Crystal structure of polysaccharide lyase family 20 endo-β-1,4-glucuronan lyase from the filamentous fungus Trichoderma reesei

The crystal structure of endo-β-(1→4)-glucuronan lyase from Trichoderma reesei (TrGL) has been determined at 1.8 Å resolution as the first three-dimensional structure of polysaccharide lyase (PL) family 20. TrGL has a typical β-jelly roll fold, which is similar to glycoside hydrolase family 16 and PL7 enzymes. A calcium ion is bound to the site far from the cleft and appears to contribute to the stability. There are several completely conserved residues in the cleft. Possible catalytic residues are predicted based on structural comparison with PL7 alginate lyase A1-II′.     

Phylogeny of the Rhizobium–Allorhizobium–Agrobacterium clade supports the delineation of Neorhizobium gen. nov.

The genera Agrobacterium, Allorhizobium, and Rhizobium belong to the family Rhizobiaceae. However, the placement of a phytopathogenic group of bacteria, the genus Agrobacterium, among the nitrogen-fixing bacteria and the unclear position of Rhizobium galegae have caused controversy in previous taxonomic studies. To resolve uncertainties in the taxonomy and nomenclature within this family, the phylogenetic relationships of generic members of Rhizobiaceae were studied, but with particular emphasis on the taxa included in Agrobacterium and the “R. galegae complex” (R. galegae and related taxa), using multilocus sequence analysis (MLSA) of six protein-coding housekeeping genes among 114 rhizobial and agrobacterial taxa. The results showed that R. galegae, R. vignae, R. huautlense, and R. alkalisoli formed a separate clade that clearly represented a new genus, for which the name Neorhizobium is proposed. Agrobacterium was shown to represent a separate cluster of mainly pathogenic taxa of the family Rhizobiaceae. A. vitis grouped with Allorhizobium, distinct from Agrobacterium, and should be reclassified as Allorhizobium vitis, whereas Rhizobium rhizogenes was considered to be the proper name for former Agrobacterium rhizogenes. This phylogenetic study further indicated that the taxonomic status of several taxa could be resolved by the creation of more novel genera.     

Observations on the histopathology of a systemic ciliate (Paranophrys sp.?) disease in the Dungeness crab, Cancer magister

Infections of the hemolymph of crabs by ciliates have been known for almost a century. Originally placed in the genus Anophrys, these crab-parasitizing ciliates have been recently transferred to the genus Paranophrys. Infections were long thought to be confined to the hemolymph in living crabs, with death caused by consumption of all hemocytes. Histological examination of heavily infected, but living, Dungeness crabs demonstrate that the ciliates actively invade and probably consume many tissues of the host prior to death rather than saprophytically feeding on the decomposing tissues subsequent to death as previously reported.     

En route to a genome-based classification of Archaea and Bacteria?

Given the considerable promise whole-genome sequencing offers for phylogeny and classification, it is surprising that microbial systematics and genomics have not yet been reconciled. This might be due to the intrinsic difficulties in inferring reasonable phylogenies from genomic sequences, particularly in the light of the significant amount of lateral gene transfer in prokaryotic genomes. However, recent studies indicate that the species tree and the hierarchical classification based on it are still meaningful concepts, and that state-of-the-art phylogenetic inference methods are able to provide reliable estimates of the species tree to the benefit of taxonomy. Conversely, we suspect that the current lack of completely sequenced genomes for many of the major lineages of prokaryotes and for most type strains is a major obstacle in progress towards a genome-based classification of microorganisms. We conclude that phylogeny-driven microbial genome sequencing projects such as the Genomic Encyclopaedia of Archaea and Bacteria (GEBA) project are likely to rectify this situation.     

Chaperone-mediated native folding of a β-scorpion toxin in the periplasm of Escherichia coli

Background

Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria.

Methods

The β-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the Escherichia coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined.

Results

Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold.

Conclusions

The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method.

General significance

We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors.     

