Enhancing Nitrogen Fixation Gene Expression by Modifying the DNA-Binding Function of Rhizobium meliloti NifA

Abstract

In Rhizobium meliloti, the endosymbiont of alfalfa, genetic control of free living (nif) and symbiotic (fix) nitrogen fixation gene expression is exerted by NifA. This protein belongs to the Enhancer-Binding Protein (EBP) family of regulators that activate promoters recognized by the RNA polymerase with the sigma factor σ⁵⁴ (Eσ⁵⁴) (reviewed in Kustu et al, 1989). NifA is composed of three structural, functional and evolutionarily different domains (Morett, Segovia, 1993). The function of the NH₂-terminal domain has not been unambiguously determined and in R. trifolii, it is truncated. The central domain has the determinants for ATP hydrolysis, which is required to activate transcription, multimerization, and contacting Eσ⁵⁴. The COOH-terminal domain is involved in DNA-binding and has a helix-turn-helix (hth) motif. By directed mutagenesis the functionality of this motif has been assessed (Morett, Buck, 1988). To activate transcription, NifA binds to DNA sites typically between −100 to −200bp upstream. These Upstream Activator Sequences (UAS) have the consensus sequence TGTN₁₀ACA. The number of UAS, their sequence and location are critical for nif gene expression. The promoters transcribed by Eσ⁵⁴ differ from the canonical bacterial promoters in having conserved nucleotides around −24 and −12 bp from the transcription start site. The sequence of the promoters is critical for their level of expression. Promoters with high sequence similarity to the consensus (TGGCACN₄TTGCA) are expressed at high levels and are less dependent on NifA bound at the UAS (Morett, Buck, 1989). The R. meliloti nifH promoter, which is almost identical to the −24/−12 consensus promoter, is the prototype of a strong promoter. How the appropriate level on nif and fix gene expression is attained in this organism is not known.

We thank K. Nevairez for technical assistance. Partially supported by grants from DGAPA, UNAM (IN208894) and The European Community (CI1 * CT94-0060).