Science, Faculty of
Microbiology and Immunology, Department of
DSpace
UBCV
Jones, Michael D.
Doctor of Philosophy - PhD
University of British Columbia
Classically, outer membranes are half lipid, half protein, and the outmost layers of Gram- negative bacteria. For Caulobacter crescentus the outer membrane is the penultimate layer beneath a protein surface layer (S-layer). The S-layer of the caulobacter cell envelope is an exciting platform for high density peptide display and biotechnology development. We focused on elucidating the structure of the outer membrane by crystallizing the S-layer protein, RsaA; solving the structure of the the lipopolysaccharide; and characterizing a newly discovered porin, OmpW. S-layer proteins are highly resistant to crystallization, because wo-dimensional S-layer formation out competes three-dimensional crystal formation. To achieve a crystallisable form of RsaA, a C-terminal truncation version was constructed and expressed in the native host, C. crescentus. The secreted protein was prone to aggregation, so low agitation and slow concentration protocols had to be developed. The RsaA truncate produced large crystals that diffracted to <2.5 Å. Solving the phases proved to be a serious hurdle and the final protein structure remains unsolved. The lipopolysaccharide of C. crescentus is the anchor that supports the S-layer. The structure of the lipid A portion was solved previously but structures for the core oligosaccharide and the O-polysaccharide had not been deduced. In collaboration with Dr. Evgeny Vinogradov, these remaining structures were solved. The core oligosaccharide has a branched heptasaccharide structure. The O-polysaccharide is a heptasaccharide containing the dideoxy sugar N-acetylperosamine. Additionally, a rhamnan polysaccharide was discovered and its structure was determined. Porins, non-specific passive protein channels, are significant components of classical Gram-negative outer membranes. Despite this, no porin had ever been identified in C. crescentus. We report the identification and characterization of the porin OmpW in C. crescentus. OmpW has low conductance of 125 pSv in 1 M KCl. That is interesting because homologous porins in other bacteria have no detectable pore-forming activity. The cell envelopes of bacteria are remarkable structures; the work here illuminates the unique structures present in the caulobacter envelope.
Studies on the structure and composition of the outermembrane of Caulobacter crescentusbyMichael D JonesB. Science, Specialization in Biotechnology, University of Alberta, 2006M. Science, Pharmaceutical Sciences, University of Alberta, 2008A THESIS SUBMITTED IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFDoctor of PhilosophyinTHE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES(Microbiology and Immunology)The University of British Columbia(Vancouver)October 2015c©Michael D Jones, 2015AbstractClassically, outer membranes are half lipid, half protein, and the outmost layers of Gram-negative bacteria. For Caulobacter crescentus the outer membrane is the penultimate layerbeneath a protein surface layer (S-layer). The S-layer of the caulobacter cell envelope is anexciting platform for high density peptide display and biotechnology development. We focusedon elucidating the structure of the outer membrane by crystallizing the S-layer protein, RsaA;solving the structure of the the lipopolysaccharide; and characterizing a newly discovered porin,OmpW.S-layer proteins are highly resistant to crystallization, because wo-dimensional S-layerformation out competes three-dimensional crystal formation. To achieve a crystallisable formof RsaA, a C-terminal truncation version was constructed and expressed in the native host,C. crescentus. The secreted protein was prone to aggregation, so low agitation and slowconcentration protocols had to be developed. The RsaA truncate produced large crystals thatdiffracted to <2.5 Å. Solving the phases proved to be a serious hurdle and the final proteinstructure remains unsolved.The lipopolysaccharide of C. crescentus is the anchor that supports the S-layer. The struc-ture of the lipid A portion was solved previously but structures for the core oligosaccharideand the O-polysaccharide had not been deduced. In collaboration with Dr. Evgeny Vinogradov,these remaining structures were solved. The core oligosaccharide has a branched heptasac-charide structure. The O-polysaccharide is a heptasaccharide containing the dideoxy sugarN-acetylperosamine. Additionally, a rhamnan polysaccharide was discovered and its structureiiwas determined.Porins, non-specific passive protein channels, are significant components of classical Gram-negative outer membranes. Despite this, no porin had ever been identified in C. crescentus. Wereport the identification and characterization of the porin OmpW in C. crescentus. OmpW haslow conductance of 125 pSv in 1 M KCl. That is interesting because homologous porins inother bacteria have no detectable pore-forming activity.The cell envelopes of bacteria are remarkable structures; the work here illuminates theunique structures present in the caulobacter envelope.iiiPreface“Plenum ingenni pudoris fateri per quos profeceris”(To own up to those who were the means of one’s own achievements)Pliny the Elder, Natural HistoryThe contents contained herein are original work by or in collaboration with me, the primaryauthor, except as specified below.Chapter 2 Portions of chapter 2 have been used for the preparation of a manuscript for sub-mission for publication. The work was done in collaboration with Dr. Anson Chan. I performedall the experiments and wrote the majority of the manuscript. Dr. Chan assisted in the X-raydiffraction data collection, the data evaluation and processing, and edited and provided feedbackon the writing of the manuscript.Chapter 3 Portions of chapter 3 have been published; M. D. Jones, E. Vinogradov, J. F.Nomellini, and J. Smit. The core and O-polysaccharide structure of the Caulobacter crescentuslipopolysaccharide. Carbohydrate Research, 402: 111–117, 2015.The work was done as a collaboration with Dr. Evgeny Vinogradov. Dr. Vinogradov wasresponsible for the majority of the structural work: monosaccharide analysis, methylationanalysis, and NMR analysis, as well as the written summaries of those methods. Dr. JohnNomellini constructed the strain used, JS1025. The cytokine and cell culture experiments werenew additions not present in our publication of the LPS structures. The execution of the HEK-Blue assay was performed by Gyles Ifill. I was responsible for the preparation of all the LPSivsamples, experimental design, and writing the manuscript.Chapter 4 A version of chapter 4 has been submitted for publication. I was responsible forthe sample preparation, purification, and strain construction. The laboratory of Dr. Roland Benzwas responsible for synthetic membrane conductance experiments. The writing of the resultingmanuscript was a joint effort.vTable of ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iiPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ivTable of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viList of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiList of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiiList of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvAcknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 S-layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.1 S-layer structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.2 S-layer proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.1.3 Functions of S-layers . . . . . . . . . . . . . . . . . . . . . . . . . . 81.1.4 RsaA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.1.5 Biotechnology applications of S-layers . . . . . . . . . . . . . . . . 141.2 Lipopolysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171.2.1 Lipid A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18vi1.2.2 Core oligosaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . 231.2.3 O-polysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251.3 Porins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281.4 Aims and Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 Crystallization and X-ray diffraction of the S-layer protein RsaA . . . . . . 312.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332.2.1 Strain and plasmid construction . . . . . . . . . . . . . . . . . . . . 332.2.2 Protein production . . . . . . . . . . . . . . . . . . . . . . . . . . . 352.2.3 Dynamic light scattering spectroscopy . . . . . . . . . . . . . . . . 352.2.4 Crystallization of RsaA constructs . . . . . . . . . . . . . . . . . . . 362.2.5 Data collection and processing . . . . . . . . . . . . . . . . . . . . . 362.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372.3.1 Initial crystallization and optimization . . . . . . . . . . . . . . . . 372.3.2 Properties of the RsaA ∆0–222 crystals . . . . . . . . . . . . . . . . 422.3.3 Crystallization of RsaA ∆0–222 GSCC723 . . . . . . . . . . . . . . 432.3.4 Properties of the RsaA ∆0–222 GSCC723 crystals . . . . . . . . . . 442.3.5 X-ray diffraction and phasing . . . . . . . . . . . . . . . . . . . . . 452.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493 The core and O-polysaccharide structure of the Caulobacter crescentuslipopolysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553.2.1 Bacterial strain construction and growth conditions . . . . . . . . . 553.2.2 Lipopolysaccharide isolation . . . . . . . . . . . . . . . . . . . . . . 563.2.3 Bligh Dyer extraction . . . . . . . . . . . . . . . . . . . . . . . . . . 56vii3.2.4 Silver stain and gel electrophoresis . . . . . . . . . . . . . . . . . . 573.2.5 Preparation of RsaA ∆277–784 probe . . . . . . . . . . . . . . . . 573.2.6 Western blotting and far-western blotting . . . . . . . . . . . . . . 583.2.7 Matrix assisted laser desorption/ionization-time of flight massspectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593.2.8 Periodic acid-Schiff stain . . . . . . . . . . . . . . . . . . . . . . . . 603.2.9 NMR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613.2.10 LPS chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . 613.2.11 Monosaccharide analysis . . . . . . . . . . . . . . . . . . . . . . . . 623.2.12 Determination of absolute configurations of monosaccharides . . . 623.2.13 Methylation analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 623.2.14 Periodate oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . 633.2.15 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633.2.16 HEK-Blue hTLR4 assay . . . . . . . . . . . . . . . . . . . . . . . . . 633.2.17 THP-1 derived macrophage assay . . . . . . . . . . . . . . . . . . . 643.2.18 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663.3.1 Characterization of whole LPS . . . . . . . . . . . . . . . . . . . . . 663.3.2 Identification of similar glycan structures by RsaA binding . . . . . 703.3.3 Initial assessment and component analysis . . . . . . . . . . . . . . 713.3.4 O-specific polysaccharide structure determination (PS1) . . . . . . 723.3.5 Minor component determination . . . . . . . . . . . . . . . . . . . 763.3.6 Rhamnan polysaccharide determination (PS2) . . . . . . . . . . . 783.3.7 Core oligosaccharide determination . . . . . . . . . . . . . . . . . . 793.3.8 Cellular response to C. crescentus LPS . . . . . . . . . . . . . . . . . 813.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84viii4 OmpWofCaulobacter crescentus functions as an outermembrane chan-nel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 934.2.1 Growth and maintenance of bacteria . . . . . . . . . . . . . . . . . 934.2.2 Generation of C. crescentus ∆ompW . . . . . . . . . . . . . . . . . . 944.2.3 Outer membrane-enriched preparations . . . . . . . . . . . . . . . 954.2.4 Crude membrane preparations . . . . . . . . . . . . . . . . . . . . . 964.2.5 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis . . . . 964.2.6 Isolation and purification of the channel-forming protein fromouter membrane-enriched preparations . . . . . . . . . . . . . . . . 964.2.7 Tryptic digestion and peptide sequencing . . . . . . . . . . . . . . . 974.2.8 Lipid bilayer experiments . . . . . . . . . . . . . . . . . . . . . . . . 974.2.9 Modeling of the OmpW structure . . . . . . . . . . . . . . . . . . . 984.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 984.3.1 Protein composition of the outer membrane-enriched prepara-tions of C. crescentus . . . . . . . . . . . . . . . . . . . . . . . . . . 984.3.2 Identification of channel-forming activity in outer membrane-enrichedpreparations of C. crescentus . . . . . . . . . . . . . . . . . . . . . . 994.3.3 Identification of the purified channel-forming protein from theenriched outer membranes . . . . . . . . . . . . . . . . . . . . . . . 1004.3.4 Partial sequencing of the 22 kDa protein of C. crescentus . . . . . . 1014.3.5 Analysis of the channels formed by OmpW channel-forming pro-tein of C. crescentus . . . . . . . . . . . . . . . . . . . . . . . . . . . 1024.3.6 The OmpW channel of C. crescentus is highly cation-selective . . . 1064.3.7 Knocking out ompW removes channel-forming activity . . . . . . . 1064.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107ix5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1175.1 Relevance and contribution to the field . . . . . . . . . . . . . . . . . . . . 1175.2 Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .125Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154xList of TablesTable 2.1 Summary data from the X-ray diffraction of RsaA ∆0–222 . . . . . . . . . . 52Table 3.1 LPS silver stain procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . 58Table 3.2 MALDI-TOF settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Table 3.3 NMR data for C. crescentus PS1 . . . . . . . . . . . . . . . . . . . . . . . . 77Table 3.4 NMR data for the minor components of the double oxidized non-deacylated PS 79Table 3.5 NMR data for C. crescentus PS2 and its NaIO4 oxidation product OS1 . . . . 81Table 3.6 NMR data for the core oligosaccharide . . . . . . . . . . . . . . . . . . . . 83Table 4.1 Average conductance through OmpW . . . . . . . . . . . . . . . . . . . . . 105xiList of FiguresFigure 1.1 Cross-sectional diagrams of S-layer containing cell envelopes . . . . . . . 3Figure 1.2 The first published image of a S-layer . . . . . . . . . . . . . . . . . . . . 4Figure 1.3 A simple overview of S-layer symmetries . . . . . . . . . . . . . . . . . . 5Figure 1.4 Electrotomograph of C. crescentus . . . . . . . . . . . . . . . . . . . . . . 6Figure 1.5 Reconstructed surface of the C. crescentus S-layer . . . . . . . . . . . . . . 12Figure 1.6 The structure of RTX motifs . . . . . . . . . . . . . . . . . . . . . . . . . 14Figure 1.7 Simplified structure of LPS from Salmonella minnesota . . . . . . . . . . . 19Figure 1.8 Kdo2-Lipid A from E. coli K12 . . . . . . . . . . . . . . . . . . . . . . . . 20Figure 1.9 A generalized formula for core oligosaccharides . . . . . . . . . . . . . . . 24Figure 1.10 A simple overview of the Wzy-dependent pathway . . . . . . . . . . . . . 26Figure 1.11 A simple overview of LPS transport across the periplasm . . . . . . . . . . 27Figure 1.12 TonB-mediated transport versus transport througha porin . . . . . . . . . . 29Figure 2.1 Example of a large cluster of ‘plate’ RsaA ∆0–222 crystals . . . . . . . . . 37Figure 2.2 Edge plots for useful anomalous dispersion elements . . . . . . . . . . . . 38Figure 2.3 Purification of RsaA ∆0–222 shown by SDS-PAGE . . . . . . . . . . . . . 40Figure 2.4 DLS plots of RsaA constructs . . . . . . . . . . . . . . . . . . . . . . . . . 41Figure 2.5 Panel of well diffracting ‘plate’ crystals of RsaA ∆0–222 . . . . . . . . . . 44Figure 2.6 A panel of ‘pencil crystals of RsaA ∆0–222’ . . . . . . . . . . . . . . . . . 45Figure 2.7 Examples of unusable ‘dendritic’ RsaA ∆0–222 GSCC723 crystals . . . . . 45xiiFigure 2.8 ‘Dendritic’ RsaA ∆0–222 GSCC723 crystals that were used for X-raydiffraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Figure 2.9 A diffraction pattern of RsaA ∆0–222, ‘plate’ crystal . . . . . . . . . . . . 48Figure 3.1 The lipid A of C. crescentus . . . . . . . . . . . . . . . . . . . . . . . . . 54Figure 3.2 Four different visualisations of C. crescentus LPS . . . . . . . . . . . . . . 68Figure 3.3 Visual comparison of E. coli O122 LPS and C. crescentus LPS . . . . . . . 69Figure 3.4 MALDI-TOF analysis of intact, whole LPS from C. crescentus . . . . . . . . 70Figure 3.5 1H NMR spectra of C. crescentus OPS . . . . . . . . . . . . . . . . . . . . 73Figure 3.6 Monosaccharide analysis of LPS . . . . . . . . . . . . . . . . . . . . . . . 74Figure 3.7 The structure of the C. crescentus OPS . . . . . . . . . . . . . . . . . . . . 75Figure 3.8 2D NMR of C. crescentus PS1 . . . . . . . . . . . . . . . . . . . . . . . . . 76Figure 3.9 The structure of minor component, the end caps of the OPS. . . . . . . . . . 78Figure 3.10 The structure of the C. crescentus rhamnan. . . . . . . . . . . . . . . . . . 80Figure 3.11 The structure of the C. crescentus core oligosaccharide. . . . . . . . . . . . 82Figure 3.12 Fragment of 1H-13C HSQC spectrum of the core. . . . . . . . . . . . . . . 84Figure 3.13 NF-κB Assay for cellular activation by LPS . . . . . . . . . . . . . . . . . 85Figure 3.14 Cytokine profile comparing Caulobacter crescentus and Salmonella enter-ica var. Typhimurium LPS . . . . . . . . . . . . . . . . . . . . . . . . . . 86Figure 4.1 Coomassie stained SDS-PAGE of different protein preparations of C. crescentus. 99Figure 4.2 SDS-PAGE showing OmpW from C. crescentus obtained by elution of the20 kDa band . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101Figure 4.3 Single-channel measurements with detergent treated outer membrane of C.crescentus and purified 20 kDa protein . . . . . . . . . . . . . . . . . . . . 104Figure 4.4 SDS-PAGE of C. crescentus ∆ompW . . . . . . . . . . . . . . . . . . . . . 108Figure 4.5 Single-channel conductance of OmpW in 1 M salt solution as a function ofthe ionic radii of monovalent cations . . . . . . . . . . . . . . . . . . . . . 111xiiiFigure 4.6 Amino acid sequence alignment of OprG of Pseudomonas aeruginosa,OmpW of E. coli, and OmpW of C. crescentus . . . . . . . . . . . . . . . . 113Figure 4.7 Structural properties of OmpW of C. crescentus . . . . . . . . . . . . . . . 115Figure 4.8 Comparison of porin channel radii . . . . . . . . . . . . . . . . . . . . . . 116Figure 5.1 The complete structure of the C. crescentus LPS . . . . . . . . . . . . . . . 121Figure A.1 RsaA, amino acid sequence . . . . . . . . . . . . . . . . . . . . . . . . . . 154Figure A.2 OmpW, amino acid sequence . . . . . . . . . . . . . . . . . . . . . . . . . 155xivList of AbbreviationsGeneral abbreviationsAA Amino acidABC ATP binding cassetteBLAST Basic local alignment searchCFG Consortium of Functional GlycomicsCLS Canadian Light SourceCOSY Correlation spectroscopyDLS Dynamic light scatteringDMEM Dulbecco’s modified Eagle’s mediumEDTA Ethylenediaminetetraacetic acidEPS Extracellular polysaccharideESI Electrospray ionizationFBS Fetal bovine serumGC-MS Gas chromatography-mass spectroscopygCOSY Gradient correlation spectroscopygHMBC Gradient heteronuclear multiple bond coherencegHSQC Gradient heteronuclear singe quantum coherenceHMBC Heteronuclear multiple bond coherenceHMQC Heteronuclear multiple-quantum correlation spectroscopyxvHSQC Heteronuclear single quantum coherenceIFN-β Interferon βIL InterleukinJCSG Joint Centre for Structural GenomicsKD Dissociation constantLDAO Lauryldimethylamine-oxideLPS LipopolysaccharideMAD Multi-wavelength anomalous dispersionMALDI-TOF Matrix assisted laser desorption/ionization-time of flight mass spectroscopyMAMP Microbe-associated molecular patternMCP-1 Monocyte chemotactic protein 1MS Mass spectrometryMW Molecular weightNF-κB Nuclear factor kappa-light-chain-enhancer of activated B cellsNMR Nuclear magnetic resonance spectroscopyNOE Nuclear Overhauser enhancementNOESY Nuclear Overhauser enhancement spectroscopyOD600 Optical density at 600 nmOPS O-specific polysaccharideOS OligosaccharidePBS Phosphate-buffered salinePC PhosphatidylcholinePCR Polymerase chain reactionPEG Polyethylene glycolpI Isoelectric pointPMA Phorbol 12-myristate 13-acetatePS PolysaccharidexviPYE Peptone-yeast extract mediumROESY Rotating frame nuclear Overhauser effect spectroscopyRPMI Roswell Park Memorial Institute mediumRTX Repeat-in-toxinS-layer Protein surface layerSAD Single-wavelength anomalous dispersionSDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresisSSRL Stanford Synchotron Radiation Light SourceT1SS Type 1 secretion systemTFA Trifluoroacetic acidTLC Thin-layer chromatographyTLR4 Toll-like receptor 4TNF-α Tumor necrosis factor αTCOSY Total correlation spectroscopyUV Ultraviolet LightSugar abbreviationsGalA Galacturonic acidGalNAc N-acetylgalactosamineGlc GlucoseGlcA Glucuronic acidGlcN GlucosamineGlcNAc N-acetylglucosamine3-O-MeGlc 3-O-methylglucoseKdo 3-deoxy-D-mannooctulosonic acidxviiLDHep L-glycero-D-mannoheptoseLLHep L-glycero-L-mannoheptoseMan MannosePerN PerosamineRha RhamnosexviiiAcknowledgmentsFirst and most of all I’d like to thank my family. My parents, J.C. and Dorothea, your support hasbeen completely indispensable. I am lucky to have such fantastic parents. My wife, Elizabeth,I started this PhD before I met you but I could not have finished it without you.I do believe I have had the best supervisory committee of anyone I know. Dr. Beatty,Dr. Murphy, and Dr. Fernandez you have been the exactly perfect level of supportive, critical,and congratulatory. Thank you.I have been incredibly fortunate to have been in the Smit lab. It has been said that choosingyour supervisor is the most important choice you have to make in grad school—I chose well inJohn Smit. Dr. Smit, your patience with my crazy ideas let me grow as a scientist but your lackof patience with my crazy ideas kept me in line and prevented my PhD from lasting a decade.Dr. Nomellini, I have been fortunate to have you nearby to fix my mistakes, hear me complainabout my mistakes, and showing me how to prevent future mistakes. The few other membersof our lab that I have shared time with, Lyngrace, Jan, Christina, you have all been great alliesin the lab.Beyond the official credits that the are given, I want to acknowledge the incredible collab-orators I have had since day one. Dr. Evgeny Vinogradov is the world’s preeminent expert onbacterial polysaccharide analysis, there is quite literally no one that would have been a bettercollaborator for our lipopolysaccharide work. The Consortium for Functional Glycomics CoreH at Emory University were incredibly easy to work with and even though our results with themwere ultimately negative, the service they provide is an invaluable asset to the field. Dr. MartinexixCaroff has been a surprisingly helpful source of expertise and critique, especially critique.The Murphy lab in our department of Microbiology and Immunology is the go to collabora-tors for protein X-ray crystallography, my experiences are evidence to why that is. All of them(Angele, Meghan, Slade, Jason, Catherine, Stephanie, Marek, Michael, Mariko) accepted melike a lab member from the start. Dr. Anson Chan was an exceptional partner for crystallography,his expertise kept us moving forward against a tough project. Especially I would like to thankDr. Chan for staying up all night many times on our long data collection runs. Staying up lateis not so bad, but staying up late for poor results deserves commendation.Dr. Roland Benz was my only collaborator to initiate a collaboration with us. The porinproject started as a side-project but it has become a nice little story that I am happy to have beenapart of. Dr. Benz, I hope to actually meet you one day.Lastly, I would like to acknowledge my department as a whole. This department has beenincredibly supportive and warm. With people like Darlene, Sue, and Dr. Gold guiding the ship,I never felt like we were going to crash. A special acknowledgment goes to Dr. Jan Burian, whowas always around with encouragement and advice but especially for his organization of manydepartmental events and teams that brought together a cadre of scientists on the dodgeball court,on the softball diamond, and beyond the confines of campus.xxFor my Oma, Agnes Bergen...She pushed everyone in our family to get as much education aspossible. She supported me even when she thought a PhD mightbe a bit too much education.Your prayers for my sugars and crystals helped me along the way,I love you Oma!xxiChapter 1Introduction“But during the writing of this review, I learned how little I knew in this area, andthis was a humbling and sobering experience. I am certain that I have made manymistakes due to my ignorance, and I hope that the review will be useful despite itsmany faults”— Hiroshi Nikaido, 2003, my grand supervisor.1.1 S-layers1.1.1 S-layer structureCELLULAR envelopes are the interface between a cell and its environment. Many differentorgansims have evolved extra coatings and membrane adaptations to enhance or providenew functionality to their cell envelopes. For example, capsules can enhance the virulence ofstreptococci2 and mycolic acids increase resistance to antibiotics for mycobacteria.3 One suchadaptation widespread in prokaryotic life is the protein surface layer (S-layer). S-layers areproteinaceous, para-crystalline coatings that surround some bacteria and archaea.4,5. A good re-view on the subject of S-layer structure was written by Tea Pavkov-Keller, Stefan Howorka and2F. Griffith. J Hygiene, 27: 113–159, 1928.3L. Nguyen and C. J. Thompson. TRENDS Microbiol, 14: 304–312, 2006.4T. J. Beveridge and L. L. Graham. Microbiol Rev, 55: 684, 1991.5M. Sára and U. B. Sleytr. J Bacteriol, 182: 859–868, 2000.1Walter Keller.6 S-layers are two-dimensional sheets, being a thin layer of protein or glycoproteinsitting exterior to the outer membrane for Gram-negative bacteria, the peptidoglycan layer inGram-positive bacteria, and the cell membrane of most archaea. Figure 1.1 shows the general,cross-sectional structure of cell envelopes of bacteria and archaea that possess an S-layer.S-layers are composed of one or only a few proteins or glycoproteins. In most knownexamples, S-layers are composed of one repeated protein. In a few cases S-layers are composedof a few separate proteins, which are stacked one on the other. The S-layer from Clostridiumdifficile is composed of two proteins (HMW and LMW) that arise from the cleavage of a singleprecursor protein (SlpA) that is cleaved during secretion.8,9 Bacillus anthracis has a S-layerthat is composed of two proteins (EA1 and Sap) that are individually expressed and assembletogether on the surface of the bacterium.10 Interestingly, Brevibacillus brevis appears to havetwo S-layers stacked on top of each other; the outer layer having oblique symmetry and theinner layer having hexagonal symmetry.11For S-layers that are composed of a single protein, that protein is repeated thousands oftimes across the surface of the cell and is arranged in a regular geometric pattern. The geo-metric patterns can be observed by electron microscopy. The traditional electron microscopytechniques of freeze fracture and negative stain have been the most successful techniques his-torically to see S-layers and S-layer geometries.12 The discovery of S-layers and the first imageof an S-layer published was produced by electron microscopy.13 That first S-layer image can beseen in fig. 1.2. For a historical overview of S-layer discoveries, refer to Sleytr et al., 2014.14The five major geometric symmetries found in S-layers are oblique (P1 and P2), triangular(P3), tetragonal (P4), and hexagonal (P6). Figure 1.3 features simple examples of the five6T. Pavkov-Keller, S. Howorka, and W. Keller. “Chapter 3 - The Structure of Bacterial S-Layer Proteins”in: Molecular Assembly in Natural and Engineered Systems ed. by S. Howorka. vol. 103 Progress in MolecularBiology and Translational Science Academic Press, 2011. 73–1308E. Calabi et al. Mol Microbiol, 40: 1187–1199, 2001.9R. P. Fagan et al. Mol Microbiol, 71: 1308–1322,10S. Mesnage et al. Mol Microbiol, 23: 1147–1155, 1997.11M. Sára et al. Arch Microbiol, 153: 209–214, 1990.12J. Smit et al. J Bacteriol, 146: 1135–1150, 1981.13A. Houwink. Biochim Biophys, 10: 360–366, 1953.14U. B. Sleytr et al. FEMS Microbiol Rev, 38: 823–864, 2014.2PeptidoglycanCytoplasmic MembraneS-layerCytoplasmic MembraneS-layerPeptidoglycanOuter MembraneCytoplasmic MembraneS-layerLipopolysaccharideABCFigure 1.1: Cross-sectional diagrams of the cell envelopes of (A) Gram-negative bacteria,(B) Gram-positive bacteria, and (C) archaea. In all known cases the S-layer sits onthe outer surface of the cell. The left side is a cartoon, artistic interpretation. Theright side is a simpler, structural diagram.