A method for DNA extraction from the desert cyanobacterium chroococcidiopsis and its application to identification of ftsZ

Abstract

A method was developed for extraction of DNA from Chroococcidiopsis that overcomes obstacles posed by bacterial contamination and the presence of a thick envelope surrounding the cyanobacterial cells. The method is based on the resistance of Chroococcidiopsis to lysozyme and consists of a lysozyme treatment followed by osmotic shock that reduces the bacterial contamination by 3 orders of magnitude. Then DNase treatment is performed to eliminate DNA from the bacterial lysate. Lysis of Chroococcidiopsis cells is achieved by grinding with glass beads in the presence of hot phenol. Extracted DNA is further purified by cesium-chloride density gradient ultracentrifugation. This method permitted the first molecular approach to the study of Chroococcidiopsis, and a 570-bp fragment of the gene ftsZ was cloned and sequenced.

FIG. 1

Electron micrographs of ultrathin sections of Chroococcidiopsis strain (29)N6904 stained with periodic acid-thiosemicarbazide-silver proteinate for 2 h, for detection of polysaccharides (26). (A) Chroococcidiopsis cell from a 4-month-old culture surrounded by a thick electron-dense envelope with a division septum visible in the cytoplasm. (B) Chroococcidiopsis cell from an 8-month-old culture showing a thick multilayered envelope with a bacterium inside (arrow). Bars, 0.5 μm.

FIG. 2

Protocol for DNA extraction from Chroococcidiopsis.

FIG. 3

Absorption spectra of the supernatants recovered from about 10⁸ cells of Chroococcidiopsis strain (29)N6904 and Anabaena cylindrica Lemm after lysozyme treatment and osmotic shock. The absence of phycobiliproteins (——) indicates the cell integrity of Chroococcidiopsis; lysis occurred in Anabaena cylindrica (–––). Supernatant recovered from A. cylindrica was diluted 15-fold.

FIG. 4

(a) HindIII (lane 1) and EcoRI (lane 2) digestions of CsCl-purified DNA from Chroococcidiopsis strain (29)N6904. Lane 3, 1-kb DNA ladder (Gibco BRL). (b) Southern transfer of gel in panel A. The DNA blot was hybridized with the 570-bp fragment of ftsZ. Lanes: 4, 6-kb EcoRI fragment; 5, 1-kb HindIII fragment.