Corynebacterium glutamicum - systemic understanding of an industrial workhorse

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 signal transduction systems described in the following section, C. glutamicum possesses more than 120 one-component transcriptional regulators, which alter transcription of their target genes in response to intracellular signals. About half of them have been studied experimentally since 2003. An overview of one-component regulators characterised by our group is given in Table 1. Together with research groups in Germany, France, Japan, and Korea we are aiming at a functional characterization of all transcriptional regulators of C. glutamicum. Fig. 1 shows two examples of regulators that have been characterised by us. The structure of AcnR was solved in cooperation with the YSBL at the University of York and helped to identify the ligand that binds to an unusual binding site (Figs. 1A and 1B). The regulator IpsA is involved in a regulatory network important both for cell wall biogenesis and mycothiol formation (Fig. 1C).

Table 1: Selection of transcriptional regulators which have been characterised in our institute.

Transcriptional regulation via two-component systems

In order to survive, bacteria must adapt to changing environmental conditions, such as carbon source availability, oxygen concentration, or pH. Therefore, extracellular changes have to be sensed (input), transmitted across the membrane into the cytoplasm, and converted into an appropriate cellular response (output). In bacteria, this process is often mediated by two-component signal transduction systems. The C. glutamicum genome encodes 13 of such regulatory systems (17). By now six of these two-component systems have been functionally characterised (Tab. 1). In all cases the output is an altered gene expression. 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 (Fig. 2). 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.

Table 2: Selection of two component systems which have been characterized by us.

cAMP in C. glutamicum

The second messenger cyclic AMP (cAMP) plays an important role in the metabolism of C. glutamicum, as the global transcriptional regulator GlxR, a homolog of Crp of E. coli, requires complex formation with cAMP to become DNA-binding competent and thus active. We are interested in the factors influencing the intracellular cAMP concentration, as this is crucial for a full understanding of GlxR function. After description of cyaB as cAMP-generating enzyme by Cha et al. (27), we identified and characterized the cAMP phosphodiesterase CpdA as an important cAMP-degrading enzyme for C. glutamicum (28). Furthermore, we showed that cpdA expression is activated by cAMP-GlxR, representing a further, so far unknown, GlxR target gene. Based on this data we proposed a model for a feedback loop formed by GlxR and CpdA that is important for the control of the cellular cAMP level (Fig. 3). To search for further proteins involved in cAMP homeostasis, we developed a cAMP biosensor (29) that is described in detail in the biosensor section.

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 subject to a complex transcriptional control, thereby allowing the cell to adjust the respective enzyme activities precisely to the specific metabolic requirements. An overview of the current knowledge concerning the transcriptional regulation of the TCA cycle genes is shown in Fig. 4. Our group has studied the regulation of gltA (30), acn (2,3,15,31) and sdhCAB (32).

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

Besides transcriptional regulation, posttranslational regulation by covalent protein modification represents another important level of cellular regulation. We have analysed the role of serine/threonine phosphorylation in C. glutamicum. Genome analysis revealed four genes for serine/threonine protein kinases (STPKs) named PknA, PknB, PknL, and PknG (according to their homologs from the pathogenic relative Mycobacterium tuberculosis) and one phospho-serine/threonine protein phosphatase Ppp. Except PknG, all other STPKs and Ppp are membrane-integral enzymes. Our studies on PknG uncovered that this kinase plays a key role in the regulation of the 2-oxoglutarate dehydrogenase (ODH) complex via the target protein OdhI (Fig. 5) (34). In the unphosphorylated state, the 15-kDa OdhI protein binds with high affinity (Kd approx. 15 nM) to the OdhA subunit of the ODH complex and inhibits the activity of this enzyme. As a consequence, the conversion of 2-oxoglutarate in the TCA cycle is diminished and conversion of 2-oxoglutarate to glutamate by glutamate dehydrogenase is increased. However, if OdhI is phosphorylated by PknG at Thr-14, binding to OdhA is prevented due to a conformational change of the OdhI protein, which thus functions as a phosphorylation-dependent molecular switch. The structural change is caused by binding of the phosphothreonine residue to the carboxyterminal FHA domain of OdhI (35). Deletion of the odhI gene in C. glutamicum almost completely inhibits L-glutamate production triggered by biotin limitation or addition of penicillin, Tween-40, or ethambutol. Thus, OdhI is essential for efficient glutamate production (36). Besides PknG, also PknA, PknB and PknL can phosphorylate OdhI, allowing the integration of multiple, currently unknown stimuli sensed by the different STPKs (37). Dephosphorylation of OdhI was shown to be catalysed by Ppp (37).

