Optimization of lipase production in Burkholderia glumae
Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie an der Internationalen Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum
angefertigt am Institut für Molekulare Enzymtechnologie
vorgelegt von Anke Beselin aus Frankfurt a. Main
Bochum August 2005
Optimierung der Lipaseproduktion in Burkholderia glumae
Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie an der Internationalen Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum
angefertigt am Institut für Molekulare Enzymtechnologie
vorgelegt von Anke Beselin aus Frankfurt a. Main
Bochum August 2005
Die vorliegende Arbeit wurde im Rahmen des Europäischen Graduiertenkollegs der Ruhr- Universität Bochum (EGC 795): Regulatory Circuits in Cellular Systems: Fundamentals and Biotechnological Applications angefertigt.
Referent: Prof. Dr. K.-E. Jäger Korreferent: Prof. Dr. W. J. Quax Tag der mündlichen Prüfung: 28.10.2005
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Danksagungen
Herrn Prof. Dr. K.-E. Jäger danke ich für die Überlassung des interessanten und aktuellen Themas, für das rege Interesse am Fortschritt meiner Arbeit, die konstruktiven Diskussionen und die mir gebotene Möglichkeit, die experimentelle Arbeit frei und selbständig zu gestalten.
I would like to thank Prof. Dr. W. J. Quax, Laboratory of Pharmaceutical Biology- Rijksuniversität Groningen (NL) for agreeing to co-supervise this project.
Herrn Dr. M. Breuer und Herrn Prof. Dr. B. Hauer, BASF AG (Ludwigshafen), danke ich für die sehr gute Kooperation, die finanzielle Unterstützung im Rahmen des Projektes und die ausgesprochen freundliche, zeitweilige Aufnahme in die Arbeitsgruppe der Abteilung „Forschung Feinchemikalien & Biokatalyse“ in Ludwigshafen.
Des Weiteren danke ich Herrn Prof. Dr. M. Rögner am PhD-Programm des Europäischen Graduiertenkollegs EGC 795 teilnehmen zu können, sowie allen Organisatoren (innen) für die Gestaltung zahlreicher interessanter Kurse und Tagungen. An dieser Stelle auch ein herzlicher Dank an alle Mitglieder des Kollegs für die interessanten Diskussionen und die nette Atmosphäre auf den Tagungen.
Herrn Dr. F. Rosenau danke ich für die freundschaftliche Unterstützung bei der Planung und Durchführung dieser Promotionsarbeit, für die Hilfe bei der Lösung großer und kleiner wissenschaftlicher Probleme und für die aufmerksame Durchsicht des Manuskriptes.
A special thanks goes to Dr. M. Tsoli (Sydney, Australia) for good advices during writing my thesis and the critical reading of the manuscript. Thanks for the funny times we had in Germany and for always being a very good friend.
Den Mitarbeitern des Instituts für Molekulare Enzymtechnologie danke ich für das angenehme und freundschaftliche Arbeitsklima während meiner gesamten Promotionszeit. Ein besonderer Dank an alle, die dazu beigetragen haben, den „kleinen und großen Rückschlägen“ im Labor mit Heiterkeit und Optimismus entgegenzutreten. Für die Unterstützung bei der Durchführung einiger Experimente danke ich Frau K. Range, Frau I. Frindi-Wosch und Frau V. Svensson. Außerdem, danke ich Herrn Dr. T. Drepper für die zahlreichen, wissenschaftlichen Diskussionen und hilfreichen Anregungen beim Zusammenschreiben der Arbeit. Herrn ´Dr.` C. Leggewie danke ich ebenfalls für die aufmerksame Durchsicht des Manuskriptes.
Last but not least, möchte ich mich ganz herzlich bei meiner Familie bedanken, für die stetige Unterstützung und den Rückhalt während meiner gesamten Studien- und Promotionszeit. Insbesondere geht ein ganz großer Dank an Ulrich, für seine liebevolle Unterstützung und für seinen unermüdlichen Optimismus, mit dem er mich in „schlechten Zeiten“ aufgemuntert und sich in „guten Zeiten“ mit mir gefreut hat.
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Veröffentlichungen im Rahmen der Promotion:
Boukema, B., Beselin A., Rosenau, F., Jaeger, K.-E. and Tommassen, J. (2005) Improvement of lipase production by addition of inert compounds to Burkholderia glumae cultures. In Vorbereitung
Beselin, A., Breuer, M., Hauer, B., Rosenau, F. and Jaeger, K.-E. (2005) Erhöhung der Expression und/oder Faltung und Sekretion der Lipase LipA in Burkholderia glumae durch Co-Expression einer putativen, cytoplasmatischen Protease. Patent-Nr. 56817, BASF AG (Ludwigshafen)
Tagungsbeiträge: Beselin, A., Rosenau, F. and Jaeger, K.-E. (2003) Identification of bottlenecks in lipase production of Burkholderia glumae. Pseudomonas 2003, Québec, Canada (Posterbeitrag)
Beselin, A., Rosenau, F. and Jaeger, K.-E. (2003) Overexpression of lipase in Burkholderia glumae. EGC-Symposium, Groningen, The Netherlands (Posterbeitrag)
Beselin, A., Rosenau, F., Breuer, M., Hauer, B. and Jaeger, K.-E. (2005) Construction of a Burkholderia glumae strain suitable for lipase overexpression. Pseudomonas 2005, Marseille, France (Posterbeitrag)
Beselin, A., Rosenau, F., Breuer M., Hauer B., Jaeger, K.-E. (2005) Optimization of lipase production in Burkholderia glumae. EGC-Symposium, Groningen, The Netherlands (Vortrag)
______Contents
Contents
Abbreviations and units List of Figures List of Tables
1. Introduction………………………………………………….…….. 1 1.1 Lipolytic enzymes: Lipases………………………………………………..……. 2 1.1.1 Structure and enzymatic reactions of lipases……………………………. 3 1.1.2 Biotechnological applications of lipases………………………………… 5 1.2 Regulation of gene expression, folding and secretion of lipases………………... 6 1.2.1 Regulation of lipase gene expression……………………………………. 6 1.2.2 Periplasmic folding of lipases…………………………………………… 8 1.2.3 Secretion of lipases……………………………………………………… 11 1.3 Burkholderia glumae - A lipase-producing plant pathogen…………………….. 13 1.4 Expression systems……………………………………………………………… 15 1.4.1 General aspects………………………………………………………….. 15 1.4.2 The T7 RNA polymerase-based expression system………...…………... 17 1.5 Aims of this study………………………………………………………………. 19
2. Materials……………………………………………………………. 21 2.1 Chemicals and enzymes…………………………………………………………. 21 2.2 Strains and Plasmids…………………………………………………………….. 21 2.3 Oligonucleotides……………………………………………………………….... 26 2.4 Culture media and plates………………………………………………….…….. 26 2.5 Buffers and solutions……………………………………………………………. 27 2.6 Molecular weight standards……………………………………………….…….. 28 2.7 Kits………………………………………………………………………………. 28 2.8 Laboratory instruments………………………………………………………….. 28
3. Methods…………………………………………………………….. 30 3.1 Bacterial strain and growth conditions………………………………………….. 30 3.1.1 Cultivation of E. coli strains…………………………………………….. 30 3.1.2 Cultivation of B. glumae strains…………………………………….…... 30 3.1.3 Table of applied antibiotics……………………………………………... 30 3.1.4 Storage of microorganisms………………………………………….…... 30 3.2 Isolation of nucleic acid…………………………………………………………. 30 3.2.1 Isolation of plasmid and cosmid DNA………………………………….. 30 3.2.2 Isolation of chromosomal DNA……………………………………….... 31 3.2.3 Isolation of RNA………………………………………………...…….... 31 3.3 Agarose gel electrophoresis…………………………………………………….. 31 3.4 In vitro recombination of DNA…………………………………………………. 31 3.5 Construction of a cosmid library……………………………………..………… 31 3.6 Polymerase chain reaction (PCR)………………………………………………. 31 3.7 Primer extension analysis………………………………………………………. 32 3.8 DNA sequencing………………………………………………………………... 32 3.9 Transformation of bacteria……………………………………………………… 33 3.9.1 Chemical transformation of E. coli……………………………………... 33 3.9.2 Electroporation of B. glumae……………………………………………. 33 3.9.3 Conjugational transfer of plasmids or cosmids into B. glumae…………. 33 3.10 Determination of protein concentration…………………………………………. 34 ______I Contents
3.11 TCA precipitation of proteins…………………………………………………… 34 3.12 Polyacrylamide gel electrophoresis…………………………..…………………. 34 3.12.1 SDS-polyacrylamide gel electrophoresis...... …. 34 3.12.2 Native polyacrylamide gel electrophoresis………………………...……. 34 3.13 Western blot……………………………………………………………………... 34 3.14 Protein chromatography…………………………………………………………. 34 3.15 Preparative gel filtration…………………………………………………………. 35 3.16 Enzyme activity assays…………………………………………………………... 35 3.17 Fluorescence measurements……………………………………………………... 35 3.18 Computational methods………………………………………………………….. 36
4. Results………………………………………………………………. 37 4.1 Development of a T7 RNA polymerase-based expression system in B. glumae……………………………………………………………… 37 4.1.1 Expression of plasmid-encoded T7 RNA polymerase in B. glumae…….. 37 4.1.2 Construction of a T7-expression strain of B. glumae……………………. 38 4.1.3 Construction of expression vectors for high-level production of lipase in B. glumae………………………………………….……………. 40 4.2 Identification of bottlenecks for an improved lipase production in B. glumae….. 42 4.2.1 Construction of two cosmid libraries of B. glumae PG1 and B. glumae LU8093………………………………………………………. 42 4.2.2 Screening of the cosmid library of B. glumae PG1 led to the identi- fication of 15 cosmids influencing lipase production in B. glumae.…….. 43 4.2.3 Homologous expression of subcloned genomic DNA of B. glumae PG1.………………………………………………………….. 44 4.2.4 Co-expression of a gene encoding a putative protease increases lipase production in B. glumae………………………………………….. 48 4.2.5 The putative protease does not affect foldase production in B. glumae…. 49 4.3 Characterization of the putative protease………………………………………... 50 4.3.1 Heterologous overexpression of the putative protease gene in E. coli BL21(DE3) and purification of the protein by affinity chromatography… 51 4.3.2 Determination of the molecular weight of the putative protease….……... 53 4.3.3 The putative protease displays amino peptidase activity………………… 53 4.4 Elimination of bottlenecks for an improved lipase production in B. glumae……. 55 4.4.1 Tn5 mutagenesis of B. glumae...... 55 4.4.2 Inactivation of a putative tripartite efflux system in B. glumae………….. 57 4.5 Analysis of regulation of lipase gene expression in B. glumae………………….. 59 4.5.1 Emulsifiers and detergents enhance extracellular lipase production in B. glumae……………………………………………………………… 60 4.5.2 Use of the green fluorescent protein for the analysis of lipase gene expression in B. glumae………………………………………………….. 65 4.5.3 Effect of hexadecane and detergents on lipase gene expression in B. glumae………………………………………………………………… 68 4.5.4 Determination of the transcription start of lipA in B. glumae……………. 73 4.5.5 Effect of a in the upstream region of the lipAB-operon in B. glumae LU8093 on lipase gene expression…………………………… 74
5. Discussion…………………………………………………………… 77 5.1 Development of a T7 RNA polymerase-based expression system in B. glumae………………………………………………………………………… 77
______II Contents
5.2 Identification and elimination of bottlenecks for an improved lipase production in B. glumae…………………………………………………...……. 80 5.3 Regulation of lipase gene expression in B. glumae……………………………… 89 5.4 Optimization of lipase production in B. glumae – A perspective for future developments…………………………………………………………………….. 100
6. Summary…………………………………………………………..... 101
7. Zusammenfassung………………………………………………….. 103
8. References…………………………………………………………… 105
9. Appendix……………………………………………………………. 119
______III Contents
Abbreviations and units
A Ampere A. dest Distilled water Amp Ampicillin APS Amino peroxide disulfide ATP Adenosine 5`-triphosphate bp Base pair BSA Bovine serum albumin
°C Degree of Celsius Cm Chloramphenicol
Da Dalton DMF Dimethyl formamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxyribonucleoside triphosphat
EDTA Ethylendiamine tetra acetic acid EP Eppendorf tube EtOH Ethanol
Fig Figure g Gram gfp Green fluorescence protein h Hour HD Hexadecane
IPTG Isopropyl-β-D-thiogalactoside
K Kilo kb Kilobases kDa Kilodalton Km Kanamycin l Liter LB Luria Broth
M Molarity (mol/l) m Milli µ Micro mcs Multiple cloning site min Minutes mRNA Messenger RNA n Nano NCBI National Center for Biotechnology Information
______IV Contents
Ω Ohm OD Optical density ON Over night orf Open reading frame ori Origin of replication p.A. Per Analyse PAGE Polyacrylamid gel electrophoresis PEG Polyethylen glycol PBS Phosphate buffered saline PCR Polymerase chain reaction PDB Protein database bank PG Pseudomonas glumae pI Isoelectric points of proteins rbs Ribosome binding site RFU Relative fluorescence units RNA Ribonucleic acid RNase Ribonuclease Rpm Rounds per minute RT Room temperature
SAP Shrimp alkaline phosphatase sec Seconds SDS Sodium dodecyl sulfate t Time Tab Table TBE Tris/Borat/EDTA Tc Tetracycline TCA Trichlor acetic acid TCF Transcriptional fusion TE Tris/EDTA TEMED N, N, N‘, N‘-Tetramethyl diamine TLF Translational fusion Tm Melting Temperature Tris Tris(hydroxyl methyl)amino methane
U Units UV Ultra violet light
V Volt v/v Volume per volume w/v Weight per volume
X-Gal 5-Chlor-4-brom-3-indolyl-β-D-galactoside
______V Contents
List of figures
Fig. 1 General folding pattern of α/β hydrolases……………………………….. 3 Fig. 2 General enzymatic reaction of a lipase…………………………………… 4 Fig. 3 Different types of synthesis reactions catalyzed by lipases……………… 4 Fig. 4 Regulation of lipase gene expression in P. aeruginosa………………….. 8 Fig. 5 Genetic organization of the lipAB-operon of B. glumae…………………. 14 Fig. 6 Strategy for regulating gene expression using the pET-expression system…………………………………………………... 18 Fig. 7 Western blot analysis of the expression of plasmid-encoded T7 RNA polymerase in B. glumae PG1and B. glumae LU8093….……... 38 Fig. 8 Schematic representation of the construction of an inducible T7-expression strain of B. glumae……………………………………….. 39 Fig. 9 Expression of B. glumae lipase in E. coli BL21(DE3)…………………... 42 Fig. 10 Screening of the cosmid library of B. glumae PG1 using lipase indicator plates…………………………………………………………… 44 Fig. 11 Effect of two cosmids on lipase activity of B. glumae LU8093 and B. glumae PG1………………………………………………………. 45 Fig. 12 Restriction analysis of the plasmids pBBRPG 5/1, 5/3, 5/7, 8/1 and 8/3 harboring subcloned genomic DNA of B. glumae PG1…………. 45 Fig. 13 Schematic representation of the identified open reading frames of the plasmids pBBRPG 5/7 and 8/1 and 8/3…………………………… 47 Fig. 14 Lipolytic activity of B. glumae PG1and B. glumae LU8093 expressing the plasmid-encoded putative protease………………………. 49 Fig. 15 Western blot analysis of the supernatants and cell extracts of B. glumae PG1 and B. glumae LU8093 expressing the plasmid- encoded putative protease ……………………………………………… 50 Fig. 16 Overexpression of the pro gene in E. coli BL21(DE3)…………………. 51 Fig. 17 SDS PAGE analysis of purification of putative protease by affinity chromatography…………………………………………………. 52 Fig. 18 Native polyacrylamide gel electrophoresis of the purified putative protease…………………………………………………………. 53 Fig. 19 Determination of enzyme activity of the putative protease using a microtiter plate assay…………………………………………………... 54 Fig. 20 Transposon mutagenesis of B. glumae…………………………………... 56 Fig. 21 Schematic representation of the inactivation of the chromosomal ompC and mfpB genes in B. glumae PG1 and B. glumae LU8093……… 58 Fig. 22 Lipolytic activity and growth of B. glumae PG1 and B. glumae LU8093 under different physiological conditions……………………….. 61 Fig. 23 Effect of carbon sources and chemical agents on lipase and foldase production in B. glumae PG1……………………………………………. 63 Fig. 24 Effect of carbon sources and chemical agents on lipase and foldase production in B. glumae LU8093………………………………………... 64 Fig. 25 Expression of Gfpmut3 and GfpLAA in B. glumae PG1 and B. glumae LU8093……………………………………………………….. 67 Fig. 26 Determination of the stability of Gfpmut3 and GfpLAA in B. glumae PG1 and B. glumae LU8093………………………………….. 68 Fig. 27 Schematic representation of transcriptional and translational reporter gene fusions……………………………………………………... 69
______VI Contents
Fig. 28 Schematic representation of the constructed transcriptional and translational lipA::gfp(LAA)-fusions B. glumae PG1 and B. glumae LU8093……………………………………………………….. 70 Fig. 29 Fluorescence measurements of transcriptional lipA::gfp-fusion of B. glumae PG1 and B. glumae LU8093………………………………. 71 Fig. 30 Fluorescence measurements of translational lipA::gfp(LAA)- fusion of B. glumae PG1 and B. glumae LU8093………………………. 72 Fig. 31 Localization of the transcription start and σ54-dependent promoter sequences of lipA in B. glumae………………………………... 74 Fig. 32 Sequence alignment of a part of the upstream sequence of the lipAB-operon of B. glumae PG1 and B. glumae LU8093………………... 75 Fig. 33 Fluorescence measurements of translational lipA::gfp-fusions of B. glumae PG1 and B. glumae LU8093………………………………….. 76 Fig. 34 Schematic representation of the construction of T7-expression strain of B. glumae based on physiological induction…………………… 80 Fig. 35 Multiple sequence alignments of an open reading frame encoding a putative protease of B. glumae with proteins of the database………….. 87 Fig. 36 Hypothetical model of the regulation of lipase gene expression in B. glumae PG1 and B. glumae LU8093………………………………….. 98 Fig. 37 Schematic representation of the construction of the suicide vector pSUPT7pollacIpolup/down……………………………………………… 119 Fig. 38 Schematic representation of the construction of the expression vectors pBBR22lipABT7, pBBRlipABT7 and pBBRlipABlac…………... 120 Fig. 39 Schematic drawing of the construction of the plasmid fusions lipA::gfp….………………………………………………………………. 121 Fig. 40 DNA and amino acid sequence of the putative protease (Pro)…………... 122 Fig. 41 DNA and amino acid sequence of the putative efflux pump (EpA)……... 123 Fig. 42 DNA and amino acid sequence of the putative membrane fusion protein (MfpB)……………………………………….…………………... 124 Fig. 43 DNA and amino acid sequence of the putative outer membrane protein (OmpC)…………………………………………………………... 125
List of tables
Tab. 1 Characteristics of different expression systems………………………….. 16 Tab. 2 Overview of some existing T7 RNA polymerase-based expression systems in Gram-negative and Gram-positive bacteria……… 19 Tab. 3 Bacterial strains and plasmids used or constructed in this thesis………... 21 Tab. 4 Applied oligonucleotides for PCR amplification………………………... 26 Tab. 5 Concentrations and solving reagents of applied antibiotics……………... 30 Tab. 6 Composition of a PCR reaction mix……………………………………... 32 Tab.7 PCR program…………………………………………………………….. 32 Tab. 8 Sizes of subcloned genomic DNA of B. glumae PG1…………………… 45 Tab. 9 Lipolytic activity of B. glumae PG1 and B. glumae LU8093 harboring expression plasmids with different sizes of subcloned genomic DNA of B. glumae PG1………………………………………… 46 Tab. 10 Growth and lipolytic activity of vector-insertion mutants of B. glumae PG1 and B. glumae LU8093………………………………….. 59
______VII 1. Introduction
1. Introduction
Enzymes catalyze the conversion of a vast number of different molecules by accelerating the velocity of a reaction to its equilibrium position than would occur otherwise. Therefore, enzymes play an important role in many biological processes. Due to the fact that enzymes are environmentally benign catalysts, working under mild conditions and generating few waste products, their use in a large number of industrial processes is ever increasing. Especially, enzymes of microbial origin are attractive alternatives to expensive and mostly ecologically harmful chemical methods in food and diary, detergent, textile, cosmetic and pharmaceutical industries (Saxena et al., 1999). However, in nature enzymes have evolved to contribute to the survival and reproduction of organisms. Thus, they do not provide any transformation desired in industrial processes and are often not produced in large amounts. By the advent of recombinant DNA technology, which enables to specifically manipulate DNA sequences, it became possible to redesign existing biocatalysts at the molecular level and to produce them in large quantities (Arnold, 2001). Together with recent progress in high-throughput screening techniques and protein engineering methods, it is now possible to produce stable biocatalysts displaying almost any customized activity and selectivity in high amounts (Zaks, 2001). For instance, the so called “directed evolution” approach allows to create novel biocatalysts without requiring any knowledge of the enzyme structure or its catalytic mechanism (Arnold and Volkov, 1999; Jaeger et al., 2001; Tao and Cornish, 2002; Cherry and Fidantsef, 2003). Additionally, new experimental approaches of screening metagenomic DNA enable to identify novel biocatalysts with high biotechnological potential, which are produced by yet uncultivable microorganisms (Henne et al.,1999; Rondon et al., 2000; Voget et al., 2003; Streit et al., 2004). The application of nature’s toolset, i.e. living cells and/or their enzymes, for the production of biochemicals, biomaterials and biofuels from renewable resources, also known as “white biotechnology” (EuropaBio, 2003), has gained high priority. Biotechnological products are used in a variety of different industrial sectors with the pharmaceutical sector dominating. In 2000, the world-wide use of enzymes in biotechnology amounted U.S.$ 1.5 billion, with hydrolytic enzymes such as lipases, proteases, amylases, amidases and esterases occupying the major part (Kirk et al., 2002; Straathof et al., 2002). By 2010, the potential economic value of white biotechnology for the chemical industry alone is estimated to be € 11-22 billion per annum (McKinsey, 2003; Festel et al., 2004).
______1 1. Introduction
1.1 Lipolytic enzymes: Lipases Lipolytic enzymes catalyzing the conversion of lipids comprise lipases (triacylglycerol hydrolases) [EC 3.1.1.3], esterases [EC 3.1.1.1], phospholipases [EC 3.1.4.3] and lysophospholipases (different registrations [EC 3.1.x.x]) (IUBMB, 1992; Bairoch, 1999). Since the first description of lipases, the definition of these enzymes has been changed several times. Based on the fact that lipolytic reactions occur at the lipid-water interface where lipolytic substrates usually form an equilibrium between monomeric, micellar and emulsified states, one criterion used to classify a “true” lipase was that the enzyme should be activated by the presence of an interface. This phenomenon was termed “interfacial activation” (Sarda et al., 1958). After solving the first three-dimensional structures of two lipases, a human pancreatic lipase (Winkler et al., 1990) and a lipase of Rhizomucor miehei (Brady et al., 1990), the hypothesis of interfacial activation of all lipases seemed to be proved. Additionally, since both lipases contain a so called “lid”, a surface loop covering the active site of the enzyme which moves away upon contact with an interface, a second criterion for the classification of lipases was established. However, in the last decade, these two criteria proved to be unsuitable for classification, due to the fact that a number of exceptions were described. For example, lipases containing a loop domain but lacking the “interfacial activation” have been identified in Burkholderia glumae (Nobel, 1993), Pseudomonas aeruginosa (Jaeger et al., 1993) and Candida antarctica B (Uppenberg et al., 1994). Furthermore, enzymes neither displaying “interfacial activation” nor having a “lid” structure have also been characterized such as the two lipolytic enzymes of Bacillus subtilis (Lesuisse et al., 1993; Eggert et al., 2001) and a cutinase of Fusarium solani (Martinez et al., 1992). Today a “true” lipase is defined as a carboxyl esterase catalyzing the hydrolysis and synthesis of long-chain acylglycerols (C>9) in contrast to esterases, which hydrolyze short chain triacylglycerols. Lipases can also hydrolyze short chain triacylglycerols, but display maximum activity towards substrates forming emulsions (Verger, 1997; Chahinian et al., 2002). In nature, lipases can be found in many organisms as well as in plants. While in human and other animals, lipases are involved in the digestion, adsorption and modification of fats (Desnuelle, 1986; Carriere et al., 1994), in plants, they play an important role in jasmonic acid dependent defense signaling and in pathogen induced accumulation of salicylic acid (Mukherjee, 1994; Jakab et al., 2003). In microorganisms, the microbiologist C. Eijkmann first described lipases in 1901. Today lipases are known in bacteria, fungi and yeast, for
______2 1. Introduction example Chromobacteria, Pseudomonas, Staphylococci, Candida, Aspergillus, Mucor and Rhizopus. Based on a comparison of the amino acid sequences and some fundamental biological properties, bacterial lipolytic enzymes have been classified into eight families with family I being further divided into six subfamilies (Arpigny and Jaeger, 1999). Recently, this classification has been extended mainly affecting changes in family I, which comprises now 30 lipases subgrouped into seven subfamilies. Lipases of the genera Burkholderia and Pseudomonas belong to the subfamilies I.1, I.2 and I.3 (Jaeger and Eggert, 2002).
1.1.1 Structure and enzymatic reactions of lipases Although bacterial lipases vary in size (20-60 kDa), they all obey a so-called α/β hydrolase fold, which is a general folding pattern of different hydrolases (Ollis et al., 1992; Nardini and Dijkstra, 1999) such as acetylcholine esterase (Sussmann et al., 1991), serine carboxypeptidase (Liao and Remington, 1990), haloalkane dehalogenase (Franken et al., 1991) and dienelactone hydrolase (Pathak et al., 1988). In general, this α/β hydrolase fold consists of a central, mostly parallel β-sheet of eight strands with the second strand being antiparallel. The parallel strands β3 to β8 are connected by α-helices, which are located on either site of the central β-sheet (Fig.1). The curvature of the β-sheet as well as the positions of the α-helices vary considerably among the different lipases (Schrag and Cygler, 1997).
Fig. 1: General folding pattern of α/β hydrolases (reproduced from van Pouderoyen et al., 2001). α-Helices are indicated by bars and β-sheets by arrows. The topological position of the active site residues is shown by a solid circle.
The active site of lipases is located in the central β-sheet and consists of three catalytic amino acid residues: a nucleophilic serine, a histidine and a catalytic acid residue that is either an aspartate or glutamate. The nucleophilic serine residue is located in a highly conserved Gly-X-Ser-X-Gly pentapeptide. This consensus sequence is part of the β-ε-Ser-α motif, which can be found in all serine hydrolases playing an important role for the catalytic activity by providing the steric requirements (Ollis et al., 1992). The catalytic mechanism of lipases follows the pattern of serine hydrolases. The activated primary hydroxyl group of the activate site serine acts as a nucleophile in the formation of a
______3 1. Introduction tetrahedral intermediate. This intermediate disintegrates by elimination of the alcohol leaving behind the acyl-enzyme complex. The acyl-enzyme is the central intermediate in the different lipase catalyzed reactions. In aqueous media, lipases catalyze the hydrolysis of triacylglycerols into diglycerides, monogylcerides, glycerol and fatty acids (Fig. 2). In non- aqueous environment, lipases catalyze ester synthesis (Jaeger et al., 1994).
O
O O O Lipase O OH O + O + H2O O O HO
O O
Triacylglycerol Fatty acid Diacylglycerol
Fig. 2: General enzymatic reaction of a lipase. Under natural conditions lipases catalyze the hydrolysis of triacylglycerol into a diglyceride and fatty acid. In non-aqueous environment, lipases can also catalyze ester synthesis (reproduced from Jaeger et al., 1994).
Within ester synthesis reactions, there are different types: ester synthesis from glycerol and fatty acids, transesterification involving the transfer of an acyl group to an alcohol (alcoholysis) or glycerol (glycerolysis) and interesterification in which an acyl group is transferred to a fatty acid (acidolysis) or a fatty acid ester (Fig. 3). The last two described reactions are most important for industrial applications (Jaeger et al., 1994).
Transesterification alcoholysis O
R O C R + R2 OH O R3 OH 3 1
+ O R1 C O R2 glycerolysis
OH O C R1 + R2 OH OH OH OH OH
acidolysis Interesterification O O
R C OH R C OH O 3 1
R C O R 1 2 + O O
R3 C O R4 R3 C O R2
Fig. 3: Different types of synthesis reactions catalyzed by lipases. Transesterification involves the transfer of an acyl group to an alcohol (alcoholysis) or glycerol (glycerolysis) whereas in interesterification an acyl group is transferred to a fatty acid (acidolysis) or a fatty acid ester (reproduced from Jaeger et al., 1994).
______4 1. Introduction
1.1.2 Biotechnological applications of lipases Due to remaining enzymatically active in organic solvents, lipases have become the most widely used group of biocatalysts in organic chemistry (Jaeger and Eggert, 2002). Further characteristics of theses enzymes are that they display exquisite chemoselectivity, regioselectivity and stereoselectivity, do not require a cofactor and can be produced in high yields from microorganisms (Gupta et al., 2004). Consequently, they have gained great importance for many industrial sectors such as agrochemical (insecticide, pesticide), pharmaceutical (e.g. naproxen, ibuprofen), detergent (e.g. washing powders, industrial cleaners) and food and dairy industry (e.g. cheese ripening, flavor development) (Jaeger and Reetz, 1998; Saxena et al., 1999). Lipases in detergent industry In general, lipases are often used in household detergents and industrial cleaners together with proteases and cellulases (Pandey et al., 1999). In order to improve detergent performance, intensive screening programs followed by genetic manipulations have been performed which resulted in the development of improved enzymes such as Novo Nordisk’s Lipolase (Hoq et al., 1985). Lipases in food industry A variety of processes in food manufacture such as vegetable fermentation, dairy enrichment or the production of fruit juices and baked foods, have been improved by the use of enzymes. Main applications of lipases include lipolysis of butter fat and cream, flavor enhancement of cheese, acceleration of cheese ripening as well as production of cheese-like products (Falch, 1991). In these processes, the ability of lipases to catalyze esterification reactions is of great importance such as the production of modified acylglycerols by interesterification of fats and oils that cannot be obtained by conventional chemical reactions (Gupta et al., 2004). Lipases in polymer synthesis The stereoselectivity of lipases is useful for the synthesis of biopolymers like polyphenols, polysaccharides and polyesters. Based on the fact, that these biopolymers are biodegradable and produced from renewable natural resources, they receive increased attention in polymer synthesis (Jaeger and Eggert, 2002). Lipases in pharmaceutical and agrochemical industry Today it is well known that mostly one of two drug enantiomers displays the therapeutic activity, while the other enantiomer might cause unwanted site effects or might even be toxic. Therefore, the demand for enantiopure substances has become extremely important (Lin and Lu, 1997). Since 1992, the US Food and Drug Administration (FDA) enforces the
______5 1. Introduction pharmaceutical industry to develop enantiopure products. However, these compounds are often difficult to synthesize chemically which resulted in the application of biocatalysts that specifically catalyze enantioselective reactions (Breuer et al., 2004). Examples for the application of lipases are the kinetic resolution of racemic mixtures of secondary alcohols in hydrolysis, esterification and transesterification (Jaeger et al., 1996; Schulz et al., 2000; Turner, 2004). Also racemic amines can be resolved leading to R-amide and S-amine, which can be recovered and separated by distillation. Amines are of considerable interest as chiral building blocks or as auxiliaries for the synthesis of bioactive ingredients (Schmid et al., 2001). Lipases from Pseudomonas sp. are used for the synthesis of chiral intermediates in the production of the antimicrobial compound chaungxinmycin and the potent antitumor agent epothilone (Yoshida et al., 2001; Zhu and Panek, 2000). In agrochemical industry, lipases are applied for example, in the production of enantiopure (S)-indanofan, an herbicide applied against grass weeds in paddy fields (Gupta et al., 2004).
1.2 Regulation of gene expression, folding and secretion of lipases The regulation of lipase gene expression, folding into an active enzyme and protein secretion has been studied most intensively in Pseudomonas aeruginosa. Therefore, the next sections will mainly focus on the description of gene regulation, protein folding and mechanisms of secretion of lipases of the genera Pseudomonas and Burkholderia.
1.2.1 Regulation of lipase gene expression Under natural conditions, lipase gene expression in bacteria takes place in the late logarithmic phase of growth. Since lipases hydrolyze ester bonds of triacylglycerols, the expression of lipases is often induced or improved in the presence of fats or oil in the growth media. Additionally, chemical agents like polysaccharides or detergents can increase the production level of lipases. For example, in Serratia marcescens and P. aeruginosa ATCC 9027, it was shown that glycogen, hyaluronate and other polysaccharides enhanced the formation of extracellular lipase (Winkler and Stuckmann, 1979; Schulte et al., 1982). In Pseudomonas sp. strain 109, gene expression of lipase LipL could be increased 44-45-fold by supplementing the growth medium with either soybean oil or a non-ionic detergent (Noigen HC) and the presence of both resulted in a further 56-fold increase (Tanaka et al., 1999). In the Gram- negative soil bacterium Acinetobacter calcoacticus BD413, high levels of extracellular lipase could be detected by supplementing the growth medium with long-chain alkanes such as
______6 1. Introduction hexadecane. In contrast, fatty acids produced by the hydrolysis of the lipase substrate triolein strongly repressed lipase gene expression in this strain (Kok et al., 1996). Interestingly, although many studies have been performed that deal with chemical agents affecting lipase production, the exact molecular mechanisms often remain unknown. While polysaccharides are assumed to dislocate cell-bound lipase (Winkler and Stuckmann, 1979), in A. calcoaceticus, it has been proposed that a yet unidentified regulatory protein mediates repression of lipBA-operon transcription upon binding of a fatty acid (Kok et al., 1996). A well-studied mechanism of controlling lipase gene expression is called “quorum sensing”. This term describes the regulation of gene expression in response to cell density by producing and releasing a signal molecule, a so-called autoinducer that accumulates during cell growth in the extracellular medium. After reaching a threshold concentration, this autoinducer is bound by a cognate regulatory protein in the cell. This complex then in turn binds at specific promoter elements and activates or represses expression of downstream-located genes (Salmond et al., 1995; Fuqua et al., 1996; Swift et al., 1996). In P. aeruginosa, there are two quorum sensing systems, the LasI/R and the RhlI/R system (Ochsner et al., 1994; Brint and Ohman, 1995; Latifi et al., 1995; Chapon-Herve et al., 1997; Pesci et al., 1997; McKnight et al., 2000). In both systems, the extracellular signal molecules produced are acylated homoserine lactones. After binding to its cognate regulator LasR or RhlR, expression of a variety of genes is induced, which code for extracellular enzymes and virulence factors such as lipase, alkaline protease, elastase, exotoxinA and exoenzymeS. Additionally, bacterial motility, biofilm formation and rhamnolipid production are controlled as well (Miller and Bassler, 2001; Juhas et al., 2005). Since the quorum sensing circuits form a highly complex network, interacting at the transcriptional and posttranscriptional level as well as being subjected to regulation by global regulators (Juhas et al., 2005), the system will not be discussed in further detail. Concerning regulation of lipase gene expression, it is assumed, that the RhlI/R system is involved indirectly (Rosenau and Jaeger, 2000). After identification of a σ54-dependent promoter sequence in the upstream region of the lipAH- operon, encoding the lipase LipA and its specific foldase Lif, screening of a Tn5 transposon library of P. aeruginosa led to the identification of a two-component system LipQ/R (Duefel, 2000). Two component systems, which consist of a sensor protein kinase and a response regulator, display another important regulatory system in the adaptation of bacteria to cell density and environmental signals (Hoch and Silhavy, 1995; Dunny and Winans, 1999). Environmental signals can trigger autophosphorylation of the sensor kinase at a histidine residue. This phosphoryl group is then transferred to an aspartate residue at the response
______7 1. Introduction regulator, which in turn activates the expression of target genes (Stock et al., 1989; Parkinson and Kofoid, 1992). In the lipQ/R system of P. aeruginosa, the sensor kinase LipQ seems to be activated by either the transcriptional regulator RhlR, by so far unknown environmental stimuli or by periplasmic signals such as misfolded enzymes. The activated transcriptional regulator LipR then binds to a specific upstream activating sequence (UAS) preceding the σ54-dependent promoter of the lipAH-operon and induces lipase and foldase gene expression (Duefel, 2000). In Figure 4 general aspects of the regulation of lipase gene expression in P. aeruginosa are summarized.
