CotE Binds to CotC and CotU and Mediates Their Interaction during Spore Coat Formation in Bacillus subtilis

CotE is a morphogenic protein that controls the assembly of the coat, the proteinaceous structure that surrounds and protects the spore of Bacillus subtilis. CotE has long been thought to interact with several outer coat components, but such interactions were hypothesized from genetic experiment results and have never been directly demonstrated. To study the interaction of CotE with other coat components, we focused our attention on CotC and CotU, two outer coat proteins known to be under CotE control and to form a heterodimer. We report here the results of pull-down experiments that provide the first direct evidence that CotE contacts other coat components. In addition, coexpression experiments demonstrate that CotE is needed and sufficient to allow formation of the CotC-CotU heterodimer in a heterologous host.The spore of Bacillus subtilis is a dormant cell, resistant to harsh conditions and able to survive extreme environmental conditions (25). Spores are produced in a sporangium that consists of an inner cell, the forespore, that will become the mature spore and an outer cell, the mother cell, that will lyse, liberating the mature spore (18, 26). Resistance of the spore to noxious chemicals, lytic enzymes, and predation by soil protozoans is in part due to the coat, a complex, multilayered structure of more than 50 proteins that encases the spore (5, 8, 13). Proteins that constitute the coat are produced in the mother cell and deposited around the outer membrane surface of the forespore in an ordered manner (8).A small subset of coat proteins have a regulatory role on the formation of the coat. Those proteins, referred to as morphogenic factors, do not affect the synthesis of the coat components but drive their correct assembly outside of the outer forespore membrane (8). Within this subset of regulatory coat proteins, SpoIVA and CotE play a crucial role. SpoIVA (6, 20, 23) is assembled into the basement layer of the coat and is anchored to the outer membrane of the forespore through its C terminus that contacts SpoVM, a small, amphipathic peptide embedded in the forespore membrane (16, 21, 22). A spoIVA-null mutation impairs the assembly of the coat around the forming spore, and as a consequence, coat material accumulates in the mother cell cytoplasm (23).CotE (28) assembles into a ring and surrounds the SpoIVA basement structure. The inner layer of the coat is then formed between the SpoIVA basement layer and the CotE ring by coat components produced in the mother cell that infiltrate through the CotE ring, while the outer layer of the coat is formed outside of CotE (6). However, not all CotE molecules are assembled into the ring-like structure, and CotE molecules are also found in the mother cell cytoplasm, at least up to 8 h after the start of sporulation (3). CotE was first identified as a morphogenic factor in a seminal study in which an ultrastructural analysis indicated that a cotE-null mutation prevented formation of the electron-dense outer layer of the coat while it did not affect inner coat formation (28). A subsequent mutagenesis study has revealed that CotE has a modular structure with a C-terminal domain involved in directing the assembly of various coat proteins, an internal domain involved in the targeting of CotE to the forespore, and a N-terminal domain that, together with the internal domain, directs the formation of CotE multimers (17). More recently, formation of CotE multimers has been also confirmed by a yeast two-hybrid approach (14). In a global study of protein interactions in the B. subtilis coat, performed by a fluorescence microscopy analysis of a collection of strains carrying cot-gfp fusions, CotE has been proposed to interact with most outer coat components (12).From those and other studies, the interactions of CotE with coat structural components have been exclusively inferred on the basis of genetic experiment results, i.e., cotE mutants that failed to assemble one or more coat components. Evidence of a direct interaction between CotE and another coat component has never been provided. We addressed this issue by using as a model two coat components, CotC and CotU, known to be controlled by CotE and to form a heterodimer (10, 28).CotC is an abundant, 66-amino-acid protein known to assemble in the outer coat in various forms: a monomer of 12 kDa, a homodimer of 21 kDa, and two less abundant forms of 12.5 and 30 kDa, probably due to posttranslational modifications of CotC (9). CotU is a structural homolog of CotC of 86 amino acids. The two proteins, which share an almost identical N terminus and a less conserved C terminus, interact, originating the formation of a heterodimer of 23 kDa (10). Heterodimer formation most likely requires a B. subtilis-specific factor since it does not occur in Escherichia coli or Saccharomyces cerevisiae (10). CotC and CotU are synthesized in the mother cell compartment of the sporulating cell but do not accumulate there since they are immediately assembled around the forming spore (10). In a strain carrying a cotE-null mutation, CotC and CotU, together with all other outer coat components, do not assemble around the forming spore (10). CotC and CotU are also dependent on CotH, an additional morphogenic factor involved in coat formation (9). A cotH-null mutation prevents CotC and CotU assembly in the coat as well as their accumulation in the mother cell cytoplasm (10). Since a mutation causing cotH overexpression allows CotC and CotU accumulation in the mother cell cytoplasm (1), it has been proposed that CotH acts by stabilizing CotC and CotU in the mother cell cytoplasm (1, 10).Here we provide the first direct evidence that CotE interacts with two other coat components, CotC and CotU, and show that CotE is essential and sufficient to mediate CotC-CotU interaction to form a heterodimer.     

Analysis of locomotor activity rhythms in Drosophila

The genetic, molecular and anatomical dissection of the circadian clock in Drosophila and other higher organisms relies on the quantification of rhythmic phenotypes. Here, we introduce the methods currently in use in our laboratories for the analysis of fly locomotor activity rhythms. This phenotype provides a relatively simple, automated, efficient, reliable and robust output for the circadian clock. Thus it is not surprising that it is the preferred readout for measuring rhythmicity under a variety of conditions for most fly clock laboratories. The procedure requires at least 10 days of data collection and several days for analysis. In this protocol we advise on fly maintenance and on experimental design when studying the genetics of behavioral traits. We describe the setup for studying locomotor activity rhythms in the fruit fly and we introduce the statistical methods in use in our laboratories for the analysis of periodic data.     

