The Characterization Of Micrococcus (Deinococcus)Radiodurans Sark Plasmids

Sri Hariani Sjarief* Masahiro Kikuchi * *dan Hiroshi Watanabe"

Abstract

The Cbaraterization or Micrococcus (Deinococcus) Radiodurans Sark Plasmids.This bacterium has been classified as a new genus Deinococcus radiodurans which isresistant to gamma-rays. It can repair itself completely almost all of DNA damages includingdouble strand breaks induced by gamma-rays up to about 5 KGy. To reveal the repairmechanism, several investigations had been done to develop a cloning vector available forthe genetic analysis. For this purpose D. radiodurans Sark are to be prepared as a vectorby studying the characteristics of its plasmid. Plasmids were isolated by electrophoresisusing 0.6% low-melting-temperature agarose in TAE and run for 5.5 hours, followed bythe identification. An antibiotic marker was also carried out in this experiment to identifyits location in the genetic materials of the cell, beside making a restriction map of theplasmid. Results have shown that D. radioduralls Sark has 4 plasmids (Pi, P2, P3 andP4) and the refampicin resistant genes were not found in the plasmid.

INTRODUCTION

Appplications of ionizing radiation for sterilizing foods and othermaterials have led to a number of studies on the radio-resistance of micro­

organisms. One of the microorganisms frequently found in canned. meatirradiated with a sterilizing dose of gamma rays was Micrococcusradiomlrans [1]. This bacterium has been classified into a new genusDeinococcus by Brooks and Murray [2] in 1980. It can grow everywhere,in sewage - sludge, animal feeds, and sawdust medium of mush­rooms [3, 4]. Radioresistance in bacteria is associated in general with itssporeforming ability. However, radioresistance also occured in Deinococcusradiodllra/1S which has no sporeforming ability. It is also known to beradioresistant to both ionizing and ultra-violet radiation [1].

D. radiodllra/1S has been isolated not only from irradiated but also fromunirradiated sources [3]. This indicates that the radioresistancy is an inherentcharacteristic to the bacterium which is not acquired as a result of oneor a few exposures to radiation. It is known that the radioresistancy ofthis organism is due to the high ability of repair on DNA damages [5].

It is also known that D. radiodllrans can repair not only single- butalso double-strand breaks of DNA induced by radiation [6]. However therepair mechanism in the bacterium has not been revealed yet. To reveal

.) National Atomic Energy Agency of Indonesia (BATAN)U) Japan Atomic Energy Research Institute (JAERI)

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the repair mechanism, gene transformation using homologous chromosomalDNA is the only method so far available for the bacterial genetic analysis.Mapping of chromosomal genes containing billion of base pairs is a longsearch, besides no phages have been isolated that plaque on Deinococcusspp [7].

Further, an attemp to mobilize the chromosomal genes by introdusingplasm ids from other species of bacteria as a vector has been un­successful [8]. To overcome this problem a search of plasmids inDeinococcus spp. has been carried out by Mackay et a1. [9]. This plasmidis suspected to provide the basis for constructing a chromosome-mobilizingelement and which might also be useful as a cloning vehicle in this groupof organisms. Plasm ids known as double-stranded, covalently closed circleDNA (ccc-DNA) molecules containing several thousand of base pairs andfirst noticed as genetic elements not linked to the main chromosome werealso found [10]. They constitute a non-essential addition to the genetic infor­mation of the host bacterium, and they may confer on their host propertieswhich are essential for survival in certain environment [11].

Several species of radioresistant bacterium such as D. radio­durans Sark, D. radiophilus, D. proteolyticus, contained plasm ids, exceptD. radiodurans Rl strain [9].

Previous experiments [12] have shown that the sensitivity ofD. radiodurans Rl to irradiation damage of gamma-rays of doses morethan 10 KGy seems not to be different from D. radiodurans Sark. It issuspected that chromosomal genes may be responsible for the radio­resistance of the Rl bacterium cells. However as it has been reported Rlstrain has no plasmid, therefore plasm ids of the Sark strain are neededas a cloning vector to prove the resistancy of the genes of Rl strain whichare located in its DNA chromosome.

MATERIALS AND METHODS

Several methods have been used to isolate plasmid DNA. All of theminvolve the basis steps such as growth, harvesting and analysis of bacteria,and finally followed by amplification and purification of the plasmid DNA.

