Canonical Ordered Cosmid Library of the Symbiotic Plasmid Of

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Proc. Nati. Acad. Sci. USA Vol. 88, pp. 1923-1927, March 1991 Genetics Canonical ordered cosmid library of the symbiotic plasmid of Rhizobium species NGR234 ("contig"/fingerprinting/genome mapping/nod and nif genes) XAVIER PERRET*t, WILLIAM J. BROUGHTON*, AND SYDNEY BRENNERt *Laboratoire de Biologie Moleculaire des Plantes Superieures, Universite de Geneve, 1 chemin de l'Imperatrice, 1292 Chambesy, Geneva, Switzerland; and tMolecular Genetics Unit, Medical Research Council, Hills Road, CB2-2QH Cambridge, United Kingdom Contributed by Sydney Brenner, October 15, 1990

ABSTRACT Many of the bacterial genes involved in nod- Table 1. Bacterial strains, plasmids, and vectors used in ulation (nod) and nitrogen frxation (nif) are dispersed over the this study 500-kilobase plasmid pNGR234a of the broad host-range Strains, Rhizobium species NGR234. As a first step toward generating plasmids, and a complete physical and genetic map of the plasmid, a full vectors Relevant characteristics Ref. overlapping collection of cosmids was derived from a total E. coli genomic library. Clones were aligned by combining finger- 1046 RecA- strain 11 printing, hybridization, and pulsed-field gel electrophoresis JM101 RecA' strain 12 data. Symbiotic loci were localized by probing a representative Rhizobium set of cosmids with both homologous and heterologous genes. NGR234R NGR234, -rifampamycin 13 nodABC, nodDI, nodD2, nodSU, noifB, and region II are widely resistant dispersed over pNGR234a, while the two functional copies of ANU265 pSym- NGR234 14 niJKDH are separated by only 28 kilobases. Interestingly, Plasmids sequences homologous to nodE, nodG, nodP, and nodQ have pNGRH6 nodDi locus of NGR234 15 been assigned to another autonomously replicating element in pA16 nodSUof NGR234 16 Rhizobium species NGR234. Similarly one copy of the struc- pJSS38 nodP of Rhizobium meliloti 17 tural dctA gene is located on the symbiotic plasmid (dctAl) pJSS31 nodQ of R. meliloti 17 while the other is on what we assume to be the chromosome. pSA30 nifYKDH of Klebsiella 18 pneumoniae pGMI724 nodE of R. meliloti 19 Soil bacteria of the genus Rhizobium may form symbiotic pGMI727 nodG of R. meliloti 19 associations with leguminous plants, leading to the develop- pRmSL42 nodDABC of R. meliloti 20 ment of specialized root structures called nodules. Once pXNODAB nodAB of R. meliloti This work* within the nodules, rhizobia differentiate into bacteroids pXNODC nodC of R. meliloti This workt capable of reducing atmospheric nitrogen to ammonia. Mu- pRFDH421 nolB of Rhizobium fredii L. W. Meinhardt and tual recognition by the symbiotic partners is often highly S. G. Pueppke specific, especially in symbioses involving fast-growing pG14 Region Ila of R. meliloti E. Cervantes Rhizobium species. In contrast, Rhizobium species NGR234 pTVB60 dctAl of NGR234 T. V. Bhuvaneswari is characterized by an unusually broad host range, which Vectors includes the nonlegume Parasponia andersonii (1, 2). Early Lorist2 5.6-kilobase (kb) cosmid, 21 studies have shown that most symbiotic genes in NGR234 are kanamycin resistant located on a plasmid of310 + 20 megadaltons, pNGR234a (3, Bluescript 3.0-kb phagemid, ampicillin 22 4). To study the molecular basis of host-specificity phenom- resistant ena in MPIK3030 (a streptomycin-resistant derivative of *A 1.2-kb Sst I-BspMI fragment from pRmSL42 comprising the NGR234), a pJB8 cosmid bank of pMPIK3030a was con- complete nodAB genes of R. melilot was cloned into Bluescript. structed (3). Mobilization of individual pJB8 subclones into tA 440-base-pair (bp) BspMI-HindIII fragment from pRmSL42 in- heterologous rhizobia and screening for host-range extension ternal to nodC was cloned into Bluescript. on Vigna unguiculata allowed the identification of three distinct host-specificity loci, HsnI, -II, and -III (5, 6). Ex- MATERIALS AND METHODS tensive mapping of these loci showed that they are dispersed Bacteria and Plasmnids. Escherichia coli was grown on LB over the symbiotic plasmid but failed to link them on a medium (9) or in 2 x YT medium (9); Rhizobium species were plasmid map. To establish the relationship between HsnI, grown in/on TY medium (10) at 28°C. Lorist2 cosmid re- HsnII, and HsnIII and to study their organization in relation combinants were grown in E. coli strain 1046, while Blue- to other nodulation genes, we have applied the techniques script clones were grown in JM101. The bacterial strains as developed for mapping the genome of Caenorhabditis ele- well as the plasmids and vectors used in this work are listed gans (7, 8) to Rhizobium species NGR234. First, we gener- in Table 1. ated an ordered array of cosmid clones, which then allowed DNA Isolation. Genomic and plasmid DNAs were isolated genetic and restriction mapping analysis. This resulted in a from Rhizobium species NGR234R by standard methods (23, combined physical, genetic, and Spe I restriction map of 24). Cosmid DNA was prepared from 2.2 ml of overnight pNGR234a, which is presented below. culture grown in 2x YT medium. DNA was purified by an alkaline lysis procedure (9), followed by extraction with The publication costs of this