c-di-GMP Homeostasis Is Critical for Heterocyst Development in Anabaena sp. PCC 7120

c-di-GMP is a ubiquitous bacterial signal regulating various physiological process. Anabaena PCC 7120 (Anabaena) is a filamentous cyanobacterium able to form regularly-spaced heterocysts for nitrogen fixation, in response to combined-nitrogen deprivation in 24h. Anabaena possesses 16 genes encoding proteins for c-di-GMP metabolism, and their functions are poorly characterized, except all2874 (cdgS) whose deletion causes a decrease in heterocyst frequency 48h after nitrogen starvation. We demonstrated here that c-di-GMP levels increased significantly in Anabaena after combined-nitrogen starvation. By inactivating each of the 16 genes, we found that the deletion of all1175 (cdgSH) led to an increase of heterocyst frequency 24h after nitrogen stepdown. A double mutant ΔcdgSHΔcdgS had an additive effect over the single mutants in regulating heterocyst frequency, indicating that the two genes acted at different time points for heterocyst spacing. Biochemical and genetic data further showed that the functions of CdgSH and CdgS in the setup or maintenance of heterocyst frequency depended on their opposing effects on the intracellular levels of c-di-GMP. Finally, we demonstrated that heterocyst differentiation was completely inhibited when c-di-GMP levels became too high or too low. Together, these results indicate that the homeostasis of c-di-GMP level is important for heterocyst differentiation in Anabaena.

Effects of PatU3 peptides on cell size and heterocyst frequency of Anabaena sp. PCC 7120

patU, one of the genes specifically found in filamentous cyanobacteria, is required for the pattern formation in heterocyst-forming species. In Anabaena sp. 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 that of the wild type. Yeast two-hybrid and pull-down assays demonstrated a direct interaction between PatU3 and the cell division protein Ftn6. Without the N-terminal 16-aa portion (MQERFQAVIKRRLQIH), 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 near wild-type level. PatU3(1-16aa)-GFP, the N-terminal 16-aa sequence fused with GFP, formed polar aggregates and peripheral patches in heterocysts of Anabaena 7120, whereas PatU3(1-198aa)-GFP showed a homogeneous distribution in the cytoplasm of all cells. The N-terminal AVIKRRL-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. PCC 7120 is involved in cell size determination. The N-terminal 16-aa sequence of PatU3 is required for control of cell size and interaction with the cell division protein Ftn6, and an octapeptide (aa7 ∼ aa14) 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.

Interactions of PatA with the Divisome during Heterocyst Differentiation in Anabaena

ABSTRACT The Anabaena organismic unit is a filament of communicating cells. Under conditions of nitrogen scarcity, some cells along the filament differentiate into heterocysts, which are specialized in the fixation of atmospheric N2 and provide the vegetative cells with N2 fixation products. At a certain stage, the differentiation process becomes irreversible, so that even when nitrogen is replenished, no return to the vegetative cell state takes place, possibly as a consequence of loss of cell division capacity. Upon N-stepdown, midcell FtsZ-rings were detected in vegetative cells, but not in differentiating cells, and this was also the case for ZipN, an essential protein that participates in FtsZ tethering to the cytoplasmic membrane and divisome organization. Later, expression of ftsZ was arrested in mature heterocysts. PatA is a protein required for the differentiation of intercalary heterocysts in Anabaena. The expression level of the patA gene was increased in differentiating cells, and a mutant strain lacking PatA exhibited enhanced FtsZ-rings. PatA was capable of direct interactions with ZipN and SepF, another essential component of the Anabaena Z-ring. Thus, PatA appears to promote inhibition of cell division in the differentiating cells, allowing progress of the differentiation process. PatA, which in mature heterocysts was detected at the cell poles, could interact also with SepJ, a protein involved in production of the septal junctions that provide cell-cell adhesion and intercellular communication in the filament, hinting at a further role of PatA in the formation or stability of the intercellular structures that are at the basis of the multicellular character of Anabaena. IMPORTANCE Anabaena is a cyanobacterial model that represents an ancient and simple form of biological multicellularity. The Anabaena organism is a filament of cohesive and communicating cells that can include cells specialized in different tasks. Thus, under conditions of nitrogen scarcity, certain cells of the filament differentiate into heterocysts, which fix atmospheric nitrogen and provide organic nitrogen to the rest of cells, which, in turn, provide heterocysts with organic carbon. Heterocyst differentiation involves extensive morphological, biochemical, and genetic changes, becoming irreversible at a certain stage. We studied the regulation during heterocyst differentiation of several essential components of the Anabaena cell division machinery and found that protein PatA, which is required for differentiation and is induced in differentiating cells, interacts with essential cell division factors and destabilizes the cell division complex. This suggests a mechanism for establishment of commitment to differentiation by inhibition of cell division.

The First Proteomic Study of Nostoc sp. PCC 7120 Exposed to Cyanotoxin BMAA under Nitrogen Starvation

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, Nostoc punctiforme 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.

The CRP-family transcriptional regulator DevH regulates expression of heterocyst-specific genes at the later stage of differentiation in the cyanobacterium Anabaena sp. strain PCC 7120

SummaryHeterocysts are terminally differentiated cells of filamentous cyanobacteria, which are specialized for nitrogen fixation. Because nitrogenase, an enzyme for nitrogen fixation, 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, devHKD, was created by replacing the original promoter with the gifA promoter, which is repressed during heterocyst differentiation. Although devHKD 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 devHKD. Moreover, expression of the nif gene cluster was completely abolished. Even under anaerobic conditions, the nif gene cluster was not induced in devHKD. Thus, DevH is necessary for the establishment of a microoxic environment and induction of nitrogenase in heterocysts.

Expression from DIF1-motif promoters of hetR and patS is dependent on HetZ and modulated by PatU3 during heterocyst differentiation

HetR and PatS/PatX-derived peptides are the activator and diffusible inhibitor for cell differentiation and patterning in heterocyst-forming cyanobacteria. HetR regulates target genes via HetR-recognition sites. However, some genes (such as patS / patX ) upregulated at the early stage of heterocyst differentiation possess DIF1 (or DIF + ) motif (TCCGGA) promoters rather than HetR-recognition sites; hetR possesses both regulatory elements. How HetR controls heterocyst-specific expression from DIF1 motif promoters remains to be answered. This study presents evidence that the expression from DIF1 motif promoters of hetR , patS and patX is more directly dependent on hetZ , a gene regulated by HetR via a HetR-recognition site. The HetR-binding site upstream of hetR is not required for the autoregulation. PatU3 (3′ portion of PatU) that interacts with HetZ may modulate HetZ-dependent gene expression. These findings contribute to understanding of the mutual regulation of hetR , hetZ-patU and patS / patX in a large group of multicellular cyanobacteria.

The Integrity of the Cell Wall and Its Remodeling during Heterocyst Differentiation Are Regulated by Phylogenetically Conserved Small RNA Yfr1 in Nostoc sp. Strain PCC 7120

ABSTRACT Yfr1 is a strictly conserved small RNA in cyanobacteria. A bioinformatic prediction to identify possible interactions of Yfr1 with mRNAs was carried out by using the sequences of Yfr1 from several heterocyst-forming strains, including Nostoc sp. strain PCC 7120. The results of the prediction were enriched in genes encoding outer membrane proteins and enzymes related to peptidoglycan biosynthesis and turnover. Heterologous expression assays with Escherichia coli demonstrated direct interactions of Yfr1 with mRNAs of 11 of the candidate genes. The expression of 10 of them (alr2458, alr4550, murC, all4829, all2158, mraY, alr2269, alr0834, conR, patN) was repressed by interaction with Yfr1, whereas the expression of amiC2, encoding an amidase, was increased. The interactions between Yfr1 and the 11 mRNAs were confirmed by site-directed mutagenesis of Yfr1. Furthermore, a Nostoc strain with reduced levels of Yfr1 had larger amounts of mraY and murC mRNAs, supporting a role for Yfr1 in the regulation of those genes. Nostoc strains with either reduced or increased expression of Yfr1 showed anomalies in cell wall completion and were more sensitive to vancomycin than the wild-type strain. Furthermore, growth in the absence of combined nitrogen, which involves the differentiation of heterocysts, was compromised in the strain overexpressing Yfr1, and filaments were broken at the connections between vegetative cells and heterocysts. These results indicate that Yfr1 is an important regulator of cell wall homeostasis and correct cell wall remodeling during heterocyst differentiation. IMPORTANCE Bacterial small RNAs (sRNAs) are important players affecting the regulation of essentially every aspect of bacterial physiology. The cell wall is a highly dynamic structure that protects bacteria from their fluctuating environment. Cell envelope remodeling is particularly critical for bacteria that undergo differentiation processes, such as spore formation or differentiation of heterocysts. Heterocyst development involves the deposition of additional layers of glycolipids and polysaccharides outside the outer membrane. Here, we show that a cyanobacterial phylogenetically conserved small regulatory RNA, Yfr1, coordinates the expression of proteins involved in cell wall-related processes, including peptidoglycan metabolism and transport of different molecules, as well as expression of several proteins involved in heterocyst differentiation.