Unraveling the functional connectivity of a protein network required for cell differentiation and pattern formation in the cyanobacterium Nostoc PCC 7120 – FunDate

To address the central question of our project, we developed a methodological plan integrating several complementary experimental approaches, ranging from genetics to bioinformatics. Our initial strategy was to study the protein network PatB, HetP, and HetC using both direct genetic and biochemical analyses. To understand the effects of each player, we specifically modified the corresponding genes in the bacterium and observed the consequences on cell differentiation. We also sought to physically visualize the interaction between HetP and HetC directly within living cells, as well as to map all genes controlled by the regulator PatB using RNA sequencing methods. However, the part of the project aimed at directly characterizing the membrane protein HetC encountered technical difficulties. Due to its low natural abundance and the challenges associated with its extraction and purification, the classic biochemical experiments planned to study its interactions and conformational changes were not feasible.

Faced with this challenge, we designed and implemented an alternative and innovative approach to indirectly identify the partners and function of HetC. This new strategy relied on three pillars. First, we used advanced computational modeling tools to predict the three-dimensional structure of the HetC protein, obtaining a theoretical model of its shape. Second, we conducted extensive genetic research to identify genes whose activity is absolutely required at the key commitment stage of differentiation. Finally, in a third step, we cross-referenced these results using bioinformatic prediction methods. The goal was to analyze, using the structural model of HetC, which of the proteins identified in our genetic screening were likely to bind to it. In summary, while the core of our methodological plan was successfully executed as intended, the direct characterization of HetC required a successful strategic adaptation. We thus replaced the direct biochemical analysis with an integrative approach combining molecular modeling, genetic screens, and interaction predictions, thereby still progressing towards our goal of identifying the substrate of HetC.

Our project has succeeded in elucidating the fundamental mechanisms underlying a complex biological organization: how the cyanobacterium Nostoc reliably generates a regular pattern of specialized cells, the heterocysts. We have characterized the precise roles of three key molecular players. First, we demonstrated that the protein HetC functions as an active ABC-type transporter endowed with a proteolytic activity. Its energy-dependent operation is indispensable for triggering differentiation. Our computational modeling and targeted genetic tests allowed us to identify the specific regions of HetC involved in substrate recognition and designated the HetP protein as its likely substrate.

Second, we clarified the function of the transcriptional regulator PatB. Our exhaustive analysis of its genetic targets revealed that its role extends beyond nitrogen fixation. PatB actually coordinates distinct genetic programs in the two cell types of the filament, activating, for example, photosynthesis-related genes in vegetative cells. This discovery establishes PatB as the first identified factor that acts as a central metabolic integrator between two bacterial cell lineages.

Finally, we identified the evolutionary origin of the system responsible for the regular spacing of heterocysts. This spatial pattern is controlled by a signaling peptide whose maturation depends on an enzyme, PatP. Comparative genomic analysis shows that the gene encoding PatP is ancestral and universal in cyanobacteria, predating the emergence of differentiation. This result highlights a phenomenon of evolutionary co-option: a basic-function peptidase was co-opted to produce a new signal, transforming a simple biochemical reaction into a sophisticated system of communication and morphogenesis.

Thus, our work describes an integrated regulatory cascade where a transporter-enzyme (HetC) processes a signal (HetP) that influences a master regulator (PatB), which in turn synchronizes cellular fates, while an ancient enzymatic tool (PatP) generates the spatial pattern. It illustrates how the assembly and repurposing of pre-existing molecular modules can lead to the emergence of new multicellular properties.

Our project now paves the way for new ambitious research aimed at transforming our molecular understanding into a holistic view of the functioning and evolution of these bacteria.

First, we wish to observe how the two cell types, vegetative and specialized, cooperate in real time. Thanks to technologies that allow us to sort and analyze each cell type separately, we will be able to precisely map which genes and proteins are active in each. The goal is to understand how these two "compartments" exchange and allocate nutrients like carbon and nitrogen to ensure the harmonious growth of the entire bacterium, thereby modeling a primitive form of division of labor.

Second, we will finalize the study of the pivotal moment of differentiation. We must confirm how the HetP protein, by interacting with the regulator PatB, acts as a molecular switch to definitively initiate the specialization program. This will involve a combination of genetics, computational modeling, and direct tests in the bacterium.

Third, we will explore other key players in the system. For example, the function of the membrane protein PatN, which dictates the candidate state for differentiation, remains a mystery. By applying the methods we have developed, we will be able to determine its role and begin to reconstruct the complete network of cellular machinery that builds a specialized cell.

Finally, these discoveries offer potential perspectives in applied research. The expertise and genetic tools we have developed, particularly in genome analysis and editing, constitute a valuable toolbox. We can now use them to study many other essential processes in cyanobacteria and microalgae, organisms of major interest for biotechnology. This fundamental research pathway can thus inspire the engineering of metabolic pathways or communication systems in these photosynthetic organisms, with potential benefits in biotechnology and environmental science.

Under nitrogen-limiting conditions, the multicellular and diazotrophic cyanobacterium Nostoc PCC 7120 differentiate ~10% of its cells to become specialized nitrogen-fixing heterocysts. The decision to commit into terminal differentiated cells occurs ~13 h after the induction of differentiation. While the initiation of this process is rather well understood, the mechanisms that govern the transition to final commitment and to maturation are still unknown. In the Fun-Date project, we aim at addressing this point by elucidating the interplay between an ABC-transporter (HetC), HetP and a transcriptional regulator (PatB). Our hypothesis is that the transporter HetC modulates the activity of PatB, and therefore gene expression, through the transport of HetP which otherwise inhibits PatB. Genetic and biochemical approaches will be used to analyze the functional interactions between the three proteins, highlighting the role of this network to achieve terminal differentiation.