The chitin synthase genes chs-1 and chs-2 are essential for C. elegans development and responsible for chitin deposition in the eggshell and pharynx, respectively

It is widely accepted that chitin is present in nematodes. However, its precise role in embryogenesis is unclear and it is unknown if chitin is necessary in other nematode tissues. Here, we determined the roles of chitin and the two predicted chitin synthase genes in Caenorhabditis elegans by chitin localization and gene disruption. Using a novel probe, we detected chitin in the eggshell and discovered elaborate chitin localization patterns in the pharyngeal lumen walls. Chitin deposition in these two sites is likely regulated by the activities of chs-1 (T25G3.2) and chs-2 (F48A11.1), respectively. Reducing chs-1 gene activity by RNAi led to eggs that were fragile and permeable to small molecules, and in the most severe case, absence of embryonic cell division. Complete loss of function in a chs-1 deletion resulted in embryos that lacked chitin in their eggshells and failed to divide. These results showed that eggshell chitin provides both mechanical support and chemical impermeability essential to developing embryos. Knocking down chs-2 by RNAi caused a defect in the pharynx and led to L1 larval arrest, indicating that chitin is involved in the development and function of the pharynx.     

Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation

Fermentation of shrimp shell in jaggery broth using Bacillus subtilis for the production of chitin and chitosan was investigated. It was found that B. subtilis produced sufficient quantities of acid to remove the minerals from the shell and to prevent spoilage organisms. The protease enzyme in Bacillus species was responsible for the deprotenisation of the shell. The pH, proteolytic activity, extent of demineralization and deprotenisation were studied during fermentation. About 84% of the protein and 72% of the minerals were removed from the shrimp shell after fermentation. Mild acid and alkali treatments were given to produce characteristic chitin and their concentrations were standardized. Chitin was converted to chitosan by N-deacetylation and the properties of chitin and chitosan were studied. FTIR spectral analysis of chitin and chitosan prepared by the process was carried out and compared with spectra of commercially available samples.     

cda1+, encoding chitin deacetylase is required for proper spore formation in Schizosaccharomyces pombe

In Schizosaccharomyces pombe, a major role of chitin is to build up a complete spore. Here, we analyzed the cda1(+) gene (SPAC19G12.03), which encodes a protein homologous to chitin deacetylases, to know whether it is required for spore formation in S. pombe. The homothallic Deltacda1 strain constructed by homologous recombination was found to form a little amount of abnormal spores that contained one, two, or three asci, similar to (but not as strong as) the phenotype observed in a deletion mutant of chs1 encoding chitin synthase 1. This phenotype is reversed by expression of S. cerevisiae chitin deacetylase CDA1 or CDA2, suggesting that cda1 encodes a chitin deacetylase. To support the role of Cda1 in sporulation, the timing of expression of cda1(+) mRNA increased during sporulation process. We also found that the Cda1 protein self-associated when its binding was tested both by two-hybrid system and immunoprecipitation. Thus, these data indicated that cda1(+) is required for proper spore formation in S. pombe.     

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.     

Presence of LYM2 dependent but CERK1 independent disease resistance in Arabidopsis

Plants have the ability to detect invading fungi through the perception of chitin fragments released from the fungal cell walls. Plant chitin receptor consists of two types of plasma membrane proteins, CEBiP and CERK1. However, the contribution of these proteins to chitin signaling is different between Arabidopsis and rice. In Arabidopsis, it seems CERK1 receptor kinase is enough for both ligand perception and signaling, whereas both CEBiP and OsCERK1 are required for chitin signaling in rice. Here we report that Arabidopsis CEBiP homolog, LYM2, is not involved in chitin signaling but contributes to resistance against a fungal pathogen, Alternaria brassicicola, indicating the presence of a novel disease resistance mechanism in Arabidopsis.     

A comparison of stigmatellin conformations, free and bound to the photosynthetic reaction center and the cytochrome bc1 complex

We describe in detail the conformations of the inhibitor stigmatellin in its free form and bound to the ubiquinone-reducing (Q(B)) site of the reaction center and to the ubiquinol-oxidizing (Q(o)) site of the cytochrome bc(1) complex. We present here the first structures of a stereochemically correct stigmatellin in complexes with a bacterial reaction center and the yeast cytochrome bc1 complex. The conformations of the inhibitor bound to the two enzymes are not the same. We focus on the orientations of the stigmatellin side-chain relative to the chromone head group, and on the interaction of the stigmatellin side-chain with these membrane protein complexes. The different conformations of stigmatellin found illustrate the structural variability of the Q sites, which are affected by the same inhibitor. The free rotation about the chi1 dihedral angle is an essential factor for allowing stigmatellin to bind in both the reaction center and the cytochrome bc1 pocket.     

