Systematic comparison of eight methods for preparation of high purity sulfated fucans extracted from the brown alga Pelvetia canaliculata.

Sulfated fucans from brown algae are a heterogeneous group of biologically active molecules. To learn more on their structure and to analyze and exploit their biological activities, there is a growing need to develop reliable and cost effective protocols for their preparation. In the present study, a brown alga Pelvetia canaliculata (Linnaeus) was used as a rich source of sulfated fucans. Sulfated fucan preparation methods included neutral and acidic extractions followed by purification with activated charcoal (AC), polyvinylpolypyrrolidone (PVPP), or cetylpyridinium chloride (CPC). Final products were compared in terms of yield, purity, monosaccharide composition and molecular weight. Acidic extractions provided higher yields compared to neutral ones, whereas the AC purification provided sulfated fucan products with the highest purity. Mass spectrometry analyses were done on oligosaccharides produced by the fucanase MfFcnA from the marine bacterium Mariniflexille fucanivorans. This has provided unique insight into enzyme specificity and the structural characteristics of sulfated fucans.

Identification and Characterization of a New Type III Polyketide Synthase from a Marine Yeast, Naganishia uzbekistanensis.

A putative Type III Polyketide synthase (PKSIII) encoding gene was identified from a marine yeast, Naganishia uzbekistanensis strain Mo29 (UBOCC-A-208024) (formerly named as Cryptococcus sp.) isolated from deep-sea hydrothermal vents. This gene is part of a distinct phylogenetic branch compared to all known terrestrial fungal sequences. This new gene encodes a C-terminus extension of 74 amino acids compared to other known PKSIII proteins like Neurospora crassa. Full-length and reduced versions of this PKSIII were successfully cloned and overexpressed in a bacterial host, Escherichia coli BL21 (DE3). Both proteins showed the same activity, suggesting that additional amino acid residues at the C-terminus are probably not required for biochemical functions. We demonstrated by LC-ESI-MS/MS that these two recombinant PKSIII proteins could only produce tri- and tetraketide pyrones and alkylresorcinols using only long fatty acid chain from C8 to C16 acyl-CoAs as starter units, in presence of malonyl-CoA. In addition, we showed that some of these molecules exhibit cytotoxic activities against several cancer cell lines.

Genetic analyses unravel the crucial role of a horizontally acquired alginate lyase for brown algal biomass degradation by Zobellia galactanivorans.

Comprehension of the degradation of macroalgal polysaccharides suffers from the lack of genetic tools for model marine bacteria, despite their importance for coastal ecosystem functions. We developed such tools for Zobellia galactanivorans, an algae-associated flavobacterium that digests many polysaccharides, including alginate. These tools were used to investigate the biological role of AlyA1, the only Z. galactanivorans alginate lyase known to be secreted in soluble form and to have a recognizable carbohydrate-binding domain. A deletion mutant, ΔalyA1, grew as well as the wild type on soluble alginate but was deficient in soluble secreted alginate lyase activity and in digestion of and growth on alginate gels and algal tissues. Thus, AlyA1 appears to be essential for optimal attack of alginate in intact cell walls. alyA1 appears to have been recently acquired via horizontal transfer from marine Actinobacteria, conferring an adaptive advantage that might benefit other algae-associated bacteria by exposing new substrate niches. The genetic tools described here function in diverse members of the phylum Bacteroidetes and should facilitate analyses of polysaccharide degradation systems and many other processes in these common but understudied bacteria.

Cryo-EM study of start codon selection during archaeal translation initiation.

Eukaryotic and archaeal translation initiation complexes have a common structural core comprising e/aIF1, e/aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit. e/aIF2 plays a crucial role in this process but how this factor controls start codon selection remains unclear. Here, we present cryo-EM structures of the full archaeal 30S initiation complex showing two conformational states of the TC. In the first state, the TC is bound to the ribosome in a relaxed conformation with the tRNA oriented out of the P site. In the second state, the tRNA is accommodated within the peptidyl (P) site and the TC becomes constrained. This constraint is compensated by codon/anticodon base pairing, whereas in the absence of a start codon, aIF2 contributes to swing out the tRNA. This spring force concept highlights a mechanism of codon/anticodon probing by the initiator tRNA directly assisted by aIF2.

Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.

Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.

The family X DNA polymerase from Deinococcus radiodurans adopts a non-standard extended conformation.

Deinococcus radiodurans is an extraordinarily radioresistant bacterium that is able to repair hundreds of radiation-induced double-stranded DNA breaks. One of the players in this pathway is an X family DNA polymerase (PolX(Dr)). Deletion of PolX(Dr) has been shown to decrease the rate of repair of double-stranded DNA breaks and increase cell sensitivity to gamma-rays. A 3'-->5' exonuclease activity that stops cutting close to DNA loops has also been demonstrated. The present crystal structure of PolX(Dr) solved at 2.46-A resolution reveals that PolX(Dr) has a novel extended conformation in stark contrast to the closed "right hand" conformation commonly observed for DNA polymerases. This extended conformation is stabilized by the C-terminal PHP domain, whose putative nuclease active site is obstructed by its interaction with the polymerase domain. The overall conformation and the presence of non standard residues in the active site of the polymerase X domain makes PolX(Dr) the founding member of a novel class of polymerases involved in DNA repair but whose detailed mode of action still remains enigmatic.