article were defrayed in part by page charge phenol/chloroform/isoamyl alcohol, 50:49:1 (vol/vol). payment. This article must therefore be hereby marked "advertisement" Cosmid Library Construction. High molecular weight in accordance with 18 U.S.C. §1734 solely to indicate this fact. NGR234R DNA was partially digested with various concen- 1923 Downloaded by guest on September 30, 2021 1924 Genetics: Perret et al. Proc. Natl. Acad. Sci. USA 88 (1991) trations of Sau3AI, and the samples were loaded onto a 0.4% ping clones allowed the 227 cosmids to be grouped into 14 initial low-gelling temperature agarose (SeaKem; FMC) gel. Frag- "contigs" (sets of contiguous clones). Eleven clones that did ments ranging in size from 25 to 50 kb were excised and not match any other remained unassigned. repurified on a second 0.4% SeaKem gel. About 0.2 jug ofthis Surprisingly, the 14 contigs were estimated to cover 900 kb DNA was ligated into the BamHI site of 0.2 pug of dephos- rather than the 470 + 30 kb expected of pNGR234a (3, 4). phorylated Lorist2 vector DNA. In vitro packaging of the Neither the total size of the undetected overlaps between the ligated DNA was performed with mixes derived from E. coli existing contigs nor underestimation of the plasmid size can strains NS428 and NS433 (25). Yields of recombinants were explain this difference. Contamination of the purified sym- -2 x 105 recombinants per ,ug of insert DNA. Individual biotic plasmid probe by chromosomal DNA or the presence clones were identified following adsorption onto E. coli 1046 of dispersed chromosomal sequences homologous to strain and low-density plating on kanamycin (50 gg/ml)- pNGR234a DNA are more likely explanations ofthe discrep- containing plates. ancy. We favor the second possibility, mostly because a Fingerprinting ofCosmid DNA. Clones were characterized by contaminated pNGR234a probe would have resulted in a the fingerprinting procedure ofCoulson et al. (7). Cosmid DNA smaller number of contigs and a larger number of unlinked was first cleaved with HindIII (or alternatively with BamHI or solitary chromosomal clones. EcoRI), and the restriction fragments were simultaneously Since our long-term objective was to map the chromosome of end-labeled by using reverse transcriptase. A second cleavage NGR234, we prepared HindIII fingerprints of 800 additional was obtained by incubating the labeled DNA with Sau3A1, and clones that did not hybridize to pNGR234a DNA and stored the the resulting small fragments were separated on 6% polyacryl- information in a second data base (NGRDB). NGRDB also in- amide gels. The scanning equipment for digitizing the autora- cluded the fingerprints of75 cosmids selected to cover both the diographs of the fingerprint gels, together with the computer 14 contigs and the 11 unassigned clones of the MEGAPL data software (8) for analyzing the data were generously made base. Comparisons of all the fingerprint patterns showed that 8 available by A. Coulson and J. Sulston ofthe Medical Research ofthe 14 original MEGAPL contigs were extended by clones that Council Laboratory of Molecular Biology. did not hybridize to the symbiotic plasmid and could therefore Hybridization Procedures. Endonuclease-digested DNA be assigned to the chromosome map. was transferred to nylon membranes by standard Southern Reliable ordering of adjacent DNA fragments critically de- blotting procedures. Multiple samples of non-digested DNA pends on the amount of information that can be extracted from were analyzed by dot-blot hybridization. 32P-labeled DNA each clone. In the fingerprint approach, regions with few probes were synthesized by primer extension using random restriction sites for the endonuclease used in the primary or specific oligonucleotides. cleavage of the cosmid DNA cannot easily be assigned by the computer routines, unless the region is well represented in the RESULTS clone collection. To compensate forthe awkward distribution of A Canonical Ordered Cosmid Library of pNGR234a. A ge- restriction sites and the nonrandomness of the clone bank, we nomic library of total NGR234R DNA was established in the attempted to increase the amount of information available for Lorist2 cosmid vector, and putative symbiotic plasmid clones interesting clones. This was achieved by first characterizing the were identified in 2000 randomly picked clones by colony cosmids covering the ends of existing contigs as well as the hybridization of purified 32P-labeled pNGR234a probe. To unattached clones of the NGRDB data base with BamHI and ensure high sampling redundancy, we prepared -700C stocks, EcoRI fingerprints. This way, we confirmed two doubtful miniprep DNA, and Hindil fingerprints from each of the 227 assignments (Fig. 1, links 3 and 7) and established four addi- positive clones. The digitized data obtained by scanning the tional linkages (Fig. 1, links 1, 4, 5, and 6). autoradiographs of the fingerprint gels were stored in a com- Even then, complementary mapping methods were neces- puter data base (MEGAPL) and analyzed for possible overlaps. sary to finalize the plasmid map. Dot-blot hybridization exper- Manual comparison of the fingerprints of potentially overlap- iments confirmed two overlaps of low probability in the finger-

