During early stationary phase, NAFM5 produces capsular poly(-glutamic acid) (PGA, 2×10 Da), which contains - and -glutamate, and then degrades it during late stationary phase. The -glutamyltransferase (EC; GGT) of this strain successively hydrolysed PGA from the amino-terminal end, to yield both - and -glutamate. This enzyme was specifically synthesized during the stationary phase through transcriptional activation of the corresponding gene by the ComQXPA quorum-sensing system. A knockout mutant degraded PGA into 1×10 Da fragments, but not any further, indicating that the capsule PGA is first internally degraded by an endo-type of PGA hydrolase into 1×10 Da intermediates, then externally into glutamates via GGT. Due to its inability to generate the glutamates from the capsule, the mutant sporulated more frequently than the wild-type strain. The results show that GGT has a powerful exo--glutamyl hydrolase activity that participates in capsule PGA degradation to supply stationary-phase cells with constituent glutamates.


Article metrics loading...

Loading full text...

Full text loading...



  1. Abe, K., Ito, Y., Ohmachi, T. & Asada, Y.(1997). Purification and properties of two isozymes of γ-glutamyltranspeptidase from Bacillus subtilis TAM-4. Biosci Biotechnol Biochem 61, 1621–1625.[CrossRef] [Google Scholar]
  2. Ashiuchi, M., Soda, K. & Misono, H.(1999). A poly-γ-glutamate synthetic system of Bacillus subtilis IFO 3336: gene cloning and biochemical analysis of poly-γ-glutamate produced by Escherichia coli clone cells. Biochem Biophys Res Commun 263, 6–12.[CrossRef] [Google Scholar]
  3. Birrer, G. A., Cromwick, A.-M. & Gross, R. A.(1994).γ-Poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies. Int J Biol Macromol 16, 265–275.[CrossRef] [Google Scholar]
  4. Del Bello, B., Paolicchi, A., Comporti, M., Pompella, A. & Maellaro, E.(1999). Hydrogen peroxide produced during γ-glutamyl transpeptidase activity is involved in prevention of apoptosis and maintenance of proliferation in U937 cells. FASEB J 13, 69–79. [Google Scholar]
  5. Dubnau, D.(1999). DNA uptake in bacteria. Annu Rev Microbiol 53, 217–244.[CrossRef] [Google Scholar]
  6. Elsenhans, B., Ahmad, O. & Rosenberg, I. H.(1984). Isolation and characterization of pteroylglutamate hydrolase from rat intestinal mucosa. J Biol Chem 259, 6364–6368. [Google Scholar]
  7. Guérout-Fleury, A.-M., Shazand, K., Frandsen, N. & Stragier, P.(1995). Antibiotic-resistance cassettes for Bacillus subtilis. Gene 167, 335–336.[CrossRef] [Google Scholar]
  8. Guérout-Fleury, A.-M., Frandsen, N. & Stragier, P.(1996). Plasmids for ectopic integration in Bacillus subtilis. Gene 180, 57–61.[CrossRef] [Google Scholar]
  9. Hanigan, M. H. & Ricketts, W. A.(1993). Extracellular glutathione is a source of cysteine for cells that express γ-glutamyl transpeptidase. Biochemistry 32, 6302–6306.[CrossRef] [Google Scholar]
  10. Karp, D. R., Shimooku, K. & Lipsky, P. E.(2001). Expression of γ-glutamyl transpeptidase protects Ramos B cells from oxidation-induced cell death. J Biol Chem 276, 3798–3804.[CrossRef] [Google Scholar]
  11. Kimura, K. & Itoh, Y.(2003). Characterization of poly-γ-glutamate hydrolase encoded by a bacteriophage genome: possible role in phage infection of Bacillus subtilis encapsulated with poly-γ-glutamate. Appl Environ Microbiol 69, 2491–2497.[CrossRef] [Google Scholar]
