C4-dicarboxylate transport proteins of di-azotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ, and dctM grouped together. Its nucleotide and amino acid se...C4-dicarboxylate transport proteins of di-azotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ, and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene en-coding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii. Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively. The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobiliza-tion plasmid pSZ21. The resultant plasmid was named pSZY6. By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501, randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was con-structed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20, 10 and 5 mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A1501. This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.展开更多
基金supported by the National Basic Research Priorities Programme(Grant No.001CB108904)the National Natural Science Foundation of China(Grant No.39580001).
文摘C4-dicarboxylate transport proteins of di-azotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ, and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene en-coding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii. Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively. The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobiliza-tion plasmid pSZ21. The resultant plasmid was named pSZY6. By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501, randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was con-structed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20, 10 and 5 mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A1501. This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.
