1887

Abstract

has an -alanine export system that protects the cells from toxic accumulation of intracellular -alanine in the presence of-alanyl--alanine (-Ala--Ala). When a DadA-deficient strain was incubated with 6.0 mM -Ala--Ala, we detected -alanine and -alanine using high-performance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with-amino acid oxidase resulted in the disappearance of a signal corresponding to -alanine. Additionally, the culture supernatant enabled a -alanine auxotroph to grow without -alanine supplementation, confirming that the signal detected by HPLC was authentic -alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, or , the extracellular level of -alanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow fromalanine to -alanine enhanced -alanine secretion. When high-density DadA-deficient cells preloaded with -Ala--Ala were treated with 20 µM carbonyl cyanide -chlorophenyl hydrazone (CCCP), secretion of both -alanine and-alanine was enhanced ~twofold compared with that in cells without CCCP treatment. In contrast, the ATPase inhibitor dicyclohexylcarbodiimide did not exert such an effect on the -alanine and -alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the -alanine exporter AlaE accumulated [H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [H]D-alanine was strongly inhibited by CCCP. These results indicate that has a transport system(s) that exports -alanine and that this function is most likely modulated by proton electrochemical potential.

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2016-07-01
2022-01-28
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References

  1. Aberhart D. J., Cotting J. A., Lin H. J. 1985; Separation by high-performance liquid chromatography of (3R)- and (3S)-beta-leucine as diastereomeric derivatives. Anal Biochem 151:88–91  [PubMed] [CrossRef]
    [Google Scholar]
  2. Baba T., Ara T., Hasegawa M., Takai Y., Okumura Y., Baba M., Datsenko K. A., Tomita M., Wanner B. L., Mori H. 2006; Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology 2: [View Article]
    [Google Scholar]
  3. Cava F., Lam H., de Pedro M. A., Waldor M. K. 2011; Emerging knowledge of regulatory roles of D-amino acids in bacteria. Cell Mol Life Sci 68:817–831 [View Article][PubMed]
    [Google Scholar]
  4. Cherepanov P. P., Wackernagel W. 1995; Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158:9–14 [View Article][PubMed]
    [Google Scholar]
  5. Dassler T., Maier T., Winterhalter C., Böck A. 2000; Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway. Mol Microbiol 36:1101–1112[PubMed] [CrossRef]
    [Google Scholar]
  6. 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 97:6640–6645 [View Article][PubMed]
    [Google Scholar]
  7. Doroshenko V., Airich L., Vitushkina M., Kolokolova A., Livshits V., Mashko S. 2007; YddG from Escherichia coli promotes export of aromatic amino acids. FEMS Microbiol Lett 275:312–318 [View Article][PubMed]
    [Google Scholar]
  8. Fisher R., Tuli R., Haselkorn R. 1981; A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated. Proc Natl Acad Sci U S A 78:3393–3397[PubMed] [CrossRef]
    [Google Scholar]
  9. Guan L., Nurva S., Ankeshwarapu S. P. 2011; Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium . J Biol Chem 286:6367–6374 [View Article][PubMed]
    [Google Scholar]
  10. Hori H., Ando T., Isogai E., Yoneyama H., Katsumata R. 2011a; Identification of an L-alanine export system in Escherichia coli and isolation and characterization of export-deficient mutants. FEMS Microbiol Lett 316:83–89 [View Article][PubMed]
    [Google Scholar]
  11. Hori H., Yoneyama H., Tobe R., Ando T., Isogai E., Katsumata R. 2011b; Inducible l-alanine exporter encoded by the novel gene ygaW (alaE) in Escherichia coli . Appl Environ Microbiol 77:4027–4034 [View Article][PubMed]
    [Google Scholar]
  12. Kaback H. R., Barnes E. M. 1971; Mechanisms of active transport in isolated membrane vesicles. II. The mechanism of energy coupling between D-lactic dehydrogenase and beta-galactoside transport in membrane preparations from Escherichia coli . J Biol Chem 246:5523–5531[PubMed]
    [Google Scholar]
  13. Kaback H. R., Reeves J. P., Short S. A., Lombardi F. J. 1974; Mechanisms of active transport in isolated bacterial membrane vesicles. 18. The mechanism of action of carbonylcyanide m-chlorophenylhydrazone. Arch Biochem Biophys 160:215–222[PubMed] [CrossRef]
    [Google Scholar]