Posttranslational regulation by covalent protein modification: pupylation in C. glutamicum

Pupylation is a posttranslational protein modification occurring in the phylum Actinobacteria and some other bacterial lineages, such as Nitrospirae. It resembles eukaryotic ubiquitination and was first identified in Mycobacterium tuberculosis. Target proteins are covalently linked to the small prokaryotic ubiquitin-like protein (Pup), which, in mycobacteria, usually serves as a tag for degradation via the proteasome. However, several actinobacteria harbour genes of the pupylation machinery but lack the genes encoding the proteasome, raising the question of the fate of pupylated proteins in proteasome-free species such as C. glutamicum. In our studies, we initially demonstrated that pupylation takes place in C. glutamicum and identified 55 pupylated proteins and 66 pupylation sites (39). Subsequently, we searched for a phenotype of pupylation-deficient mutants and found a growth defect under iron limitation. Using a combination of genetic and biochemical studies, we were able to demonstrate that this growth defect is mainly due to the lacking pupylation of iron-storage protein ferritin. The available evidence indicates that pupylation is required for deoligomerization of the 24-meric ferritin in order to make the stored iron available and for preventing the assembly of ferritin shell (40). Under iron limitation, storage of iron by ferritin is counterproductive and causes the observed growth defect, as iron is lacking for the synthesis of iron-containing proteins some of which are essential for growth. Fig. 6 shows a model of the pupylation-triggered disassembly of ferritin.

Essential genes in C. glutamicum

During our basic studies of C. glutamicum metabolism and regulation we frequently come across essential genes meaning that they cannot be deleted using standard procedures. These genes are especially interesting as they perform crucial functions for life of this bacterium and are often conserved in related species. Due to the fact that we cannot characterise deletion mutants, we established alternative techniques, such as conditional downregulation, to study these proteins. One essential protein we characterised is LcpA, a membrane protein that is presumably responsible for the so far missing link in cell wall biogenesis, namely the transfer of arabinogalactan onto peptidoglycan (41). Complementation studies suggested that this function is conserved in the related pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. This article was selected as spotlight and an SEM-picture of our lcpA-silencing strain showing the loss of cell wall integrity was presented as cover picture by the Journal of Bacteriology (Fig. 7).

Respiratory chain and bioenergetics of C. glutamicum

C. glutamicum is a facultative anaerobic organism that can use oxygen and nitrate as terminal electron acceptor. Respiratory 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. The aerobic respiratory chain is composed of various dehydrogenases, such as NADH dehydrogenase type II, succinate dehydrogenase, malate:menaquinone oxidoreductase, pyruvate:menaquinone oxidoreductase, and L-lactate dehydrogenase, which transfer electrons from their respective substrates to menaquinone. The reoxidation of menaquinol is mainly catalysed by a cytochrome bc₁ complex and cytochrome aa₃ oxidase or alternatively by the cytochrome bd oxidase (42). Inspired by the finding that cytochrome c₁ is the only c-type cytochrome present in C. glutamicum and contains two heme C groups (43), we could demonstrate the existence of a cytochrome bc₁-aa₃ supercomplex by co-purification experiments (44). The kinetics of electron transfer within this supercomplex has been elucidated by the group of Peter Brezinski (45). Within the group of Carola Hunte, the redox potentials of the prosthetic groups were determined and a structural model of the supercomplex was established (Fig. 8) (46). Moreover, bioinformatic analysis of prokarytic genomes revealed that the cytochrome bc₁-aa₃ supercomplex is not unique to C. glutamicum, but obligatory in the large phylum of Actinobacteria, which includes important pathogens such as Mycobacterium tuberculosis (46).

The physiological relevance of the two branches of the aerobic respiratory chain of C. glutamicum was studied by various deletion mutants. The lack of cytochrome bd oxidase was inhibitory only under conditions of oxygen limitation, whereas the absence of the cytochrome bc₁ complex and thus of a functional supercomplex led to decreases in growth rate, biomass yield, respiration and proton-motive force (pmf) and a strongly increased maintenance coefficient under oxygen excess (47). These results show that the cytochrome bc₁–aa₃ supercomplex is of major importance for aerobic respiration.

