Analysis of fate determination of vegetative cells with reduced phycocyanin content in a multicellular cyanobacterium using Raman microscopy.

The one-dimensional multicellular cyanobacterium Anabaena sp. PCC 7120 exhibits two different cell types under nitrogen-deprived conditions. We found that the intensity of the Raman band at 1,629 cm -1 , which is associated with phycocyanin, was higher in undifferentiated cells (vegetative cells) than in differentiated cells (heterocysts). We observed cells whose band intensity at 1,629 cm -1 was statistically lower than that of vegetative cells, and named them "proheterocysts". We found that proheterocysts did not necessarily differentiate, and could divide or revert to being vegetative cells, as defined by having a higher band intensity at 1,629 cm -1 .

Draft genome sequence of the basidiomycetous yeast Mrakia hoshinonis JCM 32575T isolated from Ellesmere Island, Canadian High Arctic.

Mrakia hoshinonis JCM 32575 was isolated from glacial sediments on Ellesmere Island in the Canadian High Arctic and described as a new basidiomycetous yeast. This species does not require amino acids and vitamins for growth and can grow at sub-zero temperatures. Here, we report a draft genome sequence of this strain.

A critical role of the periplasm in copper homeostasis in Gram-negative bacteria.

Copper is essential for life, but is toxic in excess. Copper homeostasis is achieved in the cytoplasm and the periplasm as a unique feature of Gram-negative bacteria. Especially, it has become clear the role of the periplasm and periplasmic proteins regarding whole-cell copper homeostasis. Here, we addressed the role of the periplasm and periplasmic proteins in copper homeostasis using a Systems Biology approach integrating experiments with models. Our analysis shows that most of the copper-bound molecules localize in the periplasm but not cytoplasm, suggesting that Escherichia coli utilizes the periplasm to sense the copper concentration in the medium and sequester copper ions. In particular, a periplasmic multi-copper oxidase CueO and copper-responsive transcriptional factor CusS contribute both to protection against Cu(I) toxicity and to incorporating copper into the periplasmic components/proteins. We propose that Gram-negative bacteria have evolved mechanisms to sense and store copper in the periplasm to expand their living niches.

Raman spectral analysis of microbial pigment compositions in vegetative cells and heterocysts of multicellular cyanobacterium.

The one-dimensional multicellular cyanobacterium, Anabaena sp. PCC 7120, exhibits a simple topology consisting of two types of cells under the nitrogen-depleted conditions. Although the differentiated (heterocyst) and undifferentiated cells (vegetative cells) were distinguished by their cellular shapes, we found that their internal states, that is, microbial pigment compositions, were distinguished by using a Raman microscope. Almost of Raman bands of the cellular components were assigned to vibrations of the pigments; chlorophyll a, ß-carotene, phycocyanin, and allophycocyanin. We found that the Raman spectral measurement can detect the decomposition of both phycocyanin and allophycocyanin, which are components of the light-harvesting phycobilisome complex in the photosystem II. We observed that the Raman bands of phycocyanin and allophycocyanin exhibited more remarkable decrease in the heterocysts when compared to those of chlorophyll a and ß-carotene. This result indicated the prior decomposition of phycobilisome in the heterocysts. We show that the Raman measurement is useful to detect the change of pigment composition in the cell differentiation.

Dielectrophoresis-Based Selective Droplet Extraction Microfluidic Device for Single-Cell Analysis.

We developed a microfluidic device that enables selective droplet extraction from multiple droplet-trapping pockets based on dielectrophoresis. The device consists of a main microchannel, five droplet-trapping pockets with side channels, and drive electrode pairs appropriately located around the trapping pockets. Agarose droplets capable of encapsulating biological samples were successfully trapped in the trapping pockets due to the difference in flow resistance between the main and side channels. Target droplets were selectively extracted from the pockets by the dielectrophoretic force generated between the electrodes under an applied voltage of 500 V. During their extraction from the trapping pockets, the droplets and their contents were exposed to an electric field for 400-800 ms. To evaluate whether the applied voltage could potentially damage the biological samples, the growth rates of Escherichia coli cells in the droplets, with and without a voltage applied, were compared. No significant difference in the growth rate was observed. The developed device enables the screening of encapsulated single cells and the selective extraction of target droplets.