Growth of bacteria. D. radiodurans Sark used in this experiment wascultivated for years in the Research & Development Laboratory of JAERI(Japan Atomic Energy Research Institute), Takasaki. This strain wasobtained originally from Department of Microbiology, University ofEdinburgh, Scotland, through B.E.B. Moseley. These cells were grown inTGY-glycine medium containing 0.2% glycine, 5 g Bacto-tryptone, 3 gBacto-yeast extract and 1 g glucose in one litre aquadest. An amount of1.5 ml of bacteria was innoculated into the 170 ml1iquid medium mentioned

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above. The suspension was incubated in a shaking water-bath at 30°C for2 days, during which the stationary phase was reached. Bacteria cells werecollected by centrifugation with 5000 rpm for 15 minutes, then washedwith 0.01 M phosphate buffer (pH 7) solution.

Isolation of DNA plasmid. DNA plasmid of D. radiodLt.rans Sark was

prepared essentially as described by Maniatis et al [13]. In preparing cleanedlysates of plasmid, cells were digested with lysozyme-EDTA mixture tobreak the cell's walls. Lysis was completed by the addition of alkalinesodium sulfate solution (SDS, Sodium Dodecyl Sulfate) and warmed to60°C in a water-bath. For extracting the DNA plasmid, chloroform­isoamylalcohol (24:1) was added to the suspension followed by isopropanolprecipitation for recovering the plasmid from the resulting aqueous solution.Finally, 3 M sodium acetate solution of pH 5.2 was added to a finalconcentration of 1 M and then the lysate was refrigerated overnight. Thistreatment preciptitated most of the chromosomal DNA existed in thesuspension, which could be removed by centrifugation. The supernatant,which was rich of DNA plasmid, was termed a cleared lysate.

Separation of DNA plasmid. In the plasmid separation, the concentrationof agarose and duration of electrophoresis tend to take an important partin this process. The relative mobilities of plasmid DNA are dependentprimarily on the agarose concentration which are also influenced by thestrength of the applied current or voltage and the ionic strength of the buffer[13]. Therefore, for the plasmid separation from the bacterial cells, variousconditions have been examined in the previous experiment [12], such as,different growth phase, various concentrations of agarose and various kindsof pH of the buffer. In this experiment, plasm ids were recovered from thelate stationary phase (45 hours of cultivation) of bacterial growth. Theseperation Of plasmid was conducted on 0.6% low-melling-temperatureagarose (m.p. < 700q in TAE buffer with pH 8.0. The electrophoresiswas run for 5.5 hours at a constant voltage of 150 V.

Digestion of DNA plasmid by restriction enzymes. Several restrictionendonucleases were used for a single or double digestion of DNA plasmidin this experiment, such as, Sma I, Ecor I, Hind III, Pst I, Dra I,Sca I, EcoT 22 and CIa I. Sma I was conducted in this experiment foridentifying the DNA plasmid of D. radiodLt.rans, while the remainders wereused in the structuring of the restriction map of plasmid. Reaction mixturewas made up into final 15 III with 1 III of each endonuclease, 3 III of1% TAE buffer, 4 III DNA solution from the precipitated DNA plasmiddiluted with 4 ml TE, and 7 III of distilled wateSr. The digestion was carriedout at 37°C for 3 hours. An amount of 2 III of loading buffer containing

50% glycerol and 0.1 % bromophenol-blue were added to the mixture, then

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incubated at 60°C for 10 minutes. Electrophoresis was carried out in TAEbuffer (pH 8.0) with 0.9% agarose gel. The voltage used for the first30 minutes was constant at 50 V, then changed to 100 V for 3 hours.During 100 V electrophoresis, the buffer was circulated through ice to keepit cool. The molecular size of DNA frragments produced on electrophoresiswas calculated from migration of standard DNA. The standard DNA usedwas a mixture of Hind III, Eco R I and Hinf I (pBR 322).

Antibiotic resistanf gene as a marker. As vectors for recombinant DNA,plasmids with genes for antibiotic resistance offer a great advantage. Whenthey are used together with host bacteria cells that are. plasmid-free andtherefore antibiotic-sensitive, the entry of the resistance plasmid carryingthe recombinant DNA can be easily detected. To confirm the location ofrifampicin resistant gen in D. radiodurans Sark, plasmid of rifampicinresistant Sark strain was compared with a plasmid of non-resistant one.For this purpose, Sark strain was cultivated in TGY medium containingrifampicin. The restriction endonuclease used on rifampicin-resistant Sarkplasmid were ApaI, Bql II, Dra I, Eco R I, Hind III, Pvu II, Sal I,BamH I, Pst I, Sca I, Sma I and Xho 1. These enzymes were also usedin the plasmid of rifampicin non-resistant Sark strain as a control.

