Part:BBa_K535004
NifH promoter -> Rhizobium etli
This is the promoter region of one of the three nitrogenase reductase gene copies in Rhizobium etli, nitrogenase reductase a (nifHa). This region comprises a σ54-dependent promoter located between position -24 and -12 relative to the transcription start site, as well as a binding site for NifA, a positive transcriptional regulator [1]. NifA is a member of the enhancer-binding protein (EBP) family of transcriptional regulators that activate gene expression in concert with RNA polymerase containing the specialized σ54 sigma factor (RpoN) to allow the polymerase core to recognize the -24/-12 promoter [3] in response to oxygen and/or fixed nitrogen levels. The binding site for NifA is located ~200 bp upstream of the transcription start site [2].
Activation of gene expression by NifA (along with the specialized sigma factor σ54) occurs only at low oxygen concentrations [2]. In R. etli, the nifA gene is located on the symbiotic plasmid [4]. This promoter region, in addition to other similar promoter regions ensure the expression of the nitrogen fixation apparatus during symbiosis of the bacteroid with its legume host [3].
We decided to use this promoter region in our project to promote the transcription of gene constructions that, according to our design, need to be expressed in low oxygen conditions in R. etli. The genes regulated by this promoter region will generally be active in the bacteroid state of R. etli because there are low concentrations of oxygen in the nodule’s environment.
Characterization
Previous work has been done using the promoter region of the nifHa gene of Rhizobium etli. The promoter sequence used in (Balderrama, et. al., 1996) is slightly different to our sequence: an A to G substitution at position -199 relative the the last nucleotide of our promoter sequence, and a 1 bp insertion in a region that hasn’t been implicated in the regulation of transcription. Also, the promoter sequence they use has an RBS sequence [5’-AGGAAGGCGAT-3’] that we don’t report in our promoter sequence. However we use an almost identical RBS sequence in our system (we changed the last codon in the sequence from a GAT codon to a CAT codon to create a NdeI restriction site important for our design). Given that the alterations are minor, we believe that the functional characterization that has been performed on the nifHa promoter should be directly applicable to the promoter sequence we are reporting.
A characterization of this part has already been done using a β-Galactosidase reporter (Balderrama, et. al., 1996). This group constructed locus-specific fusions with the E. coli lacZ gene by gene replacement, to analyze the transcriptional regulation of the nifHa promoter. An R. etli strain containing the nifHa-lacZ was constructed and its free-living expression was estimated under different oxygen concentrations. The strain they developed was cultured in liquid minimal medium under 1, 5, and 20% oxygen concentrations. Maximal β-Galactosidase activity was always observed when the strain was grown under 1% oxygen. Furthermore, levels of activity declined as the oxygen concentration was increased (Figure 1). Specific activities were reported as nanomoles of o-nitrophenol produced per minute per miligram of protein.
Another similar characterization has been done by fusing the nifH promoter with β-Glucuronidase (Girard, unpublished data). In 1% oxygen, they quantified 230 units of β-Glucuronidase specific activity (SD ± 37) while at 20% oxygen, the value they estimated at 54 units (SD ± 19) (Figure 2).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |