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The filamentous, multicellular cyanobacterium

Bacterial cells mostly divide symmetrically. In the filamentous, multicellular cyanobacterium Anabaena, cell-division planes are aligned vertically relative to the long axis of every single cell. This observation suggests that both the placement and the angle of the division planes are controlled in every single cell so that the filament can grow in one single dimension along the long axis. In this study, we showed that inactivation of patU3 encoding a cell-division inhibitor led cells to divide asymmetrically in two dimensions leading to twisted filaments, indicating that PatU3 controls not only the position but also the angle of the division planes. Deletion of the conserved minC and minD genes affected cell division symmetry, but not the angle of the division planes. Remarkably, when both patU3 and minCD were inactivated, cells could divide asymmetrically over 360° angles in three dimensions across different cellular sections, producing not only cells with irregular sizes, but also branching filaments. This study demonstrated the existence of a system operating in a three-dimensional manner for the control of cell division in Anabaena. Such a regulation may have been evolved to accommodate multicellular behaviors, a hallmark in evolution.

The filamentous, multicellular cyanobacterium Anabaena sp. PCC 7120 ( Anabaena ) is a prokaryotic model for the study of cell differentiation and cell-cell interactions. Upon combined-nitrogen deprivation, Anabaena forms a particular cell type, heterocyst, for aerobic nitrogen fixation. Heterocysts are semiregularly spaced among vegetative cells. Heterocyst differentiation is coupled to cell division, but the underlying mechanism remains unclear. This mechanism could be mediated by the putative protease HetF, which is a divisome component and is necessary for heterocyst differentiation. In this study, by suppressor screening, we identified PatU3, as a negative regulator acting downstream of HetF for cell division and heterocyst development. The inactivation of patU3 restored the capacity of cell division and heterocyst differentiation in the Δ hetF mutant, and overexpression of patU3 inhibited both processes in the wild-type background. We demonstrated that PatU3 was a specific substrate of the protease activity of HetF. Consequently, PatU3 accumulated in the hetF -deficient mutant, which was responsible for the resultant mutant phenotype. The cleavage site of PatU3 by HetF was mapped after the Arg117 residue, whose mutation made PatU3 resistant to HetF processing, and mimicked the effect of hetF deletion. Our results provided evidence that HetF regulated cell division and heterocyst differentiation by controlling the inhibitory effects of PatU3. This proteolytic pathway constituted a mechanism for the coordination between cell division and differentiation in a prokaryotic model used for studies on developmental biology and multicellularity.

Bacterial cell shape is determined by the peptidoglycan (PG) layer. The cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a filamentous strain with ovoid-shaped cells connected together with incomplete cell constriction. When deprived of combined nitrogen in the growth medium, about 5–10% of the cells differentiate into heterocysts, cells devoted to nitrogen fixation. It has been shown that PG synthesis is modulated during heterocyst development and some penicillin-binding proteins (PBPs) participating in PG synthesis are required for heterocyst morphogenesis or functioning. Anabaena has multiple PBPs with functional redundancy. In this study, in order to examine the function of PG synthesis and its relationship with heterocyst development, we created a conditional mutant of mraY, a gene necessary for the synthesis of the PG precursor, lipid I. We show that mraY is required for cell and filament integrity. Furthermore, when mraY expression was being limited, persistent septal PG synthetic activity was observed, resulting in increase in cell width. Under non-permissive conditions, filaments and cells were rapidly lysed, and no sign of heterocyst development within the time window allowed was detected after nitrogen starvation. When mraY expression was being limited, a high percentage of heterocyst doublets were found. These doublets are formed likely as a consequence of delayed cell division and persistent septal PG synthesis. MraY interacts with components of both the elongasome and the divisome, in particular those directly involved in PG synthesis, including HetF, which is required for both cell division and heterocyst formation.

Bacterial cell division, with a few exceptions, is driven by FtsZ through a treadmilling mechanism to remodel and constrict the rigid peptidoglycan (PG) layer. Yet different organisms may differ in the composition of the cell division complex (divisome). In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, hetF is required for the initiation of the differentiation of heterocysts, cells specialized in N2 fixation under combined-nitrogen deprivation. In this study, we demonstrate that hetF is expressed in vegetative cells and necessary for cell division under certain conditions. Under nonpermissive conditions, cells of a ΔhetF mutant stop dividing, consistent with increased levels of HetF under similar conditions in the wild type. Furthermore, HetF is a membrane protein located at midcell and cell-cell junctions. In the absence of HetF, FtsZ rings are still present in the elongated cells; however, PG remodeling is abolished. This phenotype is similar to that observed with the inhibition of the septal PG synthase FtsI. We further reveal that HetF is recruited to or stabilized at the divisome by interacting with FtsI and that this interaction is necessary for HetF function in cell division. Our results indicate that HetF is a member of the divisome depending mainly on light intensity and reveal distinct features of the cell division machinery in cyanobacteria that are of high ecological and environmental importance. IMPORTANCE Cyanobacteria shaped the Earth’s evolutionary history and are still playing important roles for elementary cycles in different environments. They consist of highly diverse species with different cell shapes, sizes, and morphologies. Although these properties are strongly affected by the process of cytokinesis, the mechanism remains largely unexplored. Using different approaches, we demonstrate that HetF is a new component of the cell division machinery under certain environmental conditions in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. The common and diverged characteristics of cell division in prokaryotes reflect the evolutionary history of different bacteria as an adaptive measure to proliferate under certain environmental conditions. As a protein for cell differentiation, the recruitment of HetF to the septum illustrates such an adaptive mechanism in cyanobacteria.

This study is the first to determine the transcriptomes of eight colonial volvocine algae: Pandorina morum, P. colemaniae, Volvulina compacta, Eudorina elegans, E. cylindrica, Yamagishiella unicocc...

The eukaryotic phytoplankton community in the Three Gorges Reservoir (TGR) of the Yangtze River in China was investigated, based on marker gene analysis of the 18S rRNA V4 region. Community composition was investigated in four seasons (to assess seasonal variation) and in the mainstream and tributary of the river (to assess spatial variation). Chlorophyll a concentration was used to estimate eukaryotic phytoplankton biomass. A significant negative correlation of biomass and evenness was found in the TGR; this relationship may be due to a few highly competitive species dominating the eukaryotic phytoplankton community, suggesting that eukaryotic phytoplankton bloom events tend to occur in such communities. Our results showed that eukaryotic phytoplankton is more likely to bloom in the tributary when compared with mainstream, and we identified two unreported algal blooms, Tetraselmis and Mychonastes. Phylogenetic analysis revealed a novel lineage in Cryptophyta and substantially more diverse species in Bacillariophyta, two high relative abundance operational taxonomic units in Bacillariophyta classified to Peridiniopsis diatom endosymbionts; their high relative abundance may result from a high relative abundance of the genus Peridiniopsis. We found that Cryptomonas was the most widely distributed genus and had the highest relative abundance, and species in TGR that have higher relative abundance tend to be distributed more widely.

The colonial volvocine algae span the full range of organizational complexity, from four-celled species to multicellular species, and this group of algae is often used for the study of evolution. In recent years, many organelle genomes have been sequenced using the application of next generation sequencing technology; however, only a few organelle genomes have been reported in colonial volvocine algae. In this study, we determined the organelle genomes of Eudorina elegans and Eudorina cylindrica and analysed the organelle genome size, structure and gene content between these volvocine species. This provided useful information to help us understand the composition of colonial volvocine organelle genomes. Based on the chloroplast genome protein-coding genes, we conducted a phylogenomics analysis of the volvocine algae. The result revealed an unexpected phylogenetic relationship, namely, E. elegans is more closely related to Pleodorina starrii than to E. cylindrica. The substitution rate of volvocine algae was then calculated based on organelle genome protein-coding genes; our analysis suggested the possibility that the two Eudorina species may be under similar evolutionary pressure. Lastly, the synteny analysis of the mitochondrial genome showed that gene arrangements and contents are highly conserved in the family Volvocaceae, and the synteny analysis of the chloroplast genome indicated that the genus Eudorina may have experienced genomic changes.

