Endocytosis in different lifestyles of protozoan parasitism: role in nutrient uptake with special reference to Toxoplasma gondii

A fundamental property of any eukaryotic cell is endocytosis, that is the ability to take up external fluid, solutes and particulate matter into membrane-bound intracellular vesicles by various mechanisms. Toxoplasma gondii is an intracellular protozoan parasite of the phylum Apicomplexa with a wide geographical and host range distribution. Significant progress in studying the cell biology of this parasite has been accomplished over the last few years. Only recently endocytic compartments and endocytic trafficking have come to a closer dissection in T. gondii. In this review, we discuss the evidence for an endocytic compartment and present a model for an endocytic pathway in Toxoplasma against a background of endocytosis in kinetoplastida and the extensive insights gained from mammalian and yeast cells.     

Discovery of the novel candidate phylum "Poribacteria" in marine sponges

Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named "Poribacteria", to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with "Poribacteria"-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.     

Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes

In eukaryotic cells, it is generally accepted that protein synthesis is compartmentalized; soluble proteins are synthesized on free ribosomes, whereas secretory and membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes. The partitioning of mRNAs that accompanies such compartmentalization arises early in protein synthesis, when ribosomes engaged in the translation of mRNAs encoding signal-sequence-bearing proteins are targeted to the ER. In this report, we use multiple cell fractionation protocols, in combination with cDNA microarray, nuclease protection, and Northern blot analyses, to assess the distribution of mRNAs between free and ER-bound ribosomes. We find a broad representation of mRNAs encoding soluble proteins in the ER fraction, with a subset of such mRNAs displaying substantial ER partitioning. In addition, we present evidence that membrane-bound ribosomes engage in the translation of mRNAs encoding soluble proteins. Single-cell in situ hybridization analysis of the subcellular distribution of mRNAs encoding ER-localized and soluble proteins identify two overall patterns of mRNA distribution in the cell-endoplasmic reticular and cytosolic. However, both partitioning patterns include a distinct perinuclear component. These results identify previously unappreciated roles for membrane-bound ribosomes in the subcellular compartmentalization of protein synthesis and indicate possible functions for the perinuclear membrane domain in mRNA sorting in the cell.     

Discovery of the Novel Candidate Phylum “Poribacteria” in Marine Sponges

Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named “Poribacteria”, to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with “Poribacteria”-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.     

In or out? Regulating nuclear transport

The compartmentalization of proteins within the nucleus or cytoplasm of a eukaryotic cell offers opportunity for regulation of cell cycle progression and signalling pathways. Nuclear localization of proteins is determined by their ability to interact with specific nuclear import and export factors. In the past year, substrate phosphorylation has emerged as a common mechanism for controlling this interaction.     

The cell cycle of the planctomycete Gemmata obscuriglobus with respect to cell compartmentalization

Background  

Gemmata obscuriglobus is a distinctive member of the divergent phylum Planctomycetes, all known members of which are peptidoglycan-less bacteria with a shared compartmentalized cell structure and divide by a budding process. G. obscuriglobus in addition shares the unique feature that its nucleoid DNA is surrounded by an envelope consisting of two membranes forming an analogous structure to the membrane-bounded nucleoid of eukaryotes and therefore G. obscuriglobus forms a special model for cell biology. Draft genome data for G. obscuriglobus as well as complete genome sequences available so far for other planctomycetes indicate that the key bacterial cell division protein FtsZ is not present in these planctomycetes, so the cell division process in planctomycetes is of special comparative interest. The membrane-bounded nature of the nucleoid in G. obscuriglobus also suggests that special mechanisms for the distribution of this nuclear body to the bud and for distribution of chromosomal DNA might exist during division. It was therefore of interest to examine the cell division cycle in G. obscuriglobus and the process of nucleoid distribution and nuclear body formation during division in this planctomycete bacterium via light and electron microscopy.     

Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation methods

By means of fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH), it has been shown that members of the phylum Planctomycetes represent a numerically significant bacterial group in boreal Sphagnum peat bogs. The population size of planctomycetes in oxic layers of the peat bog profile was in the range of 0.4-2.0 x 10(7) cells per g of wet peat, comprising 4 to 13% of the total bacterial cell number. A novel effective approach that combined a traditional cultivation technique with FISH-mediated monitoring of the target organism during the isolation procedure has been developed for the isolation of planctomycetes. Using this approach, we succeeded in isolating several peat-inhabiting planctomycetes in a pure culture. Sequencing of the 16S rRNA genes from two of these isolates, strains A10 and MPL7, showed that they belonged to the planctomycete lineages defined by the genera Gemmata and Planctomyces, respectively. The 16S rRNA gene sequence similarity between strains A10 and MPL7 and the phylogenetically closest organisms, namely, Gemmata obscuriglobus and Planctomyces limnophilus, was only 90%. These results suggest that the indigenous planctomycetes inhabiting Sphagnum peat bogs are so far unknown organisms.     

