Fingerprinting bacterial chromosomal DNA with restriction endonuclease EcoRI: comparison of Rhizobium spp. and identification of mutants
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JONATHANR . M I E L E N Z , ' L. E. JACKSON,' F. ~ ' G A R AAND , ~ K . T. S H A N M U G A M ~ Pl(r11fGt.o~~,rlr Lohot~crtot:\~,Dcpot.rrtrort c~f'Agrotrot?lv curt/ R o t t ~ S'(.;OIIL.(,. r Utlii.ot..sit!' (~f'C'(rl~fi~r.tli(r. D(Ic~.Y. CA. U.S.A. Y5616 Accepted March 30. 1979 MIELI:.NZ,J . R . . L . E. JACKSON. F . O'GARA.i~ndK. T. S H ~ ~ I U C A 1979. M . Fingerprinting bz~cterial chromosomal DNA with ~.estriction endonuclcasc L'coKI: cornparison of Illtizobicrttr spp. and identification of mutants. Can. J. Microbiol. 25: 803-807. Total cellula~.DNA f~.oniR/~izo/~irrttt rr[fi)lii, R.r~roliloti.and ~ . , ~ t l p f ~ t l i lstwins ~ / / t ? i I lo and 1 17 wel-c pi-epared. DNA fragments generated with ~'estrictionendonuclease Ec.oR1 from these DNA samples wele compared in agarose gels afterelectrophoresis. DNA cleavage patterns genelxtcd f~.omR.,jol)otric~trt~t strain 110. R. tr.ifi11ii. and R. rttc,liloti were clearly distinguishable f~.omeach other. Restriction endonuclease cleavage patterns of DNA fi.on1 R. .j(l/~O~~i~'/ltll strain 110 and presumptive R . rrifi~liimuti1nt strains that nodulatc soybean were found to be similn~..R1rizol)irrrrl trifi)lii mutant strains were also lysed by a phage specific f o ~R. ' ,jciportic.rrrrr strain 110. These strain 110 results show that " R . trifi~liimutant strains" are indccd derivatives of R. jtrpotlic~rrt~l and not Il. tt.[lolii. M I ~I:NZ. I .I. R.. I.. E. J..\(.KsoN.F. O ' G ~ i l ; \ c tK. T. S I . I A N > ~ U C ;1979. A \ I . Fingcrp~'intingb:~cteri:~l chromosomal DNA with r'cstriction endonucleasc Ec.oKI: compa~.isonofR/ri:obillrrr spp. and iclentification of niutants. Can. J . Microbiol. 25: 803-807. Nvus avons extrait dans sa totalite I'ADN dcs cellules de Xhbobirrt?~trififolii. Il. trlrliloti et des souches 110 qt 117 dc R. jtrporric.rrrrl. A p k s klectrophorese, nous avons comp;lre, sur gel d'agarose, les fragments d'ADIU obtenusgrice i I'action de I'endonuclease restrictiveG.o-RI sur chacun deb Cchi~ntillonsd'ADN. Les profils de rupture obtenus chez la souche 110 d e R. ,jc~pottic,rr~rr de mi-me que chez R. trifilii et R. trlcliloti etiiient trks distinctifs. Le pl.ofil de rupture r.Csult:~ntde I'action de I'endonuclease restrictive sur I'ADN de In souche 110 de R . ,jcrpor~ic.rrrrr s'est revele semblable i~celui des souches mutantes presumPes de R. trifolii capebles d e PI-ovoquerla nodulation du soja. Les souches mutantes de R. trifi~liiOtaient aussi lysees par un phage spkcifique b I;\ souche 1 10 de R.,ioporri~.rrt~r. Ces resultuts demontrent que "les souches rnutilntes dc R. tr[fi~lii" sont effectivement derivees de la souche 110 de R.,jcrpotiic.rrr?le t non de R. tr('11ii. [Trad~ritpar lejournal]
Materials and Methods Introduction There is increasing interest in the molecula~.biol- 5'tr.trirr.s Rlti:ohirrr17 trifi~liistrain DT-6, as well 21s the presumptive ogy of Rhizohilim spp.. symbiotic nitrogen-fixing bacteria that form root nodules on leguminous mutant sttxins DT-72, DT-125. DT-128. and DT-130 \rere described previously (O'Gnra and Shanrnugam 1977, 1978). plants such as soybean, clovel-, alfalfa, and Illrizohiro~r,jopottic~rttt~ stl'ains 31 1h 1 10 (1 10) and 31 1 b 117 ( 117) peanuts. The mechanism of host specificity is of werc obtained from Dr. D. Weber, USDA, Beltsville, MD. particulal- interest because of the possibility of ex- Derivatives of strain 1 10 which differ in their colony morphology panding the host range of Rl~izohirrtnspp. I n a pre- and c;tl.bohydrate utilization, strains L I , L2, and I (Kuykendall vious paper. we have reported that strains of and Elkan 1976). were obtained from Dr. D. Kuykendall. USDA, Beltsville. MD. Strain DJ-I is ;I spontaneous mutant of Rl~izohiri~n trtfblii can be genetically altered to pro- strain 110 resistant to 100 pg/mL spectinomycin. All other R. duce nodules on soybeans and mungbeans (O'Gar-a ,jcrl~orric~rrr~r strains used in this study were descl-ibed previously 1978). R/7i:ohilrr11 rtrrliloti strain U45 was obtained from and Shanrnugam - 1978). Further analvsis of these (Lim strains sing I-estriction endonuclease analysis of Dr. D. Munns. University of California, Davis. total cellular DNA shows that the pattern observed Gr.o~~.rh Cotlt1itior1.s All cultures for isolation of DNA were grown in glutamate in agarose gels aftel- electrophoresis is similar to that of R. jcrpot1ic.11111and q ~ ~ i distinct te from R . (10.7 mA4) salts medinm containing glutamate as sole source of carbon and nitrogen. For experiments involving bacteriophage ttYfifo1ii. D l , mannitol salts- yeast extract medium described before was 'P~.esent;idd~-ess:Department of Microbial Genetics. Corn P ~ o d u c t Corporation s Internation;rl, Argo, IL. U.S.A. 60501 lPresent address: Department of Microbiology. Weber State College. Ogden, UT, U.S.A. 84408. 3Present address: University College COI-k.Cork, Ireland. JAuthor to whom reprint requests should be k~ddressed.
