Journal of Bacteriology, January 1999, p. 666-669, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Targeted Mutagenesis by Duplication Insertion in the
Radioresistant Bacterium Deinococcus radiodurans: Radiation
Sensitivities of Catalase (katA) and Superoxide
Dismutase (sodA) Mutants
Lye Meng
Markillie,
Susan M.
Varnum,
Preston
Hradecky, and
Kwong-Kwok
Wong*
Pacific Northwest National Laboratory,
Molecular Biosciences, Richland, Washington 99352
Received 21 August 1998/Accepted 27 October 1998
 |
ABSTRACT |
Deinococcus radiodurans R1 is extremely resistant to
both oxidative stress and ionizing radiation. A simple and general
targeted mutagenesis method was developed to generate catalase
(katA) and superoxide dismutase (sodA) mutants.
Both mutants were shown to be more sensitive to ionizing radiation than
the wild type.
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TEXT |
Deinococcus
(Micrococcus) radiodurans R1 has extreme
resistance to genotoxic chemicals, oxidative damage, high levels of
ionizing and UV radiation, and desiccation (4, 7, 12, 19).
It has been suggested that the extreme resistance to ionizing radiation is attributable to an effective DNA repair system and a special chromosome structure (8, 13, 14, 16-18). However,
protective mechanisms against oxidative damage may also be involved in
this extreme radiation resistance. The lethal effect of ionizing
radiation is known to be oxygen enhanced (15) by generating
hydrogen peroxide and oxygen free radicals, which damage cell
membranes, proteins, and nucleic acids. It has been shown that a
pretreatment of D. radiodurans at early
exponential-growth phase with hydrogen peroxide enhances its
resistance to radiation (22). Moreover, D. radiodurans expresses relatively high levels of
catalase and superoxide dismutase activities (5). However,
the importance of the catalase and superoxide dismutase activities in
the extreme radiation resistance of D. radiodurans has
never been tested.
In this study, we have developed a general and simple method to
inactivate any targeted gene in D. radiodurans. We have
successfully constructed both catalase (katA) and superoxide
dismutase (sodA) mutants for investigating their roles in
the extreme resistance to ionizing radiation.
Construction of katA and sodA mutants.
The plasmids and strains used in this study are listed in Table
1. Based on the available sequence of the
katA gene from D. radiodurans, three
different plasmids were constructed carrying different parts of the
gene. A 705-bp DNA fragment from codon 154 to 386 of the deinococcal
katA gene (accession no. D63898) was PCR amplified with
katA-F (5'-GGACTTCGTCGTCAACAACCTC-3') and katA-B
(5'-ATCGGCAGTTGCAGGTAGTTGG-3') primers. A 2,970-bp PCR fragment containing the complete regulatory and coding regions of
katA was PCR amplified with katA-F1
(5'-GCTCTTCCATCCCGATCAC-3') and katA-B6
(5'-CCAGAAAAGCACCGTACTGG-3') primers. Amplified PCR fragments were blunt end cloned into a pCR-Blunt cloning vector (Kmr; Invitrogen, Carlsbad, Calif.). D. radiodurans was transformed with the plasmid constructs, selecting
for kanamycin resistance at 25 µg/ml. The transformation protocol was
described previously (20). Since pCR-Blunt is a ColE1
plasmid derivative and does not replicate as a plasmid in D. radiodurans, kanamycin resistance in D. radiodurans was produced by duplication insertion of the plasmid
into the chromosome and subsequent amplification of the plasmid
(20). To confirm that the plasmid constructs had integrated into the chromosome, high-molecular-weight DNA plugs were prepared and
analyzed by pulsed-field gel electrophoresis (PFGE) as described previously (23). DNA plugs were digested with
NotI restriction enzyme and resolved by PFGE (Fig.
1). Because the pCR-Blunt vector has a
unique NotI restriction site, its integration into the
chromosome creates an extra NotI site in the 485-kb
NotI fragment (Fig. 1A, lane 2) of wild-type R1, yielding
290- and 195-kb fragments (Fig. 1A, lanes 3 to 5). The integration of
the plasmid constructs was further confirmed by Southern blot analysis
(Fig. 1B) with a nonradioactive probe generated from pKKW1 plasmid DNA
by a method described previously (24).
