Identification and Localization of Cyanophycin in Bacteria Cells via Imaging of the Nitrogen Distribution Using Energy-Filtering Transmission Electron Microscopy

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.

<i>Ralstonia eutropha</i> Strain H16 as Model Organism for PHA Metabolism and for Biotechnological Production of Technically Interesting Biopolymers

“…strain PCC7120 led to the accumulation of CGP in R. eutropha H16 and in the PHAnegative mutant PHB -4 [Aboulmagd et al, 2001;Voss et al, 2004]. The presence of CGP in the cells of these recombinant strains and its distribution in the cytoplasm was also demonstrated by energy-filtering transmission electron microscopy also distinguishing the CGP inclusions from PHA inclusions [Koop et al, 2007]. CGP yields of about 20% of cellular dry matter were achieved in flask experiments.…”

A new simple method for quantification and locating P and N reserves in microalgal cells based on energy-filtered transmission electron microscopy (EFTEM) elemental maps

“…Cyanophycin granules were also successfully mapped in recombinant strains of Ralstonia eutropha expressing the cyanophycin synthetase of Anabaena sp. [26]. EFTEM mapping was also successfully applied to eukaryotic microalgae for identification and localization of their PRIs and non-protein NRIs [27].…”

Iron Sources Used by the Nonpathogenic Lactic Acid Bacterium Lactobacillus sakei as Revealed by Electron Energy Loss Spectroscopy and Secondary-Ion Mass Spectrometry

“…EELS also allowed identification of phosphorus and thus DNA in an extracellular matrix produced by a Gram-negative bacterial isolate involved in biofilm formation (2). The nitrogen distribution map created through EELS image analysis allowed the localization of cyanophin, a cyanobacterial protein known for functioning as temporary nitrogen, carbon, and energy stock (27). Information on the cellular status of metals in bacteria is difficult to obtain, since conventional preparations used for electron-microscopic observations can alter structures, relocate, and/or remove metal ions and probably other labile cytoplasmic constituents when processing at room temperature (16).…”