Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors

Abstract

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...

The exbD2 gene as well as the iron-uptake genes tonB, exbB and exbD1 of Xanthomonas campestris pv. campestris are essential for the induction of a hypersensitive response on pepper (Capsicum annuum)

“…Restriction-endonuclease-generated fragments of total DNA were separated by gel electrophoresis and subsequently vacuum blotted on nylon filters as described by Simon et al (1991). Labelling of the probe (vector and DNA region) and hybridization were done as described elsewhere (Simon et al, 1991). Transformation of E. coli cells and electroporation of X. campestris pv.…”

“…Restriction enzymes and other commercially available enzymes were obtained from Pharmacia or BRL and used in accordance with the manufacturer's recommendations. Restriction-endonuclease-generated fragments of total DNA were separated by gel electrophoresis and subsequently vacuum blotted on nylon filters as described by Simon et al (1991). Labelling of the probe (vector and DNA region) and hybridization were done as described elsewhere (Simon et al, 1991).…”

Identification and analysis of aarP, a transcriptional activator of the 2'-N-acetyltransferase in Providencia stuartii

“…To recombine the aarP::Cm disruption on plasmid pKS.SF2::Cm into the P. stuartii chromosome, a 5-kb XbaI-BamHI fragment was excised from pSK.SF2::Cm and ligated to pKNG101 (22) cut with the same enzymes, resulting in plasmid pKNG101.SF2::Cm. pKNG101 is an R6K-based plasmid containing a streptomycin resistance gene, the sacB gene conferring sucrose sensitivity (17,34), and an origin of replication that is dependent on the pir gene product being provided in trans. E. coli SM10 pir (27) was used as a donor strain to introduce pKNG101.SF2::Cm into the chromosome of P. stuartii PR50 by a conjugal mating.…”

Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans

“…In addition, this enzyme is capable of adding fructosyl residues to a wide range of acceptor molecules (3). In Escherichia coli and other gram-negative bacteria such as Rhizobium, Agrobacterium, or Cyanobacterium species, expression of sacB is lethal in the presence of sucrose (2,4,15,17). Therefore, in these gram-negative bacteria sacB has been widely used as a positive selection marker to entrap transposable elements by screening for sacB inactivation (4,15).…”