Expression of mercuric reductase from Bacillus megaterium MB1 in eukaryotic microalga Chlorella sp. DT: an approach for mercury phytoremediation

A eukaryotic microalga, Chlorella sp. DT, was transformed with the Bacillus megaterium strain MB1 merA gene, encoding mercuric reductase (MerA), which mediates the reduction of Hg2+ to volatile elemental Hg0. The transformed Chlorella cells were selected first by hygromycin B and then by HgCl2. The existence of merA gene in the genomic DNA of transgenic strains was shown by polymerase chain reaction amplification, while the stable integration of merA into genomic DNA of transgenic strains was confirmed by Southern blot analysis. The ability to remove Hg2+ in merA transgenic strains was higher than that in the wild type. The merA transgenic strains showed higher growth rate and photosynthetic activity than the wild type did in the presence of a toxic concentration of Hg2+. Cultured with Hg2+, the expression level of superoxide dismutase in transgenic strains was lower than that in the wild type, suggesting that the transgenic strains faced a lower level of oxidative stress. All the results indicated that merA gene was successfully integrated into the genome of transgenic strains and functionally expressed to promote the removal of Hg2+.

[1]  R. Schoeny,et al.  Mercury study report to Congress. Volume 5. Health effects of mercury and mercury compounds , 1997 .

[2]  R. Twyman,et al.  Selectable and Screenable Markers for Rice Transformation , 2002 .

[3]  Lewis M. Brown,et al.  Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea , 1991, Current Genetics.

[4]  G. Krause,et al.  Chlorophyll fluorescence as a tool in plant physiology , 1984, Photosynthesis Research.

[5]  R. Burlingame,et al.  Stable Transformation of Chlorella: Rescue of Nitrate Reductase-Deficient Mutants with the Nitrate Reductase Gene , 1997, Current Microbiology.

[6]  J. Benemann,et al.  Bioremoval of heavy metals by the use of microalgae. , 1993, Biotechnology advances.

[7]  D. Inthorn,et al.  Sorption of mercury , cadmium and lead by microalgae , 2002 .

[8]  Susan M. Miller,et al.  Bacterial mercury resistance from atoms to ecosystems. , 2003, FEMS microbiology reviews.

[9]  D. Morse,et al.  HEAVY METAL–INDUCED OXIDATIVE STRESS IN ALGAE 1 , 2003 .

[10]  P. Juneau,et al.  Evaluation of different algal species sensitivity to mercury and metolachlor by PAM-fluorometry. , 2001, Chemosphere.

[11]  R. Meagher,et al.  Toward detoxifying mercury‐polluted aquatic sediments with rice genetically engineered for mercury resistance , 2003, Environmental toxicology and chemistry.

[12]  Pei-Chung Chen,et al.  Changes in the Productivity and Nuclear Divisions in Synchronous Chlorella and Circadian Rhythm , 1986 .

[13]  Wenbin Li,et al.  Highly efficient expression of rabbit neutrophil peptide-1 gene in Chlorella ellipsoidea cells , 2001, Current Genetics.

[14]  R. Moreno-Sánchez,et al.  Mercury uptake and removal by Euglena gracilis , 2000, Archives of Microbiology.

[15]  K. Chow,et al.  Electrotransformation of Chlorella vulgaris , 1999, Plant Cell Reports.

[16]  I. Hwang,et al.  Stable Integration and Functional Expression of Flounder Growth Hormone Gene in Transformed Microalga, Chlorella ellipsoidea , 2002, Marine Biotechnology.

[17]  H. Linskens,et al.  Testing for Genetic Manipulation in Plants , 2002, Molecular Methods of Plant Analysis.

[18]  Richard T. Sayre,et al.  Molecular Mechanisms of Proline-Mediated Tolerance to Toxic Heavy Metals in Transgenic Microalgae , 2002, The Plant Cell Online.

[19]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[20]  Richard L. Hawkins,et al.  Expression of Human Growth Hormone by the Eukaryotic Alga, Chlorella , 1999, Current Microbiology.

[21]  Scott A. Merkle,et al.  Development of transgenic yellow poplar for mercury phytoremediation , 1998, Nature Biotechnology.

[22]  R. Wu,et al.  Isolation of an efficient actin promoter for use in rice transformation. , 1990, The Plant cell.

[23]  A O Summers,et al.  Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Physiological adaptation during cell dehydration and rewetting of a newly‐isolated Chlorella species , 1996 .

[25]  D. Allemand,et al.  Characterization of superoxide dismutases in anoxia- and hyperoxia-tolerant symbiotic cnidarians. , 2003, Biochimica et biophysica acta.

[26]  M. Harada,et al.  Minamata disease: methylmercury poisoning in Japan caused by environmental pollution. , 1995, Critical reviews in toxicology.

[27]  G. Endo,et al.  Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive Bacterium bacillus megaterium MB1, a strain isolated from minamata bay, Japan. , 1999, Gene.

[28]  D. Arnon COPPER ENZYMES IN ISOLATED CHLOROPLASTS. POLYPHENOLOXIDASE IN BETA VULGARIS. , 1949, Plant physiology.

[29]  Chin-Bum Lee,et al.  Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber : in gel enzyme activity assays , 2000 .

[30]  I. Fridovich,et al.  Superoxide dismutase and chilling injury in Chlorella ellipsoidea. , 1984, Archives of biochemistry and biophysics.

[31]  K. Niyogi,et al.  Photo-oxidative Stress in a Xanthophyll-deficient Mutant of Chlamydomonas* , 2004, Journal of Biological Chemistry.

[32]  R. Meagher,et al.  Phytodetoxification of hazardous organomercurials by genetically engineered plants , 2000, Nature Biotechnology.

[33]  S. Wilkinson,et al.  Mercury removal by immobilized algae in batch culture systems , 1990, Journal of Applied Phycology.

[34]  Xianzhong Feng,et al.  Differential mercury volatilization by tobacco organs expressing a modified bacterial merA gene , 2001, Cell Research.

[35]  D. Inthorn,et al.  SORPTION OF MERCURY, CADMIUM AND LEAD BY MICRO ALGAE , 2002 .

[36]  W. Kabsch,et al.  Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607 , 1991, Nature.