Identification and localization of cyanophycin in bacteria cells via imaging of the nitrogen distribution using energy-filtering transmission electron microscopy

Biomacromolecules. 2007 Sep;8(9):2675-83. doi: 10.1021/bm0611230. Epub 2007 Aug 22.

Abstract

In this study the technique of energy-filtering transmission electron microscopy was applied to localize cyanophycin (CGP) in recombinant strains of Ralstonia eutropha. Since CGP is a polymer consisting of the amino acids aspartate and arginine, which functions as a temporary nitrogen reserve that is deposited as insoluble inclusions in the cytoplasm of the cell, its nitrogen content is significantly higher than that of the other cell matter. In this study, we recorded nitrogen distribution maps, which represent the location of CGP in ultrathin sections of resin-embedded cells of recombinant strains of R. eutropha expressing the cyanophycin synthetase of Anabaena sp. strain PCC 7120. Furthermore, the existence of nitrogen in CGP granules was additionally proven by recording electron energy-loss spectra. The samples of R. eutropha H16 (pBBR1MCS-2::cphA1(7120)) revealed a second type of granule, which does not show nitrogen in the corresponding maps and which can be identified as an inclusion containing poly(3-hydroxybutyric acid). The methods applied in this study are suitable to identify storage compounds with elevated nitrogen contents and to reveal their location in the bacterial cell. The methods are also very helpful to distinguish between inclusions of different chemical compositions that occur both at the same time in the cells but cannot or only hardly be distinguished by other methods.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins
  • Cupriavidus necator / chemistry
  • Cupriavidus necator / metabolism*
  • Cupriavidus necator / ultrastructure*
  • Microscopy, Energy-Filtering Transmission Electron*
  • Nitrogen / analysis*
  • Plant Proteins / analysis*
  • Plant Proteins / chemistry
  • Plant Proteins / metabolism
  • Plant Proteins / ultrastructure*
  • Plasmids

Substances

  • Bacterial Proteins
  • Plant Proteins
  • cyanophycin
  • Nitrogen