Rhizobium etli Genetically Engineered for the Heterologous Expression of Vitreoscilla sp. Hemoglobin: Effects on Free-Living and Symbiosis

Oxygen concentration is an environmental signal that regulates nitrogen fixation in the Rhizobium-legume symbiosis. We investigated the effect of the heterologous expression of Vitreoscilla sp. hemoglobin (VHb), which is an oxygen-binding protein, in Rhizobium etli. The vhb gene and its native promoter were subcloned in the plasmid pMR4 and transformed into the R. etli strain CE3. Free-living cultures of engineered R. etli CE3 expressed the vhb gene, as shown by the CO-difference spectral and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of cell extracts. The expression of vhb in free-living R. etli grown under most limiting oxygen concentrations resulted in an increase in respiratory activity, chemical energy content, and expression of the nitrogen-fixation gene nifHc. Bacteroids isolated from nodules of bean plants inoculated with the engineered R. etli CE3 expressed the vhb gene, as shown by RNA slot-blot analysis. Bean plants inoculated with the engineered strain exhibi...

[1]  C. Quinto,et al.  Nitrogenase reductase: A functional multigene family in Rhizobium phaseoli. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Wakabayashi,et al.  Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla , 1986, Nature.

[3]  E. Morett,et al.  Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes , 1996, Journal of bacteriology.

[4]  D. Webster,et al.  The binding of cyanide and carbon monoxide to cytochrome o purified from Vitreoscilla. Evidence for subunit interaction in the reduced protein. , 1978, The Journal of biological chemistry.

[5]  J. Bailey,et al.  Intracellular Expression of Vitreoscilla Hemoglobin Alters the Aerobic Metabolism of Saccharomyces cerevisiae , 1994, Biotechnology progress.

[6]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[7]  S. Encarnación,et al.  Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate , 1996, Journal of bacteriology.

[8]  D. Webster,et al.  Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E. coli through transcriptional fusion. , 1990, Nucleic acids research.

[9]  D. Helinski,et al.  A region of the broad-host-range plasmid RK2 causes stable in planta inheritance of plasmids in Rhizobium meliloti cells isolated from alfalfa root nodules , 1992, Journal of bacteriology.

[10]  D. Layzell,et al.  Oxygen and the regulation of nitrogen fixation in legume nodules , 1990 .

[11]  A. Mendoza,et al.  The enhancement of ammonium assimilation in Rhizobium etli prevents nodulation of Phaseolus vulgaris. , 1995, Molecular plant-microbe interactions : MPMI.

[12]  C. Appleby.  LEGHEMOGLOBIN AND RHIZOBIUM RESPIRATION , 1984 .

[13]  D. Webster,et al.  Presence of the bacterial hemoglobin gene improves α-amylase production of a recombinantEscherichia coli strain , 1990 .

[14]  H. Noller,et al.  Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. , 1981, Journal of molecular biology.

[15]  J. Bailey,et al.  Transgenic tobacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolite production , 1997, Nature Biotechnology.

[16]  J. Wittenberg.  Facilitated oxygen diffusion. The role of leghemoglobin in nitrogen fixation by bacteroids isolated from soybean root nodules. , 1974, The Journal of biological chemistry.

[17]  D. Helinski,et al.  Transcription and autoregulation of the stabilizing functions of broad‐host‐range plasmid RK2 in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa , 1992, Molecular microbiology.

[18]  R. Poole,et al.  Isolation of a Rhizobium phaseoli cytochrome mutant with enhanced respiration and symbiotic nitrogen fixation , 1989, Journal of bacteriology.

[19]  J. Bailey,et al.  Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli , 1988, Nature.

[20]  M. Gilles-Gonzalez,et al.  A haemoprotein with kinase activity encoded by the oxygen sensor of Rhizobium meliloti , 1991, Nature.

[21]  M. Foussard,et al.  Oxygen as a key developmental regulator of Rhizobium meliloti N2-fixation gene expression within the alfalfa root nodule. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[22]  D. Webster,et al.  Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. , 1988, Gene.

[23]  J. Bailey,et al.  Actinorhodin Production By Streptomyces coelicolor and Growth of Streptomyces lividans Are Improved by the Expression of a Bacterial Hemoglobin , 1991, Bio/Technology.

[24]  J. Bailey,et al.  Expression of Intracellular Hemoglobin Improves Protein Synthesis in Oxygen-Limited Escherichia coli , 1990, Bio/Technology.

[25]  D. Webster,et al.  Reduced nicotinamide adenine dinucleotide cytochrome o reductase associated with cytochrome o purified from Vitreoscilla. Evidence for an intermediate oxygenated form of cytochrome o. , 1974, The Journal of biological chemistry.

[26]  R. Hardison,et al.  Hemoglobins from bacteria to man: evolution of different patterns of gene expression. , 1998, The Journal of experimental biology.

[27]  M. R. O'Brian,et al.  Molecular aspects of the energetics of nitrogen fixation in Rhizobium-legume symbioses. , 1989, Biochimica et biophysica acta.

[28]  J. Guest,et al.  Adaptive responses to oxygen limitation in Escherichia coli. , 1991, Trends in biochemical sciences.

[29]  J. Martín,et al.  The Production of Cephalosporin C by Acremonium chrysogenum is Improved by the Intracellular Expression of a Bacterial Hemoglobin , 1993, Bio/Technology.

[30]  J. Leemans,et al.  Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions , 1984, Journal of bacteriology.

[31]  D. Helinski,et al.  Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[32]  D. Webster,et al.  Genetic engineering of Serratia marcescens with bacterial hemoglobin gene: effects on growth, oxygen utilization, and cell size. , 1998, Biotechnology and bioengineering.

[33]  P. Wood.  Bacterial proteins with CO-binding b- or c-type haem. Functions and absorption spectroscopy. , 1984, Biochimica et biophysica acta.

[34]  J. Bailey,et al.  Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions. , 1994, European journal of biochemistry.

[35]  James E. Bailey,et al.  Chemical engineering of cellular processes , 1995 .

[36]  H. Boyer,et al.  A complementation analysis of the restriction and modification of DNA in Escherichia coli. , 1969, Journal of molecular biology.