LasI/R Global Signal regulator - Environment RhlR RhlI/R + - + -
+
lipQ lipR PE
LipQ P P LipR
+ + σ54 lipA lipH Signal UAS Plip -Environment -Secretion/Folding
Fig. 4: Regulation of lipase gene expression in P. aeruginosa. Global or additional regulators can activate or inhibit the quorum sensing circuits, LasI/R and RhlI/R. RhlR may in turn induce transcription of the lipQ/R two- component system (PE: promoter element). The sensor kinase may also be activated by so far unknown environmental stimuli or by periplasmic signals such as misfolded proteins. Finally, the activated transcriptional regulator LipR binds to an upstream activating sequence (UAS) preceding the σ54-dependent promoter and induces transcription of the lipAH-operon. Activation of transcription is indicated by ⊕, inhibition by \ (modified from Rosenau and Jaeger, 2000).
1.2.2 Periplasmic folding of lipases Besides regulation of gene expression, subsequent steps of folding and secretion of the protein also play an important role in the production of enzymatically active lipase and involve a variety of cellular and periplasmic proteins. Since most lipases of the genera Pseudomonas and Burkholderia are secreted into the extracellular medium by the type II secretion pathway, transport across the inner membrane by the Sec machinery only occurs for the unfolded protein whereas transport across the outer membrane requires the protein to be folded in an enzymatically active confirmation. Consequently, folding of the periplasmic intermediates is essential for further secretion (Frenken et al., 1993b; Bortoli-German et al., 1994).
______8 1. Introduction
In 1973, Anfinsen studied protein folding in vitro for the first time, suggesting that the amino acid sequence of a protein contains all information to reach its three-dimensional structure. However, today it is well known that two classes of folding modulators, which have mainly been studied in bacteria, provide correct folding of proteins in vivo. The first class is designated as molecular chaperons including proteins such as DnaK-DnaJ- GrpE or GroEL-GroES. These proteins bind their folding intermediate, suppress off-pathway aggregations and provide correct folding of the protein by controlled binding and release. The required energy for this process is gained by hydrolysis of ATP (Thomas and Ayling, 1997; Hartl and Hayer-Hartl, 2002; Schlieker and Bukau, 2002; Walter and Buchner, 2002). The second class includes folding catalysts and foldases whose function it is to accelerate slowly steps in the folding pathway of proteins. One slow process is for example the cis-trans isomerization of peptidyl-prolyl bonds, which is catalyzed by peptidyl-prolyl cis-trans isomerases (PPIases) (Schiene and Fischer, 2000). PPIases are found in the cytoplasm as well as in the periplasm (Hayano et al., 1991). On the contrary, disulfide bond forming proteins (Dsb proteins) only occur in the periplasm, where they catalyze the formation or isomerization of disulfide bonds of proteins. Dsb proteins are widely distributed among Gram-negative bacteria and have well been studied in Escherichia coli as well as P. aeruginosa (Andersen et al., 1997; Liebeton et al., 2001; Urban et al., 2001; Collet and Bardwell, 2002). In P. aeruginosa, deletion of the gene dsbA encoding a thiol:disulfide oxidoreductase led to a dramatic decrease of lipase activity in the supernatant (Urban et al., 2001). Further studies on lipase variants in which either one or both cysteine residues were replaced, showed that the disulfide bond has a stabilizing function in the protein and is not essential for the activity of the enzyme. Thus, the reduced lipase activity in the supernatant of the dsbA-deletion mutant indicated a rapid proteolysis of destabilized lipase in the periplasm (Liebeton et al., 2001). Almost 15 years ago, Jorgensen et al. identified a gene in Burkholderia cepacia, which was organized in one operon together with lipA encoding the extracellular lipase of the Gram- negative bacterium. Furthermore, they found out that the protein of this second gene, designated lipB, was essential for the production of active lipase. Today many more LipB-like proteins have been identified in other Gram-negative bacteria. They all provide correct folding of their cognate lipase and are usually encoded in one operon together with the lipase gene (Iizumi et al., 1991; Jorgensen et al., 1991; Frenken et al., 1993; Kok et al., 1995; Ogierman et al., 1997; Sullivan et al., 1999; Kim et al., 2001). Consequently, these proteins were named Lif for Lipase specific foldase (Jaeger et al., 1994).
______9 1. Introduction
A common feature of all Lif-proteins is that they are anchored in the inner membrane by a N- terminal hydrophobic domain whereas the larger C-terminal domain is exposed to the periplasm (Frenken et al., 1993). Since N-terminal truncated or modified Lif proteins were still able to catalyze folding of their cognate lipase into an enzymatically active conformation, the membrane anchor seems not to be required for the activity of the foldase. Instead it is suggested that the function of the membrane anchor is to prevent secretion of the foldase into the extracellular medium (Shibata et al., 1998; Quyen et al., 1999; El-Khattabi et al., 1999, 2000). By performing a trypsin digestion of a B. glumae lif-lipase complex in vitro, a 26 kDa fragment of the C-terminal domain of the foldase could be identified to interact with its lipase, thus being protected from trypsin digestion (El-Khattabi et al., 1999). Meanwhile, further experiments have been performed in vitro demonstrating that the C-terminal domain of Lifs and their cognate lipases form a stable complex, which can be co-purified or co- immunoprecipitated (Hobson et al., 1995; Shibata et al., 1998; El-Khattabi et al., 2000). One aspect, which still remains to be solved, deals with the ratio of Lif and its cognate lipase since in vitro and in vivo studies led to contrary results. While in vitro experiments and heterologous expression studies in E. coli revealed a 1:1 ratio of lipase and foldase, in vivo studies in B. glumae, P. aeruginosa and P. alcaligenes showed that the Lif protein was produced in significant lower amounts than the lipase suggesting Lifs are multi-turnover catalysts (Gerritse et al., 1998; El-Khattabi et al., 1999). Interestingly, in B. glumae and P. aeruginosa, the amount of available Lif protein in the cell has an effect on lipase production since overexpression of the lif gene in trans resulted in an significant increase of produced lipase in the extracellular medium (El-Khattabi, 2001; Rosenau, 2001). Finally, apart from folding modulators, another interesting group of proteins, the periplasmic proteases, also play an important role in the production of extracellular enzymes such as lipases. In general, proteases are widely distributed in nature, where they execute a large variety of physiological functions. They are degradative enzymes catalyzing the cleavage of peptide bonds in other proteins. While extracellular proteases hydrolyze large proteins to smaller molecules for subsequent absorption, intracellular proteases play an important role in the regulation of metabolism. Their proteolytic activity comprises for example the activation of zymogene forms of enzymes by limited proteolysis, processing and transport of secretory proteins across the membranes, or the regulation of gene expression by degradation of regulatory proteins or modification of ribosomal proteins (Rao et al., 1998). While cytoplasmic proteases are mostly ATP-dependent, periplasmic proteases do not require ATP for their activity since ATP is not available in the periplasm (Maurizi, 1992). Most
______10 1. Introduction periplasmic and membrane associated proteases described today have been identified in E. coli (Gottesman, 1996). A well-studied example is the periplasmic protease DegP. This serine protease is involved in the digestion of unfolded proteins and is essential for growth at high temperatures (Kolmar et al., 1996; Miyadai et al., 2004). Furthermore, Prc (Hara et al., 1991), Protease III (Finch et al., 1986; Betton et al., 1998) and SohB (Baird et al., 1991) have been described to be involved in proteolytic degradation of proteins in E. coli. In 1996, Boucher et al. described the two proteases MucD and AlgW in P. aeruginosa as being homologous to DegP. Additionally, five more genes encoding proteases with a significant homology to the proteases Prc, Protease III and SohB of E. coli have also been identified in the genome of P. aeruginosa. After inactivation of the corresponding genes in the genome of P. aeruginosa, three mutants showed a higher lipase activity while one mutant displayed a lower lipase activity in the supernatant. Further experiments revealed that gene expression of the lipAH-operon was not affected, confirming that these proteases influence folding and/or secretion of lipase LipA in P. aeruginosa (Windgassen, 2000).
1.2.3 Secretion of lipases While Gram-positive bacteria possess only one membrane, the cell envelope of Gram- negative bacteria is composed of two membranes, a cytoplasmic membrane and a lipopolysaccharide-containing outer membrane. In order to secrete metabolic products, extracellular enzymes and in some cases toxins, Gram-negative bacteria have evolved various secretion mechanisms. Overall, there are three systems to translocate molecules across both membranes (type I, III and IV), four systems for the transport across the cytoplasmic membrane (Sec, Tat, MscL and Holins) and four secretion systems for the outer membrane [main terminal branch (MTB), fimbrial usher porin (FUP), autotransporter (AT) and two- partner secretion families (TPS)]. As there are various secretion pathways known today, there are as many excellent articles dealing with this topic (Tommassen et al., 1992; Pugsley, 1993; Binet et al., 1997; Filloux et al., 1990, 1998; Berks et al., 2000; Saier et al., 2000; Thanassi and Hultgren, 2000; Cao and Saier, 2001; Lee and Schneewind, 2001; Sandkvist, 2001; Voulhoux et al., 2001; Baron et al., 2002; Ochsner et al., 2002; Yen et al., 2002; Ma et al., 2003; Desveaux et al., 2004). Consequently, the following section will only focus on the type II secretion pathway by which lipases of various Pseudomonas and Burkholderia species are secreted.
______11 1. Introduction
Secretion across the inner membrane Transport of lipases across the cytoplasmic membrane mostly occurs via the Sec-pathway, which is best studied in E. coli. This pathway includes several ubiquitous proteins (SecY, SecE, SecG, YidC, FtsY and Ffh) as well as several additional proteins, which are not present in every organism (SecA, SecB, SecD, SecF, YajC) (Cao and Saier, 2003). All proteins that are translocated across the inner membrane by the Sec-machinery possess an N-terminal signal sequence (Fekkes and Driessen, 1999). These signal sequences display a common overall composition containing a positively charged N-terminus, a hydrophobic core and a recognition site for the leader peptidase (von Heijne, 1985). In the first step of secretion, the ATP-independent chaperon SecB binds the secretory protein in an unfolded state and targets it to SecA, an ATPase. Upon ATP hydrolysis, SecA drives the transport of the unfolded preprotein through the cytoplasmic membrane. The channel through which the preprotein is translocated, is built up by the three proteins SecYEG (Manting and Driessen, 2000). Before the transported protein is released into the periplasm, a signal peptidase, which is also located in the inner membrane, cuts of the N-terminal signal peptide. So far, two types of signal peptidases) have been identified in Gram-negative and Gram-positive bacteria, designated as SPases I and II (Ma et al., 2003). Secretion across the outer membrane After folding into an enzymatically active conformation in the periplasm, lipases of Gram- negative bacteria are then transported across the outer membrane by the secreton, a large complex consisting of up to 14 proteins. This complex is part of the type II secretion pathway, also named the main terminal branch (MTB) of the general secretion pathway (Pugsley, 1993). So far, a targeting sequence has not been identified for the MTB, assuming that the overall three-dimensional structure of the secretory protein plays an important role since this machinery only translocates folded proteins. Interestingly, there are two complete systems (Xcp and Hxc) as well as one incomplete system (HpI) present in P. aeruginosa whereas in Pseudomonas fluorescence, a complete Hxc system and a partial Xcp system have been identified (Ma et al., 2003). The Xcp machinery in P. aeruginosa is encoded by 12 xcp genes that are located in two divergently transcribed operons (Tommassen et al., 1992; Filloux et al., 1998). The Xcp proteins are located in the inner and outer membrane with XcpQ forming a multimeric outer membrane pore (Bitter et al., 1998). In 1997, Chapon-Herve et al. showed that expression of the xcp genes is controlled by quorum sensing whereas the Hxc system is shown to be regulated by the pho gene product (Ball et al., 2002). Additionally, two genes encoding homologous proteins to the secretins have been identified in P. aeruginosa: XphA is
______12 1. Introduction homologous to the essential protein XcpP, while XqhA can substitute XcpQ in an xcpQ mutant strain (Martinez et al., 1998). Another interesting aspect is that the Xcp system and the type IV pili show extensive homology in sequence and organization suggesting that these two systems are evolutionary related (Lory 1998; Nunn, 1999; Koster et al., 2000). For example, the N-termini of XcpT, XcpU, XcpV and XcpW are similar to the N-terminus of the type IV pili subunit PilA. The leader peptides of these proteins are cut off by a prepilin peptidase encoded by xcpA, also named pilD (Bally et al., 1992; Lory and Strom, 1997). Consequently, due to their similarity to pilin subunits, the five Xcp proteins XcpT-X are also designated as pseudopilins, which might be organized in a pilus-like structure supporting secretion in an indirect or direct way (Lory and Strom, 1997; Bleves et al., 1998; Lory, 1998).
1.3 Burkholderia glumae – A lipase-producing plant pathogen In 1992, Yabuuchi et al. proposed the new genus Burkholderia for the RNA homology group II of the genus Pseudomonas. Based on 16S rRNA sequences, DNA-DNA homology values, cellular lipid and fatty acid composition as well as phenotypic characteristics, they suggested to transfer seven species of the Pseudomonas group to the new genus, including for example Pseudomonas cepacia, Pseudomonas pseudomallei and Pseudomonas gladioli. In subsequent years, some more species such as Pseudomonas glumae (Urukami et al., 1994), Pseudomonas thailandensis (Brett et al., 1998), Pseudomonas caribensis (Achouak et al., 1999) and Pseudomonas glathei (Viallard et al., 1998) were transferred to the genus Burkholderia. In general, members of the genera Burkholderia and Pseudomonas are Gram-negative proteobacteria. While Pseudomonas belongs to the RNA homology group I of γ-subdivision, Burkholderia belongs to the β-subdivision of proteobacteria. Interestingly, many members of the genus Burkholderia are important for the soil microbial community. For example, some species of the B. cepacia complex produce compounds with antimicrobial activity and thus are useful as biocontrol agents with activity against phytopathogens (Cartwright et al., 1995; El Banna and Winkelmann, 1998; Hu and Young, 1998; Kang et al., 1998). Burkholderia vietnamiensis, among other plant-growth-promoting rhizobacteria, is able to fix atmospheric nitrogen and is thus beneficial in rice inoculation in low fertility sulphate acid soil (Tran Van et al., 2000). Likewise, introduction of some Burkholderia species in crops such as maize and sorghum resulted in increases in root and shoot dry weight (Bevivino et al., 1998; Chiarini et al., 1998). However, there are also several pathogenic Burkholderia strains affecting humans, animals as well as plants. For instance, some species
______13 1. Introduction of the B. cepacia complex cause severe problems in cystic fibrosis patients (Govan et al., 1996; Vandamme et al., 1997), B. pseudomallei is recognized as a biothreat agent and causative agent of melioidoso (Dance, 1991) whereas B. glumae and Burkholderia plantarii are involved in plant pathogenesis (Iwai et al., 2002). B. glumae was first described by Kurita and Tabei in 1967 as a 0,5-0,7 x 1,5-2,5 µm large rod bacterium that is motile by means of 2-4 polar flagella. The temperature limits for growth are 11-40°C with an optimum between 30-35°C. The bacterium is pathogenic for rice plants causing seedling rot in rice nursery boxes and rice grain rot in paddy fields. Moreover, Jeong et al. (2003) reported that B. glumae not only destroys rice plants but also causes wilting symptoms in tomato, sesame, perilla, eggplant and hot pepper. The key factor in wilt symptom development is a toxoflavin produced by B. glumae. This toxoflavin is a 7-azapteridine antibiotic, which has antibacterial, antifungal and herbicidal activities and is also toxic to mice causing haematuria, diarrhea and lachrymation (Nagamatsu, 2001). Just recently, Kim et al. (2004) reported for the first time that the toxoflavin biosynthesis and transport is controlled by quorum sensing and the LysR-type transcriptional activator ToxR. Due to the fact that B. glumae causes severe wilt symptoms on many field crops leading to dramatic yield loss in agriculture each year, especially in South-East Asian countries such as Japan and Korea, research on B. glumae mainly focuses on the biosynthesis, regulation and mode of action of the phytotoxin produced by the bacteria. Nevertheless, B. glumae also produces an extracellular lipase, which turned out to be useful for a variety of different biotechnological applications (Schmid et al., 2001). Like in many other Gram-negative bacteria, the lipase-encoding gene lipA is organized in a bicistronical operon together with the lipB gene encoding the lipase specific foldase (Fig. 5). As it is typical for species of the genera Burkholderia and Pseudomonas, the GC-content of both genes is significantly high, amounting 70% in the case of lipA and 78% in the case of lipB. Interestingly, the stop codon of lipA overlaps with the start codon of lipB (Frenken et al., 1993a), which is in contrast to P. aeruginosa, where an intergenic region could be identified between the two genes (Rosenau, 2000). In general, gene overlaps are assumed to be important for regulation of gene expression (Krakauer, 2000).
GGGCGTGTGATGGCGCAGG Fig. 5: Genetic organization of the lipAB-operon of B. glumae. The DNA sequence represents the last base pairs of the lipA gene and the first base pairs of the lipB gene indicating the overlap of stop and start codon (black).
lipA lipB
______14 1. Introduction
The mature lipase has a molecular weight of 33 kDa (Frenken et al., 1993). In 1993, Nobel et al. solved a 3.0 Å structure of the lipase and in 1996, Lang et al. improved the resolution to 1.6 Å. The serine protease-like catalytic triad, composed of the amino acid residues Ser87- His285-Asp263, is completely buried under a lid-like structure formed by one α-helix consisting of 13 amino acid residues. However, the lipase does not show the phenomenon of “interfacial activation”. Stabilization of the structure is provided by a calcium ion, which is bound close to the active site and a disulfide bond involving the only two cysteine residues present in the sequence (Cys190/Cys269) (Nobel et al., 1993; Lang et al., 1996). As described before, secretion of the lipase occurs via the type II secretion pathway. Interestingly, the enzyme is synthesized with an unusual long signal sequence of 39 amino acid residues containing an extended C-region with four potential cleavage sites for signal peptidases I (SPaseI) (Frenken et al., 1992). So far, not many studies have been performed dealing with the regulation of lipase gene expression in B. glumae. Although it is assumed that there are some parallels to P. aeruginosa based on the relationship, they might also be several differences. One difference is for example that in B. glumae, lipase gene expression is subjected to catabolite repression, which is not the case in P. aeruginosa (Frenken, 1993). Based on amino acid sequence analysis, the Lif protein of B. glumae has been classified into family II of the four existing families (Rosenau et al., 2004). The protein is composed of 353 amino acid residues of which the largest part is exposed to the periplasm whereas about 76 amino acids are anchored in the inner membrane. As described in chapter 1.2.2, the function of Lif is to fold its cognate lipase in the periplasm into an enzymatically active conformation (Frenken et al., 1993; Khattabi, 2001).
1.4 Expression systems 1.4.1 General aspects The isolation of genes, their manipulation in vitro and their expression in different organisms are powerful and versatile tools in modern biology. In the past two decades, a broad spectrum of different expression systems applicable in different expression hosts have been developed, which is reflected by the numerous articles and laboratory manuals that are available (Jacobs et al., 1989; Studier et al., 1990; Hannig and Makrides, 1998; Tao and Zhang, 1998; Matthey et al., 1999; Newman and Fuqua, 1999; Grabherr and Ernst, 2001; Sørensen and Mortensen, 2004; Byrne et al., 2005).
______15 1. Introduction
Dependent on the target protein, desired posttranslational modification or expression level and further application of the protein, one has to consider which expression system to choose. Thus, there is no universal expression system and for each protein the single steps of protein expression, folding and if necessary secretion have to be optimized. In Table 1 some characteristics of different expression systems are listed.
Tab. 1: Characteristics of different expression systems
Yeast and Features Bacteria Insects Mammalian Fungi
Cell growth fast middle-fast slow slow Cultivation in yes/yes yes/yes yes/yes no/yes flasks/bioreactor Defined media yes yes no no Costs for cultivation low low high high Expression level high low-high low-high low Secretion of target yes yes yes yes proteins yes, but often inclusion Protein folding yes yes yes bodies/in vitro refolding N-Glykosylation no yes, mannose yes, except sialine acid yes O-Glykosylation no yes yes yes Phosphorylation, no no yes yes Acetylation, Acylation
Based on the fact that bacteria are easy to handle, grow fast and allow producing of a large number of proteins at high levels, these microorganisms are most likely used in the production of industrial biocatalysts. Nevertheless, in some cases eukaryotic systems are more favorable, especially if the target protein origins from a eukaryotic organism and needs to be posttranslational modified. In addition, heterologous expression of the corresponding gene as well as protein folding in bacteria often fails. In general, expression of recombinant proteins is performed in trans using an expression vector, which harbors central genetic elements. These elements include an origin of replication (ori), an antibiotic resistance marker, promoters, translational initiation regions (TIRs) as well as transcriptional terminators. Approximately 80% of all proteins submitted to the protein data bank (PDB) were expressed in E. coli, which is still the most favored bacterial host strain for recombinant protein expression (Sørensen and Mortensen, 2004). The most used expression system in E. coli is the T7 RNA polymerase-based pET expression system (commercialized by Novagen) followed by other common expression systems using λPL promoter/cI repressor (e.g. Invitrogen, pLEX), Trc promoter (e.g. Amersham Biosciences, pTrc), Tac promoter (e.g. Amersham Biosciences, pGEX) and hybrid lac/T5
______16 1. Introduction
(Qiagen pQE) (Hannig and Makrides, 1998). The next section will only focus on the T7 RNA polymerase-based expression system and its application in Gram-negative bacteria other than E. coli.
1.4.2 The T7 RNA polymerase-based expression system In 1990, Studier and colleagues first described the pET expression system as being suitable for a variety of different expression applications. This system depends on the strictly selective nature of the bacteriophage T7 RNA polymerase for respective promoters (Studier and Moffat, 1986; Studier et al., 1990). Moreover, the bacterial host RNA polymerase does not recognize T7 promoters. As a consequence, no transcription of the target gene occurs in the absence of a source of T7 RNA polymerase and the cloning step is thus effectively uncoupled from the expression step. Another advantage is that the T7 RNA polymerase does not recognize bacterial transcriptional termination signals properly enabling the co-expression of functionally clustered genes. In the pET system, the gene encoding T7 RNA polymerase (gene1) is integrated into the genome of the host strain by lysogenization with the DE3 phage fragment containing the gene under the control of the IPTG (Isopropyl-β-D-thiogalactoside) inducible lacUV5 promoter. LacI represses the lacUV5 promoter and the T7/lac hybrid promoter located on the expression plasmid. After adding IPTG to the cell culture, it binds to the repressor protein and triggers the release of the tetrameric LacI from the lac operator, which leads to the transcription of gene1. Finally, transcription of the target gene, which has been cloned under the control of the T7/lac promoter, is subsequently initiated by the T7 RNA polymerase (Fig. 6) (Studier et al., 1990). Today more than 40 different pET vectors including hybrid promoters, multiple cloning sites for the incorporation of different fusion proteins and protease cleavage sites as well 12 different E. coli expression hosts are commercially available (Novagen Inc. Madison, USA). This elucidates that the T7 RNA polymerase-based overexpression system is a powerful tool for the production of recombinant proteins.
______17 1. Introduction
Fig. 6: Strategy for regulating gene expression using the pET-expression system. The gene encoding the T7 RNA polymerase (gene1) is inserted into the chromosome of E. coli and transcribed from the lac promoter. Therefore, it is expressed only if the inducer IPTG is added to the growing culture. The T7 RNA polymerase then transcribes the target gene cloned into the pET vector. The presence of a lac operator between T7 promoter and the cloned gene reduces transcription of the cloned gene in the absence of the inducer IPTG (reproduced from Novagen Catalogue, Madison USA, 1995).
Although E. coli is still the most preferred bacterial host strain for high-level expression of many different recombinant proteins, in some cases other bacteria are more suitable, because they provide special physiological properties for gene expression, protein folding or secretion. For instance, for the production of extracellular enzymes, E. coli is not the strain of choice, because under standard conditions it does not secrete proteins. In this case, bacteria of the genera Pseudomonas or Burkholderia are more suitable, because they harbor several secretion pathways. Another example is the overexpression of inner membrane proteins or proteins carrying complex redox co-factors. In both cases, the non-sulphur purple bacterium Rhodobacter capsulatus was shown to be particularly suitable. Under phototrophic growth conditions, formation of an intracytoplasmic membrane system is induced, thus providing a large surface for protein incorporation in combination with efficient membrane protein assembly machinery. In addition, R. capsulatus is able to synthesize many types of metal- containing prosthetic groups allowing the production of several active redox enzymes (Drepper et al., 2005). Finally, in some cases expression of a target gene or folding of the corresponding protein requires the presence of additional proteins such as chaperons and folding catalysts, so that heterologous overexpression in E. coli would require the additional expression of a whole set of genes. Consequently, in recent years, the T7 RNA polymerase- based expression system has been adapted to other Gram-negative and Gram-positive bacteria. In Table 2, some of the existing T7 RNA polymerase-based expression systems are summarized.
______18 1. Introduction
Tab. 2: Overview of some existing T7 RNA polymerase-based expression systems in Gram-negative and Gram-positive bacteria.
Gram-negative bacteria Reference P. aeruginosa Brunschwig and Darzins, 1992; Rosenau et al., 1998 Ralstonia eutropha Barnard et al., 2004 R. capsulatus Drepper et al., 2005 Salmonella enterica seorvar typhi Santiago-Machuca et al., 2002 Gram-positive bacteria Reference Bacillus megaterium Jahn (unpublished)
With one exception, a common feature of these systems is that the gene encoding the T7 RNA polymerase has been integrated into the genome of the host cell whereas the integration sites are different. Besides, not all of these systems use the lacUV5 promoter to control expression of gene1 and thus do not contain the lacI gene, which is necessary for repression of the lac promoter. For instance, in R. eutropha the T7 RNA polymerase gene was cloned behind the strong pha promoter (Barnard et al., 2004) whereas in S. enterica seorvar typhi, the gene was cloned under the control of the anaerobically inducible nirB promoter (Santiago-Machuca et al., 2002). In R. capsulatus, not gene1 was integrated into the chromosome of the host cell, but an interposon cassette Ω-T7, containing the omega fragment from plasmid pHP45 Ω and the T7 promoter from pET22b. This cassette was integrated into the hupV gene, which codes for the large subunit of the regulatory sensor hydrogenase. Thus, overexpression of the downstream located hydrogenase genes can be induced after transformation of this strain with a plasmid harboring the T7 RNA polymerase-encoding gene under the control of an inducible promoter (Drepper et al., 2005).
1.5 Aims of this study Lipases constitute the most important class of biocatalysts used for a variety of different industrial processes (Jaeger and Eggert, 2002). The Gram-negative bacterium Burkholderia glumae is a plant pathogen, which is useful for biotechnological applications. Moreover, it secretes a lipase with favorable enzymatic properties (Schmid et al., 2001). Therefore, high- level production of this lipase in B. glumae is required. This thesis has focused on the following aims:
______19 1. Introduction
1) Development of a T7 RNA polymerase-based expression system First, it had to be explored whether the establishment of a T7 RNA polymerase-based expression system in B. glumae is feasible. Second, this thesis aimed at constructing an expression strain of B. glumae, which carries the T7 RNA polymerase gene in the chromosome. Additionally, the chromosomal lipAB-operon was attempted to be deleted in this expression strain in order to prevent unwanted recombination of chromosomal and plasmid-encoded lipase during overexpression studies. Finally, a suitable expression vector had to be constructed that contains the lipAB-operon of B. glumae under the control of the T7 promoter. This expression vector would also allow for cloning of lipase variants.
2) Identification and elimination of bottlenecks for an improved lipase production In order to specifically improve lipase production, potential bottlenecks in the production pathway needed to be identified. Therefore, a cosmid library of B. glumae had to be constructed and screened. By either inactivating the chromosomal genes of limiting components or by additionally integrating genes of rate-limiting steps into the genome of B. glumae, an optimal production strain was finally aimed at being constructed.
3) Analysis of regulation of lipase gene expression With respect to obtaining an improved lipase production, the effect of physiological conditions on lipase production in B. glumae had to be examined. Furthermore, it was intended to elucidate the underlying molecular mechanisms of these effects, in order to obtain a more detailed understanding of the regulation of lipase gene expression in B. glumae.
______20 2. Materials
2. Materials
2.1 Chemicals and enzymes All used chemicals and enzymes have been obtained in p.a. quality from the following companies. Antibiotics: Serva (Heidelberg) Sigma-Aldrich (Taufkirchen) Chemicals: Fluka (Sternheim) Merck (Darmstadt) Roth (Karlsruhe) Sigma-Aldrich (Taufkirchen) Enzymes: Lysozyme Sigma-Aldrich (Taufkirchen) Omniscript Reverse Transcriptase Qiagen (Hilden) Pfu-DNA Polymerase Stratagene (Heidelberg) Restrictions enzymes MGI Fermentas (St. Leon-Roth) RNase Sigma-Aldrich (Taufkirchen) T4 DNA Ligase MGI Fermentas (St. Leon-Roth) T4 DNA Polymerase MGI Fermentas (St. Leon-Roth)
2.2 Strains and Plasmids
Tab. 3: Bacterial strains and plasmids used or constructed in this thesis
Strain Genotype Reference/Source E. coli BL21(DE3) f- dcm ompT hsdS(rB- mB-) Studier and Moffat, 1986 gal λ(DE3) Novagen, Madison (USA) E. coli DH5α F-, ø80dlacZ∆M15, Hanahan, 1983 ∆(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk-, mk+), phoA, supE44, λ-, thi-1, gyrA96, relA1 E. coli JM101 supE thi-1 ∆(lac-proAB) [F´ Stratagene, Heidelberg traD36 proAB lacIqZ.M15] E. coli S17-1 thi pro hsdR-M+ [RP4-2- Simon et al., 1983 Tcr::Mu:Kmr::Tn7, Tra+TrirStrr] E. coli XL1blue recA1endA1 gyrA96 thi-1 Stratagene, Heidelberg hsdR17 supE44 relA1 lac [F´ proAB lacIqZ.M15 Tn10 (Tetr)] B. glumae PG1 wild type University of Utrecht, Utrecht B. glumae LU8093 derivative of wild type, lipase BASF AG, Ludwigshafen production strain q B. glumae PGT7lacI lipAB:pSUPlacI PlacUV5 T7 This study RNA polymerase gene (gene1) B. glumae PgompC- ompC:pSUPompC´ This study B. glumae LuompC- ompC:pSUPompC´ This study B. glumae PGmfpB- mfpB:pSUPmfpB´ This study
______21 2. Materials
B. glumae LumfpB- mfpB:pSUPmfpB´ This study
Plasmid Genotype Reference/Source r pUC19 Plac lacZα, Amp Yanisch-Perron et al., 1985 pBluescriptKS+ ColE1 PT7Φ10PT3Plac lacZα Stratagene, Heidelberg (pBKS) Ampr q r pET22b ColE1 PT7Φ10lacI Amp Novagen, Madison (USA) q r pBBR22b rep mob PT7Φ10lacI , Cm Rosenau, 2001 r pBBR1mcs rep mob lacZα Plac PT7 Cm Kovach et al., 1994 r pBBR1mcs-2 rep mob lacZα Plac PT7 Km Kovach et al., 1994 pLAFR3 IncP1 λcos rlx Tcr Staskawicz et al., 1982 pSUP202 ColE1 rep mob Cmr Tcr Simon et al., 1983 Ampr pRK2013 ColE1 RK2-mob, RK2-tra+ Figurski et al., 1979 Kmr , helper plasmid
Recombinant plasmid Genotype Reference/Source pBKST7pollacI pBKS containing a 2,8 kb Brookmann, University of EcoRI/BamHI fragment Bochum, unpublished q comprising lacI PlacUV5 gene1 pML5-T7 pML5 containing a 2,8 kb Drepper et al., 2005 EcoRI/BamHI fragment q comprising lacI PlacUV5 gene1 pol 1 pUC18Kmr containing a Rosenau, Ruhr-University 2,8 kb PaeI/EcoRI fragment Bochum, unpublished comprising gene1 pSWgfp pTZ110 derivative containing Wilhelm, Ruhr-University a 0,75 kb EcoRI/HindIII Bochum, unpublished fragment comprising the gfpmut3 gene pSWgfpLAA pTZ110 derivative containing Wilhelm, Ruhr-University a 0,75 kb EcoRI/HindIII Bochum, unpublished fragment comprising the gfp(LAA) gene pBP1500 pML130 derivative BASF AG, Ludwigshafen containing a 2,9 kb EcoRI fragment comprising the lipAB-operon and 0,5kb of the upstream region pSUP2021 pSUP202 containing the Simon et al., 1989 transposon Tn5 pBKSlipAB pBKS containing a 2,8 kb This study EcoRI fragment of pBP1500 comprising the lipAB-operon pBBR22lipABT7 pBBR22b containing a 2,2 kb This study HindIII fragment of pBKSlipAB comprising q lipAB-operon, PT7Φ10lacI
______22 2. Materials
pBBRlipABT7 pBBR1mcs containing a This study 2,2 kb HindIII fragment of pBKSlipAB comprising the lipAB-operon, PT7 pBBRlipABlac pBBR1mcs containing a This study 2,2 kb HindIII fragment of pBKSlipAB comprising the lipAB-operon, PT7 pLAFRPG 1-15 pLAFR3 containing genomic This study DNA of B. glumae PG1, BamH1/SauA3 pBBRPG 5/1 pBBR1mcs containing a This study 4,0 kb HindIII/BamHI fragment of subcloned genomic DNA of pLAFRPG 5, Plac pBBRPG 5/3 pBBR1mcs containing a This study 4,7 kb HindIII/BamHI fragment of subcloned genomic DNA of pLAFRPG 5, Plac pBBRPG 5/7 pBBR1mcs containing a This study 5,2 kb HindIII/BamHI fragment of subcloned genomic DNA of pLAFRPG 5, Plac pBBRPG 8/1 pBBR1mcs containing a This study 1,2 kb HindIII/BamHI fragment of subcloned genomic DNA of pLAFRPG 8, Plac pBBRPG 8/3 pBBR1mcs containing a This study 3,8 kb HindIII/BamHI fragment of subcloned genomic DNA of pLAFRPG 8, Plac pETpro pET22b containing a This study NdeI/XhoI PCR fragment of pro pBBRpro pBBR1mcs containing a This study HindIII/BamHI PCR q fragment of pro, PT7Φ10lacI pSUPT7pollacI pSUP202 containing a 3,8 kb This study HindIII/BamHI fragment q comprising lacI PlacUV5 gene1 pSUPT7pollacIup pSUPT7pollacI containing a This study 0,5 kb fragment of the upstream region of the lipAB- operon, cloned blunt-end into
______23 2. Materials
the blunted HindII-site pSUPT7pollacIup/down pSUPT7pollacIup containing This study a 0,5 kb BamHI/SalI fragment comprising the downstream region of the lipAB-operon pSUPT7pol pSUP202 containing a 2,8 kb This study EcoRI fragment of pol1 comprising gene1 pSUPT7polup pSUPT7pol containing a This study 0,5 kb fragment of the upstream region of the lipAB- operon, cloned blunt-end into the blunted EcoRI-site pSUPT7polup/down pSUPT7polup containing a This study 0,5 kb NcoI fragment of the downstream region of the lipAB-operon pSUPompC` pSUP202 containing a 0,5 kb This study HindIII/BamHI PCR fragment comprising part of the ompC gene pSUPmfpB` pSUP202 containing a 0,5 kb This study HindIII/BamHI PCR fragment comprising part of the mfpB gene pSUPTn5pro pSUPTn5 containing the pro This study gene of pETpro cloned blunt- end into the SmaI site pSWgfpupPG1 (TCF) pSWgfp containing a This study 0,45 kb EcoRI/SmaI fragment (lipAPG1::gfp, transcriptional fusion) pSWgfpupLU (TCF) pSWgfp containing a This study 0,45 kb EcoRI/SmaI fragment (lipALU::gfp, transcriptional fusion) pSWgfpupPG1 (TLF) pSWgfp containing a This study 0,45 kb EcoRI/NdeI fragment (lipAPG1::gfp, translational fusion) pSWgfpupLU (TLF) pSWgfp containing a This study 0,45 kb EcoRI/NdeI fragment (lipALU::gfp, translational fusion) pSWgfpLAAupPG1 (TLF) pSWgfp containing a This study 0,45 kb EcoRI/NdeI fragment (lipAPG1::gfp(LAA), translational fusion)
______24 2. Materials
pSWgfpLAAupLU (TLF) pSWgfp containing a This study 0,45 kb EcoRI/NdeI fragment (lipALU::gfp(LAA), translational fusion) pBBKgfpPlac pBBRmcs-2 containing a This study 0,7 kb HincII/EcoRI fragment of pSWgfp comprising the gfpmut3 gene, Plac pBBKgfpLAAPlac pBBRmcs-2 containing a This study 0,7 kb HincII/EcoRI fragment of pSWgfpLAA comprising the gfp(LAA)gene, Plac pBBKgfpPT7 pBBRmcs-2 containing a This study 0,7 kb EcoRI/HindIII fragment of pSWgfp comprising the gfpmut3 gene, PT7Φ10 pBBKgfpLAAPT7 pBBRmcs-2 containing a This study 0,7 kb EcoRI/HindIII fragment of pSWgfpLAA, comprising the gfpLAA gene PT7Φ10 pBBKgfpupPG (TCF) pBBRmcs-2 containing a This study 1,2 kb EcoRI/HindIII fragment comprising gfpupPG of pSWgfpupPG (TCF), PT7Φ10 pBBKgfpupLU (TCF) pBBRmcs-2 containing a This study 1,2 kb EcoRI/HindIII fragment comprising gfpupLU of pSWgfpupLU (TCF), PT7Φ10 pBBKgfpupPG (TLF) pBBRmcs-2 containing a This study 1,2 kb EcoRI/HindIII fragment comprising gfpupPG of pSWgfpupPG (TLF), PT7Φ10 pBBKgfpupLU (TLF) pBBRmcs2 containing a This study 1,2 kb EcoRI/HindIII fragment comprising gfpupLU of pSWgfpupLU (TLF), PT7Φ10 pBBKgfpLAAupPG (TLF) pBBRmcs-2 containing a 1,2 This study kb EcoRI/HindIII fragment comprising gfpLAAupPG of pSWgfpLAAupPG (TLF), PT7Φ10
______25 2. Materials
pBBKgfpLAAupLU (TLF) pBBRmcs-2 containing a This study 1,2 kb EcoRI/HindIII fragment comprising gfpLAAupLU from pSWgfpLAAupLU (TLF), PT7Φ10
2.3 Oligonucleotides All used oligonucleotides for PCR amplification were synthesized at Thermoelectron and were obtained as high purified salt free and lyophilized. The primers were dissolved in A. dest resulting in a concentration of 100 pmol/µl. These samples were stored at -20°C.
