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2. Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and polyphosphate kinase

Author

Gonzalez-Ruiz Gloriene, Alvarez Derry, Ruiz Oscar N, and Torres Cesar

Subjects
Biotechnology, TP248.13-248.65
Abstract

Abstract Background The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1) and polyphosphate kinase (ppk) genes in bacteria in order to provide high mercury resistance and accumulation. Results In this study, bacterial transformation with transcriptional and translational enhanced vectors designed for the expression of metallothionein and polyphosphate kinase provided high transgene transcript levels independent of the gene being expressed. Expression of polyphosphate kinase and metallothionein in transgenic bacteria provided high resistance to mercury, up to 80 μM and 120 μM, respectively. Here we show for the first time that metallothionein can be efficiently expressed in bacteria without being fused to a carrier protein to enhance mercury bioremediation. Cold vapor atomic absorption spectrometry analyzes revealed that the mt-1 transgenic bacteria accumulated up to 100.2 ± 17.6 μM of mercury from media containing 120 μM Hg. The extent of mercury remediation was such that the contaminated media remediated by the mt-1 transgenic bacteria supported the growth of untransformed bacteria. Cell aggregation, precipitation and color changes were visually observed in mt-1 and ppk transgenic bacteria when these cells were grown in high mercury concentrations. Conclusion The transgenic bacterial system described in this study presents a viable technology for mercury bioremediation from liquid matrices because it provides high mercury resistance and accumulation while inhibiting elemental mercury volatilization. This is the first report that shows that metallothionein expression provides mercury resistance and accumulation in recombinant bacteria. The high accumulation of mercury in the transgenic cells could present the possibility of retrieving the accumulated mercury for further industrial applications.

Published
2011
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13. Characterization of heterologous multigene operons in transgenic chloroplasts. Transcription, processing, and translation (1)

Author

Quesada-Vargas, Tania, Ruiz, Oscar N., and Daniell, Henry

Subjects
Genetically modified plants -- Research, Genetically modified plants -- Genetic aspects, Chloroplasts -- Research, Chloroplasts -- Genetic aspects, Tobacco (Plant) -- Research, Tobacco (Plant) -- Genetic aspects, Plant genetics -- Research, Biological sciences, Science and technology
Published
2005

36. Engineering Cytoplasmic Male Sterility via the Chloroplast Genome by Expression of β-Ketothiolase1

Author

Ruiz, Oscar N. and Daniell, Henry

Subjects
Cytoplasm, Chloroplasts, Acinetobacter, Transcription, Genetic, Gene Expression Regulation, Plant, Reproduction, food and beverages, Breakthrough Technologies, Cloning, Molecular, Acetyl-CoA C-Acyltransferase, Plants, Genetically Modified, Gene Expression Regulation, Enzymologic, Genome, Plant
Abstract

While investigating expression of the polydroxybutyrate pathway in transgenic chloroplasts, we addressed the specific role of beta-ketothiolase. Therefore, we expressed the phaA gene via the chloroplast genome. Prior attempts to express the phaA gene in transgenic plants were unsuccessful. We studied the effect of light regulation of the phaA gene using the psbA promoter and 5' untranslated region, and evaluated expression under different photoperiods. Stable transgene integration into the chloroplast genome and homoplasmy were confirmed by Southern analysis. The phaA gene was efficiently transcribed in all tissue types examined, including leaves, flowers, and anthers. Coomassie-stained gel and western blots confirmed hyperexpression of beta-ketothiolase in leaves and anthers, with proportionately high levels of enzyme activity. The transgenic lines were normal except for the male-sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Floral developmental studies revealed that transgenic lines showed an accelerated pattern of anther development, affecting their maturation, and resulted in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium, and vacuolation affected pollen grains and resulted in the irregular shape or collapsed phenotype. Reversibility of the male-sterile phenotype was observed under continuous illumination, resulting in viable pollen and copious amount of seeds. This study results in the first engineered cytoplasmic male-sterility system in plants, offers a new tool for transgene containment for both nuclear and organelle genomes, and provides an expedient mechanism for F(1) hybrid seed production.