(This diagram was inspired by Fig. 1 fromU. B. Sleytr and P. Messner. Crystalline surface layers on bacteria. Annual Reviewsin Microbiology, 37: 311–339, 1983.)major symmetries found in S-layers. Examples of bacteria with oblique S-layers are Bacillusstearothermophilus NRS2004/3a15 and Brevibacillus (Lactobacillus) brevis.16 Examples of bac-teria with tetragonal S-layers are Corynebacterium diptheriae17 and Aeromonas salmonicida15P. Messner, D. Pum, and U. B. Sleytr. J Ultra Mol Struct Res, 97: 73–88, 1986.16K. Masuda and T. Kawata. Microb Immun, 24: 299–308, 1980.17T. Kawata and K. Masuda. Jpn J Microbiol, 16: 515–523, 1972.3Figure 1.2: The first published image of a S-layer. The hexagonal S-layer on the surfaceof the bacterium — probably Spirillum sp. — is visible along the edges of the cellbody (centre right). The scale bar denotes one micrometre. (This image is Figure 1from A. Houwink. A macromolecular mono-layer in the cell wall of Spirillum spec.Biochimica et Biophysica Acta, 10: 360–366, 1953. Reused with full permissionfrom the publisher, Elsevier.)4A450.18 An example of a bacterium with a triangular S-layer is Sulfolobus acidocaldarius.19 Ex-amples of bacteria with hexagonal S-layers are Bacillus anthracis20 and Caulobacter crescentusCB15.12p1 p2 p4p6p3Figure 1.3: A simple overview of S-layer symmetries. P1 and P2 are oblique symme-tries. P4 is a tetragonal symmetry. P3 is a triangular symmetry. P6 is a hexagonalsymmetry. Bacterial examples are given on page 2In addition to the traditional transmission electron microscopy techniques that have beenused to visualize bacterial cell surfaces in the past, a new method of cryo-electron tomographyhas been used recently to observe and dissect bacterial cells. Cryo-electron tomography has theadvantage that it can be used to observe unfixed and unstained intact cells. Being a tomograph-ical technique, cross sections can be reconstructed to observe the fine structure within and ona bacterial cell. No deliberate study has been taken to use cryo-electron tomography to studyS-layers, but their presence is easily observed in the fantastic images produced by other studiesutilizing the technique. This technique has been pioneered for use in bacteria and specificallyin Caulobacter crescentus by the laboratories of Dr. Grant Jensen at the California Institute ofTechnology and Dr./,Sriram Subramaniam at the National Cancer Institute. Figure 1.4 featuresa tomographical cross-section of a C. crescentus cell; visible are the inner and outer membranesand the S-layer composed of the protein RsaA.18E. E. Ishiguro et al. J Bacteriol, 148: 333–340, 1981.19R. L. Weiss. J Bacteriol, 118: 275–284, 1974.20S. Holt and E. Leadbetter. Bacteriol Rev, 33: 346–378, 1969.5Figure 1.4: The location of the S-layer is around the exterior of the cell, as can be seenin this image. The figure is a 19 nm cross-section through the three-dimensionalreconstruction of one representative C. crescentus cell: SL, surface layer; OM,outer membrane; PG, peptidoglycan layer; IM, inner membrane; St, stalk; Rib,probable ribosome; GF, gold fiducial used to align images; Phb, putative poly-β -hydroxybutyrate granule. The scale bar indicates 200 nm. (This figure is a portionof Figure 1 from A. Briegel et al. Multiple large filament bundles observed inCaulobacter crescentus by electron cryotomography. Molecular Microbiology, 62:5–14, 2006. Reused with full permission from the publisher, Wiley. The dottedwhite lines highlight the location of filaments of the protein Crescentin, the focus ofthe originating paper. )1.1.2 S-layer proteinsDiscussing S-layer proteins as a whole can be difficult because S-layer proteins do not representa monophyletic group of related molecules; S-layer proteins seem to be unrelated proteins thatshare a common cellular location and gross structure through convergent evolution. One of thebest reviews on S-layer proteins was published in 2000 by Margit Sára and Uwe B. Sleytr.5S-layer proteins do tend to share similar properties and overall composition. S-layer proteinsare relatively large proteins. For the S-layer proteins whose sequences are known, all are largerthan 400 amino acids (AAs) and many of them larger than 1000 AAs.5 The amino acids inS-layer proteins are rich in hydrophobic and negatively charged residues, low in methionines,and cysteines are almost completely absent.5,22,23The most well studied S-layer proteins are RsaA from C. crescentus, SbsB, and SbsC, from22A. Gilchrist, J. A. Fisher, and J. Smit. Can J Microbiol, 38: 193–202, 1992.23H. Vilen et al. J Bacteriol, 191: 3339–3349, 2009.6Geobacillus stearothermophilus. RsaA is studied in part for its potential as a biotechnologyplatform. SbsB and SbsC also have potential for applications but they are well studied becausethey are easy to work with; they can be expressed in Escherichia coli and will readily renaturefrom dissociation in strong chaotrope solutions to form S-layers on almost any flat surfaceavailable. The ease of working with SbsB has paid off in the form of the first, and only, atomic-level resolution crystal structure of a bacterial S-layer protein with the symmetry determiningdomains intact.24 The protein SbsB was crystallized by inhibiting its S-layer forming ability bybinding it with a nanobody, a monoclonal single domain camelid antibody. The crystal studyreveals that calcium ions play an integral role in the structure of the S-layer protein. Thesecalcium ions ‘trigger’ the folding of the protein. This mirrors the hypothesized role of repeat-in-toxin (RTX) motifs in the C. crescentus S-layer protein RsaA discussed in section 1.1.4. Forour efforts to crystallize RsaA, see chapter 2.S-layer proteins are almost universally glycosylated in archaea, commonly glycosylated inGram-positive bacteria, and very rarely glycosylated in Gram-negative bacteria.5,25 For somemicrobes, the S-layer’s main function may be as a platform for oligo/polysaccharide chains.One study by Schäffer et al. asked the question about the glycosylation of S-layer proteins“Are S-layer Glycoproteins and Lipopolysaccharide Related?”26 The study identified structuralsimilarities between the carbohydrates in O-specific polysaccharide (OPS) and the carbohydrateson glycosylated S-layer proteins and pointed out they have more in common with each otherthan with eukaryotic polysaccharides. The study makes flawed leaps from sugar structures tophylogenetic and taxonomic conclusions, but the study did highlight the concept that supportinga highly glycosylated surface is a widespread feature in microbial life and it is a role that S-layerproteins serve in some organisms.24E. Baranova et al. Nature, 487: 119–122, 2012.25J. Lechner and F. Wieland. Annu Rev Biochem, 58: 173–194, 1989.26C. Schäffer et al. Microbiol Drug Resist, 2: 17–23, 1996.71.1.3 Functions of S-layersAs mentioned before, S-layers are not a single family of related proteins—likewise they do notfill the same needs in all species. Interestingly, despite the apparent convergent evolution inmany disparate organisms pushing towards a widespread similarity in structure and composi-tion, S-layers seemingly perform many different functions. Further, S-layers require a largemetabolic commitment to produce and maintain. Despite their cost, they are still retained inmany species where their functions are enigmatic. Due to their location on the cell surface,S-layers are undoubtedly involved with the cell’s interaction with the environment. Whilethe classification of S-layer-possessing bacteria into pathogenic versus non-pathogenic can besomewhat arbitrary, it is a convenient distinction to compare S-layer functions.Pathogenic bacteria For pathogenic bacteria, an S-layer is often a virulence factor. In Bacillusanthracis the S-layer consists of the protein BslA and it functions as an adhesin.27 BslA issufficient and required for B. anthracis to adhere to host cells and mutants that lack the S-layerhave much reduced virulence and increased time-to-death for animal models. Due to theirlocation on the surface of the cell, S-layers functioning as adhesins is a common theme inpathogens. A. salmonicida has a S-layer that also functions as an adhesin.28 The A. salmonicidaS-layer binds to both fibronectin and laminin on host cell surfaces29 but is not involved orrequired for host cell invasion, which is probably mediated by the lipopolysaccharide (LPS).28The A. salmonicida S-layer, composed of the protein VapA, is sufficient to confer host celladhesion to the bacterial cells and also to nylon microbeads in vitro.For Campylobacter fetus, its S-layer is central to its success as a pathogen of ungulates.The S-layer of C. fetus is involved in disseminating the bacteria throughout the host’s body andevading the host immune system.30 The manner in which C. fetus uses its S-layer to escape27J. Kern and O. Schneewind. Mol Microbiol, 75: 324–332, 2010.28R. A. Garduño et al. Can J Microbiol, 46: 660–668, 2000.29P. Doig, L. Emödy, et al. J Biol Chem, 267: 43–49, 1992.30S. A. Thompson. Annal Periodontol, 7: 43–53, 2002.8the immune system is remarkable. The S-layer provides the cells with an innate resistance tocomplement; strains with an intact S-layer bound less C3 protein than cells lacking a S-layer.By binding less C3, S-layer positive C. fetus cells prevent complement-mediated killing andopsonization by C3b. The S-layer would be a natural target for antibody-mediated adaptiveimmunity, but C. fetus has also evolved a strategy to escape antibody recognition. C. fetuspossess the ability to undergo antigenic conversion by expressing different versions of its S-layer. The C. fetus cells can invert portions of their genomic DNA to express alternate S-layerproteins and cognate OPS anchor molecules. As an example, the C. fetus serotype A strain 23Dencodes for eight different S-layer proteins and can switch between expressing each by movinga single active promoter in front of the S-layer protein gene to be expressed. These S-layers arenot just different antigenically, they can even form different symmetry patterns (see fig. 1.3).Non-pathogenic bacteria The roles of S-layers in non-pathogenic bacteria can often be mys-terious in lab settings. While virulence can be a clear, quantifiable phenotype, the fitness gainderived from an S-layer in the wild is not often abundantly obvious in a test tube. S-layershave been shown to act as barriers to the outside world. S-layers acting as a physical barrier,protecting the cell, is a function primarily exemplified by C. crescentus and other Gram-negativebacteria possessing an S-layer. Susan Koval of the University of Western Ontario has doneextensive research into the biology and life cycle of Bdellovibrio spp. and Bdellovibrio-likespecies, predatory bacteria that prey on Gram-negative bacteria such as C. crescentus. Kovalshowed that the S-layer of C. crescentus had a protective effect against Bdellovibrio predation.31This protective effect was seen for A. salmonicida, Aquaspirillum serpens, Aquaspirillum sinuo-sum, C. fetus, and C. crescentus. On the other hand, S-layers do not confer any protection frompredation by protozoans (e.g. Tetrahymena thermophila and Paraphysomonas vestita).32 Thebarrier capacity of S-layers extends to blocking abiotic threats, as was demonstrated by De la31S. Koval and S. Hynes. J Bacteriol, 173: 2244–2249, 1991.32S. F. Koval. “Predation on bacteria possessing S-layers” in: Advances in Bacterial Paracrystalline SurfaceLayers. Springer, 1993. 85–929Fuente-Núñez et al. in C. crescentus in 2012. C. crescentus cells that possessed an S-layer werefound to have protection against the effects of antimicrobial peptides compared to cells that lackan S-layer.33 The authors note that the peptides were smaller than the pores in C. crescentusS-layer (see fig. 1.5) and yet the protective effect existed. The S-layer may act as a sponge, notjust a physical barrier, adsorbing harmful molecules before they can reach the cell. The S-layerof C. crescentus is not glycosylated but many other species’ S-layers are glycosylated and thosecarbohydrates would add another layer of coating to the cell to ward off intrusion.A protein coating will impart to a cell surface with different properties than a ‘naked’ cell;these differences may be a conserved function of an S-layer. It has been noted that manybacteria with an S-layer are more hydrophobic than isogenic variants lacking an S-layer.34 Anoted exception to the hydrophobicity observations was for C. crescentus strains CB2A andCB2NY66R; its S-layer covers beneath it a more hydrophobic outer membrane (see chapter 3for our research on the C. crescentus LPS). Beyond changing the general chemical properties ofthe cell surface, S-layers also change the cell surface to provide a specific site for extracellularproteins to anchor. Enzymes that are secreted, but remain associated with the cell, can use S-layers as display platforms. The enzyme pullulanase in Clostridium thermosulfurogenes anchorsitself into the S-layer with three C-terminal regions that have a significant sequence homologyto the C. thermosulfurogenes S-layer protein.35 It is thought that the C. thermosulfurogenespullulanase integrates itself in the lattice of the S-layer in order to anchor itself. Integration intothe S-layer is not the strategy used by G. stearothermophilus DSM2358 and its amylases. Thehigh molecular weight amylase from G. stearothermophilus binds the cell surface but withoutany detectable interaction with peptidoglycan, instead it associates with S-layer in a mannerthat is specifically not lattice integration.3633C. de la Fuente-Núñez et al. App Environ Microbiol, 78: 5452–5456, 2012.34T. J. Beveridge et al. FEMS Microb Rev, 20: 99–149, 1997.35M. Matuschek et al. J Bacteriol, 176: 3295–3302, 1994.36E. Egelseer et al. J Bacteriol, 177: 1444–1451, 1995.101.1.4 RsaARsaA, named for regular surface array,37 is a 1026 AA protein from C. crescentus with an averagemolecular weight (MW) of 98001.70 Da and an isoelectric point (pI) of 3.85. The amino acidsequence of RsaA can be found in the appendix on page 154. The S-layer of C. crescentuscomprises roughly 45000 copies38 of RsaA arranged in an p6 symmetry (see fig. 1.3). RsaA isa type 1 secretion system (T1SS) secreted protein containing five to six RTX motifs.Figure 1.5 displays a computer reconstructed view of the surface of the S-layer of C. crescentusas determined by tilt-tomography electron microscopy.39 This figure is the highest resolutionimage available for the structure of RsaA and the S-layer, a resolution of about 20 Å. In a veryelegant series of experiments in 2010, Amat et al. were able to determine the configuration ofthe RsaA monomers in S-layer structure.38 Different constructs of RsaA were produced thatcontained single cysteine residues at different locations throughout the protein. Labelling thosecysteines with nano-gold particles in situ in the S-layer, led to noticeable changes in electrondensity in the hexagonal unit cell of the S-layer. These densities corresponded to the physicallocations of the engineered cysteine residues. This work proved that the N-termini of the RsaAmonomers are located in the axis of three-fold symmetry, while the C-termini are centeredaround the axis of the six-fold symmetry (refer to fig. 1.5)Type 1 secretion RsaA is secreted by a T1SS. T1SS secreted proteins are secreted in one stepacross both the inner and outer membranes of Gram-negative bacteria.40 A T1SS requires threecomponents: an inner membrane spanning ATP binding cassette (ABC) transporter, a periplasmspanning membrane fusion protein, and an outer membrane protein. For RsaA’s secretionfrom C. crescentus, these proteins are RsaD, RsaE, and RsaF (either RsaFa or RsaFb).41,42 Like37A. Gilchrist and J. Smit. J Bacteriol, 173: 921–925, 1991.38F. Amat et al. J Bacteriol, 192: 5855–5865, 2010.39J. Smit et al. J Bacteriol, 174: 6527, 1992.40I. B. Holland, L. Schmitt, and J. Young. Mol Membrane Biol, 22: 29–39, 2005.41P. Awram and J. Smit. J Bacteriol, 180: 3062–3069, 1998.42M. C. Toporowski et al. J Bacteriol, 186: 8000–8009, 2004.11A B12345Figure 1.5: The surface structure of the S-layer from C. crescentus, reconstructed from tilt-series electron microscopy. A. A section of reconstructed surface of the C. crescentusS-layer. The image shown is in a left-handed configuration, except the handednessof the S-layer could not be determined in the original study, therefore this imagemay be a mirror image of the true configuration of the S-layer. B. Interpretationsof the micrograph. A monomer of RsaA is highlighted in green (B.1). The unitcell of the S-layer is six copies of RsaA arranged in a hexagonal pattern (B.2). Thecenter of three-fold symmetry, red, is comprised of the N-termini of RsaA (B.3). TheC-termini of the RsaA monomers face towards each other in the center of the pointof six-fold symmetry, blue (B.4). Possible pores through the S-layer are highlightedin orange (B.5). (This image is derived from Figure 6 from J. Smit et al. The S-layerof Caulobacter crescentus: three-dimensional image reconstruction and structureanalysis by electron microscopy. Journal of Bacteriology, 174: 6527, 1992.)all T1SS secreted proteins, RsaA is secreted starting from the C-terminus to the N-terminus.As proteins are translated from the N-terminus to the C-terminus, all T1SS secreted proteins(including RsaA) exist in the cytoplasm as fully translated polypeptides prior to secretion. Thisis in contrast to protein secreted by N-terminus first means, which allow for the possibilityof secretion to begin on a nascent peptide before translation is complete. The diameter ofT1SS pore is not wide enough to accommodate the secretion of a fully folded protein, so T1SSsecreted proteins often have a C-terminus that is intrinsically disordered prior to secretion; thisdisordered state is mediated by RTX motifs.4343A. Chenal et al. J Biol Chem, 284: 1781–1789, 2009.12Repeat-in-toxin motifs First identified in the E. coli protein α-haemolysin (HlyA),44 RTX mo-tifs are a nearly ubiquitous component of T1SS secreted proteins. Despite the name, repeat-in-toxin are not exclusively found in toxins, they are found in many classes of protein, lipases,proteases, S-layers,etc. RTX motifs are small peptide sequences, typically 6–9 AA in length, theamino acid sequence is GGXGXD45 or GGXGXDXLX(where G is glycine, D is aspartate, X can beany amino acid, and L is leucine but is often substituted by valine, isoleucine, phenylalanine, ortyrosine).43,46 The short sequences are always present in multiples (6–50+), often found in tan-dem repeats. Each RTX motif forms two half sites for binding Ca2+, which can collaborate withother neighbouring RTX sites to coordinate ions and stabilize a connection between disparateregions of a protein. Upon binding Ca2+, RTX motifs assume a parallel β -roll fold,47 rollingaround Ca2+ ions at the turns. An example of a RTX β -roll structure can be found in fig. 1.6.RTX motifs are always on the C-terminal side of T1SS proteins, and are thus are in theleading part of the polypeptide to be loaded into the secretion machinery and the first part to besecreted. It should be noted that bacterial intracellular Ca2+ concentrations are extremely low,90±10 nM, and extracellular Ca2+ concentrations are higher, 0.1–1 mM .49,50 It is thought thatthe roles of RTX motifs in T1SS proteins are to induce an intrinsically disordered C-terminusprior to secretion and to quickly bind Ca2+ and begin folding once the protein has started toemerge from the secretion machinery.51,52 The calcium-induced folding may help the thermo-dynamics of secretion, possibly assisting by pulling the protein out of the pore. Despite theapparent importance of RTX motifs in T1SS proteins, they are not sufficient nor even necessaryfor secretion44 but in at least the case of HlyA, in E. coli, the RTX repeats are required for properfolding, activity, and maximal secretion.44 It has been thought that the folding of the RTX motifscan act as an ‘intramolecular’ chaperone; they fold quickly first then act as a nucleation point44T. Felmlee and R. Welch. PNAS, 85: 5269–5273, 1988.45S. Létoffé and C. Wandersman. J Bacteriol, 174: 4920–4927, 1992.46T. Rose et al. J Biol Chem, 270: 26370–26376, 1995.47R. Meier et al. J Biol Chem, 282: 31477–31483, 2007.49P. Gangola and B. Rosen. J Biol Chem, 262: 12570–12574, 1987.50V. Norris et al. J Bacteriol, 178: 3677, 1996.51A.-C. Sotomayor-Pérez et al. J Mol Biol, 397: 534–549, 2010.52A.-C. Sotomayor-Pérez, D. Ladant, and A. Chenal. Toxins, 7: 1–20, 2014.13Figure 1.6: The structure of RTX motifs. This view is down the barrel of an RTX β -fold. The blue spheres are calcium ions coordinated by aspartate residues whichare shown. This structure is of MIS38, an extracellular lipase from P. aeruginosa(PDB:2Z8X). The structure here depicts five RTX sites binding five Ca2+ ions, RsaAfrom C. crescentus also has five RTX motifs in close vicinity and my have a similarstructure the presented structure above. See the sequence of RsaA on page 154,RTX sequences are bolded. Generated using the program Chimera (E. F. Pettersenet al. UCSF Chimera—a visualization system for exploratory research and analysis.Journal of Computational Chemistry, 25: 1605–1612, 2004.).for the rest of the protein to continue to fold on.521.1.5 Biotechnology applications of S-layersS-layers’ ability to self-assemble into large, complex structures makes them an attractive plat-form for biotechnological applications. Their density and regular arrangement over relativelylarge distances is unique in biology. There have been several groups pursuing technologiesthat utilize S-layers assembled off of the surface of the cell and groups that focus on S-layertechnologies on the cell surface. Many different technologies have been developed. One of thesignificant defining differences among all S-layer applications is the location where the S-layeris assembled. The potential sites for assembly are:• In suspension• On a solid surface14• On a liquid interface of lipid membrane• On a lipid nanoparticle• On a cell surfaceVery little interest or work has been done on developing applications for S-layers that as-semble and exist in liquid suspension. S-layers are inherently two-dimensional and unrestrictedfreedom in liquid suspension does not highlight their unique structure. Assembling S-layersin solution is most often a means to getting the S-layers onto a solid surface. Once an assem-bled S-layer is on a solid surface it is a potent structure for biotechnology development. TheS-layer itself can act as a filter or the S-layer can be a platform for functionalized moleculesand peptides.Using S-layers as ultrafilters has been a tempting idea since not long after they were discov-ered. As mentioned in section 1.1.3, one of the possible natural functions of S-layers is to act asa physical barrier against large, intrusive foreign entities. Unlike conventional ultrafilters madeof inorganic polymers, S-layers are isoporous with very stringent molecular weight cutoffs. Inits most basic inception, an S-layer ultrafilter could be constructed by assembling an S-layer onthe inner surface of a traditional ultrafiltration membrane. These S-layer ultrafiltration mem-branes (sometimes called SUMs) do have exquisite selectivity of filtering and when they arecrosslinked with glutaraldehyde, they are highly resistant to solvents, acids, and bases.53 TheseS-layer filters were covered by at least three US patents, the last of which expired in 2011. Theapparent lack of enthusiasm in the field of ultrafiltration using S-layers in the last few yearssuggests that the early results were not easily translatable into a large-scale commercial product.Having the S-layer function as a platform for functional molecules and peptides is the mostpopular approach to using S-layers. The most uncomplicated way to introduce a functionalstructure into an S-layer is to genetically clone it into the S-layer gene itself. Molecules ofinterest can always be chemically linked to an intact S-layer,54 but the opportunity for S-layer to53M. Sára and U. B. Sleytr. J Membrane Sci, 33: 27–49, 1987.54U. Weinert et al. Molecules, 20: 9847–9861, 2015.15inherently display peptides of interest dominates the field. The best few examples of moleculesof interest being attached to an S-layer after it is assembled are cases where coupling domainswere first cloned into the S-layer protein. One such example is streptavidin cloned into SbsB(an S-layer protein from G. stearothermophilus) so that biotinylated molecules could be loadedon to it.55 Another example is from the S-layer of C. crescentus, where protein G has beenexpressed so that thousands of copies of any IgG antibody can be loaded across its surface.56As mentioned above, genetically modifying S-layer genes so that the proteins contain valu-able peptides and proteins is the showpiece technology for S-layers. These exogenous peptidescan be displayed at a high density across a relatively large distance (e.g. the size of a cellor larger) by being displayed in an S-layer. This approach has been successfully pursued inC. crescentus’ S-layer. The C. crescentus S-layer has displayed vaccines,57 enzymes,58 HIV-binding proteins,59,60 and peptides for biosorption of heavy metals.61All S-layers are biological entities that are ultimately tethered to a cell membrane, but ifthat arrangement is flipped, a lipid membrane can be formed and supported by an S-layer.62Two-dimensional lipid-membrane structures can also be turned into three-dimensional S-layer-coated nanoparticles.63 The two-dimensional structures can be assembled across the surface ofa Langmuir-Blodgett apparatus. The majority of the interest in these structures is in the lipidlayer,64 the S-layer only acts as a stabilizer. On the other hand, lipid nanoparticles coated in S-layers garner a great deal of excitement for the S-layer itself. The promise of lipid nanoparticles(i.e. vesicles, liposomes and solid-cored particles) is that all the above technology alreadydeveloped or being currently developed can be translated to an abiotic particle that highlightsthe strengths of S-layers. Some S-layers will readily assemble on nearly any surface, those S-55D. Moll et al. PNAS, 99: 14646–14651, 2002.56J. F. Nomellini et al. AEM, 73: 3245–3253, 2007.57W. H. Bingle, J. F. Nomellini, and J. Smit. Mol Microbiol, 26: 277–288, 1997.58W. H. Bingle, H. D. Kurtz Jr, and J. Smit. Can J Microbiol, 39: 70–80, 1993.59J. F. Nomellini et al. PLoS One, 5: e10366, 2010.60C. Farr et al. PLoS One, 8: e65965, 2013.61J. Patel et al. App Biochem Biotech, 160: 232–243, 2010.62D Pum et al. J Bacteriol, 175: 2762–2766, 1993.63S. Küpcü, M. Sára, and U. B. Sleytr. BBA-Biomembranes, 1235: 263–269, 1995.64B. Schuster and U. B. Sleytr. Rev Molec Biotech, 74: 233–254, 2000.16layers will coat standard lipid vesicles that are present,65 while other S-layers require specializedlipids to assemble upon a nanoparticle. For instance, C. crescentus S-layer can be reassembledon to lipid vesicles containing C. crescentus LPS.66Two good reviews on the subject of S-layer applications were written by Smit (2008)67and Ilk, Egelseer, and Sleytr (2011).68 The long-range uniformity of S-layers whet the interestfor the structures that can be built on top of them. In this vein, using S-layers to supportmineralization, nanoparticle arrangement, and lipid membranes all share in common using S-layers as physical structures. Many S-layers can self-assemble into long-range regular structuresin vitro, unattached to a cell surface. These “in vitro S-layers” are a hotbed for biotechnologydevelopment because they provide a uniquely dense and patterned protein surface that can bedivorced from contaminants of whole cells.1.2 LipopolysaccharideThe most abundant non-protein component of the Gram-negative outer membrane is LPS. TheLPS of E. coli and Salmonella enterica var. Typhimurium are regarded as the “canonical” exam-ples. Those LPS are the molecules that all others are generally compared to and this thesis willdo the same. An excellent review of LPS was written by Raetz and Whitfield (2002).69LPS resides in the outer leaflet of the Gram-negative outer membrane. In physiological con-ditions, in wild-type coliform bacteria LPS is the only lipid component in the outer leaflet.70,71LPS is synthesized in the inner membrane. While in wild type cells there exists detectablelevels of LPS in the inner membrane, the vast majority of LPS resides in the outer membrane.72,7365M. H. Ucisik et al. Colloid Surface B, 128: 132–139, 2015.66J. F. Nomellini et al. J Bacteriol, 179: 6349–6354, 1997.67J. Smit. Structure, 16: 1151–1153, 2008.68N. Ilk, E. M. Egelseer, and U. B. Sleytr. Curr Opinion Biotech, 22: 824–831, 2011.69C. R. H. Raetz and C. Whitfield. Annu Rev Biochem, 71: 635–700, 2002.70J. Smit, Y. Kamio, and H. Nikaido. J Bacteriol, 124: 942–958, 1975.71Y. Kamio and H. Nikaido. Biochemistry, 15: 2561–2570, 1976.72A. Polissi and C. Georgopoulos. Mol Microb, 20: 1221–1233, 1996.73D. Ding and S Kaplan. Prep Biochem, 6: 61–79, 1976.17Fully formed LPS is transported across the periplasm to the outer membrane via the proteinsLptABCDE.74,75 The