Tab. 4: Applied oligonucleotides for PCR amplification
Name DNA-sequence (5`-3`) Restriction sites/Modification lipABup CGATGAATTCACCTTGAACGCAGGCG EcoRI lipABTF CGTCATATGTTTATCTCCATCGTTAAAGC NdeI lipABdn1 GCATGTCGACGGCGGGCGCCGTGT SalI lipABdn2 GTCAGGATCCGGCGCGGGCGGTTAGGG BamHI lipABdnN1 CAGACCATGGATCCGGCGCGGGCGGTTAG NcoI lipABdnN2 CAGACCATGGGGCGGCGCGGTGTG NcoI lipAPE1 CAATCTGACCATGTTTATCTCCATCG Cy5 lipAPE2 GCCCATGCCACCGCCCTCGCCGCCA Cy5 T7polup CGATCGATGAACACGATTAACATCGC PaeI T7poldn GAGAGAATTCCTTTAGCCGGAAGTGCT EcoRI proup GGAAGCTTGTAAGAAGCAGCTATGCAGC HindIII prodn CGCAGGATCCTTCACGCGCCGGCCCCCA BamHI proNde GTACATATGCAGCAGCGTGGCGGCAG NdeI proXho CGATCTCGAGTCACGCGCCGGCCCCCA XhoI ompCup GGAAGCTTGTCGCGCTCAATCGCCGCAT HindIII ompCdn GTCAGGATCCTCACGCGGCGGCGTCCGC BamHI mfpBup GGAAGCTTAACAGGAAACCTTTGGCAC HindIII mfpBdn GTCAGGATCCTCATTGCACGCCTCCGACC BamHI epAup GGAAGCTTGCACGTTTCTTCATCGAG HindIII epAdn GTCAGGATCCTCAATGCGAACCCTCATC BamHI
2.4 Culture media and plates LB Medium 10 g/l Tryptone, 10 g/l NaCl, 5 g/l Yeast extract LB Agar LB Medium, 15 g/l Agar 2x LB Medium 20 g/l Tryptone, 10 g/l NaCl, 10 g/l Yeast extract NB Medium 8g/l Peptone, 4g/l NaCl PG Medium Solution1: 6 g/l (NH4)2SO4, 0,02 g CaCl2, 1 g/l MgSO4, 2 g/l Yeast extract Solution 2: 3,5 g/l K2HPO4, 3,5 g/l KH2PO4 Solution 1 and 2 were autoclaved separately and combined as follows: 800 ml Solution1 200 ml Solution 2 The following C-sources were added to the PG Medium: 1% (v/v) Olive oil, 0,5% (w/v) Glucose, 0,5% (w/v) Sucrose, 0,5% (w/v)
______26 2. Materials
Maltose, 10 mM Tributyrin or 1% (v/v) Gum arabic emulsion (1,5g Gum arabic, 15 ml A. dest) PG Tributyrin Agar PG Medium, 15 g Agar, Tributyrin emulsion (1,5 g Gum arabic, 15 ml Tributyrin, 15 ml A. dest) M9 Medium Solution1: 40 g/l Glucose Solution 2: 25 g/l MgSO4 x 7H2O Solution 3: 2 g/l CaCl2 Solution 4: 70 g/l Na2HPO4 x 2H2O, 30 g/l KH2PO4, 5 g/l NaCl, 10 g/l NH4C The solutions were autoclaved separately and mixed as follows: 100 ml Solution1 10 ml Solution 2 10 ml Solution 3 100 ml Solution 4 ad 1000 ml A. dest M9 Agar M9 Medium, 15g/l Agar M12 Medium 1 g/l (NH4)SO4 0,1 g/l NaCl 0,2 g/l MgSO4 x 7 H2O 0,02 g/l CaCl2 x 2 H2O 0,05 g/l Yeast extract ad 900 ml A. dest After autoclaving the following solutions are added: 100 ml Vitamin solution (BASF AG) 20 ml KH2PO4 (10 g/100 ml pH 6,7) 4 ml Glucose (50 g/100 ml) Skim Milk Agar 30 g/l Skim milk powder 5 g/l Yeast extract 10 g/l NaCl 10 g/l Tryptone 15 g/l Agar α-Complementation Agar 4 ml IPTG (100 mM solved in 70% EtOH) 16 ml X-Gal (2% w/v solved in DMF) 1000 ml LB Agar
2.5 Buffers and solutions Bradford Solution 100 mg Coomassie Brillant Blue G-250, 50 ml Ethanol, 100 ml 85% (v/v) Phosphoric acid, ad 1 l A. dest Coomassie Staining Solution 0,2% (w/v) Coomassie Brillant Blue R-250, 40% (v/v) Ethanol, 10% (v/v) Acetic acid Coomassie Discoloring Solution 40% (v/v) Ethanol, 10% (v/v) Acetic acid DNA Electrophoresis Buffer TBE: 89 mM Tris, 89 mM, Boric acid, 2,5 mM Na2-EDTA TAE: 89 mM Tris, 40 mM Acetic acid, 2,5 mM Na2-EDTA DNA Loading Buffer (5x) 100 mM Na2-EDTA, 43% (v/v) Glycerol, 0,5% (w/v) Bromphenol blue
______27 2. Materials
Dunn Carbonat Buffer 10 mM NaHCO3 3 mM Na2CO3 20% (v/v) Methanol Mix I 50 mM Tris-HCl (pH 8,0), 10 mM Na2-EDTA Mix II 200 mM NaOH, 1% (w/v) SDS Mix III 3 M Potassium acetate (pH 5,2) 2+ Mg -Mix 500 mM MgCl2, 500 mM MgSO4 x 7 H2O Native Loading Buffer 10% Glycerol, 0,125 M Tris-HCl pH 6,8, 0,1% (w/v) Bromphenol blue Phosphate Buffered Saline (PBS) 150 mM NaCl, 20 mM NaH2PO4 (pH 7,2) SDS Electrophoresis Buffer 25 mM Tris, 0,129 M Glycin, 0,1% (v/v) SDS SDS Loading Buffer 10% Glycerol, 0,2% (v/v) SDS, 0,125 M Tris- HCl pH 6,8, 0,1% (w/v) Bromphenol blue, 2% (v/v) ß-Mercaptoethanol Sφrensen Phosphate Buffer Solution A: 8,9 g/l Na2HPO4 x 2 H2O Solution B: 0,68 g/l KH2PO4 Solution A and B are autoclaved separately and mixed in a ratio of 17:1 TBST Buffer 10 mM Tris-HCl, 150 mM NaCl, 0,2% (v/v) Tween 80 Tris-HCl-Buffer (TE) 10 mM Tris-HCl, 1mM Na2-EDTA (pH 8,0) Transformation Buffer (TMF) 100 mM CaCl2, 50 mM RbCl2, 40 mM MnCl2
2.6 Molecular weight standards 1 kb DNA Ladder Invitrogen (Kalsruhe) Prestained Protein Marker Fermentas (St. Leon-Roth) M12 Protein Standard Invitrogen (Kalsruhe)
2.7 Kits DNAeasy Tissue Kit Qiagen (Hilden) ECL-Western Blotting Detection System Amersham-Biosciences (Freiburg) Gigapack® III XL Packaging Extract Stratagene (Heidelberg) Omniscript RT Kit Qiagen (Hilden) Plasmid DNA prep Kit Eppendorf AG (Hamburg) QIAprep Spin Miniprep/Midiprep Qiagen (Hilden) QIAquick PCR Purification Kit Qiagen (Hilden) QIAquick Gel Extraction Kit Qiagen (Hilden) RNeasy Mini Kit Qiagen (Hilden)
2.8 Laboratory instruments ALF Express-DNA Sequencer A.L.F., Pharmacia Biotech (Freiburg) Centrifuges Mikro 22 R, Hettich AG (Baech) Rotina 35 R, Hettich AG (Baech) Sorvall RC5B, Kendro Laboratory Products GmbH (Duesseldorf) Chromatography Aekta explorer, Pharmacia Biotech (Freiburg) Ni-NTA superflow, 30 ml volume, Qiagen, (Hilden) Sephadex G-25, Amersham-Biosciences, (Freiburg) Superdex 200 prep grade Hi Load 16/60, Amersham-Biosciences, (Freiburg)
______28 2. Materials
Agarose gel electrophoresis BioRad Laboratories GmbH (Munich) Electroporator MicroPulser or Gene Pulser, BioRad Laboratories GmbH (Munich) Fluorescence Spectrometer Perkin Elmer Luminescence spectrometer LS 50 B (Duesseldorf) Luminograph EG&G Luminograph LB 980 (Bad Wildbad) PCR Mastercycler gradient, Eppendorf AG (Hamburg) Photosystem for DNA-Gels Eagle Eye II Videosystem, Stratagene (Amsterdam) Gel Jet Imager Plus, INTAS (Göttingen) Protein gel electrophoresis Mini Protean II Dual Slap Cell, Bio-Rad Laboratories GmbH (Munich) Mini Trans-Blot Cell, Bio-Rad Laboratories GmbH (Munich) Scanner HP Precisionsscan Pro 3.1, Hewlett Packard (Böblingen) Spectrophotometer UV/VIS Spectrophotometer 16A, Shimadzu Germany GmbH (Duisburg) UV/VIS Spectrophotometer DU 650, Beckmann Instruments GmbH (Munich) Speed-Vac Univapo 150, Unicryo MC1L Sonificator Sonificator UP200S, Dr. Hielscher (Teltow) Thermocycler Thermocycler, Eppendorf AG (Hamburg)
______29 3. Methods
3. Methods
3.1 Bacterial strain and growth conditions
3.1.1 Cultivation of E. coli strains E. coli DH5α, JM101 and XL1blue were used for routine cloning. Conjugational transfer of cosmids into B. glumae PG1 or B. glumae LU8093 were performed as triparental spot matings with E. coli JM101 as the donor and E. coli pRK2013 as the helper strain. In order to perform conjugational transfer of broad host range plasmids, E. coli S17-1 was applied in biparental matings with B. glumae strains. Heterologous overexpressions were performed using E. coli BL21(DE3). All E. coli strains were grown in LB medium (2.4) at 37°C. Over night cultures were grown for at least 16 h and were diluted 1:100 to inoculate expression cultures. Overexpression of the putative protease was induced by adding 0,4 mM IPTG during the log- phase (OD580 0,5). For blue/white screening of recombinant E. coli clones, X-Gal (Roth, at final concentration of 0,4 mM) was added to LB agar plates. Adding the respective antibiotics to the growth media ensured maintenance of the plasmids.
3.1.2 Cultivation of B. glumae strains B. glumae PG1 and B. glumae LU8093 were grown at 30°C in PG medium or LB medium. The wild type strain B. glumae PG1 was maintained in M9 medium whereas for the production strain B. glumae LU8093 M12 medium was used. Over night cultures were grown for at least 16 h and were inoculated to an OD580 of 0,05 for expression studies. Adding the respective antibiotics to the growth media ensured maintenance of the plasmids.
3.1.3 Table of applied antibiotics
Tab. 5: Concentrations and solving reagents of applied antibiotics
Antibiotic Abbreviation Final concentration Solving [µg/ml] reagents E. coli B. glumae Ampicillin Amp 100 - A. dest Carbenicillin Cb - 150 A. dest Chloramphenicol Cm 50 200 70% EtOH Cycloserine Cy - 200 A. dest Kanamycin Km 50 50 A. dest Tetracycline Tc 25 50 70% EtOH
3.1.4 Storage of microorganisms Grown E. coli clones on agar plates were stored up to 6 weeks, grown B. glumae clones up to 7 days at 4°C. For longer storage 1 ml of liquid culture was mixed with DMSO (8% v/v end concentration) and stored at – 80°C.
3.2 Isolation of nucleic acid
3.2.1 Isolation of plasmid and cosmid DNA Isolation of plasmid and cosmid DNA was basically performed as described by Sambrook et al. (1989) in a modified protocol. After adding Mix I, II, III (2.5) and centrifugation, the supernatant was directly mixed with 0,7 volume isopropanol and centrifuged again (RT, 13000 rpm, 30 min). After washing the pellet with 70% (v/v) ethanol, the plasmid DNA was resuspended in 30-50 µl TE buffer (2.5). For DNA sequencing, PCR amplifications and
______30 3. Methods electroporations, plasmid DNA was isolated using the respective Kits of Qiagen or Eppendorf (2.7) according to the protocols provided.
3.2.2 Isolation of chromosomal DNA Isolation of chromosomal DNA of B. glumae strains was performed using the DNeasy Tissue Kit (2.7) according to the protocol provided.
3.2.3 Isolation of RNA Isolation of RNA of B. glumae strains was performed using the RNeasy Mini Kit (2.7) according to the protocol provided.
3.3 Agarose gel electrophoresis Agarose gel electrophoresis was used to determine concentration and size of DNA fragments as well as the efficiency of RNA isolation. In general, the method was performed according to Sambrook et al. (1989) using 0,5x TBE or 0,5x TAE buffer (2.5). For purification of DNA fragments out of agarose gels, the QIAquick Gel extraction Kit (2.7) was applied according to the protocol provided.
3.4 In vitro recombination of DNA All restriction and ligation reactions of DNA fragments as well as further modifications of DNA fragments, such as dephosphorylation and blunting ends, were performed according to Sambrook et al. (1989). The enzymes were obtained at different companies (2.1) and applied in the provided buffers.
3.5 Construction of a cosmid library Construction of a cosmid library of B. glumae was essentially performed as described by Staskawicz et al. (1987). After isolation of genomic DNA from overnight cultures, the DNA was partially hydrolyzed using Sau3A. Separation of the DNA-fragments was performed by sucrose density gradient centrifugation (Sambrook et al., 1989) and electrophoresis using a 0,5% agarose gel in standard TAE buffer (2.5) and fragments larger than 15 kb were cut out and purified (3.3). The cosmid pLAFR3 was hydrolyzed in two separate reactions with the restriction enzymes HindIII or EcoRI and dephosphorylated afterwards. In a second step, both restriction samples were digested with BamHI in order to obtain Sau3A compatible ends. Ligation of the genomic DNA and the two DNA-fragments of pLAFR3 was performed overnight at 16°C using T4 DNA ligase. Finally, the cosmids were packed in vitro using the Gigapack® III XL Packaging Extract (2.7) and transduced in E. coli JM101. Since the cosmid pLAFR3 allows blue-white screening, positives clones could be identified on LB agar plates containing tetracycline (25 µg/ml), isopropyl-1-thio-ß-D-galactopyranoside (IPTG, 250 µg/ml final concentration) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranosid (X-Gal, 40 µg/ml final concentration).
3.6 Polymerase chain reaction (PCR) For amplification of DNA fragments polymerase chain reactions were basically performed according to Saiki et al. (1988). For amplification of DNA fragments not coming from Burkholderia, standard PCR conditions were sufficient. In order to amplify genomic DNA from Burkholderia, it was necessary to optimize the conditions in the reaction mix as well as annealing temperatures for each reaction separately. Since DNA of Burkholderia is very GC- rich, the addition of 5-10% (v/v) DMSO and an increased dNTP concentration for the amplification of DNA fragments larger than 1 kb, in combination with Gradient or Touch down PCR provided reproducible results. In addition, Hot-start PCR was performed at any time, which prevents extension of nonspecifically annealed primers and primer-dimers before
______31 3. Methods the initial heating step. In order to proof vector-insertion mutants, Colony PCR was performed. Therefore, 100 µl of an overnight culture was centrifuged and resuspended in 100 µl TE buffer. After heating of the sample for 5 min at 95°C, 1µl was used as template. All PCR reactions were carried out using the Mastercycler gradient from Eppendorf (2.8).
Tab. 6: Composition of a PCR reaction mix
Components Standard PCR reaction PCR reaction mix for mix Burkholderia DNA Primer 1 50 pmol 50 pmol Primer 2 50 pmol 50 pmol Template 1 µl 1 µl Buffer (10x) 5 µl 5 µl dNTP mix (10 mM) 1 µl 1-2 µl DMSO - 3-5 µl Pfu DNA Polymerase 0,5 µl 1 µl A. dest Ad 50 µl Ad 50 µl
Tab. 7: PCR program; Tm: melting temperature
Steps Standard PCR Gradient PCR Touch down PCR 1. Denaturation 98°C 2 min 98°C 2 min 98°C 2 min 2. Denaturation 96°C 45 sec 96°C 45 sec 96°C 45 sec 3. Annealing Tm primer: -5-7°C Tm primer: - 5-7°C Tm primer: -5-7°C ± 10°C -0,2°C/cycle 4. Elongation 72°C 1 min/kb 72°C 1 min/kb 72°C 1 min/kb 5. Denaturation - - 96°C 45 sec 6. Annealing - - Tm primer: -5-7°C 7. Elongation - - 72°C 1 min/kb 8. Elongation 72°C 5 min 72°C 5 min 72°C 5 min
After the polymerase chain reaction, 10% of each reaction mix was analyzed by agarose gel electrophoresis. Purification of amplified DNA fragments was either performed by preparative agarose gel electrophoresis using the QIAquick Gel extraction Kit (2.7), or by using the QIAquick PCR Purification Kit (2.7) according to the protocols provided.
3.7 Primer extension analysis The start of transcription of the lipAB-operon was determined by primer extension analysis, which was carried out at the Ruhr-University of Bochum (Institute for Molecular Neurobiochemistry) and at IIT Biotech GmbH (Bielefeld). In principal, the localization of 5`-end of specific mRNAs was determined in a one step reaction using the enzyme omniscript reverse transcriptase (2.1) followed by detection at the ALF express DNA sequencer (2.8). Therefore total RNA of B. glumae cultures was isolated after 10-12 h of growing at 30°C using the RNeasy Mini Kit (2.7). The following steps of reverse transcription were performed according to protocol provided with the OmniscriptTM Reverse transcription Kit (2.7). After inactivation of the reaction mixture for 5 min at 93°C the samples were stored at -20°C.
3.8 DNA sequencing DNA sequencing of recombinant plasmids was carried out at Sequiserve (Vaterstetten).
______32 3. Methods
3.9 Transformation of bacteria
3.9.1 Chemical transformation of E. coli Preparation of transformation competent E. coli cells and transformation of plasmid DNA was performed according to the RbCl2 method of Hanahan (1983).
3.9.2 Electroporation of B. glumae Preparation of electroporation competent cells: Overnight cultures were grown in 10 ml LB medium (2.4) at 30°C (wild type B. glumae PG1) or 37°C (production strain B. glumae LU8093). Subsequently 2 ml of each overnight culture were inoculated in 200 ml of prewarmed LB medium and were grown at 30°C or 37°C, respectively until the cells reached an OD580 of 0,2. The cells were centrifuged at 4°C and 3000 g for 20 min. The cell pellets were resuspended in ½ Volume of ice cold 0,3 M sucrose and were centrifuged again. Since it is necessary to remove the salt completely, these washing steps were repeated twice whereby the volume of 0,3 M sucrose was divided each time. After that, two washing steps followed using 0,3 M sucrose containing 10% (v/v) glycerol. Finally, the cells were resuspended in an adequate volume of 0,3 M sucrose and 10% (v/v) glycerol leading to an OD580 of 50. Aliquots of 50 µl were dispensed into sterile Eppendorf tubes (EP) and were either directly used or stored frozen at -80°C. It should be noted however that the electroporation efficiency of cells drops rapidly after a few weeks of storage. Electroporation of B. glumae: It has been generally considered that higher efficiency of electroporation can be obtained when using plasmid DNA, which is isolated from Burkholderia rather than from E. coli. In addition, the DNA must have a very low ionic strength and thus may be additionally purified by dialysis (Nitrocellulose membrane filter, Millipore). A required number of micro- electroporation cuvettes (0,2 cm) were initially precooled on ice. Depending on the concentration and size of the plasmid, 3-10 µl of the DNA were added to the cuvettes and mixed with one aliquot of cells. Electroporation was carried out using the program EC2 (MicroPulser) or the following instrumental set up: 600 ohm, 2,5 kV, 25 µF, time constant: 12-15 (Gene Pulser). After the pulse, the cells were directly added in 1 ml 2 x LB medium (2.4) and transferred into sterile EP. After incubation of samples at 30°C for 2 h on a shaking platform, the cells were plated on selective LB agar plates and stored for 1-2 days at 30°C.
3.9.3 Conjugational transfer of cosmids or plasmids into B. glumae Conjugations of the cosmids were performed as triparental spot matings with E. coli JM101 as the donor and E. coli pRK2013 as the helper strain. Conjugations of broad-host-range plasmids were performed as biparental spot matings with E. coli S17-1 as the donor strain. Overnight cultures of the recipients and logarithmic-stage cultures of the donor and helper strains were centrifuged, washed and resuspended in LB medium. In the case of using B. glumae as a recipient, it is preferable to culture the cells two days, whereby after one day, the culture is inoculated again in fresh LB medium. Aliquots of 1ml from each culture were combined, centrifuged and resuspended in 50 µl LB medium. In the case of transposon mutagenesis of B. glumae, 10 mM MgSO4 or 0,9% (w/v) NaCl, was also used for resuspension of the cultures. These mixtures were spotted onto a membrane filter (Schleicher & Schuell, 0,2 µm) on LB agar plates and allowed to dry. The plates were incubated overnight at 30°C. The next day, the spots were resuspended in 0,9% (w/v) NaCl and plated on selective LB agar plates containing the corresponding antibiotic needed for plasmid maintenance and Irgarsan (25 µg/ml final concentration) for contra selection of E. coli.
______33 3. Methods
3.10 Determination of protein concentration Protein concentrations were determined spectrophotometrically by the Bradford assay (1976) using bovine serum albumin as a standard.
3.11 TCA precipitation of proteins 1/10 volume of 1% (w/v) sodium dodecyl sulfate was added to the supernatant and incubated for 10 min at room temperature. Proteins were then precipitated with 1/10 volume of 70% (v/v) trichloracetic acid (TCA) and incubated on ice for 1 h. After centrifugation for 10 min (RT, 13000 rpm), samples were washed twice with ice-cold 80% (v/v) acetone and dried in a vacuum dryer (2.8). Finally, proteins were resuspended in 15 µl SDS buffer and incubated for 5 min at 95°C.
3.12 Polyacrylamide gel electrophoresis
3.12.1 SDS polyacrylamide gel electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was carried out with a 12-15% polyacrylamide gel as described by Laemmli (1970). Cells were adjusted to an OD580 of 0,15 resuspended in 15 µl SDS buffer and incubated for 5 min at 95°C. Protein containing supernatants were applied as described in 3.12. Electrophoresis was carried out using the Mini Protean II Dual Slap Cell (2.8) at 10-20 mA. Afterwards, proteins were stained with SDS staining solution containing Coomassie Brilliant Blue R250 (2.5).
3.12.2 Native polyacrylamide gel electrophoresis Native polyacrylamide gel electrophoresis was performed by using a 4-12% gradient polyacrylamide gel (Invitrogen) according to the protocol provided. Proteins were stained with the native staining solution containing Coomassie Brilliant Blue G250 (2.5).
3.13 Western blot Transfer of proteins from a SDS polyacrylamide gel to a PVDF membrane was performed under semi dry conditions using the Mini Trans-Blot Cell (BioRad). First, the PVDF membrane was shortly incubated in methanol, washed for 5 min. in A. dest and then equilibrated in Dunn Carbonat buffer (2.5) for about 5 min. The SDS gel was equilibrated in Dunn Carbonat buffer as well. Blotting was then carried out for 15 min at 15 mA followed by 20 min at 300 mA using Dunn Carbonat buffer. After the transfer, blocking of the membrane was achieved by incubation in TBST (2.5) containing 5% (w/v) milk powder. The following steps were then carried out using TBST. Since all applied antibodies were conjugated with horseradish peroxidase (HRP), detection of the antibodies could be performed by using the ECL-Western Blotting Detection system according to the protocol provided.
3.14 Protein chromatography Purification of the putative protease was performed twice under native conditions using Kpi- buffer with two different pH values. Since the corresponding gene was cloned with a C-terminal His-tag, purification of the protein could be achieved by nickel-nitrilotriacetic acid metal-affinity chromatography. For this, 5 or 8 liters of liquid culture of E. coli BL21(DE3) pETpro was grown at 37°C to an optical density at 580nm of 0,5. By adding 1 mM IPTG the cells were then induced and further grown for 4 h at 37°C. After harvesting the cells by centrifugation (5000 rpm, 15 min, 4°C), the pellets were resuspended in 25 ml ice-cold buffer (50 mM Kpi, 10 mM imidazole, pH 8,0 or pH 6,5 respectively). Disruption of the cells was performed by incubation for 30 min on ice with lysozyme (0,5 mg/ml,), DNase (5 µg/ml) and RNase (10 µg/ml) followed by sonication (10 times, 30 sec). In order to remove the debris, the samples were centrifuged again (14000 rpm, 20 min, 4°C) and filtrated (Schleicher &
______34 3. Methods
Schuell, 0,45 µm). The clear lysates were then applied on a nickel-nitrilotriacetic acid column (Ni-NTA superflow, 30 ml volume, Qiagen, Hilden) and washed with Kpi-buffer (50 mM Kpi, 20mM imidazole, pH 8,0/pH 6,5). After elution of the recombinant protein with 50 mM Kpi-buffer containing 250 mM imidazole (pH 8,0/ pH 6,5), the corresponding fractions were combined and desalted using a G25-sephadex column (2.8). An aliquot of 50 µl was collected from each purification step for SDS PAGE analysis. Protein concentrations were finally determined by the Bradford assay (3.10). Storage of the purified protein was carried out at for 4°C or at -20°C containing 50% (v/v) glycerol. 10 ml of the protein solution were also tested for lyophilization and resuspended in 2 ml 10 mM Kpi buffer (pH 8,0/ pH 6,5).
3.15 Preparative gel filtration Determination of the molecular weight of the purified protein was performed by preparative gel filtration using the Superdex 200 prep grade Hi Load 16/60 (2.8). Calibration of the column was carried out using the following standards: Ribonuclease (13,7 kDa), Chymotrypsinogen A (25 kDa), Ovalbumin (43 kDa), Albumin (67 kDa) and Aldolase (158 kDa). As a filtration buffer 10 mM Kpi buffer containing 150 mM KCl (pH 8,0/ pH 6,5) was used. The concentration of the analyzed protein was determined to be about 10-15 µg.
3.16 Enzyme activity assays Lipolytic activity was determined on indicator plates containing a tributyrin emulsion (per liter: 15 ml Tributyrin, 1,5 g Gum arabic, ad 30 ml A. dest) or spectrophotometrically with p-nitrophenylpalmitate as a substrate, basically as described by Winkler & Stuckmann (1979). Aliquots of 10-50µl of the supernatants were added to a total volume of 2 ml substrate and the ∆OD410 was measured over a time of 5 min. Relative lipolytic activity was calculated by correlating ∆OD410/min to the optical density of the cultures. In this thesis, all values of lipolytic activity are presented as relative lipolytic activities or in percentage, due to the production strain being protected by patents. Proteolytic activity was determined using skim milk agar plates (2.4) containing 1 mM IPTG. Single colonies of the expression cultures or lyzed cell extracts were placed onto the agar plates and incubated over night at 30°C (B. glumae) or 37°C (E. coli). Proteolytic activity can be detected by the formation of halos around the colonies or cell extracts. Biochemical characterization of the purified putative protease was performed using a microtiter plate assay (Taxa Profile E, Merlin, Bornheim-Hersel) containing substrates for 95 amino peptidase and protease reactions, 76 substrates for glycosidase, phosphatase and esterase reactions as well as 17 classical reactions at pH 8,2, pH 7,5, pH 5,5 and pH 4,0. The assay was performed according to the protocol provided. After incubation of the plates for 24 h at 30°C, evaluation was carried out visually. As a control, 10 mM Kpi buffer pH 6,5 was used.
3.17 Fluorescence measurements Samples of the test cultures were washed and resuspended in 2 ml PBS buffer (2.5). After determination of the OD580, the cells were adjusted to an OD580 of 0,4 in the case of the stable Gfp variant and to an OD580 of 1,0 in the case of the unstable Gfp variant (GfpLAA). Cell lyses were then carried out by incubation with lysozyme (0,5 mg/ml, 30 min on ice) followed by sonication (2 times, 10 sec, 2.8). Cell extracts containing the unstable protein GfpLAA were additionally supplemented with 20 µl protease inhibitor (Complete EDTA-free, Roche Diagnostics) in order to prevent further protein degradation. After centrifugation, green fluorescence was measured with a fluorescence spectrometer (2.8) set at an excitation wavelength of 485 nm and emission detection of 460 to 560 nm. The excitation slit was set at 7,5 nm and the emission slit at 10 nm. Documentation of the spectra was performed using the
______35 3. Methods
Fluorescence Data Manager Software (Perkin Elmer), evaluation of the date was carried out using the Microsoft Excel program.
3.18 Computational methods Analyses of DNA, amino acid sequences and construction of plasmid maps were carried out using the programs Edit Sequencer and Clone Manager for Windows 7.0 (Scientific and Educational Software, Durham, USA). Sequence alignments, open reading frame and database searches were performed using the standard programs of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov): Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990, Altschul et al., 1997) or Open Reading Frame-Finder (ORF-Finder) or of the European Bioinformatics Institute (EMBL-EBI, http://www.embl-ebi.ac.uk): Washington University Basic Local Alignment Search Tool Version 2.0 (WU-BLAST2, ClustalW) (Altschul et al., 1990, Altschul et al., 1997, Higgins et al., 1994). Prediction of a possible localization of the putative protease and computational analysis were performed using the prediction programs ProtParam tool (Gill and Von Hippel, 1989), PSORT-B (Gardy et al., 2003), SignalP 3.0 Server (Nielsen et al., 1997) of the Expert Protein Analysis System (ExPASY, http://www.expasy.org/tools) and the Helix-Turn-Helix prediction server of the Pole BioInformatic Lyonnais, Network Protein Sequence Analysis (PBIL, NPS, France, http://npsa-pbil.ibcp.fr) (Dodd and Egan, 1990). Further results of this work were digitalized and embedded in the manuscript using a scanner and a photo system (2.8). During data transmission and processing, original contents have not been changed.
______36 4. Results
4. Results
4.1 Development of a T7 RNA polymerase-based expression system in B. glumae For high-level expression of lipase variants, one goal of this thesis was to construct a suitable expression system for B. glumae. For this, the T7 RNA polymerase-based expression system was chosen, since it has been shown to be a very powerful system enabling high-level gene expression. First, the use of this system had to be explored in the wild type strain B. glumae PG1. Second, for industrial application, it was aimed at establishing the expression system in the production strain B. glumae LU8093. In the first step, the expression of plasmid-encoded T7 RNA polymerase was examined in B. glumae. Subsequently, it was aimed at integrating the T7 RNA polymerase gene into the chromosome of both B. glumae strains, while the lipAB-operon, encoding the lipase (LipA) and lipase-specific foldase (LipB), was cloned under the control of the T7 promoter into a suitable expression vector.
4.1.1 Expression of plasmid-encoded T7 RNA polymerase in B. glumae In order to investigate the expression of the T7 RNA polymerase gene (gene1 from bacteriophage T7) in B. glumae the broad-host-range vector pML5-T7 was used. This plasmid harbors the T7 RNA polymerase gene under the control of the E. coli lacUV5 promoter and the lacIq gene coding for the LacI repressor. Thus, expression of gene1 is induced by adding Isopropyl-β-D-thiogalactoside (IPTG) to the culture. IPTG binds to the LacI repressor and thus inhibits further repression of gene expression. The use of the E. coli lacUV5 promoter had not to be explored, since previous studies have shown that E. coli promoters are applicable in B. glumae (Frenken et al., 1992). By biparental conjugation the plasmid was transferred into the wild type and production strain. Expression of gene1 was induced by adding different concentrations of IPTG (0 mM – 2 mM) to the logarithmic cultures. After further incubation for 3 h, samples were taken and detection of synthesized T7 RNA polymerase was performed by immunoblotting using a T7 RNA polymerase specific antibody (Novagen, Madison, USA). As a positive control, a culture of E. coli DH5α harboring the plasmid pML5-T7 was used, in which expression of gene1 was induced with 0,5 mM IPTG. As a negative control, the same strain was used, but the cells were cultured without IPTG. Thus, the T7 RNA polymerase was not produced. In addition, the wild type and production strain of B. glumae lacking the plasmid pML5-T7 were used, in order to explore possible cross-reactions of cellular proteins with the T7 RNA polymerase antibody.
