Gene amplification in Rhizobium: identification and in vivo cloning of discrete amplifiable DNA regions (amplicons) from Rhizobium leguminosarum biovar phaseoli.

Abstract

A genetic element that allows the positive selection of different genomic rearrangements was used to analyze DNA amplification in Rhizobium leguminosarum biovar phaseoli. Discrete amplifiable DNA regions (amplicons) were detected in different regions of the genome of the model strain CFN42, including the chromosome and several large plasmids. Amplicons were mobilized into Escherichia coli using a genetic approach that involves the introduction of an origin of replication active in E. coli and an origin of conjugal transfer into the amplifiable DNA regions ofthe Rhizobium genome. The strategy can be a valuable tool for studies on genome organization and function. We propose that amplicons define a structural characteristic of the genome that may play an important biological role.Rhizobium species have stimulated scientific interest due to their ability to interact with plants establishing nitrogenfixing symbioses. The genome ofdifferent Rhizobium species contains a large amount of reiterated DNA sequences that include complete operons, specific genes, regulatory sequences, and insertion sequences (for review, see ref. 1). Presumably due to the presence of reiterated DNA sequences, the genome of some Rhizobium strains is subjected to frequent genomic rearrangements (2-5).We have recently constructed a genetic element (the GDYN1 cassette) that allows the positive selection of different types of genomic rearrangements (5). This element contains the kanamycin/gentamycin and spectinomycin/ streptomycin resistance markers from plasmid pSa. In Rhizobium leguminosarum biovar phaseoli (R. phaseoli), the symbiont of the common bean plant Phaseolus vulgaris, these genes confer resistance to high levels of spectinomycin and to low levels of kanamycin. Because the level of kanamycin resistance increases with gene dosage, the element provides a positive selection system to detect variants with amplified DNA regions. The GDYN1 cassette was used to demonstrate high-frequency amplification and deletion events of a 120-kb region located in the symbiotic plasmid of R. phaseoli (5).In the present study we used a GDYN1 derivative to analyze gene amplification events in different regions of the genome ofR. phaseoli strain CFN42, including the chromosome and several large plasmids. Discrete amplifiable DNA regions, referred to here as amplicons, were identified and mobilized from R. phaseoli to Escherichia coli by an in vivo genetic procedure. MATERIALS AND METHODSConstruction of TnS-GDYNl. This transposon derivative was constructed by in vitro manipulation of the TnS insert in pSUP2021, which is a mobilizable pBR325 derivative (6). The central Bgl II-Bgl II fragment of TnS in pSUP2021 was replaced by a 5.7-kb BamHI-BamHI fragment of pGUS3 (5), carrying the GDYN1 cassette. After ligation, the mixture was transformed into E. coli HB101, selecting for recombinant plasmids in LB medium containing chloramphenicol and spectinomycin. Colonies resistant to chloramphenicol and spectinomycin that showed sensitivity to sucrose were scre...

Discrete amplifiable regions (amplicons) in the symbiotic plasmid of Rhizobium etli CFN42

“…Insertion of GDYN1 elements into the symbiotic plasmid. To screen different regions of pSym for the occurrence of amplifications and deletions, we took advantage of the GDYN1 elements (13,40). These elements, in the form of a cassette or a Tn5 derivative, carry a 5.7-kb BamHI-BamHI fragment that contains a dose-dependent Km/Sp determinant (thus providing a positive selection for amplifications) as well as the sacR-sacB genes of Bacillus subtilis, which confer sucrose-dependent lethality to gram-negative bacteria (17).…”

“…These elements, in the form of a cassette or a Tn5 derivative, carry a 5.7-kb BamHI-BamHI fragment that contains a dose-dependent Km/Sp determinant (thus providing a positive selection for amplifications) as well as the sacR-sacB genes of Bacillus subtilis, which confer sucrose-dependent lethality to gram-negative bacteria (17). This characteristic allows a positive selection for deletion events (13,40).…”

“…trifolii and viceae, and R. etli (5,9,13,15,20,23,40,42,45,48,51), thus constituting an important cause of variability in Rhizobium populations.…”

“…These rearrangements are generated presumably by recombination between reiterated sequences, thus provoking events of cointegration, translocation, deletion, and amplification of specific sequences (5,13,15,20,23,40,42,45,48,51). Using specific genetic constructs (GDYN1 elements), we have detected high-frequency tandem amplification of defined sectors in the genome of R. etli CFN42, located in the chromosome and in several large plasmids (13,40).…”

“…Using specific genetic constructs (GDYN1 elements), we have detected high-frequency tandem amplification of defined sectors in the genome of R. etli CFN42, located in the chromosome and in several large plasmids (13,40). We have proposed the term amplicons to refer to specific DNA regions that have the potential to be amplified or deleted as a unit (13).…”