Applied Microbiology and Biotechnology

, Volume 99, Issue 13, pp 5627–5637 | Cite as

The glyoxylate shunt is essential for CO2-requiring oligotrophic growth of Rhodococcus erythropolis N9T-4

  • Takanori Yano
  • Nobuyuki Yoshida
  • Fujio Yu
  • Miki Wakamatsu
  • Hiroshi Takagi
Applied microbial and cell physiology


Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth requiring atmospheric CO2 and forms its colonies on an inorganic basal medium (BM) without any additional carbon source. Screening of a random mutation library constructed by a unique genome deletion method that we established indicated that the aceA, aceB, and pckG genes encoding isocitrate lyase, malate synthase, and phosphoenolpyruvate carboxykinase, respectively, were requisite for survival on BM plates. The aceA- and aceB deletion mutants and the pckG deletion mutant grew well on BM plates containing L-malate and D-glucose, respectively, suggesting that the glyoxylate (GO) shunt and gluconeogenesis are essential for the oligotrophic growth of N9T-4. Interestingly, most of the enzyme activities in the TCA cycle were observed in the cell-free extract of N9T-4, with perhaps the most important exception being α-ketoglutarate dehydrogenase (KGDH) activity. Instead of the KGDH activity, we detected a remarkable level of α-ketoglutarate decarboxylase (KGD) activity, which is the activity exhibited by the E1 component of the KGDH complex in Mycobacterium tuberculosis. The recombinant KGD of N9T-4 catalyzed the decarboxylation of α-ketoglutarate to form succinic semialdehyde (SSA) in a time-dependent manner. Since N9T-4 also showed a detectable SSA dehydrogenase activity, we concluded that N9T-4 possesses a variant TCA cycle, which uses SSA rather than succinyl-CoA. These results suggest that oligotrophic N9T-4 cells utilize the GO shunt to avoid the loss of carbons as CO2 and to conserve CoA units in the TCA cycle.


Random mutagenesis Carbon dioxide Glyoxylate shunt TCA cycle Oligotroph Rhodococcus erythropolis N9T-4 



The authors greatly appreciate Maki Kaneoka for her technical assistance for construction and screening of the random mutation library. This work was partly supported by the Sasakawa Scientific Research Grant from The Japan Science Society to T.Y. and Global COE Program in NAIST from the Ministry of Education, Science, Culture, Sports and Technology of Japan.

