Towards understanding the molecular mechanism of the endocytosis-like process in the bacterium Gemmata obscuriglobus.

An endocytosis-like process of protein uptake in the planctomycete Gemmata obscuriglobus is a recently discovered process unprecedented in the bacterial world. The molecular mechanisms underlying this process are not yet characterized. A homolog of the MC (membrane-coating) proteins of eukaryotes has been proposed to be involved in the mechanism of this process, but its relationship to eukaryote proteins is controversial. However, a number of other proteins of G. obscuriglobus with domains homologous to those involved in endocytosis in eukaryotes can also be identified. Here we critically evaluate current bioinformatic knowledge, and suggest practical experimental steps to overcome the limits of bioinformatics in elucidating the molecular mechanism of endocytosis in bacteria. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

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.

Endocytosis-like protein uptake in the bacterium Gemmata obscuriglobus.

Endocytosis is a process by which extracellular material such as macromolecules can be incorporated into cells via a membrane-trafficking system. Although universal among eukaryotes, endocytosis has not been identified in Bacteria or Archaea. However, intracellular membranes are known to compartmentalize cells of bacteria in the phylum Planctomycetes, suggesting the potential for endocytosis and membrane trafficking in members of this phylum. Here we show that cells of the planctomycete Gemmata obscuriglobus have the ability to uptake proteins present in the external milieu in an energy-dependent process analogous to eukaryotic endocytosis, and that internalized proteins are associated with vesicle membranes. Occurrence of such ability in a bacterium is consistent with autogenous evolution of endocytosis and the endomembrane system in an ancestral noneukaryote cell.

Genomic rearrangements at the FRA2H common fragile site frequently involve non-homologous recombination events across LTR and L1(LINE) repeats.

Common fragile sites (cFSs) are non-random chromosomal regions that are prone to breakage under conditions of replication stress. DNA damage and chromosomal alterations at cFSs appear to be critical events in the development of various human diseases, especially carcinogenesis. Despite the growing interest in understanding the nature of cFS instability, only a few cFSs have been molecularly characterised. In this study, we fine-mapped the location of FRA2H using six-colour fluorescence in situ hybridisation and showed that it is one of the most active cFSs in the human genome. FRA2H encompasses approximately 530 kb of a gene-poor region containing a novel large intergenic non-coding RNA gene (AC097500.2). Using custom-designed array comparative genomic hybridisation, we detected gross and submicroscopic chromosomal rearrangements involving FRA2H in a panel of 54 neuroblastoma, colon and breast cancer cell lines. The genomic alterations frequently involved different classes of long terminal repeats and long interspersed nuclear elements. An analysis of breakpoint junction sequence motifs predominantly revealed signatures of microhomology-mediated non-homologous recombination events. Our data provide insight into the molecular structure of cFSs and sequence motifs affected by their activation in cancer. Identifying cFS sequences will accelerate the search for DNA biomarkers and targets for individualised therapies.

Making heads or tails of the HU proteins in the planctomycete Gemmata obscuriglobus.

Gemmata obscuriglobus has a highly condensed nucleoid which is implicated in its resistance to radiation. However, the mechanisms by which such compaction is achieved, and the proteins responsible, are still unknown. Here we have examined the genome of G. obscuriglobus for the presence of proteins homologous to those that have been associated with nucleoid condensation. We found two different proteins homologous to the bacterial nucleoid-associated protein HU, one with an N-terminal and one with a C-terminal extension relative to the amino acid sequence of the HU found in Escherichia coli. Sequence analysis revealed that one of these HU homologues represents a novel type with a high number of prolines in its C-terminal extension, whereas the other one has motifs similar to the N terminus of the HU homologue from the radio-resistant bacterium Deinococcus radiodurans. The occurrence of two such HU homologue proteins with these two different terminal extensions in one organism appears to be unique among the Bacteria.

