Study of an alternate glyoxylate cycle for acetate assimilation by <i>Rhodobacter sphaeroides</i>

Alber, Birgit E.; Spanheimer, Regina; Ebenau-Jehle, Christa; Fuchs, Georg

doi:10.1111/j.1365-2958.2006.05238.x

Cited by 102 publications

(122 citation statements)

References 38 publications

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“…As the intracellular concentration of acetyl-CoA is likely to be high in cells grown on acetate and as pyruvate level should be kept low through consumption by acetolactate synthase, the net flux through this enzyme is expected to favour pyruvate synthesis when the bacteria are grown on acetate. Noticeably, the high level of acetyl-CoA can be maintained in the cell through the upregulation of acetyl-CoA transferase (Rru_A1927; fold change, 1.8; P-value, 0.004; identified peptides, 34), which could be responsible for acetate activation, as mentioned by Alber et al (2006). The preference for reductive carboxylation activity is further supported by the observation of major downregulation of all subunits of the pyruvate dehydrogenase (lipoamide) complex (Rru_A1878, Rru_A1879, Rru_A1881).…”

Section: Resultsmentioning

confidence: 99%

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New insight into the photoheterotrophic growth of the isocytrate lyase-lacking purple bacterium Rhodospirillum rubrum on acetate

et al. 2015

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Purple non-sulfur bacteria are well known for their metabolic versatility. One of these bacteria, Rhodospirillum rubrum S1H, has been selected by the European Space Agency to ensure the photoheterotrophic assimilation of volatile fatty acids in its regenerative life support system, MELiSSA. Here, we combined proteomic analysis with bacterial growth analysis and enzymatic activity assays in order to better understand acetate photoassimilation. In this isocitrate lyaselacking organism, the assimilation of two-carbon compounds cannot occur through the glyoxylate shunt, and the citramalate cycle has been proposed to fill this role, while, in Rhodobacter sphaeroides, the ethylmalonyl-CoA pathway is used for acetate assimilation. Using proteomic analysis, we were able to identify and quantify more than 1700 unique proteins, representing almost one-half of the theoretical proteome of the strain. Our data reveal that a pyruvate : ferredoxin oxidoreductase (NifJ) could be used for the direct assimilation of acetyl-CoA through pyruvate, potentially representing a new redox-balancing reaction. We additionally propose that the ethylmalonyl-CoA pathway could also be involved in acetate assimilation by the examined strain, since specific enzymes of this pathway were all upregulated and activity of crotonyl-CoA reductase/carboxylase was increased in acetate conditions. Surprisingly, we also observed marked upregulation of glutaryl-CoA dehydrogenase, which could be a component of a new pathway for acetate photoassimilation. Finally, our data suggest that citramalate could be an intermediate of the branched-chain amino acid biosynthesis pathway, which is activated during acetate assimilation, rather than a metabolite of the so-called citramalate cycle.

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Section: Resultsmentioning

confidence: 99%

“…All the enzymes belonging to this pathway in Rb. sphaeroides have been identified, purified and extensively characterized (Alber et al, 2006;Erb et al, 2007Erb et al, , 2008Erb et al, , 2009bMeister et al, 2005). Schneider et al (2012) recently showed that Methylobacterium extorquens also employs the EMC pathway.…”

Section: Introductionmentioning

confidence: 99%

New insight into the photoheterotrophic growth of the isocytrate lyase-lacking purple bacterium Rhodospirillum rubrum on acetate

et al. 2015

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“…Subsequently, propionyl-CoA is transformed to malate, from which 1 glyoxylate and 1 acetyl-CoA are generated (20). More recently, a second hypothesis, the ethylmalonyl-CoA pathway (EMCP), was proposed from studies of C2 assimilation pathways in R. sphaeroides (21)(22)(23). This pathway ( Fig.…”

