Rhizobium japonicum and Bradyrhizobium japonicumt

Transcription

1 JOURNL OF CTERIOLOGY, JUlY 1985, p /85/ $02.00/0 Copyright (C 1985, merican Society for Microbiology Vol. 163, No. 1 Conservation of Symbiotic Nitrogen Fixation Gene Sequences in Rhizobium japonicum and radyrhizobium japonicumt ROERT V. MSTERSON, R. K. PRKSH, ND LN G. THERLY* Department of Genetics, Iowa State University, mes, Iowa Received 24 January 1985/ccepted 28 March 1985 Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DN probes from Rhizobium meliloti against intact plasmid DN of Rhizobium japonicum and radyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DN in most R. japonicum strains. n exception is found with R. japonicum strain USD194 and all. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USD205, both nif and nod sequences are on the same plasmid. In strain USD205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DN to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DN hybridization patterns were identical among all R. japonicum strains and with most of the. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of. japonicum. We have also found that the plasmid prjausd193, which possesses nif and nod sequences, does not possess sequence homology with any plasmid of USD194, but is homologous to parts of the chromosome of USD194. Strain USD194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined. Rhizobium japonicum is a nitrogen-fixing member of the family Rhizobiaceae that forms a symbiotic relationship with soybeans. Rhizobium species include fast growers and slow growers. The slow-growing R. japonicum has now been included in the new genus radyrhizobium (7), but recently fast-growing R. japonicum strains from the People's Republic of China have been examined (9). These strains physiologically resemble other fast-growing R. japonicum strains and can nodulate soybean plants; however, the nodulation is generally ineffective. Therefore, the physiological differences between radyrhizobium japonicum and R. japonicum strains are significant (18) and reflect a divergent genetic background. We have shown the presence of nitrogen fixation (nif) genes on the plasmids of fast-growing R. japonicum strains (12), and it was of interest to investigate the conservation of R. japonicum and. japonicum genes and surrounding sequences that may be involved in symbiotic nitrogen fixation. In this study, we report the conservation of DN sequences of symbiotic importance within and between. japonicum and R. japonicum strains. The structural organization of symbiotic genes appears highly conserved in R. japonicum and. japonicum strains isolated from diverse geographical and ecological backgrounds. The nif structural gene sequence was found to be very highly conserved in all. japonicum and R. japonicum strains examined. Nodulation genes are also conserved in R. japonicum; however, reduced stringency conditions are required to detect homologous sequences in. japonicum strains. lso, we establish herein that nod and nif genes are present on the plasmids of most R. japonicum strains. * Corresponding author. t Journal paper no. J of the Iowa griculture and Home Economics Experiment Station, mes, project no MTERILS ND METHODS acterial strains and growth media. The properties of the. japonicum and R. japonicum strains used are summarized in Table 1. For growth of cells for DN isolation, late-logphase bacteria grown in TY medium (per liter: 5 g of actotryptone [Difco Laboratories], 3 g of yeast extract, 0.93 g of CaC12 1H20) were diluted 100-fold into P medium (per liter: 4 g of acto-peptone [Difco], 0.24 g of anhydrous MgSO4) and grown to 2 x 108 cells per ml at 28 C with vigorous shaking. Escherichia coli strains were grown in L medium (2) at 37 C with appropriate antibiotic selection. Isolation of total bacterial DN. Late-log-phase cells (5 ml) were centrifuged at 7,000 rpm in a Sorvall SS-34 rotor for 10 min at 5 C. The pellet was suspended in 16 ml of 50 mm Tris-hydrochloride (ph 8.0)-20 mm EDT to which 2 ml each of a 10-mg/ml concentration of pronase E (final concentration, 1 mg/ml; Sigma Chemical Co.) and 10% sodium dodecyl sulfate (final concentration, 1%) were