Cathepsin D protects human neuroblastoma cells from doxorubicin-induced cell death.

High incidence of chemotherapy resistance is the primary cause of treatment failure in a subset of neuroblastomas with amplified MYCN. We have reported previously that ectopic MYCN expression promotes proliferation of neuroblastoma Tet21N cells and simultaneously sensitizes them to the drug-induced apoptosis. In search for genes that are involved in MYCN-dependent regulation of drug resistance, we used a function-based gene cloning approach and identified CTSD encoding for a lysosomal aspartyl protease cathepsin D. Downregulation of cathepsin D expression by RNA interference or inhibition of its enzymatic activity increased sensitivity of MYCN-expressing Tet21N cells to doxorubicin. Overexpression of cathepsin D in Tet21N cells attenuated doxorubicin-induced apoptosis. It was accompanied by activation of protein kinase B (Akt) and persistent antiapoptotic activity of Bcl-2. In primary neuroblastomas, high CTSD messenger RNA (mRNA) levels were associated with amplified MYCN, a strong predictive marker of adverse outcome. Chromatin immunoprecipitation and luciferase promoter assays revealed that MYCN protein binds to the CTSD promoter and activates its transcription, suggesting a direct link between deregulated MYCN and CTSD mRNA expression. We further show that neuroblastoma cells can secrete mitogenic procathepsin D and that MYCN expression and especially doxorubicin treatment promote procathepsin D secretion. Extracellular exogenous cathepsin D induces Akt-1 phosphorylation and doxorubicin resistance in sensitive cells. These results demonstrate an important role of cathepsin D in antiapoptotic signaling in neuroblastoma cells and suggest a novel mechanism for the development of chemotherapy resistance in neuroblastoma.

FRA1E common fragile site breaks map within a 370kilobase pair region and disrupt the dihydropyrimidine dehydrogenase gene (DPYD).

Common fragile sites represent components of normal chromosome structure that are particularly prone to breakage under replication stress. Although the cytogenetic locations of 88 common fragile sites are listed in the Genome database, the DNA at only 14 of them has been defined and characterized at the molecular level. Here, we identify the precise genomic position of the common fragile site FRA1E, mapped to the chromosomal band 1p21.2, and characterize the genetic complexity of the fragile DNA sequence. We show that FRA1E extends over 370kb within the dihydropyrimidine dehydrogenase (DPYD) gene, which genomically spans approximately 840kb. The 185kb region of the highest fragility, which accounts for 86% of all observed breaks at FRA1E, encompasses the central part of DPYD including exons 13-16. DPYD encodes dihydropyrimidine dehydrogenase (DPD), which is the first and rate-limiting enzyme in a three-step metabolic pathway involved in degradation of the pyrimidine bases uracil and thymine. Deficiency in human DPD is associated with autosomal recessive disease, thymine-uraciluria, and with severe 5-fluorouracil toxicity in cancer patients. To which extent the disruption of the DPYD gene by the fragile site break is only transient, followed by DNA repair to restore the original structure, or occasionally may result in genomic damage associated with human disease remains to be determined.

Suppression of polyploidy by the BRCA2 protein.

Mounting evidence implicates BRCA2 not only in maintenance of genome integrity but also in cell-cycle checkpoints. However, the contribution of BRCA2 in the checkpoints is still far from being understood. Here, we demonstrate that breast cancer cells MX-1 are unable to maintain genome integrity, which results in gross polyploidization. We generated MX-1 clones, stably expressing BRCA2, and found that BRCA2 acts to suppress polyploidy. Compared with MX-1, the ectopically BRCA2-expressing cells had different intracellular levels of Aurora A, Aurora B, p21, E2F-1, and pRb, suggesting a BRCA2-mediated suppression of polyploidy via stabilization of the checkpoint proteins levels.

Nuclear Pore-Like Structures in a Compartmentalized Bacterium.

Planctomycetes are distinguished from other Bacteria by compartmentalization of cells via internal membranes, interpretation of which has been subject to recent debate regarding potential relations to Gram-negative cell structure. In our interpretation of the available data, the planctomycete Gemmata obscuriglobus contains a nuclear body compartment, and thus possesses a type of cell organization with parallels to the eukaryote nucleus. Here we show that pore-like structures occur in internal membranes of G.obscuriglobus and that they have elements structurally similar to eukaryote nuclear pores, including a basket, ring-spoke structure, and eight-fold rotational symmetry. Bioinformatic analysis of proteomic data reveals that some of the G. obscuriglobus proteins associated with pore-containing membranes possess structural domains found in eukaryote nuclear pore complexes. Moreover, immunogold labelling demonstrates localization of one such protein, containing a ß-propeller domain, specifically to the G. obscuriglobus pore-like structures. Finding bacterial pores within internal cell membranes and with structural similarities to eukaryote nuclear pore complexes raises the dual possibilities of either hitherto undetected homology or stunning evolutionary convergence.

Low-frequency common fragile sites: link to neuropsychiatric disorders?

Common fragile sites are unstable chromosomal regions that predispose chromosomes to breakage and rearrangements. Recombinogenic DNA sequences encompassing these sites may contribute to both germinal and somatic genomic mutations, and the genomic instability at these regions might cause severe inherited disorders or predispose to cancer. In this review, we discuss the characterization of common fragile site FRA13A within the neurobeachin gene, which is involved in development and function of the central nervous system. We raise the possibility of an implication of common fragile sites in neuropsychiatric disorders and overview previous and recent reports concerning individual variability of expression of common fragile sites in human populations.

Crosstalk between sugarcane and a plant-growth promoting Burkholderia species.

Bacterial species in the plant-beneficial-environmental clade of Burkholderia represent a substantial component of rhizosphere microbes in many plant species. To better understand the molecular mechanisms of the interaction, we combined functional studies with high-resolution dual transcriptome analysis of sugarcane and root-associated diazotrophic Burkholderia strain Q208. We show that Burkholderia Q208 forms a biofilm at the root surface and suppresses the virulence factors that typically trigger immune response in plants. Up-regulation of bd-type cytochromes in Burkholderia Q208 suggests an increased energy production and creates the microaerobic conditions suitable for BNF. In this environment, a series of metabolic pathways are activated in Burkholderia Q208 implicated in oxalotrophy, microaerobic respiration, and formation of PHB granules, enabling energy production under microaerobic conditions. In the plant, genes involved in hypoxia survival are up-regulated and through increased ethylene production, larger aerenchyma is produced in roots which in turn facilitates diffusion of oxygen within the cortex. The detected changes in gene expression, physiology and morphology in the partnership are evidence of a sophisticated interplay between sugarcane and a plant-growth promoting Burkholderia species that advance our understanding of the mutually beneficial processes occurring in the rhizosphere.

Structural studies of planctomycete Gemmata obscuriglobus support cell compartmentalisation in a bacterium.

Members of phylum Planctomycetes have been proposed to possess atypical cell organisation for the Bacteria, having a structure of sectioned cells consistent with internal compartments surrounded by membranes. Here via electron tomography we confirm the presence of compartments in the planctomycete Gemmata obscuriglobus cells. Resulting 3-D models for the most prominent structures, nuclear body and riboplasm, demonstrate their entirely membrane - enclosed nature. Immunogold localization of the FtsK protein also supports the internal organisation of G.obscuriglobus cells and their unique mechanism of cell division. We discuss how these new data expand our knowledge on bacterial cell biology and suggest evolutionary consequences of the findings.

Nested bacterial boxes: nuclear and other intracellular compartments in planctomycetes.