The origin of the eukaryotic cell. III. Principles of the morphofunctional organization of the eukaryotic cell

The eukaryotic plasmalemma, eukaryotic cytoplasm with its usual cytomembranes, and eukaryotic nucleus are obligatory components of the eukaryotic cell. All other structural elements (organelles) are only derivates of the aforesaid cell components and they may be absent sometimes. There are protozoans having simultaneously no flagelles, mitochondria and chloroplasts (all the representatives of phylum Microspora, amoeba Pelomyxa palustris, and others). The following five general principles play the main role in the morphofunctional organization of the cell. The principle of hierarchy of block organization of living systems. Complex morphofunctional blocks (organelles) specific for the eukaryotic cell are formed. The compartmentalization principle. The main cell organelles (nuclei, flagellae, mitochondria, chloroplasts, etc.) undergo a relative morphological isolation from each other and other cell organelles by means of the total or partial surrounding by membranes; this may ensure the originality of their evolution and function. The principle of poly- and oligomerization of morphofunctional blocks. It permits the cell to enlarge its sizes and to raise the level of integration. The principle of heterochrony, including three subprinciples: conservatism of useful signs; a strong acceleration of evolutionary development of the separate blocks; simplification of the structure, reduction or total disappearance of some blocks. It explains a preservation of prokaryotic signs in the eukaryotic cell or in its organelles. The principle of independent origin of similar morphofunctional blocks in the process of evolution of living systems. The parallelism of the signs in unrelated groups of cells (or protists) arises due to this principle.     

Isolation and diversity of planctomycetes from the sponge Niphates sp., seawater, and sediment of Moreton Bay, Australia

Planctomycetes are ubiquitous in marine environment and were reported to occur in association with multicellular eukaryotic organisms such as marine macroalgae and invertebrates. Here, we investigate planctomycetes associated with the marine sponge Niphates sp. from the sub-tropical Australian coast by assessing their diversity using culture-dependent and -independent approaches based on the 16S rRNA gene. The culture-dependent approach resulted in the isolation of a large collection of diverse planctomycetes including some novel lineages of Planctomycetes from the sponge as well as sediment and seawater of Moreton Bay where this sponge occurs. The characterization of these novel planctomycetes revealed that cells of one unique strain do not possess condensed nucleoids, a phenotype distinct from other planctomycetes. In addition, a culture-independent clone library approach identified unique planctomycete 16S rRNA gene sequences closely related to other sponge-derived sequences. The analysis of tissue of the sponge Niphates sp. showed that the mesohyl of the sponge is almost devoid of microbial cells, indicating this species is in the group of ‘low microbial abundant’ (LMA) sponges. The unique planctomycete 16S rRNA gene sequences identified in this study were phylogenetically closely related to sequences from LMA sponges in other published studies. This study has revealed new insights into the diversity of planctomycetes in the marine environment and the association of planctomycetes with marine sponges.     

Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation approaches

By means of fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH), it has been shown that members of the phylum Planctomycetes represent a numerically significant bacterial group in boreal Sphagnum peat bogs. The population size of planctomycetes in oxic layers of the peat bog profile was in the range of 0.4–2.0 × 10⁷ cells per g of wet peat, comprising 4 to 13% of the total bacterial cell number. A novel effective approach that combined a traditional cultivation technique with FISH-mediated monitoring of the target organism during the isolation procedure has been developed for the isolation of planctomycetes. Using this approach, we succeeded in isolating several peat-inhabiting planctomycetes in a pure culture. Sequencing of the 16S rRNA genes from two of these isolates, strains A10 and MPL7, showed that they belonged to the planctomycete lineages defined by the genera Gemmata and Planctomyces, respectively. The 16S rRNA gene sequence similarity between strains A10 and MPL7 and the phylogenetically closest organisms, namely, Gemmata obscuriglobus and Planctomyces limnophilus, was only 90%. These results suggest that the indigenous planctomycetes inhabiting Sphagnum peat bogs are so far unknown organisms.     