!OTIG3 r- CONT-G2 _.>CONTIG 1 , ...... _..._,..../ i 11 i".#,do T'' rL 754 r 1423 .. 95 14~~ ~~ " ~315 ri 901 . Li. 1312 ; 9 ______+ 901 110 390 iL 18 ;, 5;30, . ~ 110 - iii 6 , 1686 1027 43 r -- 64 _ .il 1459 64 1771 L .11. 738L *6 ; ; 1696 ;_-_-l--- _ 564 1533 hJl'i W-6 182 ,. 452 '2L ; . 1357 1LL LL 5 137 IIi5 j9i L.' 177 _ 464 -. 6 L-L-'iL 8693 3 56 605 3 368 1353 IIIII'U 285 , 17C 586 459 5 r 807 1. 5 740 i I*23 1661 i =-!i -,:66.6. _. pWA76 P_-WA46 E pWA58 IN -p------W------WA88------M OS S(: ------pWA54 - SO stj sl.) S19 LIS S916ts sf - r N I IN I v I ' JL - _ 1. - .-- P-1 nodD1 nodC nodSU RS.1 dctAl nodD2 nodAB nifHDK nolB 10 units RegionIl niftHDK FIG. 1. Decomposition of the physical map of pNGR234a into four levels (boxes A-D). Level A: the contig organization. Level B: selected overlapping cosmids. The minimal subset of clones necessary to cover the symbiotic plasmid is marked by thicker lines and boldface numbers. Linkages that were difficult to establish are delimited by black numbered boxes (boxes 1-7). Level C: position of relevant pJB8 clones from the pMPIK3030a (3, 4) bank relative to the Lorist2 map. Level D: approximate locations of the symbiotic genes as well as dctAl and the Spe I restriction sites (marked by numbered capital S) on segments of pNGR234a. Note: one map unit corresponds to one band in the HindIII fingerprint. Variation in the clone*,, size depends on the number of HindIII sites present in the insert (average insert size is -35 kb). Downloaded by guest on September 30, 2021 Genetics: Perret et al. Proc. Nati. Acad. Sci. USA 88 (1991) 1925 print data (Fig. 1, links 1 and 5). To avoid high background on in size from 4.4 to 290 kb (sized relative to comigrating A autoradiographs of dot-blot membranes due to Lorist2 vector concatemers) were identified (data not shown). The corre- cross-hybridization, we used specially designed oligonucleo- sponding restriction sites were localized on the Sym-plasmid tides as primers for the synthesis of probes homologous to the physical map by digesting the DNA of the 25 representative left and right side of the insert DNA. In order to exclude clones (Fig. 1, level D) with Spe I (data not shown). Then, false-positive signals due to repeated sequences, linkages es- using the clones with an Spe I site(s) as a linking library (27), tablished through dot-blot hybridization were always verified we were able to confirm the contig organization obtained by by examinination of the restriction patterns of the relevant the fingerprint approach as well as to derive the Spe I cosmids for logical distribution of fragments. restriction map of pNGR234a (Fig. 2 Upper). Summation of The last gap was filled by "walking." Two supplementary the sizes of the Spe I restriction fragments yielded a molec- clones, pXBS4 and pXBS23, were obtained by successive ular mass of pNGR234a of -500 kb. screening of the 800 additional colonies from the genomic Lcalization of Symbiotic Loci and Genes. Depending on the bank, first with pXB285 then with the