used (O'Garn and Shanmugam 1976). Bacteriophage was titered in minimal medium using soft-agar overlay procedures described for coliphage lambda (Miller 1972). I.soltrtior7 of' DNA High molecular weight cellular DNA was isolated using the procedure described by Berns and Thomas (1965) with minor
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804
C A N . J . MICROBIOL. VOL. 25, 1979
rnoclific;itions SI; described below. Cells were collected from a stationary phase c u l t ~ ~ rby e cent~.ifi~g:rtionand washed twice with st;~ndardsaline citl-ate (SSC; N;rCI. 0. I5 M; sodil~mc i t ~ i t c . 0.015 M ; p H 7.0)and storctl at -20°C. T o isolate the D N A . cells \vc~.csuspended in SSC at about 0. I g (wet wcight) pel- millilitre o f SSC. Pronxse wxs nddetl to this cell suspension ( I mg/mL) followetl by sodium I:ruryl aulfiite (final concentration o f I%) with quick, gentle mixing. This m i x t ~ ~ W:I~ ~ . c incubated at 37°C for 7 h ant1 then extlxcted twice with phenol s : ~ t ~ ~ r a twith cd buffel-. The aqueous phase was removed and the D N A wxs p~.ecipitatedwith ethanol. The D N A was ~.es~rspended in SSC ;lntl dii~lyzcdagainst 0. I x SSC f o ~ . a h o24 ~ ~ht with three changes o f the buffer. The concentration o f totkil nucleic acids was determined by U V nbso~ption.Average molecul;~rwcight o f D N A in these prepa~.:~rions was ;tbout 30 x IOh and K N A in these p ~ . e p ~ ~ ~ i t was i o n sfount1 to vary from 30 to 50% o f the total n ~ ~ c l eacitl i c content. Arroly.\i.c ~ ~ . i ! l Rc,s!ric./ioir r Ell,-yitrc Twenty microgfiims o f D N A was s~ispcndetlin 0.05 m L o f digestion buffer(fo1. EcmRI: tris(hyd~.oxymethyl)arninometh~ine (Tris), 0.05 M: MgCI,. 0.01 M: NLICI, 0.05 M. p H 7.0: for Wiird 111: Tris. 0.006 hl: MgCI,. 0.006 M: N;iCI. 0.05 M. p H 7.0). One huntlretl units o f the appropriate enzyme (Miles L;~bo~.atories. E1kh~11.t. Indiana) wcl-e added (about 10-fold excess: one unit of the enzyme digested only partially I D N A in I h at 37'C) and the niixtl~rewas incub:itctl ;it 37°C. Aliql~otscontaining 1-2 ly o f D N A were removed :it va~.ioustime intervals and elect~-ophorehctlinto 0.7% ngarose gels (9 x 9 cni and 3 nim thick) at 10 V per centimctre to detel-mine the extent o f digestion of D N A . When the D N A was completely digested (about 2 h) a l i q ~ ~ o ot sf the samples were electrophoresed at 4 V per centinictre fol- 6 to 10h. The slow electropho~.etic migration is ci-iticnl to obtain good resolution. Electrophoresis was continllctl until the b~.omophenolblue dye indicatol.re>ichetl theend o f the gel. Gels were photog~xphetlas dchcribed by Bolivarc~/ol. (1977). Sc~l(,c.!iotrc!f'/lrc, Eri;yrrrc, Over 100 restriction endonuclcases have hcen reported (Roberts 1978) and about 30 o f these enzymes are :tvnil:ible comrnerci;~lly. We have used the following 1-21tion:ilein selecting an enzyme for ourexpcrinients. I f the recognition and cleavage C). site of !I given enzyme is low i n r/r guanine-cytosine (G then t h ~ senzyme should hydrolyze n D N A sample with 21 high C/o G C infrequently and yield a sm;ill number of high molecular wcight fragments. I f the same D N A is hydrolyzed by nnothcl. enzyme. whose Irecognition site is high in 5% G C. :I compam tivcly I;lrge number of sm;iller fragments should result. A small number o f fragments can be separated easily with better resolution in gels after electl-ophorcsis. Since Rhizohirrt?r D N A has :I 60-68% G C (Gibbins ant1 Gregol-y 1972). the enzymes Ec.oKI and Hi,rdIll (recognition I-egion o f G AATTC and A 1 AGCTT. respectively) wel-e used in preliminary experi ments to hydrolyze the D N A . Both these enzymes produced 1:ll.g~ fragments from R. .joporric.rrt!r strain 110 D N A and the avel-age molecul;lr weight o f the In!-ge fragment was about 14 x 106. In contrast, Sol1 (recognition region. G J TCGAC) was found to hydrolyze this D N A more frequently yielding large numbers o f smaller fragments (nver;ige maximum size ofabout 6 x 10Valtons). This rationale did not hold true SorBrrrrr H I which produced larger fragments (similar to G w R I or W i t ~ d111) although the recognition site is high in % G x C ( G 4 GATCC). Hydrolysis at the recognition site is known to be influenced by the neighboring sequences also (Roberts 1976). and this could cxpli~inthe results obtained with Btr~rrHI.We have used the enzyme EcoKI in our experiments since highly active prepariitions are available commcrci;illy and this enzyme produces large