exact details of how a large LPS molecule can traverse the periplasm andbe inserted into the outer leaflet are still uncertain; the difficulty is in no small part due to thefact that all the required proteins are essential for viability.Figure 1.7 highlights a simplified LPS structure from Salmonella minnesota as reported byVukajlovich, Hoffman, and Morrison (1987).76 There are three fundamental components of LPS:lipid A, the core oligosaccharide, and the O-polysaccharide.1.2.1 Lipid AStructure Lipid A is the ‘base’ of the LPS molecule. The backbone of canonical LPS is twoglucosamine sugars bound to each other by an β1→6 bond. The diglucosamine molecule hastwo O-linked beta hydroxy acyl chains at the C-3 and C-3′ positions and two N-linked betahydroxy acyl chains at the C-2 and C-2′ positions. The two ‘prime’ acyl chains are modifiedwith subsequent acyl chains attached at the beta hydroxy groups. All these acyl chains are fullysaturated.The C-1 and C-4′ positions of the diglucosamine are phosphorylated. These phosphatesgive the lipid A a net negative charge and they are thought to bind divalent cations which formelectrostatic bridges with neighbouring lipid A molecules and stabilize the outer membrane.77,78The C-6′ position is ligated to the core oligosaccharide. The canonical structure has the C-6′position occupied with an α2→6 bond to a Kdo sugar. Figure 1.8 shows this canonical structureas it is found in E. coli K12.There are numerous Lipid A structures that differ from the canonical structure. Examplesexist of modified structures at all the possible locations: the phosphates, the glucosamines, and74P. Sperandeo et al. J Bacteriol, 189: 244–253, 2007.75N. Ruiz et al. PNAS, 105: 5537–5542, 2008.76S. W. Vukajlovich, J. Hoffman, and D. C. Morrison. Mol Immun, 24: 319–331, 1987.77L. Leive. Biochem Biophys Res Commun, 21: 290–296, 1965.78L. Leive. Ann NY Acad Sci, 235: 109–129, 1974.18P PPPPPEtNEtNGlcN GlcNKdo KdoHepHepGlcGalGalGlcHepKdoGlcNAcGlcNAcGalNAcGalNAcGalGalO-polysaccharideCore oligosaccharideLipid Ax 5-40Figure 1.7: The wild type LPS from Salmonella minnesota. The lipid A is at the bot-tom of the figure (in blue). It comprises a backbone of two glucosamine residues.Each glucosamine has an attached phosphate moiety and three fully saturated acylchains. Above the lipid A is the core oligosaccharide (in green). This core oligosac-charide is a hendecasaccharide. Extending from the core oligosaccharide is theOPS (in purple). The structure of this OPS is a variable number of pentasaccharidesubunits (5–40). Abbreviations: Glc, glucose; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; Gal, galactose; GalNAc, N-acetylgalactosamine; Kdo, 3-deoxy-D-mannooctulosonic acid; Hep, L-glycero-D-mannoheptose; P, phosphate; and EtN,ethanolamine.19C-3ʹC-4ʹ C-6ʹC-3C-2C-1C-2ʹβ1 6Figure 1.8: Kdo2-Lipid A from E. coli K12. The lipid A portion comprises the phosphates(green), the glucosamines (blue), and the acyl chains (black). The two Kdo sugarspresent are the start of core oligosaccharide.the lipids. An excellent review on lipid A modifications is by Raetz et al. (2007).79The phosphate modifications are often extra chemical groups linked directly to the phos-phates. Cationic attachments are common, such as ethanolamine (e.g. Salmonella minnesota,see fig. 1.7) and glucosamine (e.g. Bordetella pertussis80). Neutral attachments to the phosphateare also possible such as methyl groups (e.g. Leptospira interrogans81). The phosphates can alsobe absent; examples of lipid A molecules missing a phosphate are Rhizobium etli,82 Bacteroidesthetaiotaomicron,83 Prevotella intermedia,83 Helicobacter pylori,84 and Francisella tularensis.85Removed phosphates can be replaced with ethanolamine (e.g. H. pylori84) or anionic sugarssuch as galactosamine (e.g. C. crescentus86 and R. etli82).79C. R. Raetz et al. Ann Rev Biochem, 76: 295, 2007.80N. Marr et al. J Bacteriol, 190: 4281–4290, 2008.81M. B. Hinckley et al. J Biol Chem, 280: 30214–30224, 2005.82N. L. Que, A. A. Ribeiro, and C. R. Raetz. J Biol Chem, 275: 28017–28027, 2000.83P. M. Chilton et al. Infect and Immun, 81: 3317–3325, 2013.84A. X. Tran et al. J Biol Chem, 279: 55780–55791, 2004.85X. Wang et al. Biochemistry, 45: 14427–14440, 2006.86J. Smit et al. Innate Immun, 14: 25–36, 2008.20Modifications to the diglucosamine backbone are less common than other lipid A modi-fications. The most frequent alteration seen in the backbone is the replacement of the eitherone or both glucosamines for a D-2,3-diamino-2,3-dideoxy-glucose. Di-D-2,3-diamino-2,3-dideoxy-glucose backbones are seen in C. crescentus ,86 One of the most extreme modificationsto the diglucosamine backbone is the unique example of Rhizobium leguminosarum.87 R. legu-minosarum’s lipid A contains no phosphate, instead a galacturonate sugar is present at the 4′position; and a 28 carbon long acyl chain. The extra long acyl chain is attached to the C-5 position which is only possible because the reducing glucosamine’s ring has been openedup, converting the glucosamine into a linear 2-amino-2-deoxygluconate. Another uniquelymodified lipid A is found in Bdellovibrio bacteriovorus. The B. bacteriovorus lipid A’s back-bone consists is 2,3-diamino-2,3-dideoxy-D-glucose disaccharide and contains no charges; thecanonical phosphates are replaced with D-mannose moieties.88Lipid modifications come in the form of the number of acyl chains, the length of the acylchains, and the inner structure of the acyl chains. Six acyl chains is the standard found incanonical lipid A molecules in enteric bacteria, but the number of fatty acid groups can be ashigh as eight (e.g. Vibrio cholerae O13989) and as low as four (e.g. H. pylori84). The lengthsof the acyl chains in lipid A do not vary as much as in other lipids due to more stringentacyltransferases, but they do vary among bacteria.90 An extreme example is the 28 carbon longacyl chain mentioned above in R. leguminosarum.91 The acyl chains in lipid A are usually fullysaturated but unsaturated examples are not uncommon. Bacteria with unsaturations in theirlipid A include C. crescentus,86 Yersinia pestis,92 and Rhodobacter capsulatus.93The canonical lipid A biosynthesis pathway has been fully determined. The best sourcesfor information about lipid A synthesis are reviewed by the field’s most prominent investigator,87U. R. Bhat, L. S. Forsberg, and R. W. Carlson. J Biol Chem, 269: 14402–14410, 1994.88D. Schwudke et al. J Biol Chem, 278: 27502–27512, 2003.89S. Chatterjee and K. Chaudhuri. , 1639: 65–79, 2003.90T. J. Wyckoff et al. J Biol Chem, 273: 32369–32372, 1998.91S. S. Basu, M. J. Karbarz, and C. R. Raetz. J Biol Chem, 277: 28959–28971, 2002.92R. Rebeil et al. Mol Microb, 52: 1363–1373, 2004.93J. H. Krauss et al. Euro J Bioch, 180: 519–526, 1989.21Christian Raetz.69,94.Effects of LPS on the immune system LPS is a potent immune stimulatory molecule and it isthe lipid A that is the primary structure responsible for the innate immune response. It was thisinflammatory activity of LPS that was identified before the molecule was isolated,95 giving LPSits alternate, original name “endotoxin”.Lipid A is a microbe-associated molecular pattern (MAMP), a highly conserved structurethat is specifically recognized by the vertebrate innate immune system.96 There are a number ofexcellent papers and reviews on the immunological aspects of lipid A. The review by Alexanderand Rietschel (2001)97 is a good general review of the innate immunity against LPS. The LPS-toll-like receptor 4 (TLR4)- MD2, complex has been solved by Park et al. (2009).98 The reviewby Lu, Yeh, and Ohashi (2008)99 covers the signal transduction pathways, downstream ofLPS/TLR4 recognition.In the immune system, two proteins, LPS-binding protein and CD14, are responsible for theinitial binding of LPS and delivery of it to the recognition machinery. That recognition machin-ery, once it binds LPS, dimerizes and is comprised of two copies of TLR4 and two copies of theprotein MD2. The LPS is held in the cup of MD2 and two of these complexes are sandwichedbetween two TLR4s. All of the canonical lipid A acyl chains a buried within MD2 exceptfor the acyl chain attached at the C-2 position that is positioned in the hydrophobic interfacebetween MD2 and TLR4. This ‘outside’ acyl group aids in the dimerization of TLR4 and islikely the reason that lipid A molecules with fewer acyl chains are less immunogenic.100 Thephosphates present on the lipid A interact with positively charged residues on both TLR4s andMD2. The presence of phosphates increases the immunogenicity of lipid A about a 100 fold.10094C. R. Raetz et al. J Lipid Res, 50: S103–S108, 2009.95H. Zinsser. J Exp Med, 17: 117–131, 1913.96F. M. Ausubel. Nature Immun, 6: 973–979, 2005.97C. Alexander and E. T. Rietschel. J Endotoxin Res, 7: 167–202, 2001.98B. S. Park et al. Nature, 458: 1191–1195, 2009.99Y.-C. Lu, W.-C. Yeh, and P. S. Ohashi. Cytokine, 42: 145–151, 2008.100E. T. Rietschel et al. FASEB J, 8: 217–225, 1994.22The crystal structure of the LPS-TLR4-MD2 complex revealed space around the phosphates thatmay accommodate the presence of bulkier anionic substitutions.98Once LPS is bound and the TLR4-MD2 receptor dimerizes, a signal transduction pathwayis activated. The two signaling pathways are the MyD88-dependent pathway and the TRIFpathway (MyD88-independent). Briefly, the MyD88 pathway leads to the expression of proin-flammatory cytokines and activation of acquired immunity. The TRIF pathway leads to theexpression of type 1 interferons and the activation of the innate immune system.99 These twopathways’ signals do overlap at a few places, specifically in the activation of the nuclear factorkappa-light-chain-enhancer of activated B cells (NF-κB). Modulating the immune response toLPS is an avenue for vaccine/adjuvant development. The two pathways can be differentiallyactivated depending on the structure of the bound lipid A molecules.83 See section 3.2.16 forour investigation into the immunogenicity of LPS from C. crescentus.1.2.2 Core oligosaccharideStructure On the subject of core oligosaccharide structures there are excellent reviews by Frir-drich and Whitfield (2005)101 and Holst (1999).102 Core oligosaccharides, relative to lipid A, arehighly variable, even within a species. As an example, there are five known core oligosaccha-rides in E. coli .103 Yet compared to OPSs, core oligosaccharides are highly conserved; there are186 known OPS structures in E. coli .104 Figure 1.7 shows the LPS from S. minnesota; its coreoligosaccharide has many “canonical” features (e.g. Kdos, heptoses, hexoses, and phosphates)that are used as comparators for other core oligosaccharides.The only universally conserved feature of core oligosaccharides is the eight carbon sugarthat sits at the base of the structure, Kdo. Kdo is always found as the sugar bonded to the C-6′position of the lipid A by a α2→6 bond (see fig. 1.8). The only known exception to the Kdo101E. Frirdich and C. Whitfield. J Endo Res, 11: 133–144, 2005.102O. Holst. Endotoxin Health Disease, 1: 115–154, 1999.103D. E. Heinrichs, J. A. Yethon, and C. Whitfield. Mol Microb, 30: 221–232, 1998.104R. Stenutz, A. Weintraub, and G. Widmalm. , 30: 382–403, 2006.23rule is Acinetobacter calcoaceticus, where the Kdo is replaced with a 2-mannooctulosonic acid,Ko.105 This Kdo-lipid A bond is a ketosidic bond, usually the only one present in an LPS, and itis easily cleaved by heating in the presence of a mild acid. This cleavage make the separationof the poly/oligosaccharides and the lipid A a simple procedure.There are often multiple Kdo sugars present in the core, as seen in fig. 1.7. These ad-junct Kdo sugars are bonded to the central Kdo by an α2→4 bond. That C-4 position on thecentral Kdo is often bonded to a anionic moiety, such as Kdo, Ko (Y. pestis106), phosphate (V. cholerae107), or galacturonic acid (Rhizobium trifolli108). The C-5 position on the centralKdo is usually bonded to a manno-configured sugar; the canonical and common example isLD-heptose. The manno-sugar is usually the first sugar in an elongated oligosaccharide. Threeheptoses are attached to that C-5 position in enteric bacterial core oligosaccharides, these hep-toses along with the Kdos represent the ‘inner’ core of enteric cores. The ‘inner’ and ‘outer’core delineations do not apply well to non-canonical LPSs. Figure 1.9 shows these general coreoligosaccharide characteristics. Many core oligosaccharides are phosphorylated at one or manylocations. Presumably, the phosphates and other negative charges fulfill similar functions as thecanonical lipid A phosphates. LPSs that terminate at the core oligosaccharide, or before, aretermed “rough,” while those that possess an OPS are called “smooth.”5)-Kdo-(α2 6)-lipid A4)-RR12Figure 1.9: This is a general formula for core oligosaccharide structures. R1 is usually amanno sugar, specifically it is most often a heptose. R2, when present, is substitutedwith a negatively charged moiety, examples are Kdo and phosphate. (This figureis a recreation of fig 2 from O. Holst. Chemical structure of the core region oflipopolysaccharides. Endotoxin in Health and Disease, 1: 115–154, 1999.)105K. Kawahara et al. Euro J Biochem, 163: 489–495, 1987.106E. V. Vinogradov et al. Carbohyd Res, 337: 775–777, 2002.107Y. A. Knirel et al. Euro J Biochem, 247: 402–410, 1997.108R. W. Carlson, R. L. Hollingsworth, and F. B. Dazzo. Carbohyd Res, 176: 127–135, 1988.24Synthesis The synthesis of the core oligosaccharide happens in the inner leaflet of the innermembrane. The structure is built on to lipid A, one sugar at a time.101 It is worth noting that thetransfer of the central Kdo is the first step for the core oligosaccharide and a required step forthe complete synthesis of lipid A.109 The completed core-lipid A is then flipped across the innermembrane by the flippase MsbA.691.2.3 O-polysaccharideStructure OPSs are highly variable in structure between species and even strains. For anexamples of this variability, an exhaustive review of the OPSs in E. coli has been assembled byStenutz, Weintraub, and Widmalm (2006).104 There are 186 known OPSs in E. coli alone.OPSs are synthesized with a repeating structure, formed from subunits. For E. coli, theOPSs are built of subunits that range in size from 2–7 monosaccharides (3 examples of 2 sugarsubunits, 1 example of a 7 sugar subunit). Even smaller configurations exist, such as that arehomopolymer of a single repeating sugar in a single configuration (e.g. V. cholerae110). Thereare also OPSs in E. coli that are comprise a single repeating sugar (O8, O101) but they havemultiple configurations or intersaccharide bonds. These ‘simpler’ OPSs are often characteristicof an OPS assembled by the ABC transporter-dependent pathway (see below).OPSs can be synthesized from a large range of sugars. Well known sugars, such as glucoseand mannose are regular components of OPSs but so are rare sugars such as talose, colitose,and pseudaminic acid.104 See chapter 3 for our determination of the structure of the OPS fromC. crescentus which contains the rare sugar N-acetylperosamine.Synthesis Unlike the core oligosaccharide, the OPS is produced on a lipid carrier in the innermembrane and then ligated onto the nascent LPS as a fully completed polymer. There are109C. Belunis and C. Raetz. J Biol Chem, 267: 9988–9997, 1992.110L. Kenne et al. Carbohyd Res, 100: 341–349, 1982.25three known biosynthetic pathways that produce OPS: the Wzy-dependent pathway, the ABCtransporter-dependent pathway, and the synthase-dependent pathway.69Figure 1.10: A simple cartoon overview of the Wzy-dependent pathway of OPS synthesis.Wzx is the ‘O-antigen/OPS subunit flippase.’ Wzy is the ‘O-antigen polymerase.’Wzz is the ‘O-antigen length determining protein.’ WaaL is the ‘O-antigen ligase.’MsbA is the ‘LPS ABC flippase.’The Wzy-dependent pathway is the classic subunit based synthesis pathway for OPSs. Thesubunits, consisting of 2–7 sugars, are assembled on a undecaprenyl-pyrophosphate carrierlipid in the inner leaflet of the inner membrane. The complete subunits are flipped across theinner membrane by the protein Wzx. The subunits are then polymerized by the enzyme Wzyin the outer leaflet of the inner membrane. The length of the growing OPS polymer can varybut it is influenced by the chain length regulator protein Wzz. When the OPS has reached asuitable length, the enzyme WaaL transfers the entire OPS from the carrier lipid and ligates iton to the core oligosaccharide, completing LPS synthesis. Figure 1.10 is an overview of thispathway. LPSs with OPSs made via the Wzy-dependent pathway have a characteristic ladderpattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Theladder pattern comes from the variable chain length introduced during subunit polymerization.The separation between the bands is equivalent to the size of one OPS subunit. Figure 3.3 hasan example of that Wzy-dependent pathway ladder pattern.The defining feature of the ABC transporter-dependent pathway is that it assembles OPSs26one sugar at a time by glycosyltransferase enzymes. Because the polymer grows one sugarat a time there is no specific polymerase enzyme (like Wzy) and the chains are non-branched.The OPS polymer is fully assembled in the inner leaflet of the inner membrane before it istransported across the inner membrane by a ABC transporter. Once the OPS is in the periplasmit is ligated on to the core via WaaL. A unique feature of ABC transporter-dependent pathwaysynthesized OPSs is the addition of a unique end cap on the non-reducing end.111Figure 1.11: A simple cartoon overview of LptABCDE mediated transport of LPS acrossthe periplasm. LptA is depicted here as shuttling the LPS across the periplasm butit is possible that LptA forms filaments across the periplasm.The synthase-dependent pathway is a rare pathway only found in the plasmid-encodedOPS:54 of Salmonella enterica serovar Borreze.112 The synthase-dependent pathway involves asingle protein that processively assembles the OPS in the inner membrane.Once the LPS is fully formed in the outer leaflet of the inner membrane it is transportedacross the periplasm to the outer membrane by the LptABCDE proteins. Ruiz, Kahn, andSilhavy (2009) covers the subject of LPS transport.113 Figure 1.11 is an overview of LPS transport111E. Vinogradov et al. J Biol Chem, 277: 25070–25081, 2002.112W. J. Keenleyside et al. Mol Microb, 11: 437–448, 1994.113N. Ruiz, D. Kahne, and T. J. Silhavy. Nature Rev Microb, 7: 677–683, 2009.27across the periplasm.1.3 PorinsThe outer membrane of Gram-negative bacteria is a particularly impermeable membrane tolipophilic and hydrophilic molecules. In 1976 a protein complex was first isolated from theouter membrane of S. enterica that produced transmembrane channels which non-specificallytransported solutes; this complex was the first to be called a ‘porin’.114 Today, the term porinrefers to outer membrane proteins that transport molecules across a membrane passively. Thisdefinition is for non-specific protein channels115, such as OmpF from E. coli, but sometimes thedefinition is widened to include structure-specific transport channels, such as LamB in E. coli.116,117Porins act as sieves in the outer membrane allowing small solutes to pass from the envi-ronment to the periplasm. Without a channel for them to pass, the outer membrane would beimpermeable to these usually hydrophilic nutrient molecules. In their simplest form, porins canbe thought of as water filled holes in the membrane, allowing anything that will fit through. Inreality, these holes are complex structures with changing shapes and charges. The activity ofthese holes is defined by their size and selectivity.The size of a pore can be an absolute measure of the pore diameter, as measured by X-raycrystallography, or as a derived figure based on permeability studies. The selectivity is judgedon the relative diffusion rates of different molecules and how they are discriminated by size,charge, and hydrophobicity. An excellent example of these properties, as seen in E. coli porins,is assembled in a review by Nikaido.115 In E. coli the porins OmpF and OmpC both have apreference for cationic molecules over anionic molecules, but both will allow the passage ofuncharged molecules like sugars. On the other hand, the E. coli porin PhoE strongly prefers114T Nakae. J Biol Chem, 251: 2176–2178, 1976.115H. Nikaido. Microb Mol Biol Rev, 67: 593–656, 2003.116R. Benz. Ann Rev Micrbiol, 42: 359–393, 1988.117R. Hancock. J Bacteriol, 169: 929, 1987.28OMPPorinExbBTonBExbDH+Figure 1.12: A simple cartoon overview of how TonB-mediated active transport differsfrom passive transport through a porin. On the left is a simplified TonB-mediateduptake system consisting of a TonB-dependent outer membrane protein (OMP), andthe TonB inner membrane complex (TonB and ExbBD). Energy consumed at theinner membrane is transduced to the outer membrane through the action of TonB.Not shown is the globular domain of the TonB-dependent outer membrane proteincalled the ‘plug.’ On the right is a porin, passively allowing solutes to pass throughit.anions over cations. There are theoretical models that suggest that ion selectivity is related toionic concentration, the lower the concentration the greater the selectivity.118 One importantdifference between OmpF and OmpC is the size; OmpF has a larger pore diameter, allowingmore solutes to be transported and a broader range of solutes. This discrimination in size isan important aspect of porin function. Porins give outer membranes a characteristic molecularweight cutoff of permeability, also known as an exclusion limit.117 This exclusion limit can beanalyzed by measuring the rate of diffusion of differing sized carbohydrates through the poresin a liposome swelling assay.119 OmpF and OmpC have severe reductions in permeability forsolutes above a size around 200 Da.Porins increase the communication between the periplasm and the outside world allowingselective permeability take place at the cytoplasmic membrane by proteins that have access118W. Im and B. Roux. J Mol Biol, 322: 851–869, 2002.119H. Nikaido and E. Y. Rosenberg. J Bacteriol, 153: 241–252, 1983.29to cellular energetics. There does exist active transport systems in the outer membrane, suchas TonB-dependent transporters. These active transport systems are often associated with theuptake of scarce nutrients like iron and vitamin B12. The alphaproteobacterium C. crescentusencodes for 67 such TonB-dependent outer membrane proteins,120 possibly as an adaptationto living in nutrient poor environments.121 TonB-mediated transport systems utilize energyconsumed at the inner membrane to power specific uptake systems in the outer membrane.Figure 1.12 illustrates TonB-mediated transport systems and a porin. To date no published studyhas characterized a general porin in C. crescentus. See chapter 4 for our work to characterizeOmpW in C. crescentus.1.4 Aims and ObjectivesThe bacterium C. crescentus is a well studied model organism for its dimorphic life cycle,122cell shape,123 and regulatory pathways.124 Despite having a unique S-layer, an unsolved LPSstructure, and no known porins in its outer membrane, the cellular envelope of C. crescentus hasnot attracted widespread attention. This thesis covers my work to illuminate the compositionand the structures of the three different components of the C. crescentus cell envelope: theS-layer protein, RsaA (chapter 2); the LPS (chapter 3); and the porin, OmpW (chapter 4).120W. C. Nierman et al. PNAS, 98: 4136–4141, 2001.121S Lohmiller et al. Micrbiol, 154: 1748–1754, 2008.122J. M. Skerker and M. T. Laub. Nature Rev Microb, 2: 325–337, 2004.123M. T. Cabeen and C. Jacobs-Wagner. Nature Rev Microb, 3: 601–610, 2005.124M. T. Laub, L. Shapiro, and H. H. McAdams. Annu Rev Genet, 41: 429–441, 2007.30Chapter 2Crystallization and X-ray diffraction ofthe S-layer protein RsaA“Please forgive me for presenting, on such a great occasion, results which are stillin the making, but the glaring sunlight of certain knowledge is dull and one feelsmost exhilarated by the twilight and expectancy of the dawn.”— Max Perutz, 1962 Nobel lectureThe father of protein X-ray crystallography.2.1 IntroductionPROTEIN SURFACE LAYERS (S-layer) are para-crystalline coatings that surround micro-bial cells.34 They have various functions, such as adhesion and immune evasion inpathogenic bacteria;27,125 protection against phage and predation;31,32 and as a pseudo-membranein archaea.126 S-layers are composed of either one or a small number of secreted proteins thatself assemble into a contiguous sheet in the extracellular environment.Despite their unique features, S-layers have not received the intensive structural investiga-tions that most other protein families have received, in no small part due to their resistanceto 3D-crystallization. The reason why S-layers resist crystallization is not completely un-34T. J. Beveridge et al. FEMS Microb Rev, 20: 99–149, 1997.27J. Kern and O. Schneewind. Mol Microbiol, 75: 324–332, 2010.125S. Dallaire-Dufresne et al. Vet Microbiol, 169: 1 –7, 2014.31S. Koval and S. Hynes. J Bacteriol, 173: 2244–2249, 1991.32S. F. Koval. “Predation on bacteria possessing S-layers” in: Advances in Bacterial Paracrystalline SurfaceLayers. Springer, 1993. 