added. The mixture was incubated at 37 C for 2 h, and the viscous solution was sheared twice through a 21-gauge needle. The solution was treated with phenol, the phenol was removed with chloroform, and the DN was precipitated by adding 3 M sodium acetate (final concentration, 0.3 M) and 2.5 volumes of 95% ethanol. DN was pelleted by centrifugation at 10,000 rpm for 10 min at 5 C, washed twice with 70% ethanol, and suspended in 0.5 ml of 10 mm Tris (ph 8.0)-1.0 mm EDT. Isolation of plasmid DN. The relatively gentle plasmid isolation method of Hirsch et al. (5) was used in the isolation of large plasmids from both slow-growing and fast-growing R. japonicum strains. Increased yields in plasmid DN from slow-growing strains resulted from washing cells with cold 3% NaCl before lysis. ll plasmid DN was purified in CsCl-ethidium bromide gradients to eliminate chromosomal contamination in subsequent hybridization assays. fter

2 22 MSTERSON, PRKSH, ND THERLY dialysis in 10 mm Tris (ph 8.0)-i mm EDT, samples were routinely concentrated by dialysis versus 10 mm Tris (ph 8.0)-50% polyethylene glycol at 4 C. Restriction enzyme analysis. R. japonicum plasmids and total DN were routinely digested with 10 U of EcoRI restriction enzyme (New England iolabs) per jig of DN for 2 h at 37 C. Samples were loaded onto a 0.7% horizontal agarose gel (0.04 M Tris M EDT, ph 8.0), and electrophoresis was performed at 3 V/cm for 18 h at room temperature. Lambda bacteriophage DN digested with EcoRI or HindIII was included in all experiments for molecular weight determinants and for negative controls in subsequent Southern hybridization (19). Several digestions were done with R. japonicum DN, and band patterns were compared to determine that complete digestion occurred. Hybridization conditions. The Southern (19) procedure, as modified by Thomashow et al. (20), was used in the preparation and blotting of suitable gels. Gene Screen (New England Nuclear Corp.) was used as a transfer membrane such that repeated hybridizations could be performed with a single blot. High-stringency hybridizations were performed at 42 C for 48 h in 50% formamide-1% sodium dodecyl sulfate-1 M NaCI-10% dextran sulfate. Low-stringency hybridization conditions were done at 37 C for 48 h in 10x Denhardt solution (19)-3x SSC (lx SSC is 0.15 M NaCl plus M sodium citrate)-0.5% sodium dodecyl sulfate-35% deionized TLE 1. Properties of strains and plasmids Hybrid- Strain Source, yr Plasmid size ization to: (megadaltons) nod nif. japonicum 6176 Mississippi, ± a102 Iowa, ± b31 Wisconsin, ± b71a rizona, ± b74 California, ± b94 North Carolina, ± ± bllO Florida, 1959 No plasmids 311b123 Iowa, 1960 NDa b143 India, ± R. japonicum USD191 People's Republic of China,b USD USD USD USD USD a ND, Not determined. b The R. japonicum strains were isolated from the East Central Provinces of Shansi, Honan, Shandong, and Shanghai. J. CTERIOL. formamide. 32P-labeled dctp and nick translation chemicals were obtained from New England Nuclear Corp. and used according to the manufacturer's specifications; 0.5 to 1.0 plg of DN probe was labeled to a specific activity between 5 x 106 and 1 x 107 cpm. Hybridized filters were exposed to Kodak X-Omat R film for 1 to 5 days with or without a Dupont Cronex Lighting-Plus intensifying screen. For reusing DN-blotted hybridization filters, each filter was washed with 60% formamide-2 x SSC at 70 C for 2 h and then washed twice with 2 x SSC at room temperature. Filters were exposed to film overnight with an intensifying screen to confirm the removal of the 32p probe. RESULTS In an earlier report from our laboratory we demonstrated that nif genes are located on large plasmids of R. japonicum (12). Table 1 summarizes the results of hybridization with nif and nod genes against intact plasmid DN from R. japonicum and. japonicum strains. The nif probe used in our study is prmr2, which contains nif structural genes D and H of Rhizobium meliloti (17). The nod probe is a 3.5-kilobase (kb) EcoRI fragment known to carry essential nod genes (S. Long, Stanford University, personal communication) and was isolated from a cosmid clone prmsl26 of R. meliloti (11). It is evident from the data in Table 1 that