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confidence: 99%

Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

Peyraud

Kiefer

Christen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

217

242

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The assimilation of one-carbon (C1) compounds, such as methanol, by serine cycle methylotrophs requires the continuous regeneration of glyoxylate. Instead of the glyoxylate cycle, this process is achieved by a not yet established pathway where CoA thioesters are known to play a key role. We applied state-of-the-art metabolomics and 13 C metabolomics strategies to demonstrate how glyoxylate is generated during methylotrophic growth in the isocitrate lyase-negative methylotroph Methylobacterium extorquens AM1. High-resolution mass spectrometry showed the presence of CoA thioesters specific to the recently proposed ethylmalonyl-CoA pathway. The operation of this pathway was demonstrated by short-term 13 C-labeling experiments, which allowed determination of the sequence of reactions from the order of label incorporation into the different CoA derivatives. Analysis of 13 C positional enrichment in glycine by NMR was consistent with the predicted labeling pattern as a result of the operation of the ethylmalonyl-CoA pathway and the unique operation of the latter for glyoxylate generation during growth on methanol. The results also revealed that 2 molecules of glyoxylate were regenerated in this process. This work provides a complete pathway for methanol assimilation in the model methylotroph M. extorquens AM1 and represents an important step toward the determination of the overall topology of its metabolic network. The operation of the ethylmalonyl-CoA pathway in M. extorquens AM1 has major implications for the physiology of these methylotrophs and their role in nature, and it also provides a common ground for C1 and C2 compound assimilation in isocitrate lyase-negative bacteria.13 C labeling ͉ CoA ester ͉ methylotroph ͉ one-carbon metabolism ͉ glyoxylate regeneration M ethylotrophic bacteria are organisms capable of using reduced carbon compounds, such as methanol or methane, as sole sources of carbon and energy, and they play a key role in carbon cycling in their environment. They also represent promising organisms in biotechnology for the conversion of one-carbon (C1) substrates to value-added products (1). The elucidation of the mechanisms enabling growth on reduced C1 compounds of Methylobacterium extorquens AM1, one of the most studied methylotrophs, has been a longstanding goal, and although great progress has been made (2-5), it is still not fully achieved. A key point has been to understand how the bacterium incorporates C1 units into cell material. The serine cycle was elucidated in this organism during the early 1960s by Quayle and coworkers (6-9). The assimilation of C1 units by this pathway requires continuous regeneration of glyoxylate from acetyl-CoA and can be achieved, in principle, via the well-known glyoxylate cycle (10). However, Dunstan and coworkers (11)(12)(13)(14) showed in 1972 and 1973 that M. extorquens AM1 lacks the key enzyme of the glyoxylate cycle, isocitrate lyase, but has an alternative route involving oxidation of acetate to glyoxylate that functions during growth on both C1 and C2 co...

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“…Table 3) support this latter observation. Whatever the nature of the complete anaplerotic pathway (see Alber et al, 2006), the apparent malate synthase in crude extract shows the cycle to be present at high levels in the taurine(carbon)-ammoniumgrown cells, but only in traces in taurine(nitrogen)-succinate-grown cells (Table 3). Presumably, when taurine is supplying nitrogen in the presence of a C 4 carbon source in excess (in our case succinate), the anaplerotic sequence is not necessary, and is regulated independently of the operation of the taurine degradative pathway.…”

Section: Discussionmentioning

confidence: 99%

Genome-enabled analysis of the utilization of taurine as sole source of carbon or of nitrogen by Rhodobacter sphaeroides 2.4.1

2006

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In the present study, R. sphaeroides 2.4.1 was found to grow exponentially with taurine as the sole source of carbon and energy for growth. When taurine was the sole source of nitrogen in succinate-salts medium, the taurine was rapidly degraded, and most of the organic nitrogen was excreted as the ammonium ion, which was then utilized for growth. Most of the enzymes involved in dissimilation, taurine dehydrogenase (TDH), sulfoacetaldehyde acetyltransferase (Xsc) and phosphate acetyltransferase (Pta), were found to be inducible, and evidence for transcription of the corresponding genes (tauXY, xsc and pta), as well as of tauKLM, encoding the postulated TRAP transporter for taurine, and of tauZ, encoding the sulfate exporter, was obtained by reverse-transcription PCR. An additional branch of the pathway, observed by Novak et al. (2004) (Microbiology 150, 1881-1891) in R. sphaeroides TAU3, involves taurine : pyruvate aminotransferase (Tpa) and a presumptive ABC transporter (NsbABC). No evidence for a significant role of this pathway, or of the corresponding alanine dehydrogenase (Ald), was obtained for R. sphaeroides 2.4.1. The anaplerotic pathway needed under these conditions in R. sphaeroides 2.4.1 seems to involve malyl-CoA lyase, which was synthesized inducibly, and not malate synthase (GlcB), whose presumed gene was not transcribed under these conditions.

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Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides

Abstract: SummaryOrganisms, which grow on organic substrates that are metabolized via acetyl-CoA, are faced with the problem to form all cell constituents from this C2-unit.

Cited by 102 publications

References 38 publications

New insight into the photoheterotrophic growth of the isocytrate lyase-lacking purple bacterium Rhodospirillum rubrum on acetate

New insight into the photoheterotrophic growth of the isocytrate lyase-lacking purple bacterium Rhodospirillum rubrum on acetate

Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

Genome-enabled analysis of the utilization of taurine as sole source of carbon or of nitrogen by Rhodobacter sphaeroides 2.4.1

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Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides

Abstract: SummaryOrganisms, which grow on organic substrates that are metabolized via acetyl-CoA, are faced with the problem to form all cell constituents from this C2-unit.

Cited by 102 publications

References 38 publications

New insight into the photoheterotrophic growth of the isocytrate lyase-lacking purple bacterium Rhodospirillum rubrum on acetate

New insight into the photoheterotrophic growth of the isocytrate lyase-lacking purple bacterium Rhodospirillum rubrum on acetate

Demonstration of the ethylmalonyl-CoA pathway by using 13 C metabolomics

Genome-enabled analysis of the utilization of taurine as sole source of carbon or of nitrogen by Rhodobacter sphaeroides 2.4.1

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Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics