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Analyzing the properties and biosynthesis of β-glucans from Gluconacetobacter and poplar
KTH, School of Biotechnology (BIO), Glycoscience. (Bulone)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glucans are polysaccharides integral to many materials and biological functions. Under the umbrella of Biomime, the Swedish Center for Biomimetic Fiber Engineering, this work has aimed to improve basic understanding of the biosynthesis of such glucans. This has been achieved through direct investigation of cellulose structure, and by developing the tools to analyze glucan biosynthesis. Notably we have identified a novel chemical effector of glucan synthesis processes and developed a proteomic toolkit useful for analyzing membrane-bound glycosyltransferases, the enzyme group responsible for glucan biosynthesis. During this work, glucan synthesis has been studied using both Gluconacetobacter and Populus cell suspension cultures.

Publication I. Gluconacetobacter cellulose (BC) was used as a base to create a novel and well characterized nano-material with improved mechanical properties. This novel composite of BC and hydroxyethylcellulose (HEC) had improved tensile strength compared to pure BC. Through thorough study utilizing dispersion measurements, electron microscopy, nuclear magnetic resonance and X-ray diffraction it was shown that the improved properties derived from a layer of HEC coating each fibril.

Publication II. Bacterial cellulose was labeled in specific positions with 13C (C4 and C6). These samples were analyzed by CP/MAS NMR along with cellulose samples from cotton and Halocynthia sp. For each sample spectral fitting was performed and general properties of crystal allomorph composition and fibril widths were determined. Calculations were also made for water accessible surfaces of the fibrils. The results showed that water accessible C4 surface signals are reflective of the allomorph composition of the sample, along with a distorted signal that derives due to fibril imperfections. Water accessible surface signals from the C6 region are instead derived from rotamer conformations of the C6 hydroxymethyl groupsfrom glucose residues.

In Publication III, a high-throughput screen was used to identify an inhibitor of Golgi-derived glycosyltransferase activity, termed chemical A. The structural basis for inhibition was determined and in vitro assays of callose synthesis were performed. The in vitro assays revealed chemical A to also be an activator of callose synthesis. To understand this activation kinetic studies were performed, showing that chemical A is a mixed type of activator, which can bind either the free enzyme or the enzyme-substrate complex. Chemical A has uses in chemical genetics for dissecting processes involving callose synthesis, such as stress response and cell-plate formation.

In publication IV, we present an in-house developed platform for proteomics with a distributed processing model. This in-house system has been central to many proteomics tasks, including for those presented in publication V, and is being distributed as the Automated Proteomics Pipeline (APP).

In publication V, conditions for enrichment of Detergent-Resistant Microdomains (DRM) have been optimized for Populus trichocarpa cell cultures. The proteins enriched in DRM were identified using mass spectrometry based proteomics, and a functional model for DRM was proposed. This model involves proteins specialized in stress response, including callose synthase, and cell signaling. This further strengthens the arguments for DRMs as sites of specific cellular functions and confirms they play a role in glucan synthesis.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , 66 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:8
Keyword [en]
Cellulose synthesis, Bioinformatics, Proteomics, Cell wall
National Category
Biological Sciences Bioinformatics and Systems Biology
Identifiers
URN: urn:nbn:se:kth:diva-147932ISBN: 978-91-7595-184-3 (print)OAI: oai:DiVA.org:kth-147932DiVA: diva2:733483
Public defence
2014-09-05, FR4, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20140710

Available from: 2014-07-10 Created: 2014-07-09 Last updated: 2014-07-10Bibliographically approved
List of papers
1. Nanostructured biocomposites based on bacterial cellulosic nanofibers compartmentalized by a soft hydroxyethylcellulose matrix coating
Open this publication in new window or tab >>Nanostructured biocomposites based on bacterial cellulosic nanofibers compartmentalized by a soft hydroxyethylcellulose matrix coating
Show others...
2009 (English)In: Soft Matter, ISSN 1744-683X, E-ISSN 1744-6848, Vol. 5, no 21, 4124-4130 p.Article in journal (Refereed) Published
Abstract [en]