resulting pXBS4 (Fig. source ofDNA, two different strategies were used: (i) discrete 1, link 2). Whereas most clones were redundant, cosmid loci were localized using standard hybridization procedures pXBS23 remained unique among the 2800 screened. with NGR234 subclones or heterologous probes internal to Alignment of the Physical and the Spe I Restriction Map. described genes and (ii) DNA molecules large enough to yield Confirmation of the order of overlapping cosmids presented a significant number of bands were mapped by matching their in Fig. 1 came from parallel studies on the chromosome. One HindIll fingerprints with those in the NGRDB data base. Clones way of grouping adjacent contigs is by hybridizing selected were analyzed from different libraries and included the original clones to large DNA restriction fragments separated by pJB8 cosmids ofpMPIK3030a (= pNGR234a) (3) that cover the pulsed-field gel electrophoresis (PFGE). In this combined three host-specificity regions (pWA76 and pWA88 for HsnI, "bottom-up" (i.e., the piecing together of larger and larger pWA58 for HsnII, and pWA54 for HsnIII), as well as pWA46 contigs) and "top-down" (the development oflow-resolution (which shows homology to region Ila of R. meliloti; P. Roch- maps) approach, the number of fragments generated by epeau, personal communication). Of the five pJB8 clones, the endonucleases that cleave the DNA infrequently is critical for fingerprints of three cosmids, pWA46, pWA58, and pWA76, ordering the contigs. As a first step toward this goal, Spe I matched perfectly those of Lorist2 clones from pNGR234a. (5'. . . A/CTAGT ... 3') was found to be the most suitable However, the fingerprints ofpWA54 and pWA88 revealed that restriction enzyme for the Rhizobium species NGR234 ge- both were made oftwo segments that are not contiguous on the nome [=62% G+C (26); X.P. and I. St. Girons, unpublished map. This was confirmed by hybridizing pWA54 and pWA88 to work]. By comparing the resulting PFGE restriction patterns digests of the relevant Lorist2 clones (data not shown). of NGR234R and ANU265 (a symbiotic plasmid-cured de- A two-step hybridization procedure was used to map rivative ofNGR234), eight Spe I restriction fragments ranging symbiotic genes as well as small loci such as the 6.7-kb HindIll fragment containing the nodDi gene (15). First, an Region II approximate location on the physical map was obtained by / nodABC hybridizing 32P-labeled probes to dot-blot filters representing the symbiotic plasmid (Fig. 3 A and B). When positive clones did not overlap, an initial estimate of the probable number of copies of the labeled gene was obtained (Fig. 3 C and D). Then, a more-precise assignment to distinct restriction fragments was obtained by hybridization against a Southern blot ofrestricted DNA of the same cosmid(s) (Fig. 4 and Table 2). DISCUSSION Using the fingerprint method developed for mapping the C. elegans genome, we have constructed a canonical ordered library of cosmid clones covering the entire 500-kb symbiotic

