Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4932-4936, June 1993 Genetics Gene amplification in Rhizobium: Identification and in vivo cloning of discrete amplifiable DNA regions (amplicons) from Rhizobium leguminosarum biovar phaseoli (genomic rearrangements/genome dynamics/genome plasticity/symbiosis/nitrogen fixation) MARGARITA FLORES, SUSANA BROM, TOMASZ STEPKOWSKI, MARiA DE LOURDES GIRARD, GUILLERMO DAVILA, DAVID ROMERO, AND RAFAEL PALACIOS Departamento de Gendtica Molecular, Centro de Investigaci6n sobre Fijaci6n de Nitr6geno, Universidad Nacional Aut6noma de M6xico, Apartado postal 565-A, Cuernavaca, Mor. Mexico Communicated by Donald R. Helinski, January 25, 1993 ABSTRACT A genetic element that allows the positive MATERIALS AND METHODS selection of different genomic rearrangements was used to analyze DNA amplification in Rhizobium leguminosarum bio- Construction of TnS-GDYNl. This transposon derivative var phaseoli. Discrete amplifiable DNA regions (amplicons) was constructed by in vitro manipulation of the TnS insert in were detected in different regions of the genome of the model pSUP2021, which is a mobilizable pBR325 derivative (6). The central Bgl II-Bgl II fragment of TnS in pSUP2021 was strain CFN42, including the chromosome and several large replaced by a 5.7-kb BamHI-BamHI fragment ofpGUS3 (5), plasmids. Amplicons were mobilized into Escherichia coli using carrying the GDYN1 cassette. After ligation, the mixture was a genetic approach that involves the introduction of an origin transformed into E. coli HB101, selecting for recombinant ofreplication active in E. coli and an origin ofconjugal transfer plasmids in LB medium containing chloramphenicol and into the amplifiable DNA regions oftheRhizobium genome. The spectinomycin. Colonies resistant to chloramphenicol and strategy can be a valuable tool for studies on genome organi- spectinomycin that showed sensitivity to sucrose were zation and function. We propose that amplicons define a screened for plasmids with the desired restriction pattern, structural characteristic of the genome that may play an and one of these was named pDR21. important biological role. Strains and Genetic Manipulations. All R. phaseoli strains used were derived from wild-type CFN42, which is naturally Rhizobium species have stimulated scientific interest due to resistant to nalidixic acid (7). Derivatives containing TnS- their ability to interact with plants establishing nitrogen- GDYN1 insertions were obtained by mating E. coli S-17 fixing symbioses. The genome ofdifferent Rhizobium species strain containing plasmid pDR21, which carries the transpo- contains a large amount of reiterated DNA sequences that son, with R. phaseoli strain CFN42 and selecting transcon- include complete operons, specific genes, regulatory se- jugants resistant to nalidixic acid and spectinomycin. Strains quences, and insertion sequences (for see ref. CFNX201, CFNX207, CFNX209, and CFNX211 contain review, 1). TnS-GDYN1 in plasmid a (pa). Strains CFNX203 and Presumably due to the presence of reiterated DNA se- CFNX205 contain the transposon in plasmid d (pd) and in the quences, the genome of some Rhizobium strains is subjected chromosome, respectively. to frequent genomic rearrangements (2-5). To isolate derivatives carrying DNA amplifications, strains We have recently constructed a genetic element (the containing Tn5-GDYN1 insertions were serially cultured in GDYN1 cassette) that allows the positive selection of differ- liquid PY medium (0.5% peptone/0.3% yeast extract/10 mM ent types of genomic rearrangements (5). This element con- CaCl2) at 30°C for periods of 24 hr as follows: once in the tains the kanamycin/gentamycin and spectinomycin/ presence of spectinomycin at 75 ,.g/ml, twice with kanamy- streptomycin resistance markers from plasmid pSa. In Rhizo- cin at 50 pg/ml, and twice with kanamycin at 100 pg/ml. bium leguminosarum biovar phaseoli (R. phaseoli), the Cells were then plated on PY medium containing kanamycin symbiont of the common bean plant Phaseolus vulgaris, at 125 jig/ml, and individual colonies were isolated. Strains these genes confer resistance to high levels of spectinomycin CFNX202, CFNX204, CFNX206, CFNX208, CFNX210, and to low levels of kanamycin. Because the level of kan- and CFNX212 are amplified derivatives from strains amycin resistance increases with gene dosage, the element CFNX201, CFNX203, CFNX205, CFNX207, CFNX209, provides a positive selection system to detect variants with and CFNX211, respectively. Strain CFNX206A is a deriva- amplified DNA regions. The GDYN1 cassette was used to tive of CFNX206 resistant to kanamycin at 1 mg/ml. demonstrate high-frequency amplification and deletion The procedure to clone in E. coli DNA sequences from R. events of a 120-kb region