Bacterial cell division, with a few exceptions, is driven by FtsZ through a treadmilling mechanism to remodel and constrict the rigid peptidoglycan (PG) layer. Yet, different organisms may differ in the composition of the cell-division complex (divisome). In the filamentous cyanobacterium Anabaena PCC 7120, hetF is required for the initiation of the differentiation of heterocysts, cells specialized in N2-fixing cells under combined nitrogen deprivation. In this study, we demonstrate that hetF is expressed in vegetative cells and necessary for cell division in a conditional manner. Under non-permissive conditions, cells of a ΔhetF mutant stop dividing, consistent with increased level of HetF under similar conditions in the wild type. Furthermore, HetF is a membrane protein located at midcell and cell-cell junctions. In the absence of HetF, FtsZ rings are still present in the elongated cells; however, PG remodelling is abolished. This phenotype is similar to that observed with the inhibition of septal PG synthase FtsI. We further reveal that HetF is recruited to or stabilized at the divisome by interacting with FtsI and this interaction is necessary for HetF function in cell division. Our results indicate that HetF is a member of the divisome, and reveal distinct features of the cell-division machinery in cyanobacteria that are of high ecological and environmental importance.Significance StatementCyanobacteria shaped the Earth’s evolutionary history, and are still playing important roles for elementary cycles in different environments. They are consisted of highly diverse species with different cell shape, size and morphology. Although these properties are strongly affected by the process of cytokinesis, the mechanism remains largely unexplored. Using different approaches, we demonstrate that HetF is a new component of the cell division machinery in the filamentous cyanobacterium Anabaena PCC 7120. The common and diverged characteristics of cell division in prokaryotes reflect the evolutionary history of different bacteria, as an adaptive measure to proliferate under certain environmental conditions. As a protein for cell differentiation, the recruitment of HetF to the septum illustrates such an adaptive mechanism for cyanobacteria.

The filamentous cyanobacterium Anabaena sp. PCC 7120 develops N2-fixing heterocyst cells under condition of combined-nitrogen deprivation and constitutes an excellent model for studying cell differentiation. The mechanism of heterocyst development has been extensively investigated and a network of regulating factors has been identified. A few studies have showed that the process of heterocyst differentiation relates with cell cycle events, but further investigation is still required to understand this relationship. In a previous study, we created a conditional mutant of PolI encoding gene, polA, by using a CRISPR/Cpf1 gene-editing technique. Here, we were able to create another conditional mutant of a PolIII encoding gene dnaENI using a similar strategy and subsequently confirmed the essential roles of both polA and dnaENI in DNA replication. Further investigation on the phenotype of the mutants showed that lack of PolI caused defects in chromosome segregation and cell division, while lack of DnaENI (PolIII) prevented bulk DNA synthesis, causing significant loss of DNA content. Our findings also suggested the possible existence of a SOS-response like mechanism operating in Anabaena PCC 7120. Moreover, we found that heterocyst development was differently affected in the two conditional mutants, with double heterocysts/proheterocysts found in PolI conditional mutant. We further showed that formation of such double heterocysts/proheterocysts are likely caused by the difficulty in nucleoids segregation, resulting delayed, or non-complete closure of the septum between the two daughter cells. This study uncovers a link between DNA replication process and heterocyst differentiation, paving the way for further studies on the relationship between cell cycle and cell development.

CRISPR systems, such as CRISPR-Cas9 and CRISPR-Cpf1, have been successfully used for genome editing in a variety of organisms. Although the technique of CRISPR-Cpf1 has been applied in cyanobacteria recently, its use was limited without exploiting the full potential of such a powerful genetic system. Using the cyanobacterium Anabaena PCC 7120 as a model strain, we improved the tools and designed genetic strategies based on CRISPR-Cpf1, which enabled us to realize genetic experiments that have been so far difficult to do in cyanobacteria. The development includes: (1) a "two-spacers" strategy for single genomic modification, with a success rate close to 100%; (2) rapid multiple genome editing using editing plasmids with different resistance markers; (3) using sacB, a counter-selection marker conferring sucrose sensitivity, to enable the active loss of the editing plasmids and facilitate multiple rounds of genetic modification or phenotypic analysis; (4) manipulation of essential genes by the creation of conditional mutants, using as example, polA encoding the DNA polymerase I essential for DNA replication and repair; (5) large DNA fragment deletion, up to 118 kb, from the Anabaena chromosome, corresponding to the largest bacterial chromosomal region removed with CRISPR systems so far. The genome editing vectors and the strategies developed here will expand our ability to study and engineer cyanobacteria, which are extensively used for fundamental studies, biotechnological applications including biofuel production, and synthetic biology research. The vectors developed here have a broad host range, and could be readily used for genetic modification in other microorganisms.