Genome Sequence of Basidiomycetous Yeast Mrakia gelida MGH-2, Isolated from Skarvsnes Ice-Free Area, East Antarctica.

Basidiomycetous yeast Mrakia gelida MGH-2 has been reported from Surikogi Ike in the Skarvsnes ice-free area, East Antarctica. Here, we report on the high-quality genome sequence of the Mrakia gelida MGH-2 strain analyzed by PacBio Sequel and HiSeq 2500 instruments.

Position-dependent changes in phycobilisome abundance in multicellular cyanobacterial filaments revealed by Raman spectral analysis.

The one-dimensional filamentous cyanobacterium, Anabaena sp. PCC 7120, shows a simple morphological pattern consisting of two distinct cell types under nitrogen-deprived conditions. We found that microbial pigment composition in differentiated (heterocyst) and undifferentiated cells (vegetative cells) can be distinguished using Raman microscopy. The Raman bands associated with phycocyanin and allophycocyanin were of higher intensity in vegetative cells than those in heterocysts. However, these bands had statistically lower intensity in vegetative cells located further away from heterocysts. That is, the pigment composition in individual cells is affected by locational information in a filament.

High-Quality Genome Sequence of Cystobasidium tubakii JCM 31526T, Isolated from East Ongul Island, Antarctica.

Cystobasidium tubakii has been reported as a new basidiomycetous yeast species from East Ongul Island, East Antarctica. This species does not require amino acids and vitamins for growth and can grow at subzero temperatures. Here, we report a high-quality genome sequence of Cystobasidium tubakii strain JCM 31526T, which was isolated from East Antarctica.

Draft Genome Sequences of Five Cystobasidium ongulense Strains Isolated from Areas near Syowa Station, East Antarctica.

Cystobasidium ongulense has been reported from East Ongul Island near Syowa Station, East Antarctica, as a new basidiomycetous yeast species. This species has cold active lipases and cellulases that are active even at subzero temperatures. We report draft genome sequences of five Cystobasidium ongulense strains isolated from East Antarctica.

ATG8 is conserved between Saccharomyces cerevisiae and psychrophilic, polar-collected fungi.

Autophagy is a conserved catabolic process by which eukaryotic cells respond to stress by targeting damaged or unneeded molecules or organelles for sequestration into specialized vesicles known as autophagosomes. Autophagosomes ultimately facilitate the digestion and recycling of their contents by fusing with the degradative organelle of the cell. Studies of the budding yeast Saccharomyces cerevisiae have revealed various types of stress that can regulate autophagy, including starvation and extreme temperatures. While autophagy has not yet been directly shown to confer the ability to survive extreme cold or freeze-thaw stress in yeast, upregulation of autophagy has been directly implicated in the ability of arctic insects to survive cold temperatures. We are interested in investigating the potential role of autophagy in polar habitat survival by cold-loving (psychrophilic) yeast like Mrakia blollopsis. To begin to examine the conservation of Atg machinery in polar-collected yeast, we focused on Atg8, a small, ubiquitin-like protein that plays an important role in autophagy. We report that Atg8 is conserved between S. cerevisiae and polar-collected yeast, using Atg8 from Mrakia blollopsis (strain TGK1-2) as an example. This study represents the first direct examination of autophagy machinery conservation across mesophilic and psychrophilic species of yeast.

DIpartite: A tool for detecting bipartite motifs by considering base interdependencies.