+
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A
molecular wcight fragments after hyd~mlysiso f K1ti;oI~irrrrr D N A . Howevel-.Ec.oKI pl.cp:r~-;~tionsare known to h:iveE~~oKI:': ;ictivity (Polisky 1.1 (11. 1975: Tikchonenko e!, (11.. 1978) and this should be kept in mind while sing thisenzyme. Isolo!iorr c(o Plrogcjiir. K.,jerl~orric.rri~r S~roitt110 Samples o f City o f I>;ivis sewage effluent were cl;ir.ilied by filtration ant1 ccntrifi~gation.i\ Millipore (0.45 piM) filtrate was passed through :I 5-n1L hydl.oxy;lp:itite column to concentrate the phage (Primrose ant1 Day 1977). The phage was eliltetl with 0.2M Na2HP0,. The conccnt~.;~ted phage was niixctl with 1 to 7 m L o f an exponentially growing c u l t i ~ ~o' ef H.,joporric.rrr~rstrain 110 in I .0% soft agar ( M S Y m c d i ~ ~ mant1 ) plated onto M S Y niedi~~m Aftcr . 3 d;iys. a single p l ~ i q ~was r e visible. I'hage particles wc1.e picked from this plaq~leand p;~sscd thl-ough R. ,jopotti~-rrtrrstr;rin I I 0 three times. The purified phage was stored over chloroform.
Results In Fig. 1 , agal-ose gel elect~.opherog~-xms of DNA fragments obtained after complete digestion of total cellular DNA by the I-estriction endonuclease EcoRI are presented. DNA fragments from fastgt-owing organisms, K . t~.(fi)liistrain DT-6 and R. ~?lclilofistrain U45 (Fig. 1 . wells 8 and 9, respectively), and the slow growing organism R. ,jr~po~iicrr~n strain 110 showed clear and distinct banding patterns that are distinguishable and different from each other-. These differences extend th~.oughoutthe range of molecular weights of the DNA fragments seen in this figure (fi-om about 15 LIPto aboi~t0.6 x loh). In compal-ison. DNA fl-on1 the presumptive I?. t~.(fhliistrains (strain DT-72, DT- 125. DT- 128. and DT- 130) (Fig. 1 , wells 4-7. ~.espectively)yielded different sizes as well 21s quantities of fragments after- the endonuclease EcoRI digestion. If 21 mutation was introduced into a central cont1.01 system, that mutation may account for the physiological differences descr.ibed in the PI-eviouspapers (O'Gal-a and Shanmugam 1977, 1978). But such a mutation should have little or no visible effect on the number as well as the positions of the restriction endonuclease sites in the cellul:~r genomic DNA, which are sequence-specific. EcoRI digestion of DNA from the two R. ,jrrpo~lic.rrtl~ strains. strains 110 and 117 (wells 2 and 3), yielded cleavage patterns which were essentially similar, although somequalitative differences could be observed. The clearest difference was visible among the largest bands with molecular weights of 10 to 14 x 10% Among the fragments from the DNA from strain 117, a bright band at a molecular weight of about 3.2 x IOh ant1 another band at 1.6 x 106 can be easily observed. which were undetectable among the DNA fragments from strain 110. Restriction endonuclease digestion patterns of strain 110 and strains DT-72, 125, 128, and 130 were all similar. both qualitatively as well as quantitatively. These I-esultsshowed that the DNA
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M I E L E N Z ET A L .
805
FIG. I. Agu-osc slab gel electrophoresis of E(.oKI clcavetl total cellular DNA from 12lrizohiii1,1.From left to light: AIINA cleavcd with Hi~ltlI11 (moleculal. weight stand;i~-ds.from top to bottom. 14.78, 5.54. 4.3 I . 7.77, 1.54. 1.23, and 0.62 x 106):R. ,jo/xuric.li~lr5train 110. strain 117: PI-esumptiveR. tr(fi11ii mutant \tr;iins DT-72, DT-125. DT-128. DT-130: R . tr(fi11ii stl.iiin DT-6: 12. ~j~cliloti strain U45. See Materials and Mcthotl section fo~.cxperimentnltlct;iils.