Novel protein domains and motifs in the marine planctomycete Rhodopirellula baltica

The planctomycetes are a phylum of bacteria that have a unique cell compartmentalisation and yeast-like budding cell division and peptidoglycan-less proteinaceous cell walls. We wished to further our understanding of these unique organisms at the molecular level by searching for conserved amino acid sequence motifs and domains in the proteins encoded by Rhodopirellula baltica. Using BLAST and single-linkage clustering, we have discovered several new protein domains and sequence motifs in this planctomycete. R. baltica has multiple members of the newly discovered GEFGR protein family and the ASPIC C-terminal domain family, whilst most other organisms for which whole genome sequence is available have no more than one. Many of the domains and motifs appear to be restricted to the planctomycetes. It is possible that these protein domains and motifs may have been lost or replaced in other phyla, or they may have undergone multiple duplication events in the planctomycete lineage. One of the novel motifs probably represents a novel N-terminal export signal peptide. With their unique cell biology, it may be that the planctomycete cell compartmentalisation plan in particular needs special membrane transport mechanisms. The discovery of these new domains and motifs, many of which are associated with secretion and cell-surface functions, will help to stimulate experimental work and thus enhance further understanding of this fascinating group of organisms.     

Assessment of planctomycetes cell viability after pollutants exposure

In this study, the growth of six different planctomycetes, a particular ubiquitous bacterial phylum, was assessed after exposure to pollutants. In addition and for comparative purposes, Pseudomonas putida, Escherichia coli and Vibrio anguillarum were tested. Each microorganism was exposed to several concentrations of 21 different pollutants. After exposure, bacteria were cultivated using the drop plate method. In general, the strains exhibited a great variation of sensitivity to pollutants in the order: V. anguillarum > planctomycetes > P. putida > E. coli. E. coli showed resistance to all pollutants tested, with the exception of phenol and sodium azide. Copper, Ridomil^® (fungicide), hydrazine and phenol were the most toxic pollutants. Planctomycetes were resistant to extremely high concentrations of nitrate, nitrite and ammonium but they were the only bacteria sensitive to Previcur N^® (fungicide). Sodium azide affected the growth on plates of E. coli, P. putida and V. anguillarum, but not of planctomycetes. However, this compound affected planctomycetes cell respiration but with less impact than in the aforementioned bacteria. Our results provide evidence for a diverse response of bacteria towards pollutants, which may influence the structuring of microbial communities in ecosystems under stress, and provide new insights on the ecophysiology of planctomycetes.     

The PVC superphylum: exceptions to the bacterial definition?

The PVC superphylum is a grouping of distinct phyla of the domain bacteria proposed initially on the basis of 16S rRNA gene sequence analysis. It consists of a core of phyla Planctomycetes, Verrucomicrobia and Chlamydiae, but several other phyla have been considered to be members, including phylum Lentisphaerae and several other phyla consisting only of yet-to-be cultured members. The genomics-based links between Planctomycetes, Verrucomicrobia and Chlamydiae have been recently strengthened, but there appear to be other features which may confirm the relationship at least of Planctomycetes, Verrucomicrobia and Lentisphaerae. Remarkably these include the unique planctomycetal compartmentalized cell plan differing from the cell organization typical for bacteria. Such a shared cell plan suggests that the common ancestor of the PVC superphylum members may also have been compartmentalized, suggesting this is an evolutionarily homologous feature at least within the superphylum. Both the PVC endomembranes and the eukaryote-homologous membrane-coating MC proteins linked to endocytosis ability in Gemmata obscuriglobus and shared by PVC members suggest such homology may extend beyond the bacteria to the Eukarya. If so, either our definition of bacteria may have to change or PVC members admitted to be exceptions. The cases for and against considering the PVC superphylum members as exceptions to the bacteria are discussed, and arguments for them as exceptions presented. Recent critical analysis has favoured convergence and analogy for explaining eukaryote-like features in planctomycetes and other PVC organisms. The case is made for constructing hypotheses leaving the possibility of homology and evolutionary links to eukaryote features open. As the case of discovery of endocytosis-like protein uptake in planctomycetes has suggested, this may prove a strong basis for the immediate future of experimental research programs in the PVC scientific community.     

The Candidate Phylum Poribacteria by Single-Cell Genomics: New Insights into Phylogeny,Cell-Compartmentation,Eukaryote-Like Repeat Proteins,and Other Genomic Features

The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake.     

Subcellular compartmentalization by local differentiation of cytoplasmic structure

The compartmentalization of eukaryotic cells by internal membranes and the subcellular localization of endogenous macromolecules by specific binding mechanisms are familiar concepts. In this report we present evidence that the cytoplasmic ground substance, which surrounds and contains the membrane-bound compartments, may also be compartmentalized by local differentiations of its submicroscopic structure that sort subcellular particles on the basis of size. The subcellular distribution of size-fractionated, fluorescent tracer particles was studied in living cells by ratio imaging and fluorescence recovery after photobleaching (FRAP). Large and small particles showed different distributions within the cytoplasmic volume, suggesting that the large particles were relatively excluded from some domains. While the structural basis for this phenomenon is not yet understood in detail, ratio imaging of large and small particles can be used as an empirical tool to identify cytoplasmic compartments for further study. The cytoplasmic diffusion coefficient (Dcyto) and % mobile fraction of the large particles showed considerable spatial variation over the projected area of the cell, while Dcyto and % mobile fraction of the small particles did not. A model is presented to account for this difference. Based on this model, a method is proposed by which FRAP can be used to detect sol-gel transitions in the cytoplasmic ground substance of living cells.     