85–92126A. E. Blaurock et al. J Cell Biol, 71: 1–22, 1976.31derstood, but it is definitely related to their proclivity to self-assemble into large polymerictwo-dimensional structures (i.e. S-layers). That S-layer formation plays a role in the crystal-lization problems seemed confirmed by the fact that the only groups to successfully crystallizean S-layer protein have done so by specifically inhibiting or removing the S-layer formationability of the proteins24,127. It is thought that perhaps the 2D-S-layer formation out competes the3D-crystallization or maybe the 2D-S-layer structure is incompatible with 3D-crystal growth.Possibly there are conditions that exist that would allow for the crystallization of an intactS-layer protein, but those conditions are currently unknown.The Gram-negative bacterium Caulobacter crescentus possesses an S-layer that is composedof thousands of copies of a single 98.6 kDa protein, RsaA.12 This particular S-layer has beenpursued as a potent platform for peptide display and protein expression.67 RsaA is secreted fromthe cell via a type 1 secretion system.128RsaA shares little to no sequence similarity to any other proteins, except for in the conservedrepeat-in-toxin (RTX) motifs canonically found in all type 1 secreted proteins.43,129 When theentire RsaA protein sequence is compared against other proteins with a basic local alignmentsearch (BLAST) analysis the highest hits returned are unknown proteins from Skermanella aero-lata (Score: 1063, Identity: 33.7%) and Pseudomonas syringae (Score: 958, Identity: 31.5%).Interestingly, the protein BLAST also returns hits to the S-layer protein from Campylobacterfetus, SapA (Score: 598, Identity: 29.0%). Closer investigation of the sequence alignmentbetween RsaA and C. fetus SapA shows few extended regions of sequence identity. Of the fewregions of consistent homology most were RTX motifs. There are no tantalizing clues to belearned from BLAST analysis at this point, but it may be good platform to translate any futurestructural information to other unsolved proteins.24E. Baranova et al. Nature, 487: 119–122, 2012.127T. Pavkov et al. Structure, 16: 1226 –1237, 2008.12J. Smit et al. J Bacteriol, 146: 1135–1150, 1981.67J. Smit. Structure, 16: 1151–1153, 2008.128S. G. Walker et al. J Bacteriol, 176: 6312–6323, 1994.43A. Chenal et al. J Biol Chem, 284: 1781–1789, 2009.129W. H. Bingle, J. F. Nomellini, and J. Smit. J Bacteriol, 182: 3298–3301, 2000.32RsaA further isolates itself in its sequence by featuring a uniquely limited amino acidcomposition that is biased towards small, simple residues. This bias towards amino acids thatare energetically less costly to synthesize is thought to be an evolutionary strategy for secretedproteins conserved across microbial life and especially in C. crescentus.130All crystallographic studies of S-layer proteins appear to require inhibition of S-layer forma-tion. In the past co-crystallization with nanobodies24 and truncation127 approaches have resultedin monodisperse, crystallizable proteins. Here we describe the expression of an 804 amino acid(AA) C-terminal fragment of RsaA (RsaA ∆0–222) from its native host, C. crescentus. Theuntagged protein was purified from culture supernate and crystallized by hanging-drop vapordiffusion. Crystals were grown that diffracted to 2.5 Å. Diffraction data were collected at boththe Canadian Light Source (CLS) and the Stanford Synchotron Radiation Light Source (SSRL).RsaA is the first Gram-negative S-layer protein to be crystallized.Crystallization and X-ray diffraction are the two essential first steps of any structural determi-nation but the next hurdle of solving the phasing problem is an equally crucial and fundamentalproblem in crystallography. Solving the phases has been the hurdle with which this project hadparticular difficulties. Many strategies have been pursued to complete the structural solutionof RsaA. No successful solution to the phase problem has been determined. The atomic-levelstructure of RsaA remains ellusive.2.2 Methods2.2.1 Strain and plasmid constructionExpression strain The strain used to produce RsaA truncates was JS4032.131 This strain canalso be labeled C. crescentus CB2A ∆sapA ∆manB ::repABC.130D. R. Smith and M. R. Chapman. MBio, 1: e00131–10, 2010.131J. H. Y. Lau, J. F. Nomellini, and J. Smit. Can J Microbiol, 56: 501–514, 2010.33Expression vectors The plasmid used initially in this study, pUC8CVX-RsaA ∆0–222, wasa construct of the plasmid pUC8CVX56 containing an open-reading frame encoding for theC-terminal 804 AA of RsaA. pUC8CVX, digested with BamHI and HinDIII, was ligated to the2819 bp fragment from a similarly digested plasmid containing an open-reading frame encodingfor RsaA with a BamHI endonuclease recognition sequence at the 666 bp position created in aprevious study.132 The plasmid, pUC8CVX-RsaA ∆0–222, was then electroporated37 into theexpression strain.The same protocol as above was followed to generate an expression vector to produce RsaA∆0–551, a smaller C-terminal truncate. To construct this plasmid, pUC8CVX-RsaA ∆0–551,the rsaA-truncate gene was swapped for a version encoding for only the C-terminal 475 AAof RsaA. An annotated sequence of full-length, wild-type RsaA is found in the appendix onpage 154.The plasmid that was used to produce RsaA ∆0–222 GSCC723, pUC8CVX-RsaA ∆0–222GSCC723, was produced by replacing the rear portion of RsaA ∆0–222 in pUC8CVX-RsaA ∆0–222 with a preexisting engineered RsaA fragment. The fragment contained an in-frame insertionof the C-Myc-tag antigen at the 723rd codon.56 This replacement was done by exchanging 2630bp BstXI–HinDIII fragments. The C-Myc-tag is a decapeptide, EQKLISEEDL, that was derivedfrom the oncogenic human protein Myc. In RsaA we predicted that the added charges mightimprove the ability of the protein to bind ions useful for the phasing problem. Our ‘GSCC’cassette contains the C-myc-tag as well as a few other restriction sites that can be utilized forthe instillation of future peptide cassettes, specifically a BglII site and a PstI site. The translatedpeptide sequence of the GSCC cassette is GSRSVNNASEQKLISEEDLRPSADGS.56J. F. Nomellini et al. AEM, 73: 3245–3253, 2007.132M. J. Ford, J. F. Nomellini, and J. Smit. J Bacteriol, 189: 2226–2237, 2007.37A. Gilchrist and J. Smit. J Bacteriol, 173: 921–925, 1991.342.2.2 Protein productionThe same protocol, once it was finalized, was used for all the different constructs of RsaA(∆0–222, ∆0–551, and ∆0–222 GSCC). The cells were grown in M16HIGG medium133 at 30◦Cfor 72 hr. Cultures, 250 ml, were used in 2800 ml wide-bottom Fernbach flasks, resulting inshallow cultures that did not require shaking for sufficient aeration, as agitation of the cultureled to macroaggregation of the secreted protein.The cultures were centrifuged at 6300 rpm in JA-10 bottles for 35 min, the supernates wererecovered and recentrifuged. The supernates were then filtered through a 0.22 µm microfilterto ensure no cells remained. The protein-containing supernate was initially concentrated bylyophilization and rehydration to 1/10 the original volume. The rehydrated protein solutionwas dialyzed against distilled water and microfiltered. Purification was performed over a 26/60S100 Sephadex size-exclusion chromatography column and mobile phase of 200 mM NaCl and10 mM Tris pH 7.5. The first major elution peak was pooled and dialyzed against distilled waterand concentrated by placing the dialysis bags on polyethylene glycol (PEG) with an averagemolecular weight of 20 000 Da (Sigma-Aldrich). The protein solution was slowly concentratedto a final concentration of 3.5–4 mgml−1. Dialysis against PEG was slow and gentle enoughto prevent the protein from aggregating during concentration. Other methods of concentration,such as ultrafiltration caused RsaA ∆0–222 to come out of solution as unusable aggregates.2.2.3 Dynamic light scattering spectroscopyDynamic light scattering (DLS) was used as a qualitative measure of protein quality. DLShas an accessible and rapid ability to determine the degree of aggregation and monodispersityof a purified protein sample. Prior to DLS analysis all protein samples were concentrated toat least 1 mgml−1 and filtered through a 0.22 µm syringe filter. Sixty microlitres of filteredprotein solution was pippetted into a glass-bottomed 384-well plate well. Standard instrumentprocedures were followed (Wyatt, DynaPro Plate ReaderTM).133J. Smit, M. Hermodson, and N. Agabian. J Biol Chem, 256: 3092–3097, 1981.352.2.4 Crystallization of RsaA constructsRsaA ∆0–222 was initially crystallized at a concentration of 9 mgml−1. Sparse-matrix screen-ing was performed using the Joint Centre for Structural Genomics (JCSG) Core I, II, III and IVscreens (Qiagen).Inital crystal formation conditions were not reproducible in a hanging drop vapor apparatus.Contrary to conventional wisdom, thin plates readily formed once the concentration of theprotein and the PEG 8000 were both reduced. Thicker, more robust crystals formed whenMgCl2 was replaced with other Group 2, alkaline earth metal salts, specifically CaCl2, SrCl2,or BaCl2. Although calcium is almost certainly the natural ion involved with RsaA folding,SrCl2 was ultimately deemed the most successful at producing crystals. Final crystallizationconditions were 8.5% (w/v) PEG 8000, 100 mM Tris pH 7.4, and 150 mM SrCl2. Two crystalsforms were observed, planar ‘plate’ crystals and hexegonal ‘pencil’ crystals. Occasionally the‘plate’ crystals would grow in clusters where it was advantageous to break off a section ofthe cluster for diffraction (like the crystal in fig. 2.1). The ‘pencil’ crystals always formed asindividual crystals.Mother liquor (i.e. the crystallization well solution) amended with 25% glycerol was usedas a cryo-protectant for all crystals prior to flash freezing in liquid nitrogen or a cryo-nitrogenstream.2.2.5 Data collection and processingX-ray diffraction data was collected, multiple times, at two synchrotron light sources: the CLSin Saskatoon, Saskatchewan and SSRL at Stanford University, California. Both facilities provedsuitable for our data collection needs. The beamlines at CLS offered a slightly larger spectrum ofavailable X-ray wavelengths; some of the high energy wavelengths proved useful for accessingthe K-edges of strontium (0.7699 Å) and bromine (0.9202 Å). For our best diffracting crystal,diffraction was performed at the ‘CMCF 08ID-1’ beamline at CLS. A wavelength of 1.54 Å36Figure 2.1: An example of a large cluster of ‘plate’ crystals of RsaA ∆0–222. The clusterwas just over one millimeter in width from tip to tip. The largest crystal, on the right,was broken off and diffracted using synchrotron radiation, resulting in a resolution>10 Å. The color is imparted by a polarizing filter on the microscope, the crystalsare naturally colourless.(equal to the copper K-edge) was used to maximize the overall anomalous signal of iodide. Asreference, fig. 2.2 shows theoretical anomalous scattering coefficients for most of the elementsused in this study. Data were processed using HKL3000134 or XDS135 ( see table 2.1); phasingattempts were performed using Shelx136 or AutoSol in Phenix.137 Data were collected througha full 360◦ of rotation, due the low symmetry found in the ‘plate’ crystals2.3 Results2.3.1 Initial crystallization and optimizationProtein was produced in JS4032. The strain’s genomic copy of rsaA is inactivated by a nonsenseframe-shift mutation at the 353rd codon. The gene sapA has been deleted; it encodes for anS-layer associated protease (SapA) that has been implicated in degrading modified versions ofRsaA.138 The surface polysaccharides, extracellular polysaccharide (EPS), polyrhamnan, and134W. Minor et al. Acta Crystallogr D, 62: 859–866, 2006.135W. Kabsch. Acta Crystallogr D, 66: 125–132, 2010.136G. M. Sheldrick. Acta Crystallogr D, 66: 479–485, 2010.137P. D. Adams et al. Acta Crystallogr D, 66: 213–221, 2010.138E. Umelo-Njaka et al. J Bacteriol, 184: 2709–2718, 2002.3720−−15−10 −5 0 5 10 15 6000 8000 10000 12000 14000 16000 18000 20000 2.47Å 1.80Å 1.41Å 1.16Å 0.99Å 0.86Å 0.76Å 0.68ÅX-ray energy (eV)X-ray wavelength (Å)Cu KSe K Br KEu L1Ba L1I L1 Sr KI f "I f ’Eu f "Eu f ’Ba f "Ba f ’Br f "Br f ’Sr f "Sr f ’Se f "Se f ’Figure 2.2: Theoretical plots of anomalous scattering coefficients for various elementsused in this study. The K-edges and L1-edges that are present within the rangeof this graph were indicated. Selenium was included because it is often used as alandmark to compare with other elements. The data presented here was obtained andplotted by the Edgeplots web tool provided by the Biomolecular Structure Center atthe University of Washington (http://skuld.bmsc.washigton.edu/scatter)O-specific polysaccharide (OPS), were all eliminated by disrupting manB. manB encodes forphosphomannomutase, an important enzyme in the bio-synthesis of many deoxysugars (seechapter 3 for our investigation on the structure of the lipopolysaccharide (LPS)). Removingthese carbohydrate structures causes RsaA to no longer associate with the cell surface and aidsin downstream processing, giving the cells favorable centrifugability. The operon repABC hasalso been knocked into this strain to allow for the replication of small broad-host range plasmidsthat we use for protein expression.139139E. Umelo-Njaka et al. Plasmid, 46: 37–46, 2001.38The protein construct, RsaA ∆0–222, was expressed in both a traditional Escherichia coliexpression system and in our lab’s C. crescentus protein expression system.57,140,141 All attemptsto produce protein in E. coli resulted in highly insoluble inclusion bodies. Expression levels ofunmodified RsaA in its native host is notably high.131 After numerous efforts to produce proteinin an appropriate form for crystallization, that is monodisperse, soluble protein, we were forcedto conclude that other approaches were needed. A number of counter-intuitive methods had tobe used to eventually produce a crystallizable form of RsaA from C. crescentus. To begin withthe protein encoding gene had to be genetically altered to produce a truncated form of RsaA,the cells had to be cultured under low agitation, and the resulting secreted protein had to beconcentrated very slowly.We had observed that cells cultured in shaken test tubes produced aggregated protein inthe form of large insoluble floating masses, while cells cultured in rolled test tubes producedsoluble protein.The reasons for an N-terminal truncation of RsaA are two-fold. Deletion of the N-terminusresults in protein that no longer anchors itself to outer membrane, resulting in soluble super-natant protein; and the N-terminus has been identified as the center of three-fold symmetry; itsdeletion should prevent wide-scale S-layer formation and aggregation. This is our strategy forpreventing S-layer formation as discussed in section 2.1.Attempts to crystallize RsaA ∆0–222 started with the pre-assembled crystallization screensJCSG Core I, II, III, and IV. Higher protein concentrations were sought because it is generallybelieved that higher protein concentrations would lead to better results and literature sourcesrecommended protein concentration ranges centering on 10 mgml−1 .142 The RsaA ∆0–222truncate was readily concentrated to 5–7 mgml−1, but when concentrated beyond 7–9 mgml−1aggregation began to occur, either visual macroaggregation or microaggregation assessed byDLS. DLS was used as a qualitative measure of protein quality and guided our protein production57W. H. Bingle, J. F. Nomellini, and J. Smit. Mol Microbiol, 26: 277–288, 1997.140E. Umelo-Njaka et al. Vaccine, 19: 1406 –1415, 2001.141W. H. Bingle and J. Smit. Plasmid, 24: 143 –148, 1990.142J. Jancarik and S.-H. Kim. J Appl Crystal, 24: 409–411, 1991.39A B97.4 kDa66.2 kDa45.0 kDaFigure 2.3: The purification of RsaA ∆0–222 as monitored by SDS-PAGE. A. 7 µl ofculture supernate of our C. crescentus expression strain expressing RsaA ∆0–222.B. Each lane has 15 µl from separate size-exclusion chromatography elutions fromthe major peak containing RsaA ∆0–222. The contents of the tubes represented bythese 4 bands were pooled, dialyzed, concentrated, and crystallized.and purification protocols. Higher concentrations of RsaA ∆0–222 would exhibit lower levelsof monodispersity and larger fractions of high-molecular weight aggregates, both predictivesigns of reduced crystallizability.143 Figure 2.4 shows DLS plots of RsaA ∆0–222 at 9 mgml−1and 3 mgml−1.For RsaA ∆0–222, initial screens were assembled at 9 mgml−1. Small crystalline shapeswere observed after one week at room temperature. Those first crystals grew in JCGS Core I,well 28 with conditions of 20% PEG 8000, 200 mMMgCl2, and 100 mM Tris pH 7.143A. George and W. W. Wilson. Acta Crystallogr D, 50: 361–365, 1994.4001020304010010.01.00Radius (nm) Percentage (%)Radius (nm): MW (kDa):Polydispersity (%):Mass (%):4.2962399.9Radius (nm): MW (kDa):Polydispersity (%):Mass (%):28.1823417.500.101020304010010.01.00Radius (nm) Percentage (%)Radius (nm): MW (kDa):Polydispersity (%):Mass (%):3.14863.310001020304010010.01.00Radius (nm) Percentage (%)Radius (nm): MW (kDa):Polydispersity (%):Mass (%):4.410812.599.9Radius (nm): MW (kDa):Polydispersity (%):Mass (%):39.11793311.100.101020304010010.01.00Radius (nm) Percentage (%)Radius (nm): MW (kDa):Polydispersity (%):Mass (%):9.259832.482.9Radius (nm): MW (kDa):Polydispersity (%):Mass (%):46.82725446.217.1A CDBFigure 2.4: The DLS plots of various constructs and preparations of RsaA. The data areshown as histograms representing the population of particle sizes observed in theprotein solutions. A. RsaA ∆0–222 at 3.5 mgml−1. This protein sample was usedsuccessfully to produce well-diffracting crystals. B. RsaA ∆0–222 at 9 mgml−1. C.RsaA ∆0–551 at 5 mgml−1. D. Full-length wild-type RsaA at 2 mgml−1.41The first attempts to transfer from a sitting-drop strategy, as used in initial screening, to ahanging-drop strategy, to be used in optimization, were unsuccessful due to widespread proteinprecipitation/aggregation. Shifting our divalent cation from Mg2+ to Ca2+ led to small crystals inour drops. Crystals that were grown with Ca2+ were very thin, but sizable (>0.5 mm) in the othertwo dimensions. The biggest improvements came from shifting the cation again to Sr2+ andlowering the concentrations of the protein and precipitants. Due to low protein concentrations,larger drop sizes (6–10 µl) were utilized and larger ratios between protein and mother liquorwere (1.5:1–3:1) used so that initial conditions would not cause protein precipitation but proteinconcentrations would eventually get high enough to be supersaturated. Fine tuning of proteinconcentration and drop ratios were mostly used to optimize nucleations in the drops. RsaA∆0–222 always nucleated well, leading to more drops full of small crystals than clear dropswith no crystals. A protein concentration in the range of 3.5–4.5 mgml−1, mixed in a ratio of1.5:1 with mother liquor, had the best results, averaging around 3 nucleations per drop. Furtheroptimization to the crystallization conditions, such as lowering salt concentration (200 mM to150 mM) and raising the pH (7 to 7.4), were of arguable efficacy.For RsaA ∆0–551, no crystal formation was observed in any crystallization screening con-dition or in any protein concentration used. Once successful crystallization conditions wereobserved for RsaA ∆0–222, those conditions were used with RsaA ∆0–551 but no crystals wereobserved. Figure 2.4 shows the DLS plot of a representative RsaA ∆0–551 protein sample.2.3.2 Properties of the RsaA ∆0–222 crystalsOur first diffracting crystals of RsaA ∆0–222 were ‘plate’ crystals having a rhomboid shape.Figure 2.5 shows examples of these rhomboid crystals. The proportional dimensions of thecrystals roughly match the relative dimensions of the crystal unit cell (see Table 2.1), althoughthe thickness seemed variable. The crystals grew often as clusters or stacks, which did not aidin diffraction. Figure 2.1 shows one example of how many of these crystal clusters appeared.The rhomboid ‘plate’ crystals were very strong, resisting crushing so well that we were initially42concerned that they were salt crystals. The ‘plates’ also resisted X-ray induced damage well—often diffracting acceptably well after two or three full data collections.Later crystallization efforts for RsaA ∆0–222 started to yield crystals that were not rhom-boid, rather they were hexagonal prisms, like pencils. Drops identical to and next to dropsproducing ‘plate’ crystals would often grow ‘pencil’ crystals. A few drops even producedboth ‘plate’ crystals and ‘pencil’ crystals. These ‘pencil’ crystals looked like protein crystalsunder a polarizing filter on the microscope and they were confirmed to be protein by X-raydiffraction. The ‘pencil’ crystals were very fragile and often broke or cracked under gentlemanipulation. The ‘pencils’ grew as solitary crystals often on contact with the lower surface ofthe crystallization drop. Figure 2.6 shows a few examples of the hexagonal prism crystals ofRsaA ∆0–222.Low pH solutions would readily crack and destroy any form of RsaA ∆0–222 crystal. Un-fortunately, this pH limitation precluded us from using some soaking agents like uraniumcompounds and higher concentrations of tantalum bromide. The ‘plate’ crystals were generallymore stable in soaking conditions with high salt concentrations than the ‘pencil’ crystals. The‘pencil’ crystals would quickly crack in high concentrations of KI and KBr.2.3.3 Crystallization of RsaA ∆0–222 GSCC723RsaA ∆0–222 GSCC723 readily crystallized in the successful conditions used for RsaA ∆0–222.However, the crystals that formed had a different morphology. The crystals looked dendritic,like trees or snowflakes. Extensive optimizations were performed to improve the shape and sizeof the crystals but no improved conditions could be found. The final conditions used were 9%PEG, 100 mM tris pH 7.4, and 150 mM SrCl2. The protein concentration was 3.5 mgml−1, 6µl of protein solution was mixed with 3 µl of mother liquor.A few of the dendritic crystal clusters had branches that were clean, individual crystals. Inthose cases, the crystal branches were gently broken off of the main cluster and used for X-raydiffraction.43Figure 2.5: A panel of ‘plate’ crystals of RsaA ∆0–222. The left figure show an individualcrystal with the common rhomboid shape and relatively thin width. The center figureshows a wide-view of a drop containing multiple ‘plate’ crystals. The right figureshows three crystals of RsaA ∆0–222 that were grown in the presence of 5 mMEuNO3. The color in the first two images is imparted by a polarizing filter on themicroscope, the crystals are naturally colourless. The colour of the right figure isunadjusted and representative of the natural colour of the crystals.2.3.4 Properties of the RsaA ∆0–222 GSCC723 crystalsThe only crystals that formed when using the RsaA ∆0–222 GSCC723 protein were ‘dendritic’crystals that resembled trees or snowflakes. Figure 2.7 shows a few examples of these crystals.Most of these crystals were too clustered to be useful for diffraction but a few of the clustershad branches that could be removed and used for diffraction. Figure 2.8 shows a few of thedendritic clusters that were broken apart and diffracted.The RsaA ∆0–222 GSCC723 crystals had similar properties to the RsaA ∆0–222 ‘plate’crystals; the GSCC723 crystals were very strong and stable in most soaking conditions. Unfor-tunately due to the poor diffraction, the GSCC crystals’ potential ability to bind more heavyions for phasing was never realized.44Figure 2.6: A panel of ‘pencil crystals of RsaA ∆0–222. The left figure shows how someof the ‘pencils’ grew in the presence of ‘plate’ crystals, note the ‘plates’ in that panelwere not suitable for X-ray diffraction. The right figure highlights a particularly large‘pencil’ crystal. These crystals were very fragile. The color in the images is impartedby a polarizing filter on the microscope, the crystals are naturally colourless.Figure 2.7: Examples of ‘dendritic’ crystals formed when the RsaA ∆0–222 GSCC723protein construct was crystallized. The color is imparted by a polarizing filter on themicroscope, the crystals are naturally colourless.2.3.5 X-ray diffraction and phasingCrystal variability impeded the determination of the structure of RsaA. The majority of thecrystals of RsaA that were grown had very poor X-ray diffraction. There was not an effectiveway to predict which crystal would diffract well. Our best results came from a large crystal thatwas relatively thin—two features that seemed to be important because small crystals did notdiffract well and overly thick crystals were plagued with smeared, messy reflections. Figure 2.9shows one frame of data from our best dataset. An interesting aspect of the data, specifically45Figure 2.8: A panel of better ‘dendritic’ crystals of RsaA ∆0–222 GSCC723. These areexamples of some of the GSCC723 crystals having branches that were suitable forX-ray diffraction. A few of the side crystals were broken off and used for diffraction.The color in the images is imparted by a polarizing filter on the microscope, thecrystals are naturally colourless. The colour of the right figure is unadjusted andrepresentative of the natural colour of the crystals.from ‘plate’ and ‘dendrite’ crystals, is the anisotopy we observed in our diffraction. Due to theoften poor quality of the data, we would always collect our data through 360◦ which would leadto long collection strategies and increased radiation damage to the crystal. Radiation damagewas never too much of an issue though, as the ‘plate’ crystals were remarkably resistant todamage. Table 2.1 gives an overview of the X-ray diffraction data.Once we had crystals that diffracted to a suitable extent the final step in generating anelectron density map would be phasing. Molecular replacement strategies were not possibledue to a lack of homologous structures in the Protein Data Bank. The RTX motifs in RsaAprovide a tempting region for structural homology, such as the structure in fig. 1.6. Neither thestructures of solved RTX motifs as molecular replacement nor the generated Phyre144 structurefor RsaA’s RTX motifs were effective molecular replacement search models. To try and solve thephase problem we turned to the X-ray fluorescence techniques of multi-wavelength anomalousdispersion (MAD) and single-wavelength anomalous dispersion (SAD).144L. A. Kelley et al. Nature Protocols, 10: 845–858, 2015.46Our main two candidates for MAD/SAD phasing elements were the halides, iodine andbromine.145 These halides were introduced into the crystals by soaking the crystals in KI andKBr. Iodine does not have an accessible K or L edge but provides a SAD suitable anomaloussignal at achievable wavelengths. Refer back to fig. 2.2 for the theoretical profiles of anomaloussignal. Iodide-soaked ‘plate’ crystals, among the first conditions ever tested, diffracted well andan anomalous signal could be detected in their datasets but not enough to allow for densities tobe calculated. A great deal of effort went into trying to improve our initial datasets with more ofan anomalous component from iodide. Very few subsequent iodide-soaked crystals diffractedat all and those that did were of low resolution. Bromine has an accessible Ka edge at 0.9202 Åand so it is useful for MAD. Unlike iodide, bromide-soaked crystals did not have the high rateof crystals with no diffraction but like iodide, few bromide-soaked crystals diffracted beyond 4Å.Many other phasing compounds were tested. The lanthanides all provide a strong anomaloussignal and have been successfully used in the past for SAD.24 lanthanum, lutetium, and europiumwere all used as soaking agents but none produced any detectable anomalous dispersion in theresulting datasets. Europium was also successfully used in the mother-liquor of the crystalsto be co-crystallized. Unfortunately, the co-crystallized crystals had no detectable anomaloussignal, despite good diffraction. Europium’s L1 edge is accessible with synchrotron radiation(see fig. 2.2), and MAD fluorescence scans were done to confirm that there was no detectableEuropium in the crystals. Figure 2.5 features a few of the europium co-crystallized crystals. Inthe vein of co-crystallization, strontium and barium were effective components of the motherliquor, and possibly potent phasing elements, but neither provided an effective solution to thephase problem when tried. The more exotic compounds Tantulum bromide (Ta6Br12) andtriiodide were also tried without resulting in the necessary data.There were only ever a few ‘dendritic’ crystals that were suitable for X-ray diffraction andnone of those few diffracted to an extent or in a manner that was conducive to solving the145Z. Dauter, M. Dauter, and K. Rajashankar. Acta Crystallogr D, 56: 232–237, 2000.47Figure 2.9: This figure shows a frame from a X-ray diffraction data collection. This crystalwas a large ‘plate’ crystal that was our best diffracting crystal to date. The crystalwas soaked in 0.5 M KI for 30 sec. The wavelength of the incident X-ray beamwas set to 1.541 Å (the copper Ka edge). The resolution from this data was 2.50Å. The crystal used here was soaked in an iodide solution. Notice the anisotropy indiffraction, i.e. the spots reach further from the center to the upper right and lowerleft.structure of RsaA ∆0–222 GSCC723. One ‘dendritic’ crystal did diffract reliably to 3.5 Å butexhibited multiple overlapping signals, probably due to separate crystals being fused together.The ‘pencil’ crystals never diffracted to any significant extent, at most to 6–10 Å. Initialprocessing of one of the ‘pencil’ diffraction patterns suggested that its space group was P6. A P648space group has higher symmetry than the P21 space group we observed in our ‘plate’ crystals.We had hopes that the higher symmetry would provide better quality data, unfortunately no wellperforming ‘pencil’ crystals were ever isolated. The only pencil that we did try to perform acomplete data collection on quickly lost diffraction due to radiation damage.2.4 DiscussionOur protein construct, RsaA ∆0–222, was expressed our in C. crescentus protein expressionsystem.57,140,141 All attempts to produce protein in E. coli resulted in highly insoluble inclusionbodies that resisted refolding (data not shown). Expression levels of RsaA in its native host isnotably high131 but the system did not initially produce protein in an appropriate form. Theproblem of aggregation persisted in C. crescentus secreted RsaA; the problem was assuagedby the N-terminal deletion construct, RsaA ∆0–222, but was only completely solved whenwe started to culture the cells without agitation in wide, shallow flasks. Our shallow cultureexpression system proved successful at producing 130–150 mg of purified, concentrated proteinper litre of culture supernate. RsaA is the natural product of C. crescentus but this system couldbe useful in producing exogenous protein for crystallization by fusion to the required type 1section signal.129The reasons for an N-terminal truncation of RsaA are two-fold: (1) deletion of the N-terminus produces protein that no longer anchors itself to outer membrane, resulting in free-secreted supernatant protein and (2) the N-terminus has been identified as the center of three-foldsymmetry.38 Therefore, its deletion results in soluble protein that no longer leads to wide-scaleS-layer formation or aggregation and is freely secreted into the supernate. We can visualizethe success in preventing aggregation and S-layer formation by measure the particle sizesof the RsaA constructs in situ. Figure 2.4 shows how the RsaA ∆0–222 protein behaves insolution compared to full-length RsaA. The latter exhibits a much higher particle size and rateof aggregation. The success of structurally characterizing any S-layer protein impinges upon38F. Amat et al. J Bacteriol, 192: 5855–5865, 2010.49overcoming such issues and our strategies resulted in large well-diffracting crystals.Our ‘plate’ crystals diffracted to 2.5 Å with unit-cell parameters of a=210.79 Å, b=80.74Å, c=221.9 Å, and β=117.46 degrees. These crystal unit-cell sizes almost exactly match the2D unit size of native S-layer repeating unit, a hexamer of RsaA monomers.39 These resultssuggest that the unit-cell of our crystals is six copies of RsaA ∆0–222, corresponding to asolvent content of about 60%. Our hexagonal columnar crystals have never diffracted to asuitable resolution, but they are promising because they have higher symmetry and that mayresult in improved datasets. As our protein has no close matches in the structural databases touse for the purposes of molecular replacement, solving the phase problem was attempted withiodine derivitization with SAD and bromide with MAD. Neither of these approaches gave us therequired anomalous data that we would need for a solution. RsaA does not have many chargedamino acids, even after we installed the GSCC cassette, and that is why the halides have beenso strongly pursued as they do not require specific residues for activity.145 Patterson analysis ofthe dataset suggests issues with pseudotranslational symmetry confounding phasing attempts,with the largest Patterson peak at a height of about 30%. Twinning analyses indicate that notwinning is suspected.As of this writing, the crystallography portion of this work is still ongoing. Only the phasingproblem remains to be solved in this structural determination project; we hope that a solutionexists and that we will find it soon. The fact remains that we have developed techniques tocrystallize the S-layer protein and generally that is the most important step in a crystallizationproject. To conclude this chapter, below is a quote from Alexander McPherson’s 2004 reviewon macromolecular crystallography:146“Presently, and in the foreseeable future, the only technique that can yield atomiclevel structural images of biological macromolecules is X-ray diffraction analysisas applied to single crystals. While other methods may produce important structuraland dynamic data, for the purposes described above, only X-ray crystallography is39J. Smit et al. J Bacteriol, 174: 6527, 1992.146A. McPherson. Methods, 34: 254–265, 2004.50adequate. As its name suggests, application of X-ray crystallography is absolutelydependent on crystals of the macromolecule, and not simply crystals, but crystalsof sufficient size and quality to permit accurate data collection. The quality of thefinal structural image is directly determined by the perfection, size, and physicalproperties of the crystalline specimen, hence the crystal becomes the keystoneelement of the entire process, and the ultimate determinant of its success.”51Table 2.1: A summary of the X-ray diffraction data from a planar crystal of RsaA ∆0–222RsaA ∆0–222Space group P21Cell dimensionsa, b, c (Å) 210.79, 80.74, 221.9β (◦) 117.46Resolution (Å) 50.00–2.50 (2.54–2.50)Rmerge 0.13 (0.54)I / δ I 13.7 (2.8)Completeness (%) 99.8 (97.6)Redundancy 3.852Chapter 3The core and O-polysaccharidestructure of the Caulobactercrescentus lipopolysaccharide“And so, progressively, the veil behind which Nature has so carefully concealed hersecrets is being lifted where the carbohydrates are concerned.”