  12. Kimura, K., Tran, L.-S. P. & Itoh, Y.(2004). Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis. Microbiology 150, 2911–2920.[CrossRef] [Google Scholar]
  13. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G. & 146 other authors(1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249–256.[CrossRef] [Google Scholar]
  14. Lazazzera, B. A., Palmer, T., Quisel, J. & Grossman, A. D.(1999). Cell density control of gene expression and development in Bacillus subtilis. In Cell–Cell Signalling in Bacteria, pp. 27–46. Edited by G. M. Dunny & S. C. Winans. Washington, DC: American Society for Microbiology.
  15. Lieberman, M. W., Wiseman, A. L., Shi, Z. Z. & 10 other authors(1996). Growth retardation and cysteine deficiency in γ-glutamyl-transpeptidase-deficient mice. Proc Natl Acad Sci U S A 93, 7923–7926.[CrossRef] [Google Scholar]
  16. Makino, S., Sasakawa, C., Uchida, I., Terakado, N. & Yoshikawa, M.(1988). Cloning and CO2-dependent expression of the genetic region for encapsulation from Bacillus anthracis. Mol Microbiol 2, 371–376.[CrossRef] [Google Scholar]
  17. Makino, S., Uchida, I., Terakado, N., Sasakawa, C. & Yoshikawa, M.(1989). Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis. J Bacteriol 171, 722–730. [Google Scholar]
  18. Makino, S., Watarai, M., Cheun, H.-I., Shirahata, T. & Uchida, I.(2002). Effect of the low molecular capsule released from the cell surface of Bacillus anthracis on the pathogenesis of anthrax. J Infect Dis 186, 227–233.[CrossRef] [Google Scholar]
  19. Mehdi, K. & Penninckx, M. J.(1997). An important role for glutathione and γ-glutamyltranspeptidase in the supply of growth requirements during nitrogen starvation of the yeast Saccharomyces cerevisiae. Microbiology 143, 1885–1889.[CrossRef] [Google Scholar]
  20. Meijer, W. J. J., de Boer, A. J., van Tongeren, S., Venema, G. & Bron, S.(1995). Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a novel type of replicon. Nucleic Acids Res 23, 3214–3223.[CrossRef] [Google Scholar]
  21. Meijer, W. J. J., Wisman, G. B. A., Terpstra, P., Thorsted, P. B., Thomas, C. M., Holsappel, S., Venema, G. & Bron, S.(1998). Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analysis of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparison with related plasmids from Gram-positive bacteria. FEMS Microbiol Rev 21, 337–368.[CrossRef] [Google Scholar]
  22. Minami, H., Suzuki, H. & Kumagai, H.(2003). Salt-tolerant γ-glutamyltranspeptidase from Bacillus subtilis 168 with glutaminase activity. Enzyme Microb Technol 32, 431–438.[CrossRef] [Google Scholar]
  23. Minami, H., Suzuki, H. & Kumagai, H.(2004).γ-Glutamyltranspeptidase, but not YwrD, is important in utilization of extracellular glutathione as a sulfur source in Bacillus subtilis. J Bacteriol 186, 1213–1214.[CrossRef] [Google Scholar]
  24. Nagai, T., Koguchi, K. & Itoh, Y.(1997). Chemical analysis of poly-γ-glutamic acid produced by plasmid-free Bacillus subtilis (natto): evidence that plasmids are not involved in poly-γ-glutamic acid production. J Gen Appl Microbiol 43, 139–143.[CrossRef] [Google Scholar]
  25. Nakada, Y. & Itoh, Y.(2002). Characterization and regulation of the gbuA gene, encoding guanidinobutyrase in the arginine dehydrogenase pathway of Pseudomonas aeruginosa PAO1. J Bacteriol 184, 3377–3384.[CrossRef] [Google Scholar]