one plasmid from all R. japonicum strains, except strain USD194 and USD205, has homology to nif and nod sequences of R. meliloti. In R. japonicum strain USD205 the nif genes are on a 112-megadalton plasmid and the nod genes are on a 195-megadalton plasmid, and in strain USD194 nif and nod sequences are on the chromosome. Similar to the results shown for R. japonicum strain USD194 (12), the plasmid DN from. japonicum strains does not have any sequence homology with nif and nod probe DN of R. meliloti. nif sequence conservation in R. japonicum and. japoinicum. To investigate the sequence conservation and organization of symbiotic nitrogen fixation genes, we purified total DN from. japonicum strains and from R. japonicum strain USD194; however, we used only plasmid DN from the other R. japonicum strains. The DNs were digested with EcoRI, separated by electrophoresis on 0.7% agarose gel, and transferred to nitrocellulose paper (19). The Southern blots containing the EcoRI-digested DNs were hybridized with 32P-labeled prmr2 DN or the 3.5-kb nod fragment DN of R. meliloti. The hybridization results with nifdh structural gene DN of R. meliloti are presented in Fig. 1. The nifstructural genes in every R. japonicum strain are located on the 4.2- and 4.9-kb EcoRI fragments of plasmid DN (Fig. 1). Furthermore, the 32P-labeled nif DN hybridized to the same sized fragments from total DN of R. japonicum strain USD194. These data indicate that nif structural genes are highly conserved and organized in all R. japonicum strains, even though their location may be on plasmid DN or on chromosomal DN as in strain USD194. When Southern blots of EcoRI-digested total DN from. japonicum strain were hybridized with prmr2 DN, six of the eight strains examined had identical hybridization patterns with relatively strong hybridization to 11- and 4.2-kb fragments (Fig. 1, lanes 2 through 7). weak signal was also observed with 5.4- and 3.3-kb EcoRI fragments. The 1.8-kb band was due to homology with the vector. The weak signals observed with the total DN from strain 311b74 (Fig. 1, lane 3) is due to a lesser amount of DN in the lane. The two other strains examined, 311bllO and 102,

3 VOL. 163, 1985 SYMIOTIC NITROGEN FIXTION GENE SEQUENCES S 7 5 ' I6 NJL FIG. 1. () Hybridization of 32P-labeled prmr2 (containing nifdh from R. meliloti) to EcoRI-digested total DN from slow-growing strains. Lanes: 1, 311b110; 2, 311b94; 3, 311b74; 4, 311b143; 5, 311b71a; 6, 6176; 7, 311b31; 8, 102. () Hybridization of 32P-labeled prmr2 to EcoRI-digested total DN from fast-growing strains. Lanes: 1, USD201; 2, USD205; 3, USD206; 4, USD191; 5, USD193; 6, USD194. Shown are the sizes in kb of the EcoRI fragments calculated by using lambda DN as a marker. had different nif hybridization patterns. Strain 311b110 showed strong hybridization with two EcoRI fragments of 2.8 and 6.1 kb (Fig. 1, lane 1). Strain 102 (Fig. 1, lane 8) has two large bands of 12 and 13.7 kb homologous to the nif DN probe. These results show that the organization of nif genes is highly conserved in. japonicum strains. nod sequence conservation in R. japonicum and. japonicum. When the 3.5-kb EcoRI nod fragment of R. 11i2 :L 'MM _ :.*. -.9 meliloti was hybridized to EcoRI-digested plasmid DN from R. japonicum strains, both common and unique bands hybridized (Fig. 2). The 3.5-kb nod probe of R. meliloti hybridized to the same three EcoRI fragments from strains USD201, USD191, and USD193 (Fig. 2, lanes 2, 5, and 6). The common sized fragments were 11.2, 5.3, and 2.8 kb. In strain USD206, only the larger hybridization band (11.2 kb) was common to the other R. japonicum strains. I -b 5 c FIG. 2. () Hybridization of 32P-labeled nod probe to EcoRI-digested plasmid DN from fast-growing strains. Lanes: 1, total DN of USD194; 2, prjausd201a,b; 3, prjausd205a,b,c; 4, prjausd206a,b,c; 5, prjausd19la,b; 6, prjausd193. () Hybridization of the same nod probe to EcoRI-digested total DN from slow-growing strains (under reduced stringency conditions). Lanes: 1, 311b31; 2, 6176; 3, 311b71a; 4, 311b143; 5, 311b74; 6, 3I1b94; 7, 3I1b11O; 8, 311b123; 9, 102. Shown are the sizes in kb of the EcoRI fragments calculated by using lambda DN as a marker.