— H. Emil Fischer, 1902 Nobel lectureMy great-great-great-great-great-great-great-grand adviser.3.1 IntroductionCAULOBACTER CRESCENTUS is an aquatic alphaproteobacterium well known for a stalked,crescent cell morphology, asymmetric cell division, and a protein surface layer (S-layer).C. crescentus is a widely studied model organism but despite this, the structure of its lipopolysac-charide (LPS) has not previously been fully determined.Interest in the LPS of C. crescentus is focused on its immunological profile86 and its struc-tural role as an anchor for the self-assembled, para-crystalline S-layer.128 The LPS of C. crescentuspossesses a much reduced immunogenic activity, most likely due to its lipid A structure, whichis significantly different from that of LPS from enteric bacteria. The lipid A structure hasbeen reported.86 It is a unique molecule containing a di-diaminoglucose backbone (instead ofdi-glucosamine) and two galacturonate moieties that replace the canonical phosphates that are86J. Smit et al. Innate Immun, 14: 25–36, 2008.128S. G. Walker et al. J Bacteriol, 176: 6312–6323, 1994.53on each end of the disaccharide in most lipid A molecules. Figure 3.1 shows the structure ofthe C. crescentus lipid A.Core oligosaccharideNNNNCOOH HOOC α-GalAp-5-β-DAGp-6-α-DAGp-1-α-GalAp |3 |2 |3 |2 A A A A 11223 344A : 12:0[3-O[∆5-12:1(3-OH)]]A : 12:0[3-O(∆5-12:1)]A : 12:0(3-OH) A : 12:0[3,6-(OH)] Figure 3.1: The structure of the C. crescentus lipid A.The C. crescentus S-layer non-covalently attaches to the O-specific polysaccharide (OPS).128However, the OPS structure has not been resolved. Genetic analyses have pointed towardsthe unusual dideoxyamino sugar N-acetylperosamine being a major component.147 A notable147P. Awram and J. Smit. Microbiology, 147: 1451–1460, 2001.54feature of this OPS is that it exists completely hidden beneath the S-layer, inaccessible to theenvironment by at least such tools as specific antibodies.128 Carbohydrate structures from non-pathogenic bacterial LPS are rarely studied and an LPS that is sequestered beneath an S-layer isnot represented in the literature.In the present study our data has determined the core oligosaccharide structure from C. crescentusCB15 NA1000 (advancing an earlier report of core composition148), as well as the central back-bone and non-reducing ends of its OPS. Unexpectedly, we identified a previously unknownrhamnan polysaccharide. Along with previous reports on lipid A86 and extracellular polysaccha-ride (EPS),149 we believe that all the major carbohydrate structures in C. crescentus cell envelopehave now been solved.3.2 Methods3.2.1 Bacterial strain construction and growth conditionsThe strain used for the preparation of LPS was JS1025, a derivative of C. crescentus CB15NA1000. The salient features are that it has an engineered amber mutation in rsaA leading tothe loss of the S-layer and the gene CCNA_00471 has been inactivated by a partial deletion.CCNA_00471 encodes a putative GDP-L-fucose synthase.150 The knockout (∆471) confersa deficiency in an EPS that was previously found to contain L-fucose.149 CCNA_00471 wasdisrupted in the same manner as previously in JS4038,60 except the starting strain used here wasJS1023.38Cells were grown to mid-to-late log phase (OD600 = 0.9) in M16HIGG defined mediumat 30◦C in 2.8 l Fernbach flasks containing 1250 ml of medium, with shaking at 100 rpm.148N. Ravenscroft et al. J Bacteriol, 174: 7595–7605, 1992.149N. Ravenscroft et al. J Bacteriol, 173: 5677–5684, 1991.150M. E. Marks et al. J Bacteriol, 192: 3678–3688, 2010.60C. Farr et al. PLoS One, 8: e65965, 2013.38F. Amat et al. J Bacteriol, 192: 5855–5865, 2010.55M16HIGG is a modification of M6HIGG medium,133 containing 0.31% glucose, 0.09% gluta-mate, 1.25 mm sodium phosphate, 3.1 mm imidazole, 0.05% ammonium chloride, and 0.5%modified Hutner’s Mineral Base.1513.2.2 Lipopolysaccharide isolationLPS was isolated from the cells via disrupting the outer membrane by chelation. The protocol weused was a modification of the procedure reported by Walker et al. 128 Cells were centrifuged at12 400 x g for 10 min. The pellets were suspended with distilled water and recentrifuged. Thesepellets were resuspended in 1/10 original culture volume in phosphate-buffered saline (PBS)152amended with 35 mm ethylenediaminetetraacetic acid (EDTA), agitated at room temperaturefor 10 min and then centrifuged at 15 300 x g for 15 min. The supernatant was retrieved andre-centrifuged, as before, to ensure clarity and then dialyzed against 5 mm MgCl2. DNase andRNase were added to final concentrations of 10 µgml−1 and 100 µgml−1, respectively, andincubated at 37◦C for 2 h. Proteinase K was added to a final concentration of 0.3 mgml−1 andthe preparation was incubated at 50◦C overnight. The sample was then ultracentrifuged at 184000 x g for 3 h. Glassy pellets formed which were suspended in distilled water to 1/100 originalculture volume. A Bligh-Dyer extraction was performed to reduce contaminating lipids.1533.2.3 Bligh Dyer extractionA Bligh Dyer extraction was performed on all LPS preparations to reduce the presence ofcontaminating lipids. The extraction was performed as first published.153 In short, to onevolume of aqueous LPS sample, 3.75 volumes of chloroform/methanol (1:2 v/v) was added andthe sample was vortexed for 30 sec. Chloroform, 1.25 volumes, was added and the sample wasvortexed again for 30 sec. Water, 1.25 volumes, were added and the sample was vortexed a final133J. Smit, M. Hermodson, and N. Agabian. J Biol Chem, 256: 3092–3097, 1981.151G. Cohen-Bazire, W. Sistrom, and R. Stanier. J Cell Comp Physiol, 49: 25–68, 1957.152T. Maniatis, E. F. Fritsch, and J. Sambrook. Molecular cloning: a laboratory manual. vol. 545 Cold SpringHarbor, NY, 1982.153E. G. Bligh and W. J. Dyer. Can J Biochem Physiol, 37: 911–917, 1959.56time for 30 sec. This mixture was then centrifuged at 15 300 x g for 10 min. After centrifugation,the mixture separated into two phases, a lower organic phase and an upper aqueous phase. TheLPS partitions into the aqueous phase, while other lipids partition into the organic phase. Theaqueous phase was retrieved. Samples were stored at 4◦C for short term use or frozen at -20◦Cfor long term storage. Occasionally, NaN3 was added to a concentration of 3 mM to preventmicrobial growth and it was never found to effect analysis.3.2.4 Silver stain and gel electrophoresisDiscontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed with a 13% separating gel.154 Detection of LPS was done by periodate oxidationand silver staining as described by Zhu et al. 155 The silver stain protocol is a newer techniquethat uses vitamin C (ascorbic acid) in place of formaldehyde in traditional LPS stains.156 Table3.1 give the details of the silver stain procedure.All samples were boiled for 5 min in samplebuffer prior to loading them to a gel. Sample buffer is made as a 4X concentrate comprising200 mM Tris pH 6.8, 8% sodium dodecyl sulfate, 40% glycerol, 4% β -mercaptoethanol, 0.25%bromophenol blue.3.2.5 Preparation of RsaA ∆277–784 probeThe plasmid, p4BRsaA∆277–784, was in our laboratory from the work of Bingle and Nomellini.In short, various versions of rsaA were engineered to have BamHI sites at locations throughoutthe gene.157 The BamHI–HinDIII fragment from a version of rsaA with a BamHI site at the831 bp from the start codon was replaced with a BamHI–HinDIII fragment from a version ofrsaA with a BamHI site at the 2352 bp position. In effect this made a rsaA gene missing 1521bp from the center, corresponding to amino acids 277–784 within the resulting protein. This154U. K. Laemmli. Nature, 227: 680–685, 1970.155Z.-X. Zhu et al. Electrophoresis, 33: 1220–1223, 2012.156C.-M. Tsai and C. E. Frasch. Anal Biochem, 119: 115–119, 1982.157W. H. Bingle, J. F. Nomellini, and J. Smit. J Bacteriol, 179: 601–611, 1997.57Table 3.1: LPS silver stain procedureStep Solution TimeOxidation 30% EtOH, 10% HOAc, 0.7% periodic acid 10 minRinse dH2O 5 min x2Silver 0.2% AgNO3 5 minRinse dH2O 20 sec x2Develop 3% NaCO3, 0.04% Na2S2O3, 0.02% ascorbic acid, 0.05% NaOH 8 minStop 10% HOAc 1 min‘internally truncated’ gene was then moved into the context of our expression vector ‘p4B’131 asa EcoRI–HinDIII fragment. p4BRsaA∆277–784 was electroporated into C. crescentus JS1030.JS1030 is the same strain used for protein production in chapter 2; its construction is detailedin section 2.2.2. Isolation and concentration of the RsaA ∆277–784 probe followed the sameprotocol used for RsaA ∆0–222, as found in section 2.2.2, but unpurified and unconcentratedcell culture supernate, containing secreted RsaA ∆277–784, was also to be an effective probefor far-western blotting.For the protein probe used in the glycan array screening, the RsaA ∆277–784 was labeledwith fluorescein isothiocyanate (Life Technologies); manufacturer’s instructions were followedfor the labeling procedure.3.2.6 Western blotting and far-western blottingWestern blotting of LPS followed standard laboratory blotting techniques.158 SDS-PAGE gelsof LPS were electro-blotted on to a nitrocellulose membrane that was subsequently agitatedin Tris-buffered milk blocking buffer for one hour. Occasionally, spot-blot immunoblots wereperformed. Spot-blots are like western blots that forgo the SDS-PAGE; the sample is spotted131J. H. Y. Lau, J. F. Nomellini, and J. Smit. Can J Microbiol, 56: 501–514, 2010.158H. Towbin, T. Staehelin, and J. Gordon. PNAS, 76: 4350–4354, 1979.58directly on to the membrane and allowed to dry.The primary probe was rabbit serum raised against a preparation of C. crescentus holdfast.The holdfast is a complex carbohydrate ‘glue’ that is present on the stalk of C. crescentus. Theholdfast acts to anchor the cells to surfaces. This so-called anti-holdfast serum was fortuitouslyfound to have specific activity against the C. crescentus LPS.128 AlexaFluor680 goat anti-rabbitIgG secondary antibody (Invitrogen) was used at 50 000−1 and was detected and quantified bythe Odyssey Infrared Imaging System using Odyssey 2.0 software (Licor Biosciences, Lincoln,NE), similar to previously published methods.131 Two 5 min washes were performed betweeneach step.Far-western blotting was originally developed to screen protein expression libraries with32P-labeled glutathione-S-transferase-fusion proteins,159 but the idea has developed as a quickmethod to test for in vitro protein interactions on the surface of a western blot membrane.The idea to use far-western blotting to check for RsaA–LPS interaction was spurred by therealization that the denaturing conditions in standard SDS-PAGE would have no detrimentaleffect on the primary structure of LPS. Our far-western blot protocol started as a standardwestern blot, except the primary probe was replaced with RsaA∆277–784 in distilled water.Primary probe concentration was not a critical variable in the range tested, 0.1–4 mgml−1.Anti-RsaA∆277–784 rabbit serum was used as a secondary probe, followed by AlexaFluor680goat-anti rabbit IgG as a tertiary probe. Controls checking non-specific binding between theLPS and the secondary probes and tertiary probes showed were performed (data not shown).3.2.7 Matrix assisted laser desorption/ionization-time of flight massspectroscopyMALDI-TOF analysis was done with the UBC Proteomics Core Facility. For our initial anal-ysis we used LPS purified from SDS-PAGE gels, but the resulting acrylamide/polyacrylamidecontamination proved insurmountable (data not shown).159E. M. Blackwood and R. N. Eisenman. Science, 251: 1211–1217, 1991.59The MALDI-TOF procedure used our purified LPS mixed with a sinapinic acid matrix andanalyzed on a Applied Biosystems Voyager System 4311. Table 3.2 highlights the machinesetting used for the MALDI-TOF.Table 3.2: The settings of the Applied Biosystems Voyager 4311 that were used to doMALDI-TOF analysis of the C. crescentus LPSMode of operation: LinearExtraction mode: DelayedPolarity: NegativeAccelerating Voltage: 25 kVExtraction Delay Time: 670 nsecNumber of Laser Shots: 100 per spectrumLaser Intensity: 2933Laser Rate: 20 HzCalibration Matrix: Sinapinic AcidLow Mass Gate: 2000 Da3.2.8 Periodic acid-Schiff stainPeriodic acid-Schiff stain was performed on LPS samples that were run on SDS-PAGE.The staining procedure starts by soaking the gel on a slow shaker in 0.7% periodic acid indistilled water for 15 min. The gel is then rinsed in distilled water and placed in Schiff reagentand shaken slowly for 15 min. The gel is then washed in distilled water for 5 min, at whichtime pink bands should appear that are corresponding to polysaccharides/LPS in the gel.The protocol to prepare Schiff reagent started by dissolving 5 g of basic fuchsin in 900 ml ofboiling distilled water. Once dissolved the fuchsin solution was removed from heat and allowedto cool for a few minutes, then 100 ml of 1 M HCl was slowly added. After the solution wasallowed to cool to room temperature, 10 g of K2S2O5 was added. This solution was thoroughly60mixed, then incubated in the fume hood overnight. The next day, a quantity of activated charcoalwere added to the Schiff reagent to decolourize the solution. The solution was stirred and thenfiltered through Watman paper # 1. The now clear Schiff stain solution was ready for use. Atest for Schiff reagent activity is to add a few drops of Schiff reagent to 5 ml of formaldehydein a test tube. The mixture should instantly turn a bright pink colour, indicating activity.3.2.9 NMR spectroscopyNuclear magnetic resonance spectroscopy (NMR) experiments were carried out on a VarianINOVA 600 MHz (1H) spectrometer with 5 mm gradient probe at 25–50◦C with acetone internalreference (2.225 ppm for 1H and 31.45 ppm for 13C), using standard pulse sequences gradientcorrelation spectroscopy (gCOSY), total correlation spectroscopy (TCOSY)(mixing time 120ms), rotating frame nuclear Overhauser effect spectroscopy (ROESY) (mixing time 300 ms),gradient heteronuclear singe quantum coherence (gHSQC), and gradient heteronuclear multiplebond coherence (gHMBC)(100 ms long range transfer delay), heteronuclear multiple-quantumcorrelation spectroscopy (HMQC) for 1H-31P correlation, JHX set to 10 Hz. Acquisition time waskept at 0.8–1 sec for H-H correlations and 0.25 sec for heteronuclear single quantum coherence(HSQC). 256 increments were acquired for t1 in all 2D spectra, except 512 for gCOSY.3.2.10 LPS chromatographyGel chromatography was performed on a Sephadex G-15 column (1.5 x 60 cm) or a Bio-gelP6 column (2.5 x 60 cm) in pyridine-acetic acid buffer (4 ml:10 ml:1 l water), and monitoredby refractive index detector (Gilson). Anion exchange chromatography was done on an HitrapQ column (2x5 ml size, Amersham), with ultraviolet Light (UV) monitoring at 220 nm in alinear gradient of NaCl (0–1 M, 1 h) at the 3 mlmin−1. Fractions of 1 min were collectedand additionally tested for carbohydrates, by spotting on an SiO2 thin-layer chromatography(TLC) plate, dipping them in 5% H2SO4 in ethanol and heating with a heat-gun. All fractions of61interest were dried in a Savant drying centrifuge and 1H spectra were recorded for each fractionwithout desalting. For 2D NMR, desalting was performed on a Sephadex G15 column.3.2.11 Monosaccharide analysisSamples with added inositol standard were hydrolysed with 3 M trifluoroacetic acid (TFA) at120◦C. Monosaccharides were converted to alditol acetates by conventional methods and iden-tified by gas chromatography-mass spectroscopy (GC-MS) on a Varian Saturn 2000 instrumenton a DB17 capillary column (30 m x 0.25 mm ID x 0.25 µm film) with helium carrier gas, usinga temperature gradient 170◦C (3 min)–250◦C at 5◦Cmin−1.3.2.12 Determination of absolute configurations of monosaccharidesTo the polysaccharide sample (0.2 mg) (R)-2-BuOH (0.2 ml) and acetyl chloride (0.02 ml) wereadded at room temperature, heated at 90◦C for 2 h, dried by air stream, acetylated, analysedby GC-MS as described above. Standards were prepared from monosaccharides of knownconfiguration with (R)- and (S)-2-BuOH.3.2.13 Methylation analysisFor the methylation analysis core sample (2 mg) was dephosphorylated with 50 µl of 48% HFfor 20 h at +10◦C, diluted with 2 ml of ethanol, the precipitate collected by centrifugation,washed with 2 ml of ethanol, and dried.Methylation was performed by Ciucanu-Kerek procedure.160 Half a milligram of the samplewas dissolved in 0.5 ml of dry DMSO with heating at 100◦C for 5-10 min until completedissolution. Powdered NaOH (about 50 mg) was added and the mixture was stirred for 30 min.0.2 ml of MeI was added and the mixture was stirred for a subsequent 30 min. The samplewas then flushed with air to remove the MeI and diluted to 10 ml with water. The sample was160I. Ciucanu and F. Kerek. Carbohyd Res, 131: 209–217, 1984.62passed through a C18 Seppak cartridge, washed with 10 ml of water, and then the methylatedcompound was eluted with 5 ml of methanol. The methylated product was hydrolysed with 3M TFA (120◦C, 3h), dried, reduced with NaBD4, and the reagent destroyed with 0.5 ml of 4 MHCl. The solution was dried under a stream of air and dried twice more with the addition ofMeOH (1 ml). The sample was acetylated with 0.4 ml Ac2O and 0.4 ml pyridine for 30 min at100◦C. It was then dried and analysed by GC-MS.3.2.14 Periodate oxidationPolysaccharide (PS) (10 mg) was dissolved in water (2 ml). NaIO4 (20 mg) was added and thesolution was incubated at room temperature for 24 h. Ethylene glycol (0.2 ml) and an excessNaBD4 were added. The solution was then kept for 1 h before being treated with 0.2 ml ofAcOH and desalted on a Sephadex G-15 column. The product was hydrolysed with 2% AcOH,2 h at 100◦C, and separated on a Sephadex G-50 column to give oligosaccharide (OS)1.3.2.15 Cell cultureThe HEK-Blue htoll-like receptor 4 (TLR4)TM (InVivoGen) cells were grown in Dulbecco’smodified Eagle’s medium (DMEM) (Gibco) with 10% heat-inactivated fetal bovine serum (FBS)(Thermo Scientific), 2 mM GlutaMAX, 25 mM HEPES buffer, and 50 U ml−1 penicillin and50 µgml−1 streptomycin (GIBCO, Invitrogen). The THP-1 cells were grown in Roswell ParkMemorial Institute medium (RPMI) 1640 (Gibco) with 10% heat-inactivated FBS (ThermoScientific). Cells were incubated at 37◦C in humid air with 5% CO2. The FBS was heatinactivated by incubating it at 56◦C for 30 minutes.3.2.16 HEK-Blue hTLR4 assayTo assay the stimulatory effect C. crescentus LPS has via TLR4, we used a cell line that ex-presses a secreted phosphatase reporter under the regulation of nuclear factor kappa-light-chain-63enhancer of activated B cells (NF-κB). The NF-κB assay was done in collaboration with GylesIfill from the Fernandez lab at UBC. HEK-Blue hTLR4TM were cultured and maintained accord-ing to the supplier’s instructions. Cells were grown in standard cell culture flasks then seededinto 96-well plates with 5x104 cells per well. When the cells were established in the 96-wellplates, their growth medium were replaced with fresh media containing LPS at concentrations of0, 1, 10, and 100 µgml−1. After 24 hr the supernatant medium from all the wells were assayedfor phosphatase activity. All experimental conditions were mirrored with cells not expressingTLR4, Null2 cellsTM. Fernandez lab at UBC. HEK-Blue hTLR4TM cells were cultured andmaintained according to the supplier’s instructions. Cells were grown in standard cell cultureflasks then seeded into 96-well plates with 5x104 cells per well. When the cells were establishedin the 96-well plates, their growth medium were replaced with fresh media containing LPS atconcentrations of 0, 1, 10, and 100 µgml−1. After 24 hr the supernatant medium from all thewells were assayed for phosphatase activity. All experimental conditions were mirrored withcells not expressing TLR4, Null2 cellsTM.3.2.17 THP-1 derived macrophage assayTo measure the cytokine stimulating ability of C. crescentus LPS, we treated macrophage cells,derived from a THP-1 cell line, with LPS from C. crescentus or Salmonella enterica var. Ty-phimurium.The C. crescentus LPS was isolated from C. crescentus JS1013, a derivative of NA1000.The LPS isolation protocol can be found in section 3.2.2. The S. enterica LPS was S. entericaserotype minnesota Re 595 and it was purchased from Sigma-Aldrich.The initial starting cultures of THP-1 cells were generous gifts from the Fernandez lab andthe Finlay lab. For cell culture, we followed previously published protocols.161 We maintainedtwo separate cell lines from the start from the two sources to ensure neither was non-viableor contaminated; both were found to have identical growth rates and both were clean of con-161N. Marr et al. Infection and Immun, 78: 2060–2069, 2010.64tamination. Upon the recommendation of Dr. Joris Van Der Heiden, the initial passage of thecells was in medium containing 20 µgml−1 of kanamycin. The cells with kanamycin seemedto achieve maximal growth rate earlier than cells that did not have any antibiotic added. TheFernandez lab cell line was expanded and used for all subsequent assays. No antibiotics wereadded the medium other than during that first initial passage.When the THP-1 cells were in their third passage and at a density of 1.4x106 live cells ml−1they were pelleted by centrifugation and resuspended in fresh medium containing 50 ngml−1phorbol 12-myristate 13-acetate (PMA). One hundred thousand THP-1 cells, in PMA, wereplaced into the wells of 24-well plates and allowed to differentiate. After 48 hr, all the cellswere found to have become adherent to the well surfaces, the PMA-containing medium wasremoved, the cells were washed once with fresh medium, and then fresh medium was appliedto all the wells and the cells allowed to rest. After a subsequent 48 hr, all the growth mediumwas replaced with medium containing LPS at concentrations of 0, 1, 10, 100 ngml−1 of eitherC. crescentus or S. enterica origin. At the time points of 4 hr and 24 hr, culture supernateswere removed and frozen at -80◦C for cytokine assay. Biological replicates were done inquadruplicate. When the THP-1 cells were in their third passage and at a density of 1.4x106live cell per millilitre they were pelleted by centrifugation and resuspended in fresh mediumcontaining 50 ngml−1 PMA. 1x105 THP-1 cells, in PMA, were placed into the wells of 24-wellplates and allowed to differentiate. After 48 hr, all the cells were found to have become adherentto the well surfaces, the PMA-containing medium was removed, the cells were washed oncewith fresh medium, and then fresh medium was applied to all the wells and the cells allowedto rest. After a subsequent 48 hr, all the growth medium was replaced with medium containingLPS and concentrations of 0, 1, 10, 100 ngml−1 of either C. crescentus or S. enterica origin. Atthe time points of 4 hr and 24 hr, well solutions were removed and frozen at -80◦C for cytokineassay. Biological replicates were done in quadruplicate.Kits for assaying various cytokines were purchased from Meso Scale Discovery: tumornecrosis factor α (TNF-α), a proinflammatory cytokine released by macrophages in response65to LPS162; interleukin (IL) 6, an important cytokine for generating adaptive immunity163; IL10, an anti inflammatory signal,“Master Regulator of Immunity to Infection”164; and monocytechemotactic protein 1 (MCP-1), a key chemokine for macrophage trafficking.165 The assays weredone following the supplier’s instructions and the data readouts were produced on a QuickPlexSQ 120 imager (courtesy of the Timothy Kieffer’s lab, UBC).3.2.18 Statistical AnalysisStatistical analyses were done with Microsoft Excel. An unpaired t-test was used to comparethe means between treatment groups at all time points or dosage levels. A P value of <0.05 wastaken as a statistically significant difference.3.3 Results3.3.1 Characterization of whole LPSThe protocol we developed for isolating the LPS from C. crescentus was unconventional com-pared to the majority of existing literature. Our EDTA chelation method was found to be a greatfit for our needs and effective at producing LPS. The negative charges in Gram-negative bac-terial LPS are stabilized and bridged by available divalent cations (i.e. Ca2+ and Mg2+). Lievefirst noted in 196577 that one can strip the counter ions from the outer membrane to disrupt itand cause it to shed into the medium. We chose a chelation strategy because we found it easyto scale up, it did not require noxious organic solvents, and because our lab’s first attempt toisolate and analyze C. crescentus LPS148 using the Darveau extraction method166 only resultedin the isolation of core+lipidA (rough LPS).162H. Wajant, K. Pfizenmaier, and P. Scheurich. Cell Death & Diff , 10: 45–65, 2003.163J. Scheller et al. Biochem et Biophys Acta, 1813: 878–888, 2011.164K. N. Couper, D. G. Blount, and E. M. Riley. J Immuno, 180: 5771–5777, 2008.165S. L. Deshmane et al. J Interferon Cytokine Res, 29: 313–326, 2009.77L. Leive. Biochem Biophys Res Commun, 21: 290–296, 1965.166R. P. Darveau and R. E. Hancock. J Bacteriol, 155: 831–838, 1983.66Whole C. crescentus LPS was examined using a number of different techniques. Elec-trophoresis and staining was used as routine checks for sample quality. Multiple methodsof staining were used to visualize the LPS. The traditional silver stain method of Tsai andFrasch156 is very susceptible to errors and it is not convenient for day-to-day sample checks,so the periodic acid-Schiff stain was co opted from common histology protocols to visualizeour carbohydrates. The Schiff reagent based stain was a very quick and reliable method thatwas used until the improved silver stain technique of Zhu et al. was published in 2012.155 Zhuet al.’s protocol was superior to the traditional silver stain methods in our hands in both interms of reliability and difficulty. Despite their established use, LPS silver stains may not beacting on the PS portion of the molecule as often thought,167 but none the less silver stainswere the primary tool used for checking sample quality and accuracy. Zinc-imidazole stain168was marginally successful at visualizing our LPS (data not shown) but the results were weakand poorly recordable. Zinc-imidazole stains were only used during attempts to purify LPS byelectrophoresis. Figure 3.2 demonstrates how the LPS appears on an SDS-PAGE.C. crescentus LPS runs as two separate bands; a high molecular weight smooth-LPS bandand a low molecular weight rough-LPS (see fig. 3.2 E.). The C. crescentus smooth-LPS bandruns as a single band, which is in contrast to the canonical laddering pattern of most other wellstudied smooth-LPS. The laddering pattern of LPS is from the heterogeneity in length that ariseswhen OPS subunits are polymerized. Figure 3.3 shows C. crescentus LPS next to the LPS fromEscherichia coli (E. coli) O122.Figure 3.2 also demonstrates how our LPS samples appeared when analyzed by westernblotting and far-western blotting using RsaA as the primary probe. Both western blotting andfar-western blotting primarily identified the smooth-LPS band on the blot, indicating that theOPS is probably the antigenic portion of the LPS and the primary anchor molecule for RsaA andthe C. crescentus S-layer. For the far-westerns, controls were run to test for LPS cross reactivityfrom the anti-RsaA antibodies and the anti-rabbit antibodies. None were detected.167A. M. Kropinski, D. Berry, and E. P. Greenberg. Current Microb, 13: 29–31, 1986.168E. Hardy et al. Anal Biochem, 244: 28–32, 1997.671309555433426(kDa)A B C D E9555724334261710(kDa)Figure 3.2: Four different visualisations of C. crescentus LPS. A. Western blot with rabbitanti-OPS serum used as the primary probe. B. Far-western blot with RsaA ∆277–784 used as the primary probe and rabbit anti-RsaA serum used as the secondaryprobe. C. Schiff stained SDS-PAGE. D. Silver stained SDS-PAGE. The smooth-LPS(i.e. containing a complete OPS) runs as a single band at roughly the equivalent rateof a 50 kDa protein and is visible in all lanes. The fainter band visible at 30 kDa inthe western blot (A) is of unknown source. The faint 30 kDa band is not seen in byfar western (B), Schiff stain (C), or silver stain (D). E. A differently resolved silverstained SDS-PAGE. The high molecular weight smooth-LPS is present at 50 kDaand less well resolved band at around 10 kDa is present which is the low molecularweight rough-LPS.We attempted to isolate the core oligosaccharide-OPS fraction from C. crescentus LPS priorto pursuing NMR analysis. Hydrolysis of the ketosidic bond between Kdo and lipid A (seefig. 