Abstract
C4-dicarboxylate transport proteins of diazotrophPseudomonas stutzen were encoded bydctPQM genes. Nucleotide sequence analysis indicated thatdctP, dctQ, anddctM grouped together. Its nucleotide and amino acid sequence shared high homology with that ofdctP gene encoding periplasmic C4-dicarboxylate-binding protein anddctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixingAotobacter vinelandii. Structural analysis showed that DctP ofP. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively. The fragment containing the completedctPQM genes was cloned into the Tn5 transposon region of suicide mobilization plasmid pSZ21. The resultant plasmid was named pSZY6. By triparental mating, Tn5 transposon carrying thedctPQM genes inserted into the genome of the wild type strain A1501, randomly. The recombinant strain A-142 which harboured an extra copy ofdctPQM genes was constructed and identified by PCR amplification ofnpt 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 ofdctPQM genes could increase the activity of nitrogen fixation ofP. stutzeri strain A1501.
Published Version
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