from these presumptive R. t~.[fi)liistrains is simil211. especially R. jnponic.rrt?~strain 110, n phage that to ii.,jl~l>otlic.~r~?~ strain 110 and quite distinct from infects strain I10 was tested for its ability togrow in the parent strain DT-6. the presumptive R . tt.ifolii strains. All the slowT o determine that the strain 110 used in the growing D T stl-ains were lysed 21s efficiently a s R. above experiment was really R. jcrponiclrm strain ,jrrponic.rrm strain DJ- I, a spectinomycin-resistant 110, this strain was compared with another culture mutant of strain 3 I l b 110 (data not presented). of strain 1 10 (obtained from Dl-. Weber, USDA) a s Other auxotrophic and drug-resistant mutants of well a s strains L1. L2, and I (Kuykendall and Elkan strain 3 I l b 110 were also lysed efficiently by the 1976). Endonuclease EcoRI digestion of the total phage D l . In contrast, none o f the other R. cellulai- DNA from all five organisms showed es- ,jrrponic.rrnl stl-ains tested (USDA strains 6, 3 1, 76, sentially similar patterns after electrophoresis (Fig. 83, 117. 123, 129, 61A96, 505, CB1809, cowpea 2). It is also interesting to note that the strains strain 32H I) was sensitive to the phage D I. differing in their colony morphology, stl-ains L1, A final test was run to determine if there was n L2, and I, were not distinguishable from each other -restrictionv b,~u- I-.lei. between the slow-growing or other 31 1 b 110 strains. Thus any genetic differ- presumptive mutant strains of R. tt.$o/ii and strain ences among them did not alter the restriction pat- 110. Phage lysates grown on strain DT-125, DTtern significantly. 128, DT-129, o r DT-130 were able t o lyse strain Since the EcoRI-cleavage pattern of the DNA of DJ- I a s efficiently a s the source strain and one other the presumptive R. trififolii strains resembles that of strain belonging t o the DT series indicating n o rethe slow-growing species of root nodule bacteria, striction barrier was active among these strains.
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C A N . J. MICROBIOL.. VOL. 2 5 . 1979
F I G .2. Ag:~roscslabgel electrophoresis of EcoRI clcavcd total cellular D N A from different strains of R.,jopirweight standard). S e e Matel-ials and Methods section for othel-experimental clctails.
Discussion One of the main criteria used in the classification of Rhizobil111lstrains has been the ability of these strains to nodulate specific host plants. 'To understand the genetic and biochemical mechanism of host specificity and symbiosis, isolation and chn~.acte~'ization of mutants altered in these pathways is essential. Pleiotropic contl-ol mutants can alter the cellulal metabolism in such a way that a numbel- of physiological and biochemical properties of the parent and the mutant strains can be markedly different. The presence or introduction of auxotrophic requirements to identify strains may not provide a conclusive characteristic since several auxotrophic mutants are known to have pleiotropic defects in establishing symbiotic associations with the host plant (Schwinghnmer 1977). The percentage G C composition of the total DNA is another property which can be used to distinguish
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several strains. if a clear difference exists (Gibbins and Gregory 1972). The most sensitive means of C is the determination of the analysis of % G buoyant density of DNA in gradients of CsCI. Unfortunately, the ability of this method to detect small buoyant density differences depends on conditions of analysis, such as base composition heterogeneity, molecula~ weight of the isolated DNA, and conditions ofcentrifugation (Atchison et rrl. 1976). In this communication, a method of classification is developed which is based on the linear sequence of the total DNA from the organism. Analysis with restriction endonucleases can determine strain differences not detectable by buoyant density analysis. Clear and distinct cleavage patterns can be used as a tool to identify viral or organelle DNA which are much smaller than bacterial genome (Nathans and Smith 1975; Roberts 1976). Restriction endonuclease cleavage patterns
+
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S . 1965. Ih~li~tion of h i- ~ h of DNA from E~yc.c./lcl-ic~/lirr (.o/; 21nd B(lc.i//lrs .yl[/)fi/j.y BEHNS.K. I.. ant1 C. A. T H O ~ I AJK.