1.8) was carried out with one of the classical protocols, 0.1 M HCl at 100◦C for one hour.The reaction caused the expected cleavage and precipitation of lipid A, but the supernate had nodetectable OPS. We used standard silver stain SDS-PAGE, western blot, spot-blot immunoblot,and far-western blot methods to detect the presence of free-OPS. None of the methods gave apositive result. Milder hydrolysis conditions, acetic acid or SDS accelerated,169 also producedno detectable free-OPS. Addressing the concern that OPS might not bind to nitrocellulosemembrane, we tried to chemically link the polysaccharide to amine-coated surfaces using169M. Caroff, A. Tacken, and L. Szabó. Carbohyd Res, 175: 273–282, 1988.68Figure 3.3: Comparison of silver stained E. coli O122 LPS and C. crescentus LPS. LPSsamples were run on a SDS-PAGE and silver stained. Lanes 1 and 3 contain equalamounts of LPS from E. coli O122. Lanes 2 and 4 contain our isolated LPS fromC. crescentus. Lane 4 has twice the loaded amount compared to lane 2 to demonstratethat there are no minor, hidden bands. Notice the canonical ‘laddering’ pattern ofE. coli LPS and the distinctly singular band of C. crescentus LPS.carbodiimide chemistry170; this method also failed to immobilize any detectable free-OPS.We had no evidence from the literature to indicate that the OPS could be destroyed bythe mild hydrolysis conditions. At the time the only clue about the structure of the OPS wasthe presence of N-acetylperosamine; perosamine containing OPSs have no known inherentinstability.171,172 We were left to conclude that either the carbohydrates were in fact beingdegraded during hydrolysis or that the carbohydrates we were interested in were unable tomigrate in gel electrophoresis, unable to bind to nitrocellulose, and unsuitable for chemicallinkage. It was this curious situation, where procedures that should be routine consistently gavenegative results, that led us to pursue a collaboration with Dr. Evgeny Vinogradov enabling abroader and hopefully more comprehensive analysis of the core and OPS regions of the LPS byNMR.Attempts to accurately measure the molecular weight of our intact smooth-LPS were partlysuccessful. Getting accurate MALDI-TOF results for large molecular weight polysaccharides isat best difficult and the technology is most often used for the analysis of smaller oligosaccharides(as below) and lipid A molecules. Figure 3.4 shows our best results. Our mass spectrum showsa tight clustering of m/z peaks at or near 10.8 kDa (assuming a single negative charge). Likeour electrophoresis data, our MALDI-TOF data demonstrate that C. crescentus LPS is essentiallyhomogenous in length. The small differences in m/z between the peaks in fig. 3.4 are around170E. S. Jauho et al. J Immuno Meth, 242: 133–143, 2000.171M Caroff et al. Infection and Immun, 46: 384–388, 1984.172M. B. Perry, L. MacLean, and D. W. Griffith. Biochem Cell Biol, 64: 21–28, 1986.69180 Da, the size range of a single hexose molecule, not an entire OPS subunit.2.008068.991144.995120.9991Mass (m/z)0102030405060708090100% Intensity2493.852136.263137.605105.8610834.902.993218.231114.66890.0068Mass (m/z)0102030405060708090100% Intensity10834.9011013.3610648.80Figure 3.4: MALDI-TOF analysis of intact, whole LPS from C. crescentus. The inserthighlights the peaks attributed to LPS. The major LPS peak is labelled 10834.90 m/z3.3.2 Identification of similar glycan structures by RsaA bindingThe successful demonstration that RsaA ∆277–784 could bind C. crescentus OPS in vitro, led usto hypothesize that we could probe in vitro libraries of carbohydrates to identify the structure ofour OPS. A service available at Consortium of Functional Glycomics (CFG) Core H at Emory70University is glycan array screening.173,174 Fluorescently labeled lectins can be screened overan microarray of 465 known glycan structures commonly found in mammals. The array can beused to identify targets of known carbohydrate-binding proteins, such as RsaA. We hoped thatRsaA may bind a glycan on the array and that the structure of that carbohydrate may informus on the structure of the OPS from C. crescentus. Due to our initial component analysis, knewthat our OPS differed significantly from any of the glycans in the array, at least compositionally(see below). RsaA (specifically RsaA ∆277–784) was found to not measurably bind any of theglycan structures found on the array.3.3.3 Initial assessment and component analysisThe PS was released from the LPS by hydrolysis with acetic acid. 1H NMR spectrum of the PS(see fig. 3.5) contained a large number of partially overlapping signals of various intensities inthe anomeric region. It was obviously not a regular polymer with well-defined repeating units. Itwas nevertheless clear that an intact polymer was released by hydrolysis. The free-OPS was de-tectable by NMR but not by the other methods that typically do allow monitoring of purification(see above, section 3.3.1). Attempts to separate this material by anion-exchange chromatogra-phy led to the isolation of a number of fractions from neutral to slightly retained, but all of themhad virtually identical NMR spectra. Methylation of the polysaccharide led to the identificationof 3- and 3,4-substituted mannopyranose, terminal glucopyranose (derived from side-chain 3-O-MeGlc), terminal, 3-, 4-, and 2,4-substituted rhamnopyranose, 3-substituted PerNAc, andan unidentified derivative resembling methylated PerN that eluted between dimethylhexosederivatives and 3-substituted PerNAc. To identify the position of the methyl groups in natu-rally methylated monosaccharides, methylation was conducted with CD3I. This confirmed theidentification of tetramethylglucitol as originating from 3-O-MeGlc, but did not identify anyother naturally methylated monosaccharides, visible in NMR spectra. An unknown derivative173O. Blixt et al. PNAS, 101: 17033–17038, 2004.174R. A. Alvarez and O. Blixt. Meth Enzymol, 415: 292–310, 2006.71received two deuterated methyl groups.3.3.4 O-specific polysaccharide structure determination (PS1)A set of 2D spectra [gCOSY, TCOSY, nuclear Overhauser enhancement spectroscopy (NOESY),1H-13C gHSQC, gHMBC] was obtained for the PS. There were many (more than 20) lines ofcorrelations from the anomeric signals. Later, after the analysis of PS degradation products,most of them could be assigned to particular structures (figs. 3.7, 3.9, and 3.10). Polysaccharideheterogeneity was not caused by random acetylation, as was first hypothesized. Monosaccharideanalysis revealed L-Rha, D-Man, D-PerN (perosamine, 4-aminodeoxyrhamnose), and 3-O-MeGlc. Other methylated monosaccharides were not identified by GC-MS as alditol acetates,possibly due to low content or degradation during hydrolysis. The GC-MS data is presented infig. 3.6.In an attempt to simplify the structure, PS was oxidized with NaIO4, reduced with NaBD4,hydrolysed with 2% AcOH, and the products were separated on a Biogel P6 column to givea polymer and an oligosaccharide, OS1. Analysis of OS1 will be described below. For somereason not all of the rhamnan was oxidized, and some of its signals persisted in the spectra ofthe remaining polymer (without side-chain Rha F). To remove the rest of it, the oxidation wasrepeated to produce PS1. Spectra still contained some signals of minor components that wereanalyzed later. Assignment of the spectra of the non-oxidizable polymer PS1 was difficult dueto complete or partial overlap of the H-2,3,4,5 signals of PerNAc. To improve signal spread, PS1was deacylated with 4 M NaOH. At this point the major polymer became positively chargedand an attempt was made to separate it from the minor components using cation-exchangechromatography. However, all material was eluted together at high salt concentration, thusindicating that all components were chemically bound together. Assignment of the spectra(fig. 3.8, table 3.3) became possible at this stage due to better signal spread (H-4 signals ofPerN moved to high field due to deacylation) and the sequence shown on fig. 3.7 was proposed.Spectra contained the signals of two β -mannopyranose, α-3-O-MeGlc, and two -Per4N. The721.01.52.02.53.03.54.04.55.05.5ppmPolysaccharidePolysaccharide oxidizedPolysaccharide oxidized deacylatedAcetoneOMe3 minor OMeFigure 3.5: 1H NMR spectra of the intact C. crescentus OPS (bottom trace), double oxi-dized polysaccharide (middle trace) and N-deacylated double oxidized polysaccha-ride (upper trace).730.025.710.515.210.015.7 Minutes8.818 min13.543 min13.777 min17.756 min19.016 min7.879 min12.078 min12.297 min13.362 minRhaPerN3-O-methyl-GlcManGlcGal DD-HepLD-HepFigure 3.6: Monosaccharide analysis of C. crescentus LPS. Alditol acetates derivatives ofthe LPS component monosaccharides were separated and identified by GC-MS. Theprotocol used is described on page 62.following interresidual nuclear Overhauser enhancement (NOE) and heteronuclear multiplebond coherence (HMBC) correlations were used to determine the sequence: R1:L3, L1:Z3,Z1:Q3, Q1:W3, W1:X3, A1:X4. PS1 had trisaccharide repeating units composed of β -mannoseand two α-PerNAc residues, and every second repeating unit carried a side branch of 3-O-MeGlc. It seems that side-chains were present quite regularly at each second trisacchariderepeat of the main chain, because NOE correlations were observed between the repeating unitswith and without 3-O-MeGlc, and not between units of the same structure. Thus altogether, therepeating unit contained seven monosaccharides.74AB2222-3-α-PerNAcp-3-α-PerNAcp-3-β-Manp-3-α-PerNAcp-3-α-PerNAcp-3-β-Manp- |4 α-Glc3Mep-3-α-PerNp-3-α-PerNp-3-β-Manp-3-α-PerNp-3-α-PerNp-3-β-Manp- |4 α-Glc3MepR L Z Q W XR L Z Q W XAAFigure 3.7: The structure of the C. crescentus OPS. A. The intact repeating unit, PS1. B.The deacylated product of PS1.754.4 4.2 4.0 3.8 3.6 3.4ω2 - 1H (ppm)5.45.25.04.8ω1-1H (ppm)ABJDERQL WW2 P2 P WXZB1-MeJ1-MeFigure 3.8: Overlap of COSY (green), TCOSY (red) and ROESY (black) correlations fromanomeric protons of double oxidized deacylated C. crescentus PS1.3.3.5 Minor component determinationPS and PS1 spectra contained signals of minor components, which could not be removed bychromatography, as described above. They probably represented the non-reducing ends of themajor chain, PS1 (fig. 3.9). The minor components contained methylated Rha (2-O-Me-Rharesidue J and 2,3-Me2-PerN residue B). The position of the methyl groups were found fromHMBC correlations between protons of methyl groups and carbon atoms bearing OMe groups,which all were well visible and did not overlap with other signals due to their low field position.Thus, two independent structural fragments, 1 and 2, were found and are shown in fig. 3.9.Mannose residues Z’ and Z" at the non-reducing ends of these fragments were further linked toPerN residues, indistinguishable from the PerN of the main chain. PerN residue D had upfieldshifted C-2 and downfield shifted C-3 signals (table 3.4)), which have not been explained. Itappears that its O-3 was phosphorylated, producing typical phosphorylation signal shifts and76Table 3.3: NMR data for C. crescentus PS1 (40◦C) and deacylated PS1 (50◦C). Me at3.62/61.3 ppm.PS11 2 3 4 5 6PerNAc RH 4.97 3.98 4.13 3.92 3.91 1.21C 103.3 68.2 75.5 52.4 69.4 18.0PerNAc QH 4.98 3.97 4.11 3.92 3.91 1.21C 103.3 68.2 75.5 52.4 69.4 18.0PerNAc LH 5.05 4.12 3.99 3.98 3.98 1.21C 103.3 70.4 78.2 53.0 69.4 18.0PerNAc WH 5.02 4.12 3.96 3.98 3.98 1.21C 104.0 70.4 77.8 53.0 69.4 18.0β -Man XH 4.74 4.09 3.98 3.90 3.54 3.80; 3.96C 97.8 72.1 85.4 72.1 75.8 62.6β -Man ZH 4.72 4.06 3.70 3.68 3.41 3.77; 3.96C 98.2 71.9 82.3 67.2 77.3 62.4Glc3Me AH 5.36 3.51 3.40 3.44 3.68 3.75; 3.85C 100.0 72.2 84.1 70.1 74.1 61.6Deacylated PS1PerN RH 5.13 4.34 4.29 3.30 4.18 1.38C 103.5 67.4 75.0 53.4 67.8 18.0PerN QH 5.11 4.34 4.29 3.30 4.18 1.38C 103.5 67.4 75.0 53.4 67.8 18.0PerN LH 5.08 4.22 4.06 3.11 4.09 1.35C 103.9 69.9 79.9 53.6 69.5 18.0PerN WH 5.06 4.22 4.06 3.11 4.09 1.35C 103.9 69.9 79.9 53.6 69.5 18.0β -Man XH 4.91 4.18 4.03 3.96 3.59 3.83; 3.96C 98.0 71.7 85.0 71.7 75.9 62.1β -Man ZH 4.90 4.18 3.75 3.75 3.46 3.83; 3.96C 98.0 71.7 82.0 67.0 77.4 62.1Glc3Me AH 5.39 3.57 3.40 3.48 3.68 3.76; 3.86C 100.2 72.2 84.0 70.0 74.1 61.777broadening of the H-3 signal, but 1H-31P HMQC NMR spectrum showed no signals, possibly dueto the low abundance of this residue. Possibly Rha residues inserted in the structure resemblingPS1 represented the attachment point of the rhamnan (PS2) to PS1, if they were linked together.α-PerNAc2,3Mep-3-β-Manp-PSα-PerNAcp-3-α-Manp-3-α-Manp-3-α-Rha2Mep-3-β-Rhap-3-β-Manp-PSABD E P J U Z”B Z’Figure 3.9: The structure of minor component, the end caps of the OPS. A. Fragment 1.B. Fragment 2.3.3.6 Rhamnan polysaccharide determination (PS2)Periodate oxidation of the PS produced an OS1, which was analyzed by NMR and its structure,as shown on fig. 3.10, was determined using standard 2D NMR methods. Signal assignmentis shown in the table 3.5. It contained three rhamnopyranose units and 4-deoxy-1-deutero-erythritol, produced by the oxidation-reduction of 4-substituted rhamnose. Formation of thisoligosaccharide could be explained by oxidation of the side chain Rha F and 4-substituted Rha Gin the PS1 (letter labels for monosaccharides were given using anomeric signals in the whole PSspectra starting from low-field). The unoxidized 4-substituted residue T in the oligosaccharideoriginally carried side-chain Rha F at position 2. Knowing the OS1 structure the signals of acorresponding polymer (PS2) were identified in the spectra of the whole PS and are given in the78Table 3.4: NMR data for the minor components of the double oxidized non-deacylatedPS (50◦C). Methyl group signals: B2: 3.48/59.5; B3: 3.42/57.9; J2: 3.45/59.6 ppm(H/C)1 2 3 4 5 6PerN2,3Me BH 5.25 3.96 3.71 3.81 3.91 1.17C 100.2 76.2 78.2 53.2 69.5 18.0α-Rha2Me JH 5.25 3.95 4.02 3.45 3.90 1.30C 100.2 75.9 75.4 71.8 69.5 18.0α-PerN DH 5.14 4.27 4.20 3.95 3.97 1.23C 100.1 67.4 75.2 52.2 69.4 18.0α-Man EH 5.13 4.17 3.99 3.75 3.84C 100.1 70.9 79.8 67.1 74.4α-Man PH 5.05 4.14 4.00 3.85 3.96C 97.6 71.0 79.5 66.9 74.0β -Rha UH 4.78 4.16 3.69 3.48 3.44 1.32C 100.9 71.8 82.0 72.4 73.1 18.0β -Man Z"H 4.72 4.06 3.75 3.75 3.46 3.83; 3.96C 98.2 71.9 86.0 67.0 77.4 62.1β -Man Z’H 4.72 4.06 3.73 3.75 3.46 3.83; 3.96C 98.2 71.9 82.3 67.0 77.4 62.1table 3.5.3.3.7 Core oligosaccharide determinationThe core oligosaccharide of the C. crescentus LPS isolated after AcOH hydrolysis containedone non-degraded Kdo, two LD-Hep, one DD-Hep, mannose, galactose, and glucuronic acid inpyranose form. 2D NMR analysis led to the structure shown on fig. 3.11 (NMR assignments are79ABα-Rhap-3-β-Rhap-4-β-Rhap-2-(4-deoxy)-Ery-ol1d-3-α-Rhap-3-β-Rhap-4-β-Rhap-4-α-Rhap- |2 α-RhapN V T GFN V T GFigure 3.10: The structure of the C. crescentus rhamnan. A. the intact rhamnan, PS2. B.the oxidized rhamnan product, OS1.in table 3.6, the HSQC spectrum is in fig. 3.12). The sequence followed from the observed NOE:E1:C5,C7,F5; F1:E2; G1:F3; H1:C7,E2; K1:C4; L1:K4. Correlation E1:C7 is always observedin the α-Hep-5-Kdo fragment. E1:F5 was due to the α-Man-2-Hep linkage. H1E2 indicatesspatial proximity of the residues E and H, linked to the same Kdo C. All expected transglycosidecorrelations were observed in HMBC spectrum, together with intra-ring correlations H-1:C-3and H-1:C-5 for all α-pyranoses. Methylation analysis revealed terminal DD-Hep, terminaland 2-substituted LD-Hep, 3-substituted Man and terminal Gal. The structure agreed withmass spectral data, electrospray ionization (ESI) mass spectrometry (MS) in negative ion mode,[M-H]- = 1314.9, [M-2H]/2- = 656.7, calculated exact mass Hex x 2 + Hep x 3 + HexA x 1 +Kdo x 1 = 1314.4 Da.80Table 3.5: NMR data for C. crescentus PS2 and its NaIO4 oxidation product OS1 (40◦C).1 2 3 4 5 6α-Rha N, OS1H 5.04 4.07 3.85 3.47 3.85 1.30C 103.1 71.1 71.1 72.9 69.9 17.6α-Rha N, PSH 5.02 4.02 3.79 3.45 3.72 1.28C 103.0 71.5 78.8 73.3 70.4 17.5β -Rha T, OS1H 4.78 4.13 3.71 3.54 3.53 1.33C 100.7 71.1 72.3 83.4 71.7 17.5β -Rha T, PSH 4.79 4.22 3.82 3.62 3.61 1.36C 101.7 76.7 73.5 83.4 73.2 17.5β -Rha V, OS1H 4.75 4.12 3.67 3.49 3.49 1.34C 101.1 71.4 81.3 71.9 72.9 17.5β -Rha V, PSH 4.77 4.13 3.66 3.66 3.50 1.35C 101.4 71.2 81.7 67.2 73.3 17.5X (ox. G), OS1H 3.69; 3.71 3.75 3.99 1.20C 61.8 84.8 67.7 18.0α-Rha G, PSH 5.10 4.13 3.94 3.58 3.90 1.35C 103.1 71.5 70.3 84.5 17.5α-Rha F, PSH 5.11 4.09 3.87 3.45 4.07 1.27C 102.4 72.1 71.3 73.3 17.53.3.8 Cellular response to C. crescentus LPSWe analyzed the immune/cellular response against C. crescentus LPS by two methods, assayingNF-κB activation in HEK293 cells and measuring cytokine production levels from THP-1derived macrophages. Figure 3.13 presents our data from the NF-κB assays. When comparedto S. enterica LPS, C. crescentus LPS is much less stimulatory. We found a significantly lower81Lipid A α-DLHepp |7α-DDHepp-3-α-Manp-2-α-DLHepp-5-α-Kdop- |4 α-Galp-4-α-GlcApHCKLEFGFigure 3.11: The structure of the C. crescentus core oligosaccharide.level of NF-κB activation by C. crescentus LPS at 1, 10, and 100 ngml−1.The cytokine production assays were a more in depth analysis of the profile of clear-cut asthe NF-κB data. We hypothesized in light of the NF-κB experiment and past work with lipid A,that the C. crescentus LPS would significantly stimulate less cytokine production than entericLPS. Figure 3.14 compiles the data from all the cytokine assays. At 4 hr after treating the cellwith only 1 ngml−1 of LPS added, all the cells that were treated with C. crescentus LPS hadnotably lower production levels of all cytokines tested than the cells that were treated with S.enterica LPS. At 24 hr, and also with 1 ngml−1 of LPS added, C. crescentus LPS stimulatedless cytokine production than S. enterica LPS for all cytokines other than MCP-1. At the 1082Table 3.6: NMR data for the core oligosaccharide (25◦C).1 2 3 4 5 6 7 8Kdo CH 2.05; 2.35 4.23 4.28 4.09 3.91 3.81; 3.93C 98.2 34.7 74.9 74.2 71.0 79.2 61.5DLHep EH 5.24 3.85 4.12 3.89 3.88 4.04 3.73; 3.73C 101.1 80.2 71.1 67.3 74.1 70.0 63.9Man FH 5.03 4.30 4.00 3.79 3.80 3.80; 3.91C 103.7 70.6 78.1 67.5 74.5 62.0DDHep GH 5.15 4.05 3.86 3.77 3.84 4.06 3.76; 3.84C 103.1 71.0 71.8 68.7 74.5 72.9 62.9DLHep HH 5.07 4.14 3.89 3.89 3.80 4.10 3.79; 3.81C 102.7 73.7 71.8 67.3 73.2 70.2 64.9GlcA KH 5.22 3.66 4.00 3.99 4.15C 98.8 72.4 74.9 73.6Gal LH 5.49 3.80 3.85 3.99 3.94 3.69; 3.69C 99.9 69.7 70.3 70.0 71.8 61.6and 100 ngml−1 LPS treatment levels, the differences between C. crescentus and S. entericabecome less clear. At the 100 ngml−1 LPS treatment conditions C. crescentus LPS actuallystimulated more cytokine production in almost every test (except MCP-1, 4 hr). clear-cut as theNF-κB data. We hypothesized in light of the NF-κB experiment and past work with lipid A,that the C. crescentus LPS would significantly stimulate less cytokine production than entericLPS. Figure 3.14 compiles the data from all the cytokine assays. At four hr after treating thecell with only 1 ngml−1 of LPS added, all the cells that were treated with C. crescentus LPS hadnotably lower production levels of all cytokines tested than the cells that were treated with S.enterica LPS. At 24 hr, and also with 1 ngml−1 of LPS added, C. crescentus LPS stimulated lesscytokine production than S. enterica LPS for all cytokines other than MCP-1. At the 10 and 10083ppm3.84.04.24.44.64.85.05.25.45.65.865707580859095100105ppmppmFigure 3.12: Fragment of 1H-13C HSQC spectrum of the core.ngml−1 LPS treatment levels, the differences between C. crescentus and S. enterica becomeless clear. At the 100 ngml−1 LPS treatment conditions C. crescentus LPS actually stimulatedmore cytokine production in almost every test (except MCP-1, 4 hr).3.4 DiscussionThe LPS of C. crescentus has an unusually complicated structure with two different polysaccha-rides, irregular substituents, resulting in NMR spectra that were especially difficult to analyze.Presented data show structures of the core, two polymers, and putative terminal structures. Thepolysaccharides could not be separated by size exclusion or anion-exchange chromatography840.01.20.40.81.60 1 10 100LPS (ng/ml) Salmonella LPSCaulobacter LPSResponse (A )655 nmp<0.05p<0.05 p<0.05nsFigure 3.13: The activation of the NF-κB promoter as measured by the HEK-BluehTLR4TM cell line (InVivoGen) treated with LPS from either S. enterica orC. crescentus. Error bars signify ±standard deviation, n=3 for all groups, p-valuesless than 0.05 are noted.and so are probably linked together through the same core. The core of the C. crescentus LPShas been studied previously and an initial assessment of its composition was made,148 but thecomplete structure had not been determined. The structure of the OPS had not been studiedbefore. In our view the polysaccharide structure of the C. crescentus LPS represents one of themost complicated bacterial LPS polysaccharide structures identified so far.An early goal in working with the C. crescentus LPS was to isolate the S-layer anchoring por-tion of the molecule and use it to assemble the S-layer onto an abiotic surface. The hypothesiswe were following was that a simple mild-acid hydrolysis of the LPS would produce a free-OPSthat could be chemically linked to aminated surfaces by virtue of a free carboxyl on the Kdoresidue exposed by hydrolysis. We were unable to demonstrate the presence of a free-OPS afterhydrolysis using biochemical methods, such as SDS-PAGE and western immunoblotting. For-tunately, NMR analysis following mild acid hydrolysis did demonstrate an intact OPS structure.851 10 10005104 hours 24 hoursTNF-αIL 6IL 10 MCP-11 10 100050100LPS (ng/ml)1 10 1001 10 1001 10 1001 10 1001 10 1001 10 10002.254.50816040080003.5708001600060120Salmonella LPSCaulobacter LPS(pg/ml)(pg/ml)(pg/ml)(pg/ml) ************* **** ****** ** ****** *****p<0.01p<0.05*** ≈≈Figure 3.14: Cytokine production levels produced by THP-1 derived macrophages in re-sponse to treatment with LPS from C. crescentus or S. enterica. All data points arepresent, n=4 for all groups, p-values less than 0.05 are marked with a star.86None of the determined carbohydrate structures give clues as to why we were unable to detectfree-OPS by spot-blot immunoblot (see section 3.3.1). We do not have much information aboutwhich particular structures in the C. crescentus LPS are bound by RsaA or the antibodies used inwestern blotting. Genetic knock outs point towards the requirement of N-acetylpersoamine, butnow that the complete structure has been determined it is not clear why the N-acetylpersoaminecontaining structures (OPS and end caps) were not previously detectable.The Kdo present in the LPS core structure (see fig. 3.11) has the typical substitutions atC-4 and C-5 of a manno-configured sugar and a negatively charged sugar, respectively.102,175It also has a rarely observed third substitution at C-7 with a heptose moiety. The Kdo C-7position is known to be occupied by a galactose moiety in the core of Rhizobium leguminosarumbv. Viciae VF39,176 and the secondary Kdo in the core oligosaccharide from Acinetobacterbaumannii ATCC 19606 has an C-7 substituted with a glucosamine.177 This arrangement of atriple substituted Kdo is extremely rare in known structures. The core oligosaccharide of Vibriocholerae O139 has triple substituted Kdo with the core oligosaccharide elongating off the C-5position, a phosphate on the C-4 position, and a phosphoethanolamine on the C-7 position.107Our core structure is, as far we are aware, the only core oligosaccharide with a triple substitutedKdo with sugars on all three sites.In the traditional model LPS occupies the outer leaflet of the outer membrane of a Gram-negative bacterium, and so (excepting the presence of cell associated EPS) is the outermost layerof the cell. For C. crescentus, however, LPS is the penultimate barrier below the protein S-layer.The C. crescentus OPS serves as the anchor for the S-layer and is likely not accessible to theenvironment.128 The carbohydrates found in the OPS are particularly hydrophobic, marked by anabundance of deoxy-sugars, acetyl groups, and methyl groups. This hydrophobicity is possiblya result of particular sugars needed for S-layer anchoring, as these carbohydrate structures likely102O. Holst. Endotoxin Health Disease, 1: 115–154, 1999.175H. Brade. Endotoxin in health and disease. New York: Marcel Dekker, 1999. xviii, 950176Y. Zhang, R. I. Hollingsworth, and U. B. Priefer. Carbohyd Res, 231: 261–271, 1992.177E. V. Vinogradov et al. Eur J Biochem, 243: 122–127, 1997.107Y. A. Knirel et al. Euro J Biochem, 247: 402–410, 1997.87evolved as the cognate ligands for the S-layer protein, RsaA. The distance between the S-layerand the outer membrane is about 17–19 nm.178 It is possible the hydrophobicity aids in packingthe polysaccharides between the S-layer and the LPS. Further determination of RsaA’s structureshould help illuminate the interaction between the S-layer and OPS.Knowledge of the structure of C. crescentus OPS and LPS will facilitate the determinationand characterization of their biosynthetic enzymes and mutant variants. Already, the enzymesLpxI179 and GDP-L-perosamine acetylase180 from C. crescentus have been characterized. Oneuncharacterized enzyme, WbqL, is necessary for proper OPS synthesis. Disruption of wbqLleads to the accumulation of aberrant OPS precursors in the inner membrane which inhibitsCrescentin-mediated cell curvature.181 Many genes, such as wbqL, have been identified asessential for OPS synthesis147 but most if not all have not yet been characterized. Other genes,that must be essential for OPS synthesis, have yet to be identified or characterized, such as theOPS polymerase and ligase.The subunit-based repeating nature and branching structure of the C. crescentus OPS sug-gests that a Wzy-dependent pathway synthesizes the polymer.69 The previous study that aimedto identify genes essential for OPS did not identify many of the canonical genes in the Wzy-dependent pathway,147 such as the O-unit transporter, Wzx, OPS polymerase, Wzy; the chain-length determinate protein, Wzz; and the OPS ligase, WaaL. Genes that have been annotated asputative OPS synthesis genes do appear in the sequenced genomes for C. crescentus CB15, butthey have not been experimentally confirmed.An additional aspect to this LPS is the fact that its OPS is of homogeneous length. Whileother LPSs vary in size due to the number of OPS repeat groups, appearing as a laddering ofbands by SDS-PAGE, the LPS from C. crescentus appears as a single band.128 MALDI-TOF anal-ysis of the entire LPS indicates a size of about 10.8 kDa (see fig. 3.4). After accounting for the178A. Moll et al. Mol Microbiol, 77: 90–107, 2010.179L. E. Metzger IV et al. Nat Struct Mol Biol, 19: 1132–1138, 2012.180J. B. Thoden et al. Biochemistry, 51: 3433–3444, 2012.181M. T. Cabeen et al. J Bacteriol, 192: 3368–3378, 2010.69C. R. H. Raetz and C. Whitfield. Annu Rev Biochem, 71: 635–700, 2002.88solved structures for the lipid A and core regions, this suggests the LPS contains approximately5 repeats of the proposed heptameric OPS structure. There is not currently a known mechanismfor the regulation and synthesis of a strictly homogeneous length OPS. Another example ofa homogenous length OPS is Aeromonas hydrophila AH-1.182 The Aeromonas OPS is formedby an ATP binding cassette (ABC) transporter-dependent pathway. The homogenous lengthand unique non-reducing end caps suggest that the C. crescentus OPS is synthesized via anABC-transporter-dependent pathway69, but there may also be another heretofore undiscoveredmechanism.Previously, our lab showed that alone the C. crescentus lipid A fraction of the LPS stim-ulates less than a hundredth of the TNF-α production than E. coli D31m4 rough-LPS.86 Wehypothesized that intact C. crescentus LPS would have a similar effect because lipid A is gener-ally considered the innate immune stimulating portion of the LPS molecule. Our first immunecell-based assay used a cell line constructed with a TLR4–NF-κB reporter system. The NF-κB results clearly support the previous reports that C. crescentus LPS is less inflammatorythan enteric LPS. Expanding our investigation into cytokine production, using THP-1 derivedmacrophages, produced some results that did not entirely fit with our hypotheses. Figure 3.14shows that in some circumstances C. crescentus LPS causes the production of less cytokinesthan S. enterica LPS (for IL 6, IL 10, and TNF-α) but at highest doses it stimulates more, atleast in this assay. Perhaps the data show that the stimulatory effect of C. crescentus LPS isnot always less than its enteric counterparts, but there are enough examples to the contrary thatweaknesses in the experimental design may be at fault. Because TLR4 is the major sensor forLPS in both our assays and because NF-κB activation is a major contributor to many cytokineproduction levels,183 our two assays are not completely independent of one another. If theycompletely contradict each other, then one of them is wrong or there are more complexitiesinvolved than are obvious.The conditions in our assays that contradict our hypotheses were most often at the high182S. Merino et al. Mar Drugs, 13: 2233–2249, 2015.183A. S. Baldwin Jr. Ann Rev Immun, 14: 649–681, 1996.89LPS concentrations (100 ngml−1). The C. crescentus LPS, at higher concentrations, may beactivating alternate receptors than just TLR4. If it is not the LPS stimulating in a non-TLR4manner, then something else is. One difference in our assays is the cells used. The THP-1cells express a full complement of toll-like receptors, in addition to TLR4. If our purifiedC. crescentus LPS contained any microbe-associated molecular pattern (MAMP) contaminants(e.g. flagellin, peptidoglycan) then they may have stimulated THP-1 cells in addition to thesignal from the LPS. These possible contaminants would not have affected the results fromthe HEK-Blue hTLR4 cells because they only expressed the LPS sensor, TLR4. Possibly, thecommercially sourced S. enterica LPS was depleted of any contaminants by the manufacturer.There were no contaminants in the C. crescentus LPS that were abundant enough to affect (or bedetected by) the NMR or MS analysis, but it is possible that the cell assays are more sensitive andit is possible any contaminants present were not in a detectable form with those other methods.If contamination is the culprit of our surprising cytokine results, then repeating the experimentswith a cell line only expressing TLR4 should show what we expect: C. crescentus LPS is lessstimulatory than enteric LPS.90Chapter 4OmpW of Caulobacter crescentusfunctions as an outer membranechannel“Now in the 21st century, the boundaries separating chemistry, physics, andmedicine have become blurred, and as happened during the Renaissance, scientistsare following their curiosities even when they run beyond the formal limits of theirtraining”— Peter Agre, 2003 Nobel Prize Banquet Speech.Ion channel researcher4.1 IntroductionGRAM-NEGATIVE bacterial cell envelopes consist of different layers or membranes. Theinner, or cytoplasmic, membrane contains the respiration chain components, proteins forthe transport of nutrients, and proteins involved in the synthesis and transport of phospholipids,peptidoglycan, and lipopolysaccharide (LPS).184,185 The periplasm contains the peptidoglycanand a large number of proteins. The outer membrane is composed of protein, lipid and LPS.184 Ittypically contains many fewer proteins than the cytoplasmic membrane. Normally at least oneof the constitutive outer membrane proteins is a porin, a general diffusion pore with a definedexclusion limit for hydrophilic solutes.115 In addition to constitutive porins, an outer membrane184T. Beveridge. Internat Rev Cytology, 72: 229–317, 1981.185H. Nikaido and M. Vaara. Microbiol Rev, 49: 1, 1985.115H. Nikaido. Microb Mol Biol Rev, 67: 593–656, 2003.91may contain porins that are induced under special growth conditions.186 They often form solute-specific channels and contain binding sites for neutral substrates such as carbohydrates,187,188or nucleosides,187 and phosphate.189,190 Many of the specific porins are part of uptake anddegradation systems, such as the maltose uptake system of Escherichia coli.191Caulobacter crescentus, an alphaproteobacterium found in oligotrophic aquatic environments,192is an unusual Gram-negative bacterium in that genes coding for typical general diffusionporins of the OmpF/C type of enteric Gram-negative bacteria have not been identified in itsgenome.120,121,193 Similarly, genes coding for specific porins such as Tsx or LamB are also ab-sent. To date, no definitive porin has been demonstrated in C. crescentus. Instead, the genomeof C. crescentus contains a large number of genes that code for TonB-dependent receptors.121,193More than 60 of these receptors have been identified,121 which probably means that most of thenutrients from dilute environments are taken up actively by these systems. Examples for thisare the uptake of maltose and N-acetylglucosamine (GlcNAc) into the cells,121,194, as opposedto the passive mechanisms of porins.In this study we investigated whether the outer membrane of C. crescentus also contained aporin-like channel. The results suggest that a porin-like channel could be detected in artificialmembranes with a single-channel conductance of about 125 pSv in 1 M KCl. The proteinresponsible for channel formation has been identified to be a member of the large OmpWfamily of outer membrane proteins. OmpW analogue proteins are found in many Gram-negativebacteria. Two members of this family, OmpW of E. coli and OprG of Pseudomonas aeruginosahave been crystallized and their 3D-structures are known at high resolution (2.7 and 2.4 Å,186R. Benz. Micrb Transp Sys, 227–246, 2001.187R. Benz et al. J Bacteriol, 165: 978–986, 1986.188T. Ferenci et al. J Bacteriol, 142: 521–526, 1980.189R. Benz and R. Hancock. J Gen Physiol, 89: 275–295, 1987.190R. Hancock, K Poole, and R Benz. J Bacteriol, 150: 730–738, 1982.191M Schwartz. The maltose regulon. American Society for Microbiology, Washington, DC, 1987. 1482–1502192P. D. Curtis and Y. V. Brun. Microb Molec Biol Rev, 74: 13–41, 2010.120W. C. Nierman et al. PNAS, 98: 4136–4141, 2001.121S Lohmiller et al. Micrbiol, 154: 1748–1754, 2008.193H. Neugebauer et al. J Bacteriol, 187: 8300–8311, 2005.194S. Eisenbeis et al. J Bacteriol, 190: 5230–5238, 2008.92respectively).195–197 OmpW of C. crescentus functions as a channel for cations, which is insharp contrast to OprG of P. aeruginosa and OmpW of E. coli, which are believed to be pluggedcompletely or involved in the transport of small, yet unknown hydrophobic molecules acrossthe cell wall of these bacteria.195–197 The 3D structure of OmpW of C. crescentus was modeledon the basis of the known structures of OmpW of E. coli and OprG of P. aeruginosa.196,197 Theresults indicated that OmpW of C. crescentus could have a larger diameter and more hydrophilicinterior than the two crystallized members of the OmpW family.4.2 Methods4.2.1 Growth and maintenance of bacteriaC. crescentus NA1000 353Φ (JS1013), a strain that carries an amber mutation in the genersaA resulting in S-layer deficiency,132 was used for initial identification and characterization ofOmpW. Wildtype C. crescentus NA1000 was used for generating an ompW-knockout strain andas a comparison against the knockout strain. The bacteria were grown to mid log phase (opticaldensity at 600 nm (OD600) ≈ 0.8) in peptone-yeast extract medium (PYE)198 at 30◦C in 5 mlcultures, which were used to start large cultures in 2.8 l Fernbach flasks containing 1250 mlM16HIGG medium, shaken at 100 rpm. M16HIGG is a modification of M6HIGG medium,12containing 0.31% glucose, 0.09% glutamate, 1.25 mM sodium phosphate, 3.1 mM imidazole,0.05% ammonium chloride and 0.5% modified Hutner’s Mineral Base.195R. Albrecht et al. Acta Crystallogr F, 62: 415–418, 2006.196H. Hong et al. J Biol Chem, 281: 7568–7577, 2006.197D. S. Touw, D. R. Patel, and B. Van Den Berg. PLoS One, 5: e15016, 2010.132M. J. Ford, J. F. Nomellini, and J. Smit. J Bacteriol, 189: 2226–2237, 2007.198J. S. Poindexter. Bacteriol Rev, 28: 231, 1964.12J. Smit et al. J Bacteriol, 146: 1135–1150, 1981.934.2.2 Generation of C. crescentus ∆ompWThe gene ompW, CCNA_01475 (CC_1409), which encodes for OmpW, was knocked out inwildtype C. crescentus NA1000 via a two step method, derived from previously publishedprotocols132, resulting in an dysfunctional copy of ompW with a large internal deletion. Theamino acid sequence for OmpW can be found in the appendix on page 155.The suicide plasmid ‘pKMOBsacB-ompW-A/B’ was constructed by combining two frag-ments of DNA homologous to the 5′ and 3′ ends of the gene ompW into a suicide vector con-taining both positive and negative selection elements. The ‘A-fragment’ was polymerase chainreaction (PCR) amplified using the primers F-ompW-A (5′– TAC CGG AAT TCT CGG GCGCTG GGC CTG TCT GTT GAG –3′) and R-ompW-A (5′– GTC CCA AGC TTG CGG AAGATC TAT TGG CGC CGG CGG CAG TCA GGA TG -3′), resulting in a 994 bp productwith a 5′ EcoRI cleavage site and 3′ BglI and HinDIII cleavage sites. The ‘A-fragment’ PCR prod-uct was blunt ligated into pBSK-ESH,42 resulting in pBSK-ompW-A. The ‘B-fragment’ PCRproduct was amplified using the primers F-ompW-B (5′– CGG GAT CCA CGT CAA GAAGGT CTA TTT CAG CAC –3′) and R-ompW-B (5′– GTC CCA AGC TTG CGT CGA TGCTAG TGC GCT GCG ATG –3′), resulting in a 1090 bp product with a 5′ BamHI cleavage siteand a 3′ HinDIII cleavage site. The ‘B-fragment’ PCR product was similarly blunt subclonedinto pBSK-ESH, then excised by BamHI and HinDIII digestion, and ligated into pBSK-ompW-A, digested with BglII and HinDIII, resulting in the plasmid pBSK-ompW-A/B. The plasmidspBSK-ompW-A/B and pKMOBsacB were digested with EcoRI and HinDIII, the 2070 bp frag-ment from pBSK-ompW-A/B was ligated into the pKMOBsacB fragment creating the plasmidpKMOBsacB-ompW-A/B.The plasmid pKMOBsacB-ompW-A/B was electroporated into C. crescentus NA1000 cells.37The resulting transformants that were kanamycin resistant were re-passaged through kanamycin-free PYE medium three times and plated on kanamycin-free PYE agar plates that contain 3%42M. C. Toporowski et al. J Bacteriol, 186: 8000–8009, 2004.37A. Gilchrist and J. Smit. J Bacteriol, 173: 921–925, 1991.94sucrose. All colonies that were found to be sucrose resistant were screened for kanamycinsensitivity to confirm that the pKMOBsacB-ompW-A/B plasmid had crossed out of the genome.Colonies that were both sucrose resistant and kanamycin sensitive had their ompW genes PCRamplified using the primers F-ompW (5′– CGC ACT GGG CTT GCT GGC CTT TTT C –3′) and R-ompW (5′– GGA GCC AGA GGA CGG ACG ACC GGG G –3′); intact ompW re-sulted in a roughly 800 bp product, and knocked-out ompW resulted in a roughly 500 bpproduct.4.2.3 Outer membrane-enriched preparationsThe protocol for preparing outer membrane extracts enriched in OmpW was derived from ourLPS isolation protocol (on page 56). The crucial difference here is the omission of proteinaseK. The lack of a nuclease step was primarily for convenience, but also it was deemed unesse-cary. Nucleic acids can be significant contaminants in nuclear magnetic resonance spectroscopy(NMR) analysis, even in trace quantities, but they are not a serious concern in the these bi-layer experiments or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)analyses.Cells were pelleted by centrifugation at 12,400 x g for 10 min. Cell pellets were washedby suspension in distilled water and repelleted. The pellets were resuspended in 1/10 originalculture volume of phosphate-buffered saline (PBS)152 amended with 10 mM ethylenediaminete-traacetic acid (EDTA), agitated at room temperature for 5 min and then centrifuged at 15,300 xg for 15 min. The supernatant was retrieved and re-centrifuged to clarify. The supernatant wasthen ultracentrifuged at 184,000 x g for 2 h. Glassy pellets formed which were suspended in1/100 original culture volume in 10 mM Tris pH 8.0. This treatment led preferentially to thedisruption of the outer membrane and periplasmic contents were released without significantlyreleasing cytoplasmic contents.152T. Maniatis, E. F. Fritsch, and J. Sambrook. Molecular cloning: a laboratory manual. vol. 545 Cold SpringHarbor, NY, 1982.954.2.4 Crude membrane preparationsFor comparison to the PBS-EDTA membrane enrichment method, crude membrane preparationswere prepared from 5 ml of mid logarithmic culture. The culture was sonicated at 50% intensityfor 5 x 30 sec bursts. DNAse and RNAse were added to final concentrations of 0.06 mgml−1and 0.60 mgml−1 respectively, and incubated at 37◦C for 1 h. The preparation was then ultra-centrifuged for 2 h at 107 000 x g. A glassy pellet formed which was resuspended in 200 µl ofdistilled water.4.2.5 Sodium dodecyl sulfate-polyacrylamide gel electrophoresisElectrophoretic analyses of protein samples were done by the standard methods of discountin-uous SDS-PAGE,154 in a similar fashion to the methods used to analyze LPS in chapter 3 onpage 57. Initially, samples were boiled in sample buffer for 5 min prior to running on the gel.When we wanted to probe our samples for heat-modifiabilitiy, samples were not boiled; to meltthe sample buffer, it was heated to 37◦C prior to mixing with the protein samples. Gels werestained to visualize protein with Coomassie brilliant blue as previously described.1994.2.6 Isolation and purification of the channel-forming protein fromouter membrane-enriched preparationsThe outer membrane-enriched samples were prepared by PBS-EDTA extraction were inspectedfor channel-forming activity by treatment with the detergent lauryldimethylamine-oxide (LDAO).The detergent extracts of the enriched OM showed rapid channel formation in the lipid bilayerassay. The protein responsible for channel formation was identified by preparative SDS-PAGE.Highest channel-forming activity was observed in the molecular mass range between 20 and 25kDa.154U. K. Laemmli. Nature, 227: 680–685, 1970.199J Bennett and K. Scott. Anal Biochem, 43: 173–182, 1971.96Analytical and preparative SDS-PAGE was performed according to Laemmli.154 The gelswere stained with Coomassie brilliant blue or Colloidal Coomassie blue.2004.2.7 Tryptic digestion and peptide sequencingThe pure 22 kDa protein eluted from preparative SDS-PAGE was subjected to amino acidsequence analysis. Direct sequencing was not possible presumably because of blocking of theN-terminus. The 22 kDa protein was then cleaved with trypsin as described.201 The peptideswere separated by reverse phase HPLC on a Purospher RP18 encapped 5 µm column (Merck,Darmstadt, Germany) using a solvent gradient from 0 to 60% acetonitrile in 0.1% trifluoroaceticacid/water (v/v). The flow rate was 60 µlmin−1 and UV-detection was performed at 206 nm.The amino acid sequence analysis of the tryptic peptides was performed using an ABI 472Aprotein sequencer (Applied Biosystems, Langen, Germany).4.2.8 Lipid bilayer experimentsThe method used for the reconstitution experiments using black lipid bilayer membranes hasbeen described previously.202 The membranes were formed from a 1% (w/v) solution of diphytanoyl-phosphatidylcholine (PC) (Avanti Polar Lipids, Alabaster, AL, U.S.A.) in n-decane. The mem-brane current was measured with a pair of calomel electrodes switched in series with a voltagesource and an electrometer (Keithley model 617). For single-channel recordings the electrom-eter was replaced by a highly sensitive current amplifier (Keithley model 427). Zero-currentmembrane potentials were measured by establishing a salt gradient across membranes contain-ing 100–1000 channels, as has been described earlier203,204 using a high impedance electrometer(Keithley model 617).200G. Candiano et al. Electrophoresis, 25: 1327–1333, 2004.201C. Eckerskorn and F Lottspeich. Chromatographia, 28: 92–94, 1989.202R. Benz et al. Biochim Biophys Acta Biomembr, 511: 305–319, 1978.203R Benz, K Janko, and P Läuger. Biochim Biophys Acta Biomembr, 551: 238–247, 1979.204R. Benz, A Schmid, and R. Hancock. J Bacteriol, 162: 722–727, 1985.974.2.9 Modeling of the OmpW structureThe possible 3D-structure of OmpW of C. crescentus was derived using the homology modelingapproach. Three dimensional model of C. crescentus OmpW was built using the programModeller205 taking E. coli OmpW as a template structure.1964.3 Results4.3.1 Protein composition of the outer membrane-enriched preparationsof C. crescentusCrude membranes from C. crescentus CB15A NA1000 353Φ cells that were disrupted by soni-cation and membrane material released by PBS-EDTA treatment were analyzed by SDS-PAGE.The membranes from the sonicated cells (presumably a mixture of the cytoplasmic and outermembranes) (fig. 4.1, lane 1) and the membranes from the PBS-EDTA extract (fig. 4.1, lane2) showed numerous proteins present, whereas the PBS-EDTA extracted cell membranes (notboiled before analysis) showed an enrichment of two protein bands at about 20 and 22 kDa.When the PBS-EDTA extract preparations were boiled prior to SDS-PAGE, the two enrichedbands resolved as a single band of about 22 kDa in size (fig. 4.1, lane 3). This ‘heat modi-fiability’ of the enriched protein (i.e. lower mobility when boiled in the presence of SDS) isoccasionally observed for Gram-negative bacterial outer membrane proteins and is probablycaused by their unfolding.206205N. Eswar et al. “Protein structure modeling with MODELLER” in: Structural Proteomics. Springer, 2008.145–159206E. Sugawara et al. J Bacteriol, 178: 6067–6069, 1996.9897.4 kDa66.2 kDa45.0 kDa31.4 kDa21.5 kDa1 231 12Figure 4.1: Coomassie stained SDS-PAGE of different protein preparations ofC. crescentus. Lane 1, 7.5 µl of crude membrane preparation. Lane 2, 15 µlof PBS-EDTA extract, not boiled prior to loading. Lane 3, 15 µl of PBS-EDTAextract, boiled prior to loading. The star highlights the position of OmpW.4.3.2 Identification of channel-forming activity in outermembrane-enriched preparations of C. crescentusThe outer membrane-enriched preparations were treated with 0.5% LDAO. When small amountsof this detergent extract of the outer membranes was added to one or both sides of a black PC/n-decane membrane a remarkable increase of membrane conductance was observed, indicatingthat the enriched outer membranes contained membrane-active components. After an initialrapid increase of conductance for 5–10 min, the increase slowed down and saturated at 30–60min after addition of the detergent solution to the black membranes. When the detergent alonewas added in the same concentration as with the PBS-EDTA extracted membranes, it had no99influence on the conductance of the lipid bilayer membranes. When the detergent extract ofthe outer membranes was added at much lower concentrations to the aqueous phase bathingthe black lipid membrane, the current increased in a step-wise fashion (see fig. 4.3A, upperpanel). A histogram of the channel distribution demonstrated that most of the conductance stepshad a conductance of about 125 pSv in 1M KCl (see fig. 4.3A, lower panel). Besides the 125pSv channel, some fluctuations with higher conductance (250 and 350 pSv) were also obtainedwhich probably represented oligomers of the 125 pSv channel that could not be separated at thetime scale of the experimental conditions. It is significant that the conductance of these stepswas notably much lower than that of general-diffusion pores from enteric bacteria, which is atleast 10 times higher at the same conditions (1–4 nSv in 1 M KCl207). This result suggested thatthe PBS-EDTA extracted membranes of C. crescentus contained a porin-like channel of smallconductance.4.3.3 Identification of the purified channel-forming protein from theenriched outer membranesTo identify the protein responsible for the channel-forming activity, the PBS-EDTA extractedmembranes were dissolved in detergent and subjected to preparative SDS-PAGE. The gel was cutinto thin slices, corresponding to defined molecular mass ranges, and each was eluted overnightwith a buffer containing 1% Genapol. The eluted molecular mass fractions were examined forchannel-forming activity in the lipid bilayer assay. Extremely high channel-forming activitywas exclusively localized within the molecular mass range around 20 to 22 kDa, correspondingto the protein bands enriched by PBS-EDTA extraction (see fig. 4.1). The other bands from thegel, in particular the 66 kDa band, had no activity in the lipid bilayer assay. When excisedand eluted, the 20–22 kDa band was again subjected to SDS-PAGE. Without heating, the gelshowed a single protein band of about 20 kDa suggesting that the excised protein was essentiallypure (fig. 4.2, lane 2). When the excised 20 kDa band was heated to 100◦C prior to addition to207R. Benz. New Compr Biochem, 27: 397–423, 1994.100SDS-PAGE, most of the protein ran at an apparent molecular mass of about 22 kDa (fig. 4.2, lane2), with the indication that some of the protein ran at 20 kDa (fig. 4.2, lane 2). This suggestedagain that the 20 kDa protein from C. crescentus is heat modifiable.66 kDa45 kDa29 kDa36 kDa24 kDa20 kDa14 kDa11 212Figure 4.2: 15% SDS-PAGE showing OmpW of C. crescentus obtained by elution of the20 kDa band from preparative SDS-PAGE Lane 1: 5 µg of OmpW solubilized atroom temperature for 10 min in 5 µl of sample buffer. Lane 2: 5 µg of OmpWsolubilized at 100◦C for 10 min in 5 µl of sample buffer. The gel was stained withCoomassie.4.3.4 Partial sequencing of the 22 kDa protein of C. crescentusThe 20 kDa protein was subjected to sequencing using Edman-degradation. In a first run theprotein could not be sequenced starting from the N-terminus, presumably because of N-terminal101blockage. Following trypsin treatment one stretch corresponding to the N-terminal end witha molecular mass of 1764.8 Da could be resolved by sequencing. The sequence of the partialpeptide was QDFTPNAKGDLIVHAR, which suggested that the N-terminus was blocked bythe formation of pyroglutamate. A basic local alignment search (BLAST) analysis208,209 ofthe sequenced peptide unambiguously demonstrated that the 20 kDa protein was OmpW ofC. crescentus and was a member of the extensive OmpW-family of outer membrane proteinsof Gram-negative bacteria. To ensure that the higher molecular mass band observed in boiledsamples was also OmpW, this protein band (about 22 kDa) was also subjected to sequencingfollowing trypsin treatment. Its N-terminal end was identical to that of the 20 kDa protein(OmpW), indicating again that OmpW existed in two different configurations where one washeat-modifiable.4.3.5 Analysis of the channels formed by OmpW channel-formingprotein of C. crescentusFigure 4.3B (upper panel) shows a single-channel recording of a PC membrane in the presenceof the purified OmpW protein of C. crescentus, which was added to a black lipid membrane at aconcentration of about 20 ngml−1. The single-channel recording demonstrates that the proteinformed the same defined channels as found in PBS-EDTA extracted membranes of C. crescentus.The average single-channel conductance was about 125 pSv in 1 M KCl (almost 40% of allconductance steps). Only a minor fraction of channels with other conductance was observed(see the histogram in fig. 4.3B, lower panel) suggesting that conductance steps with morethan one unit conductance were less frequent for the purified OmpW than for the crude outermembrane fraction. It is noteworthy that the channels formed by OmpW of C. crescentus had along lifetime, similar to those that have been detected previously for porins of Gram-negative207and Gram-positive bacteria.210,211 All these channel-forming proteins from the cell walls of208S. F. Altschul et al. J Mol Biol, 215: 403–410, 1990.209J. Zhang and T. L. Madden. Genome Res, 7: 649–656, 1997.210J Trias and R Benz. J Biol Chem, 268: 6234–6240, 1993.211J. Trias and R. Benz. Mol Microbiol, 14: 283–290, 1994.102bacteria form channels in lipid bilayer membranes with long lifetimes at small transmembranepotential (mean lifetime at least 5 min). Furthermore, no voltage-dependence closure wasobserved in KCl solution voltages up ±120 mV (data not shown).Single-channel experiments were also performed with salts other than KCl to obtain infor-mation on the size and selectivity of the channels formed by OmpW of C. crescentus. Theresults are summarized in table 4.1. The conductance sequence of the different salts within thechannel was KCl ≈ KOAc ≈ NH4Cl >RbCl >NaCl >CsCl >LiCl. The influence of cations ofdifferent size and mobility on the conductance was substantial (see table 4.1) suggesting indeedhigh cation-selectivity of the OmpW channel. For more bulky cations such as N(CH3)4+ orTris+ we observed a very low conductance of much less than 10 pSv suggesting that the size ofthe OmpW channel was indeed very small.An additional significant result of the single-channel measurements was the extreme highconductance of OmpW in CaCl2 (see table 4.1). In 1 M CaCl2, the conductance was about 250pSv; this was again close to saturation because in 0.5 M CaCl2 the conductance was only a littlelower than in 1 M solution. Interestingly, conductance traces in CaCl2 solutions were very noisysuggesting a strong interaction between the divalent cations and the OmpW channels. Also,similarly, channel-forming activity in salt solutions containing divalent cations was much lowerthan that in monovalent cation solutions, indicating that OmpW could be a channel for divalentcations, such as Ca2+ or Mg2+. Table 4.1 also shows the average single-channel conductance,G, as a function of the KCl concentration in the aqueous phase. Similarly, as in the case ofsome channels of Gram-positive bacteria,210–212 the conductance was not a linear function ofthe KCl-concentration, which means that OmpW did not form a wide, water-filled channel.The saturation with increasing salt concentration could be caused by point net charges and/or abinding site for ions.212F. G. Rieß et al. Mol Microbiol, 29: 139–150, 1998.10301020300 100 200 300 400Single-channel conductance (pSv)Probability of occurrence (%)0102040300 100 200 300 400Single-channel conductance (pSv)Probability of occurrence (%)250 pSv5 pA15 sec250 pSv5 pA15 secA BFigure 4.3: Single-channel measurements with detergent treated outer membrane ofC. crescentus and purified 20 kDa protein.A Upper panel: Single-channel recording of a PC/n-decane membrane in the pres-ence of enriched outer membranes of C. crescentus. The aqueous phase contained 1M KCl and 100 ngml−1 protein from enriched outer membranes treated with 0.5%LDAO. The applied membrane potential was 20 mV; T = 20◦C.Lower panel: Histogram of the probability P(G) for the occurrence of a given con-ductivity unit observed with membranes formed of PC/n-decane in the presence ofenriched outer membranes of C. crescentus. P(G) is the probability that a givenconductance increment G is observed in the single-channel experiments. It wascalculated by dividing the number of fluctuations with a given conductance incre-ment by the total number of conductance fluctuations. The aqueous phase contained1 M KCl. The applied membrane potential was 20 mV; T = 20◦C. The averagesingle-channel conductance was 125 pSv for 105 single-channel events (left-handmaximum).B Upper Panel: Single-channel recordings of a PC/n-decane membrane in the pres-ence of purified OmpW of C. crescentus. The aqueous phase contained 1 M KCland 20 ngml−1 OmpW dissolved in 1% Genapol. The applied membrane potentialwas 20 mV; T = 20◦C.Lower panel: Histogram of the probability P(G) for the occurrence of a given con-ductivity unit observed with membranes formed of PC/n-decane in the presence ofOmpW of C. crescentus The applied membrane potential was 20 mV; T = 20◦C.The average single-channel conductance was 125 pSv for 95 single-channel events.104Table 4.1: Average single-channel conductance of OmpW of C. crescentus in differentsalt solutions and radii, hydrated radii, and limiting molar conductivity of the cations.This data is also presented in a graphical figure, fig. 4.5Salt Molarity Single-channel Ion Hydrated Ion Limiting Molarityconductance Radius Radius ConductivityM pSv nm nm mSvM−1LiCl 1.0 15 ± 3.0 0.059 0.216 38.68NaCl 1.0 40 ± 3.5 0.100 0.163 50.10KCl 0.01 30 ± 3.2 0.137 0.110 73.50KCl 0.03 40 ± 3.4KCl 0.1 55 ± 4.9KCl 0.3 80 ± 6.2KCl 1.0 125 ± 8.1KCl 3.0 150 ± 8.9NH4Cl 1.0 125 ± 9.5 0.147 0.110 73.55RbCl 1.0 100 ± 7.6 0.152 0.105 77.81CsCl 1.0 30 ± 2.9 0.167 0.106 77.26N(CH3)4Cl 