Wide distribution of Phycisphaera-like planctomycetes from WD2101 soil group in peatlands and genome analysis of the first cultivated representative

Phycisphaera-like WD2101 ‘soil group’ is one of the as-yet-uncultivated phylogenetic clades within the phylum Planctomycetes. Members of this clade are commonly detected in various terrestrial habitats. This study shows that WD2101 represented one of the major planctomycete groups in 10 boreal peatlands, comprising up to 76% and 36% of all Planctomycetes-affiliated 16S rRNA gene reads in raised bogs and eutrophic fens respectively. These types of peatlands displayed clearly distinct intra-group diversity of WD2101-affiliated planctomycetes. The first isolate of this enigmatic planctomycete group, strain M1803, was obtained from a humic lake surrounded by Sphagnum peat bogs. Strain M1803 displayed 89.2% 16S rRNA gene similarity to Tepidisphaera mucosa and was represented by motile cocci that divided by binary fission and grew under micro-oxic conditions. The complete 7.19 Mb genome of strain M1803 contained an array of genes encoding Planctomycetal type bacterial microcompartment organelle likely involved in l -rhamnose metabolism, suggesting participation of M1803-like planctomycetes in polysaccharide degradation in peatlands. The corresponding cellular microcompartments were revealed in ultrathin cell sections. Strain M1803 was classified as a novel genus and species, Humisphaera borealis gen. nov., sp. nov., affiliated with the formerly recognized WD2101 ‘soil group’.     

Shuffling the cards in signal transduction: Calcium, arachidonic acid and mechanosensitivity

Cell signaling is a very complex network of biochemical reactions triggered by a huge number of stimuli coming from the external medium. The function of any single signaling component depends not only on its own structure but also on its connections with other biomolecules. During prokaryotic-eukaryotic transition, the rearrangement of cell organization in terms of diffusional compartmentalization exerts a deep change in cell signaling functional potentiality. In this review I briefly introduce an intriguing ancient relationship between pathways involved in cell responses to chemical agonists (growth factors, nutrients, hormones) as well as to mechanical forces (stretch, osmotic changes). Some biomolecules (ion channels and enzymes) act as "hubs", thanks to their ability to be directly or indirectly chemically/mechanically co-regulated. In particular calcium signaling machinery and arachidonic acid metabolism are very ancient networks, already present before eukaryotic appearance. A number of molecular "hubs", including phospholipase A2 and some calcium channels, appear tightly interconnected in a cross regulation leading to the cellular response to chemical and mechanical stimulations.     

Characterization of a Planctomycetal Organelle: a Novel Bacterial Microcompartment for the Aerobic Degradation of Plant Saccharides

Bacterial microcompartments (BMCs) are organelles that encapsulate functionally linked enzymes within a proteinaceous shell. The prototypical example is the carboxysome, which functions in carbon fixation in cyanobacteria and some chemoautotrophs. It is increasingly apparent that diverse heterotrophic bacteria contain BMCs that are involved in catabolic reactions, and many of the BMCs are predicted to have novel functions. However, most of these putative organelles have not been experimentally characterized. In this study, we sought to discover the function of a conserved BMC gene cluster encoded in the majority of the sequenced planctomycete genomes. This BMC is especially notable for its relatively simple genetic composition, its remote phylogenetic position relative to characterized BMCs, and its apparent exclusivity to the enigmatic Verrucomicrobia and Planctomycetes. Members of the phylum Planctomycetes are known for their morphological dissimilarity to the rest of the bacterial domain: internal membranes, reproduction by budding, and lack of peptidoglycan. As a result, they are ripe for many discoveries, but currently the tools for genetic studies are very limited. We expanded the genetic toolbox for the planctomycetes and generated directed gene knockouts of BMC-related genes in Planctomyces limnophilus. A metabolic activity screen revealed that BMC gene products are involved in the degradation of a number of plant and algal cell wall sugars. Among these sugars, we confirmed that BMCs are formed and required for growth on l-fucose and l-rhamnose. Our results shed light on the functional diversity of BMCs as well as their ecological role in the planctomycetes, which are commonly associated with algae.