1887

Abstract

Nitrogen assimilation is important during solvent production by NCP262, as acetone and butanol yields are significantly affected by the nitrogen source supplied. Growth of this bacterium was dependent on the concentration of organic nitrogen supplied and the expression of the assimilatory enzymes, glutamine synthetase (GS) and glutamate synthase (GOGAT), was shown to be induced in nitrogen-limiting conditions. The regions flanking the gene encoding GS, , were isolated from genomic DNA, and DNA sequencing revealed that the structural genes encoding the GS () and GOGAT ( and ) enzymes were clustered together with the gene in the order . RNA analysis showed that the and the genes were co-transcribed on 2.3 and 6.2 kb RNA transcripts respectively, and that all four genes were induced under the same nitrogen-limiting conditions. Complementation of an mutant, lacking a GOGAT small subunit, was achieved only when both the and genes were expressed together under anaerobic conditions. This is believed to be the first functional analysis of a gene cluster encoding the key enzymes of nitrogen assimilation, GS and GOGAT. A similar gene arrangement is seen in NCIMB 8052, and based on the common regulatory features of the promoter regions upstream of the operons in both species, we suggest a model for their co-ordinated regulation by an antitermination mechanism as well as antisense RNA.

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2007-09-01
2024-03-29
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References

  1. Aiba H., Adhya S., de Crombrugghe B. 1981; Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem 256:11905–11910
    [Google Scholar]
  2. Allcock E. R., Reid S. J., Jones D. T., Woods D. R. 1982; Clostridium acetobutylicum protoplast formation and regeneration. Appl Environ Microbiol 43:719–721
    [Google Scholar]
  3. Amine J., Marczak R., Maazouzi N., Masion E. 1990; Regulation of ammonium assimilation by Clostridium acetobutylicum. J Basic Microbiol 30:635–642
    [Google Scholar]
  4. Belitsky B. R. 2002; Biosynthesis of amino acids of the glutamate and aspartate families, alanine and polyamines. In Bacillus subtilis and its Closest Relatives: from Genes to Cells pp 203–210 Edited by Sonenshein A. L., Hoch J. A., Losick R. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  5. Bogdahn M., Kleiner D. 1986; N2 fixation and NH4 + assimilation in the thermophilic anaerobes Clostridium thermosaccharolyticum and Clostridium thermoautotrophicum. Arch Microbiol 144:102–104
    [Google Scholar]
  6. Bohannon D. E., Rosenkrantz M. S., Sonenshein A. L. 1985; Regulation of Bacillus subtilis glutamate synthase genes by the nitrogen source. J Bacteriol 163:957–964
    [Google Scholar]
  7. Brown N. L., Stoyanov J. V., Kidd S. P., Hobman J. L. 2003; The MerR family of transcriptional regulators. FEMS Microbiol Rev 27:145–163
    [Google Scholar]
  8. Castano I., Bastarrachea F., Covarrubias A. A. 1988; gltBDF operon of Escherichia coli. J Bacteriol 170:821–827
    [Google Scholar]
  9. Castano I., Flores N., Valle F., Covarrubias A. A., Bolivar F. 1992; gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression. Mol Microbiol 6:2733–2741
    [Google Scholar]
  10. Chai W., Stewart V. 1998; NasR, a novel RNA-binding protein, mediates nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader in vitro. J Mol Biol 283:339–351
    [Google Scholar]
  11. Covarrubias A. A., Sanchez-Pescador R., Osorio A., Bolivar F., Bastarrachea F. 1980; ColE1 hybrid plasmids containing Escherichia coli genes involved in the biosynthesis of glutamate and glutamine. Plasmid 3:150–164
    [Google Scholar]
  12. Dainty R. H. 1972; Glutamate biosynthesis in Clostridium pasteurianum and its significance in nitrogen metabolism. Biochem J 126:1055–1056
    [Google Scholar]
  13. Dürre P. 1998; New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation. Appl Microbiol Biotechnol 49:639–648
    [Google Scholar]
  14. Fierro-Monti I. P., Reid S. J., Woods D. R. 1992; Differential expression of a Clostridium acetobutylicum antisense RNA: implications for regulation of glutamine synthetase. J Bacteriol 174:7642–7647
    [Google Scholar]
  15. Fisher S. H. 1992; Glutamine synthesis in Streptomyces – a review. Gene 115:13–17
    [Google Scholar]
  16. Fisher S. H. 1999; Regulation of nitrogen metabolism in Bacillus subtilis: vive la difference!. Mol Microbiol 32:223–232
    [Google Scholar]
  17. Fisher S. H., Sonenshein A. L. 1991; Control of carbon and nitrogen metabolism in Bacillus subtilis. Annu Rev Microbiol 45:107–135
    [Google Scholar]
  18. Galperin M. Y. 2006; Structural classification of bacterial response regulators: diversity of output domains and domain considerations. J Bacteriol 188:4169–4182
    [Google Scholar]
  19. Gottschal J., Morris J. G. 1981; Non-production of acetone and butanol by Clostridium acetobutylicum during glucose- and ammonium-limitation in continuous culture. Biotechnol Lett 3:525–530