4 24 MSTERSON, PRKSH, ND THERLY The smaller band in strain USD206 (4.8 kb), which hybridized strongly with nod probe, may or may not be common to the fragment found in total DN of strain USD194 (Fig. 2, lanes 1 and 4). Even though the size is identical, the weaker signal in Fig. 2, lane 1 (USD194), suggests low homology. The sizes of the other two bands (11.2 and 5.3 kb) from the total DN of strain USD194 are similar to those found for strains USD201, USD191, and USD193. Similarly, in strain USD205 (Fig. 2, lane 3), the 5.3- and 2.8-kb fragments are common bands. The largest fragment appeared after longer exposure (data not shown), but was 9.4 kb instead of 11.2 kb. These results indicate that some diversity exists in the EcoRI restriction enzyme sites for nodulation genes in R. japonicum strains. In contrast, the 3.5-kb nod fragment hybridized very weakly to total DN from. japonicum strains and was only seen after long exposures with intensifying screens (Fig. 2). Reduced hybridization stringency conditions (described above) were necessary to determine related nodulation gene sequences in. japonicum strains. Total DN isolated from. japonicum strains was digested with EcoRI and probed with the 3.5-kb nodulation sequence. Similar to that observed with R. japonicum strains, the sizes of the hybridizing EcoRI bands are generally the same within. japonicum strains, but with some diversity.. japonicum strain 102 has a distinctly different pattern (Fig. 2, lane 9) with four EcoRI bands (5.5, 5.2, 3.0 and 1.8 kb). Only strain 311b143 has two bands that hybridize in common with strain 102 (Fig. 2, lane 4). Overall the other strains fall into four classes as represented by lanes 1 through 4, lanes 5, 6 and 8, lane 7, and lane 9. Conservation of prjausd193 sequences within R. japonicum and.japonicum. Since the nifand nod sequences were found to be organized similarly on plasmids of the R. japonicum strains, it was of interest to determine whether the sequence of total plasmid DN was organized similarly within different strains. For this, we first compared the EcoRI restriction patterns of plasmid DNs from R. japonicum strains and then examined sequence conservation between the plasmid DNs by hybridization. Figure 3 shows the gel electrophoresis pattern of EcoRIdigested plasmid DN isolated from six different R. japonicum strains obtained from different locations in the People's Republic of China. Each strain contains one to three large plasmids (Table 1), and each strain possesses a unique restriction digest pattern (Fig. 3). However, they all share some common bands. Variable amounts of the largest plasmid DN were obtained from each strain because of plasmid fragility and loss during CsCl gradient centrifugation. The only R. japonicum strain from which we obtained pure plasmid, which is homologous to nif and nod sequences of R. meliloti, is strain USD193 (Fig. 3, lane 7). The involvement of plasmid prjausd193 in symbiotic nitrogen fixation has been confirmed by genetic experiments carried out in our laboratory (N. DuTeau and. G. therly, unpublished data). Therefore, the 240-megadalton plasmid prjausd193 was chosen as a hybridization probe to determine the sequence conservation between plasmid DNs of R. japonicum. Figure 3 is a Southern blot of plasmid DN fragments hybridized to 32P-labeled prjausd193. Even though the restriction pattern between the plasmids of R. japonicum strains was variable (Fig. 3), examination of the hybridization pattern in Fig. 3 shows distinct conservation of many similar sized fragments. This relatedness is particularly evident between the labeled plasmid prjausd193 and the plasmid(s) in strain USD201, Kbp 4.?.i USD205, USD206, and USD191 (Fig. 3, lanes 3 through 6). The digest of the two large plasmids from USD194, which do not harbor nif structural genes (Table 1), showed very limited homology to the labeled plasmid (Fig. 3, lane 2). Consequently these results indicate that, although plasmid DN from R. japonicum strains share similar nifand nod hybridization patterns (Fig. 1 and 2), they do not share total plasmid DN sequence conservation. In R. japonicum strain USD194, the nif and nod sequences are found on the chromosome; as expected, no significant hybridization was observed between the plasmid DN from USD193 and plasmid prjausd194 (Fig. 3, lane 2). We also looked for sequence conservation of prjausd193 in total DN of R. japonicum strain USD194. Rather surprisingly, prjausd193 plasmid DN sequences are present in strain USD194 in either the megaplasmid or the chromosome. comparison of the hybridization of prjausd193 with strain USD194 total DN (Fig. 4, lane 6) as opposed to just plasmid DN (Fig. 3, lane 2) shows a significant increase in hybridization to total DN with this strain. These data prove that many of the sequences of prjausd193 are present on the chromosome of USD194. The data presented in Fig. 4 also show that these same sequences are present in all of the strains examined. large degree of conservation of prjausd193 se- _ J. CTERIOL. w IIMIs FIG. 3. () Gel electrophoresis of 1,ug of EcoRI-digested plasmids from fast-growing strains (lanes 2 through 7). Lanes: 1, prja6176; 2, prjausd194a,b; 3, prjausd201a,b; 4, prjausd205a,b,c; 5, prjausd206a,b,c; 6, prjausd191a,b; 7, prjausd193. Molecular weight markers are bacteriophage lambda DN digested with HindIlI. () Hybridization of 32P-labeled prjausd193 to a Southern blot of the gel in. The lane contents are as in panel. I :I

