I. Growth of Cells
D. radiodurans grows on nutrient rich medium (TGY: 1% bactotryptone, 0.5% yeast extract; and 0.1% glucose), defined minimal medium (DMM) or defined rich medium (DRM). For solid medium, 1.5% w/v Bacto-agar (Difco) (for TGY) and 1.5% w/v Nobel agar is added.
II. Carbonyl Assay (protocol)
III. 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 (250-1000 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 µg/ml; USB, OH) or chloramphenicol (Cm) selection (3 µg/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
IV. 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 32oC on TGY plate, is transferred to 5 ml TGY and grown at 32oC with aeration to OD600 = 0.9 (~18 hours), diluted in 1:10 ratio into fresh TGY broth and incubated at 32oC 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, 4oC) and resuspended in 7.7 ml filter-sterilized TGY/CaCl2/glycerol (65% (v/v) TGY/ 25 mM CaCl2 /10% 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 32oC for 30 minutes with gentle shaking. To the transformation mixture, 900 µl sterile TGY is added followed by incubation at 32oC with vigorous shaking. After 18 hours of incubation, 100 µl of the transformation mixture is spread on appropriate selective agar.
V. Genomic DNA Extraction
Single colonies of D. radiodurans pre-grown at 32oC on selective TGY plates, are transferred to 5 ml selective TGY and grown at 32oC 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 µl of TE buffer (10 mM Tris-HCl, pH 8; 1 mM EDTA) containing 15 µl of 20% sodium dodecyl sulphate (SDS) (Quality Biological Inc., MD) and 10 µl of 20mg/ml proteinase K (Sigma, MO), and incubated at 37oC 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 65oC 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 10,000 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 µl of TE buffer (Quality Biological Inc., MD). To obtain RNA-free genomic DNA, 50 µl of each genomic DNA sample is treated with 1 µl DNase-free RNase (500 µg/ml, Roche, CH). The concentration and purity of the DNA samples are determined by spectrophotometric ratio assay at 260 nm and 280 nm.
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 (0oC), 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].
VII. Irradiation (April 2015)
Typically, bacterial cells are grown on rich or defined medium plates in the presence of chronic irradiation at 36.5 Gy/hour (137Cs Gammacell 40 irradiation unit [Atomic Energy of Canada Limited]).
For high-level acute irradiation exposures, liquid cultures are irradiated without change of broth on ice at 10 kGy/hour (60Co Gammacell irradiation unit [J.L. Shepard and Associates; Model 109]).
VIII. Preparation of samples for ICP analysis
NOTE: analytical grade water used for all work with cell/sample prep.
Fluka analytical grade water, cat# 95305
Cell pellet prep
Prepare PBS using analytical grade water. Add 5 ml of PBS to cell pellets. Resuspend by vortexing. Measure volume with volumetric pipet, and add sufficient PBS to bring to 11 ml volume. Remove 10 ml for iron analysis, and transfer to acid washed, glass, 25 ml centrifuge tube. Pellet cells from 10 ml volume at 6000 X g for 20 minutes and remove as much supernatant as possible without disturbing pellet.
Resuspend pellet in 2 ml of trace metal grade nitric acid by vortexing. Incubate in water bath for 1 hour at 80oC. Remove from incubation and transfer to fume hood. Loosen cap carefully, and allow tube and contents to cool. Transfer 10 ml of analytical grade water to an acid washed, glass tube. Carefully, transfer acidified cell suspension into water. Measure volume of water with serological pipet, and bring volume to 20 ml with MQ water. Dilute volume 1:5 in analytical grade water for 2% solution.
Use remaining 1 ml of cells in PBS for protein analysis using Pierce Micro BCA Protein Assay Kit (#23235). Cells can be re-frozen until analysis. For blank media and supernatant, add 0.1 ml concentrated HNO3 to 4.9 ml sample.
Thaw cells for analysis.
Dilute cell suspensions in 1:10 measures, and use 10-2 and 10-3 dilutions for protein analysis.
Samples/standards for analysis:
- Standards are prepared in the same buffer as the samples are in using BSA standards and following protocol in Pierce kit instructions.
- Prepare 4 reps for each sample to be quantified.
- Prepare several (10) media blanks to check pH adjustment after HCl neutralization step.
NaOH treatment and HCl neutralization:
- Transfer 1 ml of each blank, standard, and cell suspension into 13x100 mm glass tubes.
- Add 0.11 ml 10 M NaOH immediately prior to adding samples to water bath
- Put samples into water bath for 10 minutes at 90oC.
- Remove samples and place in pan of cold water
- Add ~0.170 ml of 6N HCl to neutralize samples. Determine exact amount of HCl to add by pH'ing the blanks that have had HCl added. Start neutralization step using the blanks, and then move on to the standards and samples.
- Prepare appropriate amount of protein reagent per kit instructions.
- Add 0.5 ml protein reagent to labeled, 4 ml plastic tubes
- Dispense 0.5 ml of standards and blanks into appropriate tubes
- Dispense 0.5 ml volumes of each cell of the 2 cell samples to appropriate tubes
- Cover tubes with plastic wrap and parafilm to minimize evaporation
- Incubate at 60 C for exactly 1 hour
- Remove samples from water bath and allow to cool at room temp
- Measure OD at 562 nm using the appropriate blank for each standard and cell suspension measurement.