molecula~.weight DNA from Hc~tto~)hilo.t it~fit~c,ttioc.. .I. Mol. have been reported to be complex (Helling ct (11. Biol. 11: 476-490. 1974: Harris-Warrick ct (11.1975). As presented in BOI.IV.\K,F.. R. L . RODKI(;UI..%. M. C . BI:~I.,ZICI~. and H. W. Fig. 1 , although Rhizohinl~l DNA restriction enB O Y ~ R1977. . Const~.uction and chnr.nctcriz:~tit,n of ncw cloning vehicles. 1. Anipicillin-rcsistk~n~del-ivatives of the donuclease cleavage patterns are found to be complasmid pMB 9. Gene. 2: 75-93. plex. thcy can still be used to distinguish one strain R R C I NA. S . M., and K. F. GKI:C;ORY. 1972. Relatedness anlong of Rlli~ohillt~~ from itnother. A case i n point is that G IRl~i:oI~i~rtr~ and ilgr.oI~trc.rc~rit~~t~ species drtcrmincd by three the subtle differences between R. jrlponic.111~1 methods of nucleic acid hyb~.itlization.J . Bacteriol. 111: strains 110 and 117 w e detectable by restr-iction 139-141. n ~M.. c ~ .Y . EI.K:\N,\, S . D. E I I R L . I Cand H. J . analysis. although no buoyant density difference H I \ ~ ( ~ ~ s - W , \ nK. Lr-r~ri~ ~ L*. \ L LD.. . . anti G. H . [
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JONATHANR . M I E L E N Z , ' L. E. JACKSON,' F. ~ ' G A R AAND , ~ K . T. S H A N M U G A M ~ Pl(r11fGt.o~~,rlr Lohot~crtot:\~,Dcpot.rrtrort c~f'Agrotrot?lv curt/ R o t t ~ S'(.;OIIL.(,. r Utlii.ot..sit!' (~f'C'(rl~fi~r.tli(r. D(Ic~.Y. CA. U.S.A. Y5616 Accepted March 30. 1979 MIELI:.NZ,J . R . . L . E. JACKSON. F . O'GARA.i~ndK. T. S H ~ ~ I U C A 1979. M . Fingerprinting bz~cterial chromosomal DNA with ~.estriction endonuclcasc L'coKI: cornparison of Illtizobicrttr spp. and identification of mutants. Can. J. Microbiol. 25: 803-807. Total cellula~.DNA f~.oniR/~izo/~irrttt rr[fi)lii, R.r~roliloti.and ~ . , ~ t l p f ~ t l i lstwins ~ / / t ? i I lo and 1 17 wel-c pi-epared. DNA fragments generated with ~'estrictionendonuclease Ec.oR1 from these DNA samples wele compared in agarose gels afterelectrophoresis. DNA cleavage patterns genelxtcd f~.omR.,jol)otric~trt~t strain 110. R. tr.ifi11ii. and R. rttc,liloti were clearly distinguishable f~.omeach other. Restriction endonuclease cleavage patterns of DNA fi.on1 R. .j(l/~O~~i~'/ltll strain 110 and presumptive R . rrifi~liimuti1nt strains that nodulatc soybean were found to be similn~..R1rizol)irrrrl trifi)lii mutant strains were also lysed by a phage specific f o ~R. ' ,jciportic.rrrrr strain 110. These strain 110 results show that " R . trifi~liimutant strains" are indccd derivatives of R. jtrpotlic~rrt~l and not Il. tt.[lolii. M I ~I:NZ. I .I. R.. I.. E. J..\(.KsoN.F. O ' G ~ i l ; \ c tK. T. S I . I A N > ~ U C ;1979. A \ I . Fingcrp~'intingb:~cteri:~l chromosomal DNA with r'cstriction endonucleasc Ec.oKI: compa~.isonofR/ri:obillrrr spp. and iclentification of niutants. Can. J . Microbiol. 25: 803-807. Nvus avons extrait dans sa totalite I'ADN dcs cellules de Xhbobirrt?~trififolii. Il. trlrliloti et des souches 110 qt 117 dc R. jtrporric.rrrrl. A p k s klectrophorese, nous avons comp;lre, sur gel d'agarose, les fragments d'ADIU obtenusgrice i I'action de I'endonuclease restrictiveG.o-RI sur chacun deb Cchi~ntillonsd'ADN. Les profils de rupture obtenus chez la souche 110 d e R. ,jc~pottic,rr~rr de mi-me que chez R. trifilii et R. trlcliloti etiiient trks distinctifs. Le pl.ofil de rupture r.Csult:~ntde I'action de I'endonuclease restrictive sur I'ADN de In souche 110 de R . ,jcrpor~ic.rrrrr s'est revele semblable i~celui des souches mutantes presumPes de R. trifolii capebles d e PI-ovoquerla nodulation du soja. Les souches mutantes de R. trifi~liiOtaient aussi lysees par un phage spkcifique b I;\ souche 1 10 de R.,ioporri~.rrt~r. Ces resultuts demontrent que "les souches rnutilntes dc R. tr[fi~lii" sont effectivement derivees de la souche 110 de R.,jcrpotiic.rrr?le t non de R. tr('11ii. [Trad~ritpar lejournal]
Materials and Methods Introduction There is increasing interest in the molecula~.biol- 5'tr.trirr.s Rlti:ohirrr17 trifi~liistrain DT-6, as well 21s the presumptive ogy of Rhizohilim spp.. symbiotic nitrogen-fixing bacteria that form root nodules on leguminous mutant sttxins DT-72, DT-125. DT-128. and DT-130 \rere described previously (O'Gnra and Shanrnugam 1977, 1978). plants such as soybean, clovel-, alfalfa, and Illrizohiro~r,jopottic~rttt~ stl'ains 31 1h 1 10 (1 10) and 31 1 b 117 ( 117) peanuts. The mechanism of host specificity is of werc obtained from Dr. D. Weber, USDA, Beltsville, MD. particulal- interest because of the possibility of ex- Derivatives of strain 1 10 which differ in their colony morphology panding the host range of Rl~izohirrtnspp. I n a pre- and c;tl.bohydrate utilization, strains L I , L2, and I (Kuykendall vious paper. we have reported that strains of and Elkan 1976). were obtained from Dr. D. Kuykendall. USDA, Beltsville. MD. Strain DJ-I is ;I spontaneous mutant of Rl~izohiri~n trtfblii can be genetically altered to pro- strain 110 resistant to 100 pg/mL spectinomycin. All other R. duce nodules on soybeans and mungbeans (O'Gar-a ,jcrl~orric~rrr~r strains used in this study were descl-ibed previously 1978). R/7i:ohilrr11 rtrrliloti strain U45 was obtained from and Shanrnugam - 1978). Further analvsis of these (Lim strains sing I-estriction endonuclease analysis of Dr. D. Munns. University of California, Davis. total cellular DNA shows that the pattern observed Gr.o~~.rh Cotlt1itior1.s All cultures for isolation of DNA were grown in glutamate in agarose gels aftel- electrophoresis is similar to that of R. jcrpot1ic.11111and q ~ ~ i distinct te from R . (10.7 mA4) salts medinm containing glutamate as sole source of carbon and nitrogen. For experiments involving bacteriophage ttYfifo1ii. D l , mannitol salts- yeast extract medium described before was 'P~.esent;idd~-ess:Department of Microbial Genetics. Corn P ~ o d u c t Corporation s Internation;rl, Argo, IL. U.S.A. 60501 lPresent address: Department of Microbiology. Weber State College. Ogden, UT, U.S.A. 84408. 3Present address: University College COI-k.Cork, Ireland. JAuthor to whom reprint requests should be k~ddressed.
