Spatial periodic signal for cell differentiation in some multicellular organisms is generated according to Turing\'s principle for pattern formation. How a dividing cell responds to the signal of differentiation is addressed with the filamentous cyanobacterium Nostoc sp. PCC 7120, which forms the patterned distribution of heterocysts. We show that differentiation of a dividing cell was delayed until its division was completed and only one daughter cell became heterocyst. A mutant of patU3, which encodes an inhibitor of heterocyst formation, showed no such delay and formed heterocyst pairs from the daughter cells of cell division or dumbbell-shaped heterocysts from the cells undergoing cytokinesis. The patA mutant, which forms heterocysts only at the filament ends, restored intercalary heterocysts by a single nucleotide mutation of patU3, and double mutants of patU3/patA and patU3/hetF had the phenotypes of the patU3 mutant. We provide evidence that HetF, which can degrade PatU3, is recruited to cell divisome through its C-terminal domain. A HetF mutant with its N-terminal peptidase domain but lacking the C-terminal domain could not prevent the formation of heterocyst pairs, suggesting that the divisome recruitment of HetF is needed to sequester HetF for the delay of differentiation in dividing cells. Our study demonstrates that PatU3 plays a key role in cell-division coupled control of differentiation.

patU, one of the genes specifically found in filamentous cyanobacteria, is required for the pattern formation in heterocyst-forming species. In Anabaena sp. strain PCC 7120, patU is split into patU5 and patU3, and only patU3 is involved in heterocyst patterning. Here, we report that PatU3 is also involved in control of cell size. A patU3 deletion mutant showed remarkably smaller cell size and much higher heterocyst frequency than the wild type. Yeast two-hybrid and pulldown assays demonstrated a direct interaction between PatU3 and the cell division protein Ftn6. Without the N-terminal 16-amino-acid (aa) portion (MQERFQAVIKRRLQIH [the identified octapeptide is underlined]), PatU3 was no longer able to interact with Ftn6. This portion of PatU3 is also required for the interaction with PatN, a protein related to heterocyst differentiation/patterning. Addition of the 16-aa peptide or AVIKRRLQ-containing peptides restored the cell size and heterocyst frequency of a patU3 deletion mutant to normal or nearly wild-type levels. PatU3(1-16aa)-GFP, the N-terminal 16-aa sequence fused with green fluorescent protein (GFP), formed polar aggregates and peripheral patches in heterocysts of Anabaena sp. strain PCC 7120, whereas PatU3(1-198aa)-GFP showed a homogeneous distribution in the cytoplasm of all cells. The N-terminal AVIKRRLQ-containing sequence may function in intact PatU3, as a separate peptide, or both. IMPORTANCE PatU (or split into PatU5 and PatU3) is distributed in almost all filamentous cyanobacteria, including those that do not form heterocysts (except Pseudanabaena); however, its functions other than heterocyst differentiation/patterning have not been reported before. In this study, we found that PatU3 in Anabaena sp. strain PCC 7120 is involved in cell size determination. The N-terminal 16-aa sequence of PatU3 is required for the control of cell size and interaction with the cell division protein Ftn6, and an octapeptide (aa 7 to aa 14) within the 16-aa sequence can restore the cell size (and heterocyst frequency) of a patU3 deletion mutant to normal. Such a peptide, if generated from PatU or PatU3 in vivo, may promote intercellular coordination in filamentous cyanobacteria.