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.     

Marine glycosyl hydrolases in the hydrolysis and synthesis of oligosaccharides

The marine ecosystem can be considered a rather unexplored source of biological material (e.g. natural substances with therapeutic activity) and can also be a surprising source of enzymes carrying new and interesting catalytic activities to be applied in biocatalysis. The use of glycosyl hydrolases from marine environments dates back to the end of the 1960s and was mainly focused on the development of sensitive and reliable hydrolytic methods for the analysis of sugar chains. As a result not all the benefits of a particular enzymatic activity have been investigated, especially regarding the transglycosylation potential of these enzymes for the synthesis of glycosidic bonds. In this review, the potential of marine sources will be demonstrated reporting on the few examples found in literature for the synthesis and hydrolysis of biologically relevant oligosaccharides catalyzed by glycosyl hydrolases of marine origin. Particular emphasis is given to the synthesis of glycosidic bonds, which is easy by the use of glycosyl hydrolases. Further aspects considered in this review are applications of these biocatalysts for vegetal waste treatment in recovering useful materials, for structural identification and for preparation of target materials from new purified polysaccharides, for the synthesis or modification of food-related compounds and for glycobiology related studies.     

The promoter region of the adiponectin gene is a determinant in modulating insulin sensitivity in childhood obesity

We investigated the association of the -11,391G>A, -11,377G>C, +45T>G, and +276G>T adiponectin single-nucleotide polymorphisms (SNPs) and expected haplotypes with the insulin resistance (IR) state in overweight/obese children; by using the haplotype background analysis, we also assessed the effect of each SNP independently. GG genotype at the -11,391 locus was associated with higher fasting insulin levels and homeostasis model assessment-IR index and lower adiponectin levels compared with GA + AA genotypes (p = 0.01, 0.002, and 0.03, respectively). Those heterozygous and homozygous for G allele at the -11,377 locus showed higher fasting glucose (p = 0.001 for both), fasting insulin (p = 0.001 for both), homeostasis model assessment-IR index (p < 0.001 for both), and triglyceride levels (p = 0.02 and 0.03, respectively) and lower adiponectin levels (p = 0.002 and 0.02, respectively) compared with C homozygotes. The +45G carriers showed higher fasting and 2-hour glucose levels (p = 0.01 for both) and lower adiponectin levels (p = 0.02) compared with non-carriers. Haplotype analysis suggested that, considering the same haplotypic background, each of the three polymorphisms exerted an independent effect on investigated parameters. The -11,391G>A, -11,377C>G, and +45T>G SNPs are associated with IR syndrome in overweight/obese children; they independently influence the investigated variables. The effect of +45T>G SNP seems to be marginal compared with the promoter SNPs. The GGT haplotype is associated with the highest degree of IR.     

Planococcus rifietensis sp. nov, isolated from algal mat collected from a sulfurous spring in Campania (Italy)

The taxomony of strain M8, isolated from algal mat formed at the origin of a sulfurous spring in Rifieto (Savignano Irpino, Campania, Italy), was investigated in a polyphasic approach. The morphological, physiological and genetic characteristics were compared with of Planococcus and Planomicrobium species. The isolate grew optimally at pH 9.0, 1.8 M NaCl at 37 degrees C. The cells were Gram-positive cocci that form pairs, tetrads and aggregates of several cells. The isolate was aerobic/microaerophilic and accumulated glycine-betaine, as a major osmolyte, with minor components glutamate and an unknown compound. M8 was able to hydrolyse X-Glc (5-bromo-4-chloro-3-indoyl beta-d-glucopyranoside). The polar lipid profile consisted of phosphatidylglycerol and diphosphatidylglycerol as major components, and phosphocholine as a minor compound. MK8 was the only quinone found and the fatty acid composition was dominated by branched acids, mainly aiC15:0. The G+C content of DNA was 47.9% and its phylogenetic position was established by 16S rRNA gene sequencing as a member of the genus Planococcus. The DNA/DNA similarity of M8 to the type species Planococcus citreus was less than 55%. For this reason and for physiological and chemotaxonomic features, it is proposed to create a new species Planococcus rifietensis sp. nov.     

Effects of defoliation caused by the leaf miner<Emphasis Type="Italic"> Cameraria ohridella</Emphasis> on wood production and efficiency in<Emphasis Type="Italic"> Aesculus hippocastanum</Emphasis> growing in north-eastern Italy

The leaf miner Cameraria ohridella causes premature defoliation of Aesculus hippocastanum trees. Repeated defoliation has been reported to cause decrease in radial growth of trees and a progressive decline due to reduced production and allocation of photosynthates. Our study represents an attempt to estimate the impact of C. ohridella on annual wood increments and the hydraulic properties of the wood as well as on the dry mass of seeds. Twenty-two adult horse chestnut trees were selected, four of which had been chemically treated to prevent attack (controls). All other trees were heavily infested. The ground cover (GC) of each tree, measured from monthly hemispherical photographs, revealed that infested trees were completely defoliated in September and the slope of the GC-to-measurement dates relationship (named GC decrease index) was positively related to the number of mines per leaf. Anatomical observations showed that infested trees produced more wood per year than controls through more false rings with wider xylem conduits and, hence, with higher conductive area and theoretical flow. In fact, the theoretical flow was positively related to the defoliation intensity. In contrast, the allocation of photosynthates to seeds was strongly reduced in infested trees with respect to controls (up to 50% less). The hypothesis was advanced that horse chestnut trees reacted to C. ohridella attacks by increasing the hydraulic efficiency of the wood, thus ameliorating the water and nutrient supply to leaves between the spring and mid-summer and, therefore, compensating, at least partly, the reduced leaf lifespan.     

Activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH

We have previously shown that the hyperthermophilic glycosynthase from Sulfolobus solfataricus (Ssbeta-glyE387G) can promote the synthesis of branched oligosaccharides from activated beta-glycosides, at pH 6.5, in the presence of 2 M sodium formate as an external nucleophile. In an effort to increase the synthetic potential of hyperthermophilic glycosynthases, we report a new method to reactivate the Ssbeta-glyE387G glycosynthase and two novel mutants in the nucleophile of the beta-glycosidases from the hyperthermophilic Archaea Thermosphaera aggregans (Tabeta-gly) and Pyrococcus furiosus (CelB). We describe here that, at pH 3.0 and low concentrations of sodium formate buffer, the three hyperthermophilic glycosynthases show k(cat) values similar to those of the wild-type enzymes and 17-fold higher than those observed at the usual reactivation conditions in 2 M sodium formate at pH 6.5. Moreover, at acidic pH the three reactivated mutants have wide substrate specificity and improved efficiency in the synthetic reaction. The data reported suggest that the reactivation conditions modify the ionization state of the residue acting as an acid/base catalyst. This new reactivation method can be of general applicability on hyperthermophilic glycosynthases whose intrinsic stability allows their exploitation as synthetic tools at low pH.     

The stimulation of photophosphorylation and ATPase by artificial redox mediators in chromatophores ofRhodopseudomonas capsulata at different redox potentials

(1) Inhibition of cyclic phosphorylation in chromatophores ofRhodopseudomonas capsulata by antimycin A can be fully reversed by artificial redox mediators, provided the ambient redox potential is maintained around 200 mV. The redox mediator need not be a hydrogen carrier in its reduced form, N-methyl-phenazonium methosulfate and N,N,N,N-tetramethyl-p-phenylenediamine being equally effective. However, the mediator needs to be lipophilic. Endogenous cyclic phosphorylation is fastest around 130 mV. A shift to 200 mV can also be observed if high concentrations of artificial redox mediator are present in the absence of antimycin A. (2) ATPase activity ofRhodopseudomonas capsulata, in the light as well as in the dark, activated or not activated by inorganic phosphate, can also be stimulated by N-methylphenazonium methosulfate. This stimulation is highest at redox potentials between 60 to 80 mV and is sensitive to antimycin A. In this case N,N,N,N-tetramethyl-p-phenylenediamine is much less effective.Abbreviations PES N-methyl-phenazonium ethosulfate - PMS N-methyl-phenazonium methosulfate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - DAD diaminodurene (2,3,5,6-tetramethyl-p-phenylenediamine) - Bchl bacteriochlorophyll - FCCP carbonylcyanide-p-trifluoromethoxy-phenylhydrazone - E _h redox potential - E _m midpoint redox potential     

Standardization Initiatives in the (eco)toxicogenomics domain: a review

The purpose of this document is to provide readers with a resource of different ongoing standardization efforts within the 'omics' (genomic, proteomics, metabolomics) and related communities, with particular focus on toxicological and environmental applications. The review includes initiatives within the research community as well as in the regulatory arena. It addresses data management issues (format and reporting structures for the exchange of information) and database interoperability, highlighting key objectives, target audience and participants. A considerable amount of work still needs to be done and, ideally, collaboration should be optimized and duplication and incompatibility should be avoided where possible. The consequence of failing to deliver data standards is an escalation in the burden and cost of data management tasks.     

Detection of SV40 DNA sequences in malignant mesothelioma specimens from the United States, but not from Turkey

The incidence of malignant mesothelioma (MM) shows a strong epidemiological association with exposure to asbestos fibers. Recently, simian virus 40 (SV40) DNA sequences have been reported in MM tumor specimens from the United States and several European countries, and the SV40 tumor virus has been implicated as a potential co-factor in the etiology of this disease. However, several large studies from the US, Finland, and Turkey did not detect SV40 sequences in MM samples. To address this discrepancy, MM specimens from Turkey and the US were analyzed in the same laboratory under identical conditions to detect the presence of SV40 DNA. We detected SV40 sequences in 4 of 11 specimens from the United States, but in none of the 9 Turkish samples examined. These findings suggest that geographical differences exist with regard to the involvement of SV40 in human tumors.