The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c

Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b(5) is relatively slow (k=6 x 10(2)M(-1)s(-1) at pH 7.0) and thus is unlikely to be of physiological significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2 x 10(7)M(-1)s(-1)) with an apparent overall equilibrium constant of 1 microM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis.     

Fingerprint of Biomphalaria arabica, the intermediate host of Schistosoma mansoni in Saudi Arabia, using RAPD-PCR

In the time schistosomisis control programs are implemented in many countries, schistosomiasis continues to spread throughout the world. Among these control strategies is the vector control. Within this context, analysis of the genetic variability of the intermediate host snails is important because it allows identification of specific sequences of the genome of this mollusk related to determine their fingerprint. We investigated Biomphalaria arabica, which is found in Saudi Arabia, the intermediate host of Schistosoma mansoni infection. Genetic fingerprint was studied by RAPD-PCR using our own different random primers as well as published primers. The electrophoretic patterns resulting from amplification showed specific polymorphic markers of B. arabica. This information will be helpful in the identification of the snails and demonstrating that RAPD-PCR is an appropriate and efficient methodological approach for establishment of genetic barcode development.     

On Hypolycaena from Maluku, Indonesia, including the first description of male Hypolycaena asahi (Lepidoptera, Lycaenidae)

The taxonomy and distribution of the five species of Hypolycaena in Maluku are discussed and new locality records given. Corrections are made to the published taxonomy and distribution of Hypolycaena phorbas (Fabricius, 1793). This clarification enables a better understanding of the biogeography of the genus. Hypolycaena asahi Okubo, 2007, was originally described from a single female from Ambon and is here recorded from Seram. The male is described for the first time.     

Development of Multi-Component Transplant Mixes for Suppression of Meloidogyne incognita on Tomato (Lycopersicon esculentum)

The effects of combinations of organic amendments, phytochemicals, and plant-growth promoting rhizobacteria on tomato (Lycopersicon esculentum) germination, transplant growth, and infectivity of Meloidogyne incognita were evaluated. Two phytochemicals (citral and benzaldehyde), three organic amendments (pine bark, chitin, and hemicellulose), and three bacteria (Serratia marcescens, Brevibacterium iodinum, and Pseudomonas fluorescens) were assessed. Increasing rates of benzaldehyde and citral reduced nematode egg viability in vitro. Benzaldehyde was 100% efficacious as a nematicide against juveniles, whereas citral reduced juvenile viability to less than 20% at all rates tested. Benzaldehyde increased tomato seed germination and root weight, whereas citral decreased both. High rates of pine bark or chitin reduced plant growth but not seed germination, whereas low rates of chitin increased shoot length, shoot weight, and root weight; improved root condition; and reduced galling. The combination of chitin and benzaldehyde significantly improved tomato transplant growth and reduced galling. While each of the bacterial isolates contributed to increased plant growth in combination treatments, only Brevibacterium iodinum applied alone significantly improved plant growth.     

Complete genome sequence of Jonesia denitrificans type strain (Prevot 55134)

Jonesia denitrificans (Prevot 1961) Rocourt et al. 1987 is the type species of the genus Jonesia, and is of phylogenetic interest because of its isolated location in the actinobacterial suborder Micrococcineae. J. denitrificans is characterized by a typical coryneform morphology and is able to form irregular nonsporulating rods showing branched and club-like forms. Coccoid cells occur in older cultures. J. denitrificans is classified as a pathogenic organism for animals (vertebrates). The type strain whose genome is described here was originally isolated from cooked ox blood. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a member of the genus for which a complete genome sequence is described. The 2,749,646 bp long genome with its 2558 protein-coding and 71 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Purification,crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus

Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18 equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge this is the first report of dimerization of AFP type III proteins.     

