Symbiotic nitrogen fixation by a nifA deletion mutant of Rhizobium meliloti: the role of an unusual ntrC allele

Abstract

In the N2-fixing alfalfa symbiont Rhizobium meliloti, the three r,4 (NTRA)-dependent positively acting regulatory proteins NIFA, NTRC, and DCTD are required for activation of promoters involved in N2 fixation (pnifHDKE and pfixABCX), nitrogen assimilation (pglnII), and C4-dicarboxylate transport (pdct4), respectively. Here, we describe an allele of ntrC which results in the constitutive activation of the above NTRC-, NIFA-, and DCTD-regulated promoters. The expression and activation of wild-type NTRC occur in response to nitrogen availability, whereas in cells carrying the ntrC283 allele, the NTRC283 protein appears constitutively active and is constitutively expressed. The ntrC283 allele was shown to carry a single mutation resulting in the replacement of an Asp by a Tyr residue in the helix-turn-helix motif of ntrC283. Introduction of the ntrC283 allele into a nifA deletion mutant restores the N2-fixation ability to 70 to 80%o of the wild-type level. Thus, the nifA gene is dispensable for symbiotic N2 fixation.Nitrogen-fixing root nodules develop through an intimate interaction between bacteria (rhizobia) and plant cells. In one of the early steps in this interaction, flavonoids produced by the plant induce the expression of a set of nodulation (nod) genes in the bacteria (31,40,43). In Rhizobium meliloti and Rhizobium leguminosarum, which induce root nodules on alfalfa and pea plants, respectively, the nod gene products function in the synthesis of a lipooligosaccharide signal molecule which triggers root nodule formation (62). The bacteria enter the root via tube-like structures, or infection threads, and are released into the plant cortical cells via an endocytosis-like process in which they are surrounded by the host plasma membrane. Both the bacteria and plant cells then undergo many biochemical changes, one of which is the expression of the plant globin genes whose products are involved in oxygen transport in the nodule. In the bacteria, the nitrogenase (nif) and other genes required for N2 fixation are induced. Expression of many nif genes and associated genes in nodules is regulated positively by the nifA gene product. Thus, nifA null mutants show no symbiotic N2-fixation activity. Expression of nifA is, in turn, regulated in response to oxygen concentration by a two-component environmental sensor-regulator pair, fixL and fixi, and by fixK (4,11,12). Recent data have shown that, within a nodule, the expression of the nifgenes in the bacteria and the globin genes in the plant cells occurs synchronously within a few layers and thus shows a high level of control (10, 69).The N2-fixing bacteria within nodules (called bacteroids) reduce N2 gas to ammonia which is then supplied to the plant. Because the bacteroids are within the plant cells, they are totally dependent on the plant for a source of energyreduced carbon, which is necessary for the high-energyrequiring N2-fixation reaction. The supply of a carbon source thus potentially represents a key point in control of N2 fixation and bacterial proliferation...

Functional analysis of the nifQdctA1y4vGHIJ operon of Sinorhizobium fredii strain NGR234 using a transposon with a NifA-dependent read-out promoter

“…Although not unexpected given the inability of rhizobial nifA mutants to perform symbiotic nitrogen fixation, this phenotype became our reference Fix 2 phenotype. These results also confirmed that in the parent NGR234 strain no other RpoN-dependent transcriptional regulator was capable of complementing the absence of nifA for symbiotic nitrogen fixation (Labes et al, 1993).…”

Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes

“…It has been shown that certain 54 -dependent promoters whose sequences greatly resemble the consensus sequence, like the R. meliloti nifH promoter, are prone to be partially activated by other proteins of the NifA family, such as NtrC (30,33,55). This results from the strong binding of E 54 to the promoter.…”

Identification, nucleotide sequence, and characterization of PspF, the transcriptional activator of the Escherichia coli stress-induced psp operon

“…All EBP 54 transcriptional activators have a conserved HTH motif located within a COOH-terminal DNA-binding domain of variable length, and each subfamily of EBPs recognizes a specific enhancer-like element or UAS (10,13,14,18,38,43,44). Binding to these elements confers specificity to the initiation of transcription (18,30,36). The first helix in the PspF HTH motif has a significant degree of homology to the other 54 -dependent activators, while the second helix (YHQFRALLK) of the putative DNA-binding motif is unique to the PspF protein.…”

“…Typically, activator proteins bind to a specific upstream activation sequence (UAS) located 80 to 200 bp upstream of the 54 core promoter (10,14,44). The function of the UAS is to tether the activator in the right position and to bring it in the vicinity of the promoter in order to increase the fidelity and efficiency of interaction between the 54 -RNAP and the activator (13,14,18,30,35,36,44). Expression from the psp promoter requires a UAS (located between positions Ϫ125 and Ϫ76) which has the ability to activate at a distance (57).…”