Biomimetic approaches involving environmentally-friendly synthetic pathways provide an opportunity to elaborate novel high-performance biocomposites. Here we have developed a low-energy biosynthetic system for the production of a high-strength composite material consisting of self-assembled and nanostructured cellulosic nanofibers. This biocomposite is analogous to natural composite materials with high strength and hierarchical organization such as wood or tendon. It was generated by growing the bacterium Acetobacter, which naturally produces cellulosic nanofibers, in the presence of hydroxyethylcellulose (HEC). Individual cellulose fibrils were coated by HEC and exhibited a smaller lateral dimension than pure bacterial cellulose (BC) fibrils. They self-assembled to form compartmentalized nanofibers and larger cellulose fibril aggregates compared to pure BC. The tensile strength of nanocomposite films prepared from the compartmentalized cellulosic nanofibers was 20% higher than that of pure BC sheets and wood cellulose nanopapers, and 60% higher than that of conventional BC/HEC blends, while no strain-to-failure decrease was observed. The thin nanoscale coating consisting of hydrated HEC significantly increased the mechanical performance of the nanocomposite films by provoking compartmentalization of individual fibrils.

Keyword
cp/mas c-13 nmr, different polymeric additives, cell-wall, polysaccharides, in-situ crystallization, acetobacter-xylinum, native, cellulose, mechanical-properties, composites, microfibrils, spectroscopy
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-18869 (URN)10.1039/b907838j (DOI)000270837900008 ()2-s2.0-70350117835 (Scopus ID)
Note

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
2. The surface structure of well-ordered native cellulose fibrils in contact with water
Open this publication in new window or tab >>The surface structure of well-ordered native cellulose fibrils in contact with water
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2010 (English)In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 345, no 1, 97-100 p.Article in journal (Refereed) Published
Abstract [en]

CP/MAS C-13 NMR spectroscopy was used in combination with spectral fitting to examine the surface structure of hydrated cellulose I fibrils from Halocynthia and Gluconoacetobacter xylinus. To increase the spectral intensities and minimize signal overlap, G. xylinus celluloses site-specifically enriched in C-13 either on C4 or on both C1 and C6 were examined. The experimental data showed multiple C4 and C6 signals for the water accessible fibril surfaces in the highly crystalline celluloses. These signal multiplicities were attributed to structural features in the surface layers induced by the fibril interior, and could not be extracted by spectral fitting in celluloses with a lower degree of crystallinity such as cellulose from cotton.

Keyword
Cellulose, CP/MAS C-13 NMR, Spectral fitting, Halocynthia, Gluconoacetobacter xylinus, cp/mas c-13-nmr spectroscopy, conformations, assignment
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-19131 (URN)10.1016/j.carres.2009.10.020 (DOI)000273865100014 ()2-s2.0-71649106584 (Scopus ID)
Note

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
3. Identification and Preliminary Characterization of a New Chemical Affecting Glucosyltransferase Activities Involved in Plant Cell Wall Biosynthesis
Open this publication in new window or tab >>Identification and Preliminary Characterization of a New Chemical Affecting Glucosyltransferase Activities Involved in Plant Cell Wall Biosynthesis
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2008 (English)In: MOLECULAR PLANT, ISSN 1674-2052, Vol. 1, no 6, 977-989 p.Article in journal (Refereed) Published
Abstract [en]

