Cloning and sequencing of draTG genes and their downstream region of Azospirillum brasilense Yu62.

An 8-kb fragment was cloned by probing the gene library of Azospirillum brasilense Yu62 with the 4.0-kb draTG fragment of A. brasilense Sp7. DNA hybridization of this fragment demonstrated that draTG genes were located in a 3.0-kb EcoR I-Kpn I fragment, and were contiguous to the nifH gene. This 3.0-kb fragment was completely sequenced on both strands. Sequence analysis of the fragment revealed that it included the full-length draTG genes and two ORFs downstream of draG (ORF3 and incomplete ORF4). The draTG and downstream ORF3 are presumed to be cotranscribed as a single operon. Promoter element analysis of the sequenced region showed that there were some elements of the sigma 54-dependent promoter (DPEs and UASs) in the upstream region of draG and ORF3. This suggests that the draG and ORF3 might be transcribed independently in addition to being cotranscribed with draT. Both the DNA and amino acid sequences of the draTG genes from A. brasilense Yu62 were compared with those of other nitrogen-fixing bacteria. The results showed the draTG genes were highly conservative. There were only a few changes among strains and/or species. Homology analysis of ORF revealed that the ORF3 immediately downstream of draG was homologous not only with the ORF at the same position in Rhodospirillum rubrum and A. lipoferum, but also with the ORF14 of Azotobacter vinelandii and the ORF4 showed extensive similarity to yafJ of Escherichia coli.

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