Interestingly, a C. glutamicum strain with a completely inactivated aerobic respiratory chain could be constructed lacking both the cytochrome bc₁ complex and cytochrome bd oxidase. This mutant named DOOR for “devoid of oxygen respiration” was able to grow aerobically in glucose minimal medium after supplementation with peptone. Under these conditions, the DOOR strain displayed a fermentative type of catabolism with L-lactate as major and acetate and succinate as minor products (47). In subsequent studies we showed that C. glutamicum wild type is also capable of reasonable anaerobic growth by mixed acid fermentation with L-lactate, acetate, and succinate as products when the glucose minimal medium was supplemented with tryptone, peptone, or other amino acid mixtures (48). Besides glucose, also other carbon sources allowing substrate level phosphorylated such as fructose, sucrose, or ribose supported anaerobic growth (48).

During aerobic growth, proton motive force is established by the cytochrome bc₁–aa₃ supercomplex, which presumably transports 6 H⁺/2 e^- out of the cell, and by the cytochrome bd oxidase, which translocates only 2 H⁺/2 e^-. The proton motive force is used by the F₁F_O-ATP synthase as driving force for ATP synthesis. Although textbook knowledge usually states that during aerobic growth the majority of ATP is synthesized by ATP synthase, we observed that a ΔF₁F_O mutant of C. glutamicum reached 47% of the growth rate and 65% of the biomass of the wild type during shake-flask cultivation in glucose minimal medium (49). As the ATP requirement for biomass synthesis is the same for the wild type and the ΔF₁F_O mutant, the growth properties of the ΔF₁F_O mutant suggest that under in vivo conditions substrate level phosphorylation and electron transport phosphorylation contribute about equally to ATP synthesis.

References

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2. Krug, A., Wendisch, V. F., and Bott, M. (2005) Identification of AcnR, a TetR-type repressor of the aconitase gene acn in Corynebacterium glutamicum. J. Biol. Chem. 280, 585-595

3. Garcia-Nafria, J., Baumgart, M., Turkenburg, J. P., Wilkinson, A. J., Bott, M., and Wilson, K. S. (2013) Crystal and solution studies reveal that the transcriptional regulator AcnR of Corynebacterium glutamicum is regulated by citrate-Mg²⁺ binding to a non-canonical pocket. J. Biol. Chem. 288, 15800-15812

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

5. Engels, S., Schweitzer, J. E., Ludwig, C., Bott, M., and 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

6. Russo, S., Schweitzer, J.-E., Polen, T., Bott, M., and Pohl, E. (2009) Crystal structure of the caseinolytic protease gene regulator, a transcriptional activator in actinomycetes. J. Biol. Chem. 284, 5208-5216

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9. Frunzke, J., Engels, V., Hasenbein, S., Gätgens, C., and 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. Townsend, P. D., Jungwirth, B., Pojer, F., Bussmann, M., Money, V. A., Cole, S. T., Puhler, A., Tauch, A., Bott, M., Cann, M. J., and Pohl, E. (2014) The crystal structures of apo and cAMP-bound GlxR from Corynebacterium glutamicum reveal structural and dynamic changes upon cAMP binding in CRP/FNR family transcription factors. PLoS One 9, e113265

11. Klaffl, S., Brocker, M., Kalinowski, J., Eikmanns, B. J., and Bott, M. (2013) Complex regulation of the phosphoenolpyruvate carboxykinase genepck and characterization of its GntR-type regulator IolR as a repressor of myo-inositol utilization genes in Corynebacterium glutamicum. J. Bacteriol. 195, 4283-4296

12. Baumgart, M., Luder, K., Grover, S., Gätgens, C., Besra, G. S., and Frunzke, J. (2013) IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria. BMC Biol. 11, 122

13. Lange, C., Mustafi, N., Frunzke, J., Kennerknecht, N., Wessel, M., Bott, M., and Wendisch, V. F. (2012) Lrp of Corynebacterium glutamicum controls expression of the brnFE operon encoding the export system for L-methionine and branched-chain amino acids. J. Biotechnol. 158, 231-241

14. Baumgart, M., and Frunzke, J. (2015) The manganese-responsive regulator MntR represses transcription of a predicted ZIP family metal ion transporter inCorynebacterium glutamicum. FEMS Microbiol. Lett. 362, 1-10

15. Wennerhold, J., Krug, A., and 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

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

17. Bott, M., and Brocker, M. (2012) Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets. Appl. Microbiol. Biotechnol. 94, 1131-1150

18. Brocker, M., Schaffer, S., Mack, C., and 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

19. Schelder, S., Zaade, D., Litsanov, B., Bott, M., and Brocker, M. (2011) The two-component signal transduction system CopRS of Corynebacterium glutamicum is required for adaptation to copper-excess stress. PLoS One 6, e22143

20. Kleine, B., Chattopadhyay, A., Polen, T., Pinto, D., Mascher, T., Bott, M., Brocker, M., and Freudl, R. (2017) The three-component system EsrISR regulates a cell envelope stress response in Corynebacterium glutamicum. Mol. Microbiol., In press

