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The Arabidopsis ALDP protein homologue COMATOSE is instrumental in peroxisomal acetate metabolism.

ABSTRACT: The Arabidopsis acn (acetate non-utilizing) mutants were isolated by fluoroacetate-resistant germination and seedling establishment. We report the characterization of the acn2 mutant. Physiological analyses of acn2 showed that it possessed characteristics similar to those of the mutants cts (COMATOSE)-1 and pxa [peroxisomal ABC (ATP-binding-cassette) transporter]1. The acn2 locus was mapped to within 3 cM of the CTS gene on the bottom arm of chromosome IV using CAPS (cleavage amplification polymorphism) and SSLP (simple sequence-length polymorphism) markers. Crossing acn2 and cts-1 failed to restore the fluoroacetate-sensitive phenotype, suggesting that these mutations were allelic. Sequencing of the ACN2 locus revealed a C-->T nonsense mutation in exon 13, which would have resulted in the elimination of the C-terminal hemitransporter domain of the encoded protein. Neither the full-length CTS protein nor the truncated protein was detected on immunoblots using either C-terminal- or N-terminal-specific anti-CTS antibodies respectively, demonstrating the absence of the entire CTS protein in acn2 mutants. Emerged seedlings of both cts-1 and pxa1 alleles displayed increased resistance to FAc (monofluoroacetic acid) compared with the corresponding wild-type seedlings. Complementation studies showed that mutation of the CTS gene was responsible for the FAc-resistant phenotype, as when the wild-type protein was expressed in both the cts-1 and pxa1 mutant lines, the strains became FAc-sensitive. Feeding studies confirmed that both acn2 and cts-1 mutants were compromised in their ability to convert radiolabelled acetate into soluble carbohydrate. These results demonstrate a role for the ABC protein CTS in providing acetate to the glyoxylate cycle in developing seedlings.

SUBMITTER: Hooks MA

PROVIDER: S-EPMC2049035 | biostudies-other | 2007 Sep

REPOSITORIES: biostudies-other

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Project description:During aerobic growth on glucose, Escherichia coli excretes acetate, a mechanism called "overflow metabolism." At high concentrations, the secreted acetate inhibits growth. Several mechanisms have been proposed for explaining this phenomenon, but a thorough analysis is hampered by the diversity of experimental conditions and strains used in these studies. Here, we describe the construction of a set of isogenic strains that remove different parts of the metabolic network involved in acetate metabolism. Analysis of these strains reveals that (i) high concentrations of acetate in the medium inhibit growth without significantly perturbing central metabolism; (ii) growth inhibition persists even when acetate assimilation is completely blocked; and (iii) regulatory interactions mediated by acetyl-phosphate play a small but significant role in growth inhibition by acetate. The major contribution to growth inhibition by acetate may originate in systemic effects like the uncoupling effect of organic acids or the perturbation of the anion composition of the cell, as previously proposed. Our data suggest, however, that under the conditions considered here, the uncoupling effect plays only a limited role.IMPORTANCE High concentrations of organic acids such as acetate inhibit growth of Escherichia coli and other bacteria. This phenomenon is of interest for understanding bacterial physiology but is also of practical relevance. Growth inhibition by organic acids underlies food preservation and causes problems during high-density fermentation in biotechnology. What causes this phenomenon? Classical explanations invoke the uncoupling effect of acetate and the establishment of an anion imbalance. Here, we propose and investigate an alternative hypothesis: the perturbation of acetate metabolism due to the inflow of excess acetate. We find that this perturbation accounts for 20% of the growth-inhibitory effect through a modification of the acetyl phosphate concentration. Moreover, we argue that our observations are not expected based on uncoupling alone.

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The Arabidopsis ALDP protein homologue COMATOSE is instrumental in peroxisomal acetate metabolism.

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