A% _ IC I a |740 13571568614231 231 V |4 2851182%M96|55|3WC 196 lw 66 Mr74641 43 315 296 368 6~4 VO1 190 nifHDK nodD2 no/B

,~ . _ I,,1'1 pXB1357 @ t::iSS B D

I § I 110 296, * *0 * II I 901 368 & 0:0** I 564 740 ApB* - 64 .1 .*-

- 0 kb pXB1027 pXB64 pXBllO0

FIG. 2. (Upper) Spe I restriction map of pNGR234a. The Spe I FIG. 3. Dot-blot hybridization. (A) Dot-blot filter identifying the sites correspond to those in Fig. 1. (Lower) Detailed Xho I restriction 24 clones selected to represent the symbiotic plasmid. (B-D) Results map of an 80-kb region containing the two nifKDH operons, the of hybridization with 32P-labeled pA16 (B), with the 6.7-kb HindIl dctAl gene, and the loci showing homology to nodD (nodD2) and fragment containing the nodDi locus (C), and with a 1.0-kb Sph I nolB._, fragment (internal to nodDi) to the dot-blot filter. Downloaded by guest on September 30, 2021 1926 Genetics: Perret et al. Proc. Natl. Acad. Sci. USA 88 (1991)

A Ng,0 Z 911 "I' Kr+.p p (kb) (kb)

23 9.4 - 6.6 -5.9 - 4.4

-2.9 - 2.3 -2 -

FIG. 4. Example showing assignment of symbiotic genes to specific restriction fragments: Xho I-restricted Lorist2 clones cover- ---0.6 ing about one-third of pNGR234a (Left) were "Southern" blotted and probed with 32P-labeled pA16 (Right).

plasmid of Rhizobium species NGR234. As is common in (only one of which is on pNGR234a). As RS.1 gives four projects ofthis type, 95% coverage ofthe plasmid was rapidly characteristic HindIII-Sau3AI bands in the fingerprint pat- achieved with only the HindIII fingerprint data. At this stage, tern, it interferes with the normal contig assembly and only the six plasmid contigs that would later be part ofthe plasmid the analysis of the restriction patterns of the clones contain- map, were already included in the 14 contigs with homology ing the RS.1 region gave accurate overlap assignment. Al- to pNGR234a DNA. The only real gap corresponded to the though the origin and the role of RS.1 and other repeated section of pNGR234a covered by the supplementary clones DNA families in the NGR234 genome are unknown, they pXBS4 and pXBS23. Despite this apparent efficiency, link- could generate genomic rearrangement through homologous age of the contigs into a circular unit was achieved only by recombination, as has been shown in other prokaryotes additional fingerprinting and complementary mapping meth- (29-31). If the genomic rearrangements detected in Rhizo- ods. New procedures for characterizing DNA segments such bium phaseoli under laboratory conditions (32) also occur as DNA fingerprinting by sampled sequencing (28) should frequently in other rhizobia, the pNGR234a map should no reduce the need for additional data. longer be considered as immutable but rather as a "frozen Interestingly, by starting with a cosmid bank of total image" of part of a plastic NGR234 genome. genomic DNA, we were able to identify sequences homolo- Nevertheless, an organized library facilitates the indexing gous to symbiotic plasmid DNA in eight contigs not located of data and provides immediate access to any segment of the on pNGR234a. The RS. 1 reiterated sequence, present in four symbiotic plasmid. This way, loci of interest can be localized copies in the genome, was assigned to four distinct contigs on the map and characterized rapidly (Table 2). Interestingly, Table 2. Assignment of the symbiotic genes Symbiotic plasmid Restriction fragment Gene Template DNA for probe synthesis location Clone assignment assignment Ref(s). nodAB pXNODAB + pXB285/pXB182 7.3-kb EcoRI/6-kb Xho 1 33, 34 nodC pXNODC + pXB285/pXB182 7.3-kb EcoRI/1.8-kb Xho I 33, 34 nodDI 6.7-kb HindIII fragment of pNGRH6 + pXB1357 7.5-, 2.9-, 1-, 0.8-, 0.4-kb EcoRI 15, 34-36 nodD 1-kb internal Sph I fragment of pNGRH6 + pXB1357 2.9-kb EcoRI (nodDI) 15, 34-36 + pXB64/pXB110 6.2-kb EcoRI (nodD2) 37 nodE pGMI724 (internal M13 clone) - pXB1116-6 8-kb EcoRI nodG pGM1727 (internal M13 clone) - pXB1116-6 8-kb EcoRI Region 11 pG14 (internal M13 clone) + pXB182 2.9-kb EcoRI/1.7-kb Xho 1 34 nodP 0.45-kb Sal I-Sst I fragment of pJSS38 - pXB559/pXB1130 3.1-kb EcoRI 17 - pXB1112 4.3-kb EcoRI 17 nodQ 1.2-kb Sal I fragment of pJSS31 - pXB559/pXB1130 1.6-, 3.1-kb EcoRI 17 - pXB1112 4.3-kb EcoRI nodSU pA16 + pXB1027 5.9-, 2.9-kb Xho I 16 nolB 4.2-kb BamHI fragment of pRFDH421 + pXB110/pXB901/pXB740 4.2-kb BamHI nifKDH pSA30 + pXB368 13.3-kb HindIII 38 + pXB564/pXB64/pXB110 8.3-kb HindIII 38 dctA 1.8-kb Sst I-BamHI fragment of pTVB60 + pXB296/pXB368 5.5-kb EcoRI - pXB202/pXB247 3.5-kb EcoRI Downloaded by guest on September 30, 2021 Genetics: Perret et al. Proc. Natl. Acad. Sci. USA 88 (1991) 1927

