Nickel-Resistance-Based Minitransposons: New Tools for Genetic Manipulation of Environmental Bacteria

Abstract

The ncc and nre nickel resistance determinants from Ralstonia eutropha-like strain 31A were used to construct mini-Tn5 transposons. Broad host expression of nickel resistance was observed for the nre minitransposons in members of the ␣, ␤, and ␥ subclasses of the Proteobacteria, while the ncc minitransposons expressed nickel resistance only in R. eutropha-like strains.Several nickel resistance determinants have been identified in Ralstonia eutropha (Alcaligenes eutrophus) (24) strains isolated from different biotopes heavily polluted with heavy metals. The cnrYXHCBA operon of R. eutropha CH34 plasmid pMOL28 (12), which mediates medium levels of nickel resistance (up to 10 mM) and cobalt resistance, is the most thoroughly studied determinant (3,11,17,18,20). The resistance mechanism mediated by cnr is inducible and is due to an energy-dependent efflux system driven by a chemo-osmotic proton-antiporter system (6,18,22,23). A 14.5-kb BamHI fragment of plasmid pTOM9 from R. eutropha-like strain 31A (Alcaligenes xylosoxidans 31A) (10) and a similar BamHI fragment of plasmid pGOE2 from R. eutropha-like strain KTO2 were also found to encode Ni resistance. On both fragments a locus mediating high-level nickel resistance (up to 20 to 50 mM) and a locus mediating low-level nickel resistance (3 mM) were identified and designated ncc and nre, respectively (15, 16). The nccYXHCBAN determinant, which except for the nccN gene is very similar to cnr, causes high levels of nickel and cobalt resistance and a low level of cadmium resistance in R. eutropha. Neither cnr nor ncc is expressed in Escherichia coli. On the other hand, the 1.8-kb nre locus causes low levels of nickel resistance in both Ralstonia and E. coli (16). An nre-like determinant, which could be expressed in E. coli and Citrobacter freundii, was also found in Klebsiella oxytoca CCUG15788 (19, 20).Recently, amplified ribosomal DNA restriction analysis was used to determine the phylogenetic position of zinc-and nickel-resistant Ralstonia-like strains (2). The ncc operon was found in many nickel-resistant R. eutropha-like strains and in environmental strains in the direct vicinity of the genus Burkholderia (2), a member of the ␤ subclass of the class Proteobacteria like the genus Ralstonia. This might indicate that ncc has range of expression broader than the genus Ralstonia.Heavy metal resistance markers with broad host expression ranges have been shown to be useful for genetic manipulation of Pseudomonas strains potentially designated for environmental release (14). Broad-host-range expression of ncc-nre was recently confirmed by Dong et al. (7), who found ncc-nre-based Ni resistance in Comamonas, Sphingobacterium heparinum, flavobacteria, and even gram-positive bacteria related to Arthrobacter. However, it was not clear from this study which of the Ni resistance determinants was responsible for the broadhost-range Ni resistance. In addition, plasmid instability problems were encountered with some of the transconjugants. In order to study the range of expression of ...

Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants

“…(Ni R , Km R ), a derivative of the endophytic bacterium B. cepacia L.S.2.4, was used 15,16 . B. cepacia G4 (pTOM, Tol + ) 26 served as a donor strain for toluene degradation.…”

“…B. cepacia G4 (pTOM, Tol + ) 26 served as a donor strain for toluene degradation. After conjugation 16 , transconjugants that were resistant to nickel plus kanamycin and could grow on toluene as sole carbon source were selected on '284' minimal medium 27 supplemented with 1 mM NiCl 2 plus 100 µg/ml kanamycin, while the plates were incubated under a toluene atmosphere to provide the carbon source. The 284 medium contains per liter distilled water: 6.06g Tris-HCl, 4.68 g NaCl, 1.49 g KCl, 1.07 g NH 4 Cl, 0.43 g NaSO 4 and G4 were used.…”

Genome Survey and Characterization of Endophytic Bacteria Exhibiting a Beneficial Effect on Growth and Development of Poplar Trees

“…In order to follow endophytic colonization of poplar trees, we successfully labeled Enterobacter sp. strain 638, Pseudomonas putida W619 (36), and Serratia proteamaculans 568 with gfp using Escherichia coli S17-1/pir (pUT::miniTn5-Km-gfp) (37) as a donor in conjugation (35). Kanamycin (100 g/ml)-resistant transconjugants were selected on 284 minimal medium (32) complemented with a carbon mix (a mixture of glucose, gluconate, lactate, succinate, and acetate was added at 0.05% [wt/vol] each) and subsequently tested for gfp expression under UV light.…”

Nickel Resistance Determinants in Bradyrhizobium Strains from Nodules of the Endemic New Caledonia Legume Serianthes calycina

“…strain BTAi1, respectively. It also shares 51% amino acid identity with NccA from A. xylosoxidans, 49% amino acid identity with CnrA from C. metallidurans, and 47% amino acid identity with CzcA from C. metallidurans; all of the latter resistance determinants were functionally demonstrated in these organisms (7,18,27). As we observed a clear correlation between high nickel resistance and the presence of the cnrA gene, but not the presence of the nreB gene, we performed a functional analysis of the former marker.…”