______37 4. Results
As shown in Figure 7 A, already a low concentration of 0,2 mM IPTG induces expression of the T7 RNA polymerase gene in B. glumae PG1, whereas higher concentrations of the inducer do not increase the level of protein production. Furthermore, in the absence of IPTG, the T7 RNA polymerase could not be detected, which indicated a very low or no background activity of the lacUV5 promoter in B. glumae. Thus, it is possible to strictly control expression of gene1 in B. glumae. Furthermore, it could be shown, that there is no cross- reaction of cellular proteins with the T7 RNA polymerase antibody. As expected, in the production strain B. glumae LU8093, expression of gene1 is feasible as well (Fig. 7 B). Interestingly, in all samples of B. glumae PG1 and LU8093, the detected amounts of T7 RNA polymerase were lower than in the corresponding cultures of E. coli, indicating a lower expression level of gene1 or a more rapid degradation of the T7 RNA polymerase in B. glumae.
A IPTG [mM] B IPTG [mM]
0 0,5 0 0,2 0,5 1 2 0 0,5 1
7 7 7 7 7 7 7 7 7 3 1 7 T T T T T T T T 9 - - - - G T - - T - - - - 0 5 5 5 5 5 P 5 5 5 5 8 5 L L L L L L L L L L U L M M M M M M M M M M p p p p p p p p p p 3 3 1 1 3 α α 1 1 1 9 9 9 5 5 G G G G G 0 0 0 H H P P P P P 8 8 8 D D U U U L L L
Fig. 7: Western blot analysis of the expression of plasmid-encoded T7 RNA polymerase in B. glumae PG1 (A) and B. glumae LU8093 (B). Expression of the T7 RNA polymerase gene was performed using the broad- host-range vector pML5-T7. Samples were taken 3 h after induction of gene expression with different concentrations of IPTG. Detection of synthesized T7 RNA polymerase was achieved using a T7 RNA polymerase specific antibody (Novagen, Madison, USA). As a control, E. coli DH5α harboring the plasmid pML5-T7 was used. Overall, expression of the T7 RNA polymerase gene in B. glumae was performed three times, leading to the same results.
Expression of the T7 RNA polymerase gene in B. glumae is possible and can be strictly controlled by using the E. coli lacUV5 promoter. Thus, establishment of a T7 RNA polymerase-based expression system in B. glumae is feasible.
4.1.2 Construction of a T7-expression strain of B. glumae In order to establish a stable T7 RNA polymerase-based expression system, the T7 RNA polymerase gene was integrated into the chromosome of B. glumae. Since a further aim of this study was to delete the chromosomal lipAB-operon of B. glumae, integration of gene1 into the chromosome was combined with the deletion of these two genes. As shown in
______38 4. Results
Figure 8, integration of gene1, under the control of the lacUV5 promoter, and the lacIq gene into the lipAB-operon was achieved by homologous recombination. Therefore, about 500 bp of the upstream and downstream region flanking the operon were used. Following this strategy, overexpression of plasmid-encoded lipase can be strictly controlled and performed without background activity of lipase. Additionally, the use of a selective marker was avoided, in order not to deal with the problem of secondary effects due to the presence of antibiotics. For integration of gene1 into the chromosome of B. glumae, the suicide vector pSUP202 was used. This vector can be mobilized in conjugational mating experiments, but does not replicate in non-enteric bacteria. Thus, after cloning a homologous DNA sequence into the vector, integration into the chromosome by homologous recombination occurs.
P up lacIq lac U V 5 gene1 down pSUPT7pollacIup/down pSUP202 pSUP202
Homologous recombination up lipA lipB down Chromosome of B. glumae PG1 / LU8093
PlacUV5 up lacIq gene1 down Chromosome of B. glumae PGT7lacI / LUT7lacI
Fig. 8: Schematic representation of the construction of an inducible T7-expression strain of B. glumae. Integration of the T7 RNA polymerase gene (gene1) was combined with the deletion of the chromosomal lipAB- operon of B. glumae. Therefore a suitable suicide vector was constructed containing gene1 under the control of the lacUV5 promoter, the lacIq gene, 460 bp of the upstream and 490 bp downstream region of the lipAB-operon of B. glumae (Fig. 34, Appendix). After integration of gene1 into the chromosome of B. glumae by homologous recombination, expression of the T7 RNA polymerase can be induced by the addition of IPTG.
A detailed scheme of the cloning strategy of the suicide vector pSUPT7polup/down is presented in Figure 34 (Appendix). Transfer of this vector into B. glumae PG1 and LU8093 was performed by biparental mating. For efficient mutant isolation, transconjugants were firstly selected for the integration of the complete suicide vector by single cross-over using chloramphenicol (200 µg/ml). At this point, integration of the suicide vector pSUPT7pollacIup/down into the chromosomal lipAB-operon of B. glumae PG1 was achieved. Four mutant strains, designated B. glumae PGpSUPT7lacI 1-4, were selected and integration of the T7 RNA polymerase gene was proven by colony PCR (data not shown). In the next steps, one of these mutants will be used to perform enrichment procedures in order to provide a second cross-over and therefore leading to the loss of the vector DNA.
______39 4. Results
4.1.3 Construction of expression vectors for high-level production of lipase in B. glumae For high-level production of lipase, a suitable expression vector was required, which harbors the wild type lipAB-operon of B. glumae under the control of the T7 promoter. This expression vector can first be used to perform overexpression studies in the recombinant strains. Subsequently, it is intended to use this vector to express lipase variants. In order to be able to exchange the wild type lipA gene with genes encoding lipase variants, the presence of suitable restriction sites in the lipAB-operon and the vector is required. Furthermore, it was necessary to use a mobilizable vector since conjugation is most efficient to transfer plasmids into B. glumae. In order to provide these requirements, two expression vectors were chosen. In the first case, the mobilizable broad-host-range vector pBBR1mcs was used. In the second case, a derivative of the pBBR1mcs was used, designated pBBR22b (Rosenau and Jaeger, 2004). The latter had been constructed by exchanging the multiple cloning site of pBBR1mcs with a PshAI/PpuMI-fragment of the commercially available expression vector pET22b (Novagen). Thus, the resulting vector pBBR22b can replicate in different Gram-negative bacteria as does the pBBR1mcs, but it additionally harbors some advantageous features of pET22b. It provides a lower background expression under non-inducing conditions due to the presence of a lac operator in combination with the T7 promoter as well as a constitutively expressed copy of the lac repressor gene. A strong ribosomal binding site enables an efficient translation initiation of target genes cloned into the polylinker region. Furthermore, in-frame fusions with the pelB signal sequence allow for a Sec-dependent translocation of overexpressed target proteins into the periplasm of several bacterial strains (Rosenau and Jaeger, 2004). Cloning of the lipAB-operon was performed using the plasmid pBP1500 (provided by BASF AG). This plasmid harbors a 2,9 kb EcoRI-fragment containing the lipAB-operon and 500bp of the upstream region of the lipAB-operon of B. glumae. Initially, this fragment was excised and cloned into the EcoRI-site of the vector pBluescriptKS (pBKS). Subsequently, the lipAB- operon was hydrolyzed by the restriction endonuclease HindIII and was cloned into the two expression vectors pBBR1mcs and pBBR22b, which were designated pBBRlipABT7, pBBRlipABlac and pBBR22lipABT7. A detailed scheme of the cloning steps is illustrated in Figure 35 (Appendix). Following this strategy, the 2,9 kb fragment was shortened at the upstream region, while still remaining its own ribosome binding site. Since only one restriction endonuclease was used to insert the lipAB-operon into the expression vectors, two
______40 4. Results possible orientations of the operon were achieved. In the case of pBBR22b, only one orientation was suitable, but in the case of pBBR1mcs both orientations could be used, since either the T7 or the lac promoter were located on each site of the polylinker. Thus, besides overexpression of lipase using the T7 RNA polymerase-based expression system, also constitutive expression from the lac promoter can be performed. Replacement of the lipA gene with genes encoding lipase variants can be performed using the restriction endonucleases PstI, which is located at the end of the lipA gene and SalI, which is located in the multiple cloning site of the vector pBBR1mcs (Fig. 35). These two restriction enzymes can also be used for cloning genes of lipase variants into the vector pBBR22lipABT7. As described above, one advantage of the plasmid pBBR22lipABT7 is that it provides an efficient initiation of lipA translation due to the presence of a strong ribosomal binding site. Furthermore, an in-frame fusion with the pelB signal sequence increases transport of the protein across the inner membrane into the periplasm of other host cells such as E. coli. In order to examine the functionality of the expression vectors, the three plasmids were transformed into E. coli BL21(DE3). Using lipase indicator plates containing tributyrin and 0,4 mM IPTG, expression of enzymatically active lipase could be detected by halo formation around the colonies. As illustrated in Figure 9, all three expression vectors showed a clear halo formation, while the control strains did not show any halo formation. As expected, in the case of a constitutive expression of the lipA gene, lipase production and thus halo formation was less compared to the overexpression of lipase using the T7 RNA polymerase-based expression system. Comparing the two plasmids in which the lipAB-operon was cloned under the control of the T7 promoter, the vector pBBR22lipABT7 displayed a larger halo formation than pBBRlipABT7, due to the above described advantages of the vector pBBR22b. Determination of lipolytic activity of lyzed E. coli cells revealed that lipase activity in all three cultures containing an expression vector was very low indicating that the largest fraction of expressed lipase forms insoluble inclusion bodies. Since this effect has already been reported in previous studies (Frenken, 1993), no further examinations were performed. With this experiment, a qualitative measurement of lipase production was aimed, which confirmed the correct cloning of the lipAB-operon.
______41 4. Results
Fig. 9: Expression of B. glumae lipase in E. coli BL21(DE3). The constructed expression vectors pBBRlipABT7, pBBRlipABlac and pBBR22- lipABT7 were transferred into E. coli BL21(DE3) and plated onto lipase indicator plates containing
7 s 7 tributyrin and 0,4 mM IPTG. Expression of 22b lac R mc BT B enzymatically active lipase in the periplasm of E. B pABT R1 B li B pA pA p 2 li li 2 B R coli BL21(DE3) was observed by halo formation p B BR BR B around the colonies. As controls, the expression B pB p p vectors pBBR1mcs and pBBR22b were transferred into E. coli BL21(DE3). The plates were incubated for 1 day at 37°C and 1 day at 4°C.
For the overexpression or constitutive expression of lipase, three different expression vectors were constructed, which contain the lipAB-operon of B. glumae under the control of the T7 or lac promoter, respectively. In addition, two of these vectors can be used to exchange the lipA gene with genes coding for lipase variants in one cloning step.
4.2 Identification of bottlenecks for an improved lipase production in B. glumae Apart from the construction of a suitable expression system, another aim of this study was to further improve lipase production in B. glumae. In the production strain B. glumae LU8093, the expression of lipase had already been increased by classical strain improvement. Since this method is limited with the increasing number of mutations, one way to further enhance lipase production is to specifically eliminate bottlenecks in the production pathway. In order to identify potential bottlenecks, which can be expected at the levels of gene expression, transport of the protein across the inner membrane, folding in the periplasm and secretion of the active enzyme into the extracellular medium, a cosmid library of B. glumae had to be constructed and screened. After identification of clones with increased or decreased lipase production, the corresponding genes should either be additionally integrated into the chromosome or deleted in the chromosome of B. glumae. In the case of identifying a new, but yet uncharacterized protein of B. glumae, a first characterization of this protein should be carried out.
4.2.1 Construction of two cosmid libraries of B. glumae PG1 and B. glumae LU8093 Since it was not known how many mutations had been introduced into the genome of B. glumae LU8093 and what had been changed in the production pathway of lipase, firstly, potential bottlenecks had to be identified in the genome of the wild type strain. Nevertheless,
______42 4. Results a cosmid library of the production strain was also constructed which enables to perform comparative analysis. Thus, a protein identified in B. glumae PG1 can be compared to the homologous protein of B. glumae LU8093. In this way, interesting mutations that had been introduced into the production strain may be identified. Construction of the two genomic libraries was performed using the cosmid pLAFR3, which allows cloning of DNA fragments of 15-30 kb. Cosmid vectors are plasmids containing one or two cos-sites from bacteriophage λ. They have the advantage of the replication mode of a plasmid and the ability to be packaged in vitro into bacteriophage λ heads. Depending on the size of the genomic DNA, it can be calculated, how many clones are necessary to cover the genome at least once by using the following equation (Seed et al., 1982):
Clones [N] = ln (1 – 0,99) / ln (1-size of cloned fragments/size of genome)
With the assumption of a genome size of 5-6 Mb, which is characteristic for species of the genera Burkholderia and Pseudomonas, and considering the smallest size of cloned DNA- fragments of 15 kb, a minimum of 2000 clones was calculated to be required for covering the genome of B. glumae at least once. A detailed protocol describing the cloning of genomic DNA of B. glumae into the cosmid pLAFR3 is given in chapter 3.5. The presence of the α-peptide of the ß-galactosidase in the cosmid allowed for blue-white screening of clones on selective agar plates. After transferring of all white clones into microtiter plates, randomly isolated cosmids were checked by restriction analysis revealing an insert size ranging from 20-25 kb (data not shown). In the case of the cosmid library of the wild type B. glumae PG1, a total number of about 9300 clones were selected resulting in a four-fold coverage of the genome of B. glumae PG1. The cosmid library of B. glumae LU8093 contains about 4500 clones, which covers the genome about two fold.
Two cosmid libraries of B. glumae PG1 and LU8093 are available covering the genome of B. glumae four-fold and two-fold, respectively.
4.2.2 Screening of the cosmid library of B. glumae PG1 led to the identification of 15 cosmids influencing lipase production in B. glumae In order to find bottlenecks in lipase production, cosmid clones displaying an improved or decreased lipase activity were identified. Therefore, screening of the two cosmid libraries ______43 4. Results was performed in B. glumae PG1 and B. glumae LU8093 using tributyrin agar plates, on which lipase producing strains could be identified by halo formation around the colonies (Fig. 10 A). Transfer of the cosmids into both B. glumae strains was carried out as triparental spot matings with E. coli JM101 as the donor and E. coli pRK2013 as the helper strain. Colonies displaying a larger or smaller halo formation than the control strain carrying only the cosmid pLAFR3 were isolated and reconjugated (Fig. 10 B). After screening of about 2500 clones of the cosmid library B. glumae PG1, 15 cosmids were found influencing lipase production in both B. glumae strains.
A B pLAFR3
Fig. 10: Screening of the cosmid library of B. glumae PG1 using lipase indicator plates. A: Lipase indicator plate containing tributyrin with various B. glumae LU8093 transconjugants is shown as an example. B: B. glumae LU8093 transconjugants harboring cosmids, which were reconjugated and picked on lipase indicator plates. As a control B. glumae LU8093 pLAFR3 was used.
Screening of the cosmid library of B. glumae PG1 led to the identification of 15
different cosmids influencing lipase production in B. glumae.
4.2.3 Homologous expression of subcloned genomic DNA of B. glumae PG1 After identification of cosmids, which influenced lipase production when being co-expressed in B. glumae, next step aimed at identifying the genes whose products caused the observed effects. Since the genomic DNA-fragments cloned into the cosmid pLAFR3 had a size of 20-25 kb, it was necessary to subclone the genomic DNA into a broad-host-range vector and to repeat homologous expression in B. glumae. In this way, it was possible to restrict the observed effects to a specific region of the genomic DNA and to identify the corresponding genes. After reconjugation of the 15 cosmids, two cosmids were finally selected for further analysis. While pLAFRPG 5 led to a decreased lipase activity, pLAFRPG 8 led to an increased lipase activity in the supernatant of B. glumae (Fig. 11 A+B).
______44 4. Results
Fig. 11: Effect of two cosmids A B on lipase activity of B. glumae LU8093 pLAFRPG 8 PG1 pLAFRPG 8 LU8093 (A) and B. glumae PG1 (B). The two cosmids pLAFRPG 5 and pLAFRPG 8 were reconjugated and the PG1 transconjugants were transferred pLAFRPG 5 on lipase indicator plates PG1 containing tributyrin. As a LU8093 control the cosmid pLAFR3 was pLAFRPG 5 pLAFR3 LU8093 conjugated. The plates were pLAFR3 incubated for 2 days at 30°C and one day at 4°C.
In the following steps, the genomic DNA present in the two cosmids was subcloned into the broad-host-range vector pBBR1mcs. Subsequent restriction analyses of several plasmids confirmed the presence of different sizes of genomic DNA of B. glumae PG1 (Fig. 12). Five plasmids containing different insert sizes were chosen and designated as pBBRPG 5/1, 5/3, 5/7, 8/1 and 8/3. Where, the first number refers to the cosmid of which the genomic DNA was subcloned, the second number refers to the number of the positive clone that was identified by restriction analysis.
Tab.8: Sizes of subcloned kb genomic DNA of B. glumae PG1 pBBR1mcs 4 3 Plasmids Size of inserts [kb] genomic 2 1,6 DNA pBBRPG 5/1 4,0 pBBRPG 5/3 4,7 pBBRPG 5/7 5,8 pBBRPG 8/1 1,5 pBBRPG Marker pBBRPG 8/3 4,3 5/1 5/3 5/7 8/1 8/3
Fig. 12: Restriction analysis of the plasmids pBBRPG 5/1, 5/3, 5/7, 8/1 and 8/3 harboring subcloned genomic DNA of B. glumae PG1. 1 µg DNA samples were hydrolyzed using the restriction enzymes XbaI and HincII and loaded onto a 0.8% agarose gel. In Table 8, the sizes of the subcloned genomic DNA-fragments of the two cosmids are summarized.
Subsequently, all five plasmids were transferred into B. glumae PG1 and LU8093 by biparental conjugation. Homologous expression studies were performed using PG medium supplemented with 1% (v/v) olive oil. After 24 hours, 2 ml samples were taken and used to determine the optical density of the cells (OD580) and lipolytic activity in the supernatants using p-nitrophenylpalmitate as a substrate. While the expression of pBBRPG 5/1 and pBBRPG 5/3 had no significant effect on lipase activity compared to the culture harboring the control vector pBBR1mcs, expression of pBBRPG 8/1 increased extracellular
______45 4. Results lipase activity of B. glumae PG1 about 87% and in B. glumae LU8093 about 28%. Expression of pBBRPG 5/7 significantly decreased lipase activity in both strains up to 80%, whereas expression of pBBRPG 8/3 led to a decreased lipase activity in the supernatant of B. glumae PG1 and LU8093 of 33-42% (Tab. 9).
Tab. 9: Lipolytic activity of B. glumae PG1 and B. glumae LU8093 harboring expression plasmids with different sizes of subcloned genomic DNA of B. glumae PG1. Lipase activity in the supernatant was determined spectrophotometrically using p-nitrophenylpalmitate as a substrate and is shown as relative lipolytic activity [OD410/OD580] and in percentage [%] according to the control strain harboring the vector pBBR1mcs.
Relative lipolytic Relative lipolytic B. glumae PG1 activity [%] B. glumae LU8093 activity [%] [OD410/OD580] [OD410/OD580 pBBR1mcs 0,3 ± 0,04 100 pBBR1mcs 4,2 ± 0,3 100 pBBRPG 5/1 0,27 ± 0,02 92 pBBRPG 5/1 4,1 ± 0,3 89 pBBRPG 5/3 0,26 ± 0,02 87 pBBRPG 5/3 4,5 ± 0,4 107 pBBRPG 5/7 0,04 ± 0,01 19 pBBRPG 5/7 0,7 ± 0,05 17 pBBRPG 8/1 0,56 ± 0,05 184 pBBRPG 8/1 5,4 ± 0,4 128 pBBRPG 8/3 0,2 ± 0,01 67 pBBRPG 8/3 2,4 ± 0,02 58
In order to identify genes located in the subcloned genomic DNA, the inserts of the three plasmids pBBRPG 5/7, 8/1 and 8/3 were subjected to DNA sequencing. By using the open- reading-frame search program (ORF Finder) of the National Center for Biotechnology Information (NCBI) followed by BlastP search (NCBI) or WU-Blast2 (EMBL-EBI) respectively, the following identities on the amino acid sequence level to other proteins in the database were found (Fig. 13):
______46 4. Results
EcoRI pBBRPG 5/7 HindIII
ORF 1 ORF 2 ORF 3 ORF 1: 889 aa, 76% identity to putative silver efflux pump AmrA of Burkholderia pseudomallei, 62% identity to putative outer membrane copper and drug transport protein of E. coli. pBBRPG 8/3
ORF 2: 408 aa, 77% identity to membrane fusion protein AmrB EcoRI ORF 7 ORF 8 HindIII of B. pseudomallei, 44% identity to membrane fusion protein Pseudomonas fluorescence. ORF 3: 458 aa, 71% identity to outer membrane protein OprA of ORF 9 B. pseudomallei, 45% identity to outer membrane protein OprM ORF 7: 250 aa, 37% identity to tetracycline resistance of P. aeruginosa. protein of Salmonella enterica, 40% identity to tetracycline efflux protein of E. coli. pBBRPG 8/1 ORF 8: 276 aa, 42% identity to hypothetical protein of ORF 5 EcoRI ORF 4 HindIII B. pseudomallei ORF 9:170 aa, 44% identity to putative sugar transporter of B. vietnamiensis ORF 6 ORF 4: 116 aa, 62% identity to conserved hypothetical protein of Burkholderia vietnamiensis ORF 5: 148 aa, 58% identity to hypothetical protein B. vietnamiensis ORF 6: 179 aa, 70% identity to peptidase C_56 of B. vietnamiensis, 67% similarity to YhbO of E. coli.
Fig. 13: Schematic representation of the identified open reading frames of the plasmids pBBRPG 5/7 and 8/1 and 8/3. After DNA sequencing of the subcloned genomic DNA of B. glumae PG1, the obtained data were subjected to an open reading frame search program (ORF Finder, NCBI) in order to identify open reading frames (ORF). The translated sequences of the largest ORFs were then compared to other proteins in the database by using the BlastP search program (NCBI).
Taken together, by subcloning the genomic DNA of the two cosmids pLAFRPG 5 + 8 into the broad-host-range vector pBBR1mcs and by performing subsequent homologous expression studies, it was possible to identify three plasmids, which influenced lipase production. Due to the smaller sizes of the genomic DNA-fragments, it was now possible to further analyze the existing open reading frames and to identify the genes whose products were responsible for the observed effects. Since co-expression of the plasmid pBBRPG8/1 resulted in an increase in lipase activity in the supernatant of both B. glumae strains, it was most interesting to identify the corresponding gene. Subsequent integration of additional copies of this gene into the genome of B. glumae should improve lipase production. Consequently, the following studies focused on the analysis of the genomic DNA of this plasmid.
______47 4. Results
Homologous expression studies of five plasmids containing subcloned genomic DNA- fragments of B. glumae PG1 revealed three plasmids, which influenced lipase production. While co-expression of pBBRPG8/1 led to an increased lipase activity, pBBRPG5/7 and 8/3 led to a decreased lipase activity in the supernatant of B. glumae PG1 and B. glumae LU8093.
4.2.4 Co-expression of a gene encoding a putative protease increases lipase production in B. glumae As illustrated in Figure 13, the subcloned genomic DNA of the plasmid pBBRPG8/1 contained three open reading frames of which the largest one (540 bp) coded for a protein, which was similar to bacterial proteases in the database, while the other two open reading frames did not show any significant similarity to other proteins in the database. In order to confirm that the observed effect of enhanced lipase activity could be attributed to the co- expression of the putative protease, firstly, homologous expression studies of the single gene had to be performed. With the knowledge of the DNA sequence, it was possible to design specific primers and to amplify the gene of the putative protease directly from the genome of B. glumae PG1. The resulting PCR-product, designated as pro, was cloned into the broad- host-range vector pBBR1mcs using the restriction sites HindIII and BamHI. In this way, the gene was cloned under the control of the lac promoter allowing a constitutive expression. The construct was named pBBRpro and was transferred by biparental mating into B. glumae PG1 and LU8093. Overall, four separate expression studies were performed as described in section 4.2.3. As shown in Figure 14 A+B, expression of the plasmid pBBRpro resulted in a significant increase in extracellular lipase activity of the wild type strain B. glumae PG1 of about 100%, while in the production strain B. glumae LU8093, an increase of about 30% was observed (Fig. 14 A+B). Consequently, with this experiment, it was confirmed, that the previous observed increase in lipase activity was achieved by co-expression of the putative protease.
______48 4. Results
A B
0,8 ty 10 vi 0,7 9 0,6 8 acti 580] c 7
D 0,5 yti
O 6 0,4
pol 5 i 0,3 4 D410/
ve l 0,2 3 [O ti [OD410/OD580] a 0,1 2 1 Rel 0,0 Relative lipolytic activity 0 24 h 24 h 24 h 24 h PG1 pBBR1mcs PG1 pBBR1pro LU8093 pBBR1mcs LU8093 pBBR1pro
Fig. 14: Lipolytic activity of B. glumae PG1 (A) and B. glumae LU8093 (B) expressing the plasmid- encoded putative protease. Expression studies were performed over 24 h using PG-medium containing 1% (v/v) olive oil. After 24 hours, the cells were harvested and used to determine the optical density (OD580). Lipase activity of the supernatants was determined spectrophotometrically by the hydrolysis of p-nitrophenylpalmitate, which is shown as relative lipolytic activity [OD410/OD580]. As a control, the expression vector pBBR1mcs was transformed. Since B. glumae LU8093 already produces higher amounts of lipase than B. glumae PG1, relative lipolytic activity is presented with different scales. Error bars indicate standard deviation in four separate experiments.
In order to test the putative protease for enzymatic activity, colonies obtained from the expression cultures were grown on skim milk agar plates. These indicator plates are suitable for general proteolytic activity, which can be detected by halo formation around the colonies. However, neither grown expression cultures nor lyzed cell extracts showed differences in halo formation (data not shown).
Co-expression of the gene encoding the putative protease resulted in a 2-fold increase in extracellular lipase activity in B. glumae PG1 and an increase of 30% of lipase activity in the supernatant of B. glumae LU8093.
4.2.5 The putative protease does not affect foldase production in B. glumae Based on recently published findings, which indicate the importance of a specific foldase in increased lipase activity (El-Khattabi et al., 2001; Rosenau, 2001), next aim was to examine, whether the putative protease influenced foldase production in both B. glumae strains. If co-expression of the putative protease influenced foldase production directly or indirectly, the resulting increase in lipase activity may be explained. By using samples of expression cultures in which the putative protease had been co-expressed (4.2.4) immunodetection was carried out. Samples of supernatant were treated with a lipase specific antibody, while the corresponding cell extracts were treated with a foldase specific antibody (provided by Prof. Tommassen, University of Utrecht, Netherlands).
______49 4. Results
In confirmation with the results of the enzyme activity assays, higher amounts of lipase could be detected in the supernatants of B. glumae PG1 and LU8093 in which the putative protease had been co-expressed, compared to the control strain harboring the vector pBBR1mcs (Fig. 15 A). In contrast, the corresponding cell extracts indicated no significant difference in the amount of produced foldase (Fig. 15 B). This suggested that the effect of increased lipase production is not achieved by affecting the expression of the lipase-specific foldase.
A Fig. 15: Western blot analysis of the supernatants (A) and cell extracts SN (B) of B. glumae PG1 and B. glumae Lipase LU8093 expressing the plasmid- PG1 PG1 LU8093 LU8093 encoded putative protease. Samples pBBR1mcs pBBRpro pBBR1mcs pBBRpro were taken after 24h. According to an
OD580 of 1,0, the corresponding B amounts of supernatants were used for TCA precipitation of the proteins. CE Detection of lipase and foldase was Foldase achieved using a lipase and foldase PG1 PG1 LU8093 LU8093 specific antibody (dilution 1:6000). As pBBR1mcs pBBRpro pBBR1mcs pBBRpro a control, cultures harboring the vector pBBR1mcs were used.
Co-expression of the gene encoding the putative protease does not affect foldase production in B. glumae
4.3 Characterization of the putative protease of B. glumae PG1 In order to get more information about the new protein, which had not been described in the literature yet, a further aim of this study was to perform a preliminary characterization. First, computational analysis was performed. In the second step, the pro gene was overexpressed in the heterologous host E. coli BL21(DE3) and the protein was purified by affinity chromatography. Finally, the molecular weight and the substrate spectrum of the protein were determined. When the amino acid sequence of the putative protease was compared to other proteins in the database, a conserved domain characteristic for the ThiJ/PfpI-family of proteins was found. Proteases that belong to this family are for example, the intracellular proteinase YhbO of E. coli, or the protease PfPI of Pyrococcus furiosus. As described in section 4.2.3, 67% of the amino acid residues of the putative protease were identical to YhbO of E. coli. Furthermore, 70% identity could be found to the annotated peptidase C56 of B. vietnamiensis, which also
______50 4. Results contains a conserved ThiJ/PfpI-domain. By using the ProtParam tool program provided by the Expasy-web site, a molecular weight of 19,6 kDa and a theoretical pI of 5,45 was calculated for the putative protease (Gill and von Hippel, 1989). According to the SignalP 3.0 prediction program (Nielsen et al., 1997), the amino acid sequence does not contain a signal sequence for secretion indicating a cytoplasmic localization, which was also confirmed by the prediction program for bacterial protein subcellular localization (PSORT-B; Gardy et al., 2003). Further structural comparison using the helix-turn-helix (HTH) prediction program (Network Protein Sequence Analysis; Dodd and Egan, 1990) revealed that the putative protease has no HTH-DNA binding domains. Consequently, the protein does not seem to be a transcriptional regulator. Due to the high level of amino acid sequence identity to other proteases such as the intracellular protease YhbO of E. coli or peptidase C56 of B. vietnamiensis, it is more likely, that the new protein of B. glumae also functions as a cytoplasmic protease.
4.3.1 Heterologous overexpression of the putative protease gene in E. coli BL21(DE3) and purification of the protein by affinity chromatography For heterologous overexpression of the putative protease, the gene was reamplified using primers with an NdeI and XhoI recognition site. The DNA-fragment was then cloned into the NdeI/XhoI-site of pET22b, thus being under the control of the T7 promoter and having a C-terminal 6x His-tag, which allows purification by nickel-nitrilotriacetic acid metal-affinity chromatography. The resulting plasmid was named pETpro and transformed into E. coli BL21(DE3). First, overexpression studies were performed at a small scale with 25 ml cultures as described in section 3.17 and samples were analyzed by SDS-PAGE (Fig. 16). Successful overexpression of the protein could be detected 2-6 hours after induction. According to a protein standard, the putative protease had a molecular weight of about 21 kDa, which was slightly higher than the calculated molecular weight of 19,6 kDa.
Fig. 16: Overexpression of the pro gene in
kDa E. coli BL21(DE3). Samples of the cultures M T T T 66 0 2 4 were taken 0, 2 and 4 hours (T0, T2, T4) after 55 induction with IPTG, adjusted to an OD580 of 0,15 and separated by SDS-PAGE (15%). As 36 control, the vector pET22b was transformed. 31 putative Proteins were stained with Coomassie Brillant Blue R250. M: Protein standard M12 21 protease (Invitrogen), 1: T0 E. coli BL21(DE3) pET22b, 14 2: T0 E. coli BL21(DE3) pETpro, 3: T2 E. coli BL21(DE3) pET22b, 4: T2 E. coli BL21(DE3) 1 2 3 4 5 6 pETpro, 5: T4 E. coli BL21(DE3) pET22b, 6: T4 E. coli BL21(DE3) pETpro.
______51 4. Results
When colonies obtained from the expression cultures were grown on skim milk agar plates, no differences in halo formation could be detected. Same results were obtained with lyzed cells incubated on skim milk agar plates (data not shown). Since it could be shown that overexpression of the putative protease did not lead to the formation of inclusion bodies, it was assumed that the protein was either inactive when expressed in E. coli or that skim milk was not the suitable substrate. Due to the fact that it was not possible to detect proteolytic activity, purification of the protein by nickel-nitrilotriacetic acid metal-affinity chromatography was performed twice under native conditions using Kpi-buffer with two different pH values (pH 8,0 and pH 6,5 respectively). Overexpression was performed in shaking flasks at a scale of 5 to 8 liter medium. Four hours after induction of gene expression, the cells were harvested and prepared for subsequent protein purification. After elution of the recombinant protein with 50 mM Kpi-buffer and 250 mM imidazole, the corresponding fractions were combined and desalted using a G25-sephadex column. A volume of 50 µl was saved from each purification step for SDS-PAGE analysis (Fig. 17). After desalting the eluate, the total volume of the purified protein containing fractions amounted 90 ml and 70 ml, respectively. According to the Bradford assay using bovine serum albumin as a standard, protein concentration of the first purification sample (50 mM Kpi-buffer pH 8,0) was 3 mg/ml, thus leading to an overall yield of protein of 270 mg, while the second sample (50 mM Kpi-buffer pH 6,5) revealed a concentration of 10,5 mg/ml thus leading to an overall yield of 735 mg. The higher amount of protein concentration in the second sample can be attributed to the fact that a larger amount of cells has been applied in the second purification procedure. While in the first procedure about 5 g of cells were applied, the second time about 14 g cells were used. Additionally, the total volume of the second procedure was smaller thus the protein was more concentrated.
k D a M 1 2 3 4 5 6 75 55 40 31 putative
21 protease 14
Fig. 17: SDS PAGE analysis of purification of putative protease by affinity chromatography. 10 µl of each purification step were loaded onto a 15% SDS gel. M: Prestained Protein Marker (Fermentas), 1: Cell lysates (1:5), 2: Fraction 5 (Flow through), 3: Fraction 8 (Wash fraction), 4: Fraction 42 (Wash fraction), 5: Eluat (10 µg), 6: purified protein after G25 (10 µg). The presence of a smaller band of this protein gel could be attributed to be an artifact of sample preparation.
______52 4. Results
4.3.2 Determination of the molecular weight of the putative protease In order to determine the molecular weight and possible multimerization of the native protein, preparative gel filtration was carried out. About 10-15 µg of the purified protein were loaded onto a Superdex® 200 prep grade column. Overall this procedure was performed three times using the purified protein at pH 8,0, at pH 6,5 as well as a lyophilized sample at pH 6,5. By means of calibration with standard proteins, a molecular weight of about 39 kDa was calculated, which represents the dimeric form of the putative protease. The same results were obtained by performing a native polyacrylamide gel electrophoresis. Using 10 µg of the purified protein and 20 µl of the mixture of native protein standards, which were used for the calibration of the Superdex® 200 prep grade column, two protein bands of the putative protease could be detected, which corresponded to the monomer (about 20 kDa) and dimer (39 kDa) of the protein (Fig. 18).
Fig. 18: Native polyacrylamide gel electrophoresis of the purified putative protease 10 µg of the purified putative protease and 20 µl of a kDa protein standard were loaded onto 4-12% polyacrylamide gel. Proteins were 43 stained with Coomassie Brillant Blue G250. The upper band represents the 25 putative protease having a molecular weight of about 40 kDa, which corresponds to a homodimer. The lower band represents the monomer of the protein with a molecular weight of about 20 kDa.
