Department of Bacteriology, University of Wisconsin-Madison, 1710 University Ave, Madison, WI 53726-4087, USA
Correspondence
Jorge C. Escalante-Semerena
escalante{at}bact.wisc.edu
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ABSTRACT |
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Present address: Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building Hanover, NH 03755-3844, USA.
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INTRODUCTION |
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Genetic and biochemical analyses of S. enterica strains identified the chromosomal locus required for growth of this bacterium on ethanolamine (Roof & Roth, 1988, 1989
). Sequence analysis of the ethanolamine utilization (eut) operon revealed 17 open reading frames, eutSPQTDMNEJGHABCLKR (Kofoid et al., 1999
; Stojiljkovic et al., 1995
) (Fig. 2a
). The eutR gene, located 3' to the operon and independently transcribed from it (Sheppard & Roth, 1994
), encodes the EutR transcription activator protein, which becomes active in the presence of ethanolamine and coenzyme B12 (Roof & Roth, 1988
, 1992
; Sheppard & Roth, 1994
). Several of the eut genes (e.g. eutSMNLK) encode proteins with homology to cyanobacterial carboxysomal shell proteins (Kofoid et al., 1999
; Stojiljkovic et al., 1995
). In cyanobacteria and thiobacilli, this multiprotein complex is involved in carbon dioxide fixation and concentration (Badger & Price, 2003
; Baker et al., 2000
; Friedberg et al., 1993
). The function of the metabolosome encoded by the eut operon remains unclear. In S. enterica, a similar multiprotein complex is assembled during growth on 1,2-propanediol (Havemann et al., 2002
; Havemann & Bobik, 2003
), and it was suggested that this structure might be used to contain propionaldehyde (Bobik et al., 1999
; Rondon et al., 1995
). Similarly, the metabolosome involved in ethanolamine catabolism was proposed to contain acetaldehyde (Rondon et al., 1995
).
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On the basis of the data reported here, we propose that a fraction of ethanolamine is converted to acetate via the energy-conserving phosphotransacetylase (EutD)/acetate kinase (Ack) pathway, with no apparent involvement of the housekeeping phosphotransacetylase (Pta) enzyme. Ac-CoA synthetase (Acs) and Pta are responsible for recapturing acetate for later use via the Krebs cycle and glyoxylate bypass.
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METHODS |
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PCR amplifications.
Enzyplus (ENZYPOL) was used in PCR amplification during construction of the eutD deletion strain of S. enterica. All reactions were performed in an Eppendorf Mastercycler gradient PCR thermal cycler (Brinkmann Instruments). All primers were purchased from Integrated DNA Technologies.
Construction of non-polar eutD deletion.
The deletion of eutD was constructed as described by Datsenko & Wanner (2000), except that all manipulations were performed in S. enterica. The cat+ cassette in plasmid pKD3 was amplified using PAGE-purified primers with 50 bp of perfect homology for the 5' and 3' ends of eutD: eutD5'DEL (5'-TGACGGTCGCCCAAATTCAACAACGGTTGGGAGAGAAGCCATGATCATTGAACGCGTGTAGGCTGGAGCTGCTTC-3') and eutD3'DEL (5'-CGTGTTTTCCTCTCATTAAAGGGGTCCAGAACGGGACCGTTCATTCAACCAGTGTCATATGAATATCCTCCTTA-3'). Construction of the deletion strain from this point was as described in the original protocol, except that the S. enterica strain JE6692 was used as the recipient. The presence of the deletion was confirmed using ABI PRISM non-radioactive BigDye cycle terminator DNA sequencing methodology (PerkinElmer Life Sciences) according to the manufacturer's instructions. DNA sequence was determined at the Biotechnology Center of the University of Wisconsin.
Determination of excreted acetate during growth on ethanolamine.
Overnight cultures of S. enterica grown at 37 °C in LB were subcultured 1 : 100 into 20 ml NCE minimal medium containing ethanolamine as the source of carbon and energy. Samples (1 ml) were removed at intervals, and the optical density of the culture was determined using a Klett colorimeter furnished with a red filter. Samples were centrifuged for 5 min at 13 000 g in a Microfuge 18 centrifuge (Beckman Coulter). A 500 µl sample of the supernatant was filtered through a 0·45 µm Spin-X centrifuge tube filter (Corning) and acidified by the addition of 2·5 µl of a 5 M H2SO4 solution. Acidified samples were stored at 20 °C until used. HPLC was used to analyse the composition of the samples (see below).
HPLC analysis.