  26. Ogawa, Y., Hosoyama, H., Hamano, M. & Motai, H.(1991). Purification and properties of γ-glutamyltranspeptidase from Bacillus subtilis (natto). Agric Biol Chem 55, 2971–2977.[CrossRef] [Google Scholar]
  27. Ogawa, Y., Sugiura, D., Motai, H., Yuasa, K. & Tahara, Y.(1997). DNA sequencing of Bacillus subtilis (natto) NR-1 γ-glutamyltranspeptidase gene, ggt. Biosci Biotechnol Biochem 61, 1596–1600.[CrossRef] [Google Scholar]
  28. Phillips, Z. E. & Strauch, M. A.(2002).Bacillus subtilis sporulation and stationary phase gene expression. Cell Mol Life Sci 59, 392–402.[CrossRef] [Google Scholar]
  29. Rosenberg, I. & Saini, P. K.(1980). Folylpolyglutamate endopeptidase from chicken intestine: isolation with the aid of affinity chromatography. Methods Enzymol 66, 667–670. [Google Scholar]
  30. Suzuki, T. & Tahara, Y.(2003). Characterization of the Bacillus subtilis ywtD gene, whose product is involved in γ-polyglutamic acid degradation. J Bacteriol 185, 2379–2382.[CrossRef] [Google Scholar]
  31. Suzuki, H., Kumagai, H. & Tochikura, T.(1986).γ-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties. J Bacteriol 168, 1325–1331. [Google Scholar]
  32. Suzuki, H., Hashimoto, W. & Kumagai, H.(1993).Escherichia coli K-12 can utilize an exogenous γ-glutamyl peptide as an amino acid source, for which γ-glutamyltranspeptidase is essential. J Bacteriol 175, 6038–6040. [Google Scholar]
  33. Tanaka, T., Hiruta, O., Futamura, T., Uotani, K., Satoh, A., Taniguchi, M. & Oi, S.(1993). Purification and characterization of poly(γ-glutamic acid) hydrolase from a filamentous fungus, Myrothecium sp. TM-4222. Biosci Biotechnol Biochem 57, 2148–2153.[CrossRef] [Google Scholar]
  34. Tate, S. S. & Meister, A.(1981).γ-Glutamyl transpeptidase: catalytic, structural and functional aspects. Mol Cell Biochem 39, 357–368.[CrossRef] [Google Scholar]
  35. Tate, S. S. & Meister, A.(1985).γ-Glutamyl transpeptidase from kidney. Methods Enzymol 113, 400–419. [Google Scholar]
  36. Thorne, C. B.(1993).Bacillus anthracis. In Bacillus subtilis and Other Gram-Positive Bacteria, pp. 113–124. Edited by A. L. Sonenshein, J. A. Hock & R. Losick. Washington, DC: American Society for Microbiology.
  37. Tran, L.-S. P., Nagai, T. & Itoh, Y.(2000). Divergent structure of the comQXPA quorum-sensing components: molecular basis of strain-specific communication mechanism in Bacillus subtilis. Mol Microbiol 37, 1159–1171.[CrossRef] [Google Scholar]
  38. Uchida, I., Makino, S., Sasakawa, C., Yoshikawa, M., Sugimoto, C. & Terakado, N.(1993). Identification of a novel gene, dep, associated with depolymerization of the capsule polymer in Bacillus anthracis. Mol Microbiol 9, 487–496.[CrossRef] [Google Scholar]
  39. Urushibata, Y., Tokuyama, S. & Tahara, Y.(2002). Characterization of the Bacillus subtilis ywsC gene, involved in γ-polyglutamic acid production. J Bacteriol 184, 337–343.[CrossRef] [Google Scholar]
  40. Vieira, J. & Messing, J.(1987). Production of single-stranded plasmid DNA. Methods Enzymol 153, 3–11. [Google Scholar]
  41. Xu, K. & Strauch, M. A.(1996). Identification, sequence, and expression of the gene encoding γ-glutamyltranspeptidase in Bacillus subtilis. J Bacteriol 178, 4319–4322. [Google Scholar]

Data & Media loading...

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