Science

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Now showing 1 - 10 of 41
  • (2008) Fei, W; Shui, G; Gaeta, Bruno; Du, Xi; Kuerschner, L; Brown, Andrew; Wenk, M; Parton, R; Yang, Hyuk-Seung; Li, Peng
    Journal Article
    Lipid droplets (LDs) are emerging cellular organelles that are of crucial importance in cell biology and human diseases. In this study, we present our screen of 4,700 Saccharomyces cerevisiae mutants for abnormalities in the number and morphology of LDs; we identify 17 fld (few LDs) and 116 mld (many LDs) mutants. One of the fld mutants (fld1) is caused by the deletion of YLR404W, a previously uncharacterized open reading frame. Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1 Delta cells demonstrate significantly enhanced fusion activities both in vivo and in vitro. Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fld1 Delta cells. Lipid profiling reveals alterations in acyl chain compositions of major phospholipids in fld1 Delta cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

  • (2008) Mai-Prochnow, A; Lucas-Elio, P; Egan, S; Thomas, Torsten; Webb, JS; Sanchez-Amat, A; Kjelleberg, S
    Journal Article
    The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria.

  • (2008) Egan, S; Thomas, Torsten; Kjelleberg, S
    Journal Article
    Marine sessile eukaryotic hosts provide a unique surface for microbial colonisation. Chemically mediated interactions between the host and colonising microorganisms, interactions between microorganisms in the biofilm community and surface-specific physical and chemical conditions impact differently on the diversity and function of surface-associated microbial assemblages compared with those in planktonic systems. Understanding the diversity and ecology of surface-associated microbial communities will greatly contribute to the discovery of next-generation, bioactive compounds. On the basis of recent conceptual and technological advances insights into the microbiology of marine living surfaces are improving and novel bioactives, including those previously ascribed as host derived, are now revealed to be produced by members of the surface-associated microbial community.

  • (2008) Thomas, Torsten; Evans, FF; Schleheck, D; Mai-Prochnow, A; Burke, C; Penesyan, A; Dalisay, DS; Stelzer-Braid, S; Saunders, N; Johnson, J; Ferriera, S; Kjelleberg, S; Egan, S
    Journal Article
    BACKGROUND: Colonisation of sessile eukaryotic host surfaces (e.g. invertebrates and seaweeds) by bacteria is common in the marine environment and is expected to create significant inter-species competition and other interactions. The bacterium Pseudoalteromonas tunicata is a successful competitor on marine surfaces owing primarily to its ability to produce a number of inhibitory molecules. As such P. tunicata has become a model organism for the studies into processes of surface colonisation and eukaryotic host-bacteria interactions. METHODOLOGY/PRINCIPAL FINDINGS: To gain a broader understanding into the adaptation to a surface-associated life-style, we have sequenced and analysed the genome of P. tunicata and compared it to the genomes of closely related strains. We found that the P. tunicata genome contains several genes and gene clusters that are involved in the production of inhibitory compounds against surface competitors and secondary colonisers. Features of P. tunicata's oxidative stress response, iron scavenging and nutrient acquisition show that the organism is well adapted to high-density communities on surfaces. Variation of the P. tunicata genome is suggested by several landmarks of genetic rearrangements and mobile genetic elements (e.g. transposons, CRISPRs, phage). Surface attachment is likely to be mediated by curli, novel pili, a number of extracellular polymers and potentially other unexpected cell surface proteins. The P. tunicata genome also shows a utilisation pattern of extracellular polymers that would avoid a degradation of its recognised hosts, while potentially causing detrimental effects on other host types. In addition, the prevalence of recognised virulence genes suggests that P. tunicata has the potential for pathogenic interactions. CONCLUSIONS/SIGNIFICANCE: The genome analysis has revealed several physiological features that would provide P. tunciata with competitive advantage against other members of the surface-associated community. We have also identified properties that could mediate interactions with surfaces other than its currently recognised hosts. This together with the detection of known virulence genes leads to the hypothesis that P. tunicata maintains a carefully regulated balance between beneficial and detrimental interactions with a range of host surfaces.

