GFP-tagged multimetal-tolerant bacteria and their detection in the rhizosphere of white mustard

The introduction of rhizobacteria that tolerate heavy metals is a promising approach to support plants involved in phytoextraction and phytostabilisation. In this study, soil of a metal-mine wasteland was analyzed for the presence of metal-tolerant bacterial isolates, and the tolerance patterns of the isolated strains for a number of heavy metals and antibiotics were compared. Several of the multimetal-tolerant strains were tagged with a broad host range reporter plasmid (i.e. pPROBE-NT) bearing a green fluorescent protein marker gene (gfp). Overall, the metal-tolerant isolates were predominately Gram-negative bacteria. Most of the strains showed a tolerance to five metals (Zn, Cu, Ni, Pb and Cd), but with differing tolerance patterns. From among the successfully tagged isolates, we used the transconjugant Pseudomonas putida G25 (pPROBE-NT) to inoculate white mustard seedlings. Despite a significant decrease in transconjugant abundance in the rhizosphere, the gfp-tagged cells survived on the root surfaces at a level previously reported for root colonisers

Characterization of the Metabolically Modified Heavy Metal-Resistant Cupriavidus metallidurans Strain MSR33 Generated for Mercury Bioremediation

BACKGROUND: Mercury-polluted environments are often contaminated with other heavy metals. Therefore, bacteria with resistance to several heavy metals may be useful for bioremediation. Cupriavidus metallidurans CH34 is a model heavy metal-resistant bacterium, but possesses a low resistance to mercury compounds. METHODOLOGY/PRINCIPAL FINDINGS: To improve inorganic and organic mercury resistance of strain CH34, the IncP-1β plasmid pTP6 that provides novel merB, merG genes and additional other mer genes was introduced into the bacterium by biparental mating. The transconjugant Cupriavidus metallidurans strain MSR33 was genetically and biochemically characterized. Strain MSR33 maintained stably the plasmid pTP6 over 70 generations under non-selective conditions. The organomercurial lyase protein MerB and the mercuric reductase MerA of strain MSR33 were synthesized in presence of Hg(2+). The minimum inhibitory concentrations (mM) for strain MSR33 were: Hg(2+), 0.12 and CH(3)Hg(+), 0.08. The addition of Hg(2+) (0.04 mM) at exponential phase had not an effect on the growth rate of strain MSR33. In contrast, after Hg(2+) addition at exponential phase the parental strain CH34 showed an immediate cessation of cell growth. During exposure to Hg(2+) no effects in the morphology of MSR33 cells were observed, whereas CH34 cells exposed to Hg(2+) showed a fuzzy outer membrane. Bioremediation with strain MSR33 of two mercury-contaminated aqueous solutions was evaluated. Hg(2+) (0.10 and 0.15 mM) was completely volatilized by strain MSR33 from the polluted waters in presence of thioglycolate (5 mM) after 2 h. CONCLUSIONS/SIGNIFICANCE: A broad-spectrum mercury-resistant strain MSR33 was generated by incorporation of plasmid pTP6 that was directly isolated from the environment into C. metallidurans CH34. Strain MSR33 is capable to remove mercury from polluted waters. This is the first study to use an IncP-1β plasmid directly isolated from the environment, to generate a novel and stable bacterial strain useful for mercury bioremediation