used (O'Garn and Shanmugam 1976). Bacteriophage was titered in minimal medium using soft-agar overlay procedures described for coliphage lambda (Miller 1972). I.soltrtior7 of' DNA High molecular weight cellular DNA was isolated using the procedure described by Berns and Thomas (1965) with minor
0008-4 166/791070803-05$01.0010 0 1 9 7 9 N2ttion;ll Rese;lrch C o ~ ~ n cofi l CanndaIConseil national de recherches du Canada
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804
C A N . J . MICROBIOL. VOL. 25, 1979
rnoclific;itions SI; described below. Cells were collected from a stationary phase c u l t ~ ~ rby e cent~.ifi~g:rtionand washed twice with st;~ndardsaline citl-ate (SSC; N;rCI. 0. I5 M; sodil~mc i t ~ i t c . 0.015 M ; p H 7.0)and storctl at -20°C. T o isolate the D N A . cells \vc~.csuspended in SSC at about 0. I g (wet wcight) pel- millilitre o f SSC. Pronxse wxs nddetl to this cell suspension ( I mg/mL) followetl by sodium I:ruryl aulfiite (final concentration o f I%) with quick, gentle mixing. This m i x t ~ ~ W:I~ ~ . c incubated at 37°C for 7 h ant1 then extlxcted twice with phenol s : ~ t ~ ~ r a twith cd buffel-. The aqueous phase was removed and the D N A wxs p~.ecipitatedwith ethanol. The D N A was ~.es~rspended in SSC ;lntl dii~lyzcdagainst 0. I x SSC f o ~ . a h o24 ~ ~ht with three changes o f the buffer. The concentration o f totkil nucleic acids was determined by U V nbso~ption.Average molecul;~rwcight o f D N A in these prepa~.:~rions was ;tbout 30 x IOh and K N A in these p ~ . e p ~ ~ ~ i t was i o n sfount1 to vary from 30 to 50% o f the total n ~ ~ c l eacitl i c content. Arroly.\i.c ~ ~ . i ! l Rc,s!ric./ioir r Ell,-yitrc Twenty microgfiims o f D N A was s~ispcndetlin 0.05 m L o f digestion buffer(fo1. EcmRI: tris(hyd~.oxymethyl)arninometh~ine (Tris), 0.05 M: MgCI,. 0.01 M: NLICI, 0.05 M. p H 7.0: for Wiird 111: Tris. 0.006 hl: MgCI,. 0.006 M: N;iCI. 0.05 M. p H 7.0). One huntlretl units o f the appropriate enzyme (Miles L;~bo~.atories. E1kh~11.t. Indiana) wcl-e added (about 10-fold excess: one unit of the enzyme digested only partially I D N A in I h at 37'C) and the niixtl~rewas incub:itctl ;it 37°C. Aliql~otscontaining 1-2 ly o f D N A were removed :it va~.ioustime intervals and elect~-ophorehctlinto 0.7% ngarose gels (9 x 9 cni and 3 nim thick) at 10 V per centimctre to detel-mine the extent o f digestion of D N A . When the D N A was completely digested (about 2 h) a l i q ~ ~ o ot sf the samples were electrophoresed at 4 V per centinictre fol- 6 to 10h. The slow electropho~.etic migration is ci-iticnl to obtain good resolution. Electrophoresis was continllctl until the b~.omophenolblue dye indicatol.re>ichetl theend o f the gel. Gels were photog~xphetlas dchcribed by Bolivarc~/ol. (1977). Sc~l(,c.!iotrc!f'/lrc, Eri;yrrrc, Over 100 restriction endonuclcases have hcen reported (Roberts 1978) and about 30 o f these enzymes are :tvnil:ible comrnerci;~lly. We have used the following 1-21tion:ilein selecting an enzyme for ourexpcrinients. I f the recognition and cleavage C). site of !I given enzyme is low i n r/r guanine-cytosine (G then t h ~ senzyme should hydrolyze n D N A sample with 21 high C/o G C infrequently and yield a sm;ill number of high molecular wcight fragments. I f the same D N A is hydrolyzed by nnothcl. enzyme. whose Irecognition site is high in 5% G C. :I compam tivcly I;lrge number of sm;iller fragments should result. A small number o f fragments can be separated easily with better resolution in gels after electl-ophorcsis. Since Rhizohirrt?r D N A has :I 60-68% G C (Gibbins ant1 Gregol-y 1972). the enzymes Ec.oKI and Hi,rdIll (recognition I-egion o f G AATTC and A 1 AGCTT. respectively) wel-e used in preliminary experi ments to hydrolyze the D N A . Both these enzymes produced 1:ll.g~ fragments from R. .joporric.rrt!r strain 110 D N A and the avel-age molecul;lr weight o f the In!-ge fragment was about 14 x 106. In contrast, Sol1 (recognition region. G J TCGAC) was found to hydrolyze this D N A more frequently yielding large numbers o f smaller fragments (nver;ige maximum size ofabout 6 x 10Valtons). This rationale did not hold true SorBrrrrr H I which produced larger fragments (similar to G w R I or W i t ~ d111) although the recognition site is high in % G x C ( G 4 GATCC). Hydrolysis at the recognition site is known to be influenced by the neighboring sequences also (Roberts 1976). and this could cxpli~inthe results obtained with Btr~rrHI.We have used the enzyme EcoKI in our experiments since highly active prepariitions are available commcrci;illy and this enzyme produces large
