CHARACTERIZATION, DISTRIBUTION, AND LOCALIZATION OF ISRL2, AN INSERTION-SEQUENCE ELEMENT ISOLATED FROM RHIZOBIUM-LEGUMINOSARUM BV VICIAE

An insertion sequence (IS) element, ISRl2, from Rhizobium leguminosaru m bv. viciae strain MSDJ4184 was isolated by insertional inactivation of the sacRB gene of pSUP104-sac, which allows positive selection. ISR l2 is 932 bp long, is flanked by 17-bp imperfect terminal inverted rep eats, and generated a 3-bp target site duplication. ISRl2 was found to be 63 to 77% homologous to insertion elements of the IS5 group of the IS4 superfamily. A probe incorporating a full-length copy of ISRl2 wa s used to screen genomic DNAs from a collection of strains and from tw o field populations of R, leguminosarum to detect and estimate the cop y numbers of homologous sequences. Among the collection of 63 strains representing the different species and genera of members of the family Rhizobiaceae, homology to ISRl2 was found within strains belonging to Sinorhizobium meliloti and S, fredii; within four of the six recogniz ed Rhizobium species, R. leguminosarum, R. tropici, R. etli, and R. ga legae; and within Rhizobium sp. (Phaseolus) genomic species 2. The app arent copy numbers of ISRl2 varied from one to eight. Among 139 isolat es of R. leguminosarum from two field populations, homology to ISRl2 w as detected in 91% of the isolates from one site and in 17% from the o ther. Analysis of the 95 isolates that hybridize to ISRl2 revealed a t otal of 20 distinct hybridization patterns composed of one to three ba nds. Probing blots of Eckhardt gels showed that sequences with homolog y to ISRl2 may be found on plasmids or the chromosome. Analysis of the ir genomic distribution demonstrated relationships and diversity among the R. leguminosarum isolates tested.