Supplementary information for

Extremely high intracellular concentration of glucose-6-phosphate and NAD(H) in Deinococcus radiodurans

Takumi Yamashiro,Kousaku Murata, Shigeyuki Kawai#

Disruption of G6PDH gene in D. radiodurans

Plasmids and primers used for gene disruption are shown in Supplementary Table S1and Supplementary Table S2, respectively. Genomic DNA of D. radiodurans was isolated using the DNeasy Blood & Tissue Kit (Qiagen), following the protocol for Gram-positive bacteria.

A fragment of DR_1596 (1,246 b; DR_1596_257_1502) was amplified by PCR using primers 1 and 2. DR_1596_257_1502 was inserted into the HindIII/EcoRI site of pUC119 using In-Fusion (Takara, Otsu, Japan) to yield pMK5342. The Km-resistance gene (Kmr) was PCR-amplified from pUC4K using primers 3 and 4 and inserted into the SalI site of DR_1596_257_1502in pMK5342 using In-Fusion to yield pMK5346. Kmr-inserted DR_1596_257_1502 (DR_1596::Kmr) was PCR-amplified from pMK5346 using primers 5 and 6. Introduction ofDR_1596::Kmr into D. radiodurans cell to disrupt DR_1596 was performed as previously reported (Nishida and Narumi 2002), and the resultant strain was named MK5368 (G6PDH disruptant). The PCRs described above were performed using KOD-Plus-Neo (Toyobo, Osaka, Japan). Disruption of DR_1596 was confirmed by PCR with KOD-FX-Neo (Toyobo) using primers 1 and 2.

Cycling assay for NAD(H) and NAD(H)

For NAD+ and NADH, 70 μL/well of reaction mixture [7 μL of 1 M Bicine (pH 8.0), 16.6 μL of 10 mM phenazine ethosulfate (PES; Sigma), 4.2 μL of 10 mM 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium (MTT; Dojindo, Kumamoto, Japan), 1.4 μL of 200 mM EDTA, 7 μL of 99.5% (v/v) ethanol, 0.3 μL of 1.77 U Yeast ADHII (Oriental Yeast, Tokyo, Japan), 17.5 μL of extract, and 17.5 μL of neutralizing alkali (0.1 M NaOH for NAD+) or acid (0.1 M HCl for NADH)] was placed on a microplate, and the A570 of the reaction mixture was measured every 30 seconds for 10 min. Reaction was initiated by adding ethanol and performed at 26°C. An Infinite F200 pro (TECAN, Männedorf, Switzerland) was used for measurement of A570. NADP+ and NADPH assayswere performed as described above, but using 7 μL of 10 mM G6P and 2 μL of 0.05 U/μL G6PDH instead of ethanol and ADHII. Reaction was initiated by addition of G6P. NAD(H) (1.25–80 μM) and NADP(H) (0.25–40 μM) were used as standards.

SupplementaryTable S1. Plasmids used in this study
Plasmid / Description / Procedure / Source
pUC119 / Apr, lac promoter, lacO, lacZ, M13IG / Takara
pUC4K / Apr, Kmr, lac promoter / Amersham
pMK5342 / DR_1596_257_1502/pUC119 / DR_1596_257_1502 was inserted into the HindIII–EcoRI site of pUC119. / This study
pMK5346 / Kmr/pMK5342 / Kmr was inserted into the SalI site of DR_1596 in pMK5342. / This study
Supplementary Table S2. Primers used in this study
No. / Primer / Sequencea / Note
1 / DR1596_pUC119_Fw / TGATTACGCCAAGCTCCGACGGCCAGAATCCCTTT / For insertion of DR_1596_257_1502 into the HindIII/EcoRI site of pUC119
2 / DR1596_pUC119_Rv / GACGGCCAGTGAATTTCGCGCAGCACCATCTCTTG / Same as above
3 / Km_DR1596_SalI_Fw / CCGGGGGTTTGTCGAGTTTTCCCAGTCACGACGTT / For insertion of Kmr into the SalI site of DR_1596
4 / Km_DR1596_SalI_Rv / TCTGCACGTGGTCGATGTGGAATTGTGAGCGGATA / Same as above
5 / DR1596_Km_Fw / CCGACGGCCAGAATCCCTTT / For amplification of DR_1596::Kmr
6 / DR1596_Km_Rv / TCGCGCAGCACCATCTCTTG / Same as above
7 / M13_Fw / GTAAAACGACGGCCAGT / For sequencing
8 / M13_Rv / GGAAACAGCTATGACCATG / Same as above

a Bold characters in sequences are the 15 bases required for the In-Fusion reaction.

Supplementary Fig. S1. Effect of the concentrations of NAD+and NADP+ on NAD(P)+-reducing activity. NAD(P)+-reducing activities in cell extract of D. radiodurans WT (circles) and G6PDH disruptant (squares) at NAD(P)+ concentrations of 0–500 μM. Closed and open symbols indicate NAD+-reducing and NADP+-reducing activities, respectively. n=2, Ave. ± SD.

Supplementary Fig. S2. G6PDH activity of D. radiodurans cell extract. G6PDH activity of cell extract of D. radiodurans WT was measured at various concentrations of NAD+ (closed circles) and NADP+ (open circles). n=2, Ave. ± SD.

REFERENCES

Kawai S et al (2001) Molecular characterization of Escherichia coli NAD kinase. Eur J Biochem 268: 4359-4365

Nishida H, Narumi I (2002) Disruption analysis of DR1420 and/or DR1758 in the extremely radioresistant bacterium Deinococcus radiodurans. Microbiology 148: 2911-2914