Chemical genetics as a part of chemical genomics is a powerful and fast developing approach to dissect biological processes that may be difficult to characterize using conventional genetics because of gene redundancy or lethality and, in the case of polysaccharide biosynthesis, plant flexibility. Polysaccharide synthetic enzymes are located in two main compartments-the Golgi apparatus and plasma membrane-and can be studied in vitro using membrane fractions. Here, we first developed a high-throughput assay that allowed the screening of a library of chemicals with a potential effect on glycosyltransferase activities. Out of the 4800 chemicals screened for their effect on Golgi glucosyltransferases, 66 compounds from the primary screen had an effect on carbohydrate biosynthesis. Ten of these compounds were confirmed to inhibit glucose incorporation after a second screen. One compound exhibiting a strong inhibition effect (ID 6240780 named chemical A) was selected and further studied. it reversibly inhibits the transfer of glucose from UDPglucose by Golgi membranes, but activates the plasma membrane-bound callose synthase. The inhibition effect is dependent on the chemical structure of the compound, which does not affect endomembrane morphology of the plant cells, but causes changes in cell wall composition. Chemical A represents a novel drug with a great potential for the study of the mechanisms of Golgi and plasma membrane-bound glucosyltransferases.

Keyword
cell walls, callose synthase, chemical screening and identification, glycosyltransferase, Golgi, plasma membrane, suspension-cultured-cells, arabidopsis-thaliana, golgi-apparatus, xyloglucan biosynthesis, fucosyl-transferase, molecular-cloning, cellulose, mutant, isoxaben, pea
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-18137 (URN)10.1093/mp/ssn055 (DOI)000262858000009 ()2-s2.0-70349593386 (Scopus ID)
Note

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2014-07-10Bibliographically approved
4. APP: An Automated Proteomics Pipeline for the analysis of mass spectrometry data based on multiple open access tools
Open this publication in new window or tab >>APP: An Automated Proteomics Pipeline for the analysis of mass spectrometry data based on multiple open access tools
2014 (English)In: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 15, no 1, 345Article in journal (Refereed) Published
Abstract [en]

Background: Mass spectrometry analyses of complex protein samples yield large amounts of data and specific expertise is needed for data analysis, in addition to a dedicated computer infrastructure. Furthermore, the identification of proteins and their specific properties require the use of multiple independent bioinformatics tools and several database search algorithms to process the same datasets. In order to facilitate and increase the speed of data analysis, there is a need for an integrated platform that would allow a comprehensive profiling of thousands of peptides and proteins in a single process through the simultaneous exploitation of multiple complementary algorithms. Results: We have established a new proteomics pipeline designated as APP that fulfills these objectives using a complete series of tools freely available from open sources. APP automates the processing of proteomics tasks such as peptide identification, validation and quantitation from LC-MS/MS data and allows easy integration of many separate proteomics tools. Distributed processing is at the core of APP, allowing the processing of very large datasets using any combination of Windows/Linux physical or virtual computing resources. Conclusions: APP provides distributed computing nodes that are simple to set up, greatly relieving the need for separate IT competence when handling large datasets. The modular nature of APP allows complex workflows to be managed and distributed, speeding up throughput and setup. Additionally, APP logs execution information on all executed tasks and generated results, simplifying information management and validation.

Keyword
Automation, Distributed processing, Proteomics, Validation
National Category
Bioinformatics and Systems Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-147931 (URN)10.1186/s12859-014-0441-8 (DOI)2-s2.0-84923858672 (Scopus ID)
Note

QC 20150529. Updated from manuscript to article in journal.

Available from: 2014-07-09 Created: 2014-07-09 Last updated: 2017-12-05Bibliographically approved
5. Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction, Molecular Transport, and Callose Biosynthesis
Open this publication in new window or tab >>Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction, Molecular Transport, and Callose Biosynthesis
2013 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 12, 3874-3885 p.Article in journal (Refereed) Published
Abstract [en]

The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species.

Keyword
Detergent-Resistant Membranes, Arabidopsis-Thaliana, Lipid Rafts, Statistical-Model, Phospholipase-D, Plant Biology, Proteins, Identification, Synthase, Tobacco
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-142525 (URN)10.1074/mcp.M113.029033 (DOI)000329993600034 ()2-s2.0-84890713894 (Scopus ID)
Funder
FormasSwedish Foundation for Strategic Research
Note

QC 20140306

Available from: 2014-03-06 Created: 2014-03-06 Last updated: 2017-12-05Bibliographically approved

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