5 VOL. 163, 1985 SYMIOTIC NITROGEN FIXTION GENE SEQUENCES FIG. 4. () Hybridization of 32P-labeled plasmid prjausd193 to Southern blot of EcoRI digested total DN from slow-growing strains. Lanes: 1, USD311b31; 2, 6176; 3, 311b71a; 4, 311b143; 5, 311b74; 6, 311b94; 7, 311b110; 8, 311b123; 9, 102. () Similar hybridization with fast-growing strains. Lanes: 1, USD201; 2, USD205; 3, USD206; 4, USD191; 5, USD193; 6, USD194. quences was also observed in total DN of. japonicum strains 311b31, 6176, 3I1b71, and 311b143 (Fig. 4, lanes 1 through 4). Strains 311b94, 311b123, and 102 did not show as much relatedness, but did contain bands similar in size to the bands of the other strains examined (Fig. 4, lanes 5, 6, 8, and 9). Total DN from strain 311bllO is homologous to the plasmid prjausd193 DN probe, but not to the extent suggested by the strong hybridization signal in Fig. 4, lane 7. The amount of total DN in this lane is approximately twice the amount observed in the other lanes. DISCUSSION The goal of this study was to determine the conservation and organization of R. japonicum genes involved in symbiotic nitrogen fixation. The presence of genes for symbiotic nitrogen fixation on a large plasmid DN is a common feature in various fast-growing Rhizobium species (1, 12, 14). The results presented here clearly show that in fast-growing R. japonicum, the nif and nod sequences are on plasmid DN, except in strain USD194. In R. japonicum strain USD194 and in slow-growing. japonicum strains, these sequences are either on a very large undetected plasmid or on the chromosome. In all other R. japonicum strains examined, except strain USD205, the nif and nod genes have been found to be located on one plasmid. In R. japonicum strain USD205 the nif and nod genes are on separate plasmids. esides the physical evidence presented herein, we have obtained genetic proof for the presence of symbiotic nitrogen fixation genes on plasmid prjausd193 of R. japonicum strain USD193 (N. DuTeau and. G. therly, unpublished data). It is evident from the hybridization assays that in both R. japonicum and. japonicum nif sequences are highly conserved regardless of the plasmid or chromosomal location. The sequence organization of the structural nif sequences appears identical in most R. japonicum strains. In all strains examined, two EcoRI fragments of 4.2 and 4.9 kb hybridized to R. meliloti nif DN, including that of strain USD194, which contains nif sequences on the chromosome. Hybridization between individual nif fragments has revealed that two copies of nif DH structural genes may be present in R. japonicum (13), which is in contrast to that observed in. japonicum, where single copies of nif genes per genome are present (8). Nodulation sequences of R. meliloti are also conserved in both R. japonicum and. japonicum strains, but less so in. japonicum strains, since significant hybridization with the total DN is observed only under reduced stringency conditions with. japonicum. Whether the hybridizing bands are, in fact, authentic nod gene(s) is under investigation. That such differences exist between nodulation genes of the R. japonicum and. japonicum strains suggests a possible evolutionary divergence in the nodulation properties of these bacterial symbionts. However, the sequences homologous to nod genes of R. meliloti seem to be organized more or less in a similar manner within R. japonicum and. japonicum strains. Like nif sequences, nod sequences are reiterated in R. japonicum strains (13). Using plasmid prjausd193 as a hybridization probe (which contains the symbiotic nitrogen fixation genes) to other sym plasmids of R. japonicum, we have shown that sequences within total plasmid DN are not highly conserved. In contrast, nif and nod sequences are organized in a similar fashion on the plasmid DN from different R. japonicum strains. However, almost all of the sequences of prjausd193 are conserved both on the plasmid DN and on the chromosomal DN of other R. japonicum strains, suggesting that these sequences may be essential. Similar sequence conservation has been observed between plasmid DN, containing symbiotic nitrogen fixation genes, from several fast-growing Rhizobium species (6, 14). These conserved regions have been found around the genes that are involved in symbiotic nitrogen fixation and expressed in the nodules (14-16). Similarily, highly conserved regions between tumor induction plasmid DNs of grobacterium tumefaciens are observed and are involved in tumor induction (3, 4, 10, 20), which suggests that homologous regions between DNs of R. japonicum may be involved in the control of symbiotic nitrogen fixation. erry and therly (2) have recently described genome rearrangments prompted by R-group plasmid introduction. Since in R. japonicum strain USD194 and. japonicum strains the homologous sequences of prjausd193 and nif and nod sequences of R. meliloti are located on the chromosomal DN, it is tempting to say the plasmid DN in these strains undergoes excision and integration with its host chromosome. However, this speculation remains to be proved. CKNOWLEDGMENTS We thank Steve Gradwohl for his excellent technical assistance and Linda and Mike Thomashow for helpful suggestions in the Southern blotting procedure. This project was supported in part by grant from the U.S. Department of griculture and by funds from Land O' Lakes Corp. LITERTURE CITED 1. anfalvi, Z., V. Sakanyan, C. Koncz,. Kiss, I. Dusha, and. Kondorosi Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of Rhizobium meliloti.