not all nod genes are plasmid borne; sequences homologous 6. Lewin, A., Rosenberg, C., Meyer z.A., H., Wong, C. H., to nodE, nodG, nodP, and nodQ have been assigned to three Nelson, L., Manen, J.-F., Stanley, J., Dowling, D. N., Ddna- distinct nonplasmid contigs. The remaining nod genes (nod- rid, J. & Broughton, W. J. (1987) Plant Mol. Biol. 8, 447-459. 7. Coulson, A., Sulston, J., Brenner, S. & Karn, J. (1986) Proc. ABC, nodDi, nodD2, nodSU, nolB, and region II) are widely Natl. Acad. Sci. USA 83, 7821-7825. dispersed over pNGR234a, while the two functional nifKDH 8. Sulston, J., Mallet, F., Staden, R., Durbin, R., Horsnell, T. & operons (38) are seperated by only 28 kb (Fig. 2 Lower). Coulson, A. (1988) CABIOS 4, 125-132. Surprisingly, two copies of the dctA gene were found. One is 9. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular located on the symbiotic plasmid, while a second highly Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold homologous locus was localized on a nonplasmid contig. Spring Harbor, NY), 2nd Ed. Thus, the organization of symbiotic genes on pNGR234a 10. Beringer, J. E. (1974) J. Gen. Microbiol. 84, 188-198. differs greatly from that of other well-characterized rhizobia 11. Cami, B. & Kourilsky, P. (1978) Nucleic Acids Res. 5, 2381- R. 2390. such as leguminosarum and R. meliloti (in which most 12. Messing, J. (1983) Methods Enzymol. 101, 20-78. symbiotic genes are clustered on the plasmid) or Bradyrhizo- 13. Stanley, J., Dowling, D. N. & Broughton, W. J. (1988) Mol. bium japonicum (where symbiotic genes are confined to the Gen. Genet. 215, 32-37. chromosome) (39). Our assignments of previously described 14. Morrison, N. A., Hau, C. Y., Trinick, M. J., Shine, J. & Rolfe, genes to restriction fragments agree with published data B. G. (1983) J. Bacteriol. 153, 527-531. (Table 2), except for nodD2, which was believed to be present 15. Bassam, B. J., Rolfe, B. G. & Djordjevic, M. A. (1986) Mol. only in MPIK3030 (40). There is strong evidence, however, Gen. Genet. 203, 49-57. that the second copy of the nodD gene detected on 16. Lewin, A., Cervantes, E., Wong, C. H. & Broughton, W. J. is the same as the one described in (1990) Mol. Plant-Microbe Interact. 3, 317-326. pNGR234a previously 17. Schwedock, J. & Long, S. (1989) Mol. Plant-Microbe Interact. MPIK3030 (37). 2, 181-194. Alignment of cosmids from existing clone banks [e.g., the 18. Cannon, F. C., Riedel, G. E. & Ausubel, F. M. (1979) Mol. pWA clones covering the region II, HsnII, and HsnIII regions Gen. Genet. 174, 59-66. (3, 5)] did not reveal significant differences among these and the 19. Debelld, F. & Sharma, S. B. (1986) Nucleic Acids Res. 14, Lorist2 clones. With pWA54 and pWA88, however, the corre- 7453-7472. sponding fingerprint patterns showed that each of the pJB8 20. Egelhoff, T. T., Fisher, R. F., Jacobs, T. W., Mulligan, J. T. & clones contained two nonadjacent segments, which made it Long, S. R. (1985) DNA 4, 241-248. impossible to align these clones with other overlapping pWA 21. Gibson, T. J., Coulson, A. R., Sulston, J. E. & Little, P. (1987) clones. We attribute these to Gene 53, 275-281. discrepancies cloning artifacts, 22. Stratagene (1988) Stratagene Catalogue (Stratagene, La Jolla, rather than to significant differences between pMPIK3030a and CA), pp. 108-109. pNGR234a structures. Integration ofrearranged Lorist2 clones 23. Broughton, W. J., Bohlool, B. B., Shaw, C. H., Bohnert, H. J. in the pNGR234a physical map is excluded by a high redun- & Pankhurst, C. E. (1985) Arch. Microbiol. 