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

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

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

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

25. Kocan, M., Schaffer, S., Ishige, T., Sorger-Herrmann, U., Wendisch, V. F., and Bott, M. (2005) 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

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

27. Cha, P. H., Park, S. Y., Moon, M. W., Subhadra, B., Oh, T. K., Kim, E., Kim, J. F., and Lee, J. K. (2010) Characterization of an adenylate cyclase gene (cyaB) deletion mutant of Corynebacterium glutamicum ATCC 13032. Appl. Microbiol. Biotechnol. 85, 1061-1068

28. Schulte, J., Baumgart, M., and Bott, M. (2017) Identification of the cAMP phosphodiesterase CpdA as novel key player in cAMP-dependent regulation in Corynebacterium glutamicum. Mol. Microbiol. 103, 534-552

29. Schulte, J., Baumgart, M., and Bott, M. (2017) Development of a single-cell GlxR-based cAMP biosensor for Corynebacterium glutamicum. J. Biotechnol., In press

30. van Ooyen, J., Emer, D., Bussmann, M., Bott, M., Eikmanns, B. J., and Eggeling, L. (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

31. Emer, D., Krug, A., Eikmanns, B. J., and 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

32. Bussmann, M., Emer, D., Hasenbein, S., Degraf, S., Eikmanns, B. J., and Bott, M. (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

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

34. Niebisch, A., Kabus, A., Schultz, C., Weil, B., and 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

35. Krawczyk, S., Raasch, K., Schultz, C., Hoffelder, M., Eggeling, L., and Bott, M. (2010) The FHA domain of OdhI interacts with the carboxyterminal 2-oxoglutarate dehydrogenase domain of OdhA in Corynebacterium glutamicum. FEBS Lett. 584, 1463-1468

36. Schultz, C., Niebisch, A., Gebel, L., and 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

37. Schultz, C., Niebisch, A., Schwaiger, A., Viets, U., Metzger, S., Bramkamp, M., and Bott, M. (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

38. Bott, M., and Eikmanns, B. J. (2013) TCA cycle and glyoxylate shunt of Corynebacterium glutamicum. in Corynebacterium glutamicum: Biology and Biotechnology (Yukawa, H., and Inui, M. eds.), Springer Verlag, Heidelberg. pp 281-314

39. Küberl, A., Franzel, B., Eggeling, L., Polen, T., Wolters, D. A., and Bott, M. (2014) Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis. Proteomics 14, 1531-1542

40. Küberl, A., Polen, T., and Bott, M. (2016) The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin. Proc. Natl. Acad. Sci. USA 113, 4806-4811

41. Baumgart, M., Schubert, K., Bramkamp, M., and Frunzke, J. (2016) Impact of LytR-CpsA-Psr proteins on cell wall biosynthesis in Corynebacterium glutamicum. J. Bacteriol. 198, 3045-3059

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

43. Niebisch, A., and 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

44. Niebisch, A., and Bott, M. (2003) 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. 278, 4339-4346

45. Graf, S., Fedotovskaya, O., Kao, W. C., Hunte, C., Adelroth, P., Bott, M., von Ballmoos, C., and Brzezinski, P. (2016) Rapid electron transfer within the III-IV supercomplex in Corynebacterium glutamicum. Sci. Rep. 6, 34098

46. Kao, W. C., Kleinschroth, T., Nitschke, W., Baymann, F., Neehaul, Y., Hellwig, P., Richers, S., Vonck, J., Bott, M., and Hunte, C. (2016) The obligate respiratory supercomplex from Actinobacteria. Biochim. Biophys. Acta 1857, 1705-1714

47. Koch-Koerfges, A., Pfelzer, N., Platzen, L., Oldiges, M., and Bott, M. (2013) Conversion of Corynebacterium glutamicum from an aerobic respiring to an aerobic fermenting bacterium by inactivation of the respiratory chain. Biochim. Biophys. Acta 1827, 699-708

48. Michel, A., Koch-Koerfges, A., Krumbach, K., Brocker, M., and Bott, M. (2015) Anaerobic growth of Corynebacterium glutamicum via mixed-acid fermentation. Appl. Environ. Microbiol. 81, 7496-7508

49. Koch-Koerfges, A., Kabus, A., Ochrombel, I., Marin, K., and Bott, M. (2012) Physiology and global gene expression of a Corynebacterium glutamicum DF₁F_O-ATP synthase mutant devoid of oxidative phosphorylation. Biochim. Biophys. Acta 1817, 370-380