Published
2005

39. Phytoremediation of Organomercurial Compounds via Chloroplast Genetic Engineering1

Author

Ruiz, Oscar N., Hussein, Hussein S., Terry, Norman, and Daniell, Henry

Subjects
Biodegradation, Environmental, Chloroplasts, Phenotype, Transformation, Genetic, Organomercury Compounds, Genetic Vectors, Tobacco, food and beverages, Lyases, Tetradecanoylphorbol Acetate, Genetic Engineering, Plants, Genetically Modified, Research Article
Abstract

Mercury (Hg), especially in organic form, is a highly toxic pollutant affecting plants, animals, and man. In plants, the primary target of Hg damage is the chloroplast; Hg inhibits electron transport and photosynthesis. In the present study, chloroplast genetic engineering is used for the first time to our knowledge to enhance the capacity of plants for phytoremediation. This was achieved by integrating a native operon containing the merA and merB genes (without any codon modification), which code for mercuric ion reductase (merA) and organomercurial lyase (merB), respectively, into the chloroplast genome in a single transformation event. Stable integration of the merAB operon into the chloroplast genome resulted in high levels of tolerance to the organomercurial compound, phenylmercuric acetate (PMA) when grown in soil containing up to 400 μm PMA; plant dry weights of the chloroplast transformed lines were significantly higher than those of wild type at 100, 200, and 400 μm PMA. That the merAB operon was stably integrated into the chloroplast genome was confirmed by polymerase chain reaction and Southern-blot analyses. Northern-blot analyses revealed stable transcripts that were independent of the presence or absence of a 3′-untranslated region downstream of the coding sequence. The merAB dicistron was the more abundant transcript, but less abundant monocistrons were also observed, showing that specific processing occurs between transgenes. The use of chloroplast transformation to enhance Hg phytoremediation is particularly beneficial because it prevents the escape of transgenes via pollen to related weeds or crops and there is no need for codon optimization to improve transgene expression. Chloroplast transformation may also have application to other metals that affect chloroplast function.

Published
2003

42. Graphene Oxide: A Nonspecific Enhancer of Cellular Growth

Author

Ruiz, Oscar N., primary, Fernando, K. A. Shiral, additional, Wang, Baojiang, additional, Brown, Nicholas A., additional, Luo, Pengju George, additional, McNamara, Nicholas D., additional, Vangsness, Marlin, additional, Sun, Ya-Ping, additional, and Bunker, Christopher E., additional

Published
2011
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48. Chloroplast Genetic Engineering to Improve Agronomic Traits.

Author

Walker, John M., Peña, Leandro, Daniell, Henry, Ruiz, Oscar N., and Dhingra, Amit

Abstract

Major crop losses occur annually as a result of biotic and abiotic stresses. The ability to hyperexpress foreign proteins, single-step multigene engineering, lack of positive effect and gene silencing, vector sequences and pleiotropic effects have resulted in several hundred-fold more tolerance to the environmental stresses via chloroplast genetic engineering than nuclear genetic engineering. Maternal inheritance of chloroplast expressed transgenes renders the technology environmentally safe and promotes public acceptance. This review provides protocols for engineering agronomic traits like insect, herbicide and disease resistance; salt and drought tolerance; and phytoremediation via chloroplast genome. [ABSTRACT FROM AUTHOR]

Published
2004
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50. Engineering Cytoplasmic Male Sterility via the Chloroplast Genome by Expression of ²-Ketothiolase.

Author

Ruiz, Oscar N. and Daniell, Henry

Subjects
*PLANT genetic engineering, *CYTOPLASMIC male sterility, *CHLOROPLASTS, *PLANT genomes, *GENE expression, *PLANT genetics
Abstract

While investigating expression of the polydroxybutyrate pathway in transgenic chloroplasts, we addressed the specific role of β-ketothiolase. Therefore, we expressed the phaA gene via the chloroplast genome. Prior attempts to express the phaA gene in transgenic plants were unsuccessful. We studied the effect of light regulation of the phaA gene using the psbA promoter and 5' untranslated region, and evaluated expression under different photoperiods. Stable transgene integration into the chloroplast genome and homoplasmy were confirmed by Southern analysis. The phaA gene was efficiently transcribed in all tissue types examined, including leaves, flowers, and anthers. Coomassie-stained gel and western blots confirmed hyperexpression of β-ketothiolase in leaves and anthers, with proportionately high levels of enzyme activity. The transgenic lines were normal except for the male-sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Floral developmental studies revealed that transgenic lines showed an accelerated pattern of anther development, affecting their maturation, and resulted in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium, and vacuolation affected pollen grains and resulted in the irregular shape or collapsed phenotype. Reversibility of the male-sterile phenotype was observed under continuous illumination, resulting in viable pollen and copious amount of seeds. This study results in the first engineered cytoplasmic male-sterility system in plants, offers a new tool for transgene containment for both nuclear and organelle genomes, and provides an expedient mechanism for F1 hybrid seed production. [ABSTRACT FROM AUTHOR]

Published
2005
Full Text
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