It is extremely important to identify transcription factor binding sites (TFBSs). Some TFBSs are proposed to be bipartite motifs known as two-block motifs separated by gap sequences with variable lengths. While position weight matrix (PWM) is commonly used for the representation and prediction of TFBSs, dinucleotide weight matrix (DWM) enables expression of the interdependencies of neighboring bases. By incorporating DWM into the detection of bipartite motifs, we have developed a novel tool for ab initio motif detection, DIpartite (bipartite motif detection tool based on dinucleotide weight matrix) using a Gibbs sampling strategy and the minimization of Shannon's entropy. DIpartite predicts the bipartite motifs by considering the interdependencies of neighboring positions, that is, DWM. We compared DIpartite with other available alternatives by using test datasets, namely, of CRP in E. coli, sigma factors in B. subtilis, and promoter sequences in humans. We have developed DIpartite for the detection of TFBSs, particularly bipartite motifs. DIpartite enables ab initio prediction of conserved motifs based on not only PWM, but also DWM. We evaluated the performance of DIpartite by comparing it with freely available tools, such as MEME, BioProspector, BiPad, and AMD. Taken the obtained findings together, DIpartite performs equivalently to or better than these other tools, especially for detecting bipartite motifs with variable gaps. DIpartite requires users to specify the motif lengths, gap length, and PWM or DWM. DIpartite is available for use at https://github.com/Mohammad-Vahed/DIpartite.

Mathematical study of pattern formation accompanied by heterocyst differentiation in multicellular cyanobacterium.

The filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest models of a multicellular system showing cellular differentiation. In nitrogen-deprived culture, undifferentiated vegetative cells differentiate into heterocysts at ~10-cell intervals along the cellular filament. As undifferentiated cells divide, the number of cells between heterocysts (segment length) increases, and a new heterocyst appears in the intermediate region. To understand how the heterocyst pattern is formed and maintained, we constructed a one-dimensional cellular automaton (CA) model of the heterocyst pattern formation. The dynamics of vegetative cells is modeled by a stochastic transition process including cell division, differentiation and increase of cell age (maturation). Cell division and differentiation depend on the time elapsed after the last cell division, the "cell age". The model dynamics was mathematically analyzed by a two-step Markov approximation. In the first step, we determined steady state of cell age distribution among vegetative cell population. In the second step, we determined steady state distribution of segment length among segment population. The analytical solution was consistent with the results of numerical simulations. We then compared the analytical solution with the experimental data, and quantitatively estimated the immeasurable intercellular kinetics. We found that differentiation is initially independent of cellular maturation, but becomes dependent on maturation as the pattern formation evolves. Our mathematical model and analysis enabled us to quantify the internal cellular dynamics at various stages of the heterocyst pattern formation.

Cyanobacterial cell lineage analysis of the spatiotemporal hetR expression profile during heterocyst pattern formation in Anabaena sp. PCC 7120.

Diazotrophic heterocyst formation in the filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest pattern formations known to occur in cell differentiation. Most previous studies on heterocyst patterning were based on statistical analysis using cells collected or observed at different times from a liquid culture, which would mask stochastic fluctuations affecting the process of pattern formation dynamics in a single bacterial filament. In order to analyze the spatiotemporal dynamics of heterocyst formation at the single filament level, here we developed a culture system to monitor simultaneously bacterial development, gene expression, and phycobilisome fluorescence. We also developed micro-liquid chamber arrays to analyze multiple Anabaena filaments at the same time. Cell lineage analyses demonstrated that the initial distributions of hetR::gfp and phycobilisome fluorescence signals at nitrogen step-down were not correlated with the resulting distribution of developed heterocysts. Time-lapse observations also revealed a dynamic hetR expression profile at the single-filament level, including transient upregulation accompanying cell division, which did not always lead to heterocyst development. In addition, some cells differentiated into heterocysts without cell division after nitrogen step-down, suggesting that cell division in the mother cells is not an essential requirement for heterocyst differentiation.