RESULTS AND DISCUSSIONS

Recovery of Sark strain plasmid from agarose gel

Four bands of plasm ids (PI, P2, P3 and P4) were recovered from theelectrophoresis agarose gel using 0.6% of low-melting-temperature agarosein TAE buffer, pH 8.0 (Fig. I). The electrophoresis was run for 5.5 hoursat a constant voltage of 150 V. To confirm the purity and identificationof the plasmids, a restriction endonuclease Sma I was used. Fig. 2 showsan illusitration of fragments produced from each plasmid of PI, P2, P3and P4. By comparing the density and the composition of fragmentsseparated by electrophoresis, the purity of the plasmids was determined.

The digested DNA fragments of PI, P2, P3 and P4 show differentelectrophoretic patterns (Fig. 2, lanes 2, 3 and 4). On the other hand, PIseems to be a mixture of P2, P3, and P4, because PI identical bands of

No.1, 7, 9, 12, 13, 14, 15, 16 and 17 can be seen in lane 2 of P2 plasmid.Whereas, bands, No.2, 4, 5, 6, 10, 11 and 19 are also seen in lane 3

of P3 plasmid. While No.3 and 8 bands may come from P4 plasmid. Itcan be seen that PI plasmid is being dominated by P2, then followed byP3, while P4 is contaminated slightly with PI plasmid. Thus it is clear

that P2, P3 and P4, which molecular size is 36,45 and 87 kbp, respectively,

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are the inherent plasmids in D. radioduralZS Sark. However, P4 plasmidwas first found in this investigation. Two of those plasm ids have been foundby Mackay et a1. [9] as pUElO and pUEll which corespond to P2 andP3, respectively.

Antibiotic resistant gene as a marker

A vector suitable for gene cloning must have at least the followingproperties:(1) copy number in a cell is high(2) there are many recognition sites for restrictIOn enzymes(3) it possesses distinguishable genetic markers, such as antibiotic

resistance, to allow selection on agar plates of cells containing therecombinant plasmid.

According to Watson et al. [10], plasm ids carried genes that conveyedresistance to antibiotics such as the tetracyclines or kanamycin. The plasmidof small molecular size carrying a marker such as antibiotic resistance isa favourable vector. Therefore, P2 plasmid of the smallest molecular sizewas examined to construct a vector. It is hoped that antibiotic resistantstrain would carry the resistant gene on its plasmid. So Sark strain wascultivated in TGY medium containing rifampicin to select mutant strainresistant to the drug, and then P2 plasmidswere isolated. From the digestionof this plasmid by several restriction enzymes, it was found that no differentpatterns of fragments were found on plasmid from refampicin resistant straincompared to the normal one from the sensitive strain (Fig. 3). It revealsthat the location of the antibiotic marker is probably in chromosomal DNA.Related to Nazar Agha et al [14]. M. radioduralls strain Rl was susceptibleto rifampicin antibiotic which was decreased to a certain limit when thedose of irradiation was increased. However as it has been mentioned before

Rl strain has no plasmid [9], so it could be concluded that the rifampicinantibiotic marker is located in the chromosomal DNA as well as for

D. radioduralZS Sark strain. If so, it would require a long search to transformthe antibiotic resistant gene from chromosomal DNA as a marker to theplasmid before it is used as a vector. The alternative experiment could alsobe carried out by using other resistant antibiotics marker which coulddirectly attach to the gene of the plasmid. Antibiotic resistance requiresrelatively large amounts of the enzymes that chemically neutralize theantibiotics. By being located on plasm ids, their respective genes are presentin much higher copy numbers than they would be if they were locatedon the main chromosome. One thing should also be remembered whetherthe D. radioduralZS Sark strain has episomes plasmid which has the abilityto move on amI off the main chromosomal elements.

