1887

Microbiology

Volume 147, Issue 6

Structure of the operon and formation of aspartokinase subunits in the cephamycin producer ‘’

The GenBank accession number for the sequence reported in this paper is AJ298904.

Free

Abstract

The first two genes of the lysine pathway are closely linked forming a transcriptional operon in the cephamycin producer ‘’. The gene, encoding the enzyme aspartic semialdehyde dehydrogenase, has been cloned by complementation of mutants. It encodes a protein of 355 aa with a deduced of 37109. The gene encoding the aspartokinase (Ask) is located upstream of the gene as shown by determination of Ask activity conferred to transformants. and are separated by 2 nt and are transcribed in a bicistronic 26 kb mRNA. As occurs in corynebacteria, the presence of a ribosome-binding site within the sequence suggests that this ORF encodes two overlapping proteins, Askα of 421 aa and 44108, and Askβ of 172 aa and 18145. The formation of both subunits of Ask from a single gene () was confirmed by using antibodies against the C-terminal end of Ask which is identical in both subunits. Ask activity of ‘’ is regulated by the concerted action of lysine plus threonine and this inhibition is abolished in transformants containing Ser to Tyr, or Gly to Asp mutations of the ‘’ gene.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): Asd, aspartate semialdehyde dehydrogenase , Ask, aspartokinase , aspartic semialdehyde dehydrogenase , beta-lactams , DAP, diaminopimelate and α-AAA, α-aminoadipic acid
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-6-1547
2001-06-01
2021-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/6/1471547a.html?itemId=/content/journal/micro/10.1099/00221287-147-6-1547&mimeType=html&fmt=ahah