    [Google Scholar]
  20. Gutierrez J. C., Ramos F., Otrner L., Tortolero M. 1995; nasST, two genes involved in the induction of the assimilatory nitrite-nitrate reductase operon ( nasAB) of Azotobacter vinelandii. Mol Microbiol 18:579–591
    [Google Scholar]
  21. Holdeman L. V. E., Cato E. P., Moore W. E. C. 1977 Anaerobe Laboratory Manual,, 4th edn. Blacksburg: Virginia Polytechnic Institute and State University;
    [Google Scholar]
  22. Jakoby M., Nolden L., Meier-Wagner J., Kramer R., Burkovski A. 2000; AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum. Mol Microbiol 37:964–977
    [Google Scholar]
  23. Janssen P. J., Jones D. T., Woods D. R. 1990; Studies on Clostridium acetobutylicum glnA promoters and antisense RNA. Mol Microbiol 4:1575–1583
    [Google Scholar]
  24. Jones D. T., Woods D. R. 1986; Acetone-butanol fermentation revisited. Microbiol Rev 50:484–524
    [Google Scholar]
  25. Keis S., Bennett C. F., Ward V. K., Jones D. T. 1995; Taxonomy and phylogeny of the industrial solvent-producing clostridia. Int J Syst Bacteriol 45:693–705
    [Google Scholar]
  26. Kleiner D., Fitzke E. 1979; Evidence for ammonia translocation by Clostridium pasteurianum. Biochem Biophys Res Commun 86:211–217
    [Google Scholar]
  27. Long S., Jones D. T., Woods D. R. 1984; Initiation of solvent production, clostridial stage and endospore formation in Clostridium acetobutylicum P262. Appl Environ Microbiol 20:256–261
    [Google Scholar]
  28. Meister A. 1985; Glutamate synthase from Escherichia coli, Klebsiella aerogenes, and Saccharomyces cerevisiae. Methods Enzymol 113:327–337
    [Google Scholar]
  29. Merrick M. J., Edwards R. A. 1995; Nitrogen control in bacteria. Microbiol Rev 59:604–622
    [Google Scholar]
  30. Monot F., Engasser J. M. 1983; Production of acetone and butanol by batch and continuous culture of Clostridium acetobutylicum under nitrogen limitation. Biotechnol Lett 5:213–218
    [Google Scholar]
  31. Quixley K. W. M. 1999; Studies on the regulation of nitrogen metabolism in Clostridium acetobutylicum NCP262 and Clostridium beijerinckii NCIMB 8052. MSc thesis University of Cape Town, South Africa:
    [Google Scholar]
  32. Rose R. E. 1988; The nucleotide sequence of pACYC184. Nucleic Acids Res 16:355
    [Google Scholar]
  33. Sambrook J., Russell D. 2001 Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  34. Schreier H. J. 1993; Biosynthesis of glutamine and glutamate and the assimilation of ammonia. . In Bacillus subtilis and other Gram-Positive Bacteria pp 281–298 Edited by Sonenshein A. L., Hoch J. A., Losick R. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  35. Schubert C. 2006; Can biofuels finally take center stage?. Nat Biotechnol 24:777–784
    [Google Scholar]
  36. Schulz A. A., Collett H. J., Reid S. J. 2001; Nitrogen and carbon regulation of glutamine synthetase and glutamate synthase in Corynebacterium glutamicum ATCC 13032. FEMS Microbiol Lett 205:361–367
    [Google Scholar]
  37. Shapiro B. M., Stadtman E. R. 1968; Glutamine synthetase deadenylylating enzyme. Biochem Biophys Res Commun 30:32–37
    [Google Scholar]
  38. Shu C. J., Zhulin I. B. 2002; ANTAR: an RNA-binding domain in transcription antitermination regulatory proteins. Trends Biochem Sci 27:3–5
    [Google Scholar]
  39. Stutz H. E., Reid S. J. 2004; GltX from Clostridium saccharobutylicum NCP262: glutamate synthase or oxidoreductase?. Biochim Biophys Acta 167671–82
    [Google Scholar]
  40. Usdin K. P., Zappe H., Jones D. T., Woods D. R. 1986; Cloning, expression and purification of glutamine synthetase from Clostridium acetobutylicum. Appl Environ Microbiol 52:413–419
    [Google Scholar]
  41. van Rooyen J. M., Abratt V. R., Sewell B. T. 2006; Three-dimensional structure of a type III glutamine synthetase by single-particle reconstruction. J Mol Biol 361:796–810
    [Google Scholar]
  42. Vanoni M. A., Curti B. 2005; Structure–function studies on the iron-sulfur flavoenzyme glutamate synthase: an unexpectedly complex self-regulated enzyme. Arch Biochem Biophys 433:193–211
    [Google Scholar]
  43. Wilson S. A., Wachira S. J., Drew R. E., Jones D., Pearl L. H. 1993; Antitermination of amidase expression in Pseudomonas aeruginosa is controlled by a novel cytoplasmic amide-binding protein. EMBO J 12:3637–3642
    [Google Scholar]
  44. Woods D. R., Reid S. J. 1995; Regulation of nitrogen metabolism, starch utilisation and the β-hbd-adh1 gene cluster in Clostridium acetobutylicum. FEMS Microbiol Rev 17:299–306
    [Google Scholar]
  45. Young M., Minton N., Staudenbauer W. L. 1989; Recent advances in the genetics of the clostridia. FEMS Microbiol Rev 5:301–325
    [Google Scholar]
  46. Zappe H., Jones D. T., Woods D. R. 1986; Cloning and expression of Clostridium acetobutylicum endoglucanase, cellobiase and amino acid biosynthesis genes in Escherichia coli. J Gen Microbiol 132:1367–1372
    [Google Scholar]
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