The glyoxylate shunt is essential for CO2-requiring oligotrophic growth of Rhodococcus erythropolis N9T-4
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.
KeywordsRandom 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.
- 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
- 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
- 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
- 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
- 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
- Yu F, Wakamatsu M (2011) Random genome insertion for Rhodococcus bacterium and deletion tool. Japan patent, P2011-223925AGoogle Scholar