Samples (200 µl) containing organic acids excreted by the strains of S. enterica used in these studies were resolved using a Waters HPLC system equipped with a model 600 solvent delivery system, a model 900 photodiode array detector, and a column heater. An Aminex HPX-87H organic acid analysis column (300x7·8 mm, Bio-Rads) was maintained at 45 °C and developed isocratically with 0·005 M H2SO4 at a flow rate of 0·6 ml min1. Separations were monitored at 210 nm. Under these conditions, acetate eluted 14·6 min after injection. The area under the peak corresponding to acetate was quantified using the Waters Millenium software package to determine the concentration of acetate in the sample.
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RESULTS AND DISCUSSION |
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Acetate excretion during growth on ethanolamine requires Ack activity
Parallel studies with a strain carrying an in-frame deletion of the ack gene (lacked Ack) showed a factor-of-two increase in the doubling time of the ack strain (doubling time 3·9 h) relative to that of the wild-type strain (doubling time 2·1 h) (Fig. 3a vs Fig. 4
). A more profound difference between the strains was observed when the acetate excretion/recapture pattern was determined. Unlike the wild-type strain, the ack strain excreted only a small amount of acetate (Fig. 3a
vs Fig. 4
), suggesting that acetate excretion proceeded via acetyl phosphate (Ac-P). Thus it appears that the slower growth rate of the ack strain may be due to an accumulation of Ac-P.
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Acetate excretion during growth on ethanolamine requires EutD phosphotransacetylase activity
Although the absence of EutD activity only slightly affected growth of S. enterica on ethanolamine, in the absence of this enzyme acetate excretion was abolished during growth on ethanolamine (Fig. 6a; open triangles). Inactivation of the pta gene in an eutD strain did not affect growth rate, final cell density, or acetate excretion (Fig. 6b
). Surprisingly, inactivation of the acs gene restored acetate excretion in the pta eutD strain, but no recapture was observed (Fig. 6c
), consistent with the finding that Acs and Pta activities were needed for recapture (Fig. 3d
). Based on these results, and others discussed above, we propose that ethanolamine catabolism proceeds via Ac-CoA, and that acetate is excreted via the EutD/Ack pathway.
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We used the protein sequence of Saccharomyces cerevisiae Ac-CoA hydrolase (Ach1p) (Buu et al., 2003) to search for genes of Salmonella enterica encoding homologous proteins. The search yielded open reading frame STM3118 as the only gene possibly encoding a structural orthologue of Ach1p Ac-CoA hydrolase. We constructed an in-frame deletion of STM3118 in strain JE7498 (eutD acs pta) and studied the growth behaviour and acetate excretion pattern in the resulting strain (JE7715). In addition to growing on ethanolamine to wild-type levels, strain JE7715 excreted as much acetate as did its parent strain JE7498, indicating that STM3118 was not involved in acetate excretion in a strain lacking Acs, EutD and Pta activities (data not shown). This result does not rule out the existence of non-orthologous replacements of Ach1p Ac-CoA hydrolase in S. enterica, a possibility that has not been investigated. Inactivation of either poxB or ack did not prevent acetate excretion in strains JE7832 (poxB acs pta eutD) or strain JE8326 (eutD acs ack) (data not shown). Results obtained with the poxB mutant suggested that PoxB was not responsible for the observed acetate excretion in the acs pta eutD strain JE7832. Results obtained with strain JE8326 suggested that acetate release in such a strain did not proceed via Ac-P. At present, the identity of the acetate-releasing system operating in the acs pta eutD strain remains unclear.
Why is EutD, and not Pta, function required for ethanolamine catabolism?
One plausible answer to this question may lie in part in the kinetic differences between these enzymes. Although the S. enterica Pta enzyme (SePta) has not been isolated and characterized, its close relative from Escherichia coli (EcPta) has (Brinsmade & Escalante-Semerena, 2004; Shimizu et al., 1969
; Suzuki, 1969
). The EutD enzyme has a sevenfold higher affinity for CoA than does EcPta (46 vs 320 µM), a 23-fold higher affinity for Ac-P than does EcPta (0·13 vs 3 mM), and a 10-fold higher specific activity than that reported for EcPta (3044 vs 333). Relative to EcPta (and, by extrapolation, to SePta), EutD is a very efficient enzyme (Brinsmade & Escalante-Semerena, 2004
; Shimizu et al., 1969
; Suzuki, 1969
). The high affinity of EutD for Ac-CoA and its presumable location within the ethanolamine metabolosome is proposed to prevent a non-physiological buildup of Ac-CoA, leading to a release of CoA and acetate and the concomitant production of ATP via substrate-level phosphorylation. Acetate excretion may help the cell minimize the negative effect of intracellular acetate accumulation on the pool of glutamate and other unidentified ions (Roe et al., 1998
; Russell & Diez-Gonzalez, 1998
). Relief from the toxic effects of acetate during growth on ethanolamine would be reached when the acetate switch is flipped towards acetate assimilation.