Supplementary material

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  1. Baughn AD, Garforth SJ, Vilchèze C, Jacobs WR Jr (2009) An anaerobic-type α-ketoglutarate ferredoxin oxidoreductase completes the oxidative tricarboxylic acid cycle of Mycobacterium tuberculosis. PLoS Pathog 5:e1000662PubMedCentralPubMedCrossRefGoogle Scholar
  2. Berg IA (2011) Ecological aspects of the distribution of different autotrophic CO2 fixation pathways. Appl Environ Microbiol 77:1925–1936PubMedCentralPubMedCrossRefGoogle Scholar
  3. Beste DJ, Bonde B, Hawkins N, Ward JL, Beale MH, Noack S, Nöh K, Kruger NJ, Ratcliffe RG, McFadden J (2011) 13C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation. PLoS Pathog 7:e1002091PubMedCentralPubMedCrossRefGoogle Scholar
  4. Bombar D, Heller P, Sanchez-Baracaldo P, Carter BJ, Zehr JP (2014) Comparative genomics reveals surprising divergence of two closely related strains of uncultivated UCYN-A cyanobacteria. ISME J 8:2530–2542PubMedCrossRefGoogle Scholar
  5. Bonilla-Rosso G, Peimbert M, Alcaraz LD, Hernández I, Eguiarte LE, Olmedo-Alvarez G, Souza V (2012) Comparative metagenomics of two microbial mats at Cuatro Ciénegas Basin II: community structure and composition in oligotrophic environments. Astrobiology 12:659–673PubMedCentralPubMedCrossRefGoogle Scholar
  6. Bystrykh LV, Govorukhina NI, van Ophem PW, Hektor HJ, Dijkhuizen L, Duine JA (1993) Formaldehyde dismutase activities in Gram-positive bacteria oxidizing methanol. J Gen Microbiol 139:1979–1985CrossRefGoogle Scholar
  7. de Carvalho LP, Zhao H, Dickinson CE, Arango NM, Lima CD, Fischer SM, Ouerfelli O, Nathan C, Rhee KY (2010) Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase. Chem Biol 17:323–332PubMedCentralPubMedCrossRefGoogle Scholar
  8. Goryshin IY, Jendrisak J, Hoffman LM, Meis R, Reznikoff WS (2000) Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat Biotechnol 18:97–100PubMedCrossRefGoogle Scholar
  9. Hoffelder M, Raasch K, van Ooyen J, Eggeling L (2010) The E2 domain of OdhA of Corynebacterium glutamicum has succinyltransferase activity dependent on lipoyl residues of the acetyltransferase AceF. J Bacteriol 192:5203–5211PubMedCentralPubMedCrossRefGoogle Scholar
  10. Kanao T, Kawamura M, Fukui T, Atomi H, Imanaka T (2002) Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola. Eur J Biochem 269:1926–1931PubMedCrossRefGoogle Scholar
  11. Könneke M, Schubert DM, Brown PC, Hügler M, Standfest S, Schwander T, Schada von Borzyskowski L, Erb TJ, Stahl DA, Berg IA (2014) Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO2 fixation. Proc Natl Acad Sci U S A 111:8239–8244PubMedCentralPubMedCrossRefGoogle Scholar
  12. Kuznetsov S, Dubinina G, Lapteva N (1979) Biology of oligotrophic bacteria. Annu Rev Microbiol 33:377–387PubMedCrossRefGoogle Scholar
  13. McKinney JD, Höner zu Bentrup K, Muñoz-Elías EJ, Miczak A, Chen B, Chan WT, Swenson D, Sacchettini JC, Jacobs WR Jr, Russell DG (2000) Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:735–738PubMedCrossRefGoogle Scholar
  14. Muñoz-Elías EJ, McKinney JD (2005) Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat Med 11:638–644PubMedCentralPubMedCrossRefGoogle Scholar
  15. Muro-Pastor MI, Florencio FJ (1994) NADP(+)-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. Strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene. J Bacteriol 176:2718–2726PubMedCentralPubMedGoogle Scholar
  16. Ohhata N, Yoshida N, Egami H, Katsuragi T, Tani Y, Takagi H (2007) An extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4, isolated from crude oil. J Bacteriol 189:6824–6831PubMedCentralPubMedCrossRefGoogle Scholar
  17. Pelicic V, Reyrat JM, Gicquel B (1996) Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J Bacteriol 178:1197–1199PubMedCentralPubMedGoogle Scholar
  18. Senechkin IV, Speksnijder AG, Semenov AM, van Bruggen AH, van Overbeek LS (2010) Isolation and partial characterization of bacterial strains on low organic carbon medium from soils fertilized with different organic amendments. Microb Ecol 60:829–839PubMedCrossRefGoogle Scholar
  19. Siering P, Wolfe G, Wilson M, Yip A, Carey C, Wardman C, Shapiro R, Stedman K, Kyle J, Yuan T (2013) Microbial biogeochemistry of boiling springs lake: a physically dynamic, oligotrophic, low-pH geothermal ecosystem. Geobiology 11:356–376PubMedCrossRefGoogle Scholar
  20. Steinhauser D, Fernie AR, Araújo WL (2012) Unusual cyanobacterial TCA cycles: not broken just different. Trends Plant Sci 17:503–509PubMedCrossRefGoogle Scholar
  21. Tian J, Bryk R, Itoh M, Suematsu M, Nathan C (2005a) Variant tricarboxylic acid cycle in Mycobacterium tuberculosis: identification of α-ketoglutarate decarboxylase. Proc Natl Acad Sci U S A 102:10670–10675PubMedCentralPubMedCrossRefGoogle Scholar
  22. Tian J, Bryk R, Shi S, Erdjument-Bromage H, Tempst P, Nathan C (2005b) Mycobacterium tuberculosis appears to lack α-ketoglutarate dehydrogenase and encodes pyruvate dehydrogenase in widely separated genes. Mol Microbiol 57:859–868PubMedCrossRefGoogle Scholar
  23. Usuda Y, Tujimoto N, Abe C, Asakura Y, Kimura E, Kawahara Y, Kurahashi O, Matsui H (1996) Molecular cloning of the Corynebacterium glutamicum (‘Brevibacterium lactofermentum’ AJ12036) odhA gene encoding a novel type of 2-oxoglutarate dehydrogenase. Microbiology 142:3347–3354PubMedCrossRefGoogle Scholar
  24. Wagner T, Barilone N, Alzari PM, Bellinzoni M (2014) A dual conformation of the post-decarboxylation intermediate is associated with distinct enzyme states in mycobacterial KGD (α-ketoglutarate decarboxylase). Biochem J 457:425–434PubMedCrossRefGoogle Scholar
  25. Yano T, Yoshida N, Takagi H (2012) Carbon monoxide utilization of an extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4. J Biosci Bioeng 114:53–55PubMedCrossRefGoogle Scholar
  26. Yoshida N, Yagi K, Sato D, Watanabe N, Kuroishi T, Nishimoto K, Yanagida A, Katsuragi T, Kanagawa T, Kurane R, Tani Y (2005) Bacterial communities in petroleum oil in stockpiles. J Biosci Bioeng 99:143–149PubMedCrossRefGoogle Scholar
  27. Yoshida N, Ohhata N, Yoshino Y, Katsuragi T, Tani Y, Takagi H (2007) Screening of carbon dioxide-requiring extreme oligotrophs from soil. Biosci Biotechnol Biochem 71:2830–2832PubMedCrossRefGoogle Scholar
  28. Yoshida N, Hayasaki T, Takagi H (2011) Gene expression analysis of methylotrophic oxidoreductases involved in the oligotrophic growth of Rhodococcus erythropolis N9T-4. Biosci Biotechnol Biochem 75:123–127PubMedCrossRefGoogle Scholar
  29. Yoshida N, Inaba S, Takagi H (2014) Utilization of atmospheric ammonia by an extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4. J Biosci Bioeng 117:28–32PubMedCrossRefGoogle Scholar
  30. Yu F, Wakamatsu M (2011) Random genome insertion for Rhodococcus bacterium and deletion tool. Japan patent, P2011-223925AGoogle Scholar
  31. Zhang S, Bryant DA (2011) The tricarboxylic acid cycle in cyanobacteria. Science 334:1551–1553PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Graduate School of Biological SciencesNara Institute of Science and TechnologyIkomaJapan
  2. 2.Department of Applied Chemistry and Biochemical Engineering, Graduate school of EngineeringShizuoka UniversityHamamatsuJapan
  3. 3.Yokohama Research LabolatoriesMitsubishi Rayon Co., Ltd.YokohamaJapan

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