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. 1992 May;174(9):2968–2977. doi: 10.1128/jb.174.9.2968-2977.1992

The Corynebacterium glutamicum aecD gene encodes a C-S lyase with alpha, beta-elimination activity that degrades aminoethylcysteine.

I Rossol 1, A Pühler 1
PMCID: PMC205951  PMID: 1569026

Abstract

S-(beta-Aminoethyl)-cysteine (AEC) resistance was achieved in Corynebacterium glutamicum by cloning a chromosomal 1.5-kb EcoRV-BglII DNA fragment on a multicopy plasmid. DNA sequence analysis of the 1.5-kb DNA fragment revealed an open reading frame (ORF326) which represents the AEC resistance gene, designated aecD. The aecD gene directs the synthesis of a 36-kDa protein which was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The aecD gene is a nonessential gene and mediates AEC resistance only in an amplified state. C. glutamicum strains harboring an amplified aecD gene can utilize AEC as an alternative nitrogen source, indicating that the AEC resistance mechanism is due to AEC degradation. Since the AEC degradation products analyzed by high-pressure liquid chromatography were found to be pyruvate and aminoethanethiol (cysteamine), it was concluded that the aecD gene encodes a C-S lyase with alpha, beta-elimination activity. Besides AEC, the C-S lyase was also able to use cysteine, cystine, and cystathionine as substrates.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altenbuchner J., Cullum J. DNA amplification and an unstable arginine gene in Streptomyces lividans 66. Mol Gen Genet. 1984;195(1-2):134–138. doi: 10.1007/BF00332735. [DOI] [PubMed] [Google Scholar]
  2. Arnold W., Pühler A. A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing. Gene. 1988 Oct 15;70(1):171–179. doi: 10.1016/0378-1119(88)90115-1. [DOI] [PubMed] [Google Scholar]
  3. Bartsch K., Tebbe C. C. Initial steps in the degradation of phosphinothricin (glufosinate) by soil bacteria. Appl Environ Microbiol. 1989 Mar;55(3):711–716. doi: 10.1128/aem.55.3.711-716.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Behrmann I., Hillemann D., Pühler A., Strauch E., Wohlleben W. Overexpression of a Streptomyces viridochromogenes gene (glnII) encoding a glutamine synthetase similar to those of eucaryotes confers resistance against the antibiotic phosphinothricyl-alanyl-alanine. J Bacteriol. 1990 Sep;172(9):5326–5334. doi: 10.1128/jb.172.9.5326-5334.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Belfaiza J., Parsot C., Martel A., de la Tour C. B., Margarita D., Cohen G. N., Saint-Girons I. Evolution in biosynthetic pathways: two enzymes catalyzing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region. Proc Natl Acad Sci U S A. 1986 Feb;83(4):867–871. doi: 10.1073/pnas.83.4.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berg C. M., Wang M. D., Vartak N. B., Liu L. Acquisition of new metabolic capabilities: multicopy suppression by cloned transaminase genes in Escherichia coli K-12. Gene. 1988 May 30;65(2):195–202. doi: 10.1016/0378-1119(88)90456-8. [DOI] [PubMed] [Google Scholar]
  7. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  9. Brown E. A., D'Ari R., Newman E. B. A relationship between L-serine degradation and methionine biosynthesis in Escherichia coli K12. J Gen Microbiol. 1990 Jun;136(6):1017–1023. doi: 10.1099/00221287-136-6-1017. [DOI] [PubMed] [Google Scholar]
  10. Cassan M., Boy E., Borne F., Patte J. C. Regulation of the lysine biosynthetic pathway in Escherichia coli K-12: isolation of a cis-dominant constitutive mutant for AK III synthesis. J Bacteriol. 1975 Aug;123(2):391–399. doi: 10.1128/jb.123.2.391-399.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  12. Garoff H., Ansorge W. Improvements of DNA sequencing gels. Anal Biochem. 1981 Aug;115(2):450–457. doi: 10.1016/0003-2697(81)90031-2. [DOI] [PubMed] [Google Scholar]
  13. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hermann M., Thevenet N. J., Coudert-Maratier M. M., Vandecasteele J. P. Consequences of lysine oversynthesis in Pseudomonas mutants insensitive to feedback inhibition. Lysine excretion or endogenous induction of a lysine-catabolic pathway. Eur J Biochem. 1972 Oct 17;30(1):100–106. doi: 10.1111/j.1432-1033.1972.tb02076.x. [DOI] [PubMed] [Google Scholar]
  15. Holbrook E. L., Greene R. C., Krueger J. H. Purification and properties of cystathionine gamma-synthase from overproducing strains of Escherichia coli. Biochemistry. 1990 Jan 16;29(2):435–442. doi: 10.1021/bi00454a019. [DOI] [PubMed] [Google Scholar]
  16. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  17. Ives C. L., Bott K. F. Cloned Bacillus subtilis chromosomal DNA mediates tetracycline resistance when present in multiple copies. J Bacteriol. 1989 Apr;171(4):1801–1810. doi: 10.1128/jb.171.4.1801-1810.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones B. N., Gilligan J. P. o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids. J Chromatogr. 1983 Aug 26;266:471–482. doi: 10.1016/s0021-9673(01)90918-5. [DOI] [PubMed] [Google Scholar]