Neurosecretory system of the earwig Labedura riparia, and the rôle of the aorta as a neurohaemal organ

The neurosecretory system of Labedura riparia has been described from sections and whole mounts using a variety of techniques. The pars intercerebralis contains two clusters of medial neurosecretory cells (MNC), each cluster consisting of 8 to 10 A-cells and occasional B-cells. The lateral sides of the brain have a few B-cells. The axons of the median neurosecretory cells terminate in the cephalic aorta (AO), whereas the axons of the lateral neurosecretory cells (LNC) terminate in the corpora cardiaca (CC). It appears that the neurosecretory material (NSM) elaborated in the MNC is stored in the cephalic aorta and that elaborated in the LNC is stored in the corpora cardiaca, which are two oval or elongate bodies composed of large chromophobe and small chromophil cells. Posteriorly there is the oval or elongate corpus allatum (CA) attached to the CC by thick nerves. The CA consists of one cell type only. Both CC and CA contain no A-cell neurosecretory material. It has been suggested that the neurosecretory system of L. riparia is composed of two complexes. One is formed by the medial neurosecretory cells for which the aorta functions as a neurohaemal organ, and the other is formed by lateral neurosecretory cells-lateral neurosecretory pathways-nervi corporis cardiaci-II in which the corpora cardiaca function as a neurohaemal organ.     

A novel C–S lyase from the latex-producing plant Taraxacum brevicorniculatum displays alanine aminotransferase and l-cystine lyase activity

We isolated a novel pyridoxal-5-phosphate-dependent l-cystine lyase from the dandelion Taraxacum brevicorniculatum. Real time qPCR analysis showed that C–S lyase from Taraxacum brevicorniculatum (TbCSL) mRNA is expressed in all plant tissues, although at relatively low levels in the latex and pedicel. The 1251 bp TbCSL cDNA encodes a protein with a calculated molecular mass of 46,127 kDa. It is homologous to tyrosine and alanine aminotransferases (AlaATs) as well as to an Arabidopsis thaliana carbon–sulfur lyase (C–S lyase) (SUR1), which has a role in glucosinolate metabolism. TbCSL displayed in vitrol-cystine lyase and AlaAT activities of 4 and 19 nkat mg⁻¹ protein, respectively. However, we detected no in vitro tyrosine aminotransferase (TyrAT) activity and RNAi knockdown of the enzyme had no effect on phenotype, showing that TbCSL substrates might be channeled into redundant pathways. TbCSL is in vivo localized in the cytosol and functions as a C–S lyase or an aminotransferase in planta, but the purified enzyme converts at least two substrates specifically, and can thus be utilized for further in vitro applications.     

Renalia hueberi, a new plant from the lower Devonian of Gaspé

A new genus and species, Renalia hueberi, is described from the Battery Point Formation, Gaspé Sandstone, Quebec. It consists of 1 mm wide axes, at least 11 cm long, bearing dichotomous lateral branches terminated by round to reniform sporangia. The sporangia dehisce along the distal margin and the line of dehiscence is bordered by thick-walled, rectangular cells. Spores are trilete, curvaturate and smooth to granulose. Renalia hueberi is superficially similar to the genus Cooksonia, but it differs from the presently known cooksonias in having both pseudomonopodial branching and dehiscent sporangia. It is interpreted however as being evolved from Cooksonia-like plants, and exhibits a sporangial position most comparable to that of the rhyniophytes and a mode of dehiscence characteristic of zosterophylls.     

Identification of the first deletion–insertion involving the complete structure of GAA gene and part of CCDC40 gene mediated by an Alu element

Pompe disease is an uncommon autosomal recessive glycogen storage disorder caused by deficiency of acid α-glucosidase. Classic infantile form triggers severe cardiomyopathy, hypotonia, and respiratory failure, leading to death within the first two years of life. The majority of patients with Pompe disease have been reported to have point mutations in the GAA gene. We report the first complex deletion–insertion encompassing the complete structure of GAA gene and a large fragment of the gene CCDC40 in a patient with very severe form of Pompe disease. Sequencing analysis of breakpoints allowed us to determine the potential implication of an Alu repeat in the pathogenic mechanism. We suggest that molecular strategy of Pompe disease should include systematic analysis of large rearrangements.     

