Search

Humic substances (HS) are known as stimulators of seed germination and plant growth. Here we have examined the influence of HS obtained from the extract of a mixture of brown coal and industrial waste of red seaweed, Ahnfeltia tobuchiensis (HS-Ahnfeltia), on the germination of maize seeds. Seeds of Zea mays L. were immersed in HS-Ahnfeltia (treatment) or water (control) and germinated on moist filter paper in sealed Petri dishes. After 5 days of incubation at 28°C, the results showed that treatment with HS-Ahnfeltia promoted seedling growth. While no significant differences occurred in seed emergence, an important increase in root (36%) and shoot (54%) area as well as root (100%) and shoot (54%) dry weight was observed in treated seedlings, resulting in a 100% increase of the seedling vigour. These results suggest that HS-Ahnfeltia extract can be used as an effective agent to increase seedling vigour in crops.

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.

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.

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.

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, immuno-gold labelling demonstrates localization of one such protein, containing a β-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.

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.

Molecular cloning skills are an essential component of biological research, yet students often do not receive this training during their undergraduate studies. This can be attributed to the complexities of the cloning process, which may require many weeks of progressive design and experimentation. To address this issue, we incorporated an immersive 12-week undergraduate research experience into an upper-level molecular microbiology course. Students completed two 6-week modules where the entire molecular cloning process was carried out for two sets of novel genes implicated in distinct biological processes, microbial cell division and bacterial pathogenesis. The first module trained students in molecular cloning experimental skills, and the second facilitated the application of these skills within a new biological context. Students designed and optimized primers targeted against specific genes, andthen PCR amplified, purified, and quantified the DNA of their gene of interest. Using a variety of strategies, students inserted these genes into expression plasmids to produce GFP and His-tag fusion proteins; this was tested through restriction digestion or colony PCR and verified by gel electrophoresis. At the end of each module, students were assessed on comprehension of biological concepts, professional laboratory note-keeping and scientific communication, and proficiencies in experimental procedures. The recombinant plasmids produced by the students directly contributed to ongoing research projects, and the molecular cloning skills learnt are applicable to many career and study options in molecular biology. Overall, this research experience trained students in professional molecular biology and microbiology practice and engages them in producing real-world research outcomes.

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.

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 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. VVC 2007 Wiley-Liss, Inc.

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.

Planctomycetes and their relatives in the PVC superphylum have significant implications for evolution of the diversity of bacterial and eukaryotic cell organisation. The compartmentalisation via internal membranes of an underlying plan shared by planctomycetes and by members of phyla Verrucomicrobia and Lentisphaerae within the PVC superphylum implies phylogenetic meaning to such structure. It is likely that the common ancestor of PVC superphylum members possessed such compartmentalisation. Compartmentalisation in PVC bacteria, especially within the Gemmata clade where the nucleoid is bounded by an envelope of two membranes, has implications for theories of the origin of the eukaryotic nucleus, suggesting autogenous theories should be considered seriously as major alternatives to those depending on early fusions between Archaea and Bacteria domains. Explanations for the origin of PVC compartmentalisation are considered here, as well as their implications for molecular correlates of such compartmentalisation, and their correlates with an integrated cell biology that may be an analogue or even a homologue of an ancient eukaryote cell biology. PVC bacteria can form major experimental models for exploring what such a cell biology might have looked like.

Planctomycetes are unique among the domain Bacteria in possessing cells with a complex plan defined by internal membranes forming separated compartments within the cell. They also possess other unique features such as cell walls composed of protein as a major polymer instead of the peptidoglycan typical of other bacteria. All species examined display an underlying shared cell organization in which an internal intracytoplasmic membrane separates two major cell compartments, an outer ribosome-free paryphoplasm and a more central ribosome-containing pirellulosome. Some planctomycete species have three compartments, where further membranes within the pirellulosome define another compartment, the anammoxosome in anammox planctomycetes and the membrane-bounded nuclear body in Gemmata obscuriglobus. Compartments are preserved when new cells are formed during division. Functional features which are correlated with structural compartmentalization in planctomycetes include in G. obscuriglobus the ability to take up proteins within the paryphoplasm of the cell by a mechanism similar to receptor-mediated endocytosis of eukaryotes. Novel molecular and cell biology features for bacteria can be predicted to accompany such structural and functional complexity and are discussed here.

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.

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 those of 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 fibres 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.

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.

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.

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.

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.

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 370 kb within the dihydropyrimidine dehydrogenase ( DPYD ) gene, which genomically spans approximately 840 kb. The 185 kb 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.

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.

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.