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FIG. 1.
(A) PFGE analysis of wild type and transformants.
DNA agarose plugs were digested with NotI restriction
enzyme, and digested fragments were resolved with a contour-clamped
homogeneous electric field mapper. The gel was stained with ethidium
bromide. Lane 1, yeast markers; lane 2, R1 (wild type); lane 3, KKW7001
(complete katA); lane 4, KKW7002 (katA with its
promoter region deleted); lane 5, KKW7003 (partial internal coding
region of katA). The 485-kb NotI fragment (which
disappears) is indicated by the white arrowhead. The two new
NotI fragments are indicated by black arrowheads.
The molecular sizes in kilobases are shown on the left. (B) Southern
blot from gel shown in panel A and hybridized with fluorecein-labelled
probe generated from pKKW1 plasmid DNA. The hybridization signal
was detected with a FluorImager SI as described previously
(24).
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To construct the sodA mutant, sequence data of D. radiodurans was obtained through early release from The Institute
for Genomic Research at www.tigr.org and searched for putative
sodA sequence. A putative deinococcal sodA gene,
which shares 57% identity in deduced amino acid sequence with the
Escherichia coli sodA gene, was identified (data not
shown). A 475-bp DNA fragment from codon 24 to 182 of the
putative deinococcal sodA gene was PCR
amplified with SodA-F1 (5'-AGTTGAGGTAGTAGGCGTGTTCCC-3')
and SodA-B3 (5'-GGAAATTCACCACACCAAGCATC-3') primers. Amplified PCR fragments were blunt end cloned
into pCR-Blunt cloning vector. D. radiodurans was
transformed with the plasmid construct, selecting for kanamycin
resistance at 25 µg/ml by using a previously described protocol
(20).
Enzymatic activities of katA and sodA
mutants.
Transformants with katA plasmid constructs
(pKKW1, pKKW2, and pKKW3) were tested for catalase activities by adding
10 µl of 3% hydrogen peroxide to 200 µl of overnight culture in a
microtiter plate. The robust bubbling due to the formation of oxygen
from hydrogen peroxide was an indication of catalase activity. This test indicated that only transformants from plasmid pKKW3 had lost
catalase activity. Transformants with a sodA plasmid
construct (pKKW4) were tested for loss of superoxide dismutase activity by a paraquat sensitivity test with a standardized single filter paper
disk method (1). Briefly, 10 µl of 13 mM paraquat was spotted on a 5-mm-diameter sterilized filter paper disk which was
placed on a lawn of tested bacteria. Wild-type R1 strain was resistant
to paraquat and no zone of growth inhibition was observed, but
sodA mutants were sensitive to paraquat and a zone of growth inhibition about 25 mm in diameter around the filter disk was observed.
A few selected mutant strains were further confirmed for the loss of
catalase or superoxide dismutase activities. Enzymatic activities of
the transformants were analyzed by separating total soluble protein
extract in a 7.5% nondenaturing gel and staining for catalase activity
(Fig. 2) or superoxide dismutase activity (Fig. 3). To prepare the protein extract,
D. radiodurans cultures were pelleted and resuspended
in butanol-saturated phosphate buffer to remove the outer membrane.