  • (2008) Tang, Chaka; Reyes, Josephine F; Luciani, Fabio; Francis, Andrew R.; Tanaka, Mark M
    Journal Article
    spolTools is a collection of online programs designed to manipulate and analyze spoligotype datasets of the Mycobacterium tuberculosis complex. These tools are integrated into a repository currently containing 1179 spoligotypes and 6278 isolates across 30 datasets. Users can search this database to export for external use or to pass on to the integrated tools. These tools include the computation of basic population genetic quantities, the visualization of clusters of spoligotype patterns based on an estimated evolutionary history and a procedure to predict emerging strains – genotypes associated with elevated transmission.

  • (2008) Reyes, Josephine F; Francis, Andrew R.; Tanaka, Mark M
    Journal Article
    Background: Molecular typing methods are commonly used to study genetic relationships among bacterial isolates. Many of these methods have become standardized and produce portable data. A popular approach for analyzing such data is to construct graphs, including phylogenies. Inferences from graph representations of data assist in understanding the patterns of transmission of bacterial pathogens, and basing these graph constructs on biological models of evolution of the molecular marker helps make these inferences. Spoligotyping is a widely used method for genotyping isolates of Mycobacterium tuberculosis that exploits polymorphism in the direct repeat region. Our goal was to examine a range of models describing the evolution of spoligotypes in order to develop a visualization method to represent likely relationships among M. tuberculosis isolates. Results: We found that inferred mutations of spoligotypes frequently involve the loss of a single or very few adjacent spacers. Using a second-order variant of Akaike's Information Criterion, we selected the Zipf model as the basis for resolving ambiguities in the ancestry of spoligotypes. We developed a method to construct graphs of spoligotypes (which we call spoligoforests). To demonstrate this method, we applied it to a tuberculosis data set from Cuba and compared the method to some existing methods. Conclusion: We propose a new approach in analyzing relationships of M. tuberculosis isolates using spoligotypes. The spoligoforest recovers a plausible history of transmission and mutation events based on the selected deletion model. The method may be suitable to study markers based on loci of similar structure from other bacteria. The groupings and relationships in the spoligoforest can be analyzed along with the clinical features of strains to provide an understanding of the evolution of spoligotypes.

  • (2008) Luciani, Fabio; Francis, Andrew; Tanaka, Mark
    Journal Article
    Molecular techniques such as IS6110-RFLP typing and spacer oligonucleotide typing (spoligotyping) have aided in understanding the transmission patterns of Mycobacterium tuberculosis. The degree of clustering of isolates on the basis of genotypes is informative of the extent of transmission in a given geographic area. We analyzed 130 published data sets of M. tuberculosis isolates, each representing a sample of bacterial isolates from a specific geographic region, typed with either or both of the IS6110-RFLP and spoligotyping methods. We explored common features and differences among these samples. Using population models, we found that the presence of large clusters (typically associated with recent transmission) as well as a large number of singletons (genotypes found exactly once in the data set) is consistent with an expanding infectious population. We also estimated the mutation rate of spoligotype patterns relative to IS6110 patterns and found the former rate to be about 10-26% of the latter. This study illustrates the utility of examining the full distribution of genotype cluster sizes from a given region, in the light of population genetic models. (C) 2007 Elsevier B.V. All rights reserved.

  • (2008) Venturi, Vanessa; Kedzierska, K; Tanaka, Mark; Turner, Stephen; Doherty, P; Davenport, Miles
    Journal Article
    The CD8(+) T cell response is important in the control of many viral and other infections. There have been many studies aimed at better understanding the influence of T cell receptor diversity on immune responses and the evolution of the T cell receptor repertoire over time and through the various stages of immune responses to infection. In recent years, there has been an increase in both the number of studies using T cell receptor data and the volume of T cell receptor data generated per study. Appropriate analytical tools are required to analyse this data. We present a robust approach to assessing the similarity between samples of the T cell receptor repertoire, which we demonstrate on published data of subsets of the influenza A virus (DNP366)-N-b- and D(b)PA(224)- specific CD8+ T cell responses in mice sorted on the expression of CD62L, which is a marker distinguishing central and effector memory cells. (C) 2007 Elsevier B.V. All rights reserved.

