The export of peptides or proteins is essential for a variety of important functions in bacteria. Among the diverse protein-translocation systems, peptidase-containing ABC transporters (PCAT) are involved in the maturation and export of quorum-sensing or antimicrobial peptides in Gram-positive bacteria and of toxins in Gram-negative organisms. In the multicellular and diazotrophic cyanobacterium Nostoc PCC 7120, the protein HetC is essential for the differentiation of functional heterocysts, which are micro-oxic and non-dividing cells specialized in atmospheric nitrogen fixation. HetC shows similarities to PCAT systems, but whether it actually acts as a peptidase-based exporter remains to be established. In this study, we show that the N-terminal part of HetC, encompassing the peptidase domain, displays a cysteine-type protease activity. The conserved catalytic residues conserved in this family of proteases are essential for the proteolytic activity of HetC and the differentiation of heterocysts. Furthermore, we show that the catalytic residue of the ATPase domain of HetC is also essential for cell differentiation. Interestingly, HetC has a cyclic nucleotide-binding domain at its N-terminus which can bind ppGpp in vitro and which is required for its function in vivo. Our results indicate that HetC is a peculiar PCAT that might be regulated by ppGpp to potentially facilitate the export of a signaling peptide essential for cell differentiation, thereby broadening the scope of PCAT role in Gram-negative bacteria.IMPORTANCEBacteria have a great capacity to adapt to various environmental and physiological conditions; it is widely accepted that their ability to produce extracellular molecules contributes greatly to their fitness. Exported molecules are used for a variety of purposes ranging from communication to adjust cellular physiology, to the production of toxins that bacteria secrete to fight for their ecological niche. They use export machineries for this purpose, the most common of which energize transport by hydrolysis of adenosine triphosphate. Here, we demonstrate that such a mechanism is involved in cell differentiation in the filamentous cyanobacterium Nostoc PCC 7120. The HetC protein belongs to the ATP-binding cassette transporter superfamily and presumably ensures the maturation of a yet unknown substrate during export. These results open interesting perspectives on cellular signaling pathways involving the export of regulatory peptides, which will broaden our knowledge of how these bacteria use two cell types to conciliate photosynthesis and nitrogen fixation.

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.

The Anabaena genus is a model organism of filamentous cyanobacteria whose vegetative cells can differentiate under nitrogen-limited conditions into a type of cell called a heterocyst. These heterocysts lose the possibility to divide and are necessary for the filament because they can fix and share environmental nitrogen. In order to distribute the nitrogen efficiently, heterocysts are arranged to form a quasi-regular pattern whose features are maintained as the filament grows. Recent efforts have allowed advances in the understanding of the interactions and genetic mechanisms underlying this dynamic pattern. Here, we present a systematic review of the existing theoretical models of nitrogen-fixing cell differentiation in filamentous cyanobacteria. These filaments constitute one of the simplest forms of multicellular organization, and this allows for several modeling scales of this emergent pattern. The system has been approached at three different levels. From bigger to smaller scale, the system has been considered as follows: at the population level, by defining a mean-field simplified system to study the ratio of heterocysts and vegetative cells; at the filament level, with a continuous simplification as a reaction-diffusion system; and at the cellular level, by studying the genetic regulation that produces the patterning for each cell. In this review, we compare these different approaches noting both the virtues and shortcomings of each one of them.

