Indian Journal of Experimental Biology Vol. 4\. August 2003, pp. 9 12-9 14 Curing of symbiotic plasmid of Mesorhizobium huakuii subsp. rengei isolated from Astragalus sinicus l 2 1 D Balachandar *, S Kannaiya n I, H On0 & Y Murooka IAzolla laboratory. Tamil ad u Agricultural Un iversity, Coimbatore 641003, India 2Departmellt of Biotechnology, Graduate School of Engineeri ng, Osaka Un iversity, Suita-s hi . Osaka 565-0871 . Japan Rece ived 4 July 2002: revised 30 April 2003 Astragaills sinicus (Chinese Milk vetch), a green manure leg uminous plant, harbors M e.l"o rhi ~ obi LIIIl huakllii subsp. rengei strain B3 in the root nodule. The visual izat ion of sy mbiot ic plas mid of strain B3 showed the prese nce or one SYIII plasmid of about 425 kbp. Cu rin g or .1'.)'111 plasmid by temperature and ac rydine orange was studi ed. Growing rhi zobial ce lls at hi gh temperature (37°C) or treating th e ce lls with ac rydine orange at 50 mg/I eliminated SYIII plasmid of M . IlIIakllii strain B3. whi ch \Vas confirmed by SYIII pl asm id visualizat ion and plant infect ion tes t of cu red strains. Keywords: Astragailis SilliclIS , M esorhi:obilllll hllakllii. Symbiot ic plasmid ASTragalus sinicus (Chinese milk ve tch or renge-sho iso lated from nodules of Astragalus siniclls cv. Japan in Japanese) is a common winter growing green grown in rice fields of Higashi-Hiroshi ma. Japan. B3 manure leg ume. M any strains of thi s spec ies were with pBBR I MCS2 (a broad host range vec tor for widely grown as green manurc before and after th e Rhizobium provided by Kovach7) co nferring rice crop to fertili ze th e so il in so uthern part of China, kanamycin (20 )..l g/ml) re sistance. Since, there is no Korea and Japa n. This plant harbors Mesorhi;obil.1I1/ 5\1111 - marker for se lection of s\l, m. + wild st rain or . l hllakllii or M. huakuii subsp. rengei" which resu lts in mutants, the vec tor pBBR I MCS2 was used. fo rm ation of nitrogen fi xing root nod ul es. Strains of Media and culturing condition - Rhizobiulil strai ns Rhi;obiul1l sp. harbor indigenous large plas mids and were maintained and grown in tryptone yeas t extract genes req uired for nod ul ation (/l od) and nitrogen mediums at 30°C. For visualizat ion of '<;)" 111 pl as mid of fixation (nit and fix) are located on th ese large B3, HP med ium9 was used. The hos t plant A. sinicll s plasm ids in several spec ies of Rhi ~obil /llI J.4 The was grown on nitrogen free mod ified FR medium ~ structural genes of nitrogenase enzy me (n!fKDH ) for Rhizo biul1I pl ant in fection tes t. whi ch is res ponsible for nitrogen fixation and Visuali zation of syl1l plasl1lid - T he visualiza ti on nodulation genes (nodABC) appear to be fun cti onally th rough agarose gel electrophores is was done by in­ 5 9 interchangeable among all th e spec ies of Rhizobium . gel lys is method . Rhi-::.o biul1I was grown overni ght at The M. huakuii subsp. rengei also harbors the 30°C with shaking in test tu bes comaining HP ind igenous plas mid of ap proximately 425 kbp which medium. They were diluted with TY medium and has all th ese genes, and ce ll lack ing of thi s pl as mid incubated for 2 hr. Then, 250 ~I of culture mixed with lose th e ab ility of nodulationo. Thus such large 500 ~I of 0.3% sa rkosyl so lution in 1.5 ml eppendoff plasmids, necessa ry for sy mbios is, are designated as tube. Then cells were pelleted by centrifugation at low Sylll plasmids. The prese nt study was ca rried out to temperature for 5 min, resuspended in 25 )..ll of lyti c co nfirm the presence of single sym plasmid, its so lution ( 10% sucrose; 100 ~g/ ml lysozyme and 10 stabi lity aga inst temperature and acrydine orange, and ~ g/ml of RNase in 0.5X TB buffer) and loaded into a to optimize the temperature and concentration of we ll of 0.5 % agarose gel co ntaining 1% SDS in 0.5X acryd ine orange for pl as mid curing which \V iii be TB buffer. Electrophores is was carried out at 5-10 V useful for genetic modification of Rhizobium. for 30 min and 100 V for 3 hr. Bands were visualized Rhi ~ob i a l sTraills-MesorhizobiulII huakuii subsp. on UV illuminator after ethidium bromide staining. rengei