References

  1. Barreiro C., Pisabarro A., Martı́n J. F. 2000; Characterization of the ribosomal rrnD operon of the cephamycin producer ‘ Nocardia lactamdurans ’ shows that this actinomycete belongs to the genus Amycolatopsis. . Syst Appl Microbiol 23:15–24 [CrossRef]
     [Google Scholar]
  2. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523 [CrossRef]
     [Google Scholar]
  3. Chen N. Y., Jiang S. Q., Klein D. A., Paulus H. 1993; Organization and nucleotide sequence of the Bacillus subtilis diaminopimelate operon, a cluster of genes encoding the first three enzymes of diaminopimelate synthesis and dipicolinate synthase. J Biol Chem 268:9448–9465
     [Google Scholar]
  4. Chung C. T., Niemela S. L., Miller R. H. 1989; One-step preparation of competent Escherichia coli : transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA 86:2172–2175 [CrossRef]
     [Google Scholar]
  5. Cirillo J. D., Weisbrod T. R., Pascopella L., Bloom B. R., Jacobs W. R. Jr 1994; Isolation and characterization of the aspartokinase and aspartate semialdehyde dehydrogenase operon from mycobacteria. Mol Microbiol 11:629–639 [CrossRef]
     [Google Scholar]
  6. Cohen G. N., Stainer R. Y., LeBras G. 1969; Regulation of the biosynthesis amino acids of the aspartate family in coliform bacteria and Pseudomonas. . J Bacteriol 99:791–801
     [Google Scholar]
  7. Coque J. J. R., Liras P., Laı́z L., Martı́n J. F. 1991; A gene encoding lysine 6-aminotransferase, which forms the β-lactam precursor α-aminoadipic acid, is located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans. . J Bacteriol 173:6258–6264
     [Google Scholar]
  8. Coque J. J. R., Malumbres M., Martı́n J. F., Liras P. 1993; Analysis of the codon usage of the cephamycin C producer A. lactamdurans. . FEMS Microbiol Lett 110:91–96 [CrossRef]
     [Google Scholar]
  9. Cremer J., Creptow C., Eggeling L., Sahm H. 1988; Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum. . J Gen Microbiol 134:3221–3229
     [Google Scholar]
  10. Eikmanns B. J., Eggeling L., Sahm H. 1993; Molecular aspects of lysine, threonine, and isoleucine biosynthesis in Corynebacterium glutamicum. . Antonie Leeuwenhoek 64:145–163
     [Google Scholar]
  11. Follettie M. T., Peoples O. P., Agoropoulou C., Sinskey A. J. 1993; Gene structure and expression of the Corynebacterium flavum N13 ask - asd operon. J Bacteriol 175:4096–4103
     [Google Scholar]
  12. de la Fuente J. L., Rumbero A., Martı́n J. F., Liras P. 1997; Δ-1-Piperideine-6-carboxylate dehydrogenase, a new enzyme that forms α-aminoadipate in Streptomyces clavuligerus and other cephamycin C-producing actinomycetes. Biochem J 327:59–64
     [Google Scholar]
  13. Galán J. E., Nakayama K., Curtiss R. I. 1990; Cloning and characterization of the asd gene of Salmonella typhimurium : use in stable maintenance of recombinant plasmids in Salmonella vaccine strains. Gene 94:29–35 [CrossRef]
     [Google Scholar]
  14. Ginther C. L. 1979; Sporulation and the production of serine proteases and cephamycin C by Streptomyces lactamdurans. . Antimicrob Agents Chemother 15:522–526 [CrossRef]
     [Google Scholar]
  15. Gribskov M., Devereux J., Burgess R. R. 1984; The codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression. Nucleic Acids Res 12:539–549 [CrossRef]
     [Google Scholar]
  16. Haziza C., Strategier P., Patte J. C. 1982; Nucleotide sequence of the asd gene of Escherichia coli : absence of a typical attenuation signal. EMBO J 1:379–384
     [Google Scholar]
  17. Higgins D. G., Bleasby A. J., Fuchs R. 1992; clustal v: improved software for multiple sequencing alignment. Comput Appl Biosci 8:189–191
     [Google Scholar]
  18. Hoang T. T., Williams S., Schweizer H. P., Lam J. S. 1997; Molecular genetic analysis of the region containing the essential Pseudomonas aeruginosa asd gene encoding aspartate-β-semialdehyde dehydrogenase. Microbiology 143:899–907 [CrossRef]
     [Google Scholar]
  19. Holmes D. S., Quigley M. 1981; A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197 [CrossRef]
     [Google Scholar]
  20. Hopwood D. A., Bibb M. J., Chater K. F. 7 other authors 1985 Genetic Manipulation of Streptomyces: a Laboratory Manual Norwich: John Innes Foundation;
     [Google Scholar]
  21. Jagusztyn-Krynicka E. K., Smorawinska M., Curtiss R.III. 1982; Expression of Streptococcus mutans aspartate-semialdehyde dehydrogenase gene cloned into plasmid pBR322. J Gen Microbiol 128:1135–1145
     [Google Scholar]
  22. Kalinowski J., Bachmann B., Thierbach G., Pühler A. 1990; Aspartokinase genes lysC α and lysC β overlap and are adjacent to the aspartate β-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum. . Mol Gen Genet 224:317–324
     [Google Scholar]
  23. Kalinowski J., Cremer B., Bachmann B., Eggeling L., Sahm H., Puhler A. 1991; Genetic and biochemical analysis of the aspartokinase from Corynebacterium glutamicum . Mol Microbiol 5:1197–1204 [CrossRef]
     [Google Scholar]
  24. Kirby K. S., Fox-Carter E., Guest M. 1967; Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria. Biochem J 104:258–262
     [Google Scholar]