  • (2008) Nousch, Marco
    Thesis
    Members of the eukaryotic initiation factor 4G (eIF4G) family play a central role in the translation initiation process. One member of this family is p97 (also called DAP5 and NAT1), a protein that is highly homologous to the C-terminal two thirds of eIF4G. Overexpression studies suggested that p97 is a pure translational repressor that has to be cleaved into a shorter form called p86, in order to show translational activity. In this study a series of experiments indicated that full length p97 has a number elF property such as association with active translating ribosomes, stimulatory effects in the Direct Initiation Factor assay and accumulation in stress granules. Additionally the endogenous p97 complex was isolated from HeLa cells and mRNA as well as the protein components were characterized. P97 associated mRNAs were described by a custom made 5'UTR focus array, showing that the protein binds to a broad range of mRNA. The relative lack of mRNA specificity argues for a general role of p97 in translation, which does not seems to be essential in unchallenged cells, because a down regulation of p97 protein levels has no effect on the translational status of the bulk of mRNAs. Mass spectrometry analysis revealed a novel protein-protein interaction between p97 and DNA methyltransferase 1 (Dnmt1), which does not rely on a nucleic acid. For this interaction the C- and N-terminus of p97 play a critical role. Further, Dnmt1 has the ability to interact with elF4G and the small ribosomal subunit, which might provide evidence for a novel function of Dnmt1 in RNA metabolism.

  • (2008) Mihali, Troco Kaan
    Thesis
    Freshwater cyanobacteria produce a wealth of biologically active metabolites, which can adversely affect human and animal health, and cause great economic damage to the fishing, tourism and water-management industries on a global scale. We describe the molecular genetics and biochemistry of biosynthesis for the cyanobacterial toxic alkaloids cylindrospennopsin, paralytic shellfish toxins (PST) and anatoxin-a. Characterisation of the 43 kb cylindrospennopsin biosynthesis gene cluster (cyr), in Cylindrospermopsis raciborskii AWT205 is described. Biosynthesis is initiated via an amidinotransfer onto glycine followed by five polyketide extensions. Rings are formed via Michael additions, while the uracil ring is formed by a novel mechanism. Tailoring reactions, including sulfation and hydroxylation complete the biosynthesis. We describe the characterisation of PST biosynthesis gene clusters in Anabaena circinalis, Aphanizomenon sp. and Lyngbya wollei. These gene clusters span between 28 and 36 kb and contain genes coding for the biosynthesis and export of PSTs. The Lyngbya wollei PST gene cluster represents a 'natural combinatorial biosynthesis' event, explaining its unique toxin profile. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed, and a putative insertion/excision site of the PST gene cluster in Anabaena circinalis 310F was identified. Interestingly, PSTs are produced by distantly related organisms via this unique biosynthesis pathway. We Investigated the phylogenetics of PST biosynthesis genes from four different genera of cyanobacteria. The results suggested that PST biosynthesis in cyanobacteria is an ancient trait, whereby the sporadic distribution of PST production in extant isolates of Anabaena circinalis and Aphanizomenon sp. is a result of the repeated loss of the biosynthetic gene cluster. Horizontal gene transfer also appears to have had a critical influence on PST biosynthesis in Lyngbya wollei. We additionally propose a hypothetical, mixed non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) biosynthesis scheme for anatoxin-a. Degenerate PCR primers were developed, for the specific amplification of mixed NRPSIPKS hybrid ketosynthase (KS) domains. Gene-walking distally to a novel hybrid KS domain in the anatoxin-a producer Planktothrix rubescens, revealed an orphan gene cluster, denoted pro, which spans 24 kb and codes for a mixed NRPS/PKS system, putatively producing an acetylated and sulphated dipeptide.