α-1,4-Glucan lyase,a new class of starch/glycogen-degrading enzyme

Antibodies have been raised against an -1,4-glucan lyase purified from the red alga Gracilariopsis lemaneiformis (Bory) Dawson, Acleto et Foldvik. Localization of -1,4-glucan lyase in ultra-thin sections of the red alga was performed using immunogold/transmission electron microscopy. The enzyme was found exclusively in the stroma of the chloroplasts of the algal cells, not in the cell wall, cytosol or around the cytosolic starch granules. Partial amino-acid sequences of the algal lyase, with a total length of 100 amino-acid residues, were obtained. No sequence homology was found with proteins and peptides of known sequences.This work was supported by grants from the Swedish Council for Planning and Coordination of Research (FRN), Carl Tryggers Foundation, and Hierta-Retzius Fund. We thank Ms Katrin Österlund and Anette Axén for their expert technical assistance with this work.     

Repression by the cI protein of phage λ: in vitro inhibition of RNA synthesis

We have studied the in vitro repression of RNA synthesis by the cI protein of phage λ. We find that highly purified cI protein is an effective and specific repressor of RNA synthesis from the early gene region of λ DNA. Under optimal conditions at least 95% of the early gene RNA synthesis is repressed and this repression is eliminated or severely impaired by the use of λ DNA-carrying operator-type mutations which reduce the binding affinity of the cI protein. Highly effective repression can be demonstrated only through the use of the initiation-inhibitor rifampicin, which presumably, selects “properly” initiated RNA chains; thus we can by-pass in vitro but not yet solve the problem of how the host polymerase initiates specifically in vivo from the immediate-early promoter sites.     

The light-harvesting chlorophyll a/b · protein complex of the green alga Acetabularia mediterranea isolation and characterization of two subunits

In the green alga Acetabularia mediterranea a light-harvesting chlorophyll a/b · protein complex of 67 000 daltons has been found which contains two polypeptide chains of 21 500 and 23 000 daltons. These two polypeptides were isolated on a preparative scale and were further characterized by several different methods. Both polypeptides proved to be very similar. While their amino acid and sugar compositions as well as their immunochemical properties were almost identical the tryptic peptides and the cyanogen bromide fragments of the two polypeptides revealed minor but significant differences. The 67 000-dalton chlorophyll a/b · protein complex and its two polypeptide components were compared to the light-harvesting chlorophyll a/b · protein of higher plants.     

Evaluation of a rapid molecular screening approach for the detection of toxigenic Clostridium difficile in general and subsequent identification of the tcdC Δ117 mutation in human stools

We have developed and validated a rapid molecular screening protocol for toxigenic Clostridium difficile, that also enables the identification of the hypervirulent epidemic 027/NAP1 strain. We describe a multiplex real-time PCR assay, which detects the presence of the tcdA and tcdB genes directly in stool samples. In case of positive PCR results, a separate multiplex real-time PCR typing assay was performed targeting the tcdC gene frame shift mutation at position 117. We prospectively compared the results of the screening PCR with those of a cytotoxicity assay (CTA), and a rapid immuno-enzyme assay for 161 stool samples with a specific request for diagnosis of C. difficile infection (CDI). A total of 16 stool samples were positive by CTA. The screening PCR assay confirmed all 16 samples, and gave a PCR positive signal in eight additional samples. The typing PCR assay detected the tcdC Δ117 mutation in 2/24 samples suggesting the presence of the epidemic strain in these samples. This was confirmed by PCR ribotyping and sequencing of the tcdC gene. Using CTA as the “gold standard”, the sensitivity, specificity, positive predictive value, and negative predictive value, for the screening PCR were 100%, 94.4%, 66.7%, and 100%, respectively. In conclusion, PCR may serve as a rapid negative screening assay for patients suspected of having CDI, although the low PPV hamper the use of PCR as a standalone test. However, PCR results may provide valuable information for patient management and minimising the spread of the epidemic 027/NAP1 strain.     