Cells were washed in 50 mM phosphate buffer (pH 7) and then resuspended
again in the same buffer. Cell suspensions were sonicated at 4°C (15 s pulse on, 10 s pulse off) for a total of 5 min by using a Model
60 Sonic Dismembrator set at an intensity of 8 (Fisher Scientific,
Pittsburgh, Pa.). Cell debris was removed by centrifugation
(13,000 × g, 5 min, 4°C), and the cell extracts were
either stored on ice or quick frozen at 
70°C until subsequent
analysis. Protein concentration was determined by using the Bradford
protein dye assay (Bio-Rad, Hercules, Calif.) (3). Samples
of cell extracts (5 or 10 µg per lane) were separated
electrophoretically on 7.5% nondenaturing polyacrylamide gels
(22). Gels were stained for catalase activity by
the horseradish peroxidase-diaminobenzidine method
(6) or stained for superoxide dismutase activity by the
nitroblue tetrazolium-riboflavin method (2). As predicted,
only transformants derived from the plasmid, which carries part of the
internal coding region of the katA gene (pKKW3), lost the
major catalase activity (Fig. 2). Strain KKW7003 (katA
mutant) had lost the major catalase, but a minor catalase was still
present (Fig. 2). Strain KKW7004 was also confirmed to have lost
superoxide dismutase activity (Fig. 3). These results confirmed
the validity of the developed targeted mutagenesis method.
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FIG. 2.
Analysis of catalase activity. Lane 1, R1 (wild type);
lane 2, KKW7001 (complete katA); lane 3, KKW7003
(internal coding region of katA); lane 4, KKW7002
(katA with promoter region deleted). The two catalase bands
shown are labelled KATA (catalase A) and KATB (catalase B).
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FIG. 3.
Analysis of superoxide dismutase activity. Lane 1, R1
(wild type); lane 2, KKW7003 (katA mutant); lane 3, KKW7004
(sodA mutant). SOD, superoxide dismutase.
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This is the first report to demonstrate that duplication insertion
(20) can be exploited to create insertional mutations in
D. radiodurans, although a similar strategy has been
used with budding yeast and Listeria spp. (9,
25). Unlike the previous direct insertion technique used with
D. radiodurans (10, 11), which involves
complex in vitro DNA manipulation and numerous cloning steps, the
current technique is simpler, calling for (i) PCR of the internal
fragment of the gene, (ii) a single cloning step to put the PCR
fragment into the plasmid, and (iii) transformation of the host. The
developed targeted mutagenesis method should be generally
applicable for inactivating other genes, thereby facilitating
functional studies of targeted genes with unknown function in
D. radiodurans, whose genome is almost completely sequenced.
Testing ionizing radiation resistance of katA and
sodA mutants.
Five-milliliter cultures of wild-type
(R1), katA mutant (KKW7003), and sodA
mutant (KKW7004) strains, grown in Falcon 2097 culture tubes to
exponential phase at 32°C, were placed on ice inside a 250-ml
beaker. All bacterial cultures were simultaneously exposed to
ionizing radiation with a dose of 8,000 to 32,000 Gy from a
60Co source irradiator (GammaBeam 650). The katA
mutant and wild type have similar sensitivities to ionizing radiation
when exposed to 8,000 Gy, but the katA mutant is
reproducibly more sensitive (2- to 15-fold) than the wild-type strain
to gamma radiation at doses of 16,000 Gy or higher (Fig.
4). A previous analysis of changes in
cellular proteins of D. radiodurans after gamma
irradiation by two-dimensional gel electrophoresis has identified a
number of proteins that increase in amount after irradiation, including a 60-kDa protein, RIP60 (21). The N-terminal end of RIP60,
which has been partially determined, has an amino acid sequence
of DENNKGV (21), which we were able to match perfectly
with the katA gene when the amino acid sequence was reverse
transcribed into DNA sequence, and the calculated size of deduced
katA gene product is also 60 kDa (data not shown). Thus, the
katA gene product is likely the unidentified RIP60 protein
that increases in amount after gamma radiation, which is consistent
with our finding that katA is required for extreme
radiation.
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FIG. 4.
Cell survival after gamma ray exposure. Gamma radiation
was from a 60Co source (GammaBeam 650). Circles, strain R1
(wild type); squares, strain KKW7003 (katA mutant);
triangles, strain KKW7004 (sodA mutant). The exposure rate
was 368 Gy per min.