strain B3 (referred a, B3 hereafter) was Plasmid curing by lemperatllre-The plamid *Corres ponucnl au th or: curing experiment was carried out using the strain B3 E- mail : dbalu2000@y ahoo.com containing pBBR I MCS2 co nferring Kmr. The OTES 9 13 actively growing culture of approximately 104 cells confirmed by this technique that only one sylll per ml were spread on TY plates and incubated at 35° plasmid is responsible for nodulation and nitrogen li and 37°C (Ref. 10). The plates were transferred to fixation by B3 in Chinese milk vetch . Moreover, in­ incubators (30°C) after every 24 hr up to 5 days. From gel lysis meth od is a suitable meth od to visualize such th ese plates, 100 colonies were picked up and large size plasmids without purification. This method streaked in TY plate and TY + Km (20 Ilg/ml) plates is rapid, simple and DNA shreddin g due to large size and incubated at 30°C for 2 days. Number of cells in can be avoided. TY alone and TY + Km plates were counted and The plasmid curing experiment result indicated th at number of cells lost th e plasmid were counted by incubation of Mesorhizobium cells at 35° or 37°C led detecting the number of cell s of TY + Km with TY to loss of plasmid pBBR I MCS very rapidly. After 3 plate. days of in cubation, onl y 35-40% cell s retained th e Plasmid c/lrin g by acrydine orange - TY broth in plasmid (Fig. 2) and after 5 days about 80% of cel ls test tube with acrydine orange at 0, 5, 10, 40 and 50 lost the ir plasmid. Similarly, treatment of acryd in e mgll were prepared and rhizobial cells were orange at hi gh concentrations (40 and 50 mg/l ) led to 11 inocul ated to the initial 0.0.01'0.05 • The tubes were significant loss of plasmid. The acrydine orange at 40 wrapped with black paper to avoid li ght and in cubated and 50 mg/l recorded 84 and 100% loss of plasmid at 30°C for 5 days. Cell suspensi on ( 100 Ill ) was respective ly (Table I). The colo ni es, whi ch showed spread on TY pl ates and in cubated at 30°C for 2 days. kanamycin susceptibility (loss of pBBR I MCS2). Coloni es ( 100) from the pl ates were selected and re­ were again checked for the presence of sym plasmid streaked in TY plates and TY + Km (20 Ilg/ml) plates by in-ge l lysis method as described earli er. The result incubated at 30°C for 2 days. The number of cells in confirmed the absence of sym plasmid in th e strain TY alone and TY + Km plates were counted and the which lost pBBR I MCS2, (Data not shown) means number of cell s lost the plasmid were counted by 100 detecting the number of cell s of TY + Km with TY -a E 90 plate. ~ 0. 80 Agarose gel e lectrophoresis showed the presence of -5" ~ V> 70 one symbiotic pl asmid of about 425 kbp size (Fig. I). ..9 ~ 60 Earlier studies on B3 syl1l pl asmid results were ;;; u OJ 50 15 1 2 3 4 5 0 40 ....~ 30 35°C 0 37"( E 20 8 300 kbp t; 10 50 kbp 0- 0 2 3 4 5 Chromosomal ON Days Fig. 2 - Loss of pBBRIMCS? plasmid in M. huakllii strain B3 due to incubation at elevated temperatures (35° and 37°C) Table I - Loss of pBBR I MCS2 plasmid in strain B3 due to aeryd ine orange Concentrati on No. of colonies No. of coloni es Rhi zobial cells of acrydine used for Km' showin g loss of showing loss of orange (mgll ) test' pBBR I MCS plasmid (%) a 100 5 100 35 35 10 100 42 42 40 76" 64 84 Fig. I - Visualization of SYIII plasmid (425 kbp) of Mesorhizo· 50 II .. II 100 billlll illlukuii subsp. rengei strain B3. ILane I - Uncut A DNA marker; Lane 2 - Strain B3 diluted I: I with TY broth; Lane 3 - *The colonies were streaked in TY and TY +Km (20llg/ml ) plate, Strain B3 diluted 1:2 with TY broth; Lane 4 - Strain B3 diluted and incubated at 30°C for 2 days and observed for growth . The 1:3 with TY broth; Lane 5 - M. loti with 2 sym plasmids of 300 absence of gro wth revealed the loss of pl asmid. and 150 kbp]. **The total number of cell s obtained after 5 days of in cubation. 9 14 INDIAN J EXP BIOL, AUGUST 2003 that all the pl as mid s, in va ri able of size cou ld be by plasmid curing which will help to furth er study the eliminated by elevated temperature or by acrydine functions of genes involved in the nodulation and orange treatment.