Deletion analysis of Rhizobium meliloti symbiotic promoters

Abstract

Previous examination of DNA sequences located 5' to Rhizobium meliloti niftranscription units has shown that extensive sequence homology exists among them. Here we have examined these reiterated sequences for their role in symbiotic gene regulation. Promoter deletion analysis has shown that although an extensive upstream DNA sequence (160 bp) is required for full heterologous activation of the R. meliloti nifHDK promoter by the Klebsiella pneumoniae nifA protein in Escherichia coli, this region is not required for expression of R. meliloti nifpromoters in root nodules from plants grown under greenhouse conditions. In addition, a minimum functional symbiotic promoter sequence is defined, and a DNA sequence difference affecting regulation of two symbiotic promoters is discussed.

Analysis of the expression from Rhizobium meliloti fix-promoters in other Rhizobium backgrounds

“…Data are the average of at least two independent assays. T o estimate plasmid stability Heterologous expression of R. meliloti promoters Better et al (1985) * Number of base pairs upstream of the transcription initiation point.…”

“…However, fixAp contains a 9 bp insertion at position -27 and significant variation is apparent in the region between the transcriptional and translational start sites (Better e t al., 1983). These divergences seem to lead to differences in expression in Escbericbia coli by either the Klebsiella pneumoniae NifA or the E. coli NtrC (nzfHp is activated but not fixAp, Better et al, 1985), and in R. meliloti under specific physiological conditions (Birkenhead e t al., 1990;Ditta et al, 1987). Details of this .$/fix regulation in other species of Rbixobizlm are poorly understood.…”

FixJ-regulated genes evolved through promoter duplication in Sinorhizobium meliloti

“…P1, P2 and P3 are located in the nif region of pSymA (Better et al, 1983). P1 is the original nifH promoter, while P2 controls expression of the fixABCX genes involved in electron transport to nitrogenase (Earl et al, 1987;Better et al, 1985). The function of P3 remains unknown, as it is not essential for symbiotic nitrogen fixation (Hirsch et al, 1983).…”

DNA‐binding domain mutants of sigma‐N (σN, σ54) defective between closed and stable open promoter complex formation

“…For promoter activation assays, two different systems were used: (i) the reporter promoter was carried on pMB221 as a K. pneumoniae nifH-lacZ fusion; this plasmid also supplies the K. pneumoniae NifA activator (Cannon and Buck, 1992); or (ii) on pMB210 as a R. meliloti nifH-lacZ fusion (Better et al, 1985) with plasmid pMC71A providing the K. pneumoniae NifA activator (Buchanan-Wollaston et al, 1981). E. coli TH1 (⌬rpoN2518, endA1, thi-1, hsdR17, supE44, ⌬lacU169 ) transformants were picked onto LB media with the appropriate antibiotics and grown at 30ЊC until an A 600 of 0.8-1.0 was reached.…”