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On the other hand, the sodA mutant is reproducibly more
sensitive (3- to 90-fold) than the wild-type strain and the
katA mutant to ionizing radiation at a dose of 16,000 Gy
or higher (Fig. 4). This result suggested that superoxide
dismutase encoded by sodA is more important than the
catalase encoded by katA in protecting the bacteria from
high doses of ionizing radiation. It is possible that the existence of
a secondary catalase B in the katA mutant (Fig. 2) is enough
to handle most of the hydrogen peroxide generated during ionizing
radiation. However, apparently, there does not exist a secondary
superoxide dismutase in the sodA mutant (Fig. 3) to handle
the superoxide anions generated during ionizing radiation. Through
analysis of sequence data of D. radiodurans obtained
through early release from The Institute for Genomic Research,
at www.tigr.org, we have identified other genes that encode
putative oxidative defense enzymes such as glutathione reductase
(gor) and alkyl hydroperoxide reductase (ahpA).
We are currently creating mutations in these genes and constructing
mutants with double mutations in order to decipher the oxidative
defense systems in D. radiodurans.
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ACKNOWLEDGMENTS |
We thank Ken Minton and Rita Cheng for their comments on the
manuscript. We are also grateful to Herb Schellhorn for the protocol of
catalase activity staining in a nondenaturing polyacrylamide gel and
especially to Kevin Groch for operating the 60Co gamma
radiation source.
This work was supported by the Department of Energy's Environmental
Management Science Program and Microbial Genome Program through a DOE
contract, DE-AC06-76RLO 1830, to K.K.W.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Molecular
Biosciences, P7-56, Pacific Northwest National Laboratory, P.O. Box
999, Richland, WA 99352. Phone: (509) 376-5097. Fax: (509) 376-6767. E-mail: kk.wong{at}pnl.gov.
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REFERENCES |
| 1.
|
Bauer, A. W.,
W. M. Kirby,
J. C. Sherris, and M. Turck.
1966.
Antibiotic susceptibility testing by a standardized single disk method.
Am. J. Clin. Patrol.
45:493-496.
|
| 2.
|
Beauchamp, C., and I. Fridovich.
1971.
Superoxide dismutase: improved assays and an assay applicable to acrylamide gels.
Anal. Biochem.
44:276-287[Medline].
|
| 3.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[Medline].
|
| 4.
|
Carbonneau, M. A.,
A. M. Melin,
A. Perromat, and M. Clerc.
1989.
The action of free radicals on Deinococcus radiodurans carotenoids.
Arch. Biochem. Biophys.
275:244-251[Medline].
|
| 5.
|
Chou, F. I., and S. T. Tan.
1990.
Manganese(II) induces cell division and increases in superoxide dismutase and catalase activities in an aging deinococcal culture.
J. Bacteriol.
172:2029-2035[Abstract/Free Full Text].
|
| 6.
|
Clare, D. A.,
M. N. Duong,
D. Darr,
F. Archibald, and I. Fridovich.
1984.
Effects of molecular oxygen on the detection of superoxide radical with nitroblue tetrazolium and an activity stain for catalase.
Anal. Biochem.
140:532-537[Medline].
|
| 7.
|
Dean, C. J.,
P. Feldschreiber, and J. T. Lett.
1966.
Repair of x-ray damage to the deoxyribonucleic acid in Micrococcus radiodurans.
Nature
209:49-52[Medline].
|
| 8.
|
Evans, D. M., and B. E. Moseley.
1983.
Roles of the uvsC, uvsD, uvsE, and mtcA genes in the two pyrimidine dimer excision repair pathways of Deinococcus radiodurans.
J. Bacteriol.
156:576-583[Abstract/Free Full Text].
|
| 9.
|
Guldener, U.,
S. Heck,
T. Fielder,
J. Beinhauer, and J. H. Hegemann.
1996.
A new efficient gene disruption cassette for repeated use in budding yeast.
Nucleic Acids Res.
24:2519-2524[Abstract/Free Full Text].
|
| 10.
|
Gutman, P. D.,
J. D. Carroll,
C. I. Masters, and K. W. Minton.
1994.
Sequencing, targeted mutagenesis and expression of a recA gene required for the extreme radioresistance of Deinococcus radiodurans.