located in the symbiotic plasmid of phaseoli strains carrying amplifications is presented in Re- R. phaseoli (5). sults. Strains CFNC1, CFNC2, CFNC3, CFNC4, CFNC5, In the present study we used a GDYN1 derivative (TnS- and CFNC6 are E. coli strains containing DNA from Rhizo- GDYN1) to analyze gene amplification events in different bium strains CFNX202, CFNX204, CFNX206, CFNX208, regions ofthe genome ofR. phaseoli strain CFN42, including CFNX210, and CFNX212, respectively. the DNA Electrophoresis, Filter Blot Hybridization, and Quan- chromosome and several large plasmids. Discrete ampli- tifi'cation of DNA Amplification. Total DNA or cosmid DNA fiable DNA regions, referred to here as amplicons, were was digested with BamHI, subjected to electrophoresis in 1% identified and mobilized from R. phaseoli to Escherichia coli agarose gels, blotted onto nitrocellulose, and hybridized as by an in vivo genetic procedure. described (3). Plasmid profiles were obtained by the in-gel lysis method of Eckhardt (8), blotted onto nitrocellulose, and The publication costs of this article were defrayed in part by page charge hybridized similarly. Probes were labeled with 32P by the payment. This article must therefore be hereby marked "advertisement" nick-translation procedure (9). To quantify DNA amplifica- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tion, blots were hybridized against a mixed probe containing 4932 Downloaded by guest on September 29, 2021 Genetics: Flores et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4933 the transposon and a 300-bp fragment of R. phaseoli ribo- A 1 2 3 4 5 6 7 8 somal DNA (10) obtained by PCR. Blots were subjected to Kb autoradiography, and signals were integrated by scanning 30 densitometry. 10 .... ...;K- RESULTS 3 Selection for Gene Amplification Events. The TnS-GDYN1 k4 was randomly introduced into R. phaseoli strain CFN42 as described in Materials and Methods. Derivatives containing the transposon were selected by their resistance to specti- nomycin. The genome of CFN42 contains a chromosome and six large plasmids (pa-pf) ranging in size from 150 to 600 kb; pd (390 kb) has been identified as the symbiotic plasmid (11, U*b... ; 12). The location of the transposon in different spectinomy- B cin-resistant clones was identified by hybridization of plas- Kb mid profiles with a DNA probe that recognizes the transpo- 30 son (data not shown). In two independent experiments a total 10 of 30 derivatives of CFN42 were used to search for DNA amplification events. From these derivatives, 13 carried the transposon in pa, 4 carried the transposon in pb, 2 derivatives 3 , carried it in pc, 1 carried the transposon in pd, 2 derivatives carried it in pe, 2 carried the transposon in pf, and 6 derivatives carried the transposon in the chromosome. To select for amplification events, cells were cultured in liquid medium in the presence of increased concentrations of kan- amycin (see Materials and Methods). To screen for amplification, total DNA from the original TnS-GDYN1 insertions, as well as from the kanamycin- resistant clones derived from them, was digested with restric- C tion endonuclease Southern blots oftotal DNA were Kb BamHI; 30, prepared and hybridized with a mixed probe containing a 10 .1 ribosomal DNA sequence from R. phaseoli and a DNA f * a, :... fragment that recognizes the transposon (see Materials and Aw: do Methods). The ribosomal DNA sequence hybridized to three BamHI bands. Because the TnS-GDYN1 lacks BamHI sites, 3 the insertions appeared as single fragments of different sizes containing the whole transposon and adjacent genomic se- quences. In amplification events, the intensity ofthe fragment corresponding to the insertion increased relative to those f-..I...4 corresponding to ribosomal DNA (see examples in Fig. 1, lanes 3 and 4). The level of amplification was determined by comparative densitometry. From the 30 insertions analyzed, 14 cases ofamplification were detected: 7 cases corresponded FIG. 1. Analysis of DNA amplification events in R. phaseoli. to insertions in pa, 3 cases corresponded to insertions in pb, Total DNA digests or plasmid profiles of strains harboring TnS- GDYN1 insertions and their corresponding amplified derivatives 1 case corresponded to insertions in pd, 1 case corresponded were analyzed by ethidium bromide staining and hybridized against to insertions in pe, 1 case corresponded to insertions in pf, and different probes. (A) Amplification event in pa: strain CFNX201, 1 case corresponded to insertions in the chromosome. original TnS-GDYN1 insertion; strain CFNX202, amplified variant. Characterization of DNA Amplification Events. Fig. 1 pre- (B) Amplification event in pd: strain CFNX203, original insertion; sents the analysis ofthree different amplification