The cyanobacterium Anabaena sp. PCC 7120 forms filaments of communicating cells. Under conditions of nitrogen scarcity, some cells differentiate into heterocysts, allowing the oxygen-sensitive N2-reduction system to be expressed and operated in oxic environments. The key to diazotrophic growth is the exchange of molecules with nutritional and signaling functions between the two types of cells of the filament. During heterocyst differentiation, the peptidoglycan sacculus grows to allow cell enlargement, and the intercellular septa are rebuilt to narrow the contact surface with neighboring cells and to hold specific transport systems, including the septal junction complexes for intercellular molecular transfer, which traverse the periplasm between heterocysts and neighboring vegetative cells through peptidoglycan nanopores. Here we have followed the spatiotemporal pattern of peptidoglycan incorporation during heterocyst differentiation by Van-FL labeling and the localization and role of proteins MreB, MreC and MreD. We observed strong transitory incorporation of peptidoglycan in the periphery and septa of proheterocysts and a maintained focal activity in the center of mature septa. During differentiation, MreB, MreC and MreD localized throughout the cell periphery and at the cell poles. In mreB, mreC or mreD mutants, instances of strongly increased peripheral and septal peptidoglycan incorporation were detected, as were also heterocysts with aberrant polar morphology, even producing filament breakage, frequently lacking the septal protein SepJ. These results suggest a role of Mre proteins in the regulation of peptidoglycan growth and the formation of the heterocyst neck during differentiation, as well as in the maintenance of polar structures for intercellular communication in the mature heterocyst. Finally, as previously observed in filaments growing with combined nitrogen, in the vegetative cells of diazotrophic filaments, the lack of MreB, MreC or MreD led to altered localization of septal peptidoglycan-growth bands reproducing an altered localization of FtsZ and ZipN rings during cell division.

The filamentous cyanobacterium Anabaena sp. PCC 7120 can differentiate into heterocysts to fix atmospheric nitrogen. During cell differentiation, cellular morphology and gene expression undergo a series of significant changes. To uncover the mechanisms responsible for these alterations, we built protein-protein interaction (PPI) networks for these two cell types by cofractionation coupled with mass spectrometry. We predicted 280 and 215 protein complexes, with 6322 and 2791 high-confidence PPIs in vegetative cells and heterocysts, respectively. Most of the proteins in both types of cells presented similar elution profiles, whereas the elution peaks of 438 proteins showed significant changes. We observed that some well-known complexes recruited new members in heterocysts, such as ribosomes, diflavin flavoprotein, and cytochrome c oxidase. Photosynthetic complexes, including photosystem I, photosystem II, and phycobilisome, remained in both vegetative cells and heterocysts for electron transfer and energy generation. Besides that, PPI data also reveal new functions of proteins. For example, the hypothetical protein Alr4359 was found to interact with FraH and Alr4119 in heterocysts and was located on heterocyst poles, thereby influencing the diazotrophic growth of filaments. The overexpression of Alr4359 suspended heterocyst formation and altered the pigment composition and filament length. This work demonstrates the differences in protein assemblies and provides insight into physiological regulation during cell differentiation.

Heterocysts are specialized cells that filamentous cyanobacteria differentiate for the fixation of atmospheric nitrogen when other nitrogen sources are not available. Heterocyst differentiation at semiregular intervals along the filaments requires complex structural and metabolic changes that are under the control of the master transcriptional regulator HetR. NsiR1 (nitrogen stress-induced RNA 1) is a HetR-dependent noncoding RNA that is expressed from multiple chromosomal copies, some identical, some slightly divergent in sequence, specifically in heterocysts from very early stages of differentiation. We have previously shown that NsiR1 inhibits translation of the overlapping hetF mRNA by an antisense mechanism. Here, we identify alr3234, a hetP-like gene involved in the regulation of commitment (point of no return) to heterocyst differentiation, as a target of NsiR1. A strain overexpressing one of the identical copies of NsiR1 commits to heterocyst development earlier than the wild type. The posttranscriptional regulation exerted by NsiR1 on the expression of two genes involved in heterocyst differentiation and commitment, hetF and alr3234, adds a new level of complexity to the network of transcriptional regulation and protein-protein interactions that participate in heterocyst differentiation. IMPORTANCE Heterocysts are nitrogen-fixing specialized cells that appear at semiregular intervals along cyanobacterial filaments upon nitrogen starvation. The differentiation and patterning of heterocysts is a model for the study of cell differentiation in multicellular prokaryotes. The regulation of differentiation, which is only partially understood, includes transcriptional changes, factor diffusion between cells, and protein-protein interactions. This work describes the identification of a novel target for NsiR1, a small RNA (sRNA) encoded in multiple slightly divergent copies, and shows how different copies of \"sibling\" sRNAs regulate the expression of different targets involved in one of the few examples of a differentiation process in prokaryotes.

Addition of nickel stimulates growth, heterocyst differentiation, 14 CO2 uptake and nitrogenase activity in Nostnc muscorum. The significance of this observation is discussed.