Crystal structure of an intact type II DNA topoisomerase: insights into DNA transfer mechanisms.

DNA topoisomerases resolve DNA topological problems created during transcription, replication, and recombination. These ubiquitous enzymes are essential for cell viability and are highly potent targets for the development of antibacterial and antitumoral drugs. Type II enzymes catalyze the transfer of a DNA duplex through another one in an ATP-dependent mechanism. Because of its small size and sensitivity to antitumoral drugs, the archaeal DNA topoisomerase VI, a type II enzyme, is an excellent model for gaining further understanding of the organization and mechanism of these enzymes. We present the crystal structure of intact DNA topoisomerase VI bound to radicicol, an inhibitor of human topo II, and compare it to the conformation of the apo-protein as determined by small-angle X-ray scattering in solution. This structure, combined with a wealth of experimental data gathered on these enzymes, allows us to propose a structural model for the two-gate DNA transfer mechanism.

The GTPase, CpgA(YloQ), a putative translation factor, is implicated in morphogenesis in Bacillus subtilis.

YloQ, from Bacillus subtilis, was identified previously as an essential nucleotide-binding protein of unknown function. YloQ was successfully over-expressed in Escherichia coli in soluble form. The purified protein displayed a low GTPase activity similar to that of other small bacterial GTPases such as Bex/Era. Based on the demonstrated GTPase activity and the unusual order of the yloQ G motifs, we now designate this protein as CpgA (circularly permuted GTPase). An unexpected property of this low abundance GTPase was the demonstration, using gel filtration and ultracentrifugation analysis, that the protein formed stable dimers, dependent upon the concentration of YloQ(CpgA), but independent of GTP. In order to investigate function, cpgA was placed under the control of the pspac promotor in the B. subtilis chromosome. When grown in E or Spizizen medium in the absence of IPTG, the rate of growth was significantly reduced. A large proportion of the cells exhibited a markedly perturbed morphology, with the formation of swollen, bent or 'curly' shapes. To confirm that this was specifically due to depleted CpgA a plasmid-borne cpgA under pxyl control was introduced. This restored normal cell shape and growth rate, even in the absence of IPTG, provided xylose was present. The crystal structure of CpgA(YloQ) suggests a role as a translation initiation factor and we discuss the possibility that CpgA is involved in the translation of a subset of proteins, including some required for shape maintenance.

The crystal structure of YloQ, a circularly permuted GTPase essential for Bacillus subtilis viability.

yloQ is one of 11 essential genes in Bacillus subtilis with unknown roles in the physiology of the cell. It encodes a polypeptide of 298 residues with motifs characteristic of GTPases. As a contribution to elucidating its indispensable cellular function, we have solved the crystal structure of YloQ to 1.6 A spacing, revealing a three-domain organisation. At the heart of the molecule is the putative GTPase domain, which exhibits a classical alpha/beta nucleotide-binding fold with a topology very similar to that of Ras and Era. However, as anticipated from the order in which the conserved G protein motifs appear in the sequence, the GTPase domain fold in YloQ is circularly permuted with respect to the classical GTPases. The nucleotide-binding pocket in YloQ is unoccupied, and analysis of the phosphate-binding (P) loop indicates that conformational changes in this region would be needed to accommodate GTP. The GTPase domain is flanked at its N terminus by a beta-barrel domain with an oligonucleotide/oligosaccharide-binding (OB) fold, and at its C terminus by an alpha-helical domain containing a coordinated zinc ion. This combination of protein modules is unique to YloQ and its orthologues. Sequence comparisons reveal a clustering of conserved basic and aromatic residues on one face of the OB domain, perhaps pointing to a role for YloQ in nucleic acid binding. The zinc ion in the alpha-helical domain is coordinated by three cysteine residues and a histidine residue in a novel ligand organisation. The juxtaposition of the switch I and switch II regions of the G domain and the OB and zinc-binding domains suggests that chemical events at the GTPase active site may be transduced into relative movements of these domains. The pattern of conserved residues and electrostatic surface potential calculations suggest that the OB and/or Zn-binding domains participate in nucleic acid binding consistent with a possible role for YloQ at some stage during mRNA translation.

Crystallization of YloQ, a GTPase of unknown function essential for Bacillus subtilis viability.

YloQ is a putative ATP/GTP-binding protein of unknown function identified from the complete sequence of the Bacillus subtilis genome. A gene-knockout programme established that yloQ is one of a set of some 270 indispensable genes for the viability of this organism. Crystals of YloQ have been grown from HEPES-buffered solutions at pH 7.5 containing polyethylene glycol and diffraction data have been collected extending to 2.5 A spacing.

Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis.

We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.