Aging is associated with increased clonogenic potential in rat liver in vivo

Cancer increases with age and often arises from the selective clonal growth of altered cells. Thus, any environment favoring clonal growth per se poses a higher risk for cancer development. Using a genetically tagged animal model, we investigated whether aging is associated with increased clonogenic potential. Groups of 4-, 12-, 18-, and 24-month-old Fischer 344 rats were infused (via the portal vein) with 2x10(6) hepatocytes isolated from a normal syngenic 2-month-old donor. Animals deficient in dipeptidyl-peptidase type IV (DPP-IV-) enzyme were used as recipients, allowing for the histochemical detection of injected DPP-IV+ cells. Groups of animals were sacrificed at various times thereafter. No growth of DPP-IV+ transplanted hepatocytes was present after either 2 or 6 months in the liver of rats transplanted at young age, as expected. In striking contrast, significant expansion of donor-derived cells was seen in animals transplanted at the age of 18 months: clusters comprising 7-10 DPP-IV+ hepatocytes/cross-section were present after 2 months and were markedly enlarged after 6 months (mean of 88+/-35 cells/cluster/cross-section). These results indicate that the microenvironment of the aged liver supports the clonal expansion of transplanted normal hepatocytes. Such clonogenic environments can foster the selective growth of pre-existing altered cells, thereby increasing the overall risk for cancer development associated with aging.     

Cholinesterases: New Roles in Brain Function and in Alzheimer's Disease

The most important therapeutic effect of cholinesterase inhibitors (ChEI) on approximately 50% of Alzheimer's disease (AD) patients is to stabilize cognitive function at a steady level during a 1-year period of treatment as compared to placebo. Recent studies show that in a certain percentage (approximately 20%) of patients this cognitive stabilizing effect can be prolonged up to 24 months. This long-lasting effect suggests a mechanism of action other than symptomatic and cholinergic. In vitro and in vivo studies have consistently demonstrated a link between cholinergic activation and APP metabolism. Lesions of cholinergic nuclei cause a rapid increase in cortical APP and CSF. The effect of such lesions can be reversed by ChEI treatment. Reduction in cholinergic neurotransmission–experimental or pathological, such as in AD–leads to amyloidogenic metabolism and contributes to the neuropathology and cognitive dysfunction. To explain the long-term effect of ChEI, mechanisms based on -amyloid metabolism are postulated. Recent data show that this mechanism may not necessarily be related to cholinesterase inhibition. A second important aspect of brain cholinesterase function is related to enzymatic differences. The brain of mammals contains two major forms of cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The two forms differ genetically, structurally, and for their kinetics. Butyrylcholine is not a physiological substrate in mammalian brain, which makes the function of BuChE of difficult interpretation. In human brain, BuChE is found in neurons and glial cells, as well as in neuritic plaques and tangles in AD patients. Whereas, AChE activity decreases progressively in the brain of AD patients, BuChE activity shows some increase. To study the function of BuChE, we perfused intracortically the rat brain with a selective BuChE inhibitor and found that extracellular acetylcholine increased 15-fold from 5 nM to 75 nM concentrations with little cholinergic side effect in the animal. Based on these data and on clinical data showing a relation between cerebrospinal fluid (CSF) BuChE inhibition and cognitive function in AD patients, we postulated that two pools of cholinesterases may be present in brain, the first mainly neuronal and AChE dependent and the second mainly glial and BuChE dependent. The two pools show different kinetic properties with regard to regulation of ACh concentration in brain and can be separated with selective inhibitors. Within particular conditions, such as in mice nullizygote for AChE or in AD patients at advanced stages of the disease, BuChE may replace AChE in hydrolizing brain acetylcholine.     

4-Alkyl- and 4-phenylcoumarins from Mesua ferrea as promising multidrug resistant antibacterials

Supercritical CO2 selectively extracted a series of 4-alkyl and 4-phenyl 5,7-dihydroxycoumarins from Mesua ferrea blossoms. Chemical modifications of the isolated compounds allowed us to confirm the structures elucidated by spectroscopic means and to prepare new derivatives amenable to SAR studies and potential pharmaceutical development. Biological investigations towards the screening on a number of bacteria strains and Plasmodium falciparum, identified compounds 1-9 as weak antiprotozoal agents and potent antibacterials on resistant Gram-positive strains.     

Expressionview: visualization of quantitative trait loci and gene-expression data in Ensembl

We present here a software tool for combined visualization of gene-expression data and quantitative trait loci (QTL). The application is implemented as an extension to the Ensembl project and caters for a direct transition from microarray experiments of gene or protein expression levels to the genomic context of individual genes and QTL. It supports the visualization of gene clusters and the selection of functional candidate genes in the context of research on complex traits.     

Chaos and order in plankton dynamics. Complex behavior of a simple model

The role of the diffusive interaction between fish-populated and fish-free habitats in a patchy environment in plankton pattern formation is studied by means of a minimal reaction-diffusion model of the nutrient-plankton-fish food chain. It is shown that such interaction can give rise to spatio-temporal plankton patterns. The fractal dimension of the patterns is shown dependent on the fish predation rate. The spatially averaged plankton dynamics depending on both fish predation rate and distance between fish-populated habitats can exhibit chaotic and regular behavior. The chaotic plankton dynamics is characteristic of a wide parameter range.     

Dioxygen evolution from inorganic systems. Reactions and catalytic properties of loaded TiO2 particles in photochemical dioxygen generation

The efficiency of differently prepared TiO₂ particles in photochemical water splitting through band gap irradiation of aqueous suspensions has been investigated. The effect of pH and loading with noble metals and RuO₂ has been examined. Particular attention has been devoted to dioxygen evolution and photoadsorption.