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Restriction map of plasmid

In order to construct cloning vector, the recognition sites of restrictionenzymes have to be known. Therefore, P2 plasmid was digested by 9 kindsof enzymes, i.e., BamH I, EcoR I, Hind III, Pst I, Sal I, Not I, Cia I,Xba I, and EcoT22 1. A part of the results is shown in Fig. 8. The fragmentsize after digestion with BamH I was calculated to be 0.90, 9.81 and26.62 kbp. As shown in restriction map of P2 plasmid (Fig. 4), threecleavage sites were also found by CIa I (4.04, 6.40 and 24.16 kbp), andtwo sites by Hind III (15.26 and 20,54 kbp) and Pst I (8.51 and27.29 kbp). Whereas Eco T22 I, Sal I, Not I and Xba I had introducedsingle cut. From the analysis of these datas obtained by electrophoresis,18 cleavage sites were determined.

CONCLUSION

In the isolation of plasm ids, it was found that the .agarose gelelectrophoresis showed 4 bands of plasmid DNA named as PI, P2, P3 andP4. The digestion of each plasmid by restriction enzymes provided clearevidence that P2, P3 and P4 were different plasmid and their molecularsize was 36, 45 and 87 kbp, respectively. Besides PI was a mixture ofP2, P3 and P4. The two of them have been found by Mackay et al [10]as pUElO and pUE11 which correspond to P2 and P3, respectively. P4was newly found in this study.

To obtain a better plasmid for cloning vector, plasm ids fromrifampicin-resistant Sark strain were compared with thoses fromnonresistant strain. From comparative digestion with several restrictionenzymes, it was found that no rifampicin-resistant genes were located inthe plasm ids.

The digestion of P2 plasmid by 9 kinds of restriction enzymes gave18 specific cleavage sites for the enzymes, which allowed the constructionof a restriction map.

ACKNOWLEDGEMENTS

This work was performed under the STA Program at Takasaki RadiationChemistry Research Establisment, Japan Atomic Energy Research Institute.Appreciation is expressed to all colleagues for their assistance in dealingwith this study.

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REFERENCES

1. A.W. ANDERSON, H.G. NORDON, R.F. CAIN, G. PARRISH andD. DUGGAN, Food Technol., 10 (1956) 575

2. B.W. BROOKS and R.G.E. MURRAY, Int. J. Syst. Bacteriol. 31 (1981)353

3. H. ITO, Agric. Bio. Chern. 41 (1977) 35

4. H. ITO, H. WATANABE, M. TAKEHESA, and H. IIZUKA, Agric.BioI. Chern. 47 (1983) 1239

5. D.M. SWEET, and B.E.B. MOSELEY, Mutat. Res., 34 (1976) 175

6. GJ. DEAN, P. FELDSCHREIBER, and J.T. LETT, Nature, 209 (1966)49

7. B.E.B. MOSELEY, and H.J.R. COPLAND, Mol. Genet, 160 (1978)331

8. TIRGARI, S. PhD thesis, copyright from MAKAY et ai, Arch.Microbiol., 141 (1985) 91

9. M.W., MACKAY, G.H. AL-BAKRI, and B.E.B. MOSELEY, Arch.Microbiol, 141 (1985) 91

10. J.D., WATSON, J. TOOZE, D.T. KURTZ, Recombinant DNA,Scientific American Books, W.H. Freeman & Comp. NY., (1983) 24

11. B.E.B., MOSELEY "Photochemical and Photobiological Reviews",Plenum Press, N.Y., 7 (1983) 223

12. S.H. SJARIEF, M. KIKUCHI, H. KURITA, S. KITAYAMA, andH. WATANABE, Isolation and properties of plasmids fromDeinococcus radiodurans Sark., JAERI-M (1988)

13. T. MANIATIS, E.F. FRITSCH, and J. SAMBROOK, "MolecularCloning, A. Laboratory Manual", Cold Spring Harbor, N.Y., (1985)1511

14. G.M. NAZAR AGHA, S.H. AUDA, and S. AL-BARWARI, The effectof ionizing radiation on the antibiotic sensitivity of Micrococcusradiodurans strain RI and RI15, Int. J. Appl. Rad. and Isotopes, 26(1975) 387-391

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Fig. 1. Plasmid isolation by electrophoresis using low-melting­temperature agarose with 0.6% of TAE buffer (pH 8.0),run for 5.5 hours at a constant voltage of 150 V.

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The number of fragments produced by enzyme :B (BamH I) : 3 L (Sal I) : 1E (EcoR I) : 4 N (N ot I) : 1H (Hind III) : 2 CIa (CIa I) : 3P (Pst I) : 2 Xb (Xba I) : 11'22 (Eco 1'22 I) : 1