Cryptic species and systematics of the hynobiid salamanders of the Liua–Pseudohynobius complex: Molecular and phylogenetic perspectives

Using mitochondrial DNA sequencing and allozyme electrophoresis, we examined 18 populations of the Liua–Pseudohynobius complex, endemic to China. Based on their phylogenetic affiliation and exhibited fixed allelic differences, the complex comprises at least six species, two of which are previously unknown cryptic species. The complex is clearly divided into two groups, genus Liua including Liua shihi and Liua tsinpaensis, and genus Pseudohynobius including Pseudohynobius flavomaculatus, Pseudohynobius shuichengensis and the two new species. The previously often used genus name Ranodon is inappropriate, because the type species of the genus, Ranodon sibricus, is distantly related to this complex. The species diversity among Chinese hynobiid salamanders are far from being recognized and further effort should be directed at extensive field collection in central and western China.     

In silico analysis of expression pattern of a Wnt/β-catenin responsive gene ANLN in gastric cancer

Actin-binding protein anillin (ANLN) is primarily involved in the cytokinesis and known to be dysregulated in many cancers including gastric cancer (GC). However, the regulation and clinical significance of ANLN in GC are far less clear. In the present study, we aimed to investigate the clinical significance and possible regulators of ANLN in GC. We have identified the Wnt/β-catenin associated regulation of ANLN by analyzing the in vitro perturbed β-catenin mRNA expression profiles. Investigating the gastric tumors from publicly available genome-wide mRNA expression profiles, we have identified the over expression of ANLN in gastric tumors. Association between ANLN expression and clinical characteristics of GC showed elevated expression in intestinal type GC. Performing a single sample prediction method across GC mRNA expression profiles, we have identified the over expression of ANLN in proliferative type gastric tumors compared to the invasive and metabolic type gastric tumors. In silico pathway prediction analysis revealed the association between Wnt/β-catenin signaling and ANLN expression in gastric tumors. Our results highlight that expression of a Wnt/β-catenin responsive gene ANLN in GC is a molecular predictor of intestinal and proliferative type gastric tumors.     

QM/MM investigation on the catalytic mechanism of Bacteroides thetaiotaomicron α-glucosidase BtGH97a

Bacteroides thetaiotaomicron α-glucosidase BtGH97a is an inverting enzyme. In this paper, the hydrolysis mechanism of p-nitro-phenyl α-d-glucopyranoside (pNP-Glc) catalyzed by BtGH97a was firstly studied by using quantum mechanical/molecular mechanical (QM/MM) approach. Two possible reaction pathways were considered. In the first pathway, a water molecule deprotonated by a nucleophilic base (here E439 or E508) attacks firstly on the anomeric carbon of pNP-Glc, then a proton from an acid residue (E532) attacks on the glycosidic oxygen to finish the hydrolysis reaction (named as nucleophilic attack-first pathway). In the second pathway, the proton from E532 attacks firstly on the glycosidic oxygen, then the water deprotonated by the nucleophilic base attacks on the anomeric carbon of pNP-Glc (named as proton attack-first pathway). Our calculation results indicate that the nucleophilic attack-first pathway is favorable in energy, in which the nucleophilic attack process is the rate-determining step with an energy barrier of 15.4 kcal/mol in the case of residue E508 as nucleophilic base. In this rate-determining step, the deprotonation of water and the attack on the anomeric carbon are concerted. In the proton attack-first pathway, the proton attack on the glycosidic oxygen is the rate-determining step, and the energy barrier is 24.1 kcal/mol. We conclude that the hydrolysis mechanism would follow nucleophilic attack-first pathway.