Role of the bacterial cell cycle in the symbiotic nitrogen fixation in legumes – SymbiontCellCyc

We aim to comprehend and exploit the crucial but poorly-known role of bacterial cell cycle regulators of the symbiont Sinorhizobium meliloti in the nitrogen-fixing symbiosis with Medicago legume plants. Legume plants can establish a symbiotic relationship with nitrogen fixing bacteria, known as rhizobia, forming specific root organs, called nodules, inside which the symbiotic rhizobia are housed, reduce nitrogen to ammonium and transfer it to the plant. The endosymbiotic rhizobia, called bacteroids, adapt their metabolism to intracellular life and to perform symbiotic nitrogen fixation. In some legumes, bacteroids undergo a terminal (irreversible) differentiation process, becoming strongly enlarged, with a fragilized cell envelope and a polyploid genome. This process is restricted to particular clades of legumes and is proposed to improve symbiotic efficiency and to benefit to the host plant. The amplified DNA content of bacteroids implies that during their formation, the normal cell cycle progression, consisting in an alternation of genome replication and cell division, is modified into a repeated DNA replication process without intervening division. Partner 2 (Mergaert) has shown that in M. truncatula the symbiotic nodule cells produce hundreds of Nodule-specific Cysteine-Rich (NCR) antimicrobial peptides, which induce this terminal bacteroid differentiation in S. meliloti. Strikingly, the peptide NCR247 is able to recapitulate this process in free-living rhizobia, suggesting that it targets the bacterial cell cycle machinery. Accordingly, NCR247-treated cells show a significant down-regulation of genes controlled by the master regulator of cell cycle CtrA. Partner 1 (Biondi) has shown that CtrA controls cell cycle in S. meliloti similarly to C. crescentus, the model system for cell cycle studies in Alphaproteobacteria. In C. crescentus and S. meliloti CtrA represses DNA replication and activates cell division suggesting a mechanism for bacteroid differentiation in S. meliloti by CtrA inhibition. In agreement, Partner 1 has shown that CtrA levels drop to very low levels in bacteroids and that the depletion of CtrA in S. meliloti cultures leads to bacteroid-like cells. In C. crescentus and S. meliloti, CtrA has a complex negative regulation, as it is repressed by (i) cell cycle controlled proteolysis, (ii) de-phosphorylation and (iii) post-translational cofactors. In S. meliloti particularly proteolysis of CtrA plays a crucial role in cell cycle progression, suggesting that this mechanism may also be involved in bacteroid differentiation. Nevertheless, the precise mechanistic link between NCR peptides and cell cycle regulation is still unclear and will be clarified by this project. We propose a multilevel approach to systematically investigate the role of cell cycle factors during the symbiotic infection. We will use a combination of bacterial genetics and cell biology methods on free-living bacteria, NCR247-treated cells and infected nodules. Moreover, we predict that modulating CtrA activity in planta could lead to enhanced bacteroid differentiation in Medicago or to bacteroid elongation in legumes lacking NCR peptides. This may result in an improved nitrogen fixation efficiency of the bacteroids. These hypotheses will be also tested by the project SymbiontCellCyc.