Materials and Methods for D. radiodurans

 

 

I. Growth of Cells

D. radiodurans grows on nutrient rich medium (TGY: 1% bactotryptone, 0.5% yeast extract; and 0.1% glucose) or defined minimal medium (DMM). For solid medium, 1.5% w/v Bacto-agar (Difco) (for TGY) and 1.5% w/v Nobel agar is added.

 

II. Construction and Characterization of Mutants

Typically, a ~4 kb autonomously replicating E. coli plasmid [pCR2.1 encoding kanamycin resistance (KmR) or pPCR-Script Cam SK(+) encoding chloramphenicol resistance (CmR)] that contains a fragment of D. radiodurans genomic DNA is used to knock out genes (ORFs) [Markillie L. M. et al., 1999] (Fig. 1). Disruption vectors for the selected genes are generated by cloning DNA fragments (250-700 bp) obtained by PCR amplification into position 295 of the E. coli plasmid pCR2.1 (Invitrogen, CA) or into position 728 of the E. coli plasmid pPCR-Script Cam SK(+) (Stratagene, CA) according to the manufacturer’s protocols. Purified disruption vectors are then transformed into D. radiodurans with kanamycin (Km) selection (25 mg/ml; USB, OH) or chloramphenicol (Cm) selection (3 mg/ml; USB, OH) as described previously [Daly M. J. et al., 1994] and as summarized in Fig. 1. To determine homozygosity or heterozygosity of an ORF disruption, total DNA preparations from the wild-type strain and from a transformant strain are subjected to restriction endonuclease mapping and Southern blotting with diagnostic 32P radiolabeled probes as described Liu Y. et al., 2003. This approach generates diagnostic maps of a transformant’s integration site. 

 

Figure 1. Generation of D. radiodurans mutants by duplication insertion

 

 

 

Legend Fig. 1. The segment bcd of the disruption vector is a 250-700 bp internal sequence of a D. radiodurans gene targeted for disruption. The thick gray segment represents E. coli sequences (pPCR and pCR based plasmids [Stratagene, CA and Invitrogen, CA]) including an E. coli origin of replication (Ori) and an antibiotic resistance gene (KmR or CmR). The plasmid will integrate into the recipient’s genome by a single crossover at the site of homology. Transformants are subjected to several rounds of selection on TGY plates containing antibiotics. The box surrounding the product of gene disruption represents the disrupted gene and its flanking sequence.

 

III. Competent D. radiodurans Cells and Transformation

Competent D. radiodurans cells are prepared by using the procedure originally developed by Tirgari and Moselay [Tirgari and Moselay, 1980]. Single colony of D. radiodurans pre-grown at 320C on TGY plate, are transferred to 5 ml TGY and grown at 320C with aeration to OD600 = 0.9 (~18 hours), diluted in 1:10 ratio into fresh TGY broth and incubated at 320C in a baffled 250 ml flask until an OD600 of 0.5 is obtained. Cells are harvested by centrifugation (5 min, 1,331 x g, 40C) and resuspended in 7.7 ml filter-sterilized TGY/CaCl2/glycerol (65% (v/v) TGY/ 25 mM CaCl2 / 1% glycerol). D. radiodurans competent cells thus obtained (100 μl), 1-5 μg of DNA is added in a sterile 50 ml tube. The solution is mixed gently and placed on ice for 10 minutes followed by incubation at 320C for 30 minutes with gentle shaking. To the transformation mixture, 900 μl sterile TGY is added followed by incubation at 320C with vigorous shaking. After 18 hours of incubation, 100 μl of the transformation mixture is spread on appropriate selective agar.

 

IY. Genomic DNA Extraction

Single colonies of D. radiodurans pre-grown at 320C on selective TGY plates, are transferred to 5 ml selective TGY and grown at 320C with aeration to late stationary phase.  Aliquots (1.5 ml) of these cells are pelleted in a microcentrifuge. The pellets are then resuspended in 570 ml of TE buffer (10 mM Tris-HCl, pH 8; 1 mM EDTA) containing 20 μl of 20% sodium dodecyl sulphate (SDS) (Quality Biological Inc., MD) and 15 μl of 20mg/ml proteinase K (Sigma, MO), and incubated at 370C for 1 h. To the solution, 125 ml of 4 M NaCl is added followed by 80 ml CTAB/NaCl solution [4.1% (w/v) NaCl in dH2O, 10% (w/v) Hexadecytrimethyl ammonium bromide (CTAB)]. The samples are incubated at 650C for 10 minutes to disrupt the cell wall integrity.  Genomic DNA is purified with an equal volume of 24:1 chloroform/isoamylalcohol solution. The solution is mixed by vortexing and centrifuged at 9,400 x g for 5 minutes in a microcentrifuge at room temperature to separate the aqueous and organic phases. The upper aqueous phase containing the total genomic DNA is transferred to a fresh tube and extracted with equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) to remove all the protein membrane components. The solution is mixed by vortexing and spun in a microcentrifuge for 5 minutes. The upper aqueous phase is transferred to a fresh tube and nucleic acids are precipitated with 0.6 volume isopropanol. After mixing, the DNA is allowed to precipitate. DNA is isolated by centrifugation and then air-dried before dissolving it in 50 ml of TE buffer (Quality Biological Inc., MD). To obtain RNA-free genomic DNA, 50 ml of each genomic DNA sample is treated with 1 ml DNase-free RNase (500 ng/ml, Roche, CH). The concentration and purity of the DNA samples are determined by spectrophotometric ratio assay at 260 nm and 280 nm.

 

Y. Microscopy

Transmission Electron Microscopy (TEM)
D. radiodurans suspensions are rinsed in 0.1 M cacodylate buffer (pH 7.4), fixed in 2.5% gluteraldehyde in the same buffer, and postfixed in osmium tetraoxide.  Fixed samples are embedded in Epon-araldite resin, and 50-70 nm sections are cut from these and stained with uranyl acetate followed by lead citrate [Reynolds, 1963].  Samples are examined with a Philips CM 100 electron microscope, yielding 16,000-41,000 x magnification.  Images are captured by a Kodak Megaplus camera (model 1.4) using ATM Camera software.

Confocal Laser Scanning Microscopy (CSLM)
Bacterial cells are harvested and washed with 0.1 M Tris-HCl, 0.01 M EDTA buffer (pH 8.0), fixed in 77% ethanol (00C), and stained with acridine orange.  The stained preparations are visualized with a Bio-Rad MRC-600 confocal laser scanning microscope interfaced with a Zeiss Axiovert microscope as well as a Merdian ULTIMA ACAS 570 CSL Microscope, using 100 x immersion objectives.  Images are reproduced by using a New Codonics NP1600 Postscript printer. Acridine orange-double stranded nucleic acid results in a complex that has an absorption maximum between 450-490 nm that gives rise to green fluorescence and is used to localize DNA, with a 520 nm barrier filter.  Acridine orange-single stranded nucleic acid complex has an absorption maximum between 510-560 nm that gives rise to red fluorescence and is used to localize RNA, with a 590 nm barrier filter [Darzynkiewicz, 1994].