6 26 MSTERSON, PRKSH, ND THERLY Mol. Gen. Genet. 184: , erry, J. O., and. G. therly Induced plasmid-genome rearrangements in Rhizobium japonicum. J. acteriol. 157: Costaiitino, P., M. L. Mauro, G. Micheli' G. Risuleo, P. J. J. Hooykaas, and R. ScWllperoort, Fingerprinting and sequence homology of plasmids from different virulent strains of grobacterium rhizogenes. Plasmid 5: Drummond, M. H., and M.-D. Chilton Tumor-inducing (Ti) plasmids of grobacterium share extensive regions of DN homology. J. acteriol. 130: Hirsch, P. R., M. Van Montagu,. W.. Johnston, N. J. revi4, and J. Schell Physical identification of bacteriocinogenic, nodulation and other plasmids in strains of Rhizobium leguminosarium. J. Gen. Microbiol. 120: Jarvis,. W. L., K. F. Scott, J. E. Hughes, M. Djordjevic,. G. Rolfe, and J. Shine Conservation of genetic information between different Rhizobium species. Can. J. Microbiol. 29: Jordan, D. C Tradsfer ofrhizobiumjaponicum uchanan 1980 to radyrhizobium gen nov., a genus of slow-growing, root nodule bacteria from leguminone plants. Int. J. Syst. acteriol. 32: Kaluza, K.,-M. Fuhrmann, M. Hahn,. Regensburger, and H. Hennecke In Rhizobiumjaponicum the nitrogenase genes nifh and nifdk are separated, J. acteriol. 155: Keyser, H. H.,.. ohlool, T. S. Hu, and D. F. Weber Fast-growing Rhizobia isolated from root nodules of soybean. Science 215: Kiauf, V. C., C. G. Panagopoulos, and E. W. Nester Comparison of Ti plasmids from three different biotypes of grobacterium tumefaciens. J. acteriol. 153: Long, S. L., W. J. uikema, and F. M. usubel Cloning J. CTERIOL. of Rhizobium meliloti nodulation genes by direct complementation of Nod mutants. Nature (London) 298: Masterson, R. V., P. R. Russell, and. G. therly Nitrogen fixation (nifl) genes in large plasmids of Rhizobium japonicum. J. acteriol. 152: Prakash, R. K., and. G. therly Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum. J. acteriol. 160: Prukash, R. K., R.. Schilperoort, and M. P. Nuti Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation nif genes. J. acteriol. 145: Prakash, R. K.,.. N. van russel,. Quinne,. M. Menner, and R.. Schilperoort The map position of Sym-plasmid regions expressed in the bacterial and endosymbiotic form of Rhizobium leginosarum. Plasmid 7:28i Prakash, R. K., R. van Veen, and R.. Schilperoort Restriction endonuclease mapping of a Rhizobium leguminosarium plasmid that carries nitrogen fixation (nij) genes. Plasmnid 7: Ruvkun, G.., and F. M. usubel Interspecies homology of nitrogenase genes. Proc. Natl. cad. Sci. U.S.. 77: Sadowsky, M. J., H. H. Keyser, and.. ohlool iochemical characterization of fast- and slow-growing Rhizobia that nodulate soybeans. Int. J. Syst. acteriol. 33! Southern, E. M Detection of specific sequences among DN fragments separated by gel electrophoresis. J. Mol. iol. 98: Thomashow, M. F., V. C. Knauf, and E. W. Nester Relationship between the limited and broad host range octopinetype Ti plasmids of grobacterium tumefaciens. J. acteriol. 146: Downloaded from on November 12, 2018 by guest