Vector insertion mutagenesis of Rhizobium sp. strain ORS571: direct cloning of mutagenized DNA sequences

Abstract

When the limited-host-range plasmid pVP2021 carrying Tn5 was mobilized into Rhizobium sp. strain ORS571 and stable acquisition of TnS was selected, ORS571 plasmid-genome cointegrates were exclusively obtained; direct TnS transposition was never observed. In every case, genomic cointegrates exhibited an additional (third) IS50 element that bordered VP2021 DNA sequences but maintained a single Tn5 element. Genomic cointegrates containing IS50 triplications were stable; neither phenotypic reversion nor resolution was detectable. Auxotrophic mutant strains (vector insertion mutants) were identified at expected frequencies among derivatives carrying ostensibly random genomic pVP2021 insertions; N2 fixation (Nif)-defective vector insertion mutants were observed among these derivatives at a frequency of 10-3. The presence of integrated pVP2021 in ORS571 nif::VP2021 mutant genomes enabled VP2021 to constitute an endogenous cloning vector. After EcoRI or KpnI digestions, genomic nif::pVP2021 DNA sequences contiguous with integrated pVP2021 were directly cloned as new replicons without addition of an exogenous vector. Recombinant plasmids derived from two such nif::pVP2021 mutants hybridized to previously analyzed ORS571 Nif DNA sequences. Recombinant plasmid DNA and ORS571 Nif region DNA were found to be colinear; pVP2021 insertions could be accurately mapped. pVP2021 insertion-mutagenesis thus allows the direct cloning of ORS571 gene sequences for which mutant phenotypes can be selected or screened.

Molecular biology of bacterial methanol oxidation

“…[15], Rhizobium spp. [16], Agrobacterium spp. [17] and the autotrophic organism Alcaligenes eutrophus [18].…”