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A
molecular wcight fragments after hyd~mlysiso f K1ti;oI~irrrrr D N A . Howevel-.Ec.oKI pl.cp:r~-;~tionsare known to h:iveE~~oKI:': ;ictivity (Polisky 1.1 (11. 1975: Tikchonenko e!, (11.. 1978) and this should be kept in mind while sing thisenzyme. Isolo!iorr c(o Plrogcjiir. K.,jerl~orric.rri~r S~roitt110 Samples o f City o f I>;ivis sewage effluent were cl;ir.ilied by filtration ant1 ccntrifi~gation.i\ Millipore (0.45 piM) filtrate was passed through :I 5-n1L hydl.oxy;lp:itite column to concentrate the phage (Primrose ant1 Day 1977). The phage was eliltetl with 0.2M Na2HP0,. The conccnt~.;~ted phage was niixctl with 1 to 7 m L o f an exponentially growing c u l t i ~ ~o' ef H.,joporric.rrr~rstrain 110 in I .0% soft agar ( M S Y m c d i ~ ~ mant1 ) plated onto M S Y niedi~~m Aftcr . 3 d;iys. a single p l ~ i q ~was r e visible. I'hage particles wc1.e picked from this plaq~leand p;~sscd thl-ough R. ,jopotti~-rrtrrstr;rin I I 0 three times. The purified phage was stored over chloroform.
Results In Fig. 1 , agal-ose gel elect~.opherog~-xms of DNA fragments obtained after complete digestion of total cellular DNA by the I-estriction endonuclease EcoRI are presented. DNA fragments from fastgt-owing organisms, K . t~.(fi)liistrain DT-6 and R. ~?lclilofistrain U45 (Fig. 1 . wells 8 and 9, respectively), and the slow growing organism R. ,jr~po~iicrr~n strain 110 showed clear and distinct banding patterns that are distinguishable and different from each other-. These differences extend th~.oughoutthe range of molecular weights of the DNA fragments seen in this figure (fi-om about 15 LIPto aboi~t0.6 x loh). In compal-ison. DNA fl-on1 the presumptive I?. t~.(fhliistrains (strain DT-72, DT- 125. DT- 128. and DT- 130) (Fig. 1 , wells 4-7. ~.espectively)yielded different sizes as well 21s quantities of fragments after- the endonuclease EcoRI digestion. If 21 mutation was introduced into a central cont1.01 system, that mutation may account for the physiological differences descr.ibed in the PI-eviouspapers (O'Gal-a and Shanmugam 1977, 1978). But such a mutation should have little or no visible effect on the number as well as the positions of the restriction endonuclease sites in the cellul:~r genomic DNA, which are sequence-specific. EcoRI digestion of DNA from the two R. ,jrrpo~lic.rrtl~ strains. strains 110 and 117 (wells 2 and 3), yielded cleavage patterns which were essentially similar, although somequalitative differences could be observed. The clearest difference was visible among the largest bands with molecular weights of 10 to 14 x 10% Among the fragments from the DNA from strain 117, a bright band at a molecular weight of about 3.2 x IOh ant1 another band at 1.6 x 106 can be easily observed. which were undetectable among the DNA fragments from strain 110. Restriction endonuclease digestion patterns of strain 110 and strains DT-72, 125, 128, and 130 were all similar. both qualitatively as well as quantitatively. These I-esultsshowed that the DNA
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M I E L E N Z ET A L .
805
FIG. I. Agu-osc slab gel electrophoresis of E(.oKI clcavetl total cellular DNA from 12lrizohiii1,1.From left to light: AIINA cleavcd with Hi~ltlI11 (moleculal. weight stand;i~-ds.from top to bottom. 14.78, 5.54. 4.3 I . 7.77, 1.54. 1.23, and 0.62 x 106):R. ,jo/xuric.li~lr5train 110. strain 117: PI-esumptiveR. tr(fi11ii mutant \tr;iins DT-72, DT-125. DT-128. DT-130: R . tr(fi11ii stl.iiin DT-6: 12. ~j~cliloti strain U45. See Materials and Mcthotl section fo~.cxperimentnltlct;iils.