Protease M
4.3.3 The putative protease displays amino peptidase activity First biochemical characterization comprised the determination of enzyme activity. Due to the fact that so far it had not been possible to determine proteolytic activity, a general enzyme activity assay was performed. In this assay the purified protein was tested in a microtiter plate containing 188 different substrates for amino peptidases, proteases, glycosidases, phosphatases and esterases at different pH values. The test was performed three times by applying 20 µl of the purified protein (10mM Kpi pH 6,5) in each cavity. As a control 10 mM Kpi buffer pH 6,5 was used. After incubation of the plates for 24 h at 30°C, evaluation was carried out visually. On the left site (Fig. 19), cavities A 1-H 12 as well as I 1-P 12 contained substrates for protease and peptidase activity. In the middle part, cavities A 13-F 18 and I 13-N 18 contained substrates for glucosidase and phosphatase activity at pH 7,5 and pH 8,2, whereas cavities A 19-F 23 and I 19-N 23 contained the same substrates but at pH 4,0 and pH 5,5. Additionally, substrates for some classical tests such as indolformation, nitrate-, nitrite-
______53 4. Results reduction were supplemented in the cavities G 18-H 24 and O 18-P 24. In the case of obtaining a positive reaction, substrates for protease and peptidase activity turned red, while substrates for glucosidases, phosphatases and esterases displayed a yellow color. As highlighted in the figure, distinct positive reactions could be detected with three substrates for peptidase activity: L-alanine-ß-naphtylamine (C3), lysin-ß-naphtylamine (A 4) and arginin-ß- naphtylamine (B 4), as well as with two substrates for glucosidase activity: p-nitrophenyl-a-I- arabinopyranosid pH 7,5 (A 13) and p-nitrophenyl-ß-d-galactopyranosid pH 7,5 (G 13) and pH 5,5 (C 21). While the detected proteolytic activity corresponds with the predicted function, which was based on amino acid sequence analysis in the database, the two positive reactions towards the glucosidase substrates cannot be explained at this point. In a separate experiment, 5 µg of the purified protein were tested for its activity towards p-nitrophenylpalmitate (data not shown). In this assay, no activity was detected confirming that the observed increase in lipolytic activity during co-expression of the putative protease in B. glumae was a result of an increase in extracellular lipase activity.
1 2 3 4 5 6 7 8 9 10 11 ... 13 ... 15 ... 17 ... 19 .....21...... 24 A peptidase activity B putative red = positive C protease D in 10 mM orange = negative E F Kpi G pH 6,5 H glucosidase I J activity K 10 mM yellow = positive L Kpi transparent = M pH 6,5 negative N O P
Fig. 19: Determination of enzyme activity of the putative protease using a microtiter plate assay. Overall, the microtiter plate contains 188 different substrates for amino peptidases, proteases, glycosidases, phosphatases and esterases at different pH values (see text for further explanation). In each cavity, 20µl of the purified protein (10mM Kpi pH 6,5) was applied. As a control, 10 mM Kpi buffer pH 6,5 was used. After incubation of the plates for 24 h at 30°C, evaluation was carried out visually.
______54 4. Results
Computational analysis of the amino acid sequence revealed that the putative protease of B. glumae harbors a conserved domain of the ThiJ/PfpI-family and displays highest similarity to other intracellular proteases, present in the database. The gene was successfully overexpressed in E. coli BL21(DE3) and the protein purified by affinity chromatography using the pET-overexpression system. Following this strategy, a monomer and homodimer of the protein could be detected, having a molecular weight of about 20 kDa and 39 kDa, respectively. Furthermore, a general enzyme activity assay confirmed that the putative protease displays proteolytic activity.
4.4 Elimination of bottlenecks for an improved lipase production in B. glumae After identification of potential bottlenecks in lipase production of B. glumae, a subsequent goal of this study was to eliminate these bottlenecks in order to construct an improved lipase production strain. Therefore, it was aimed at integrating genes into the chromosome of B. glumae whose proteins led to an increase in lipase production, while products of genes, which caused a decrease in lipase activity, should be deleted. Thus, in the case of the putative protease, additional copies of the gene had to be integrated into the genome of B. glumae, while in the case of the putative transport proteins, the corresponding chromosomal genes were inactivated. In order to investigate the resulting effects in the wild type and the production strain, mutagenesis was carried out in both B. glumae strains.
4.4.1 Tn5 mutagenesis of B. glumae A potent tool for integrating a DNA-fragment without extensive DNA homologies is the transposon mutagenesis. The transposon Tn5, which is extensively used in bacterial molecular genetics, is capable of transposing at a high frequency (10-2 to 10-3 in E. coli). It has a low insertional specificity and therefore can insert into a large number of locations in bacterial genomes. Furthermore, it exhibits a low probability of genome rearrangements upon transposition and a high stability once established in the genome (Berg et al., 1983; de Bruijn and Lupski, 1984). The structure of Tn5 is shown in Figure 20 A. Tn5 has an overall size of 5818 bp, consisting of two inverted repeats (IS50L and IS50R), which flank a unique region. This region contains three genes, which confer resistance to the antibiotics kanamycin (kan gene), bleomycin (ble gene) and streptomycin (str gene). Within the resistance marker, a
______55 4. Results number of convenient restriction sites enable to clone a gene of interest intoTn5 without affecting its transposition properties (Simon et al., 1989). The gene encoding the putative protease was excised from the vector pETpro and cloned into the SmaI-site of pSUP2021 as depicted in the Figure 20 B. The resulting plasmid was named pSUPTn5pro and transferred into the B. glumae strains PG1 and LU8093 by biparental conjugation or electroporation using kanamycin (25-50 µg/ml) for the selection of positive clones. However, until now, it was not possible to select any Tn5-carrying clone. Same results were obtained with the vector pSUP2021, which only carries the transposon Tn5, indicating that the conditions promoting Tn5 transposition still have to be optimized in B. glumae.
XhoI
pro XbaI PT7
Amp SphI PpuMI A BstBI BamHI pETpro SmaI PshAI XmaI XmnI SalI BstXI FseI lacI
IS50L P IS50R ori
Km Bleo Strep Tnp XhoI/XbaI Tn5 Inh XbaI XhoI B Restriction of pSUP2021 pro with SmaI blunting ends
Ligation
mob
IS50L
P Cm Km
pSUPTn5pro Bleo' Amp pro 'Bleo
Strep
TnpInh Tc´ IS50R
Fig. 20: Transposon mutagenesis of B. glumae. (A) General structure of the transposon Tn5. The two inverted repeats (IS50L and IS50R) flank an operon containing three genes, which confer resistance to the antibiotics kanamycin (kan gene), bleomycin (ble gene) and streptomycin (str gene). The genes are transcribed from a promoter located inside of IS50L. IS50R encodes the transposase responsible for Tn5 transposition (tnp gene) as well as a transposition inhibitor (inh gene). (B) Schematic drawing of the construction of pSUPTn5pro. The pro gene was excised from pETpro using XbaI +XhoI and was cloned blunt-end into the SmaI-site of pSUP2021. The resulting vector pSUPTn5pro which is used for transposon mutagenesis carries the pro gene in the orientation as depicted in this figure.
______56 4. Results
4.4.2 Inactivation of a putative tripartite efflux system in B. glumae As described in section 4.2.3, co-expression the plasmids pBBRPG5/7 and pBBRPG8/3 both caused a decrease in extracellular lipase activity in the wild type and production strain. Consequently, in this case, it should be examined whether lipase activity could be improved by inactivating these genes. While the open reading frames of the plasmid pBBRPG8/3 did not show significant similarities to other proteins in the database, the detected open reading frames present in the subcloned genomic DNA-fragment of pBBRPG5/7, revealed a high similarity to the tripartite efflux-system AmrA-AmrB-OprA of B. pseudomallei. Many studies on efflux systems in Gram-negative and Gram-positive bacteria have been performed showing that these pumps are involved in the secretion of a variety of different molecules such as ions, solvents and drugs (Nishino and Yamaguchi, 2001; Ramos et al., 2002; Nies, 2003). In P. aeruginosa, it has been reported that the efflux pumps MexAB-OprM and MexGH-OpmD also expel acylated homoserine lactones, the signal molecules of quorum sensing circuits (Schweizer, 2003). Consequently, it was of great interest to investigate the effect of inactivation the putative efflux-encoding genes in the chromosome of B. glumae. In order to find out whether the effect was caused by one gene product or whether all three gene products had the same effect, each gene should be inactivated separately. For a rapid procedure, vector insertion mutants were constructed. Therefore, the first 500 bp of the ompC and mfpB gene were amplified from the plasmid pBBRPG 5/7 and cloned separately into the HindIII/BamHI-site of the suicide vector pSUP202 (Fig. 21). In the case of the epA gene, cloning of the DNA fragment containing 500 bp of the gene was not achieved. The resulting suicide vectors pSUPompC` and pSUPmfpB` were transferred into B. glumae PG1 and LU8093 by biparental conjugations. Correct integration of the suicide vector into the corresponding gene was proven by colony PCR (data not shown). Integration of the two suicide vectors could be achieved in both B. glumae strains. The resulting mutants were designated B. glumae PGompC-, PGmfpB-, LUompC- and LUmfpB-.
______57 4. Results
PompC1 P ompC mfpB epA ompC mfpB2 mfpB epA
PompC2 Chromosome of B. glumae PmfpB1 PCR PG1 / LU8093 PCR
ompC´ mfpB´
HindIII BamHI HindIII BamHI
Tcr Tcr pSUP202 pSUP202 pSUPompC´ pSUP202 pSUP202 pSUPmfpB´ HindIII BamHI HindIII BamHI
Homologous recombination Homologous recombination
ompC mfpB epA Chromosome of B. glumae ompC mfpB epA PG1 / LU8093
ompC´ Tcr´ ompC´ mfpB epA ompC mfpB´ Tcr´ mfpB´ epA Cmr pSUP202 Cmr pSUP202
Chromosome of B. glumae Chromosome of B. glumae - - PGompC / LUompC PGmfpB- / LUmfpB-
Fig. 21: Schematic representation of the inactivation of the chromosomal ompC and mfpB genes in B. glumae PG1 and B. glumae LU8093. Therefore, 500 bp of the genes ompC and mfpB were amplified from the plasmid pBBRPG5/7. After cloning of the DNA-fragments into the suicide vector pSUP202, the resulting plasmids were transferred into B. glumae by biparental mating. Inactivation of the chromosomal genes was achieved by integration of the corresponding suicide vectors into the genes by homologous recombination.
In the next step, the recombinant strains were tested for improved lipase activity. Therefore, the mutants were grown in PG-medium containing 1% (v/v) olive oil and chloramphenicol (100 µg/ml) or carbenicillin (150 µg/ml) for the stability of the vector integration. In order to be able to evaluate extracellular lipase activities of the mutants, both parental strains B. glumae PG1 and LU8093 were grown as well. After 15, 20 and 25 hours samples were taken and prepared for the determination of the optical density and lipolytic activity as described before (4.2.3). At each time point, the observed relations between the different cultures were the same, so that only the obtained data after 25 h are presented (Tab. 10). In all cases inactivation of the two genes omp and mfp in B. glumae PG1 and LU8093 resulted in a significant decrease in extracellular lipase activity compared to the parental strains. Interestingly, the mutants also showed a significant reduced growth and seemed to be more susceptible to higher concentrations of the applied antibiotics than the parental strains. Consequently, inactivation of two genes of the putative efflux system seemed to have a negative effect on the cell metabolism and thus did not lot lead to an increase in lipase production in B. glumae.
______58 4. Results
Tab. 10: Growth and lipolytic activity of vector-insertion mutants of B. glumae PG1 and B. glumae LU8093. Lipase activity of the supernatants was determined spectrophotometrically by the hydrolysis of p-nitrophenylpalmitate, which is shown as relative lipolytic activity [OD410/OD580]. OD580: optical density of the cells. Standard deviations are presented as means of three separate experiments.
Strain Time [h] OD580 Relative lipolytic activity
[OD410/OD580] B. glumae PG1 25 9,2 ± 1,3 0,4 ± 0,15 B. glumae PGompC- 25 2,3 ± 0,5 0,1 ± 0,05 B. glumae PGmfpB- 25 2,5 ± 0,4 0,17 ± 0,05 B. glumae LU8093 25 10,6 ± 1,4 5,8 ± 1,2 B. glumae LUompC- 25 2,6 ± 0,3 0,9 ± 0,2 B. glumae LUmfpB- 25 2,9 ± 0,4 1,2 ± 0,3
While inactivation of two genes of a putative efflux system did not lead to an improved lipase production in B. glumae, integration of the gene coding for the putative protease by transposon mutagenesis, is a promising approach to optimize lipase production in B. glumae.
4.5 Analysis of regulation of lipase gene expression in B. glumae Besides the specific elimination of potential bottlenecks in the production pathway, another promising approach to improve lipase production is to create optimal growth conditions promoting lipase expression during cell growth. In the literature, several studies describe the use of growth media supplemented with lipids, detergents or alkanes as being suitable for the induction or improvement of lipase production in different bacteria. In B. glumae PG1, it was found that lipase gene expression is subjected to catabolite repression and that olive oil induces lipase expression (Frenken, 1993). Therefore, a further aim of this study was to analyze the effect of emulsifiers and detergents on lipase production in B. glumae. In order to explore differences in lipase production of the wild type and the production strain, comparative analysis was performed. Therefore, several growth studies followed by lipase activity assays and western blot analyses were carried out. In addition to the above aim, the molecular mechanisms, which were responsible for the observed effects, were examined. This investigation comprised the construction and measurement of transcriptional and translational lipA::gfp fusions, as well as the determination of the transcription start of lipA of B. glumae.
______59 4. Results
4.5.1 Emulsifiers and detergents enhance extracellular lipase production in B. glumae In the first step, the wild type B. glumae PG1 and production strain B. glumae LU8093 needed to be compared with respect to growth rate and lipase production using different media and carbon sources. Therefore, culture media were supplemented with 1% (v/v) olive oil, 5-10% (v/v) hexadecane, or 0,5% (w/v) glucose. Test cultures of 25 ml were incubated over 48 hours at 30°C. After 5, 10, 15, 24, 30 and 48 hours, 2 ml samples were taken and used to determine the optical density and lipolytic activity as described before (4.2.3). As shown in Figure 22, in both strains, highest growth rate and lipolytic activity could be obtained by adding 1% olive oil and 10% hexadecane to the culture medium. The increase in lipase activity in the wild type strain amounted up to 150%, while in the production strain an increase up to 250% was achieved. Growth rate in rich medium such as LB or NB medium was comparable to the growth rate in the defined PG medium containing 10% hexadecane and/or 1% olive oil. However, lipase activity was different in these cultures. In cultures of B. glumae PG1, no lipase activity could be observed in LB or NB medium (Fig. 22 A), confirming that lipase gene expression is subjected to catabolite repression (Frenken, 1993). In PG medium containing only 5% hexadecane as a sole carbon source, poor growth of the cells was observed, indicating, that this alkane is not a preferred C-source. The same phenomenon was observed in B. glumae LU8093. Interestingly, the production strain showed distinct lipolytic activity in cultures containing a carbohydrate as a carbon source (Fig. 22 B). Although, the values of measured lipase activity in these cultures amounted half of the values of the cultures supplemented with 1% olive oil, lipase activity was comparable to the activity of the wild type under induced conditions. Due to these findings, it was suggested, that in B. glumae LU8093 lipase gene expression was not subjected to catabolite repression anymore, probably as a consequence of the classical strain improvement.
______60 4. Results
A 1,0 0,9 580]
D 0,8
410/O 0,7 D 0,6
0,5
0,4
0,3
0,2 lative lipolytic activity [O e 0,1 R
0,0 5 5 5 5 5 5 5 10 15 24 30 48 10 15 24 30 48 10 15 24 30 48 10 15 24 30 48 10 15 24 30 48 10 15 24 30 48 10 15 24 30 48 LB NB PG + 1% Oil PG + 1% Oil + PG + 0,5% PG + 0,5% PG + 5% HD 10% HD Glu Glu + 10% HD Time [h]
14
B
580] 12 D 10 D410/O
O 8 y [
ivit 6
ic act 4 yt l o 2 ve lip i 0 5 5 5 5 5 5 5 10 15 24 30 48 10 15 24 30 48 10 15 24 30 48 10 15 24 30 48 10 15 24 30 48 10 15 24 30 48 10 15 24 30 48 Relat LB NB PG + 1% PG + 1%Oil PG + 0,5% PG + PG 5% HD Oil + 10% HD Glu 0,5%Glu + 10% HD Time [h]
LB NB PG medium PG medium PG medium PG medium PG medium medium medium + 1% Oil + 1% Oil + 0,5% Glu + 0,5% Glu + 5% HD + 10% HD + 10% HD B. glumae PG1 7,8 6,4 8,8 10,2 5,8 4,6 1,8 OD580, t=24 h ± 20% B. glumae LU8093 7,6 7,2 9,5 10,6 6,3 5,1 1,6 OD580, t=24 h ± 20%
Fig. 22: Lipolytic activity and growth of B. glumae PG1 (A) and B. glumae LU8093 (B) under different physiological conditions. Test media LB-, NB- or PG-medium were supplemented with 1% (v/v) olive oil (oil) and/or 5-10% (v/v) hexadecane (HD) or 0,5% (w/v) glucose (Glu). After 5, 10, 24, 30 and 48 hours 2 ml samples were taken. Lipolytic activity is shown as relative lipolytic activity [OD410/OD580]. Since B. glumae LU8093 already produces higher amounts of lipase than B. glumae PG1, relative lipolytic activity is presented with different scales. Error bars indicate standard deviation in three separate experiments.
The next step comprised the examination of the effect of additives other than hexadecane on lipase production in B. glumae. Therefore, further growth studies were carried out. Since preceding experiments showed that highest lipolytic activity occurred between 15 and 24 hours, 2 ml samples were taken only after 15, 20 and 25 h. After determination of the optical density (OD580) of the cells and lipolytic activity in the supernatant, samples were prepared for subsequent SDS-PAGE and Western blot analysis. Detection of lipase and foldase were performed in the supernatants as well as cell pellet by using specific antibodies against LipA and LipB from B. glumae. In conformity with the determined lipolytic activity of both strains, it was possible to detect the corresponding amounts of lipase in the supernatant and foldase in the cell pellets,
______61 4. Results respectively (Fig. 23+24 B and C). As expected, glucose and sucrose do not induce lipase gene expression in the wild type strain. Likewise, 1% (v/v) glycerol or 10mM tributyrin seem not to be suitable in liquid culture to induce lipase production. Thus, no lipase or foldase was detected in the corresponding samples (Fig. 23 B and C). In contrast, the production strain expressed the lipase gene in the presence of carbohydrates, although the resulting lipase activity was significantly lower compared to the cultures supplemented with olive oil and hexadecane or a detergent (Fig. 24 B). In the presence of olive oil, high levels of extracellular lipase activity can also be achieved by adding gum arabic, Triton X-100 or Tween 80 to the cultures. The increase in lipase activity was similar to the increase achieved by the addition of hexadecane, i.e. 150% in B. glumae PG1 and 250% in B. glumae LU8093. Consequently, it is possible to replace hexadecane by another emulsifier or detergent if olive oil is used as a carbon source. However, this effect was not observed in the presence of glucose indicating a different effect of these sugars on the mechanism of catabolite repression. Interestingly, if a carbohydrate such as sucrose replaced olive oil, Triton X-100 or Tween 80 still increased lipase activity to the same level as obtained with olive oil and the detergents. The same effect could be observed by using maltose as a carbon source (data not shown). In order to investigate the effect of the detergents and hexadecane, further Western blot analyses were performed in which samples of the supernatant were treated with the foldase antibody, while samples of the cells were treated with lipase antibody. As illustrated in Fig. 23+24 D, samples of cultures, which were supplemented with a detergent, displayed distinct amounts of foldase in the supernatant. Thus, the detergents destabilized the bacterial membranes, which led to a release of foldase. In the case of lipase, it could be shown that the addition of a detergent or hexadecane to an olive oil-containing culture led to a reduced amount of lipase in the cell extracts, indicating a solubilization of cell-bound lipase (Fig. 23+24 E). In summary, the obtained data show that under the described conditions, lipase production can be improved 2-3 fold by supplementing the growth media with 1% (v/v) olive oil and 5-10% (w/v) hexadecane. In the presence of olive oil, the stimulatory effect of hexadecane can also be achieved by using 0,1% (v/v) of a detergent or by using 1% (v/v) of an oil emulsion with gum arabic. Furthermore, in the presence of sucrose or maltose as a carbon source, the same increase in lipase activity can be obtained by supplementing the growth medium with 0,1% (v/v) of a detergent. Besides a probable detachment of cell-bound lipase by hexadecane or a detergent, it could be shown that the detergents also destabilize the cellular membranes leading to a release of periplasmic lipase and foldase.
______62 4. Results
A
1,2
1,0
0,8
0,6
0,4
0,2 Relative lipolytic acitivity [OD410/OD580]
0,0 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 123456789101112131415 Time [h] B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 SN Lipase
C CE Foldase
D SN Foldase
E CE Lipase
Fig. 23: Effect of carbon sources and chemical agents on lipase and foldase production in B. glumae PG1. 25 ml PG-medium were supplemented with different additives. After 15, 20 and 25 h, 2ml samples were taken and used to determine the optical density and lipolytic activity, which is shown as relative lipolytic activity [OD410/OD580]. For subsequent Western blot analysis, the cells were adjusted to an OD580 of 0,15, whereas proteins of supernatants were precipitated using trichlor acetic acid. Detection of lipase and foldase was achieved using lipase and foldase specific antibodies (1:6000 dilution in TBST-buffer). Error bars indicate standard deviation of three individual experiments. A: Lipolytic activity of supernatants B: Western blot analysis (SN = supernatant, t=25 h, lipase antibody) C: Western blot analysis (CE = cell extract, t =25 h, foldase antibody) D: Western blot analysis (SN = supernatant, t= 25 h, foldase antibody) E: Western blot analysis (CE = cell extract, t =25 h, lipase antibody) 1: 1% (v/v) oil 9: 0,5% (w/v) sucrose 2: 1% (v/v) oil + 10% (v/v) hexadecane (HD) 10: 0,5% (w/v) sucrose + 10% (v/v) HD 3: 1% (v/v) oil emulsion with gum arabic 11: 0,5% (w/v) sucrose + 0,1% (v/v) Tween 80 4: 0,5% (w/v) glucose 12: 10 mM tributyrin 5: 0,5% (w/v) glucose + 10% (v/v) HD 13: 10 mM tributyrin + 10% (v/v) HD 6: 1% (v/v) oil + 0,1% (v/v) Tween 80 14: 1% (v/v) glycerol 7: 1% (v/v) oil + 0,1% (v/v) Triton X-100 15: 1% (v/v) glycerol + 10% (v/v) HD 8: 0,5% (w/v) glucose + 0,1% (v/v) Tween 80
______63 4. Results
A
16
14 580]
OD 12 410/ 10 y [OD t i v 8 acti c
yti 6 pol i 4 ve l ti a l e
R 2
0 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 15 20 25 123456789101112131415 Time [h]
B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 SN Lipase
C CE Foldase
D
SN Foldase E
CE Lipase
Fig. 24: Effect of carbon sources and chemical agents on lipase and foldase production in B. glumae LU8093. Experimental procedures were analogous to the procedure described in Figure 23. Error bars indicate standard deviation of three individual experiments. A: Lipolytic activity of supernatants B: Western blot analysis (SN = supernatant, t=25 h, lipase antibody) C: Western blot analysis (CE = cell extract, t =25 h, foldase antibody) D: Western blot analysis (SN = supernatant, t= 25 h, foldase antibody) E: Western blot analysis (CE = cell extract, t =25 h, lipase antibody) 1: 1% (v/v) oil 9: 0,5% (w/v) sucrose 2: 1% (v/v) oil + 10% (v/v) hexadecane (HD) 10: 0,5% (w/v) sucrose + 10% (v/v) HD 3: 1% (v/v) oil emulsion with gum arabic 11: 0,5% (w/v) sucrose + 0,1% (v/v) Tween 80 4: 0,5% (w/v) glucose 12: 10 mM tributyrin 5: 0,5% (w/v) glucose + 10% (v/v) HD 13: 10 mM tributyrin + 10% (v/v) HD 6: 1% (v/v) oil + 0,1% (v/v) Tween 80 14: 1% (v/v) glycerol 7: 1% (v/v) oil + 0,1% (v/v) Triton X-100 15: 1% (v/v) glycerol + 10% (v/v) HD 8: 0,5% (w/v) glucose + 0,1% (v/v) Tween 80
______64 4. Results
In B. glumae PG1 lipase gene expression is subjected to catabolite repression, while in the production strain B. glumae LU8093 this repression is abolished. With respect to an improved lipase production, 15 different combinations of carbon sources and compounds in the growth medium were tested. Highest extracellular lipase activity was obtained by using 1% (v/v) olive oil and 5-10% (v/v) hexadecane, 0,1% (v/v) Triton X-100 or Tween 80 or by using 1% (v/v) of an olive oil emulsion with gum arabic. In B. glumae PG1 an increase up to 150% in extracellular lipase activity was achieved. In B. glumae LU8093 lipase activity was enhanced up to 250%. If olive oil is replaced by sucrose or maltose, Triton X-100 and Tween 80 have the same increasing effect on lipase activity in both B. glumae strains. While hexadecane seems to detach cell-bound lipase, the detergents destabilize the bacterial membranes, thus leading to a release of lipase as well as foldase.
4.5.2 Use of the green fluorescent protein for the analysis of lipase gene expression in B. glumae In order to study the regulation of gene expression, the construction of reporter fusions is required. In this thesis, the green fluorescent protein (Gfp) was chosen as a reporter. This protein of the marine invertebrate Aequorea victoria is a single autofluorescent protein, which emits green light (508nm) when excited at ultraviolet light (395nm). First, it was examined, whether the Gfp protein is a suitable reporter to measure gene fusions in vivo in B. glumae. Therefore, plasmids were constructed in which the Gfp encoding gene was under the control of the non-inducible lac promoter resulting in a constitutive gene expression in B. glumae. Altogether, two variants of the Gfp protein were tested, designated as Gfpmut3 and GfpLAA. In Gfpmut3, two mutations had been introduced into the gene, which led to the amino acid substitutions: Ser65Gly and Ser72Ala. As a consequence, the mutant protein is approximately 20 times more fluorescent than the wild type Gfp when excited at 488nm (Cormack et al., 1996). Furthermore, the protein is very stable and has a half-life of about 24 hours. On the other hand, the GfpLAA is a destabilized version of the Gfpmut3 with a half-life of about 190 minutes. This is due to three additional amino acid residues (LAA) at the C-terminus that targets the protein to degradation by specific intracellular proteases (Andersen et al., 1998).
______65 4. Results
Both genes encoding the Gfpmut3 and GfpLAA were available in two different vectors, designated as pSWgfp and pSWgfpLAA, which had recently been constructed (Wilhelm, unpublished). Since it was not possible to introduce these plasmids into B. glumae, the Gfp encoding genes were excised and cloned into HincII/EcoRI site of the vector pBBR1mcs-2, which had already been proven to be suitable for B. glumae. This derivative of pBBR1mcs harbors a gene conferring on resistance to kanamycin instead of chloramphenicol. The resulting plasmids were named pBBKgfpPlac and pBBKgfpLAAPlac and were transferred into B. glumae PG1 and LU8093 by biparental conjugation or electroporation. Expression cultures were inoculated in 25 ml PG-medium supplemented with 1% (v/v) olive oil and incubated over 48 hours at 30°C. After 10, 15, 24 and 48 h, 2 ml samples were taken and prepared for subsequent fluorescence measurements. As shown in Figure 25 A+B, both Gfp variants could be detected in both B. glumae strains PG1 and LU8093, respectively. Due to the constitutive expression, the stable Gfp protein accumulates with increasing cell density reaching values of about 258±12 relative fluorescence units [RFU]. In contrast, the intensity of fluorescence of the unstable Gfp variant was significantly lower than of the stable variant with values of about 31-36 ±2 RFU (Fig. 25 C+D).
A PG1 pBBKgfpPlac B LU8093 pBBKgfpPlac
300 300
250 250
200 200
U 150
150 RFU RF
100 100
50 50
0
0 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 0 1 1 1 2 2 3 3 4 50 5 5 51 51 5 5 52 52 5 5 52 53 5 5 53 53 5 506 508 509 511 512 514 515 517 518 520 521 523 524 526 527 529 530 532 533 535 536 538 539 Wavelength of emission [nm] Wavelength of emission [nm] t 10h t 15h t 24h t 48h t 10h t 15h t 24h t 48h
______66 4. Results
C PG1pBBKgfpLAAPlac D LU8093 pBBKgfpLAAPlac
40 40
35 35
30 30
25 25
20 U 20 RFU RF 15 15
10 10
5 5
0 0
6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 8 2 6 0 4 8 2 6 0 0 1 1 2 2 3 3 3 4 06 10 14 18 22 2 26 2 30 3 34 3 38 4 5 50 51 5 51 5 51 52 5 52 5 52 5 5 53 5 53 5 5 50 5 51 5 51 5 52 5 5 5 5 5 5 5 5 5 5
Wavelength of emission [nm] Wavelength of emission [nm]
t 10h t 15h t 24h t 48h t 10h t 15h t 24h t 48h
Fig. 25: Expression of Gfpmut3 and GfpLAA in B. glumae PG1 (A+C) and B. glumae LU8093 (B+D). The measured values of fluorescence refer to an OD580 of 1,0/ml cells and are presented as relative fluorescence units [RFU] based on the wavelength of mission. Due to the lower values of fluorescence of GfpLAA a different scale was chosen. Data are expressed as means of three different experiments having a standard deviation of less than 10%.
Next, it was investigated whether both Gfp proteins had a similar half-life in B. glumae as described for other bacteria in the literature. Therefore, the same expression cultures were prepared as described before. After incubation for 15 h, chloramphenicol (200 µg/ml) was added to the cultures. Due to the effect of the antibiotic, protein synthesis was inhibited. Incubation of the cultures harboring pBBKgfpPlac was continued for 30 h, while incubation of the cultures harboring pBBKgfpLAAPlac was continued for 5 h. 2 ml samples were taken after 24, 26 and 30 h or after 2, 3, 4 and 5 h, respectively. In both B. glumae cultures expressing the stable Gfp protein, the described half-life of 24 h could be confirmed (Fig. 26 A). In contrast, in the cultures expressing the unstable Gfp variant a decreased half-life of about 140 min was observed, compared to the half-life of about 190 min which has been described in the literature (Andersen et al., 1998). Thus, this Gfp variant seems to be rapidly degraded in B. glumae.
______67 4. Results
A pBBKgfpPlac B pBBKgfpLAAPlac
180 35
160 30 140 25 120
100 20
RFU 80 RFU 15 60 10 40 5 20
0 0 0242630 023 Time [h] Time [h]
Fig. 26: Determination of the stability of Gfpmut3 (A) and GfpLAA (B) in B. glumae PG1 and B. glumae LU8093. Inhibition of protein synthesis was achieved by adding chloramphenicol (200 µg/ml) to the cultures. Samples were taken at different time points after inhibition of protein synthesis. Standard deviation in three individual experiments was less than 10%. The measured values are presented with different scales as relative fluorescence units [RFU] at maximum emission wavelength of 515 nm.
The green fluorescent protein is a suitable reporter to measure gene expression in B. glumae. While the variant protein Gfpmut3 is very stable, having a half-life of 24 h, the variant GfpLAA is rapidly degraded in B. glumae and thus has a half-life of only 140 min.
4.5.3 Effect of hexadecane and detergents on lipase gene expression in B. glumae In order to investigate the underlying molecular mechanisms of the observed effects of the supplemented compounds to the culture media (4.4.1), transcriptional and translational reporter fusion with both Gfp variants and the lipA gene of B. glumae were constructed and analyzed. The advantage of the stable Gfp variant was, that due to its stability, it was easier to detect in the cells. On the other hand, by using the unstable Gfp variant, information about time-dependent expression was obtained. In the case of a transcriptional fusion, the reporter gene carries its own start codon and ribosome binding site for recognition of the translational machinery, whereas in a translational fusion, the reporter genes lacks its own ribosome binding site and is directly fused in frame with the start codon of the target gene (Fig. 27). As a consequence, the translational fusion employs results of transcriptional and translational regulation, whereas the transcriptional fusion only gives information about regulation at the transcriptional level.
______68 4. Results
Px gene of interest reporter gene Px gene of interest reporter gene
rbs rbs rbs
Transcriptional gene fusion Translational gene fusion
Fig. 27: Schematic representation of transcriptional and translational reporter gene fusions. In the case of a transcriptional fusion, the reporter gene carries its own start codon and ribosome binding site for recognition of the translational machinery, whereas in a translational fusion, the reporter genes lacks its own ribosome binding site and is directly fused in frame with the start codon of the interesting gene. As result one fusion protein is synthesized.
For the construction of the transcriptional and translational lipA::gfp(LAA) fusion, 460 bp of the upstream region of the lipAB-operon of B. glumae PG1 and LU8093 were used. A detailed scheme of the cloning strategy is presented in Figure 36 (Appendix). Construction of the negative controls was achieved by cloning only the genes gfpmut3 and gfpLAA into the EcoRI/HindIII-site of pBBRmcs-2. All reporter gene fusions were inserted into the vector pBBRmcs-2, under the control of the T7 promoter, which is not recognized by the bacterial RNA polymerase and therefore silent in the absence of T7 RNA polymerase. Thus, expression of the reporter gene was dependent on the promoter present in the upstream region of the lipAB-operon. Correct integration of the different gene fusions was finally confirmed by restriction analysis as well as DNA-sequencing of the inserts (data not shown). Figure 28 shows an overview of the constructed lipA::gfp fusions.
______69 4. Results
up PG1 gfp up LU gfp Plip rbs Plip rbs
TCF Transcriptional Translational pBBKmcs pBBKmcs fusion (TCF) fusion (TLF)
pBBKgfpupPG1 pBBKgfpupPG1
up PG1 gfp(LAA) up LU gfp(LAA) pBBKgfpupLU pBBKgfpupLU Plip Plip pBBKgfpLAAupPG
pBBKgfpLAAupLU TLF pBBKmcs pBBKmcs
Fig. 28: Schematic representation of the constructed transcriptional and translational lipA::gfp-fusions of B. glumae PG1 and B. glumae LU8093. The upstream sequences of the lipAB-operon of B. glumae PG1 (upPG1) and LU8093 (upLU) containing the promoter, ribosome binding site (rbs) and ATG-start codon of lipA were PCR amplified and cloned in front of the reporter genes gfpmut3 or gfpLAA, respectively. In the case of the transcriptional fusion (TCF), the upstream sequence was cloned ahead of the reporter gene maintaining its own ribosome binding site, whereas in case of the translational fusion (TLF), the upstream sequence was cloned in frame with the reporter gene. Thus, the ATG-start codon of lipA represented the ATG-start codon of the gfpmut3 or gfpLAA, respectively.
After transfer of the plasmids into B. glumae PG1 and LU8093, expression studies were performed over 24 h using under the following growth conditions: 25 ml PG-medium + 1) 1% (v/v) olive oil 2) 1% (v/v) olive oil + 10% (v/v) hexadecane, 3) 0,5% (w/v) sucrose or glucose 4) 0,5% (w/v) sucrose or glucose + 0,1% (v/v) Tween 80. According to preceding growth studies, measurements of the transcriptional reporter gene fusions in B. glumae PG1 showed that olive oil induces lipase gene expression, while in the presence of a carbohydrate lipase gene expression is not induced. Consequently, in the cultures containing olive oil (and hexadecane) relative fluorescence units [RFU] of 62-68 ±4 could be measured, while in the cultures supplemented with sucrose or glucose, less than 10 RFU were measured. No differences in the relative fluorescence could be detected in the cultures containing only 1% olive oil or 1% olive oil and 10% hexadecane, indicating that hexadecane has no effect on lipA transcription in the wild type B. glumae PG1. In contrast, a significant difference could be observed in the cultures containing 0,5% sucrose or 0,5% sucrose and 0,1% Tween 80. While in the presence of sucrose as a sole carbon source no fluorescence could be detected, the addition of a detergent led to an increase in fluorescence intensity. The measured values of fluorescence were thereby as high as the measured values of the cultures containing olive oil (and hexadecane). Thus, the addition of a detergent to the culture containing sucrose somehow induced lipase expression. Same phenomenon was
______70 4. Results observed in the cultures of the production strain B. glumae LU8093. In addition, in conformation with the results of previous growth studies, lipA transcription was also detected in the culture supplemented with glucose or sucrose only. These findings confirmed that in B. glumae LU8093 lipase gene expression is not subjected to catabolite repression anymore. Interestingly, the measured values of fluorescence in all cultures of the production strain were up to 65% higher than the corresponding values of the wild type cultures, indicating that the level of transcription of lipA in the production strain is increased.
