Culture-independent analyses reveal novel Anaerolineaceae as abundant primary fermenters in anaerobic digesters treating waste activated sludge:Genomic insights into the A6 phylotype

Anaerobic digestion for biogas production is reliant on the tightly coupled synergistic activities of complex microbial consortia. Members of the uncultured A6 phylotype, within the phylum Chloroflexi, are among the most abundant genus-level-taxa of mesophilic anaerobic digester systems treating primary and surplus sludge from wastewater treatment plants, yet are known only by their 16S rRNA gene sequence. This study applied metagenomics to obtain a complete circular genome (2.57 Mbp) from a representative of the A6 taxon. Preliminary annotation of the genome indicates these organisms to be anaerobic chemoorganoheterotrophs with a fermentative metabolism. Given their observed abundance, they are likely important primary fermenters in digester systems. Application of fluorescence in situ hybridisation probes designed in this study revealed their morphology to be short filaments present within the flocs. The A6 were sometimes co-located with the filamentous Archaea Methanosaeta spp. suggesting potential undetermined synergistic relationships. Based on its genome sequence and morphology we propose the species name Brevefilum fermentans gen. nov. sp. nov

Functional identification of a Ligase in the Red Sea Atlantis II deepest Layer

Red sea, described as one of the unique marine ecosystems, incorporates up to 25 deep-sea brine pools. These pools posses multiple extreme conditions influencing the evolution and survival of their inhabiting microbial community. The combination of maximum depth (2194 m), high temperature (68C), anoxia, high salinity (26%), high pressure and high concentrations of heavy metals in the lower convective layer (LCL) of the Atlantis II brine pool makes it an ideal environment for identification of novel enzymes with unique characteristics and potential biotechnological applications. Here we describe the identification and the preliminary in vivo functional investigation of the ligase domain of an ATP-dependent DNA ligase from the DNA of the prokaryotic community extracted from water samples of the LCL of Atlantis II brine pool. Previously, these water samples were serially filtered on different membranes and the DNA isolated from the 0.1­m filter was subjected to 454 pyrosequencing. A metagenomic dataset was initiated and used in this study to mine for genes encoding DNA ligases through Pfam search of conserved domains. The search and subsequent bioinformatic analysis resulted in the identification of a contig harboring an ORF of 915 bp (305 amino acids) that encodes a putative DNA ligase (LigATII). Homology search of the putative DNA ligase showed highest similarity to Erysiopelotrichaceae Bacterium (39% identity, 54% positive). LigATII displays modular architecture that is similar to two distinct domains-(the adenylation domain of LigD and the oligonucleotide binding (OB) fold domain)-that are conserved in ATP-dependent DNA ligases. Functional annotation of the LigATII ORF, identification of the functional conserved amino acids by the Consurf tool, 3D modeling and comprehensive phylogenetic analysis were conducted. These analyses have revealed the relatedness of LigATII to the family of ATP-dependent DNA ligases that has been recently identified through computational studies to exist in prokaryotes. This family is expected to be involved in the specialized form of genomic DNA repair through the non-homologous end joining pathway which acts to join double-stranded breaks (DSBs) or to promote genetic diversity under conditions of selection pressures. Accordingly, the putative LigATII was amplified from the whole genome DNA amplification of LCL. Sanger sequencing confirmed the sequence of the gene before cloning into pET100 Topo directional expression vector. The cloned LigATII was transformed into a temperature sensitive mutant strain of Escherichia coli; strain GR501, with mutation in the DNA ligase gene. LigATII complemented the temperature sensitive strain at the non-permissive temperature (43â—¦C) verifying the in vivo functional activity. The biochemical characteristics of the novel LigATII protein will be described

SPIDROIN N-TERMINAL DOMAIN: A PH SENSOR IN THE SPIDER SILK ASSEMBLY PROCESS

Spider silks are protein-based fibers with remarkable mechanical qualities. Perhaps even more impressive is the spinning process in which the spider silk proteins (spidroins) are assembled from a highly soluble storage state into a well-ordered and insoluble fiber. Indeed, the ordered arrangement of spidroins, which is endowed by the spinning process, is the basis of fiber strength. However, the forces driving fiber assembly and the mechanisms by which spidroins respond those forces are only poorly understood. Spidroins have a tripartite domain architecture consisting of a large and repetitive central domain flanked by small, non-repetitive N- and C-terminal domains. Both terminal domains are well conserved among different types of spidroins, and they are believed to act as coordinators of spidroin assembly. This dissertation represents an important advancement of knowledge on the sequence, structure, and biochemical behavior of the highly conserved but greatly under studied N-terminal domain of major ampullate silks (MaSp-NTD). In my analysis of MaSp-NTD sequence, I demonstrated for the first time that one of the two types of MaSp genes (MaSp1) had been duplicated in the golden orb-weaving spider (Nephila clavipes) generating MaSp1A and MaSp1B genes. My publication of this work was contemporaneous with publications by research groups finding similar MaSp gene duplications in other spider species. Characterization of the RNA transcripts from MaSp1A and MaSp1B genes indicated that they are simultaneously co-expressed in adult female spiders. It also allowed experimental verification the transcription start sites in the MaSp genes. My biochemical analysis of three recombinant MaSp-NTDs indicate that they are highly pH responsive. Upon experiencing a mild acidification, as occurs in the spider silk gland, they undergo a dramatic change in tertiary conformation. Concurrent with the pH-dependent refolding, the interaction between MaSp-NTD molecules is strengthened resulting in considerably more stable MaSp-NTD homo-dimers. Through a collaboration with another research group, we are working to determine the structure of MaSp-NTD in both conformational states by NMR. By transducing the duct pH change into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD

Genomic insights into members of the candidate phylum Hyd24-12 common in mesophilic anaerobic digesters

Members of the candidate phylum Hyd24-12 are globally distributed, but no genomic information or knowledge about their morphology, physiology or ecology is available. In this study, members of the Hyd24-12 lineage were shown to be present and abundant in full-scale mesophilic anaerobic digesters at Danish wastewater treatment facilities. In some samples, a member of the Hyd24-12 lineage was one of the most abundant genus-level bacterial taxa, accounting for up to 8% of the bacterial biomass. Three closely related and near-complete genomes were retrieved using metagenome sequencing of full-scale anaerobic digesters. Genome annotation and metabolic reconstruction showed that they are Gram-negative bacteria likely involved in acidogenesis, producing acetate and hydrogen from fermentation of sugars, and may play a role in the cycling of sulphur in the digesters. Fluorescence in situ hybridization revealed single rod-shaped cells dispersed within the flocs. The genomic information forms a foundation for a more detailed understanding of their role in anaerobic digestion and provides the first insight into a hitherto undescribed branch in the tree of life

Production and Biocompatibility of Spider Silk Proteins in Goat Milk

Due to its strength, flexibility, and biocompatibility, spider silk is a highly appealing material for applications in the medical field. Unfortunately, natural spider silk is difficult to obtain in large quantities because spiders are territorial and cannibalistic, making them impractical to farm. Synthetic spider silk proteins produced by transgenic hosts such as bacteria and goats have made it possible to obtain the quantities of spider silk needed to study it more fully and to investigate its potential uses. The spider silk proteins produced in our laboratory do not have an optimal purification method to remove all of the non-biocompatible contaminants and have not previously been tested for their biocompatibility. The first focus of this dissertation was to create goat cells that can be used to create new goats. These new goats will produce proteins that can be purified more efficiently and more completely. The second focus of this dissertation was to perform biocompatibility tests on goat-derived spider silk proteins. Prior to performing any biocompatibility tests, a method was established for removing endotoxins – an impurity that causes an immune response in the body – from the proteins. This work has shed light on areas for improvement in the silk protein purification process and laid groundwork for the production of new goat-derived proteins. These steps will help make it possible for synthetic spider silk to progress further toward becoming a viable biomaterial

Modification to the immunodominant loop of hepatitis B virus core protein to enhance vector properties of virus-like particles

Gene therapy has shown potential in alleviating a wide range of diseases, ranging from viral infections to autosomal diseases. One of the obstacles to gene therapy reaching its full potential is the inadequacy of methods to deliver therapeutic nucleic sequences. Current delivery of gene therapy entails use of viral and non-viral vectors. Viral vectors are however associated with drawbacks such as potential toxicity, high cost and labour-intensive production. Thus non-viral delivery alternatives are being developed in an attempt to overcome difficulties associated with nucleic acid delivery for gene therapy. Virus-like particles are potentially very useful delivery vehicles as their production is simple and cost effective, the particle surface is amenable to modification and the capsid interior can be altered to accommodate a variety of cargoes. One such particle is the recombinant HBV capsid, which comprises a single species of protein and is tolerant of amino acid substitutions on the surface exposed immunodominant loops. This study aimed to enhance the vector-like properties of the HBV virus-like particle by including amino acid substitutions on the particle surface. These substituted residues in turn provided a conjugation site for tropic and immuno evasive moieties. It was found that lysine substitutions resulted in poor conjugation to the capsid surface, whereas substituted cysteine residues resulted in almost all protein-moiety conjugates forming. In order to introduce lysine and cysteine substitutions, a novel method of cloning into the HBV was generated. In doing so, complicated procedures associated with cloning into the immunodominant loop of the HBV capsid have been alleviated. Ligands containing galactose were utilised on the surface of both the HBV capsid and liposomes to confer hepatotropism. The presence of the galactose moieties on the surface of the HBV capsid prevented indiscriminate cellular uptake in cultured cells, however did not improve hepatotropism. Galactose ligands on the surface of liposomes did improve transfection efficiency, however they required a short linker distance between liposome surface and galactose group. The inclusion of galactose in liposome formulations also provided a means to deliver siRNA to the liver of transgenic HBV mice. It is believed that with alterations to the ligand structure, it is possible to provide HBV capsids with hepatotropism in future experimentation. This study demonstrated that the exposed external surface of the HBV capsid is amenable to convenient conjugation, which potentially facilitates immune evasion and conferring of defined tropism

Molecular insights into the disease mechanisms of startle disease

This study describes an in-depth investigation into the pathogenic mechanisms of inherited mutations that lead to disorders of inhibitory glycinergic transmission, primarily the rare human disorder known as startle disease/hyperekplexia. I also investigated mutations causing a similar startle phenotype in cows, mice and zebrafish. Using molecular genetics techniques, I identified pathogenic mutations in the genes that encode for proteins involved in glycinergic neurotransmission, specifically the postsynaptic glycine receptor (GlyR) subunits and the presynaptic glycine transporter GlyT2. Using homology modelling and other computational biology methods, I examined the structural and functional impacts of mutations on protein function, revealing key motifs and amino acids crucial for receptor and transporter activity. Using cDNA cloning and site-directed mutagenesis, I also generated expression constructs for wild-type and mutant proteins that were used in functional tests to measure the impact of pathogenic mutations on glycine receptor and transporter function. For certain animal models of startle disease, I was also able to develop diagnostic PCR tests for pathogenic mutations, which can be used to alleviate further animal suffering by preventing ‘at risk matings’ of carrier animals. Taken together, my findings reveal several new pathogenic mechanisms of GlyR and GlyT2 mutations in startle disease in humans and animals, revealing insights into receptor and transporter function that may be applicable to other neurological disorders