Bacteria in the phylum Planctomycetes and some related phyla challenge our concept of the typical bacterium as consisting of cells without internal compartments or membrane-bounded organelles. Cells of all species of planctomycetes examined consist of at least two major compartments, and there are two other types of compartmentation in which a third compartment is formed either by a double-membrane envelope around the nucleoid in the case of the aerobic Gemmata obscuriglobus or by a single but potentially energized membrane in the case of the anaerobic ammonium-oxidizing anammox planctomycetes. We examine here the nature of these planctomycete compartments in relation to function and their relationship to the endomembranes defining them, and discuss the implications of the remarkable compartment-confined process of protein uptake in Gemmata, which resembles receptor- and clathrin-mediated endocytosis of eukaryotes. Planctomycetes have implications for our understanding of the evolution of membrane-bounded organelles, of endomembranes, transport across endomembranes and membrane trafficking, and for how the complexity of a eukaryote style of cell organization could have originated.

Keys to eukaryality: planctomycetes and ancestral evolution of cellular complexity.

Planctomycetes are known to display compartmentalization via internal membranes, thus resembling eukaryotes. Significantly, the planctomycete Gemmata obscuriglobus has not only a nuclear region surrounded by a double-membrane, but is also capable of protein uptake via endocytosis. In order to clearly analyze implications for homology of their characters with eukaryotes, a correct understanding of planctomycete structure is an essential starting point. Here we outline the major features of such structure necessary for assessing the case for or against homology with eukaryote cell complexity. We consider an evolutionary model for cell organization involving reductive evolution of Planctomycetes from a complex proto-eukaryote-like last universal common ancestor, and evaluate alternative models for origins of the unique planctomycete cell plan. Overall, the structural and molecular evidence is not consistent with convergent evolution of eukaryote-like features in a bacterium and favors a homologous relationship of Planctomycetes and eukaryotes.

Electron tomography of the nucleoid of Gemmata obscuriglobus reveals complex liquid crystalline cholesteric structure.

The nucleoid of the planctomycete Gemmata obscuriglobus is unique within the Bacteria in being both highly condensed and enclosed by a double-membrane nuclear envelope, seemingly analogous to the nucleus of eukaryotes. Here we have applied electron tomography to study high-pressure frozen, cryosubstituted cells of G. obscuriglobus and found multiple nested orders of DNA organization within the condensed nucleoid structure. Detailed examination of the nucleoid revealed a series of nested arcs characteristic of liquid crystalline cholesteric DNA structure. The finest fibers were arranged in parallel concentrically in a double-twist organization. At the highest order of nucleoid organization, several of these structures come together to form the core of the G. obscuriglobus nucleoid. The complex structure of DNA within this nucleoid may have implications for understanding the evolutionary significance of compartmentalized planctomycete cells.

Beyond the bacterium: planctomycetes challenge our concepts of microbial structure and function.

Planctomycetes form a distinct phylum of the domain Bacteria and possess unusual features such as intracellular compartmentalization and a lack of peptidoglycan in their cell walls. Remarkably, cells of the genus Gemmata even contain a membrane-bound nucleoid analogous to the eukaryotic nucleus. Moreover, the so-called 'anammox' planctomycetes have a unique anaerobic, autotrophic metabolism that includes the ability to oxidize ammonium; this process is dependent on a characteristic membrane-bound cell compartment called the anammoxosome, which might be a functional analogue of the eukaryotic mitochondrion. The compartmentalization of planctomycetes challenges our hypotheses regarding the origins of eukaryotic organelles. Furthermore, the recent discovery of both an endocytosis-like ability and proteins homologous to eukaryotic clathrin in a planctomycete marks this phylum as one to watch for future research on the origin and evolution of the eukaryotic cell.

Protein uptake by bacteria: An endocytosis-like process in the planctomycete Gemmata obscuriglobus.