Gene
141:31-37[Medline].
|
| 11.
|
Gutman, P. D.,
P. Fuchs,
L. Ouyang, and K. W. Minton.
1993.
Identification, sequencing, and targeted mutagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans.
J. Bacteriol.
175:3581-3590[Abstract/Free Full Text].
|
| 12.
|
Mattimore, V., and J. R. Battista.
1996.
Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation.
J. Bacteriol.
178:633-637[Abstract/Free Full Text].
|
| 13.
|
Minton, K. W.
1996.
Repair of ionizing-radiation damage in the radiation resistant bacterium Deinococcus radiodurans.
Mutat. Res.
363:1-7[Medline].
|
| 14.
|
Minton, K. W., and M. J. Daly.
1995.
A model for repair of radiation-induced DNA double-strand breaks in the extreme radiophile Deinococcus radiodurans.
Bioessays
17:457-464[Medline].
|
| 15.
|
Mirsa, H. P., and I. Fridovich.
1976.
Superoxide dismutase and the oxygen enhancement of radiation lethality.
Arch. Biochem. Biophys.
176:577-581[Medline].
|
| 16.
|
Moseley, B. E., and H. J. Copland.
1975.
Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans.
J. Bacteriol.
121:422-428[Abstract/Free Full Text].
|
| 17.
|
Moseley, B. E., and D. M. Evans.
1983.
Isolation and properties of strains of Micrococcus (Deinococcus) radiodurans unable to excise ultraviolet light-induced pyrimidine dimers from DNA: evidence for two excision pathways.
J. Gen. Microbiol.
129:2437-2445[Medline].
|
| 18.
|
Moseley, B. E.,
A. Mattingly, and M. Shimmin.
1972.
Isolation and some properties of temperature-sensitive mutants of Micrococcus radiodurans defective in DNA synthesis.
J. Gen. Microbiol.
70:399-409[Medline].
|
| 19.
|
Setlow, J. K., and M. E. Boling.
1965.
The resistance of Micrococcus radiodurans to ultraviolet radiation. II. Action spectra for killing, delay in DNA synthesis, and thymine dimerization.
Biochim. Biophys. Acta
108:259-265[Medline].
|
| 20.
|
Smith, M. D.,
E. Lennon,
L. B. McNeil, and K. W. Minton.
1988.
Duplication insertion of drug resistance determinants in the radioresistant bacterium Deinococcus radiodurans.
J. Bacteriol.
170:2126-2135[Abstract/Free Full Text].
|
| 21.
|
Tanaka, A.,
H. Hirano,
M. Kikuchi,
S. Kitayama, and H. Watanabe.
1996.
Changes in cellular proteins of Deinococcus radiodurans following gamma-irradiation.
Radiat. Environ. Biophys.
35:95-99[Medline].
|
| 22.
|
Wang, P., and H. E. Schellhorn.
1995.
Induction of resistance to hydrogen peroxide and radiation in Deinococcus radiodurans.
Can. J. Microbiol.
41:170-176[Medline].
|
| 23.
|
Wong, K. K., and M. McClelland.
1992.
A BlnI restriction map of the Salmonella typhimurium LT2 genome.
J. Bacteriol.
174:1656-1661[Abstract/Free Full Text].
|
| 24.
|
Wong, K. K.,
L. C. Stillwell,
C. A. Dockery, and J. D. Saffer.
1996.
Use of tagged random hexamer amplification (TRHA) to clone and sequence minute quantities of DNA application to a 180 kb plasmid isolated from Sphingomonas F199.
Nucleic Acids Res.
24:3778-3783[Abstract/Free Full Text].
|
| 25.
|
Wuenscher, M. D.,
S. Kohler,
W. Goebel, and T. Chakraborty.
1991.
Gene disruption by plasmid integration in Listeria monocytogenes: insertional inactivation of the listeriolysin determinant lisA.
Mol. Gen. Genet.
228:177-182[Medline].
|
Journal of Bacteriology, January 1999, p. 666-669, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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