Heterocysts are terminally differentiated cells of filamentous cyanobacteria, which are specialized for nitrogen fixation. Because nitrogenase is easily inactivated by oxygen, the intracellular environment of heterocysts is kept microoxic. In heterocysts, the oxygen-evolving photosystem II is inactivated, a heterocyst-specific envelope with an outer polysaccharide layer and an inner glycolipid layer is formed to limit oxygen entry, and oxygen consumption is activated. Heterocyst differentiation, which is accompanied by drastic morphological and physiological changes, requires strictly controlled gene expression systems. Here, we investigated the functions of a CRP-family transcriptional regulator, DevH, in the process of heterocyst differentiation. A devH-knockdown strain, devH-kd, was created by replacing the original promoter with the gifA promoter, which is repressed during heterocyst differentiation. Although devH-kd formed morphologically distinct cells with the heterocyst envelope polysaccharide layer, it was unable to grow diazotrophically. Genes involved in construction of the microoxic environment, such as cox operons and the hgl island, were not upregulated in devH-kd. Moreover, expression of the nif gene cluster was completely abolished. Although CnfR was expressed in devH-kd, the nif gene cluster was not induced even under microoxic conditions. Thus, DevH is necessary for the establishment of a microoxic environment and induction of nitrogenase in heterocysts.

Differentiation commitment is one of the most complex mechanisms to study in biological science. One of the model systems used for understanding differentiation complexity is heterocyst development in cyanobacteria. Cyanobacteria have the capability of biological nitrogen fixation due to highly differentiated heterocyst cells. Once the nitrogen deficiency signal is perceived by the cyanobacteria, few of its vegetative cells commit toward the development of heterocyst. Heterocyst provides a microoxic environment that is essential for the nitrogenase complex to fix the atmospheric dinitrogen. The entire process of development of heterocyst can be divided into different steps, such as (a) sensing signal and differentiation induction, (b) positional (pattern) determination of heterocyst in the filament, (c) formation of extracellular thick heterocyst-specific layers, and (d) assembly of nitrogen-fixing machinery. Many of the key regulators that are essential for heterocyst formation in these different steps have been identified. Recently, the role of small RNA and interruption DNA elements that influence the heterocyst formation and function has also been identified. In this review article, we have outlined the current understanding of the entire molecular circuit of heterocyst development in a simplistic way. This article focuses on explaining key concepts related to heterocyst development and discusses recent discoveries in this line.

The oldest prokaryotic photoautotrophic organisms, cyanobacteria, produce many different metabolites. Among them is the water-soluble neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (BMAA), whose biological functions in cyanobacterial metabolism are of fundamental scientific and practical interest. An early BMAA inhibitory effect on nitrogen fixation and heterocyst differentiation was shown in strains of diazotrophic cyanobacteria Nostoc sp. PCC 7120, Nostocpunctiforme PCC 73102 (ATCC 29133), and Nostoc sp. strain 8963 under conditions of nitrogen starvation. Herein, we present a comprehensive proteomic study of Nostoc (also called Anabaena) sp. PCC 7120 in the heterocyst formation stage affecting by BMAA treatment under nitrogen starvation conditions. BMAA disturbs proteins involved in nitrogen and carbon metabolic pathways, which are tightly co-regulated in cyanobacteria cells. The presented evidence shows that exogenous BMAA affects a key nitrogen regulatory protein, PII (GlnB), and some of its protein partners, as well as glutamyl-tRNA synthetase gltX and other proteins that are involved in protein synthesis, heterocyst differentiation, and nitrogen metabolism. By taking into account the important regulatory role of PII, it becomes clear that BMAA has a severe negative impact on the carbon and nitrogen metabolism of starving Nostoc sp. PCC 7120 cells. BMAA disturbs carbon fixation and the carbon dioxide concentrating mechanism, photosynthesis, and amino acid metabolism. Stress response proteins and DNA repair enzymes are upregulated in the presence of BMAA, clearly indicating severe intracellular stress. This is the first proteomic study of the effects of BMAA on diazotrophic starving cyanobacteria cells, allowing a deeper insight into the regulation of the intracellular metabolism of cyanobacteria by this non-protein amino acid.