The filamentous cyanobacterium Anabaena can form heterocysts specialized in N 2 fixation, mostly through a cascade of transcriptional activation in response to the nitrogen starvation signal 2-oxoglutarate. It is reported now that a transcription repressor, CalA, acts as a safety device to prevent heterocyst development under certain conditions where the 2-oxoglutarate level may touch the threshold to trigger unnecessary initiation of heterocyst development.

We sequenced the mitochondrial genome of six colonial volvocine algae, namely: Pandorina morum, Pandorina colemaniae, Volvulina compacta, Colemanosphaera angeleri, Colemanosphaera charkowiensi, and Yamagishiella unicocca. Previous studies have typically reconstructed the phylogenetic relationship between colonial volvocine algae based on chloroplast or nuclear genes. Here, we explore the validity of phylogenetic analysis based on mitochondrial protein-coding genes. We found phylogenetic incongruence of the genera Yamagishiella and Colemanosphaera. In Yamagishiella, the stochastic error and linkage group formed by the mitochondrial protein-coding genes prevent phylogenetic analyses from reflecting the true relationship. In Colemanosphaera, a different reconstruction approach revealed a different phylogenetic relationship. This incongruence may be because of the influence of biological factors, such as incomplete lineage sorting or horizontal gene transfer. We also analyzed the substitution rates in the mitochondrial and chloroplast genomes between colonial volvocine algae. Our results showed that all volvocine species showed significantly higher substitution rates for the mitochondrial genome compared with the chloroplast genome. The nonsynonymous substitution (dN)/synonymous substitution (dS) ratio is similar in the genomes of both organelles in most volvocine species, suggesting that the two counterparts are under a similar selection pressure. We also identified a few chloroplast protein-coding genes that showed high dN/dS ratios in some species, resulting in a significant dN/dS ratio difference between the mitochondrial and chloroplast genomes.

Cyanobacteria were the first oxygenic photosynthetic organisms during evolution and were ancestors of plastids. Cyanobacterial cells exhibit an extraordinary diversity in their size and shape, and bacterial cell morphology largely depends on the synthesis and the dynamics of the peptidoglycan (PG) layer. Here, we used a fluorescence analog of the PG synthesis precursor D-Ala, 7-Hydroxycoumarin-amino-D-alanine (HADA), to probe the PG synthesis pattern in live cells of cyanobacteria with different morphology. They displayed diverse synthesis patterns, with some strains showing an intensive HADA incorporation at the septal region, whereas others gave an HADA signal distributed around the cells. Growth zones covering several cells at the tips of the filament were present in some filamentous strains such as in Arthrospira. In Anabaena PCC 7120, which is capable of differentiating heterocysts for N2 fixation, PG synthesis followed the cell division cycle. In addition, an HADA incorporation was strongly activated from 12 to 15 h following the initiation of heterocyst development, indicating a thickening of the PG layer in heterocysts. The PG synthesis pattern is diverse in cyanobacteria and responds to developmental regulation. The use of fluorescent analogs may serve as a useful tool for understanding the mechanisms of cell growth and morphogenesis operating in these organisms.

The shape parameter is proved to have great influence on Radial Basis Function (RBF) interpolation. Its impacts on Multiquadric (MQ) RBF are studied in this paper. Observation suggests that a large shape parameter improves the accuracy of the approximation of high non linear problems, but may lead to Runge Phenomenon (RP). This paper proposed an algorithm to obtain the shape parameter by minimizing the cross-valid error while controlling the RP. Compared with Kriging, numerical experiments show that the MQ RBF with optimum shape parameter can achieve higher accuracy when approximate the high non linear problems, the application in the engineering problems also proved the effectiveness of the algorithm.