  19. Kalinowski J., Bachmann B., Thierbach G., Pühler A. Aspartokinase genes lysC alpha and lysC beta overlap and are adjacent to the aspartate beta-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum. Mol Gen Genet. 1990 Dec;224(3):317–324. doi: 10.1007/BF00262424. [DOI] [PubMed] [Google Scholar]
  20. Katsumata R., Ozaki A., Oka T., Furuya A. Protoplast transformation of glutamate-producing bacteria with plasmid DNA. J Bacteriol. 1984 Jul;159(1):306–311. doi: 10.1128/jb.159.1.306-311.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kumagai H., Sejima S., Choi Y., Tanaka H., Yamada H. Crystallization and properties of cysteine desulfhydrase from Aerobacter aerogenes. FEBS Lett. 1975 Apr 1;52(2):304–307. doi: 10.1016/0014-5793(75)80831-3. [DOI] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  24. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McLaughlin J. R., Murray C. L., Rabinowitz J. C. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene. J Biol Chem. 1981 Nov 10;256(21):11283–11291. [PubMed] [Google Scholar]
  26. Menkel E., Thierbach G., Eggeling L., Sahm H. Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate. Appl Environ Microbiol. 1989 Mar;55(3):684–688. doi: 10.1128/aem.55.3.684-688.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nagasawa T., Kanzaki H., Yamada H. Cystathionine gamma-lyase from Streptomyces phaeochromogenes. Methods Enzymol. 1987;143:486–492. doi: 10.1016/0076-6879(87)43087-5. [DOI] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schrumpf B., Schwarzer A., Kalinowski J., Pühler A., Eggeling L., Sahm H. A functionally split pathway for lysine synthesis in Corynebacterium glutamicium. J Bacteriol. 1991 Jul;173(14):4510–4516. doi: 10.1128/jb.173.14.4510-4516.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schwarzer A., Pühler A. Manipulation of Corynebacterium glutamicum by gene disruption and replacement. Biotechnology (N Y) 1991 Jan;9(1):84–87. doi: 10.1038/nbt0191-84. [DOI] [PubMed] [Google Scholar]
  31. Schäfer A., Kalinowski J., Simon R., Seep-Feldhaus A. H., Pühler A. High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria. J Bacteriol. 1990 Mar;172(3):1663–1666. doi: 10.1128/jb.172.3.1663-1666.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Seep-Feldhaus A. H., Kalinowski J., Pühler A. Molecular analysis of the Corynebacterium glutamicum lysl gene involved in lysine uptake. Mol Microbiol. 1991 Dec;5(12):2995–3005. doi: 10.1111/j.1365-2958.1991.tb01859.x. [DOI] [PubMed] [Google Scholar]
  33. Segal W., Starkey R. L. Microbial decomposition of methionine and identity of the resulting sulfur products. J Bacteriol. 1969 Jun;98(3):908–913. doi: 10.1128/jb.98.3.908-913.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shiio I., Miyajima R. Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum. J Biochem. 1969 Jun;65(6):849–859. doi: 10.1093/oxfordjournals.jbchem.a129089. [DOI] [PubMed] [Google Scholar]
  35. Shiio I., Miyajima R., Sano K. Genetically desensitized aspartate kinase to the concerted feedback inhibition in Brevibacterium flavum. J Biochem. 1970 Nov;68(5):701–710. doi: 10.1093/oxfordjournals.jbchem.a129403. [DOI] [PubMed] [Google Scholar]
  36. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  37. Staden R. The current status and portability of our sequence handling software. Nucleic Acids Res. 1986 Jan 10;14(1):217–231. doi: 10.1093/nar/14.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tanaka H., Esaki N., Soda K. Properties of L-methionine gamma-lyase from Pseudomonas ovalis. Biochemistry. 1977 Jan 11;16(1):100–106. doi: 10.1021/bi00620a016. [DOI] [PubMed] [Google Scholar]
  39. Tapuhi Y., Schmidt D. E., Lindner W., Karger B. L. Dansylation of amino acids for high-performance liquid chromatography analysis. Anal Biochem. 1981 Jul 15;115(1):123–129. doi: 10.1016/0003-2697(81)90534-0. [DOI] [PubMed] [Google Scholar]
  40. Thierbach G., Kalinowski J., Bachmann B., Pühler A. Cloning of a DNA fragment from Corynebacterium glutamicum conferring aminoethyl cysteine resistance and feedback resistance to aspartokinase. Appl Microbiol Biotechnol. 1990 Jan;32(4):443–448. doi: 10.1007/BF00903780. [DOI] [PubMed] [Google Scholar]
  41. Vartak N. B., Liu L., Wang B. M., Berg C. M. A functional leuABCD operon is required for leucine synthesis by the tyrosine-repressible transaminase in Escherichia coli K-12. J Bacteriol. 1991 Jun;173(12):3864–3871. doi: 10.1128/jb.173.12.3864-3871.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yoshihama M., Higashiro K., Rao E. A., Akedo M., Shanabruch W. G., Follettie M. T., Walker G. C., Sinskey A. J. Cloning vector system for Corynebacterium glutamicum. J Bacteriol. 1985 May;162(2):591–597. doi: 10.1128/jb.162.2.591-597.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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