Michael J. Daly Lab: Deinococcus radiodurans

Physiology: Microarray and Proteomic Data (Unpublished)

Microarray Experiments

I. Acute IR (Excel, 1.0Mb)

Preparation of the cells for microarray analysis:

Cells were grown at 32°C in liquid nutrient-rich medium TGY (150 ml) . Half of the culture (75 ml) was irradiated on ice to a total dose of 15 kGy. The nonirradiated control culture was incubated on ice for the same length of time (98 min) as the culture being irradiated, followed by harvesting through brief centrifugation.Control cells were washed and then frozen in RNAlater solution (Ambion, Austin, TX) that had been maintained on ice and then stored at 80°C. After exposure to 15 kGy, the irradiated cell culture was diluted 20-fold by using fresh TGY medium (at a final volume of 1.5 liters) and incubated at 32°C in an orbital shaker. At the indicated recovery time points (0, 0.5, 1.5, 3, 5, 9, 12, 16, and 24 h), cells were harvested, washed, and frozen in RNAlater solution. Cell viability and cell numbers were determined by plate assay and hemocytometer count. Three independent cell cultures and irradiation treatments of the same kind were performed and served as biological replicates for gene expression experiments, as well as for determining irradiation resistance profiles.

Protocols for genomic DNA and total cellular RNA extraction and manipulation were standard. Microarray construction was based on the genomic sequence data and annotation provided by the Institute for Genomic Research (Rockville, MD).

See also Liu et al., PNAS, 2003, V.100, N7, 4191-96.

II. Fe-treated (Excel, 0.8Mb)

Preparation of the cells for microarray analysis:

Cells were grown on DMM (defined minimal medium) plates, harvested and used to inoculate two liquid DMM variants: 1) with 10 µM Fe; and 2) without added Fe. Cells were harvested (OD600=0.6), followed by RNA isolation and cDNA synthesis as described in Liu et al., PNAS, 2003, V.100, N7, 4191-96.

III. Min_vs_rich_medium (Excel, 1.4Mb)

Preparation of the cells for microarray analysis:

Cells were grown on DMM (defined minimal medium) and TGY medium. Cells were harvested (OD600=0.6), followed by RNA isolation and cDNA synthesis as described in Liu et al., PNAS, 2003, V.100, N7, 4191-96.

IV. Growth under chronic irradiation 47 Gy/hour

Results are pending.

Proteomics Experiments

I. Updated "Accurate Mass Tag" (AMT) database for D. radiodurans (For details on AMT assignments see Lipton et al., PNAS, 2002, V.99, 17, 11049-11054)

II. Acute IR (Excel, 2.8Mb)

III. Different stresses (Excel, 0.3Mb)

Key to Proteomic Table

Protein Expression Analyzed by MS FTICR. Presence (1) or absence (0) of indicated proteins in D. radiodurans (R1) cell preparations derived from:

  • Mm: Minimal (defined) Medium Mid-log phase culture at 32oC, OD600 0.3-0.4.
  • Ml: Minimal (defined) Medium Late-log phase culture at 32oC, OD600 0.6.
  • Mp: Minimal (defined) Medium Post-stationary phase culture at 32oC, OD600 0.9.
  • Tm: TGY (rich) Medium Mid-log phase culture at 32oC, OD600 0.3-0.4.
  • Tl: TGY (rich) Medium Late-log phase culture at 32oC, OD600 0.6.
  • Tp: TGY (rich) Medium Post-stationary phase culture at 32oC, OD600 0.9.
  • Heat Shock (HT): Cells were grown to mid-log phase (OD600 0.3-0.4), at 32oC. The incubation temperature was raised to 42oC and further incubated for 1 hour before harvesting the cells.
  • Cold Shock (Cd): Cells were grown to mid-log phase (OD600 0.3-0.4), at 32oC. The incubation temperature was lowered to 0oC and further incubated for 1 hour before harvesting the cells.
  • Hydrogen Peroxide (per): Cells were grown to mid-log phase (OD600 0.3-0.4), at 32oC. H2O2 was added to a final concentration of 60mM and further incubated for 2 hours before harvesting the cells.
  • 1w: A post-stationary phase (OD600 0.9) D. radiodurans culture was incubated at 32oC for one week without the addition of fresh medium
  • 4w: A post-stationary phase (OD600 0.9) D. radiodurans culture was incubated at 32oC for four weeks without the addition of fresh medium
  • Trichloroethylene Shock (TCE) : Cells were grown to mid-log phase (OD600 0.3-0.4), at 32oC. 0.05% (v/v) TCE was added to the culture and incubated for 2 hours before harvesting the cells.
  • Xylene Shock (Xyl): Cells were grown to mid-log phase (OD600 0.3-0.4), at 32oC. 0.05% (v/v) Xylene was added to the culture and incubated for 2 hours before harvesting the cells.
  • Alkaline Shock (Alk): Cells were grown to mid-log phase (OD600 0.3-0.4), at 32oC. pH of culture was raised from 6.5 to 8.5 with 1N NaOH and incubated for 1 hour before harvesting the cells.

III. Min_vs_rich medium (Excel, 0.1Mb)

From: Lipton et al., PNAS, 2002, V.99, 17, 11049-11054