Chemical mapping of DNA and counter-ion content inside phage by energy-filtered TEM

Summary, in English

Double-stranded DNA in many bacterial viruses (phage) is strongly confined, which results in internal genome pressures of tens of atmospheres. This pressure is strongly dependent on local ion concentration and distribution within the viral capsid. Here, we have used electron energy loss spectroscopy (EELS), energy-filtered TEM (EFTEM) and X-ray energy dispersive spectroscopy to provide such chemical information from the capsid and the phage tail through which DNA is injected into the cell. To achieve this, we have developed a method to prepare thin monolayers of self-supporting virus/buffer films, suitable for EELS and EFTEM analysis. The method is based on entrapment of virus particles at air-liquid interfaces; thus, the commonly used method of staining by heavy metal salts can be avoided, eliminating the risk for chemical artifacts. We found that Mg2 + concentration was approximately 2-4 times higher in the DNA-filled capsid than in the surrounding TM buffer (containing 10 mM Mg2 + ). Furthermore, we also analyzed the DNA content inside the phage tail by mapping phosphorus and magnesium.

• Författare: Pernilla Nevsten; Alex Evilevitch; Reine Wallenberg
• Avdelning/ar: Centrum för analys och syntes; Biokemi och Strukturbiologi; NanoLund: Centre for Nanoscience
• Publiceringsår: 2012
• Språk: Engelska
• Sidor: 229-240
• Publikation/Tidskrift/Serie: Journal of Biological Physics
• Volym: 38
• Issue: 2
• Länkar: http://dx.doi.org/10.1007/s10867-011-9234-8
• Dokumenttyp: Artikel i tidskrift
• Förlag: Springer
• Ämne: Biological Sciences; Chemical Sciences
• Nyckelord: Energy-Filtered Transmission Electron Microscopy (EFTEM); Electron; Energy Loss Spectroscopy (EELS); Magnesium; Phage lambda; Spermine; Chemical mapping
• Status: Published
• ISBN/ISSN/Övrigt: ISSN: 0092-0606