Expression of β-galactosidase controlled by a nitrogenase promoter in stem nodules of Aeschynomene scabra

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Abstract
A 365-base-pair (bp) DNA fragment, containing the promoter region of the nitrogenase reductase (nifH) gene from stem Rhizobium BTAi1, was isolated and sequenced. The transcription initiation sites were localized at positions 152 (major initiation) and 114 (minor initiation) nucleotides upstream of the translation initiation codon. The 200-bp nucleotide sequence upstream of the nifH structural gene shows substantial homology to the corresponding nifH regions of cowpea Rhizobium (100%), Parasponia Rhizobium (89%) and R. japonicum (88%). The nifH promoter region of stem Rhizobium BTAi1 was fused to the lacZ gene of Escherichia coli. The fusion and a 1.6-kilobase DNA specifying neomycin phosphotransferase were inserted into a 3.4-kilobase fragment of stem Rhizobium chromosome, and the resulting construct was placed on a mobilizable vector, pREV1000. Stem Rhizobium transconjugants resistant to kanamycin contained the nifH promoter region-lacZ fusion linked to the neomycin phosphotransferase gene at the site of chromosomal homology. Analysis of the DNA from stable transconjugants showed integration of a single copy of these sequences into the chromosome by a double-reciprocal crossover event. The transconjugants formed N2-fixing nodules, indicating that the insertion occurred in a nonessential region of the stem Rhizobium chromosome. Transconjugant strain BTAi1000 grows on .beta.-galactosidase indicator plates under aerobic conditions as white colonies, whereas under microaerobic conditions as white colonies, whereas under microaerobic conditions (97% N2/3% O2), which derepress nitrogenase, the colonies (97% N2/3% O2), which derepress nitrogenase, the colonies turn blue within 15-24 h. .beta.-Galactosidase activity in derepressed cultures of BTAi1000 showed a 200-fold increase in comparison to the wild-type strain, whereas stem nodules formed by BTAi1000 exhibited 15- to 20-fold higher .beta.-galactosidase values than wild-type nodules. Nitrogenase promoter-dependent expression of .beta.-galactosidase in stem nodules was inhibited by fixed N, suggesting that the nifH promoter-lacZ fusion is controlled coordinately in trans with the native nif region.