from these presumptive R. t~.[fi)liistrains is simil211. especially R. jnponic.rrt?~strain 110, n phage that to ii.,jl~l>otlic.~r~?~ strain 110 and quite distinct from infects strain I10 was tested for its ability togrow in the parent strain DT-6. the presumptive R . tt.ifolii strains. All the slowT o determine that the strain 110 used in the growing D T stl-ains were lysed 21s efficiently a s R. above experiment was really R. jcrponiclrm strain ,jrrponic.rrm strain DJ- I, a spectinomycin-resistant 110, this strain was compared with another culture mutant of strain 3 I l b 110 (data not presented). of strain 1 10 (obtained from Dl-. Weber, USDA) a s Other auxotrophic and drug-resistant mutants of well a s strains L1. L2, and I (Kuykendall and Elkan strain 3 I l b 110 were also lysed efficiently by the 1976). Endonuclease EcoRI digestion of the total phage D l . In contrast, none o f the other R. cellulai- DNA from all five organisms showed es- ,jrrponic.rrnl stl-ains tested (USDA strains 6, 3 1, 76, sentially similar patterns after electrophoresis (Fig. 83, 117. 123, 129, 61A96, 505, CB1809, cowpea 2). It is also interesting to note that the strains strain 32H I) was sensitive to the phage D I. differing in their colony morphology, stl-ains L1, A final test was run to determine if there was n L2, and I, were not distinguishable from each other -restrictionv b,~u- I-.lei. between the slow-growing or other 31 1 b 110 strains. Thus any genetic differ- presumptive mutant strains of R. tt.$o/ii and strain ences among them did not alter the restriction pat- 110. Phage lysates grown on strain DT-125, DTtern significantly. 128, DT-129, o r DT-130 were able t o lyse strain Since the EcoRI-cleavage pattern of the DNA of DJ- I a s efficiently a s the source strain and one other the presumptive R. trififolii strains resembles that of strain belonging t o the DT series indicating n o rethe slow-growing species of root nodule bacteria, striction barrier was active among these strains.
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C A N . J. MICROBIOL.. VOL. 2 5 . 1979
F I G .2. Ag:~roscslabgel electrophoresis of EcoRI clcavcd total cellular D N A from different strains of R.,jopirweight standard). S e e Matel-ials and Methods section for othel-experimental clctails.
Discussion One of the main criteria used in the classification of Rhizobil111lstrains has been the ability of these strains to nodulate specific host plants. 'To understand the genetic and biochemical mechanism of host specificity and symbiosis, isolation and chn~.acte~'ization of mutants altered in these pathways is essential. Pleiotropic contl-ol mutants can alter the cellulal metabolism in such a way that a numbel- of physiological and biochemical properties of the parent and the mutant strains can be markedly different. The presence or introduction of auxotrophic requirements to identify strains may not provide a conclusive characteristic since several auxotrophic mutants are known to have pleiotropic defects in establishing symbiotic associations with the host plant (Schwinghnmer 1977). The percentage G C composition of the total DNA is another property which can be used to distinguish
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several strains. if a clear difference exists (Gibbins and Gregory 1972). The most sensitive means of C is the determination of the analysis of % G buoyant density of DNA in gradients of CsCI. Unfortunately, the ability of this method to detect small buoyant density differences depends on conditions of analysis, such as base composition heterogeneity, molecula~ weight of the isolated DNA, and conditions ofcentrifugation (Atchison et rrl. 1976). In this communication, a method of classification is developed which is based on the linear sequence of the total DNA from the organism. Analysis with restriction endonucleases can determine strain differences not detectable by buoyant density analysis. Clear and distinct cleavage patterns can be used as a tool to identify viral or organelle DNA which are much smaller than bacterial genome (Nathans and Smith 1975; Roberts 1976). Restriction endonuclease cleavage patterns
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S . 1965. Ih~li~tion of h i- ~ h of DNA from E~yc.c./lcl-ic~/lirr (.o/; 21nd B(lc.i//lrs .yl[/)fi/j.y BEHNS.K. I.. ant1 C. A. T H O ~ I AJK.
molecula~.weight DNA from Hc~tto~)hilo.t it~fit~c,ttioc.. .I. Mol. have been reported to be complex (Helling ct (11. Biol. 11: 476-490. 1974: Harris-Warrick ct (11.1975). As presented in BOI.IV.\K,F.. R. L . RODKI(;UI..%. M. C . BI:~I.,ZICI~. and H. W. Fig. 1 , although Rhizohinl~l DNA restriction enB O Y ~ R1977. . Const~.uction and chnr.nctcriz:~tit,n of ncw cloning vehicles. 1. Anipicillin-rcsistk~n~del-ivatives of the donuclease cleavage patterns are found to be complasmid pMB 9. Gene. 2: 75-93. plex. thcy can still be used to distinguish one strain R R C I NA. S . M., and K. F. GKI:C;ORY. 1972. Relatedness anlong of Rlli~ohillt~~ from itnother. A case i n point is that G IRl~i:oI~i~rtr~ and ilgr.oI~trc.rc~rit~~t~ species drtcrmincd by three the subtle differences between R. jrlponic.111~1 methods of nucleic acid hyb~.itlization.J . Bacteriol. 111: strains 110 and 117 w e detectable by restr-iction 139-141. n ~M.. c ~ .Y . EI.K:\N,\, S . D. E I I R L . I Cand H. J . analysis. although no buoyant density difference H I \ ~ ( ~ ~ s - W , \ nK. Lr-r~ri~ ~ L*. \ L LD.. . . anti G. H . [