Growth on ethanolamine proceeds via the glyoxylate bypass and the Krebs cycle
A strain lacking isocitrate dehydrogenase [JE4561 icd1 : : Tn10d(tet+)] did not grow on ethanolamine as the carbon and energy source on medium supplemented with glutamate, indicating that catabolism of ethanolamine proceeds via the tricarboxylic acid (Krebs) cycle. Similarly, a strain lacking malate synthase (AceB) and isocitrate lyase (AceA) activities (strain JE4173) failed to grow on ethanolamine, indicating that the glyoxylate bypass is also required for the utilization of ethanolamine as the carbon and energy source.
A working model
Fig. 7 summarizes our interpretation of the data reported in this paper. We postulate that in wild-type S. enterica, a portion of the ethanolamine-derived pool of Ac-CoA is converted to acetate and excreted into the medium for later use, with the remaining Ac-CoA entering central metabolism. Acetate excretion may be needed to regenerate free CoA. As the Acs level increases, exogenous acetate is recaptured by Acs and Ack/Pta and is catabolized via the TCA cycle and glyoxylate bypass, increasing the amount of carbon and energy available for growth. Elegant studies of acs expression indicate that the Acs level is very low in low-density cultures (Browning et al., 2004
), a fact that would explain the kinetics of acetate accumulation and recapture from the environment. The very efficient EutD phosphotransacetylase, in conjunction with acetate kinase, regenerates free CoA, a process that appears to also involve an as-yet-unidentified acetate-releasing system. It is an enigma why in the absence of EutD phosphotransacetylase the housekeeping Pta phosphotransacetylase does not compensate for EutD. We propose that the absence of EutD may trigger the diversion of acetaldehyde to ethanol, avoiding the accumulation of Ac-CoA. This and other ideas are under investigation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Badger, M. R. & Price, G. D. (2003). CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. J Exp Bot 54, 609622.
Baker, S. H., Williams, D. S., Aldrich, H. C., Gambrell, A. C. & Shively, J. M. (2000). Identification and localization of the carboxysome peptide Csos3 and its corresponding gene in Thiobacillus neapolitanus. Arch Microbiol 173, 278283.[CrossRef][Medline]
Balch, W. E. & Wolfe, R. S. (1976). New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Appl Environ Microbiol 32, 781791.[Medline]
Berkowitz, D., Hushon, J. M., Whitfield, H. J., Roth, J. & Ames, B. N. (1968). Procedure for identifying nonsense mutations. J Bacteriol 96, 215220.[CrossRef][Medline]
Blackwell, C. M. & Turner, J. M. (1978). Microbial metabolism of amino alcohols: formation of coenzyme B12-dependent ethanolamine ammonia-lyase and its concerted induction in Escherichia coli. Biochem J 176, 751757.[Medline]
Bobik, T. A., Xu, Y., Jeter, R. M., Otto, K. E. & Roth, J. R. (1997). Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase. J Bacteriol 179, 66336639.
Bobik, T. A., Havemann, G. D., Busch, R. J., Williams, D. S. & Aldrich, H. C. (1999). The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 includes genes necessary for formation of polyhedral organelles involved in coenzyme B12-dependent 1,2-propanediol degradation. J Bacteriol 181, 59675975.
Brinsmade, S. R. & Escalante-Semerena, J. C. (2004). The eutD gene of Salmonella enterica encodes a protein with phosphotransacetylase enzyme activity. J Bacteriol 186, 18901892.
Browning, D. F., Beatty, C. M., Sanstad, E. A., Gunn, K. E., Busby, S. J. & Wolfe, A. J. (2004). Modulation of CRP-dependent transcription at the Escherichia coli acsP2 promoter by nucleoprotein complexes: anti-activation by the nucleoid proteins FIS and IHF. Mol Microbiol 51, 241254.[CrossRef][Medline]
Buu, L. M., Chen, Y. C. & Lee, F. J. (2003). Functional characterization and localization of acetyl-CoA hydrolase, Ach1p, in Saccharomyces cerevisiae. J Biol Chem 278, 1720317209.
Chan, R. K., Botstein, D., Watanabe, T. & Ogata, Y. (1972). Specialized transduction of tetracycline resistance by phage P22 in Salmonella typhimurium. II. Properties of a high transducing lysate. Virology 50, 883898.[CrossRef][Medline]
Chang, G. W. & Chang, J. T. (1975). Evidence for the B12-dependent enzyme ethanolamine deaminase in Salmonella. Nature 254, 150151.[CrossRef][Medline]
Conner, C. P., Heithoff, D. M., Julio, S. M., Sinsheimer, R. L. & Mahan, M. J. (1998). Differential patterns of acquired virulence genes distinguish Salmonella strains. Proc Natl Acad Sci U S A 95, 46414645.
Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 66406645.
Davis, R. W., Botstein, D. & Roth, J. R. (1980). A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Faust, L. P., Connor, J. A., Roof, D. M., Hoch, J. A. & Babior, B. M. (1990). Cloning, sequencing and expression of the genes encoding the adenosylcobalamin-dependent ethanolamine ammonia-lyase of Salmonella typhimurium. J Biol Chem 265, 1246212466.
Friedberg, D., Jager, K. M., Kessel, M., Silman, N. J. & Bergman, B. (1993). Rubisco but not Rubisco activase is clustered in the carboxysomes of the cyanobacterium Synechococcus sp. PCC 7942: Mud-induced carboxysomeless mutants. Mol Microbiol 9, 11931201.[Medline]
Guzman, L.-M., Belin, D., Carson, M. J. & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing arabinose PBAD promoter. J Bacteriol 177, 41214130.
Havemann, G. D. & Bobik, T. A. (2003). Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2. J Bacteriol 185, 50865095.
Havemann, G. D., Sampson, E. M. & Bobik, T. A. (2002). PduA is a shell protein of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2. J Bacteriol 184, 12531261.
Kofoid, E., Rappleye, C., Stojiljkovic, I. & Roth, J. (1999). The 17-gene ethanolamine (eut) operon of Salmonella typhimurium encodes five homologues of carboxysome shell proteins. J Bacteriol 181, 53175329.
Leal, N. A., Havemann, G. D. & Bobik, T. A. (2003). PduP is a coenzyme-A-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2. Arch Microbiol 180, 353361.[CrossRef][Medline]
Roe, A. J., McLaggan, D., Davidson, I., O'Byrne, C. & Booth, I. R. (1998). Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids. J Bacteriol 180, 767772.
Rondon, M. R., Kazmierczak, R. & Escalante-Semerena, J. C. (1995). Glutathione is required for maximal transcription of the cobalamin biosynthetic and 1,2-propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, 1,2-propanediol, and propionate in Salmonella typhimurium LT2. J Bacteriol 177, 54345439.
Roof, D. M. & Roth, J. R. (1988). Ethanolamine utilization in Salmonella typhimurium. J Bacteriol 170, 38553863.[Medline]
Roof, D. M. & Roth, J. R. (1989). Functions required for vitamin B12-dependent ethanolamine utilization in Salmonella typhimurium. J Bacteriol 171, 33163323.[Medline]
Roof, D. M. & Roth, J. R. (1992). Autogenous regulation of ethanolamine utilization by a transcriptional activator of the eut operon in Salmonella typhimurium. J Bacteriol 174, 66346643.[Abstract]
Russell, J. B. & Diez-Gonzalez, F. (1998). The effects of fermentation acids on bacterial growth. Adv Microb Physiol 39, 205234.[Medline]
Schmieger, H. (1971). A method for detection of phage mutants with altered transduction ability. Mol Gen Genet 100, 378381.
Schmieger, H. & Bakhaus, H. (1973). The origin of DNA in transducing particles of P22 mutants with increased transduction frequencies (HT-mutants). Mol Gen Genet 120, 181190.[CrossRef][Medline]
Sheppard, D. E. & Roth, J. R. (1994). A rationale for autoinduction of a transcriptional activator: ethanolamine ammonia-lyase (EutBC) and the operon activator (EutR) compete for adenosyl-cobalamin in Salmonella typhimurium. J Bacteriol 176, 12871296.[Abstract]
Shimizu, M., Suzuki, T., Kameda, K. Y. & Abiko, Y. (1969). Phosphotransacetylase of Escherichia coli B, purification and properties. Biochim Biophys Acta 191, 550558.[Medline]
Stojiljkovic, I., Bäumler, A. J. & Heffron, F. (1995). Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutj eutH gene cluster. J Bacteriol 177, 13571366.
Suzuki, T. (1969). Phosphotransacetylase of Escherichia coli B, activation by pyruvate and inhibition by NADH and certain nucleotides. Biochim Biophys Acta 191, 559569.[Medline]
Way, J. C., Davis, M. A., Morisato, D., Roberts, D. E. & Kleckner, N. (1984). New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene 32, 369379.[CrossRef][Medline]
Wolfe, A. J. (2005). The acetate switch. Microbiol Mol Biol Rev 69, 1250.
Received 26 April 2005;
revised 12 August 2005;
accepted 1 September 2005.
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