On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a dear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliot strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.Insertion sequence (IS) elements are defined as relatively small mobile genetic entities which, unlike drug resistance transposons, do not contain selectable genes (5). This definition makes it clear that direct selection for the presence of an IS element is usually not possible. IS elements in procaryotes were first identified as causative agents of spontaneous mutations in Escherichia coli (21, 36). Since then, various classes of transposable elements have been found to be natural constituents of many bacterial chromosomes, plasmids, and bacteriophages. As far as gram-negative bacteria are concerned, most IS elements have been isolated from enteric bacteria (mainly E. coli; for reviews and lists of isolated elements, see references 14 and 20). However, there have been a substantial number of reports on IS elements identified in other gram-negative bacterial species, such as Agrobacterium tumefaciens (3,15,24,42), Xanthomonas campestris (22, 23), and various Rhizobium (9,28,32) and Pseudomonas (6,13,35,41,45) strains.In general, these elements have been found more or less by chance because of the capacity to inactivate or activate particular genes. However, there have been successful attempts to screen for transposable elements by means of direct selection procedures (8,15,30,35).In this report, we present the construction and use of three broad-host-range vectors designed to enable systematic searches for the presence of transposable elements in gramnegative bacteria. Advantage was taken of two direct selection systems initially constructed to simplify cloning experiments with E. coli (7, 17), as well as a system described by Gay and coworkers (15). The common principle of these systems is based upon insertional inactivation of a vectorborne dominant sensitivity marker. With pSUP104-pheS and pSUP104-rpsL, this leads to drug resistance. Inactivation of pSUP104-sac allows its host to grow on medium containing 5% sucrose. Both of these events are positively selectable phenotypes in the hosts. (A...

Rhizobium meliloti strains isolated from alfalfa plants grown in a mining recultivation field, in a model ecosystem (microcosm) and in soil core containers were characterized by two new taxonomic methods, fingerprinting and handprinting, using insertion sequence elements (IS) as hybridization probes. The diversity of strains within the field population could first be detected with IS‐fingerprinting, whereby nearly three times more groups of Rhizobium meliloti strains could be identified in comparison to the groups according to plasmid profiles. This complexity and diversity of the rhizobial population was also detected in microcosm studies. Strains identified among the field population were also detected in the microcosm studies. The persistence of rhizobia in soil was demonstrated in soil core samples held in a cold room for 2 years. A decrease in the genomic diversity of the R. meliloti population upon soil storage was observed. A novel monitoring method, IS‐handprinting, in which the presence of certain endogenous insertion elements within a strain is registered, was successfully employed to characterize genetically the field R. meliloti strains with simplicity and speed. In contrast to IS‐fingerprinting, IS‐handprinting is based on a simple plus‐or‐minus detection, which is sufficient for a taxonomic characterization. Both methods, using a non‐radioactive detection system, are sensitive enough to detect one copy of an insertion element in a strain's genome. IS‐fingerprinting, with its fine resolution, would be suitable for ecological studies of individual strains in any complex ecosystem, whereas IS‐handprinting would be suitable for monitoring strains and characterizing large numbers of strains.

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A total of 360 Rhizobium leguminosarum bv. viciae strains was isolated from three brown-coal mining restoration fields of different age and plant cover (without and in the first and second year of alfalfa, Medicago sativa, cultivation) using two host species (Vicia hirsuta and Pisum sativum) as capture plants. The strains were genetically typed by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-generated 16S-23S ribosomal DNA intergenic spacer regions (IGS-RFLP) and characterized by plasmid profiles and RFLP analysis of amplified nodABC genes. The R. leguminosarum bv. viciae population was dominated by the same group of strains (irrespective of the trap plant used). According to type richness, the genetic diversity of indigenous R. leguminosarum in the second year of restoration was lower than in the first year and it resembled that of the fallow field, except for plasmid types, in which it was higher than that of the fallow field. Some of the less frequent nodABC genotypes were associated with distinct chromosomal IGS genotypes and symbiotic plasmids (pSyms) of different sizes, indicating that horizontal transfer and rearrangements of pSym can occur in natural environments. However, the dominant pSym and chromosomal genotypes were strictly correlated suggesting a genetically stable persistence of the prevailing R. leguminosarum bv. viciae genotypes in the absence of its host plant.

A rearrangement between the symbiotic plasmid (pRleVF39d) and a nonsymbiotic plasmid (pRleVF39b) in Rhizobium leguminosarum bv. viciae VF39 was observed. The rearranged derivative showed the same plasmid profile as its parent strain, but hybridization to nod, fix, and nif genes indicated that most of the symbiotic genes were now present on a plasmid corresponding in size to pRleVF39b instead of pRleVF39d. On the other hand, some DNA fragments originating from pRleVF39b now hybridized to the plasmid band at the position of pRleVF39d. These results suggest that a reciprocal but unequal DNA exchange between the two plasmids had occurred.