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

Piotrowska-Seget Z, Beściak G, Bernaś T, Kozdrój J - Ann. Microbiol. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

ABSTRACT
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.

No MeSH data available.


Photograph of microscopic image of fluorescent green fluorescent protein (gfp)-tagged transconjugant Pseudomonas putida G25. The photograph was obtained using a confocal laser scanning microscope
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351603&req=5

Fig2: Photograph of microscopic image of fluorescent green fluorescent protein (gfp)-tagged transconjugant Pseudomonas putida G25. The photograph was obtained using a confocal laser scanning microscope

Mentions: The underlying rationale for isolating metal-tolerant bacteria from metalliferous soils is their potential application for bioaugmentation-assisted phytoremediation of these habitats. Various markers, often with different detection frequencies, have been used to track the fates of these metal-tolerant bacterial isolates following their reintroduction into the soil (Zaidi et al. 2006; Ma et al. 2009). In our study, we successfully introduced the plasmid pPROBE-NT by triparental conjugation into four strains: Pseudomonas putida G16, P. putida G20, P. putida G25 and Comamonas acidovorans. The growth of these strains on selective agar medium containing Ap (100 μg ml-1), Km (20 μg ml-1) and 3 mM Zn was used to select for the pPROBE-NT transconjugants as neither donor strains nor recipients were able to grow on the medium amended with these markers. We also checked the putative transconjugants for GFP production under the confocal microscope. All transconjugants gave positive results, with P. putida G25 (pPROBE-NT) showing the strongest green colour, indicating intensive synthesis of the protein (Fig. 2). Therefore, we used transconjugant strain G25 for further survival experiments. An approach that involves the tagging of bacterial strains with gfp genes by plasmid transfer or recombination into the chromosome has been recommended by various authors because of the efficient detectability of the tag and the low energetic burden placed on the cells. (Kendall and Badminton 1998; Errampalli et al. 1999; Kozdrój et al. 2004).Fig. 2


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

Piotrowska-Seget Z, Beściak G, Bernaś T, Kozdrój J - Ann. Microbiol. (2011)

Photograph of microscopic image of fluorescent green fluorescent protein (gfp)-tagged transconjugant Pseudomonas putida G25. The photograph was obtained using a confocal laser scanning microscope
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351603&req=5

Fig2: Photograph of microscopic image of fluorescent green fluorescent protein (gfp)-tagged transconjugant Pseudomonas putida G25. The photograph was obtained using a confocal laser scanning microscope
Mentions: The underlying rationale for isolating metal-tolerant bacteria from metalliferous soils is their potential application for bioaugmentation-assisted phytoremediation of these habitats. Various markers, often with different detection frequencies, have been used to track the fates of these metal-tolerant bacterial isolates following their reintroduction into the soil (Zaidi et al. 2006; Ma et al. 2009). In our study, we successfully introduced the plasmid pPROBE-NT by triparental conjugation into four strains: Pseudomonas putida G16, P. putida G20, P. putida G25 and Comamonas acidovorans. The growth of these strains on selective agar medium containing Ap (100 μg ml-1), Km (20 μg ml-1) and 3 mM Zn was used to select for the pPROBE-NT transconjugants as neither donor strains nor recipients were able to grow on the medium amended with these markers. We also checked the putative transconjugants for GFP production under the confocal microscope. All transconjugants gave positive results, with P. putida G25 (pPROBE-NT) showing the strongest green colour, indicating intensive synthesis of the protein (Fig. 2). Therefore, we used transconjugant strain G25 for further survival experiments. An approach that involves the tagging of bacterial strains with gfp genes by plasmid transfer or recombination into the chromosome has been recommended by various authors because of the efficient detectability of the tag and the low energetic burden placed on the cells. (Kendall and Badminton 1998; Errampalli et al. 1999; Kozdrój et al. 2004).Fig. 2

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

ABSTRACT
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.

No MeSH data available.