Endocytosis is a fundamental process of membrane-trafficking in eukaryotes, but has not been known to occur in bacteria or archaea. The origin of endocytosis is central to the understanding of evolution of the first eukaryotes and their endomembrane systems. In a recent study we have established that an endocytosis-like process for uptake of proteins into cells occurs in a bacterium, Gemmata obscuriglobus, a member of the distinctive phylum Planctomycetes of peptidoglycan-less budding bacteria. Members of this phylum characteristically possess cells divided into compartments separated by internal membranes and in the case of G. obscuriglobus these compartments include one where a double membrane envelope surrounds its nucleoid DNA, as well as an outer ribosome- free region of cytoplasm. Proteins can be internalized by cells from the external milieu and collected into this ribosome-free compartment, and this process is energy-dependent and appears to be receptor-mediated. As in eukaryote endocytosis, internalized proteins are associated with vesicles, and can be subjected to proteolytic degradation. The discovery of this process in a bacterium has significant implications for our understanding of the origins of endocytosis in eukaryotes.

Turning the table: plants consume microbes as a source of nutrients.

Interactions between plants and microbes in soil, the final frontier of ecology, determine the availability of nutrients to plants and thereby primary production of terrestrial ecosystems. Nutrient cycling in soils is considered a battle between autotrophs and heterotrophs in which the latter usually outcompete the former, although recent studies have questioned the unconditional reign of microbes on nutrient cycles and the plants' dependence on microbes for breakdown of organic matter. Here we present evidence indicative of a more active role of plants in nutrient cycling than currently considered. Using fluorescent-labeled non-pathogenic and non-symbiotic strains of a bacterium and a fungus (Escherichia coli and Saccharomyces cerevisiae, respectively), we demonstrate that microbes enter root cells and are subsequently digested to release nitrogen that is used in shoots. Extensive modifications of root cell walls, as substantiated by cell wall outgrowth and induction of genes encoding cell wall synthesizing, loosening and degrading enzymes, may facilitate the uptake of microbes into root cells. Our study provides further evidence that the autotrophy of plants has a heterotrophic constituent which could explain the presence of root-inhabiting microbes of unknown ecological function. Our discovery has implications for soil ecology and applications including future sustainable agriculture with efficient nutrient cycles.

Novel aphidicolin-inducible common fragile site FRA9G maps to 9p22.2, within the C9orf39 gene.

Common fragile sites represent a component of normal chromosome structure that form gaps and breaks on metaphase chromosomes after partial inhibition of DNA synthesis. In humans, cytogenetic locations of 89 common fragile sites are listed in the Genome Database; however, the exact number of fragile sites remains unknown. The application of high resolution mapping approaches continues to reveal new common fragile sites in the human genome. Here, we identified a novel aphidicolin-inducible common fragile site FRA9G, which maps to chromosomal band 9p22.2. We have characterized the structure of the fragile DNA sequence that extends over a genomic region of approximately 300 kb within the C9orf39 (chromosome 9 open reading frame 39) gene. Analysis of incidence in healthy individuals showed that FRA9G is commonly expressed in the population. Heterozygous BRCA2 mutation carriers exhibit an almost sevenfold increase of FRA9G expression compared to an unrelated control population group. Identification of a novel aphidicolin-inducible common fragile site at 9p22 may have implications for understanding the mechanism of genetic instability in tumorigenesis and other genetic disorders.

The neurobeachin gene spans the common fragile site FRA13A.

Common fragile sites are normal constituents of chromosomal structure prone to chromosomal breakage. In humans, the cytogenetic locations of more than 80 common fragile sites are known. The DNA at 11 of them has been defined and characterized at the molecular level. According to the Genome Database, the common fragile site FRA13A maps to chromosome band 13q13.2. Here, we identify the precise genomic position of FRA13A, and characterize the genetic complexity of the fragile DNA sequence. We show that FRA13A breaks are limited to a 650 kb region within the neurobeachin (NBEA) gene, which genomically spans approximately 730 kb. NBEA encodes a neuron-specific multidomain protein implicated in membrane trafficking that is predominantly expressed in the brain and during development.