Zebrafish Crip2 Plays a Critical Role in Atrioventricular Valve Development by Downregulating the Expression of ECM Genes in the Endocardial Cushion

The initial step of atrioventricular (AV) valve development involves the deposition of extracellular matrix (ECM) components of the endocardial cushion and the endocardialmesenchymal transition. While the appropriately regulated expression of the major ECM components, Versican and Hyaluronan, that form the endocardial cushion is important for heart valve development, the underlying mechanism that regulates ECM gene expression remains unclear. We found that zebrafish crip2 expression is restricted to a subset of cells in the AV canal (AVC) endocardium at 55 hours post-fertilization (hpf). Knockdown of crip2 induced a heart-looping defect in zebrafish embryos, although the development of cardiac chambers appeared to be normal. In the AVC of Crip2-deficient embryos, the expression of both versican a and hyaluronan synthase 2 (has2) was highly upregulated, but the expression of bone morphogenetic protein 4 (bmp4) and T-box 2b (tbx2b) in the myocardium and of notch1b in the endocardium in the AVC did not change. Taken together, these results indicate that crip2 plays an important role in AV valve development by downregulating the expression of ECM components in the endocardial cushion.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Dissecting the fungal biology of Bipolaris papendorfii: from phylogenetic to comparative genomic analysis

Bipolaris papendorfii has been reported as a fungal plant pathogen that rarely causes opportunistic infection in humans. Secondary metabolites isolated from this fungus possess medicinal and anticancer properties. However, its genetic fundamental and basic biology are largely unknown. In this study, we report the first draft genome sequence of B. papendorfii UM 226 isolated from the skin scraping of a patient. The assembled 33.4 Mb genome encodes 11,015 putative coding DNA sequences, of which, 2.49% are predicted transposable elements. Multilocus phylogenetic and phylogenomic analyses showed B. papendorfii UM 226 clustering with Curvularia species, apart from other plant pathogenic Bipolaris species. Its genomic features suggest that it is a heterothallic fungus with a putative unique gene encoding the LysM-containing protein which might be involved in fungal virulence on host plants, as well as a wide array of enzymes involved in carbohydrate metabolism, degradation of polysaccharides and lignin in the plant cell wall, secondary metabolite biosynthesis (including dimethylallyl tryptophan synthase, non-ribosomal peptide synthetase, polyketide synthase), the terpenoid pathway and the caffeine metabolism. This first genomic characterization of B. papendorfii provides the basis for further studies on its biology, pathogenicity and medicinal potential.     

Subunit a of the F1F0 ATP Synthase Requires YidC and SecYEG for Membrane Insertion

The insertion of inner membrane proteins in Escherichia coli occurs almost exclusively via the SecYEG pathway, while some membrane proteins require the membrane protein insertase YidC. In vitro analysis demonstrates that subunit a of the F₁F₀ ATP synthase (F₀a) is strictly dependent on Ffh, SecYEG and YidC for its membrane insertion but independent of the proton motive force. The insertion of the first transmembrane segment of F₀a also depends on Ffh and SecYEG but not on YidC, whereas the insertion is strongly dependent on the proton motive force, unlike the full-length F₀a protein. These data demonstrate an extensive role of YidC in the assembly of the F₀ sector of the F₁F₀ ATP synthase.     

Elevated CO2 affects shell dissolution rate but not calcification rate in a marine snail

As CO₂ levels increase in the atmosphere, so too do they in the sea. Although direct effects of moderately elevated CO₂ in sea water may be of little consequence, indirect effects may be profound. For example, lowered pH and calcium carbonate saturation states may influence both deposition and dissolution rates of mineralized skeletons in many marine organisms. The relative impact of elevated CO₂ on deposition and dissolution rates are not known for many large-bodied organisms. We therefore tested the effects of increased CO₂ levels—those forecast to occur in roughly 100 and 200 years—on both shell deposition rate and shell dissolution rate in a rocky intertidal snail, Nucella lamellosa. Shell weight gain per day in live snails decreased linearly with increasing CO₂ levels. However, this trend was paralleled by shell weight loss per day in empty shells, suggesting that these declines in shell weight gain observed in live snails were due to increased dissolution of existing shell material, rather than reduced production of new shell material. Ocean acidification may therefore have a greater effect on shell dissolution than on shell deposition, at least in temperate marine molluscs.     

X-ray crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase II (mtKasB)

Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.