  14. Kennerknecht N., Sahm H., Yen M. R., Pátek M., Saier M. H., Eggeling L. 2002; Export of L-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family. J Bacteriol 184:3947–3956[PubMed] [CrossRef]
    [Google Scholar]
  15. Kim S., Ihara K., Katsube S., Hori H., Ando T., Isogai E., Yoneyama H. 2015; Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives. Microbiologyopen 4:632–643 [View Article][PubMed]
    [Google Scholar]
  16. Kinoshita S., Udaka S., Shimono M. 1957; Studies on the amino acid fermentation. Part 1. Production of L-glutamic acid by various microorganisms. J Gen Appl Microbiol 3:193–205[PubMed] [CrossRef]
    [Google Scholar]
  17. Kitagawa M., Ara T., Arifuzzaman M., Ioka-Nakamichi T., Inamoto E., Toyonaga H., Mori H. 2005; Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12:291–299 [View Article][PubMed]
    [Google Scholar]
  18. Kolodkin-Gal I., Romero D., Cao S., Clardy J., Kolter R., Losick R. 2010; D-amino acids trigger biofilm disassembly. Science 328:627–629 [View Article][PubMed]
    [Google Scholar]
  19. Kutukova E. A., Livshits V. A., Altman I. P., Ptitsyn L. R., Zyiatdinov M. H., Tokmakova I. L., Zakataeva N. P. 2005; The yeaS (leuE) gene of Escherichia coli encodes an exporter of leucine, and the Lrp protein regulates its expression. FEBS Lett 579:4629–4634 [View Article][PubMed]
    [Google Scholar]
  20. Lam H., Oh D. C., Cava F., Takacs C. N., Clardy J., de Pedro M. A., Waldor M. K. 2009; D-amino acids govern stationary phase cell wall remodeling in bacteria. Science 325:1552–1555 [View Article][PubMed]
    [Google Scholar]
  21. Livshits V. A., Zakataeva N. P., Aleshin V. V., Vitushkina M. V. 2003; Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli . Res Microbiol 154:123–135 [View Article][PubMed]
    [Google Scholar]
  22. Lombardi F. J., Kaback H. R. 1972; Mechanisms of active transport in isolated bacterial membrane vesicles. 8. The transport of amino acids by membranes prepared from Escherichia coli . J Biol Chem 247:7844–7857[PubMed]
    [Google Scholar]
  23. Marfey P. 1984; Determination of D-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res Commun 49:591–596 [View Article]
    [Google Scholar]
  24. Nakamura J., Hirano S., Ito H., Wachi M. 2007; Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce L-glutamic acid production. Appl Environ Microbiol 73:4491–4498 [View Article][PubMed]
    [Google Scholar]
  25. Nandineni M. R., Gowrishankar J. 2004; Evidence for an arginine exporter encoded by yggA (argO) that is regulated by the LysR-type transcriptional regulator ArgP in Escherichia coli . J Bacteriol 186:3539–3546 [View Article][PubMed]
    [Google Scholar]
  26. Park J. T. 1996 The Murein Sacculus, 2nd edn. Washington, DC: ASM Press;
    [Google Scholar]
  27. Robbins J. C., Oxender D. L. 1973; Transport systems for alanine, serine, and glycine in Escherichia coli K-12. J Bacteriol 116:12–18[PubMed]
    [Google Scholar]
  28. Simic P., Sahm H., Eggeling L. 2001; L-threonine export: use of peptides to identify a new translocator from Corynebacterium glutamicum . J Bacteriol 183:5317–5324[PubMed] [CrossRef]
    [Google Scholar]
  29. Strych U., Penland R. L., Jimenez M., Krause K. L., Benedik M. J. 2001; Characterization of the alanine racemases from two mycobacteria. FEMS Microbiol Lett 196:93–98 [View Article][PubMed]
    [Google Scholar]
  30. Stäbler N., Oikawa T., Bott M., Eggeling L. 2011; Corynebacterium glutamicum as a host for synthesis and export of D-amino acids. J Bacteriol 193:1702–1709 [View Article][PubMed]
    [Google Scholar]
  31. Vrljic M., Sahm H., Eggeling L. 1996; A new type of transporter with a new type of cellular function: l-lysine export from Corynebacterium glutamicum . Mol Microbiol 22:815–826[PubMed] [CrossRef]
    [Google Scholar]
  32. Wild J., Filutowicz M., Kłopotowski T. 1978; Utilization of D-amino acids by dadR mutants of Salmonella typhimurium . Arch Microbiol 118:71–77[PubMed] [CrossRef]
    [Google Scholar]
  33. Wild J., Hennig J., Lobocka M., Walczak W., Kłopotowski T. 1985; Identification of the dadX gene coding for the predominant isozyme of alanine racemase in Escherichia coli K12. Mol Gen Genet 198:315–322[PubMed] [CrossRef]
    [Google Scholar]
  34. Zhang X., Jantama K., Moore J. C., Shanmugam K. T., Ingram L. O. 2007; Production of L-alanine by metabolically engineered Escherichia coli . Appl Microbiol Biotechnol 77:355–366 [View Article][PubMed]
    [Google Scholar]
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