The molecular puzzle of two-component signaling cascades.

Two-component systems constitute prevalent signaling pathways in bacteria and mediate a large variety of adaptative cellular responses. Signaling proceeds through His-Asp phosphorelay cascades that involve two central partners, the histidine protein kinase and the response regulator protein. Structural studies have provided insights into some design principles and activation mechanisms of these multi-domain proteins implicated in the control of virulence gene expression in several pathogens.

Inhibition of the FixL sensor kinase by the FixT protein in Sinorhizobium meliloti.

Nitrogen fixation in symbiotic rhizobia is subject to multiple levels of gene regulation. In Sinorhizobium meliloti, the alfalfa symbiont, the FixLJ two-component regulatory system plays a major role in inducing nitrogen fixation and respiration gene expression in response to the low ambient O(2) concentration of the nodule. Here we report on the mode of action of the FixT protein, a recently identified repressor of nitrogen fixation gene expression in S. meliloti. First, we provide evidence that FixT prevents transcription of the intermediate key regulatory genes nifA and fixK by counteracting the activity of the FixLJ two-component system under otherwise inducing microoxic conditions. Second, we demonstrate that FixT acts as an inhibitor of the sensor hemoprotein kinase FixL, preventing the production or the accumulation of its phosphorylated form. FixT is thus a new example of a regulatory protein that blocks signal transduction in two-component systems at the level of the sensor kinase.

Negative autoregulation of the Rhizobium meliloti fixK gene is indirect and requires a newly identified regulator, FixT.

fixK genes are crp/fnr homologues that have been discovered in diverse Rhizobium spp., in which they are usually essential for symbiotic nitrogen fixation. One recurrent function of fixK genes in rhizobia is to activate the transcription of operons required for respiration in the microoxic environment of the nodule. In a similar manner to its Escherichia coli crp and fnr homologues, R. meliloti fixK regulates its own expression negatively. However, we demonstrate here that fixK negative autoregulation is not direct and, instead, involves a newly identified gene, fixT, the expression of which depends on fixK. Inactivation of fixT resulted in derepression of fixK expression under free-living microoxic conditions. Furthermore, constitutively expressed fixT strongly repressed fixK-lacZ expression in the absence of a functional fixK gene. Several lines of evidence indicate that fixT is active via its protein product FixT. FixT does not resemble any protein present in databases so far. Nodules induced by a fixT mutant were Fix+, thus demonstrating that fixT is not essential for symbiotic nitrogen fixation.

Oxygen as a key developmental regulator of Rhizobium meliloti N2-fixation gene expression within the alfalfa root nodule.

The symbiotic pattern of expression of Rhizobium meliloti N2-fixation genes is tightly coupled with the histological organization of the alfalfa root nodule and thus is under developmental control. N2-fixation gene expression is induced very sharply at a particular zone of the nodule called interzone II-III that precedes the zone where N2 fixation takes place. We show here that this coupling can be disrupted, hereby resulting in ectopic expression of N2-fixation genes in the prefixing zone II of the nodule. Uncoupling was obtained either by using a R. meliloti strain in which a mutation rendered N2-fixation gene expression constitutive with respect to oxygen in free-living bacterial cultures or by placing nodules induced by a wild-type R. meliloti strain in a microoxic environment. These results implicate oxygen as a key determinant of the symbiotic pattern of N2-fixation gene expression.