A PG1 pBBKgfpup PG1 (TCF) B LU8093 pBBKgfpup LU (TCF)
80 140
70 120
60 100 50 80 U 40 RFU RF 60 30 40 20
10 20
0 0 1% oil 1% oil + 10% 0,5% glucose 0,5% sucrose 0,5% sucrose 1% oil 1% oil + 10% 0,5% glucose 0,5% sucrose 0,5% sucrose hexadecane + 0,1% Tween hexadecane + 0,1% Tween 80 80
Fig. 29: Fluorescence measurements of transcriptional lipA::gfp-fusion of B. glumae PG1 (A) and B. glumae LU8093 (B). B. glumae strains PG1 and LU8093 harboring plasmid-encoded a lipA::gfp fusion were grown for 24 h in PG medium supplemented with 1% (v/v) olive oil, 1% (v/v) olive oil + 10% (v/v) hexadecane, 0,5% (w/v) glucose, 0,5% (w/v) sucrose or 0,5% (w/v) sucrose and 0,1% (v/v) Tween 80. The measured values of fluorescence were calculated referring to an OD580 of 1,0/ml cells and are presented with different scales as relative fluorescence units [RFU] at maximum emission wavelength of 515 nm. The standard deviation in three individual experiments was less than 10%.
Analysis of the expression of the translational lipA::gfp-fusions of both B. glumae strains was performed under the same conditions as the transcriptional fusions. Again, in both B. glumae strains, significant fluorescence was detected in the cultures containing olive oil, olive oil and hexadecane or sucrose and Tween 80 (Fig. 30 A+B). If olive oil was replaced by sucrose or glucose, expression of lipA only occurred in the production strain B. glumae LU8093 (Fig. 30 B). In agreement with preceding growth studies in the presence of a carbohydrate, the inducing effect of Tween 80 on lipA expression could only be observed if sucrose was used as carbon source, but not glucose. Thus, both carbohydrates are involved in catabolite repression, but the effect on lipase production in conjunction with a detergent is different. Comparing the cultures, which contained olive oil or olive oil and hexadecane, only a minor difference of 23±2 RFU was detected. This confirmed the obtained data of the transcriptional reporter gene fusions that hexadecane does not have a significant effect on lipase gene expression. ______71 4. Results
As described before, by using the unstable Gfp variant (GfpLAA), information about the time-dependent expression of lipA could be obtained. Analysis of the translational lipA::gfpLAA fusions were analogous to preceding experimental procedures. As shown in Figure 30, concerning lipase expression, the relations between the different cultures were similar to the results, which were obtained with the stable Gfp variant (Gfpmut3). However, due to a rapid degradation of GfpLAA, the detected green fluorescence was generally lower than the fluorescence of Gfpmut3. Interestingly, in the presence of olive oil (and hexadecane) fluorescence could still be detected after 48 h, indicating a continuous expression of lipase under inducing conditions (data not shown).
A PG1 pBBKgfp(LAA)up PG1 (TLF) Fig. 30: Fluorescence measurements of translational lipA::gfp(LAA)- 120 fusion of B. glumae PG1 (A) and 100 B. glumae LU8093 (B). B. glumae 80 PG1 and B. glumae LU8093 harboring 60 plasmid-encoded a lipA::gfp(LAA) RFU 40 fusion were grown for 24 h in PG medium supplemented with 1% (v/v) 20 olive oil, 1% (v/v) olive oil + 10% 0 (v/v) hexadecane, 0,5% (w/v) sucrose, il ne se 80 se 80 o ca ro n co n 1% e c ee lu ee 0,5% (w/v) sucrose and 0,1% (v/v) ad su w g w ex % T % T h 0,5 % 0,5 % Tween 80, 0,5% (w/v) glucose or 0,5% % 0,1 0,1 10 + + se se il + ro co (w/v) glucose and 0,1% (v/v) Tween o c lu 1% su g % % 80. The measured values of 0,5 0,5 fluorescence were calculated referring lipA::gfp lipA::gfpLAA to an OD580 of 1,0/ml cells and are presented as relative fluorescence units B LU8093 pBBKgfp(LAA)up LU (TLF) [RFU] at maximum emission
200 wavelength of 515nm. The standard 180 deviation in three individual 160 experiments was less than 10%. 140 120 100
RFU 80 60 40 20 0 l i ne se 80 se 80 o ca ro n co n 1% e c ee lu ee ad su w g w ex % T % T h 0,5 % 0,5 1% % 0,1 0, 10 + + + se se il ro co o c lu 1% su g % 5% 0,5 0, lipA::gfp lipA::gfpLAA
Analysis of lipA::gfp fusions revealed that lipase gene expression is at least regulated on the transcriptional level, which is enhanced in the production strain B. glumae LU8093. While hexadecane does not influence lipA transcription, Tween 80 induces lipase gene expression in the presence of sucrose.
______72 4. Results
4.5.4 Determination of the transcription start of lipA in B. glumae Since the measurements of the reporter gene fusions confirmed that lipase gene expression in B. glumae is subjected to regulation, the transcription start of lipA and potential promoter sequences had to be identified. In order to determine the transcription start, primer extension analysis was performed. Therefore, the enzyme reverse transcriptase, a primer in 3´ 5´ direction and single stranded mRNA as a starting template is used in vitro for first strand cDNA synthesis. In order to get sufficient amounts of lipase-specific mRNA, B. glumae PG1 and LU8093 were grown for about 10-15h in PG-medium containing 1% (v/v) olive oil. After isolation of total RNA, samples were examined by agarose gel electrophoresis (data not shown). Determination of the transcription start was then performed using the fluorescence-labeled primers PE1 and PE2 which hybridize with its 3`OH-end at the positions +10 and +50 concerning to the start codon of lipA (Fig. 31). At the same time, the upstream region was subjected to DNA-sequencing using the same oligonucleotides, in order to be able to determine the position of the transcription start. In three individual experiments, several signals were detected, of which one signal was most significant and reproducible. Thus, one start of transcription could be determined to be located 78 bp ahead of the ATG-start codon of lipA. Subsequently, the upstream sequence was checked for putative promoter sequences. A consensus sequence for a putative σ70-dependent promoter sequence could not be detected, instead two putative sequences for the σ54-dependent promoter were identified, which were similar to the consensus sequences for σ54-dependent promoters described in the literature: GG(N8)TTGC (Barrios et al., 1999). A characteristic feature of σ54-dependent promoters is their conserved position around –24 and –12 nucleotides upstream of the transcriptional start site, instead of the typical –35 and –10 boxes. Furthermore, in contrast to the σ70-dependent promoter, the initiation of transcription at the σ54-dependent promoter cannot be initiated spontaneously, but requires the activity of a cognate activator protein (Gralla, 1990; Morett and Segovia, 1993). The first sequence in the upstream region of the lipAB-operon that was similar to this consensus sequence was identified 129-142 bp ahead of the lipA start codon. The second similar sequence was located 90-104 bp ahead of the start codon of lipA which was in agreement with the detected transcription start localized 78 bp ahead of the lipA start codon. In the following figure, the localization of the transcription start and putative σ54-dependent promoter sequences of lipA are presented schematically.
______73 4. Results
150 ATCCAAACGGCCGTCTGATTGTAGACAGGAGCCGCGCCGC 111 110 CATGTTTCACTCGGCACTTGCCGCTCGAGCGTGCCCGACGA 70
+ 1 lipA lipB -24/-12
54 PE1 σ -promoter PE2
G: start of transcription (+1) putative sequence for σ54-dependent promoter
Fig. 31: Localization of the transcription start and σ54-dependent promoter sequences of lipA in B. glumae. By performing primer extension analysis using two different oligonucleotides (PE1 and PE2), one transcription start of the lipAB-operon could be localized 78bp ahead of the lipA start codon. Further analysis of the DNA sequence led to the identification of two putative σ54-dependent promoter sequences. The start of transcription is designated +1 and the localization in the sequence is highlighted in orange. The putative σ54- dependent promoter sequences are highlighted in green.
By performing primer extension analysis, one transcription start could be determined 78 bp ahead of the ATG-start codon of lipA. Furthermore two putative σ54-dependent promoter sequences were identified. One sequence was localized in a characteristically distance of -24/-12 bp to the transcription start. The second putative σ54-dependent promoter sequence was identified to be localized 90-104 bp ahead of the lipA start codon indicating a complex regulation of lipase gene expression.
4.5.5 Effect of a mutation in the upstream region of the lipAB-operon in B. glumae LU8093 on lipase gene expression Based on the findings, that in the production strain, lipase gene expression was not subjected to catabolite repression anymore, the question was whether the upstream sequence of the lipAB-operon contained one or more mutations that could explain the observed effects. Therefore, the two upstream sequences of both strains were compared by using the program “BLAST 2 Sequences” (National Center for Biotechnology Information: Basic Local Alignment Search Tool). In this way, one mutation could be identified 129 bp upstream of the lipA start codon (Fig. 32). Interestingly, this mutation is located in one of the putative σ54-dependent promoter sequences, which were identified before (4.5.5). While the wild type sequence contains a thymine (T) at this position, the sequence of the production strain displays a cytosine (C). As a consequence, the 3´-end of the putative σ54-dependent promoter sequence is changed from a slightly different sequence compared to the consensus sequence ______74 4. Results to an exact consensus sequence for the σ54-dependent promoter: GG-N8-TTGC (Barrios et al., 1999). Another interesting finding was, that a second putative consensus sequence could be identified in this region, which was affected by this mutation. As underlined in Figure 32, the sequence CTGA(N6)ACAG is similar to the E. coli consensus sequence GTGA(N6)TCAC for the CRP protein (cAMP response protein) which is a component of the catabolite repression system (Brown and Callan, 2003). Since in the production strain, lipase gene expression is enhanced and not subjected to catabolite repression anymore, it may be possible that this mutation thereby plays an important role.
GGCCGTCTGATTGTAGACAG GGCCGTCTGATTGCAGACAG
PG1 LU8093
GG(N8)TTGC: putative sequence for the σ54-dependent promoter GTGA(N6)TCAC: putative sequence of a CRP-binding site
Fig. 32: Sequence alignment of a part of the upstream sequence of the lipAB-operon of B. glumae PG1 and B. glumae LU8093. Comparison of the DNA-sequences was performed using the program Blast2 sequences (NCBI). The illustrated DNA-sequence is located 137-197bp ahead of the lipA start codon with the mutation being located 129bp ahead of start codon. The black box frames the putative sequence of a σ54- dependent promoter (highlighted in green) and the putative sequence of the CRP-binding site (underlined)
In order to investigate whether this mutation resulted in an altered regulation of lipase gene expression, a further study was performed in which the lipA::gfp-fusions of the two strains were exchanged. Thus, the constructs containing the fused upstream region of the wild type B. glumae PG1 were transferred into the production strain B. glumae LU8093, whereas the corresponding constructs of B. glumae LU8093 were transferred into B. glumae PG1. The subsequent expression studies and fluorescence measurements were carried out in the same way as previous expression studies. When the lipA(PG)::gfp fusion was expressed in B. glumae LU8093, fluorescence could only be detected in the cultures containing 1% (v/v) olive oil or 1% olive oil (v/v) and 10% (v/v) hexadecane (Fig. 33). Thus, induction of lipase gene expression by olive oil was maintained. However, it could be observed that the measured values of fluorescence were about 55% lower compared to values of green fluorescence that were measured with the own lipA::gfp- fusion. This indicated that the upstream region of the wild type was not as well recognized as the own upstream region of the production strain. In contrast, lipase expression was not
______75 4. Results induced in the cultures containing sucrose as carbon source indicating that the effect of catabolite repression was present again, which could not be eliminated by the presence of the detergent as previously observed. In the case of the expression of lipA(LU)::gfp in B. glumae PG1, the effect was even more dramatic. As shown in Figure 33, no fluorescence could be detected in any culture. Even under inducible conditions lipase gene expression did not occur. These findings led to the assumption, that the identified mutation in the upstream sequence of the production strain plays an important role in the regulation of lipase gene expression.
PG1 pBBKgfpupLU (TLF) LU8093 pBBKgfpupPG1 (TLF)
80
70
60
50
U 40 RF 30
20
10
0 1% oil 1% oil + 10% 0,5% sucrose 0,5% sucrose + hexadecane 0,1% Tween 80
Fig. 33: Fluorescence measurements of translational lipA::gfp-fusions of B. glumae PG1 (A) and B. glumae LU8093 (B). Plasmid-encoded a lipA::gfp fusions of both B. glumae strains were exchanged and grown for 24 h in PG medium supplemented with 1% (v/v) olive oil, 1% (v/v) olive oil + 10% (v/v) hexadecane, 0,5% (w/v) sucrose or 0,5% (w/v) sucrose and 0,1% (v/v) Tween 80. The measured values of fluorescence were calculated referring to an OD580 of 1,0/ml cells and are presented as relative fluorescence units [RFU] at maximum emission wavelength of 515 nm. In three individual experiments, standard deviation was less than 10%.
The upstream region of the lipAB-operon of B. glumae LU8093 contains a mutation, which has a significant effect on the regulation of lipase gene expression.
______76 5. Discussion
5. Discussion
The Gram-negative bacterium B. glumae is a plant pathogen causing grain and seedling rot of rice plants as well as wilting symptoms in various solanaceous crops (Iwai et al., 2002; Yeonhwa et al., 2003). Since these field crops are economically important, especially in South-East Asian countries, main research on B. glumae concerns the biosynthesis, regulation and mode of action of the phytotoxin produced by the bacteria. Nevertheless, B. glumae also produces an extracellular lipase, which turned out to be useful for a variety of different industrial processes (Schmid et al., 2001). Moreover, B. glumae is not pathogenic for humans and can thus be used for biotechnological applications. Consequently, a high-level production of this lipase in B. glumae is required. Within the scope of this thesis, 1) a T7 RNA polymerase based-expression system for B. glumae was developed. 2) In order to improve lipase production in B. glumae two potential bottlenecks were identified. One protein was purified and preliminarily characterized. 3) Regulation of lipase gene expression in B. glumae was investigated.
5.1 Development of a T7 RNA polymerase-based expression system in B. glumae Besides the development of potent protein engineering methods for the improvement of existing biocatalysts at the molecular level, another important aspect of modern biotechnology comprises the production of these biocatalysts at a large scale. Therefore, the construction of a suitable overexpression system constitutes an important task (Rosenau and Jaeger, 2004). One example for a powerful expression system is the T7 RNA polymerase-based expression system, which has originally been developed for E. coli. Although, many recombinant proteins can be cloned and expressed at high levels in E. coli using this expression system, it is sometimes preferable to express the target gene in the homologous strain. Two major drawbacks of E. coli are that under standard conditions, the bacterium does not secrete proteins into the extracellular medium and that heterologous overexpression is often accompanied by misfolding and intracellular deposition of the protein in form of insoluble inclusion bodies (Gragerov et al., 1992; Whitchurch and Mattick, 1994; Hartl, 1996; Francetic et al., 2000). This is particularly true for the heterologous overexpression of lipases originating from the genera Pseudomonas and Burkholderia (Rosenau and Jaeger, 2004). As depicted in the introduction, the production of enzymatically active lipase requires not only the presence of its cognate foldase, but also the assistance of a variety of different cellular proteins (Rosenau et al., 2004). Thus, in order to achieve high-level production of enzymatic
______77 5. Discussion active lipase in E. coli a number of these proteins would have to be co-expressed. Moreover, since secretion of the enzyme into the extracellular medium is especially important for industrial down stream processes, an efficient secretion mechanism would have to be constructed in E. coli. Consequently, for large-scale production of lipase, homologous expression systems are preferred. Recently, the T7 RNA polymerase-based expression system has been adapted to P. aeruginosa enabling to specifically overexpress lipase in the homologous strain (Rosenau et al., 1998). However, P. aeruginosa is an opportunistic pathogen for animals and humans (Winkler et al., 1985, Van Delden and Iglewski, 1998) and is therefore not suitable for industrial applications. With increasing restrictions to use potential pathogenic bacteria for biotechnological applications, it is desirable to establish this overexpression system in a non- pathogenic or only plant-pathogenic lipase producing strain such as B. glumae. A further advantage of B. glumae is that E. coli promoters are applicable (Frenken et al., 1992). Thus, for the expression of the T7 RNA polymerase gene in B. glumae, the E. coli lacUV5 promoter can be used. In R. capsulatus, for example, the use of E. coli promoter is not possible. Therefore, in order to establish the T7 RNA polymerase-based expression system in this strain, firstly, a suitable promoter had to be found (Drepper et al., 2005). By using the broad-host-range vector pML5-T7, which carries the T7 RNA polymerase gene (gene1) under the control of the inducible lacUV5 promoter, successful expression of the T7 RNA polymerase in B. glumae PG1 and B. glumae LU8093 was achieved and confirmed by immunoblotting. Furthermore, it could also been shown that already a low concentration of 0,2 mM IPTG was sufficient to induce expression of gene1 (Fig. 7). In the absence of the inducer no T7 RNA polymerase was detected, which indicated a very low background activity of the lacUV5 promoter and confirmed that expression of gene1 can be strictly controlled. One interesting observation during the Western blot analysis was that the amount of T7 RNA polymerase detected in B. glumae was lower than in E. coli. One reason might be, for example, that the protein is more rapidly degraded in B. glumae than in E. coli. Furthermore, it is also possible that due to the different codon usage of B. glumae, the expression level of gene1 is lower than in E. coli. In B. megaterium for instance, it was found that the expression of gene1 is not possible at all, which is due to the different codon usage. Thus, in order to be able to establish the T7 RNA polymerase-based expression system in this strain, the T7 RNA polymerase gene had to be completely synthesized using the codon-usage of Bacillus (D. Jahn, personal communication). Nevertheless, it should be noted, that overexpression of a target gene, which is under the control of the T7 promoter, requires only low amounts of
______78 5. Discussion synthesized T7 RNA polymerase, since the polymerase specifically recognizes its promoters and is highly processive. Based on the obtained data in this experiment, it was confirmed that the establishment of a T7 RNA polymerase-based expression system in B. glumae is feasible. In subsequent steps, a suitable suicide vector was constructed, which enables the construction of a stable T7-expression host of B. glumae. Since integration of the T7 RNA polymerase gene into the chromosome of B. glumae was combined with the aim of the deletion of the chromosomal lipAB-operon, the resulting T7-expression strain of B. glumae would simultaneously be a lipAB-deletion mutant. Recently, integration of the suicide vector pSUPT7pollacIup/down into the chromosomal lipAB-operon of B. glumae PG1 was confirmed. In the following steps, this recombinant strain will be used to perform enrichment procedures in order to provide the second cross-over that leads to the loss of the vector and the deletion of the lipAB-operon. As mentioned above, one advantage of this system is that overexpression of lipase can be tightly controlled. Furthermore, this system can also be used for the overexpression of other target genes in B. glumae. Nevertheless, this system also has some disadvantages. For instance, in large-scale productions, high amounts of IPTG would be required causing an increase in the production costs. In addition, this system does not consider the physiological aspects of lipase production. Under natural conditions, lipase gene expression occurs in the late logarithmic phase of growth. Furthermore, for the successful production of enzymatically active lipase, a variety of accessory proteins are required, which are not produced until this growth phase. For example, in P. aeruginosa, it has been shown that the components of the type II secretion machinery are subjected to growth phase regulation thus being expressed only at high cell densities (Nouwens et al., 2003). Consequently, when this type of expression system is used, it is necessary to determine the most efficient time point of induction in order to achieve maximum protein yield. In the course of this thesis, it was therefore attempted to establish a second T7 RNA polymerase-based expression system. In this system, gene1 is integrated into the lipAB-operon without the lacUV5 promoter. Instead expression of the T7 RNA polymerase is controlled by the lip promoter (Fig. 34). This overexpression system enables high-level production of lipase under physiological conditions and thereby probably reduces cell stress. Moreover, it allows the study of physiological conditions influencing lipase production. Recently, it was achieved to construct a suitable suicide vector by following the same cloning strategy as for the construction of the first suicide vector pSUPT7pollacIup/down. The only differences are that the T7 RNA polymerase gene was cloned without the lacUV5 promoter and without the lacIq gene. In addition, gene1 and the
______79 5. Discussion two homologous regions flanking the lipAB-operon were cloned into the chloramphenicol resistance gene of pSUP202 instead of the tetracycline resistance gene. Using this suicide vector, the construction of a second ∆lipAB T7-expression strain of B. glumae can be constructed, following the same experimental procedure as for the construction of the IPTG- inducible T7-expression strain.
Plip up gene1 down pSUPT7polup/down pSUP202 pSUP202
Homologous recombination Plip up lipA lipB down Chromosome of B. glumae PG1 / LU8093
Plip up gene1 down Chromosome of B. glumae PGT7pol / LUT7pol
Fig. 34: Schematic representation of the construction of a T7-expression strain of B. glumae based on physiological induction. Integration of the T7 RNA polymerase gene (gene1) is intended to be combined with the deletion of the chromosomal lipAB-operon of B. glumae. In contrast to the first T7 RNA polymerase-based expression system, in this system, integration of gene1 occurs without the lacUV5 promoter and the lacIq gene. Thus, expression of gene1 is controlled by the lip promoter, which enables to perform expression studies under physiological conditions.
In summary, in the course of this thesis, important foundations for the construction of two different T7-expression strains of B. glumae have been laid. In the case of the inducible T7 RNA polymerase-based expression system, the construction of the first T7-expression strain of B. glumae PG1 is expected to be completed soon. Furthermore, a second suicide vector is available, which enables the construction of a lipase-negative T7-expression strain of B. glumae based on physiological induction. Finally, three suitable expression vectors containing the lipAB-operon of B. glumae under the control of the T7 or lac promoter were constructed, which can also be used to express lipase variants.
5.2 Identification and elimination of bottlenecks for an improved lipase production in B. glumae For high-level production of a wild type protein, which is encoded by a chromosomal gene, the most obvious approach to increase the production level is to improve the production pathway of this protein in the industrial host. Therefore, several routes come into consideration. In classical strain breeding strategies, high-level production of a target protein
______80 5. Discussion is achieved by using UV-light or mutagenic chemicals to unspecifically introduce mutations into the genomic DNA. An advantage of this method is that it can be performed without having any knowledge of the biosynthetic pathway of the target protein. On the other hand, this method is usually discontinuous and leads to significant cell damage with increasing numbers of mutations (Selifonova et al., 2001). Furthermore, since the caused mutations can have various effects, an efficient screening assay is required to detect the mutant exhibiting an improved production level. At this point, genetic engineering methods come into account. Due to the enormous progress in recombinant DNA technology in the past twenty years, genetic engineering methods have become attractive alternatives or supplements to the classical strain breeding (van der Werf, 2005). In order to be able to specifically manipulate single steps in the production pathway of the target protein, it is necessary to first identify potential bottlenecks. Subsequently, protein production can then be improved by either inactivating genes coding for limiting components or by additionally expressing genes whose products are involved in a rate limiting step of the production pathway. As protein production is a multistep process, starting with gene expression to protein folding and in some cases protein secretion, a variety of bottlenecks can thus be found and eliminated. First, one possibility to enhance protein production, is to optimize the regulation of gene expression. In P. aeruginosa, for example, an increase in extracellular lipase activity could be achieved by a moderate co-expression of an operon encoding the two-component system lipQ/R, which is suggested to directly control lipase gene expression (Düfel, 2000). For high- level production of a penicillin acylase (PAC) in E. coli, improvement of transcription of the gene was enhanced by manipulating certain DNA bases in the pac regulatory region, whereas translation was enhanced by enlarging the spacing between the ribosome binding site and the ATG initiation codon in order to increase the initiation efficiency (Chou et al., 1999). On the level of protein folding, it might be possible to improve this step by co-expression a suitable chaperone or folding catalyst leading to a reduced level of misfolded and degraded proteins. For this approach, many studies have been performed in E. coli. Classical examples describe the co-expression of cytoplasmic chaperones such as DnaK-DnaJ-GrpE or GroEL-GroES or periplasmic folding modulators such as SurA or FkpA (for review see Wuelfing and Plueckthun, 1994; Baneyx and Mujacic, 2004). But also in other microorganisms, many studies have been performed in this area. For example in a recombinant Saccharomyces cerevisiae strain, the production level of antithrombotic hirudin was increased by co- expression of the endoplasmic chaperone Bip (Kim et al., 2003). In Bacillus thuringiensis, co- expression of a 20 kDa helper protein P20 resulted in an enhanced production of two
______81 5. Discussion pesticidal proteins Cyt1A and Cyt11A (Shao et al., 2001). In P. aeruginosa and B. glumae, it has been shown that co-expression of the cognate folding catalyst, the lipase-specific foldase, results in a significantly increase in extracellular lipase formation (El-Khattabi, 2001; Rosenau, 2001). Furthermore, in P. aeruginosa, it was also possible to increase extracellular lipase activity by inactivation of three genes encoding periplasmic proteases. Based on further experiments, it was suggested that these proteases influence folding and/or secretion of the lipase (Windgassen, 2000). In the case of an extracellular protein such as lipase, also secretion of the protein might finally represent a bottleneck. Elimination of this bottleneck might be achieved by co-expressing components of the corresponding secretion pathway leading to an increased protein yield in the extracellular medium. In Pseudomonas fluorescence, it was found that co-expression of the ABC-transporter TliDEF led to enhanced secretion of lipase TliA to the extracellular medium (Ahn et al., 2001). Based on the examples described above, it can be realized that a multitude of potential bottlenecks in the production pathway of a target protein can occur. Therefore subsequent elimination might lead to a significant increase in protein yield. One approach to identify potential bottlenecks is the so called phenotype enhancement method (Gerritse et al., 1998). This method comprises the reintroduction of a cosmid library harboring random chromosomal fragments into the homologous strain of interest. By using a suitable screening assay it is possible to identify clones displaying an improved production of the target protein. Following this strategy, Gerritse et al. identified the Xcp outer membrane secretion machinery as a bottleneck in lipase production in Pseudomonas alcaligenes. Today, many more examples of screening cosmid libraries have been described in the literature. Besides the screening of a cosmid library for the identification of bottlenecks, it is also possible to identify a certain gene or operon by complementation of a negative phenotype (Visca et al., 1994). Furthermore, screening of environmental DNA libraries constructed in cosmids, plasmids or BACs enables to identify novel biocatalysts with high biotechnological potential (Rondon et al., 2000; Voget et al., 2003). For the identification of potential bottlenecks in lipase production of B. glumae, two cosmid libraries of the wild type B. glumae PG1 and production strain B. glumae LU8093 were constructed. In the first step, identification of potential bottlenecks was achieved in the wild type strain. Nevertheless, a cosmid library of the production strain was also constructed, which enables to perform comparative analysis. Thus, a protein identified in the cosmid library of B. glumae PG1 can be compared to the homologous protein of B. glumae LU8093.
______82 5. Discussion
In this way, interesting mutations, which had been introduced into the genome of the production strain by classical mutagenesis may be identified. Screening of the cosmid library of B. glumae PG1 led to the identification of 15 clones, which showed an increase or decreased lipase activity, respectively. After subsequent analysis, two cosmids were finally selected for subcloning of the genomic DNA into a broad-host-range vector, in order to perform homologous expression studies in B. glumae. While co-expression of the plasmid pBBRPG 5/7, which contained three open reading frames coding for a putative tripartite efflux system (epA/mfpB/ompC), led to a decreased lipase activity, co-expression of the plasmid pBBRPG 8/1 containing an open reading frame for a putative intracellular protease (pro), caused an increase in lipase activity in the supernatant of B. glumae PG1 and B. glumae LU8093 (Tab. 9). Consequently, two strategies were followed in order to improve lipase production in B. glumae. First, two genes of the putative tripartite efflux system were inactivated in the genome of both B. glumae strains. Second, a suitable suicide vector was constructed, which enables to integrate additional copies of the pro gene into the genome of B. glumae by transposon mutagenesis.
Inactivation of a putative efflux system resulted in a decreased lipase activity in B. glumae A comparison of the amino acid sequences of the open reading frames 1 and 2 (orf 1 and 2), designated as efflux pump (epA) and membrane fusion protein (mfpB), revealed the existence of conserved domains of AcrAB-transporters, while the amino acid sequence of the third open reading frame (ompC) contained a conserved domain of outer membrane efflux proteins (OMEP-family) (Fig. 13). Based on similarities to other efflux proteins in the database, it was suggested that the three open reading frames coded for proteins of the RND (Resistance Nodulation cell Division) family of transporters. Thus, the open reading frame designated epA might be similar to an inner membrane transporter such as AmrA of B. pseudomallei or AcrA of E. coli, while the orfs designated mfpB and ompC encode the homologous membrane fusion protein, like AmrB, and outer membrane factor such as OprA of B. pseudomallei, respectively. Multidrug efflux mechanisms are broadly distributed among Gram-negative and Gram- positive bacteria (Grkovic et al., 2002). Currently, five families of drug efflux translocases have been identified on the basis of amino acid sequence similarities: MF (Major Facilitator) family, SMR (Small Multidrug Resistance) family, RND (Resistance Nodulation Cell Division) family, ABC (ATP-Binding Cassette) family and MATE (Multidrug And Toxic
______83 5. Discussion
Compound Extrusion) family. Proteins of the RND family are usually components of tripartite efflux systems that expel a variety of different substrates including drugs, solvents, silver or copper ions and signal molecules. The driving force for the efflux mechanism is suggested to be an electrochemical potential gradient of H+ across the cell membranes (Nishino and Yamaguchi, 2001). The classical efflux system is built up by an inner- membrane transporter, a membrane fusion protein and an outer membrane factor. AcrA- AcrB-TolC of E. coli (Ma et al., 1993), MexA-MexB-OprM of P. aeruginosa (Poole et al., 1993) and AmrA-AmrB-OprA of Burkholderia pseudomallei (Moore et al., 1999) are examples of such system. Recently, also variants of this tripartite system have been described. For instance, the AcrD system of E. coli consists of only the cytoplasmic membrane associated transporter involved in efflux of aminoglycosides (Rosenberg et al., 2000). In P. aeruginosa already six RND efflux systems have been experimentally confirmed, including for example the MexCD-OprJ (Poole et al., 1996) or MexJK system (Chuanchuen et al., 2002b). In order to investigate whether inactivation of these genes could enhance lipase production, vector insertion mutants were constructed. After inactivation of two genes of the efflux system, designated as ompC and mfpB, the recombinant strains were tested for improved lipase activity. However, in all growth studies the strains displayed reduced lipase activity compared to the parental strains (Tab. 10). Interestingly, it could also be observed, that the mutants showed a significant reduced growth. Moreover, the concentrations of the antibiotics, which were applied for the maintenance of vector integrations had to be reduced to half of the standard concentrations. Similar observations have also been described in the literature. In P. aeruginosa for example, insertional inactivation of oprM homologous genes (opmG, opmI and opmH) resulted in a decrease of various degrees in the minimum inhibitory concentrations (MICs) of aminoglycosides, which could be complemented by introducing multicopy plasmids harboring the corresponding genes (Jo et al., 2003). In Salmonella enterica serovar typhimurium SL1344, deletion of acrB, acrD and acrF encoding efflux pumps was shown to lead to a hypersusceptibility of the mutant strains to antibiotics such as ciprofloxacin, chloramphenicol and tetracycline (Eaves et al., 2004). Thus, inactivation of the putative efflux pumps in B. glumae PG1 and LU8093 might have caused a susceptibility of the strains to the applied antibiotics, which would explain the poor growth in the presence of the applied antibiotics. Another interesting aspect deals with the finding that in P. aeruginosa, the MexAB-OprM efflux pump also expels acylated homoserine lactones, the signal molecules of quorum sensing systems (Poole et al., 2001). Furthermore, the expression of the
______84 5. Discussion
MexGHI-OpmD pump is regulated by quorum sensing (Aenderkerk et al., 2002) and overexpression or inactivation of the genes of the MexAB-OprM, MexGHI-OpmD and Mex EF-OprN pumps causes altered levels of extracellular acylated homoserine lactones or other phenotype tied to quorum sensing (Koehler et al., 2001; Aenderkerk et al., 2002). Consequently, for P. aeruginosa it is suggested that these efflux pumps play a role in cellular metabolism (Schweizer et al., 2003). In B. glumae, it has been observed that the cells stop growing as soon as a quorum sensing mechanism of the cells is disturbed (I. Hwang, personal communication). Assuming that the putative efflux pump of B. glumae expels acylated homoserine lactones, there might be a link between inactivation of the two genes ompC and mfpB and a more general effect on the cell metabolism as a consequence of reduced quorum sensing. The decreased lipase production and poor growth are then two of several possible phenotypes. In this respect, a further question that still remains unanswered is why also the co-expression of the putative tripartite efflux system led to a decreased lipase activity in both B. glumae strains. One explanation could be that an altered level of signal molecules again leads to a disorder of a quorum sensing mechanism, which has an effect on the cell metabolism and thus on lipase production. However, at this point, it can only be speculated whether there is a connection between inactivation of a putative efflux pump, a reduced quorum sensing and a decrease in lipase production. Hence, more research needs to be performed in order to elucidate the function and physiological role of these proteins in B. glumae.
Co-expression of a putative protease increases lipase activity in B. glumae Based on the finding that the increase in lipase activity in the supernatant of B. glumae could be attributed to the co-expression of an open reading frame encoding a putative cytoplasmic protease, it was of great interest to obtain more information about this protein. By performing database searches, it could be shown that the putative protease contained a conserved domain of the ThiJ/PfpI-family and displayed significant identity on the amino acid sequence level to other proteases or peptidases in the database such as the Peptidase C56 of B. vietnamiensis (70%), the intracellular proteinase YhbO of E. coli (67%), the Peptidase C56 PfpI of Burkholderia cenopacia (61%) or the protease PfpI of P. aeruginosa (50%). The ThiJ/PfpI-domain can be found in different proteins such as transcriptional regulators, catalases, RNA-binding proteins or proteases, which are categorized into the so called DJ-1/ThiJ/PfpI-superfamily. A recent overview of the evolutionary and functional
______85 5. Discussion relationships within this superfamily suggests the existence of eight subgroups, which were clustered on the basis of amino acid sequence similarities. Examples of these subgroups are the AraC transcriptional regulators, catalases, sigma cross reacting proteins or the human DJ-1. Furthermore, there are two subgroups that have a high similarity on the basis of amino acid sequence. The first subgroup is annotated as PfpI-proteases and includes the two proteases PfpI and PH1704 from the thermophilic bacteria Pyrococcus furiosus and Pyrococcus horikoshii, respectively (Bandyopadhyay and Cookson, 2004). Based on the presence of a cysteine residue (C100) adjacent to a histidine residue, these proteins were tentatively identified as cysteine proteases. Structure analysis of PH1704 revealed that the equivalent Cys100 is present in a nucleophilic elbow motif and forms together with the His101 part of the catalytic triad at the interfaces between three pairs of monomers (Trotter et al., 2002). As all known proteases within the two subgroups contain adjacent Cys/His residues, whereas substitution with Cys/X is found in all non-protease members, it is suggested that many proteins of the second subgroup, annotated as ThiJ/PfpI-like proteins, also display protease activity. Proteins that belong to this subgroup are for example, the intracellular protease YhbO of E. coli, Protease I of Bacillus cereus and the intracellular proteases YDR533C of S. cerevisiae. Another characteristic feature of theses proteins is the presence of a consensus sequence surrounding the Cys/His pair: AICHGP (Bandyopadhyay and Cookson, 2004). As shown in the following alignment (Fig. 35), this sequence is also present in the sequence of the putative protease. The only exception concerns the last amino acid residue. Instead of a proline, a glutamate is present at this position in the sequence of the putative protease: AICHGG. Interestingly, the peptidase C56 of B. vietnamiensis and the putative protease/amidases of B. fungorum, which were found to be similar to the putative protease of B. glumae on the basis of amino acid sequence, also contain a glutamate instead of a proline at this position. Consequently, it might be assumed that this difference in the consensus sequence is a common feature of these ThiJ/PfpI-like proteases of the genera Burkholderia.
