Resistance to tellurite as a selection marker for genetic manipulations of Pseudomonas strains - PubMed (original) (raw)
Resistance to tellurite as a selection marker for genetic manipulations of Pseudomonas strains
J M Sanchez-Romero et al. Appl Environ Microbiol. 1998 Oct.
Abstract
Resistance to the toxic compound potassium tellurite (Telr) has been employed as a selection marker built into a set of transposon vectors and broad-host-range plasmids tailored for genetic manipulations of Pseudomonas strains potentially destined for environmental release. In this study, the activated Telr determinants encoded by the cryptic telAB genes of plasmid RK2 were produced, along with the associated kilA gene, as DNA cassettes compatible with cognate vectors. In one case, the Telr determinants were assembled between the I and O ends of a suicide delivery vector for mini-Tn5 transposons. In another case, the kilA and telAB genes were combined with a minimal replicon derived from a variant of Pseudomonas plasmid pPS10, which is able to replicate in a variety of gram-negative hosts and is endowed with a modular collection of cloning and expression assets. Either in the plasmid or in the transposon vector, the Telr marker was combined with a 12-kb DNA segment of plasmid pWW0 of Pseudomonas putida mt-2 encoding the upper TOL pathway enzymes. This allowed construction of antibiotic resistance-free but selectable P. putida strains with the ability to grow on toluene as the sole carbon source through an ortho-cleavage catabolic pathway.
Figures
FIG. 1
Telr resistance cassettes in recombinant plasmids and transposon vectors. The organization of the kilA locus of the RK2Telr plasmid, spanning the kilA telAB cistrons (29) (alternatively designated klaABC [8]), is shown at the top lined up with the neighboring regions of the plasmid. The location and orientation of each gene are indicated. The three genes appear to be transcribed from a single promoter upstream of kilA. telA and telB may be translationally coupled. The 3.0-kb RK2Telr _Hin_cII segments used to construct the tellurite resistance cassettes shown at the bottom are labeled with the designations of the plasmids.
FIG. 2
Organization of Telr transposon vector mini-Tn_5_ Tel of pJMT6 and its derivatives. This plasmid is the delivery vector for the minitransposon shown at the top. The suicide donation system used (lower part of the figure) was the pUT system (11) and included the Tn_5_ transposase gene devoid of Not_I sites (tnp*), an Apr selection marker (bla), an origin of transfer for RP4-mediated mobilization (oriT), and the origin of replication of plasmid R6K, which is dependent on the π protein encoded by the pir gene carried by specialized λ_pir E. coli hosts. The top part of the figure shows the Telr cassette of pJMT4 included in the mini-Tn_5_ transposon vector portion of the plasmid, including a single _Not_I site used for cloning heterologous DNA segments. The _Not_I insertions used included the luxAB genes (3.2 kb) in the case of pJMT8 and the upper TOL segment (upp TOL) (12 kb, not to scale) in pJMT7.
FIG. 3
Phenotypes endowed by Telr in P. putida. (A) Expression of Telr in P. putida cells grown in a liquid culture. The cells were transformed with plasmid pJPS6 and grown in LB medium supplemented with different concentrations of potassium tellurite (6, 12, 50, and 80 μg/ml). Note the blackening of the culture medium during growth. (B) Selection of P. putida exconjugants containing mini-Tn_5_ Tel. Mating of E. coli S17-1λ_pir_(pJMT6) and P. putida KT2442 was performed as indicated in the text, and the preparation was plated onto M9 medium containing citrate and tellurite. The P. putida cells which received the minitransposon in their chromosome gave rise to black colonies on the selection plate. (C) Selection of Telr P. putida clones expressing the upp TOL catabolic segment. The patches show the phenotypes resulting from expression of the upper TOL pathway in P. putida cells which received the corresponding DNA segment in Telr plasmid pJPS10 (top patches) or as mini-Tn_5_ Tel (upp TOL) (middle patches). The same clones were patched on M9 medium containing citrate and tellurite (left) and on M9 medium lacking a carbon source and then exposed to saturating toluene vapor (right). Positive (C+) and negative (C−) controls were included.
FIG. 4
Organization of the minimal replication-transfer segment of the pJPS plasmid series. The DNA sequence shown (length, 2,372 bp) includes a 1,815 bp _Sfi_I-_Avr_II–_Not_I segment spanning the sequence of the repA gene and the target oriV of plasmid pMM141 (uppercase letters), as well as a 545-bp _Not_I–_Sfi_I-_Avr_II fragment with the origin of transfer (oriT) of plasmid RK2 (lowercase letters). Depending on the specific construct (Fig. 5), these two segments may appear next to each other (as shown) or may be separated by _Not_I or _Sfi_I-_Avr_II inserts.
FIG. 5
Modular assembly of pJPS plasmids. The combinations of _Not_I and _Sfi_I-_Avr_II inserts possible with the exchange of selection cassettes and cloned segments are shown. The _Not_I site is the preferred site for insertion of segments that originated in the previously reported cloning and expression vector pNot18, pNot19, and pVTR plasmid series (Table 1). The names of some of the combinations of these segments are indicated in the text.
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