141, 14-21. dancy in the cosmid coverage and by the systematic elimination 24. Stanley, J., Dowling, D. N., Stucker, M. & Broughton, W. J. of clones with a fingerprint different from those of contiguous (1987) FEMS Microbiol. Lett. 48, 25-30. cosmids. These data also show that the usual method to check 25. Sternberg, N., Tiemeier, D. & Enquist, L. (1977) Gene 1, for rearranged clones (by comparison of restriction fragment 255-280. length polymorphism of genomic and cloned DNA) is not 26. Broughton, W. J., Dilworth, M. J. & Passmore, 1. K. (1972) conclusive and Hin- Anal. Biochem. 46, 164-172. always (41). Effectively, EcoRI, BamHI, 27. Wallace, M. R., Fountain, F. W., Brereton, A. M. & Collins, dIII digests of pWA54 matched the same digests of NGR234 F. S. (1989) Nucleic Acids Res. 17, 1665-1677. genomic DNA and seemed to confirm co-linearity (41). We 28. Brenner, S. & Kenneth, J. L. (1989) Proc. Natl. Acad. Sci. attribute this erroneous result to the presence ofrestriction sites USA 86, 8902-8906. for each of the enzymes tested within a short distance of the 29. Albertini, A. M., Hofer, M., Calos, M. P. & Miller, J. H. (1982) "breaking point." Cell 29, 319-324. Finally, the combined physical/genetic map of pNGR234a 30. Edlund, T. & Normack, S. (1981) Nature (London) 292, 269- also provides a basis for studying genome organization in an 271. organism that is intermediate between the fast-growing 31. Hill, C. W. & Harnish, B. W. (1982) J. Bacteriol. 149, 449-457. Rhizobium and the 32. Florez, M., Gonzalez, V., Pardo, M. A., Leija, A., Martinez, species slow-growing Bradyrhizobium E., Romero, D., Pinero, D., Davila, G. & Palacios, R. (1988) J. (42). Methods similar to those described above could be used Bacteriol. 170, 1191-1196. to construct a combined physical/genetic map of the remain- 33. Bachem, C., Kondorosi, E., Banfalvi, Z., Horvath, B., Kon- der of the Rhizobium species NGR234 genome. dorosi, A. & Schell, J. (1985) Mol. Gen. Genet. 199, 271-278. We thank C. Cadot, E. Cervantes, A. Coulson, I. St. Girons, A. 34. Nayudu, M. & Rolfe, B. G. (1987) Mol. Gen. Genet. 206, Lewin, S. G. Pueppke, J. Sulston, andJ. A. Williams forassistance and 326-337. advice without which this work would not have been possible. Probes 35. Bassam, B. J., Djordjevic, M. A., Redmond, J. W., Batley, M. were kindly made available by C. Elmerich, J. Denarie, A. Lewin, S. G. & Rolfe, B. G. (1988) Mol. Plant-Microbe Interact. 1, 161-168. Pueppke, B. G. Rolfe, and J. Schwedock. Financial support was 36. Horvath, B., Bachem, C., Schell, J. & Kondorosi, A. (1987)EMBO provided by the Fonds National Suisse de la Recherche Scientifique J. 6, 841-848. (Project 3.180.088), the University ofGeneva, the European Molecular 37. Rodriguez-Quinones, F., Banfalvi, Z., Murphy, P. & Kondor- Biology Organization Short-Term Fellowship program, E. I. Du Pont osi, A. (1987) Plant Mol. Biol. 8, 61-75. de Nemours, and the Socidtd Acaddmique de Geneve. 38. Badenoch-Jones, J., Holton, T. A., Morrison, C. M., Scott, K. F. & Shine, J. (1989) Gene 77, 141-153. 1. Trinick, M. J. (1980) J. Appl. Bacteriol. 49, 39-53. 39. 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