______86 5. Discussion
Sequence ------MQQRGGSMSKKIAVLAVDEFEDSELVEPLRALRKAGAEVDVIS-QQAGEVKGF 52 Q4LMM4_9BURK ------MSGKLDHCKVAILAVDGFEEAELVEPQRALAAEGAQVDVIS-QKRGEIQGF 50 Q8XA99_ECO57 MGNSPHPMQQRGGSMSKKIAVLITDEFEDSEFTSPADEFRKAGHEVITIE-KQAGKTVKG 59 Q7CPQ5_SALTY ------MSKKIAVLITDEFEDSEFTSPAAEFRQAGHEVITIE-KEAGKTVKG 45 Q9I6D8_PSEAE ------MTQSLHGKVVAALVTDGFEQVELTGPKKALEDAGATVRILS-DKAGEVRGW 50 Q88JC4_PSEPK ------MMSAQLNGKRVAFLVTDGFEQVELTGPREALENSGAVVDILS-EKEGTVRGW 51 Q8P751_XANCP ------MTHSLSGKTVAVLATSGFEQSELQEPKRLLESWGATVEVIAPGDDAQIRGW 51 Q73CX9_BACC1 ------MSKKIATLITDYFEDTEYTEPAEAFKQKGYELTTIE-AKKGKTVHG 45 :* * .. **: * * : * : : . .
Sequence RHADKGEAVKVDRTFDEVREGEFDALLLPGG-----LLRGDNRMLPAAREFVTAGKPVFA 107 Q4LMM4_9BURK RHVDKGERVKVDRTFDDAREGDYDAVVLPGGVINGDAIRMVPAAREFVTAAVGAGKPVAA 110 Q8XA99_ECO57 KKGE--ASVTIDKSIDEVTPAEFDALLLPGGH-SPDYLRGDNRFVTFTRDFVNSGKPVFA 116 Q7CPQ5_SALTY KKGE--ASVTIDKAIDDVRPDEFDALLLPGGH-SPDYLRGDSRFVDFTRDFVNSGKPVFA 102 Q9I6D8_PSEAE NHHQPAEAFRVDGTFEDASLDDYDALLLPGGVINSDQIRSLAKAQELAIRAEQASKPVAV 110 Q88JC4_PSEPK NHDKPADAFSVDATFDSAQLDLYDALVLPGGVQNSDTIRLIPGAQKLVKSHDAAGRPLAV 111 Q8P751_XANCP NHTDWGDSVPVDTPLAQAKPDRYDALVLPGGVINPDNLRTNAQAIDFIRSVAASGKPVAA 111 Q73CX9_BACC1 KQGK--SEVVIDKGIDDVSPENFDALFIPGGF-SPDILRADERFVRFSKSFMDTKKPVFA 102 .: . . :* : .. :**:.:*** :* : :*: .
Sequence I CHGGWLLGSSGVINGRKLTAWPSLQDDVKNAGGEFYDQEVVRDKDQLITSRKPDDLPAF 167 Q4LMM4_9BURK ICHGGWLLVSAGLVEGKTMTSWPSLQDDIRHAGGKWVDEEVVRD-GNLITSRKPADLPAF 169 Q8XA99_ECO57 ICHGPQLLISADVIRGRKLTAVKPIIIDVKNAGAEFYDQEVVVDKDQLVTSRTPDDLPAF 176 Q7CPQ5_SALTY ICHGPQLLISADVIRGRKLTAVKPIIIDVKNAGAEFYDQEVVVDKDQLVTSRTPDDLPAF 162 Q9I6D8_PSEAE ICHGAWLLISAGLVQGRTLTSWPSLKDDINNAGGHWVDQEVAVD-GKLVSSRKPEDIPAF 169 Q88JC4_PSEPK ICHGAWLLISSGLAKGKRMTSYKTLQDDIRNAGGTWVDEQVVVD-GNLITSRQPDDIPAF 170 Q8P751_XANCP ICHGPWLLVESGLVRDRKVTSWPSVKTDLSNAGGRWEDAEVVVD-GQLITSRKPDDIPAF 170 Q73CX9_BACC1 ICHGPQLLITAQTLEGRDVTGYKSIEVDLKNAGGNFHDKEVVVCQNQLVTSRQPEDIPAF 162 **** ** : ..: :*. .: *: :**. : * :*. .:*::** * *:***
Sequence NREALRLLGAGA- 179 Q4LMM4_9BURK NGALVERLASRGA 182 Q8XA99_ECO57 NREALRLLGA--- 186 Q7CPQ5_SALTY NREALRLLGA--- 172 Q9I6D8_PSEAE NRRFIEILAG--- 179 Q88JC4_PSEPK NEQLIKALAD--- 180 Q8P751_XANCP TDAVAKALAA--- 180 Q73CX9_BACC1 IEESLRILG---- 171 . *.
Fig. 35: Multiple sequence alignments of an open reading frame encoding a putative protease of B. glumae with proteins of the database. First, the amino acid sequence of the putative protease was subjected to a sequence-similarity search using the WU-BLAST2 program of EBL-EMBI with a BLOSUM62 matrix. Proteins with a high similarity to the putative protease were then selected to perform a multiple sequence alignment by using the DbClustal of EBL-EMBI. Residues or nucleotides in one column, which are identical in all sequences in the alignment are marked with a star. A double dot means that conserved substitutions have been observed and a single dot indicates semi-conserved substitutions. The coloring of residues takes place according to the following physicochemical criteria: acidic (blue), basic (magenta), small (red), hydroxyl + amine + basic (green), others (grey). The putative consensus sequence: AICHGP of ThiJ/PfpI proteins is framed by a black box. In the first lane the amino acid sequence of the putative protease is shown. Q4LMM4: Peptidase C56 PfpI of Burkholderia cenopacia. Q8XA99: Hypothetical protein YhbO of Escherichia coli. Q7CPQ5: Putative intracellular proteinase Yhbo of Salmonella typhimurium. Q9I16D8: Protease PfpI of Pseudomonas aeruginosa. Q88JC4: Protease PfpI of Pseudomonas putida KT2440. Q8P751: Protease I of Xanthomonas campestris. Q73CX9: ThiJ/PfpI family protein of Bacillus cereus (strain ATCC 10987) BCE0935.
Although, some proteases of the group of ThiJ/PfpI-like proteins have already been identified and a few three-dimensional structures have been solved, the physiological function of these proteins still remains unknown (Bandyopadhyay & Cookson, 2004). Based on the results obtained in this study for the putative protease of B. glumae, it is possible that this protein belongs to the group of ThiJ/PfpI-like proteins. Besides the similarity on
______87 5. Discussion amino acid sequence level to other proteins of this group such as YhbO of E. coli or the intracellular proteases YDR533C of S. cerevisiae, the presence of the consensus sequence is most decisive for classifying the protein to this subgroup. Furthermore, the general enzyme activity assay supports the assumption that the protein is a protease since proteolytic activity towards certain amino peptides could be detected. By western blot analysis it could be shown that co-expression of the gene did not have an effect on the production of lipase-specific foldase (Fig. 15). The increase of lipase activity in the supernatant of B. glumae, as a result of an increased amount of Lif proteins, can therefore be excluded. One possible physiological function could be that the putative protease is involved in the regulation of lipase gene expression. Regulatory proteolysis is a well-known mechanism in Gram-negative and Gram-positive bacteria. Classical examples include the heat shock response in E. coli (Arsene et al., 2000) or the DegS protease being involved in reversible zymogenic activation (Ehrmann and Clausen, 2004). The proteolytic action of the putative protease could, for example, comprise the transformation of an activator protein from its dormant into its active state leading to an increased lipase gene expression. Furthermore, it is possible that the protein is involved in the degradation of a regulatory protein, which limits lipase gene expression under natural conditions. In addition, the putative protease might also be involved in subsequent steps of lipase expression such as targeting of the unfolded protein to the Sec-machinery for transport into the periplasm. In order to elucidate the physiological function of the protein in the cell and its effect on lipase production further studies are required. In this respect, also the exact substrate specificity needs to be determined by performing activity assays with single substrates. By co-expression of the putative protease, lipase activity in the wild type B. glumae PG1 could be increased 2 fold, while in the production strain B. glumae LU8093, an increase of 30% was observed (Fig. 14). Considering the fact that the production strain already produces at least 10 fold more lipase than the wild type, presumably co-expression of the putative protease cannot increase lipase production to the same level as obtained in the wild type strain. Therefore, one explanation is that the mechanism, in which the putative protease is involved, might already be enhanced in the production strain. Nevertheless, the increase in lipase production in B. glumae LU8093 was still significant, which allowed the application for a patent. Additionally, in order to construct an improved lipase production strain, it was desirable to integrate additional copies of the pro gene into the genome of B. glumae. In contrast to the construction of the T7-expression host, where integration of the T7 RNA polymerase gene into the chromosome of B. glumae had to occur at a specific site and was
______88 5. Discussion therefore achieved by homologous recombination, integration of the gene pro was expected to occur randomly. In order to avoid time-consuming steps like the cloning of homologous regions for the construction of a suitable suicide vector, transposon mutagenesis using Tn5 was chosen instead. Since previous studies showed that the suicide vector of the pSUP-type is suitable for mutagenesis in B. glumae, a derivative of pSUP202 was used harboring the Tn5- fragment in the tetracycline resistance gene. Recently, the pro gene has been successfully cloned into this vector, so that transposon mutagenesis can now be performed. Based on the same methodology, it was also attempted to integrate the structural gene of the lipase-specific foldase into the genome of B. glumae. Due to recent findings that co-expression of the foldase gene in trans led to a significant increase in lipase production in B. glumae and P. aeruginosa (El-Khattabi, 2001, Rosenau, 2001), the stable integration of additional copies of the gene into the chromosome is another promising approach to improve lipase production in B. glumae. Finally, the results obtained in this thesis confirm that identification and elimination of bottlenecks in the production pathway is a very promising approach to specifically improve lipase production in B. glumae.
5.3 Regulation of lipase gene expression in B. glumae In nature, microorganisms have to be able to react to environmental changes such as in pH, temperature or osmolarity quickly, in order to provide an optimal nutrient and energy supply. Therefore, cellular processes need to be tightly regulated and controlled requiring complex regulatory networks. Many Gram-negative bacteria produce extracellular, biodegradative enzymes or virulence factors in the late logarithmic phase of growth. Expression of these enzymes and virulence factors is often regulated by the cell-density determined mechanism, termed quorum sensing. For example in B. glumae, biosynthesis and transport of the virulence factor toxoflavin is controlled by quorum sensing and the LysR-type transcriptional activator ToxR (Kim et al., 2004). In Burkholderia cepacia, the quorum sensing system CepR/I regulates production of extracellular lipase, protease and the siderophore ornibactin (Lewenza et al., 1999). Likewise in P. aeruginosa, it was shown that extracellular lipase formation takes place in the late logarithmic phase of growth and that the two-component system lipQ/R, which directly controls lipA gene expression is subjected to a superior quorum sensing system (Düfel, 2000; Rosenau and Jaeger, 2000). Quorum sensing circuits can in turn be regulated by additional regulatory proteins or stimulated by environmental signals (Miller and Bassler, 2001; Juhas,
______89 5. Discussion
2005). The latter can also influence other regulatory proteins or mechanisms in the cells and thereby effect gene expression. For example, depending on substrate availability in the environment, expression of genes whose products are involved in the required metabolic pathway can be induced by activating a transcriptional regulator, while the expression of genes of other metabolic pathways may be repressed. A classical example is the carbon catabolite repression in Gram-negative and Gram-positive bacteria (Postma, 1993; Stülke and Hillen, 2000). As described in the introduction, with respect to an improved lipase production, several studies have been performed using chemical agents like polysaccharide or detergents supplemented to the growth media in order to increase lipase production (Schulte et al., 1982; Kok et al., 1996; Tanaka et al., 1999). Therefore, the effect of physiological conditions on extracellular lipase formation in B. glumae was investigated. Furthermore, the studies also aimed at elucidating some aspects of the molecular and physiological mechanisms affecting lipase production. Again, comparative analysis of the wild type B. glumae PG1 and production strain B. glumae LU8093 was carried out, in order to reveal differences between the two strains. This might also help to understand some aspects of regulation of lipase gene expression.
Determination of the transcription start of lipA Previous studies concerning lipase gene expression revealed that in B. glumae PG1 lipase production takes place in the late logarithmic phase of growth and that gene expression is subjected to catabolite repression (Frenken, 1993). Therefore, it was assumed that gene expression is regulated involving an alternative sigma factor instead of the housekeeping σ70-factor. In P. aeruginosa, for example, the involvement of the alternative σ54-factor (RpoN) in lipase gene expression has been described (Düfel, 2000). Today many examples of σ54-dependent gene expression in Gram-negative as well as in Gram-positive bacteria are known (Marques et al., 1997; Sze et al., 2002; Dasgupta et al., 2003; Arous et al., 2004; Yildiz et al., 2004). A common feature is that the promoter is located around -24 and -12 nucleotides upstream of the transcriptional start site (Morett and Segovia, 1993) and that the corresponding gene products are not essential for the survival of the bacterial cell (Shingler, 1996). Furthermore, since gene expression is regulated and does not occur spontaneously, a transcriptional activator is involved which binds to a so-called upstream activator sequence (UAS) ahead of the target gene (Morret and Buck, 1988).
______90 5. Discussion
In order to determine the transcription start of lipA and to identify a putative promoter sequence, a primer extension analysis was performed. By using two different fluorescent labeled primers, one transcription start could be localized 78 bp ahead of the lipA start codon. Subsequent analysis of the upstream sequence of the lipAB-operon led to the identification of two putative σ54-dependent promoter sequences that are localized 90-104 bp and 129-142 bp ahead of the lipA start codon. These findings confirmed the existence of the transcription start at its position, since the first putative promoter sequence identified (90-104 bp) had a characteristic distance to transcription start of 12-24 nucleotides. Due to the presence of a second putative σ54-dependent promoter sequence and the fact that during primer extension analysis further weak signals were detected, it was suggested that at least one further transcription start is located in the upstream sequence. In order to prove this assumption, further promoter studies have to be performed. Nevertheless, the obtained data indicated that lipase gene expression in B. glumae is subjected to a complex regulation, emphasizing that further studies such as analysis of transcriptional lipA::gfp-fusion are required.
Improvement of lipase production in B. glumae by emulsifiers or detergents The effect of increased lipase production by supplementing the growth medium with hexadecane has been described in several bacteria such as A. calcoacticus BD413 (Kok et al., 1996) or P. aeruginosa (Schneidinger, 1997). This effect could also be confirmed in B. glumae PG1 and B. glumae LU8093 by using 5-10% (v/v) hexadecane. The increase in extracellular lipase activity in the wild type strain amounted up to 150%, while in the production strain up to 250% increase could be achieved (4.5.1). Similar results were obtained by adding the detergents Triton X-100 or Tween 80 to media already supplemented with olive oil or by using an olive oil emulsion with gum arabic. Apart from the fact, that the addition of emulsifiers enhances substrate availability, previous studies in other bacteria have shown that supplementing the growth medium with inert compounds also affects lipase gene expression and/or secretion (Winkler and Stukman, 1979; Kok et al., 1996; Martinez and Nudel, 2002). In B. glumae cultures, it was also observed that if a carbohydrate such as sucrose or maltose replaced olive oil, the detergents still induced extracellular lipase formation. Thus, the detergents do not only increase extracellular lipase activity in the presence of olive oil, but can also induce lipase gene expression. This effect can either be directly or indirectly, as will be discussed later. The increase in lipase activity in the presence of olive oil may due to the destabilizing effect of the detergents on both membranes, leading to a release of foldase from
______91 5. Discussion the inner membrane and a release of lipase from the periplasm, as well as a detachment from the lipopolysaccharide (LPS) at the outer membrane. In contrast, hexadecane did not affect the stability of the bacterial membranes, thus no Lif protein could be detected in the supernatant of the corresponding cultures. But, likewise to the effect of the detergents, hexadecane also caused an increase in the amount of extracellular lipase and thus promotes lipase secretion or the release of cell-bound lipase (Fig. 23 and 24). Already in 1979, Winkler and Stuckmann postulated the detachment-hypothesis for the first time. In this study, the stimulatory effect of polysaccharides such as glycogen, hyaluronate and pectin B on extracellular lipase formation of Serratia marcescens was described and it was suggested that cell-bound lipase was released via specific interactions with the bacterial cell surface. Recently, Leahy et al. (2003) showed that high concentrations of hexadecane and inorganic nutrients promote the release of membrane-bound vesicles, soluble proteins and bioemulsifier in Acinetobacter venetianus RAG-1 and Acinetobacter sp. HO1-N. Nevertheless, it is also possible that hexadecane causes the release of cell-bound lipase indirectly. Recent work in the area of biosurfactants elucidates that these surface active compounds also play an important role in the solubility and uptake of hydrophobic compounds. For instance in P. aeruginosa, it was shown that the biosurfactant rhamnolipid is produced in the presence of hexadecane and enhances the degradation of organic compounds by at least two effects. First, it solubilizes hydrophobic compounds within micelle structures and thus increases the solubility of organic compounds and its availability for uptake by the cell. Second, it causes the cell surface to become more hydrophobic by releasing LPS from the outer membrane. This increases the direct physical contact between the cell and the soluble substrate (Miller, 1995; Shreve et al., 1995). The effect of rhamnolipid on LPS release can be due to two possible aspects. First, since rhamnolipid has detergency properties, it causes the direct removal of LPS by solubilization (Zhang and Miller, 1992; Al-Tahhan et al., 2000). Second, it causes the indirect removal of LPS by complexation of Mg2+ in the outer membrane, which is crucial for strong LPS-LPS interactions (Al-Tahhan et al., 2000). This mechanism is also supported by the fact that rhamnolipid has been shown to effectively complex divalent cations, such as magnesium (Herman et al., 1995). So far, it is known that B. glumae produces biosurfactants (V. Zaehringer, personal communication). However, no studies have been performed yet concerning their effect on cell surface properties and interaction with hydrophobic substrate. Taken together, based on the obtained results, it is suggested that hexadecane directly or indirectly promotes the release of cell-bound lipase, which leads to an increased lipase
______92 5. Discussion activity. Nevertheless, it should be noted, that this might not be the only effect of hexadecane. As will be discussed later on, it is also possible that there are several effects, which together cause an increase in extracellular lipase activity. Another interesting finding during the performance of growth studies was that in the production strain B. glumae LU8093, the mechanism of catabolite repression appeared to be eliminated. Thus, cultures supplemented with sucrose or glucose as a sole carbon source, displayed lipase activity in the supernatant (4.5.1). In addition, using lipase and foldase specific antibodies, lipase and foldase could be detected in the corresponding cultures by immunoblotting. In general, catabolite repression describes the regulation of genes whose products are involved in metabolic pathways, depending on carbon source availability. The underlying mechanism can thereby be different. In E. coli for example, the absence of glucose leads to the activation of the adenylate cyclase, which synthesizes cyclic-adenosine-3´-5´- monophosphate (cAMP). With increasing glucose concentrations, cAMP is bound by the cAMP reponse protein (CRP). This complex then binds to a consensus sequence in the promoter region of target genes leading to the activation of transcription (Postma et al., 1993). In the Gram-positive bacteria, the signaling intermediate is HPr (heat-stable protein, part of phosphotransferase system), which can be phosphorylated at two sites, a histidine residue and a serine residue. HPr (Ser-P) is involved in carbon catabolite repression. It binds to the transcriptional regulator CcpA (catabolite control protein), thereby inducing the binding of the complex to so-called cre sites (for catabolite responsive element) in the promoter region of the target genes, which prevents transcription of the genes (Henkin, 1996; Warner and Lolkema, 2003). In B. cepacia RR10, one example for catabolite repression control is the regulation of the alkane hydroxylase gene (alkB). In 2001, Marin et al. observed that expression of the alkB gene was repressed when the cells were cultured in the presence of several organic acids and sugars or in a complex (rich) medium. Today, many more examples of carbon catabolite repression in Gram-negative and Gram-positive bacteria have been described in the literature elucidating the versatility of this mechanism (for review see Warner and Lolkema, 2003). In B. glumae, the underlying mechanism of catabolite repression controlling lipase gene expression is not yet clear. A probable explanation will be given at the end of the chapter.
______93 5. Discussion
Lipase gene expression is regulated on the transcriptional level In order to elucidate the molecular mechanism of the observed effects of the chemical agents on lipase production, transcriptional and translational lipA::gfp fusions were constructed and analyzed using 460 bp of the upstream region of the lipAB-operon. Investigation of the regulation of lipA transcription revealed that in B. glumae LU8093, gene expression was upregulated on the transcriptional level. Thereby, the measured values of fluorescence were up to 50% higher than in the corresponding cultures of B. glumae PG1 (Fig. 29). Furthermore, it was confirmed that in the production strain lipase gene expression is not subjected to catabolite repression anymore. Thus, cultures of B. glumae LU8093, which were supplemented with a carbohydrate a sole carbon source, displayed a distinct fluorescence. Nevertheless, in accordance to the measured lipase activity in the supernatant of the corresponding samples, the values of green fluorescence in the cultures supplemented with a carbohydrate were not as high as in the cultures containing olive oil. These findings led to the assumption that two regulatory proteins control lipase gene expression in B. glumae: 1) a homologous cAMP response protein (CRP) of the catabolite repression system, which induces transcription in the absence of glucose and 2) an activator protein inducing gene expression upon the presence of olive oil in the growth medium. The latter could be a member of the NtrC/σ54-family of transcriptional activators, since two putative σ54-promoter sequences were identified ahead of the lipAB-operon. As a consequence, elimination of the mechanism of catabolite repression in B. glumae LU8093 results in lipase production in the presence as well as in the absence of a carbohydrate, because the homologous CRP protein, which might also be mutated, continuously binds to the DNA and induces transcription. However, due to the missing inducing effect of olive oil, the level of gene expression in cultures containing only carbohydrate is not as high as in the cultures supplemented with olive oil. Today several cases are known in which two divergent regulatory mechanisms act in conjunction. In E. coli for example, it was found that the expression of glnA (encoding glutamine synthetase) is transcribed from two promoters (glnAp1 and glnAp2). The glnAp1 is a σ70-dependent promoter, which is activated by CRP, while glnAp2 is a σ54-dependent promoter, which is activated by NtrC-phosphate. Additionally, it was shown that the activity of one promoter was affected by the activity of the second promoter, since glnAp2 expression was affected by different carbon sources and the CRP-cAMP complex inhibited the glnAp2 promoter activity (Tian et al., 2001). Thus, CRP can also directly interact with the RNA polymerase holoenzyme (E σ54) bound at the promoter (Wang et al., 1998).
______94 5. Discussion
Concerning the effect of hexadecane on lipase gene expression in B. glumae, the lipA::gfp- fusions showed no differences in cultures containing olive oil or olive oil and hexadecane, indicating that the alkane does not affect transcription of lipA (Fig. 29). These findings are confirmed by RT PCR experiments, which revealed that hexadecane neither affects transcription of lipA nor lipB (B. Boekema, unpublished). In contrast, the detergent Tween 80 had a positive effect on lipase expression when applied to the growth medium. This effect was most obvious in the wild type B. glumae PG1. Due to the catabolite repression, green fluorescence could not be detected in the culture containing a carbohydrate. However, by supplementing the medium with sucrose and Tween 80, the values of detected fluorescence were then as high as in the cultures containing olive oil (and hexadecane). Thus, besides the destabilizing effect discussed above, the presence of the detergent somehow led to an induction of lipase gene expression. One explanation may be that the detergent either directly induces lipase gene expression due to the presence of a fatty acid, or it causes a release of fatty acids from the membrane or lipoproteins leading to the inducing signal of lipase gene expression. Interestingly, the inducing effect of the detergents on lipase production only occurred in the presence of sucrose, but not in the presence of glucose (Fig. 30). The same phenomenon was observed during the growth studies, in which lipase activity in B. glumae PG1 was only measured in cultures containing sucrose and a detergent, but not glucose and a detergent. As was reported for other bacteria, this effect can be attributed to a different carbon utilization resulting in different catabolite repression strategies (Saier et al., 1995; Saier and Ramseier, 1996). Comparing the transcriptional and translational lipA::gfp-fusions, it was found that the detected values of green fluorescence of the translational fusions were higher than of the transcriptional fusions (Fig.30). However, with this experiment it cannot be stated whether lipase expression is also regulated at the posttranscriptional level. Since in a transcriptional fusion, the reporter gene harbors its own ribosome binding site, it is possible that the ribosome binding site of gfp is not as strong as the ribosome binding site of lipA. The reduced translation efficiency in the transcriptional fusion can then lead to a reduced level of detectable fluorescence and is therefore not a result of a lower level of gene expression. Investigation of the half-life of both Gfp proteins revealed that the stable Gfp protein has the same half-life of about 24 h as described in the literature (Andersen et al., 1998). In contrast, the unstable Gfp variant (GfpLAA) showed a reduced half-life of about 140 min in this study indicating that the protein is more rapidly degraded in B. glumae than in other bacteria such as P. putida. In this bacterium, GfpLAA has a half-life of 190 min (Andersen et al., 1998).
______95 5. Discussion
Subsequent analysis of lipA transcription using this unstable variant indicated a continuous lipase gene expression for at least 48h under inducing conditions, i.e. in the presence of olive oil. Finally, regarding the effect of hexadecane at the posttranscriptional level of lipase gene expression, a minor effect was observed, when the cultures supplemented with olive oil were compared to the cultures containing olive oil and hexadecane. Thus, there might be a slight effect on the posttranscriptional level. As mentioned above, it is possible that hexadecane causes several effects which together lead to the significant increase in lipase activity in the supernatant of B. glumae.
The mutation in the upstream sequence of the lipAB-operon in B. glumae LU8093 plays an important role in the regulation of lipase gene expression A comparison of the two DNA sequences of the upstream region of the lipAB-operon of both B. glumae strains led to the identification of a point mutation in the upstream region of the production strain, which is located 129 bp ahead of the lipA start codon. This mutation caused a substitution of a thymine (T) by a cytosine (C). As demonstrated in section 4.4.5, analysis of the DNA-sequence revealed the existence of a putative sequence for a CRP binding site surrounding the mutation. In E. coli the CRP binding site consists of 22 bp with a core sequence: GTGA-N6-TCAC (Cashel et al., 1996). Recently, Brown and Gallan (2004) analyzed various predicted CRP binding sites in E. coli and performed computational studies of sequence-depending binding energy for CRP. This analysis elucidated that single mutations in this site caused an altered binding energy for CRP. In the wild type B. glumae PG1, the putative sequence for the CRP binding site displays the following core sequence: CTGA-N6- ACAG. The identified mutation in the upstream region of B. glumae LU8093 is located at position N4. With respect to the findings of Brown and Galla, it may be that this mutation leads to an increased binding energy for CRP. On the other hand, it is also possible that concerning the catabolite repression control, this mutation does not come into account in B. glumae LU8093. If for example the homologous CRP protein is mutated resulting in a continuous binding to the DNA, it is possible that therefore the mutation does not play a role. Finally, it is also possible that another level of the mechanism of catabolite repression is affected, which leads to the observed effects. Nevertheless, by analyzing the lipA::gfp fusions of both B. glumae strains, it could be shown that this mutation has a general effect on the regulation of lipase gene expression. Since the reporter fusions were constructed on a plasmid, it was possible to analyze the fusions by
______96 5. Discussion exchanging the plasmids. Thus, the lipA::gfp fusion of B. glumae PG1 was expressed in B. glumae LU8093 and vice versa (Fig. 33). Introduction of the lipA(LU)::gfp fusion into B. glumae PG1 revealed that lipase gene expression did neither occur in the presence of a carbohydrate nor in the presence of olive oil. Considering the fact that in the wild type strain, lipase gene expression is subjected to catabolite repression and assuming the classical mechanism involving a CRP protein, these findings suggest that due to the mutation, the wild type CRP-cAMP complex cannot bind to the DNA of the production strain anymore. Furthermore, since green fluorescence could also not be detected under inducing conditions, i.e. in the presence of olive oil, it might be that the inducing effect of the CRP-cAMP complex is substantial for lipase gene expression. On the other hand, expression of the lipA(PG1)::gfp fusion in B. glumae LU8093 occurred in the presence of olive oil or olive oil and hexadecane. As mentioned above, the obtained results in this study suggest, that in B. glumae LU8093 the mechanism of catabolite repression is affected on at least two levels leading to lipase gene expression in the presence of a carbohydrate. One possible explanation may be that in the production strain the CRP protein is also mutated resulting in a continuous binding to the DNA under any condition. This could also explain why in the presence of glucose, lipase gene expression occurs. Since in the absence of glucose, the CRP-cAMP still binds, the inducing effect of olive oil can come into account as well. As a consequence, expression of the of wild type lipase (lipAPG1::gfp) in B. glumae LU8093 in the absence of glucose and presence of olive oil was induced. However, if sucrose or sucrose plus Tween 80 were present in the growth medium, no green fluorescence could be detected (Fig. 33). Thus, this carbohydrate must have a different effect than glucose. In this context, the inducing effect of the CRP-cAMP complex can for example be reduced. In the following Figure, the above discussed aspects are presented schematically.
______97 5. Discussion
CRP - glucose cAMP cAMP B. glumae PG1
P ⊕ P ? lip
UAS CRP box lipA lipB
+ oil/fatty acids ⊕
transcriptional activator
CRP -/+ glucose cAMP cAMP B. glumae LU8093
P? ⊕ Plip
UAS CRP box lipA lipB
+ oil/fatty acids ⊕
Transcriptional activator
Fig. 36: Model of the regulation of lipase gene expression in B. glumae PG1 and B. glumae LU8093. While in B. glumae PG1 lipase gene expression is subjected to catabolite repression, this mechanism seems to be eliminated in B. glumae LU8093. The mutation located in the putative CRP-binding site of B. glumae LU8093 was identified by sequence alignment with the wild type sequence. The second mutation in the cAMP repressor protein (CRP) is postulated, as well as the involvement of a second regulatory protein, which binds to an upstream activator sequence (UAS) in the presence of oil or fats in the growth medium. ⊕ = Induction of gene expression.
Finally, apart from the mechanism of catabolite repression, the mutation in the upstream sequence of the lipAB-operon of B. glumae LU8093 might also have another interesting effect. As described in section 4.4.6, in addition to the CRP binding motif, a putative sequence for a σ54-dependent promoter was identified in this region. Due to the mutation, this sequence was changed from a slightly different consensus sequence to an exact consensus sequence known for σ54-dependent promoters (Barrios et al., 1999). As a consequence, it is possible that this alteration causes an enhanced recognition of the promoter by the sigma factor and thus leads to an increase in lipase gene expression. Consequently, during expression of the lipA(PG1)::gfp fusion in B. glumae LU8093, fluorescence could be detected under inducing conditions, but the measured values were not as high as with the lipA(LU)::gfp fusion harboring the own upstream region. Thus, due to the missing mutation, the effect of increased lipase gene expression did not appear. In 1999, Wang et al. analyzed mutations in the -12 region (TTGC) of the σ54-promoter sequence. The authors found that the cytosine (C) of this tetranucleotide plays an important role, directing the RNA polymerase to
______98 5. Discussion the precise position at which the melted fork junction is created during activation. In the absence of the cytosine, the restriction is lost and deregulation can occur, leading to a reduced recognition of the promoter. In B. glumae PG1 the presence of a thymine (T) at this position would then suggest a reduced promoter activity, while in B. glumae LU8093 the presence of a cytosine (C) causes an enhance recognition of the promoter. Summarizing, the above described effects and possible explanations, elucidate that lipase gene expression in B. glumae is, as in other bacteria, highly complex involving several regulatory mechanisms. Within the scope of this thesis, it was achieved to obtain a first insight into the regulation of lipase gene expression in B. glumae, thereby laying foundations for further studies. Important aspects that have been found will be summarized as following. • Lipase production can be optimized by supplementing the growth medium with 1% (v/v) olive oil and 5-10% (v/v) hexadecane, 0,1% (v/v) Tween 80 or 0,1% (v/v) Triton X-100 or by the addition of 1% (v/v) of an olive oil emulsion with gum arabic. • If olive oil is replaced by sucrose or maltose, the same level of improved lipase production can be obtained by supplementing the medium with 0,1% (v/v) Tween 80 or Triton X-100. • The destabilizing effect of the detergents on the membranes causes a release of lipase and foldase. Furthermore lipase gene expression is induced, maybe directly or due to the release of fatty acids. • Hexadecane does not effect lipase transcription and thus cannot increase lipase activity in the supernatant in the presence of glucose. A minor effect on the posttranscriptional level could be observed by analyzing lipA::gfp fusions. The main effect is suggested to be due to a release of cell-bound lipase directly or indirectly. • Lipase gene expression is subjected to a complex regulatory network, that might involve at least two regulatory proteins of which one is a homologous cAMP response protein (CRP) of the catabolite repression mechanism. • In the production strain B. glumae LU8093 lipase gene expression is not subjected to catabolite repression anymore. In this context, the mutation in the upstream sequence of the lipAB-operon of B. glumae LU8093 plays an important role. • This mutation also changed a putative σ54-dependent promoter sequence to an exact consensus sequence, which in turn might be responsible for an enhanced lipase gene expression in the production strain B. glumae LU8093.
______99 5. Discussion
5.4 Optimization of lipase production in B. glumae – A perspective for future developments High-level production of lipase for industrial purposes requires the optimization of key issues of microbial production processes. Based on the conventionally bred strain, further improvement of lipase production can be achieved by different target approaches using modern genetic engineering methods (van der Werf, 2005). One promising approach comprises the elimination of bottlenecks in the production pathway. Within the scope of this thesis, a putative protease was identified, which led to an increase in extracellular lipase activity when being co-expressed in B. glumae. After integration of several copies of the corresponding gene into the genome of B. glumae LU8093, lipase production can be improved. Since previous studies revealed that also the lipase-specific foldase is a bottleneck in lipase production (El-Khattabi, 2001; Rosenau, 2001), integration of additional copies of the lipB gene into the chromosome of the production strain is another promising approach to improve the production level. Moreover, by analyzing the cosmids, which were identified during the screening, further bottlenecks may be identified and eliminated. For the high-level production of lipase variants, important steps for the establishment of a suitable expression system have been achieved. After providing the second cross-over in B. glumae PGpSUPT7lacI, the first lipase-negative T7-expression host of B. glumae will be available. Apart from the construction of an inducible expression system, which allows controlled gene expression, the construction of an expression system that considers physiological aspects is another attractive approach. This is especially important in the case of lipase production, which requires additional proteins and certain physiological growth conditions. The combination of such an expression system with physiological conditions that are optimal for lipase production, microbial stress can be minimized and the product yield increased. With respect to improving lipase production, physiological conditions should generally be considered to be optimized, since it could be shown that different emulsifiers and detergents have a significant effect on lipase production. At this point, metabolic engineering approaches come into account. Based on the finding that the mutation located in the upstream region of the lipAB-operon in B. glumae LU8093 plays an important role in the regulation of lipase gene expression, the introduction of other mutations in this region might enable to further optimize lipase production.
______100 6. Summary
6. Summary Lipases constitute the most important class of biocatalysts used for a variety of different industrial processes (Jaeger and Eggert, 2002). The Gram-negative bacterium Burkholderia glumae is a plant pathogen, which is useful for biotechnological applications. Moreover, it produces a lipase with favorable enzymatic properties (Schmid et al., 2001). Within the scope of this thesis, lipase production of B. glumae was investigated with special emphasis on 1) development of a T7 RNA polymerase-based expression system, 2) identification and elimination of bottlenecks for an improved lipase production and 3) analysis of regulation of lipase gene expression.
1) Development of a T7 RNA polymerase-based expression system The expression of the T7 RNA polymerase gene in B. glumae is possible and can be tightly controlled using the E. coli lacUV5 promoter and the lacIq gene. By homologous recombination, integration of a suicide vector containing the T7 RNA polymerase gene, into the chromosomal lipAB-operon of B. glumae PG1 was achieved. Furthermore, three broad- host-range expression vectors were constructed, which harbor the lipAB-operon of B. glumae either under control of the T7 or the lac promoter. These expression vectors can also be used to clone and express genes encoding lipase variants. Finally, a second suicide vector was constructed enabling to create a ∆lipAB T7-expression strain of B. glumae based on physiological induction. Thus, expression of the T7 RNA polymerase gene in this strain is controlled by the lip promoter, which allows performing overexpression studies under physiological conditions.