  25. Kumar C. V., Coque J. J. R., Martı́n J. F. 1994; Efficient transformation of the cephamycin C producer Nocardia lactamdurans and development of shuttle and promoter-probe cloning vectors. Appl Environ Microbiol 60:4086–4093
     [Google Scholar]
  26. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
     [Google Scholar]
  27. Lanzini G., Lorenzetti R. 1993 Biotechnology of Antibiotics and Other Bioactive Microbial Metabolites New York: Plenum;
     [Google Scholar]
  28. Le Y., He J., Vining L. C. 1996; Streptomyces akiyoshiensis differs from other Gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids. Microbiology 142:791–798 [CrossRef]
     [Google Scholar]
  29. Madduri K., Stuttard C., Vining L. C. 1991; Cloning and location of a gene governing lysine ϵ-aminotransferase, an enzyme initiating β-lactam biosynthesis in Streptomyces spp. J Bacteriol 173:985–988
     [Google Scholar]
  30. Malmberg L. H., Hu W. S., Sherman D. H. 1993; Precursor flux control through targeted chromosomal insertion of the lysine epsilon-aminotransferase ( lat) gene in cephamycin C biosynthesis. J Bacteriol 175:6916–6924
     [Google Scholar]
  31. Malmberg L. H., Hu W.-S., Sherman D. H. 1995; Effects of enhanced lysine epsilon-aminotransferase activity on cephamycin biosynthesis in Streptomyces clavuligerus. . Appl Microbiol Biotechnol 44:198–205 [CrossRef]
     [Google Scholar]
  32. Malumbres M. 1993 Clonación y caracterización molecular de los genes biosintéticos de treonina (thrC) y lisina de Brevibacterium lactofermentum PhD thesis, University of León; Spain:
     [Google Scholar]
  33. Malumbres M., Martı́n J. F. 1996; Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: Redirecting carbon flow. FEMS Microbiol Lett 143:103–114 [CrossRef]
     [Google Scholar]
  34. Martı́n J. F. 1998; New aspects of genes and enzymes for β-lactam antibiotic biosynthesis. Appl Microbiol Biotechnol 50:1–15 [CrossRef]
     [Google Scholar]
  35. Martı́n J. F. 2000; α-Aminoadipyl-cysteinyl-valine synthetases in β-lactam producing organisms. From Abraham’s discoveries to novel concepts of non-ribosomal peptide synthesis. J Antibiot 53:1008–1021 [CrossRef]
     [Google Scholar]
  36. Martı́n J. F., Aparicio J. F., Gutiérrez S. 2000; Secondary metabolites. In Encyclopedia of Microbiology pp 213–237 Edited by Lederberg J. San Diego: Academic Press;
     [Google Scholar]
  37. Mendelovitz S., Aharonowitz Y. 1982; Regulation of cephamycin C synthesis, aspartokinase, dihydrodipicolinic acid synthetase, and homoserine dehydrogenase by aspartic acid family amino acids in Streptomyces clavuligerus. . Antimicrob Agents Chemother 21:74–84 [CrossRef]
     [Google Scholar]
  38. Pérez-Llarena F. J., Rodrı́guez-Garcı́a A., Enguita F. J., Martı́n J. F., Liras P. 1998; The pcd gene encoding piperideine-6-carboxylate dehydrogenase involved in biosynthesis of α-aminoadipic acid is located in the cephamycin cluster of Streptomyces clavuligerus. . J Bacteriol 180:4753–4756
     [Google Scholar]
  39. Rius N., Maeda K., Demain A. L. 1996; Induction of l-lysine ϵ-aminotransferase by l-lysine in Streptomyces clavuligerus , producer of cephalosporins. FEMS Microbiol Lett 144:207–211
     [Google Scholar]
  40. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  41. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467 [CrossRef]
     [Google Scholar]
  42. Shiio I., Mijayama R. 1969; Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum. J Biochem 65:849–859
     [Google Scholar]
  43. Stadman E. R., Cohen G. W., Lebras G., Robichon-Szulmajster M. 1961; Feedback inhibition and repression of aspartokinase activity in E. coli and Saccharomyces cerevisiae. . J Biol Chem 236:2033–2038
     [Google Scholar]
  44. Thèze J., Margarita D., Cohen G. N., Borne F., Patte J. C. 1974; Mapping of the structural genes of three aspartokinases and of the two homoserine dehydrogenases of Escherichia coli K12. J Bacteriol 117:133–143
     [Google Scholar]
  45. Zhang J. J., Hu F. M., Chen N. Y., Paulus H. 1990; Comparison of the three aspartokinase isozymes in Bacillus subtilis Marburg and 168 . . J Bacteriol 172:701–708
     [Google Scholar]
  46. Zhang W., Jiang W., Zhao G., Yang Y., Chiao J. 1999; Sequence analysis and expression of the aspartokinase and aspartate semialdehyde dehydrogenase operon from rifamycin SV-producing Amycolatopsis mediterranei. . Gene 237:413–419 [CrossRef]
     [Google Scholar]
  47. Zhang W.-W., Jiang W.-H., Zhao G.-P., Yang Y.-L., Chiao J.-S. 2000; Expression in Escherichia coli , purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin SV-producing Amycolatopsis mediterranei U32. Appl Microbiol Biotechnol 44:52–58
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-6-1547
Loading
Structure of the ask-asd operon and formation of aspartokinase subunits in the cephamycin producer ‘Amycolatopsis lactamdurans’The GenBank accession number for the sequence reported in this paper is AJ298904.
Microbiology 147, 1547 (2001); https://doi.org/10.1099/00221287-147-6-1547
/content/journal/micro/10.1099/00221287-147-6-1547
/content/journal/micro/10.1099/00221287-147-6-1547
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error