activator ClgR controls transcription of genes involved in proteolysis and DNA repair in Corynebacterium glutamicum. Mol Microbiol 57: 576-591.

3. Engels S, Schweitzer JE, Ludwig C, Bott M, Schaffer S (2004) clpC and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor sigmaH. Mol Microbiol 52: 285-302.

4. Russo S, Schweitzer J-E, Polen T, Bott M, Pohl E (2009) Crystal Structure of the Caseinolytic Protease Gene Regulator, a Transcriptional Activator in Actinomycetes. J Biol Chem 284: 5208-5216.

5. Garcia-Nafria J, Baumgart M, Bott M, Wilkinson AJ, Wilson KS (2010) The Corynebacterium glutamicum aconitase repressor: scratching around for crystals. Acta Crystallogr Sect F Struct Biol Cryst Commun 66: 1074-1077.

6. Krug A, Wendisch VF, Bott M (2005) Identification of AcnR, a TetR-type Repressor of the Aconitase Gene acn in Corynebacterium glutamicum. J Biol Chem 280: 585-595.

7. Wennerhold J, Krug A, Bott M (2005) The AraC-type regulator RipA represses aconitase and other iron proteins from Corynebacterium under iron limitation and is itself repressed by DtxR. J Biol Chem 280: 40500-40508.

8. Wennerhold J, Bott M (2006) The DtxR regulon of Corynebacterium glutamicum. J Bacteriol 188: 2907-2918.

9. Frunzke J, Engels V, Hasenbein S, Gätgens C, Bott M (2008) Co-ordinated regulation of gluconate catabolism and glucose uptake in Corynebacterium glutamicum by two functionally equivalent transcriptional regulators, GntR1 and GntR2. Mol Microbiol 67: 305-322.

10. Bussmann M, Baumgart M, Bott M (2010) RosR (Cg1324), a hydrogen peroxide-sensitive MarR-type transcriptional regulator of Corynebacterium glutamicum. J Biol Chem 285: 29305-29318.

11. Brocker M, Bott M (2006) Evidence for activator and repressor functions of the response regulator MtrA from Corynebacterium glutamicum. FEMS Microbiol Lett 264: 205-212.

12. Brocker M, Mack C, Bott M (2011) Target genes, consensus binding site, and role of phosphorylation for the response regulator MtrA of Corynebacterium glutamicum. J Bacteriol 193: 1237-1249.

13. Möker N, Brocker M, Schaffer S, Krämer R, Morbach S, et al. (2004) Deletion of the genes encoding the MtrA-MtrB two-component system of Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics susceptibility and expression of genes involved in osmoprotection. Mol Microbiol 54: 420-438.

14. Kocan M, Schaffer S, Ishige T, Sorger-Herrmann U, Wendisch VF, et al. (2006) Two-component systems of Corynebacterium glutamicum: deletion analysis and involvement of the PhoS-PhoR system in the phosphate starvation response. J Bacteriol 188: 724-732.

15. Schaaf S, Bott M (2007) Target genes and DNA-binding sites of the response regulator PhoR from Corynebacterium glutamicum. J Bacteriol 189: 5002-5011.

16. Frunzke J, Gätgens C, Brocker M, Bott M (2011) Control of heme homeostasis in Corynebacterium glutamicum by the two-component system HrrSA. J Bacteriol 193: 1212-1221.

17. Brocker M, Schaffer S, Mack C, Bott M (2009) Citrate utilization by Corynebacterium glutamicum is controlled by the CitAB two-component system through positive regulation of the citrate transport genes citH and tctCBA. J Bacteriol 191: 3869-3880.

18. Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, et al. (2003) The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. J Biotechnol 104: 5-25.

19. van Ooyen J, Emer D, Bussmann M, Bott M, Eikmanns BJ, et al. (2011) Citrate synthase in Corynebacterium glutamicum is encoded by two gltA transcripts which are controlled by RamA, RamB, and GlxR. J Biotechnol 154: 140-148.

20. Emer D, Krug A, Eikmanns BJ, Bott M (2009) Complex expression control of the Corynebacterium glutamicum aconitase gene: Identification of RamA as a third transcriptional regulator besides AcnR and RipA. J Biotechnol 140: 92-98.

21. Bussmann M, Emer D, Hasenbein S, Degraf S, Eikmanns BJ, et al. (2009) Transcriptional control of the succinate dehydrogenase operon sdhCAB of Corynebacterium glutamicum by the cAMP-dependent regulator GlxR and the LuxR-type regulator RamA. J Biotechnol 143: 173-182.

22. Bott M (2007) Offering surprises: TCA cycle regulation in Corynebacterium glutamicum. Trends Microbiol 15: 417-425.

23. Niebisch A, Kabus A, Schultz C, Weil B, Bott M (2006) Corynebacterial Protein Kinase G Controls 2-Oxoglutarate Dehydrogenase Activity via the Phosphorylation Status of the OdhI Protein. J Biol Chem 281: 12300-12307.

24. Schultz C, Niebisch A, Schwaiger A, Viets U, Metzger S, et al. (2009) Genetic and biochemical analysis of the serine/threonine protein kinases PknA, PknB, PknG and PknL of Corynebacterium glutamicum: evidence for non-essentiality and for phosphorylation of OdhI and FtsZ by multiple kinases. Mol Microbiol 74: 724-741.

25. Bott M, Niebisch A (2003) The respiratory chain of Corynebacterium glutamicum. J Biotechnol 104: 129-153.

26. Niebisch A, Bott M (2003) Purification of a Cytochrome bc1-aa3 Supercomplex with Quinol Oxidase Activity from Corynebacterium glutamicum. J Biol Chem 278: 4339-4346.

27. Niebisch A, Bott M (2001) Molecular analysis of the cytochrome bc1-aa3 branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c1. Arch Microbiol 175: 282-294.

28. Kabus A, Niebisch A, Bott M (2007) Role of Cytochrome bd Oxidase from Corynebacterium glutamicum in Growth and Lysine Production. Appl Environ Microbiol 73: 861-868.

29. Schultz C, Niebisch A, Gebel L, Bott M (2007) Glutamate production by Corynebacterium glutamicum: dependence on the oxoglutarate dehydrogenase inhibitor protein OdhI and protein kinase PknG. Appl Microbiol Biotechnol 76: 691-700.

30. Kabus A, Georgi T, Wendisch V, Bott M (2007) Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves L-lysine formation. Appl Microbiol Biotechnol 75: 47-53.



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