Synthesis of the ferredoxin-like protein FdxN from Rhizobium meliloti bacteroids as a fusion protein in Escherichia coli.
To analyze the overexpression of the Rhizobium meliloti fdxN gene in Escherichia coli, different translational and transcriptional fusions were constructed. The translational signals of R. meliloti fdxN were recognized in E. coli as demonstrated by the use of in-frame lac fusions. Translational fusions consisting of the lacZ or the lpp gene fused in frame to the 3' end of the entire fdxN gene were expressed at high levels in E. coli. In contrast, the wild-type R. meliloti FdxN protein without a C-terminal fusion could only be detected using the very sensitive T7 promoter-polymerase system and not in immunoblots with antibodies against an FdxN-LacZ hybrid protein. Evidently, translational fusions to the 3' end of fdxN had a stabilizing effect on the expression of the fdxN gene. A constitutively expressed transcriptional fdxN fusion, which did not mediate detectable amounts of FdxN protein either in E. coli or in free-living R. meliloti cells, complemented the Fix- phenotype of an R. meliloti fdxN::[Tc] mutant strain to wild-type levels. Therefore, either low amounts of the wild-type FdxN protein are sufficient for symbiotic nitrogen fixation or there are stabilizing factors, which are present only in R. meliloti bacteroids but not in free-living R. meliloti cells. Fusion proteins consisting of FdxN and LacZ or a partial Lpp protein restored the Fix- phenotype of an R. meliloti fdxN mutant to 3 and 11%, respectively, indicating that a C-terminal fusion did not completely abolish the function of FdxN.
Riedel
K U
KU
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Germany.
Masepohl
B
B
Klipp
W
W
Pühler
A
A
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005288:Ferredoxins / Q000096:biosynthesis* / Q000235:genetics; D005816:Genetic Complementation Test; D015151:Immunoblotting; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D016962:Sinorhizobium meliloti / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D005288:Ferredoxins; D011993:Recombinant Fusion Proteins