1887

Abstract

The poly(amino acid)s -poly(-glutamic acid) (gPGA) and ϵ-poly(-lysine) (ePL) are known to be natural linear poly(amino acid)s secreted by spp. and spp., respectively. In this study, a strain producing both ePL and gPGA was identified. Mass spectrometry and other analyses revealed that the gPGA is a mixture of oligomers consisting of 10–13 -glutamic acid residues linked by isopeptide bonds. In contrast to the known gPGA, the glutamic acid oligomers have a cyclodehydrated structure in each molecule. We previously reported that the ePL molecules secreted by the same strain disperse only slightly in an agar culture plate, as though they were larger molecules. This phenomenon is explicable by the observed polyion complex formation between the glutamic acid oligomers and ePLs. The glutamic acid oligomers control the ePL's dispersion, which would also affect the spatial distribution of the ePL's antimicrobial activity. Therefore, gene clustering or common use of the gene was presumed for biosynthesis of the two poly(amino acid)s. However, no gene for biosynthesis of the glutamic acid oligomer was found in the neighbouring region of that for ePL biosynthesis, and the glutamic acid oligomer was produced by a mutant in which the ePL biosynthetic gene was inactivated by gene disruption.

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2009-09-01
2019-10-15
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References

  1. Ashiuchi, M. & Misono, H. ( 2002; ). Biochemistry and molecular genetics of poly γ-glutamate synthesis. Appl Microbiol Biotechnol 59, 9–14.[CrossRef]
    [Google Scholar]
  2. Ashiuchi, M., Nawa, C., Kamei, T., Song, J. J., Hong, S. P., Sung, M. H., Soda, K. & Misono, H. ( 2001; ). Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis. Eur J Biochem 268, 5321–5328.[CrossRef]
    [Google Scholar]
  3. Kawai, T., Kubota, T., Hiraki, J. & Izumi, Y. ( 2003; ). Biosynthesis of epsilon-poly l-lysine in a cell-free system of Streptomyces albulus. Biochem Biophys Res Commun 311, 635–640.[CrossRef]
    [Google Scholar]
  4. Kelly, R. W. & Taylor, P. L. ( 1976; ). tert-Butyl dimethylsilyl ethers as derivatives for qualitative analysis of steroids and prostaglandins by gas phase methods. Anal Chem 48, 465–467.[CrossRef]
    [Google Scholar]
  5. Mazodier, P., Petter, R. & Thompson, C. ( 1989; ). Intergeneric conjugation between Escherichia coli and Streptomyces species. J Bacteriol 171, 3583–3585.
    [Google Scholar]
  6. Nimura, N. & Kinoshita, T. ( 1986; ). o-Phthalaldehyde-N-acetyl cysteine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid enantiomers and its application to conventional amino acid analysis. J Chromatogr A 352, 160–177.
    [Google Scholar]
  7. Nishikawa, M. & Ogawa, K. ( 2002; ). Distribution of microbes producing antimicrobial epsilon-poly l-lysine polymers in soil microflora determined by using a novel method. Appl Environ Microbiol 68, 3575–3581.[CrossRef]
    [Google Scholar]
  8. Prentki, P. & Krisch, H. M. ( 1984; ). In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303–313.[CrossRef]
    [Google Scholar]
  9. Saimura, M., Takehara, M., Mizukami, S., Kataoka, K. & Hirohara, H. ( 2008; ). Biosynthesis of nearly monodispersed poly(epsilon-l-lysine) in Streptomyces species. Biotechnol Lett 30, 377–385.[CrossRef]
    [Google Scholar]
  10. Shima, S. & Sakai, H. ( 1981a; ). Poly l-lysine produced by Streptomyces. Part II. Taxonomy and fermentation studies. Agric Biol Chem 45, 2497–2502.[CrossRef]
    [Google Scholar]
  11. Shima, S. & Sakai, H. ( 1981b; ). Poly l-lysine produced by Streptomyces. Part III. Chemical studies. Agric Biol Chem 45, 2503–2508.[CrossRef]
    [Google Scholar]
  12. Yamanaka, K., Maruyama, C., Takagi, H. & Hamano, Y. ( 2008; ). Epsilon-Poly l-lysine dispersity is controlled using a highly unusual nonribosomal peptide synthetase. Nat Chem Biol 4, 766–772.[CrossRef]
    [Google Scholar]
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H-NMR spectroscopy of the sample containing poly(glutamic acid). After s MN-10 reached the stationary growth phase, the sample was extracted from the culture filtrate using an anion-exchanger and was subjected to NMR spectroscopy. The NMR profile shows that a small amount of aspartic acid exists in the sample as well as glutamic acid. The same sample was subjected to MALDI-TOF/MS, but no poly(glutamic acid) linking to aspartic acid was detected. Therefore, aspartic acid appears in LC analysis of the acid hydrolysis product (Fig. 2b) because the sample contains aspartic acid as an impurity. [ PDF] (33 kb) Multiple amino acid sequence alignment of ε-poly(L-lysine) synthetase from MN-10 and various Actinobacteria. Putative amino acid residues which recognize L-lysine as a substrate amino acid are shown in blue boldface characters. Underlined amino acid sequences indicate the degenerate PCR primers, 5'-cgacgcctcctgcgargaratgtg-3' and 5'-cttggcgcccagcarncknarcca-3', designed for primary gene fragment cloning. Q93H21_STRAW, non-ribosomal peptide synthetase (AB070954); B5I368_9ACTO, ' ' non-ribosomal peptide synthetase (DS570945); MN-10, MN-10 ε-poly(L-lysine) synthetase (this study, AB477240); B5BR95_9ACTO, ε-poly(L-lysine) synthetase (AB385841); A1UMK4_MYCSK, sp. non-ribosomal peptide synthetase (CP000518). A multiple sequence alignment program, CLUSTALW2, was used (European Bioinformatics Institute). [ PDF] (42 kb)

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H-NMR spectroscopy of the sample containing poly(glutamic acid). After s MN-10 reached the stationary growth phase, the sample was extracted from the culture filtrate using an anion-exchanger and was subjected to NMR spectroscopy. The NMR profile shows that a small amount of aspartic acid exists in the sample as well as glutamic acid. The same sample was subjected to MALDI-TOF/MS, but no poly(glutamic acid) linking to aspartic acid was detected. Therefore, aspartic acid appears in LC analysis of the acid hydrolysis product (Fig. 2b) because the sample contains aspartic acid as an impurity. [ PDF] (33 kb) Multiple amino acid sequence alignment of ε-poly(L-lysine) synthetase from MN-10 and various Actinobacteria. Putative amino acid residues which recognize L-lysine as a substrate amino acid are shown in blue boldface characters. Underlined amino acid sequences indicate the degenerate PCR primers, 5'-cgacgcctcctgcgargaratgtg-3' and 5'-cttggcgcccagcarncknarcca-3', designed for primary gene fragment cloning. Q93H21_STRAW, non-ribosomal peptide synthetase (AB070954); B5I368_9ACTO, ' ' non-ribosomal peptide synthetase (DS570945); MN-10, MN-10 ε-poly(L-lysine) synthetase (this study, AB477240); B5BR95_9ACTO, ε-poly(L-lysine) synthetase (AB385841); A1UMK4_MYCSK, sp. non-ribosomal peptide synthetase (CP000518). A multiple sequence alignment program, CLUSTALW2, was used (European Bioinformatics Institute). [ PDF] (42 kb)

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