2) Identification and elimination of bottlenecks for an improved lipase production in B. glumae Two cosmid libraries of the wild type B. glumae PG1 and the production strain B. glumae LU8093 were constructed. Screening the cosmid library of B. glumae PG1 led to the identification of two potential bottlenecks in lipase production. While co-expression of a putative tripartite efflux pump led to a decreased lipase activity in the supernatant of B. glumae PG1 and B. glumae LU8093, co-expression of a putative, cytoplasmic protease led to a significant increase in lipase activity in the supernatant of both B. glumae strains. The corresponding gene of the putative protease was successfully PCR amplified from the genome of B. glumae PG1, cloned and overexpressed in the heterologous host E. coli BL21(DE3). After purification of the native protein by affinity chromatography, biochemical
______101 6. Summary characterization revealed that under these conditions, the protein forms a homodimer having a molecular weight of 39 kDa. Furthermore, it could be shown that the protein displays specific proteolytic activity towards three substrates for amino peptidases.
3) Analysis of regulation of lipase gene expression Investigation of the effect of chemical agents on lipase production revealed that in the presence of olive oil, hexadecane, gum arabic and the two non-ionic detergents Triton X-100 and Tween 80 significantly increased lipase activity in the supernatant of B. glumae PG1 and B. glumae LU8093. If olive oil was replaced by sucrose, the detergents still had the same increasing effect on lipase activity. Further studies revealed that the detergents destabilized the bacterial membranes leading to a release of lipase and foldase, but also induced lipase gene expression. In contrast, hexadecane seemed to cause a detachment of cell-bound lipase directly or indirectly and had only a slight effect on the posttranscriptional level. Furthermore, it was found that lipase gene expression is regulated on the transcriptional level, which is enhanced in the production strain B. glumae LU8093. Analysis of the DNA-sequence of the upstream region of the lipAB-operon led to the identification of a single point mutation in the sequence of B. glumae LU8093. This mutation is suggested to be involved in the elimination of catabolite repression and enhancement of lipase gene expression in the production strain. Taken together, the obtained results indicate a complex regulation of lipase gene expression in B. glumae in which at least two regulatory proteins appear to be directly involved.
______102 7. Zusammenfassung
7. Zusammenfassung Das pflanzenpathogene Bakterium Burkholderia glumae sekretiert eine Lipase, die interessante biochemische Eigenschaften für verschiedene industrielle Prozesse besitzt (Schmid et al., 2001). Eine biotechnologische Anwendung dieses Enzyms erfordert somit eine Produktion des Proteins im großen Maßstab. Im Rahmen dieser Arbeit wurde die Lipaseproduktion in B. glumae untersucht. Dabei lagen die Schwerpunkte 1) in der Etablierung eines T7 RNA Polymerase-abhängigen Expressionssystems in B. glumae, 2) der Identifizierung und Beseitigung potentieller Engpässe zur Optimierung der Lipaseproduktion sowie 3) in der Untersuchung der Regulation der Lipase-Genexpression.
1) Etablierung eines T7 RNA Polymerase-abhängigen Expressionssystems in B. glumae Es konnte gezeigt werden, dass die Expression der T7 RNA Polymerase in B. glumae möglich ist und dass die Verwendung des E. coli lacUV5-Promotors und lacIq Gens eine strikte Kontrolle der Expression der T7 RNA Polymerase erlauben. Die Integration des T7 RNA Polymerase Gens in das Chromosom des Wildtyps B. glumae PG1 wurde über eine Vektorintegration erzielt. Des Weiteren wurde das lipAB-Operon von B. glumae erfolgreich in zwei Expressionsvektoren kloniert, die, je nach Orientierung des Operons, eine konstitutive Expression oder eine T7 RNA Polymerase-abhängige Expression der Lipase erlauben. Insgesamt stehen nun drei Expressionsvektoren zu Verfügung, die ebenfalls für eine homologe Expression von Lipasevarianten verwendet werden können. Die Konstruktion eines zweiten Mutagenesevektors, in dem die Expression der T7 RNA Polymerase vom Lipase Promotor reguliert wird, ermöglicht außerdem die Herstellung eines physiologisch induzierbaren T7-Expressionsstammes von B. glumae.
2) Identifizierung und Beseitigung von Engpässen zur Optimierung der Lipaseproduktion Vom Wildtyp B. glumae PG1 und Produktionsstamm B. glumae LU8093 wurden jeweils eine Cosmidbank angelegt. Die Durchmusterung der Cosmidbank von B. glumae PG1 führte zur Identifizierung von zwei potentiell limitierenden Faktoren der Lipaseproduktion in B. glumae. Im ersten Fall, wurde durch die Co-expression eines putativen Efflux-Systems für niedermolekulare Substanzen, die Lipaseaktivität im Überstand von B. glumae PG1 und B. glumae LU8093 reduziert. Dagegen führte die Co-expression einer putativen,
______103 7. Zusammenfassung cytoplasmatischen Protease zu einer signifikanten Aktivitätssteigerung der Lipase im Überstand beider B. glumae Stämme. Das Gen der putativen Protease von B. glumae PG1 wurde erfolgreich kloniert, im heterologen Wirt E. coli BL21(DE3) überexprimiert und das Genprodukt mittels Affinitätschromatographie gereinigt. Eine anschließende biochemische Charakterisierung ergab, dass das native Protein unter den gegebenen Bedingungen ein Homodimer mit einem Molekulargewicht von ca. 39 kDa ausbildet. Außerdem konnte eine spezifische Aktivität gegenüber drei Aminopeptidase-Substraten nachgewiesen werden.
3) Untersuchung der Regulation der Lipase-Genexpression Physiologische Untersuchungen ergaben, dass in Gegenwart von Olivenöl im Medium, die Zugabe von Hexadekan, Gum Arabicum, Triton X-100 oder Tween 80 eine deutliche Steigerung der Lipaseaktivität im Überstand von B. glumae PG1 und B. glumae LU8093 bewirken. In Gegenwart eines Kohlenhydrats als C-Quelle konnte diese Wirkung durch Zugabe eines Detergenz ebenfalls erzielt werden. Durch Western-Blot Analysen und mit Hilfe von gfp-Reporter-Genfusionen wurde neben der destabilisierenden Wirkung der Detergenzien auf die bakterielle Zellmembran, auch eine induzierende Wirkung der Lipase-Genexpression auf transkriptionaler Ebene nachgewiesen. Im Gegensatz dazu, zeigte Hexadekan nur eine geringe Wirkung auf posttranskriptionaler Ebene. In diesem Fall, wurde der aktivitätssteigernde Effekt auf eine direkte oder indirekte Ablösung der Lipase vom Lipopolysaccharid der äußeren Membran zurückgeführt. Mit Hilfe der Reporter-Genfusionen wurde außerdem eine Regulation der Lipase-Genexpression auf transkriptionaler Ebene nachgewiesen. Des Weiteren wurde eine Punktmutation im Stromaufwärtsbereich des lipAB- Operons von B. glumae LU8093 identifiziert, die vermutlich eine wichtige Rolle in der Aufhebung der Kontrolle der Katabolitrepression sowie der gesteigerten Lipase- Genexpression in B. glumae LU8093 spielt. Zusammenfassend lassen die Ergebnisse eine komplexe Regulation der Lipase-Genexpression in B. glumae vermuten, an der mindestens zwei regulatorische Proteine direkt beteiligt sind.
______104 8. References
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9. Appendix
BamHI
Plac Amp
6000 1000 pUC ori pBKST7pollacI
5000 2000
gene1 PT7 4000 3000
HindIII
lacI
PlacUV5
1) HindIII/BamHI restriction 2) Gelelution of 3,8 kb fragment
P H lacIq lacUV5 gene1 B
1) HindIII/BamHI restriction of pSUP202 2) Ligation with 3,8 kb fragment
HindIII
mob lacI PlacUV5
10000 Cm 2000 pSUPT7pollacI
8000 gene1 up Amp 4000
6000 460 bp
'Tc PCR fragment rep blunt-end BamHI SalI 1) HindIII restriction 2) Blunting ends using T4 DNA polymerase Ligation
up mob lacI PlacUV5
10000 Cm 2000 pSUPT7pollacIup
8000 gene1 Amp 4000 B do w n S 6000 490 bp rep 'Tc PCR fragment BamHI BamHI/SalI restriction SalI BamHI/SalI restriction
Ligation
up mob lacI PlacUV5
10000 Cm 2000 pSUPT7pollacIupdown
gene1 8000 Amp 4000
6000
down rep 'Tc
Fig. 37: Schematic drawing of the construction of the suicide vector pSUPT7pollacIup/down. B: BamHI; H: HindIII; S: SalI; up/down: upstream or downstream sequence of the lipAB-operon of B. glumae PG1.
______119 9. Appendix
EcEcoRIoRI HiHindIII
PsPsttI up lipA
lipB EcEcoRIoRI
12000 2000
PBP1500 km 10000 4000
8000 6000
1) EcoRI restriction 2) Gelelution of 2,9 kb fragment E up lipA lipB E
1) EcoRI restriction of pBKS 2) Ligation with 2,9 kb fragment
EcoRI
PT7 Amp
'up HindIII 5000
1000 pBKSlipAB
pUC ori 4000
2000 lipA
3000
Plac
lipB PstI
HindIII EcoRI 1) HindIII restriction 2) Gelelution of 2,2 kb fragment
H lipA lipB H rbs
1) HindIII restriction of pBBR22b 2) Ligation with 2,2 kb fragment and pBBR1mcs
HindIII
PT7 mob mob PstI lipA CM CM
Cm SalI SalI HindIII HindIII Plac 6000 7000 Plac 6000 1000 1000 1000 lipB pBBR22lpBBR22lipABT7 pBBRlipABT7 pBBRlipABlac 2000 6000 5000 5000 2000 2000 lipB lipA rep 5000 3000 rep 4000 HindIII 4000 3000 3000 4000
lipB rep lipA PstI mob PT7 PT7 PstI
HindIII HindIII
Fig. 38: Schematic drawing of the construction of the expression vectors pBBR22lipABT7, pBBRlipABT7 and pBBRlipABlac. E: EcoRI; H: HindIII; rbs: ribosome binding site; up: 460bp of upstream sequence of the lipAB-operon of B. glumae LU8093.
______120 9. Appendix
StuI XbaI EcoRI SphI XhoI SmaI XmaI HindIII BamHI MCS' gfp bla to
7000 CAT 1000
t1 6000pSWgfp(LAA) 2000 ori
5000 'lacZ' 3000 4000 E blunt end E blunt end E S E S upPG1 upLU upPG1 upLU rep oriT 460 bp 460 bp ori1600 460 bp 460 bp PCR fragment PCR fragment PCR fragment PCR fragment EcoRI/SmaI restriction EcoRI/SphI restriction
Ligation Ligation
SphI EcEcooRI EcoRI SphI EcoRI EcoRI HinndIIIdIII HindIII HindIII HindIII up up up up gfpgfp gfp to to 'gfpmut3* gfp to to bla bla CAT CAT bla bla 8000 8000 CAT 8000 CAT 8000
t1 t1 t1 t1 pSWgfpupPG (TCF) pSWgfpupLU (TCF) 6000 6000 pSWgfp(LAA)upPG (TLF) pSWgfp(LAA)upLU (TLF) 2000 2000 6000 6000 2000 2000 'lacZacZ'' 'lacZ' 'lacZ' 'lacZ' ori ori ori ori 4000 4000 4000 4000
oriT oriT rep oriT oriT rep ori1600 rep ori1600 rep ori1600 ori1600
1) EcoRI/HindIII restriction 1) EcoRI/HindIII restriction 2) Gelelution of gfpupPG/LU 2) Gelelution of gfp(LAA)up (TCF) PG/LU (TLF) P P Plip Plip E lip H lip E up gfp H E up gfp H up gfp E up gfp H rbs rbs 1) EcoRI/HindIII restriction 1) EcoRI/HindIII restriction of pBBR1mcs-2 of pBBR1mcs-2 2) Ligation with gfpupPG1/LU 2) Ligation with gfp(LAA)up (TCF) PG1/LU (TLF)
Km Km Km Km mob mob mob mob
5000 5000 5000 5000 1000 1000 1000 1000 pBBKgfpupLU (TCF) HindIII pBBKgfp(LAA)upLU (TLF) HindIII pBBKgfpupPG (TCF) HindIII HindIII pBBKgfp(LAA)upPG (TLF)
4000 4000 4000 4000 2000 2000 2000 2000 gfp gfp gfp gfp
3000 3000 3000 3000
rep rep rep rep 'up 'up 'up 'up SSpphIhI SphI
EcoRI EcoRI EcoRI EcoRI
Fig. 39: Schematic drawing of the construction of the plasmid fusions lipA::gfp(LAA). E: EcoRI; H:HindIII; S: SphI; gfp: green fluorescent protein; gfp (LAA): unstable variant of gfp; rbs: ribosome binding site; TLF: translational fusion, TCF: transcriptional fusion; up: 460bp of upstream sequence of the lipAB-operon of B. glumae PG1 or B. glumae LU8093.
______121 9. Appendix
Sequences of identified open reading frames of B. glumae PG1
A) ATGCAGCAGCGTGGCGGCAGCATGAGCAAGAAGATCGCCGTCCTGGCCGTGGACGAATTCGA AGACAGCGAACTGGTCGAACCCCTGCGGGCGCTGCGTAAGGCCGGCGCCGAAGTCGACGTGA TCAGCCAGCAGGCGGGCGAAGTCAAGGGGTTCCGTCACGCGGACAAGGGGGAAGCCGTCAAG GTGGACCGTACGTTCGACGAAGTCCGGGAAGGCGAATTCGACGCGCTGCTGCTGCCCGGCGGC CTGCTGCGTGGCGACAATCGGATGCTGCCCGCGGCCCGTGAATTCGTGACCGCCGGCAAGCCG GTCTTCGCGATCTGCCACGGCGGGTGGCTGCTGGGCAGCAGCGGGGTCATCAACGGCCGCAAG CTGACCGCCTGGCCCAGCCTGCAGGACGACGTGAAGAATGCGGGCGGCGAATTCTACGACCA GGAAGTGGTGCGTGACAAGGACCAGCTGATCACCAGCCGTAAGCCCGACGACCTGCCGGCCTT CAACCGGGAAGCGCTGCGGCTGCTGGGGGCCGGCGCGTGA
B) MQQRGGSMSKKIAVLAVDEFEDSELVEPLRALRKAGAEVDVISQQAGEVKGFRHADKGEAVKVD RTFDEVREGEFDALLLPGGLLRGDNRMLPAAREFVTAGKPVFAICHGGWLLGSSGVINGRKLTAW PSLQDDVKNAGGEFYDQEVVRDKDQLITSRKPDDLPAFNREALRLLGAGA
Fig. 40: A) DNA sequence and B) amino acid sequence of putative protease designated Pro. Start and stop codon are underlined.
A) ATGGCACGTTTCTTCATCGATCGCCCCGTGTTCGCATGGGTGATCGCGCTGTTCATCCTGCTCG GCGGCGGCTTCGCGATTCGTGCGCTGCCGGTCGCGCAGTATCCCGACATCGCGCCGCCCGTCG TCAGCATCTATGCGTCTTACCCGGGCGCGTCCGCGCAGGTCGTCGAGGAATCGGTGACCGCGC TGATCGAGCGCGAGATGAACGGCGCGCCGGGGCTGTTGTACACGTCGGCGAGCAGCAGCGCC GGGAGCGCATCGCTCTATCTGACCTTCAAGCAGGGCGTGAACGCCGATCTCGCGGCCGTCGAA GTGCAGAACCGGCTGAAGACGGTCGACGCGCGGCTGCCCGAACCGGTGCGGCGCGCCGGCAT CCAGGTCGAGAAGGCAGCGGACAACATCCAGCTGGTCGTGTCGCTGACGTCGGACGACGGCC GCATGACCGACGTGCAGCTCGGCGAATACGCGTCGGCGAACGTCGTGCAGGCGCTGCGCCGCG TCGACGGCGTCGGCCGCGTGCAGTTCTGGGGCGCCGAATACGCGATGCGGATCTGGCCGGACC CGGACAAGCTGGCCGGACATGGCGTCACCGCGTCGGACATCGCGTCGGCCGTGCGTGCGCACA ACGCGCGCGTGACGATCGGCGACATCGGCCGCAGCGCGGTGCCGGACAGCGCGCCGATCGCC GCGACGGTGTTCGCCGACGCGCCGCTGAAGACGCCGGCCGATTTCGGCGCGATCGCGCTGCGC ACGCAGCCGGACGGCTCCGCGCTCTATCTGCGCGACGTCGCGCGCGTCGAGTTCGGCGGCAAC GACTACAACTATCCGTCGTACGTGAACGGCAAGGTCGCGACCGGCATGGGGATCAAGCTCGCG CCCGGCTCGAACGCGGTCGCGACCGAGCGGCGCGTGCGCGCGGCGATGGACGAGCTGTCCGC GTACTTCCCGCCGGGCGTGAAGTACCAGATCCCGTACGAGACATCGTCGTTCGTGCGCGTGTC GATGAACAAGGTCGTCACGACGCTGATCGAGGCCGGCGTGCTGGTGTTCCTCGTGATGTTCCT GTTCATGCAGAACCTGCGCGCGACGCTGATCCCGACGCTCGTCGTGCCGGTCGCGCTCGCGGG CACGTTCGGCGTGATGCAGGCGCTCGGCTTCTCGATCAACGTACTGACGATGTTCGGGATGGT GCTCGCGATCGGCATCCTCGTCGACGATGCGATCGTCGTCGTCGAGAACGTCGAGCGGCTGAT GGTCGAGGAGCGGCTCGAGCCGTACGAAGCGACCGTCAAGGCGATGCAGCAGATCAGCGGCG CGATCGTCGGCATCACGGTGGTGCTGACCTCGGTGTTCGTGCCGATGGCGTTCTTCGGCGGCGC GGTGGGCAACATCTACAGGCAGTTCGCGCTCGCGCTCGCGGTGTCGATCGCCTTCTCGGCATTC CTCGCGCTGTCGCTGACGCCCGCGCTGTGCGCGACGCTGCTCAAGCCGGTGGACGGCGGCCAT CACGACAAGCGCGGCTTCTTCGGCGCGTTCAACCGCTTCGTCGCGCGCGCGACGCAGCGCTAT GCGACGCGCGTCGGCACGATGCTCGCCAGGCCGCTGCGCTGGCTCGTCGTGTACGGCGCGCTG ACCGCGGCCGCGGTGCTGATGCTCACGCAACTGCCGAGCGCGTTCCTGCCCGACGAGGATCAG GGCAACTTCATGGTGATGGTGATCCGGCCGCAGGGCACGCCGCTCGCCGAGACGATGCGCAGC GTGCGCGAGGTCGACGCGTACCTGCGCCGCGAGGAGCCGGCCGCGTACACGTTCGCGCTCGGC GGCTTCAACCTGTACGGCGAAGGGCCGAACGGCGGGATGATCTTCGTCTCGCTGAAGGACTGG CGCGCGCGCAAGGCCGCGCGCGATCACGTGCAGGCGATCGTCGCGCGCATCAACGCGCGCTTC GCGGGCACGCCGAACACGACGGTGTTCGCGATGAACGCGCCGGCCTTGCCCTATCTCGGCTCG ACGAGCGGCTTCGACTTCCGGCTGCAGAACCGCGGCGGGCTCGACTACGCGGCGTTCAGCGCC GCGCGCGAACAGTTGCTCGCGGCGGCCGGCCGCGACCCTGCGCTGACCGACGTGATGTTCGCC
______122 9. Appendix
GGCATGCAGGACGCGCCGCAACTGAAGCTCGACGTCGATCGCGCGAAGGCGTCGGCGCTCGG CGTGTCGATGGACGAGATCAACACGACGCTCGCGGTGATGTTCGGCTCCGACTACATCGGCGA CTTCATGCACGGCACGCAGGTGCGCCGCGTGATCGTGCAGGCCGACGGCCAGCATCGCGTCGA TCCCGACGACGTCAAGAAGCTGCGCGTGCGCAACGCGCGCGGCGAGATGGTGCCGCTCGCGG CGTTCACGACGCTGCACTGGACGCTCGGGCCGCCGCAGCTCACGCGCTACAACGGCTTCCCGT CGTTCACGATCAACGGCTCGGCCGCGCCGGGACACAGCAGCGGCGAAGCGATGGCCGCGCTC GAGCGGCTCGCCGCGACGCTGCCGGCGGGAATCGGCCACGCGTGGTCGGGACAGTCGTTCGA GGAGCGGCTGTCGGGCGCGCAGGCGCCGATGCTGTTCGCGCTGTCGGTGCTGGTGGTGTTCCT CGCGCTCGCGGCGCTGTACGAGAGCTGGTCGATTCCGTTCGCGGTGATGCTGGTCGTTCCGCTC GGCGTGATCGGCGCGGTGCTCGGCGTCACGTTGCGCGCGATGCCGAACGACATCTACTTCAAG GTCGGGCTGATCGCGACGATCGGCTTGTCGGCGAAGAACGCGATCCTGATCGTCGAAGTCGCG AAGGATCTGGTCGCGCAGCGCATGCCGCTGATCGACGCCGCGCGCGAGGCCGCCCGCCTGCGG CTGCGGCCGATCGTGATGACGTCGCTCGCGTTCGGCGTCGGCGTGCTGCCGCTCGCGTTCGCGT CGGGCGCGGCGTCCGGCGCGCAGATGGCGATCGGCACCGGCGTGCTCGGCGGCGTGATCACG GCCACGGTGCTCGCGGTGTTTCTCGTGCCGCTGTTTTTCGTGATGGTCGGCCGCGTGTTCGACG TCGGCCCGCGCCGGCGCGGCGCGTCGCAGCCGACGACGATGGAGGGTTCGCATTGA
B) MARFFIDRPVFAWVIALFILLGGGFAIRALPVAQYPDIAPPVVSIYASYPGASAQVVEESVTALIERE MNGAPGLLYTSASSSAGSASLYLTFKQGVNADLAAVEVQNRLKTVDARLPEPVRRAGIQVEKAAD NIQLVVSLTSDDGRMTDVQLGEYASANVVQALRRVDGVGRVQFWGAEYAMRIWPDPDKLAGHG VTASDIASAVRAHNARVTIGDIGRSAVPDSAPIAATVFADAPLKTPADFGAIALRTQPDGSALYLRD VARVEFGGNDYNYPSYVNGKVATGMGIKLAPGSNAVATERRVRAAMDELSAYFPPGVKYQIPYE TSSFVRVSMNKVVTTLIEAGVLVFLVMFLFMQNLRATLIPTLVVPVALAGTFGVMQALGFSINVLT MFGMVLAIGILVDDAIVVVENVERLMVEERLEPYEATVKAMQQISGAIVGITVVLTSVFVPMAFFG GAVGNIYRQFALALAVSIAFSAFLALSLTPALCATLLKPVDGGHHDKRGFFGAFNRFVARATQRYA TRVGTMLARPLRWLVVYGALTAAAVLMLTQLPSAFLPDEDQGNFMVMVIRPQGTPLAETMRSVR EVDAYLRREEPAAYTFALGGFNLYGEGPNGGMIFVSLKDWRARKAARDHVQAIVARINARFAGTP NTTVFAMNAPALPYLGSTSGFDFRLQNRGGLDYAAFSAAREQLLAAAGRDPALTDVMFAGMQDA PQLKLDVDRAKASALGVSMDEINTTLAVMFGSDYIGDFMHGTQVRRVIVQADGQHRVDPDDVKK LRVRNARGEMVPLAAFTTLHWTLGPPQLTRYNGFPSFTINGSAAPGHSSGEAMAALERLAATLPA GIGHAWSGQSFEERLSGAQAPMLFALSVLVVFLALAALYESWSIPFAVMLVVPLGVIGAVLGVTLR AMPNDIYFKVGLIATIGLSAKNAILIVEVAKDLVAQRMPLIDAAREAARLRLRPIVMTSLAFGVGVL PLAFASGAASGAQMAIGTGVLGGVITATVLAVFLVPLFFVMVGRVFDVGPRRRGASQPTTMEGSH
Fig. 41: A) DNA sequence and B) amino acid sequence of putative efflux pump designated EpA. Start and stop codon are underlined.
A) ATGAACAGGAAACCTTTGGCACGCGCGGTGTTGACGGTATTTGCCGGCGCGGCGCTGCTCGGC GCGGGCTATTTCGCGGGAACGCGTCACGCGGCGACCGGCACCGCAGTTGCCTCGACTGGCGCC GCCTCGCCGGGCGGCAAGATCGACCCGAAGACCGGGCGGAAGGTGCTGTACTGGCACGACCC GATGGTGCCGAACCAGCATTTCGACAAGCCGGGTAAATCGCCATTCATGGACATGCAACTTGA GCCTGTCTACGCGGATGAAGGCGAGAGCTCTTCAGGCATCAAGATCGACCCGGGGCTTGAGCA GAATCTTGGCATTCGCTATGCGACCGTGCGTCGGCAGGAAACGACCGGCGGATTCGATGCCAT CGGCACGACGCAGTTCGATGAATCGCACTCGGATGTCGTGCAGTCGCGCGTCACCGGCTACAT CGACCGGCTCTATGCGAACGCGCCGATGCAACGCATCGCAAAAGGCGCACCAGTCGCATCGCT GTTCGTCCCGGAATGGCTCGCGCCGCAGGAGGAATATCTCGCACTCAAGCGCGGCGGCATGGA CGCCACTTTGCTCGAAGCGTCGCGAGCGCGGATGCGCGCCTTGTCGATTCCTGATGGAATCATC GCGAGCCTTGACCGAACCGGCAAGGCCCAGACGCACGTCTTGCTGACGTCGCCCGAGTCCGGT GTCGTCAGCGAACTGAACGTCCGCGACGGCGCGATGGTCGCGCCCGGCCAGACACTCGCGAA GATTGCCGGCCTGTCGAAGCTGTGGCTCATCGTCGAGATTCCCGAGGCGCTCGCGCTGGGCGT ACAACCCGGTATGACCGTCGATGCGACGTTCGCAGGCGACCCGACACAACACTTCAACGGTCG CATCCGCGAAATCCTGCCGGGCATCAGCACCACCAGCCGCACGCTCCAGGCGCGTCTGGAAAT CGACAACGCCGGATTCAAGCTGACGCCGGGCATGCTGATGCGCGTGCGCGTTGCCGGCCAGAA
______123 9. Appendix
GGCTGTCTCGCGACTGCTGGTGCCCTCCGAAGCCGTGATCACAACGGGCAAGCGCTCGGTCGT CATCGTAAAGAACGGCGACGGGCGCCTTCAGCCGGCGACGGTTACCGTGGGCAATGACATTGG CAACGAAACCGAGGTGCTGAGCGGCCTGAACGACGGCGACACCGTTGTCGCGTCCGGCCAGTT CCTGATCGATTCCGAAGCGAGCCTGAAGTCCGTTCTGCCGAGGCTGGAGGGCAGCACGGGGGC AAGCGCAAGCGCACCGGCGTCCGCACCCGCCGTCGCAGCACAAACGTACGAGACCACCGGCA AGGTCGAAAAAGTGACCGCTGCGGACATCACGTTCTCCCATCAACCCGTGCCGGCGCTCGGCT GGGGCGCGATGACGATGGCGTTCAACAAGCCCGCTCCCGATGCCTTCCCCGACGTCAAGGCCG GACAGACGGTGCACTTCGTCTTCAAGCAGTCGGATGAGGGCTACCAGTTGACGAAGGTCGAAC CGGTCGGAGGCGTGCAATGA
B) MNRKPLARAVLTVFAGAALLGAGYFAGTRHAATGTAVASTGAASPGGKIDPKTGRKVLYWHDP MVPNQHFDKPGKSPFMDMQLEPVYADEGESSSGIKIDPGLEQNLGIRYATVRRQETTGGFDAIGTT QFDESHSDVVQSRVTGYIDRLYANAPMQRIAKGAPVASLFVPEWLAPQEEYLALKRGGMDATLLE ASRARMRALSIPDGIIASLDRTGKAQTHVLLTSPESGVVSELNVRDGAMVAPGQTLAKIAGLSKLW LIVEIPEALALGVQPGMTVDATFAGDPTQHFNGRIREILPGISTTSRTLQARLEIDNAGFKLTPGMLM RVRVAGQKAVSRLLVPSEAVITTGKRSVVIVKNGDGRLQPATVTVGNDIGNETEVLSGLNDGDTV VASGQFLIDSEASLKSVLPRLEGSTGASASAPASAPAVAAQTYETTGKVEKVTAADITFSHQPVPAL GWGAMTMAFNKPAPDAFPDVKAGQTVHFVFKQSDEGYQLTKVEPVGGVQ
Fig. 42: A) DNA sequence and B) amino acid sequence of putative membrane fusion protein designated MfpB. Start and stop codon are underlined.
A) ATGGTCGCGCTCAATCGCCGCATGGCGGGCCGCGTCCCGCTCGCGCTGGCCGCGGCGCTCGCG CTCGCGGGCTGCTCGCTCGCGCCGCACTACGAACGGCCGGCCGCGCCCGTGCCGGCCAGCTAC GCAGCGCCGGACGGCGGGCAGGGCGGGCAGGGTGGCGAGCCCGCTGCGTCGGCATCGGCCGA TGCAGCGCTGCTCGACGACTGGCGCGCCTACTTCACCGACCCCACGCTGCAGGCGTGGATCGA CGCCGCGCTCGCGAACAATCGCGACCTGCGGATCGCGGCCGGCCGGCTCGACGAAGCGCGCG CGCTGTACGGCGTGCAGCGCGCGGACCAGCTGCCGTCGCTCGATGCGAACCTCGCCTACGACC GCACCCGCCAGTACGACCCGGTGGTGCGCCAGAGCGCGGTGAGCGGGCTGTATCGGGCGGGC GTCGGCATCAGCGCCTACGAGCTGGACCTGTTCGGCCGCGTGCGCAGCCTGTCGGATGCCGCA CTCGCCGACTATTTCGCGACGGCGTATGCGCAGCGCACGGTGCGCATCGGCGTGATCGCCGAA GTCGCCGGCGCGTATGTCGCGCAACGATCGTTGCAGGAGCAACTGGCGCTGGCGCAGCGCACG CTCGACGCGCGCGAACGCATCGCCGCGCTCACGCGGCGCCGCTACGCGGCCGGCACGAGCGAT GCGATCGAGCTGCGCTCGGCCGAGATGCTGGTGGCGTCCGCGCGTGCGTCGCAGGCCGCGCTG CAGCGCGAGCATGCGCAAGCGGTGCGCGCGCTGCAGCTGCTCGCCGGCGATTTCGCGCGCGAG GCGCCGGCCGACGGCGCGACGCTCGACGCGCTCACCATCGCGCCGGTCGCGCCCGGCGCGCCG AGCGCGTTGCTCGAGCGGCGGCCCGACGTCCGTCAGGCCGAGTCGCGTCTGCAGGCCGCGAAC GCGCAGATCGGCGCGGCGCGCGCGGCATTCTTCCCGCGCATTGCGCTGACGACCGACTACGGC TCGGTCAGCGACGCGTTCTCGAGCCTGTTCGCGGCGGGTACCAGCGTCTGGTCGTTTGCGCCGC GCATCACGCTGCCGATCTTCGCGGGCGGGCGCAATCGCGCCAATCTCGACGTGGCCGACGCGC GCAAGTACATCGCCGTCGCCGAATACGAGAAGACCGTGCAGACGGCGTTCCGCGAAGTGGCC GACGCGTTCGCCGCACGCGGCTGGATCGATCGCCAGCTCGCGGCGCAGCAGGACGTCTACGCG GCCGACGGCGCCCGGCTGAAACTCGCCGAGCGTCGCTATGCGGCCGGCGTCGCGACCTACCTC GAGCTGCTCGACGCGCAGCGCAGCACCTACGAGTCGGGCCAGGCGCTGATCCGGCTCAAGGA GCTGCGCCTCGCGAACGCGATCGCGCTGTATCGCGCGCTCGGCGGCGGCTGGACGCCCGCCGA AGGCGCGGACGCCGCCGCGTGA
______124 9. Appendix
B) MVALNRRMAGRVPLALAAALALAGCSLAPHYERPAAPVPASYAAPDGGQGGQGGEPAASASAD AALLDDWRAYFTDPTLQAWIDAALANNRDLRIAAGRLDEARALYGVQRADQLPSLDANLAYDRT RQYDPVVRQSAVSGLYRAGVGISAYELDLFGRVRSLSDAALADYFATAYAQRTVRIGVIAEVAGA YVAQRSLQEQLALAQRTLDARERIAALTRRRYAAGTSDAIELRSAEMLVASARASQAALQREHAQ AVRALQLLAGDFAREAPADGATLDALTIAPVAPGAPSALLERRPDVRQAESRLQAANAQIGAARA AFFPRIALTTDYGSVSDAFSSLFAAGTSVWSFAPRITLPIFAGGRNRANLDVADARKYIAVAEYEKT VQTAFREVADAFAARGWIDRQLAAQQDVYAADGARLKLAERRYAAGVATYLELLDAQRSTYES GQALIRLKELRLANAIALYRALGGGWTPAEGADAAA
Fig. 43: A) DNA sequence and B) amino acid sequence of putative outer membrane protein designated OmpC. Start and stop codon are underlined.
______125 ______
Lebenslauf
Name: Beselin Vorname: Anke Geburtsdatum: 06.04.1976 Geburtsort: Frankfurt am Main Familienstand: ledig
Schulausbildung: 1982-1986 Kettler-Francke-Grundschule in Bad Homburg v. d. Höhe 1986-1987 Humboldt-Gymnasium in Bad Homburg v. d. Höhe 1987-1992 Luisen-Gymnasium in Essen 1992-1993 Senior High School in Bowie, Texas (USA) (Schüleraustausch) 1993-1995 Luisen-Gymnasium in Essen Abschluss: Allgemeine Hochschulreife
Hochschulausbildung: 10/1995-04/2000 Diplom-Studiengang der Fachrichtung Biologie an der Ruhr-Universität Bochum 03/1998 Vordiplom Biologie 03-04/2000 Diplom-Hauptprüfungen: Biol. Hauptfach: Mikrobiologie und Biotechnologie Biol. Nebenfach: Biochemie Außerbiol. Nebenfach: Immunologie 05/2000-04/2001 Diplomarbeit am Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum Thema: Modifikation des Codon-Gebrauchs von Escherichia coli seit 06/2001 Promotion am Institut für Molekulare Enzymtechnologie der Heinrich-Heine Universität Düsseldorf im Forschungszentrum Jülich Thema: Optimierung der Lipaseproduktion in Burkholderia glumae
Anstellungen: 08/2000-01/2001 Studentische Hilfskraft an der Ruhr-Universität Bochum (Lehrstuhl Biologie der Mikroorganismen) 05/2001-07/2001 Wissenschaftliche Hilfskraft an der Ruhr-Universität Bochum (Lehrstuhl Biologie der Mikroorganismen) 08/2001-07/2002 Stipendium nach dem Graduiertenförderungsgesetz NRW zur Förderung wissenschaftlichen und künstlerischen Nachwuchses an der Ruhr-Universität Bochum 08/2002-12/2002 Wissenschaftliche Hilfskraft an der Ruhr-Universität Bochum (Lehrstuhl Biologie der Mikroorganismen) 01/2003-06/2005 Wissenschaftliche Angestellte an der Heinrich-Heine Universität Düsseldorf (Institut für Molekulare Enzymtechnologie) seit 07/2005 Wissenschaftliche Hilfskraft an der Heinrich-Heine Universität Düsseldorf (Institut für Molekulare Enzymtechnologie)
______