1.0 <10 0.347 0.182 44.92KAcO– (pH 7) 0.1 60 ± 8.0KAcO– (pH 7) 1.0 125 ± 10CaCl2 0.5 225 ± 22CaCl2 1.0 250 ± 19MgCl2 1.0 275 ± 271054.3.6 The OmpW channel of C. crescentus is highly cation-selectiveAdditional information about the structure of the channel formed by OmpW of C. crescentuswas obtained from zero-current membrane potential measurements in presence of salt gradients.A fivefold KCl gradient (100 mM versus 500 mM), across a lipid bilayer membrane in whichabout 100–1000 OmpW channels were reconstituted, resulted in an asymmetry potential of35 mV at the more dilute side (mean of 3 measurements). This result indicated preferentialmovement of K+ ions over Cl– through the channel at neutral pH. The zero-current membranepotentials were analyzed using the Goldman-Hodgkin-Katz equation, see eq. (4.1).202,204 Theratio of the potassium permeability, PK , divided by the chloride permeability, PCl , was about 15(mean of 3 measurements), indicating high cation selectivity of the channel formed by OmpW(see also Discussion, page 107). This result was confirmed by measurements with LiCl andKOAc; we observed under the same conditions as for KCl, asymmetry potentials around 32to 35 mV for fivefold gradients, meaning that Pa/Pc was also in these cases also higher than10. More precise numbers cannot be expected because small errors of the asymmetry potentialresult in this range of big variations of the permeability ratio Pa/Pc. Size and mobility of thecations did not influence the cation selectivity of OmpW in contrast to the situation observedpreviously for general diffusion porins.204Em,ion =RTFln(Pion[ion+]outPion[ion+]in)(4.1)4.3.7 Knocking out ompW removes channel-forming activityTo confirm that the gene product encoded by CCNA_01475 was indeed responsible for thechannel forming activity, we removed the genomic copy of the gene with a two-step recombi-nation protocol that is standard in C. crescentus genetics. The gene loss in the resulting strain,C. crescentus ∆ompW, was confirmed by PCR (data not shown) and SDS-PAGE. The SDS-PAGEanalysis of the knockout compared to wild-type can be seen in fig. 4.4. The band that was106identified previously as comprising the channel-forming protein was found to be absent in theompW knockout. Likewise, the channel-forming activity previously found in our extracts wasnot present in our ompW knockout extracts. These results support the peptide sequencing inthat ompW, CCNA_01475, encodes for an outer membrane porin.Interestingly, all attempts to scale-down our outer membrane extractions, from large flasksto test tubes, were unsuccessful at producing a sample with a prominent SDS-PAGE band near20 kDa (the OmpW band). It is not clear what the important differences are between a flask-based extraction and a tube-based extraction. A significant amount of time was spent trying toreproduce our original results; culture volume was not considered a significant variable untilvery late. The reason for this phenomenon is unknown.Attempts to determine a phenotype for the knockout strain were inconclusive. We hypothe-sized that the OmpW was involved with Ca2+ uptake and that without OmpW the cells wouldhave reduced fitness under Ca2+ limitation. It has previously been shown that C. crescentusdoes require Ca2+ in its growth medium, but that independence from Ca2+ can be achieved and‘calcium-free’ mutants have been isolated.39 All recent attempts to produce growth media with-out Ca2+, in the manner previously published, were not successful. It seems that there may be atrace level of calcium contamination of some medium components. Nevertheless, no reductionin fitness was observed for our ompW knockout strain compared to wild-type in media repletewith Ca2+ or in media with significantly reduced Ca2+. In fact, no in vivo phenotype of any kindwas observed with our strain C. crescentus ∆ompW.4.4 DiscussionThe outer membrane-enriched preparations of C. crescentus contain a cation-permeable chan-nel, encoded by ompW Here we demonstrated the presence of a channel in the outer membrane-enriched extractions of C. crescentus. Unlike enteric Gram-negatives, the genome does not con-39J. Smit et al. J Bacteriol, 174: 6527, 1992.10797.4 kDa66.2 kDa45.0 kDa31.4 kDa21.5 kDa1 2Figure 4.4: SDS-PAGE analysis of outer membrane-enriched preparations of C. crescentus.Lane 1: C. crescentus NA1000. Lane 2: C. crescentus ∆ompW. The star highlightsthe position of OmpW, the prominent band at 22 kDa.tain genes that code for the classical outer membrane porins such as OmpC, OmpF, LamBor Tsx.120,121,193 Instead the genome of C. crescentus contains 67 genes coding for TonB-dependent receptors.193 This suggested that most nutrients from the dilute environments whereC. crescentus is typically found are actively taken up by TonB-dependent transport systems.Nevertheless, it is clear from this study that C. crescentus also contains at least one outermembrane channel. The 20 kDa band excised from preparative SDS-PAGE had a very highchannel-forming activity. No other protein bands excised from gels showed channel-formingactivity, suggesting that the outer membrane of C. crescentus may contain only one porin-likechannel. The channel had a very low single-channel conductance of about 125 pSv in 1 M KCl.The channel-forming protein was subjected to partial sequencing and was identified as OmpWof C. crescentus. The gene found to encode for the OmpW, CCNA_01475, was knocked outand the extracts of the resulting strain showed no channel-forming activity.The OmpW porin family is widespread amongst Gram-negative bacteria, but with no wellestablished function. Only for OmpW of E. coli and OprG of P. aeruginosa have channel func-tions been postulated that may have to do with the uptake of hydrophobic compounds.196,197In other studies it has been suggested that the channel function of OmpW of E. coli and OprG108of P. aeruginosa may be plugged by the tryptophans W155 and W170, respectively (see sec-tion 4.4).195–197Two big-dataset studies from the Shapiro lab at Stanford University have shed some lighton the biology of ompW in C. crescentus: the survey of essential genes by Christen et al. (2011)and the high-throughput analyses of transcription and translation by Schrader et al. (2014). Thegene ompW is non-essential for C. crescentus, at least in the rich growth medium PYE,213 Thegene lies on the forward strand of the genome surrounded by genes of seemingly unrelatedfunction. It is transcribed as a monocistronic mRNA, while the preceding and succeeding genesare both transcribed on separate polycistronic mRNAs.214 ompW is not highly transcribed, nortranslated in either rich medium (PYE) nor limiting medium (M2G), but interestingly the geneis transcribed at a much higher relative rate in rich medium than in limiting medium whileits translation rate remains relatively equal in both media. The differences in transcriptionrate and translational efficiency between medium types was relatively small within the entirestudy by Schrader et al., and so it was not focused on or discussed in the original study. Thereduction in transcriptional activity in minimal medium suggests some transcriptional control,but the translation rate (as measured by ribosome footprinting RNA-seq) remained relativelyunchanged (0.9 versus 0.8 ribosomes per kilobase per million-reads) suggesting that the changesin transcription maybe superseded by a translational control mechanism.Properties of OmpW in the C. crescentus outer membrane OmpW of C. crescentus was foundto be highly cation-selective. Its selectivity for K+ ions over Cl– was at least ten-fold; thecalculated value was about 12. However, it has to be kept in mind that the permeability ratioPa/Pc reacts in this range and is very sensitive to small changes of the asymmetry potential.189This means that we found little indication for the permeation of anions through OmpW becausethe single channel conductance in 1 M KCl was the same as in 1 M KOAc, despite the fact213B. Christen et al. Mol Sys Biol, , 2011.214J. M. Schrader et al. PLoS Gen, 10: e1004463, 2014.109that chloride in the aqueous phase has a much higher mobility than acetate. In addition, thesingle channel conductance was not a linear function of the bulk aqueous salt concentration, seetable 4.1. Instead the conductance showed strong saturation; at 3 M KCl conductance was onlyslightly higher (150 pSv) than at 1 M KCl (125 pSv). Similarly, at 0.3 M KCl, conductancewas only little smaller (80 pSv) than at 1 M KCl. Experiments with different alkaline cationsindicated clearly that OmpW does not form an aqueous pore, which would be typical for mostGram-negative and Gram-positive bacterial porins, even if they contained point charges.207,215Instead OmpW of C. crescentus appeared to be an ion channel; its single channel conductancehad a maximum for ammonium and potassium ions and became much smaller for bigger cationsindicating that they likely lose part of their hydration shell while moving through the channel.This argues for a small selectivity filter inside the channel. Presumably, the ionic radii of thedifferent cations and not the sizes of their hydration shells, play an important role for theirpermeation through OmpW as table 4.1 clearly indicates. Figure 4.5 shows the single channelconductance of OmpW as a function of the ionic radii of the ions.216–218 The conductance datafor another small substrate-specific channel (LamB of E. coli) is given for comparison.219 Therelationship for OmpW follows approximately the Eisenman series VI for carrier-mediatedtransport of cations or for the transport of monovalent cations through channels.220 This meansthat the field strength inside the channel is medium-sized. Ion transport trough general diffusionchannels of enteric bacteria follow instead Eisenman series I or II indicating low field strengthin the channels. The minimum diameter of OmpW is presumably close to that of Cs+ or smallerthan that of N(CH3)4+ ions because larger organic cations have a very low permeability throughOmpW, if any.OmpW of C. crescentus clearly functions as a cation-permeable channel, having a prefer-215F. G. Rieß and R. Benz. Biochim Biophys Acta Biomembr, 1509: 485–495, 2000.216L. Pauling. The nature of the chemical bond and the structure of molecules and crystals: an introduction tomodern structural chemistry. vol. 18 Cornell University Press, 1960.217R. Shannon. Acta Crystallogr A, 32: 751–767, 1976.218L. Smirnov et al. Physica B, 234: 66–67, 1997.219R. Benz, A. Schmid, and G. H. Vos-Scheperkeuter. J Membr Biol, 100: 21–29, 1987.220G. Eisenman and R. Horn. J Membr Biol, 76: 197–225, 1983.110040801201600 0.1 0.2Ion radius (nm) Single-channel conductance (pSv) LamBOmpWLi+ Na+ K+ Rb+ Cs+NH +4Figure 4.5: Single-channel conductance of OmpW in 1 M salt solution as a function ofthe ionic radii of monovalent cations. The data were taken from table 4.1. Thesingle-channel conductance through LamB of E. coli is given for comparison (datataken from R. Benz et al. Pore formation by LamB of Escherichia coli in lipidbilayer membranes. Journal of Bacteriology, 165: 978–986, 1986.).ence for divalent cations, since calcium and magnesium ions had a higher permeability throughOmpW than potassium ions. This could indeed mean that it is a channel for the transport ofcalcium and/or magnesium ions. This function has not been found in other homologous ver-sions of OmpW. The reason for this is that OmpW is a rather small channel with only eightbeta strands and so has little possibility for the passage of solutes. Previous data from structuralstudies of OmpW of E. coli and OprG of P. aeruginosa suggested that members of the OmpWfamily could be involved in the transport of small hydrophobic molecules across the bacterialouter membrane.196,197 It is also possible that they are blocked.195 Lipid bilayer experiments111with OmpW of E. coli suggested that it forms small ion-permeable channels with a conductanceof about 20 pSv in 1 M KCl,196 which is considerably lower than that of C. crescentus OmpW.The 3D-structure of P. aeruginosa OprG, another member of the OmpW family, was resolved at2.4 Å resolution.197 Again the structure suggested that OprG forms a channel for the diffusionof small hydrophobic molecules, although lipid bilayer experiments proposed a single-channelconductance of about 500 pSv for OprG.221 This means that OmpW of C. crescentus forms achannel that has, despite sequence homologies with OmpW of E. coli and OprG of P. aeruginosa(see below), a completely different function than other outer membrane proteins.Structure of OmpW of C. crescentus Sequence comparison, BLAST, of OmpW of C. crescentuswith other members of the OmpW family suggested that it had highest homology with OmpW ofCaulobacter segnis ATCC 21756, Asticcacaulis excentricus CB 48, Brevundimonas sp. BAL3,and Phenylobacterium zucineum HLK1.208,209 These bacteria are closely related to the genusCaulobacter, belonging to the family Caulobacteraceae, order Caulobacterales. All theseOmpW polypeptides are similar in length to C. crescentus OmpW (200–250 amino acids) andshare many strategically positioned conserved residues. The homology of the C. crescentusOmpW to the OmpW proteins with known 3D structure (OmpW of E. coli and OprG ofP. aeruginosa) is less pronounced, but still obvious (amino acid identity is approximately 32%in both cases and 19.4% for all three proteins (Hong et al., 2006; Touw et al., 2010). Fortyone amino acids, including many glycines and several prolines, appear to be highly conservedamong all three OmpW family proteins. This allows a meaningful comparison between thethree different OmpW species (see fig. 4.6) and it is also possible to build the possible structuralmodel of OmpW of C. crescentus using a homology modeling approach205 (see below).Crystal structures of E. coli OmpW196 and P. aeruginosa OprG197 suggested the formationof hydrophobic channels for these proteins and they are believed to be responsible for thepassage of hydrophobic solutes across the bacterial outer membrane. The small and hydrophobic221J. B. McPhee et al. FEMS Microbiol Lett, 296: 241–247, 2009.112Figure 4.6: Amino acid sequence alignment of OprG of P. aeruginosa, OmpW of E. coli,and OmpW of C. crescentus. The alignment was performed using Pole Bioinfor-matique Lyonnaise Network Protein Sequence Analysis (http://npsa-pbil.ibcp.fr).Amino acids identical in all three proteins are highlighted in red, strongly similaramino acids (:) are given in green and weakly similar ones (.) in blue. The replace-ment of W155 of OmpW of E. coli and W170 of OprG of P. aeruginosa by K160of C. crescentus is given in green color and is indicated by an arrow. The eightbeta strands in OprG of P. aeruginosa and in OmpW of E. coli are numbered andindicated by blue bars. The yellow highlighted sequence was found by N-terminalsequencing of OmpW of C. crescentus.E. coli OmpW channel has a conductance of approximately 20 pSv in 1 M KCl.196 In contrast,the C. crescentus OmpW formed a channel with a relatively high conductance of 125 pSv in 1M KCl in our bilayer measurements. A comparison of the structures of these OmpW familyproteins revealed factors that might lead to a larger conductance in C. crescentus OmpW. Crystalstructures suggest that both E. coli OmpW and P. aeruginosa OprG form a hydrophobic gate113(fig. 4.7A; b and c) in the central region of the channel. Both channels have an aromatic, bulkyand hydrophobic tryptophan residue as a part of a hydrophobic gate, which may make thepassage of ions through channels difficult. Similarly, W155 of OmpW of E. coli 196 and W170of OprG of P. aeruginosa,197 which are both considered as plugs of the channels are replaced inOmpW of C. crescentus by K160. This may indicate that this channel has a function differentfrom that of the homologs in E. coli and P. aeruginosa. In addition, C. crescentus OmpW has ahydrophilic and relatively less bulky lysine residue at the corresponding position (fig. 4.7A; a),which is unlikely to hinder passage of ions through the channel. Also, the C. crescentus OmpWhas a relatively more hydrophilic interior environment of the channel (fig. 4.7A; a), as shown bygreen color surface within the black box) as compared to the E. coli OmpW (fig. 4.7; b) and theP. aeruginosa OprG (fig. 4.7; c). For E. coli OmpW and P. aeruginosa OprG the interior surfaceof the channel is hydrophobic (white color surface), which could make the permeation of ionsthrough the channel energetically unfavorable. C. crescentus OmpW, on the other hand, dueto its relative hydrophilic environment may not provide the permeation barrier to ion passage,resulting in a relatively higher conductance of the channel.We also examined the contribution of the size of the to the higher conductance of C. crescentusOmpW. We observed a slightly larger radius of C. crescentus OmpW channel as compared tothe other two channels in several regions, particularly from the center of the channel towardsthe periplasmic side (fig. 4.8). Another important feature of the OmpW family channels fromE. coli and P. aeruginosa is the presence of a lateral opening in the barrel wall, which has beensuggested to allow diffusion of small hydrophobic solutes across the outer membrane by alateral diffusion mechanism.196,197 In our modeled structure of C. crescentus OmpW, we do notobserve such an opening in the channel, which further supports the view that the C. crescentuschannel may have a different function from that of E. coli OmpW and P. aeruginosa OprG.The solved structures OmpW from E. coli and OprG from P. aeruginosa provide examples ofsuccessful crystallization efforts for this class of protein and they are exciting starting pointsfor future work towards improving the structural knowledge of OmpW from C. crescentus.114A B CECPCK160 W155 L56 W170 V65I65Figure 4.7: Comparison of structural features between a) OmpW C. crescentus b) OmpWE. coli c) OprG P. aeruginosa. Residues, which are a part of a putative hydrophobicgate in E. coli (W155 and L56) and P. aeruginosa (W170 and V65) channels areshown as spheres. Corresponding residues in OmpW C. crescentus (K159 and I65)are also shown. Additionally all the channels are shown as a surface representationand color coded according to residue type (Green: hydrophilic, white: hydrophobic,red: acidic, blue: basic). Channels are cut from the front to enable visualizationof interior channel surface. Black colored box indicates a putative ion transportpathway across the channel. EC and PC denote extracellular and periplasmic sidesof the channel respectively.C. crescentus has a requirement for calcium, although it is not clear where the requirementlies. Ca2+ ions are needed for assembly of the crystalline protein surface layer (S-layer) RsaA.66.Since RsaA is secreted by a type I secretion mechanism, it is likely that calcium is also neededfor either secretion or folding of the protein following secretion, in a manner analogous toall other type I secreted proteins. However, the S-layer is a completely dispensable structure.Moreover, the type 1 secretion apparatus spans both the cytoplasmic and outer membranes;hence for S-layer there is no apparent need for a channel that enables Ca2+ ion movement to theperiplasm. There are, however, additional still undefined roles for Ca2+ ions. Mutants no longerrequiring calcium ions for growth can be isolated.128 One consequence of all these mutants isthe loss of the O-specific polysaccharide (OPS) of LPS that is used for S-layer attachment. Asa consequence, these so called ‘calcium-independent’ mutants all shed RsaA into the culture66J. F. Nomellini et al. J Bacteriol, 179: 6349–6354, 1997.128S. G. Walker et al. J Bacteriol, 176: 6312–6323, 1994.1150 4 8 12 16 20 24 28Channel Axis (Å)Radius (Å)00.511.522.53OmpW (C. crescentus)OmpW (E. coli)OprG (P. aeroginosa)PeriplasmicExtracellularFigure 4.8: Comparison of channel radii between C. crescentus OmpW (blue), E. coliOmpW (red) and OprG of P. aeruginosa (green) along the channel axis. The channelradii were calculated using the program HOLE (O. S. Smart et al. HOLE: a programfor the analysis of the pore dimensions of ion channel structural models. Journal ofMolecular Graphics, 14: 354–360, 1996.). The extracelluar side is indicted alongthe right side and the periplasmic side is indicated along the left side.medium. The causal relationship has not been deciphered, but since OPS biosynthesis involvessynthesis activities within the periplasmic space, it may be that a calcium selective OmpW porinplays a specific role in this process. Here we have provided clear evidence that OmpW couldbe responsible for the uptake of cations, especially divalent cations, like Ca2+ in C. crescentus.116Chapter 5Conclusions“We must never let ourselves fall in thinking ‘ignorabimus’ (‘We shall never know’),but must have every confidence that the day will dawn when even those processes oflife which are still a puzzle today will cease to be inaccessible to us naturalscientists.”— Eduard Buchner, 1907 Nobel lecture5.1 Relevance and contribution to the fieldTHE Caulobacter field is dominated by research groups teasing apart the fine details ofthe Caulobacter crescentus lifecycle and the panoply of regulatory networks that controlevery aspect of bacterial biology. The protein surface layer (S-layer) and the outer membraneoften are neglected by the rest of the field. The focus on laboratory cultures, and not thebacteria in the environment, is one reason for this. A pure culture living in rich medium doesnot have as complex a relationship with its environment as a wild specimen competing in ahostile setting. It is the cell envelope that acts as the barrier between the world and the cell.The cell envelope of C. crescentus was particularly suitable for inspection because it had aprotein surface layer, various polysaccharides including an O-specific polysaccharide (OPS)of unknown structure, and apparently no porins. There is the added aspect that C. crescentus,and its S-layer, are actively being pursued for biotechnology applications. This means that newfindings can often be quickly be applied towards biological products. This work presented in117chapters 2 3, and 4 present our efforts to improve the structure and composition knowledge ofthe S-layer, lipopolysaccharide (LPS), and outer membrane proteins in C. crescentus.Chapter 2 detailed effort to solve the structure of RsaA. Protein crystallography is theonly approach applicable to extend our current resolution of the C. crescentus S-layer protein.S-layer proteins are notoriously resistant to crystallography. The work of Pavkov et al.127and Baranova et al.24 demonstrated that at least portions of S-layer proteins are possible tocrystallize as long as the ability for the proteins to form S-layers is blocked. Our strategy toimpede S-layer formation was a truncation approach, removing the N-terminal 222 amino acid(AA), which mirrored the successful approach used to solve SbsC.127 Novel protein expressionand purification techniques had to be developed to produce well-behaved protein suitable forcrystallography. Once we started to grow crystals, RsaA was set to be the third bacterial S-layerprotein to be structurally solved, the second with the symmetry determining domain intact,the first from a Gram-negative, and the largest. But so far, finding a phasing solution hasbeen the issue that is insurmountable. No phasing approach was successful, not the halidesoaks that we had predicted would work, nor the lanthanide soaks that had worked for othergroups,24 nor any of a large range of compounds tried. The C-terminal 804 AAs segment ofRsaA has been crystallized, but the structure remains elusive. Solving this structure wouldprovide the field with unique window into a self-assembling protein lattice from a commonlystudied Gram-negative model organism.LPS structures solutions are not as rare as S-layer structures and yet the complete structureof the C. crescentus LPS had never been determined when we started the work covered inchapter 3. The C. crescentus lipid A structure was solved by Smit et al. in 2008,86 revealinga novel structure lacking phosphates and centered on a di-diaminoglucose backbone. Thestructures of the oligosaccharide (OS) and OPS were identified for analysis because their finestructures had never been determined and because they comprise the anchor for the C. crescentus127T. Pavkov et al. Structure, 16: 1226 –1237, 2008.24E. Baranova et al. Nature, 487: 119–122, 2012.86J. Smit et al. Innate Immun, 14: 25–36, 2008.118S-layer.128 An initial component analysis of the core OS was undergone in 1992.148 The OPShad never been studied directly but it was predicted that it was composed of perosamine.147Through a very successful collaboration with Dr. Evgeny Vinogradov the structures of theC. crescentus OS and OPS are now determined. The core OS has a three armed configuration(see fig. 3.11 on page 82). The final determined components of the core OS are different thanwere first reported,148 especially in the finding that there is no phosphate in the core OS. Thefirst arm is a disaccharide of glucuronic acid and galactose. The two negative charges in thegalacturonic acids in the lipid A, the one in the central Kdo, and the one in the glucuronic acidare the only charges present in the C. crescentus LPS. The second arm is a trisaccharide ofDD-heptose, mannose, and DL-heptose. The third arm is a single DL-heptose, linked to theC-7 position of the Kdo moiety. This triple substitution on the Kdo appears to be unique in theliterature. There are examples of sugar substitutions at all of the same positions (C-4, C-5, andC-7) in Kdo but none for all three at once. The OPS has a repeating heptasaccharide structure(see fig. 3.7 on page 75). Seven sugar OPS subunits are amongst the largest sizes that subunitsare known to exist in. For example, there is just one known example of a seven sugar OPS inEscherichia coli, and none larger.223 The C. crescentus OPS terminates in one or the other oftwo end groups (see fig. 3.9 on page 78). A complete structural view of the C. crescentus LPSis shown in fig. 5.1. Both the OPS and end groups contain an abundance of hydrophobic sugars,i.e. N-acetylperosamine, rhamnose, methylglucose, and dimethyl-N-acetylperosamine. Theremay be a role for this hydrophobicity to play in supporting an S-layer. The OPS compositionof Aeromonas hydrophila AH-1,182 another alphaproteobacterium with an S-layer, supports thishypothesis; one in two monosaccharides in its OPS is the deoxysugar acetylrhamnose. In theprocess of studying the C. crescentus LPS we discovered a previously unknown rhamnose basedpolysaccharide (see fig. 3.10 on page 80). Just the presence of the polysaccharide alone is of128S. G. Walker et al. J Bacteriol, 176: 6312–6323, 1994.148N. Ravenscroft et al. J Bacteriol, 174: 7595–7605, 1992.147P. Awram and J. Smit. Microbiology, 147: 1451–1460, 2001.223R. Stenutz, A. Weintraub, and G. Widmalm. FEMS Microbiol Rev, 30: 382–403, 2006.182S. Merino et al. Mar Drugs, 13: 2233–2249, 2015.119interest especially in light of recent investigations into the extracellular polysaccharides (EPSs)from C. crescentus by the Viollier lab at the University of Geneva.224Chapter 4 reports our characterization of OmpW, a porin in the outer membrane of C. crescentus.Previous to this work, no porin had been reported in C. crescentus. The wisdom on the subjectwas that C. crescentus primarily, if not solely, used active transport systems to take up nutrientsfrom the environment.121 We found that OmpW acts as a passive protein channel in the outermembrane (see fig. 4.3). This is particularly interesting because the homologous proteins inE. coli and Pseudomonas aeruginosa do not have any pore forming activity196,197 and no clearfunction at all. The activity we observed is possibly due to a wider pore and specifically a lackof a plug seen in the other bacteria.5.2 Future directionsEach aspect of this work, the protein crystallography, the LPS analyses, and characterizationof OmpW, opened more questions than they answered. One goal of this work has alwaysbeen to use all the gains made with basic science to improve the C. crescentus S-layer displaytechnology in the Smit lab. The rewards in biotechnology pursuits are not yet obvious, though acomplete knowledge of the LPS will undoubtedly guide efforts to assemble S-layers on abioticsurfaces.The RsaA crystallography is the obvious loose end in this dissertation. We do not have thestructure of RsaA solved to atomic resolution; this means the phase problem must be solved.It is possible that a particular compound exists or a certain soaking protocol exists that willprovide us our solution. It is possible that the next RsaA ∆0–222 crystal grown will have thebest diffraction yet and will give a nice clean signal. It is possible that we continue on our pathand we will figure this out. We will continue trying the same approaches but doing only that224S. Ardissone et al. eLife, 3: e03587, 2014.121S Lohmiller et al. Micrbiol, 154: 1748–1754, 2008.196H. Hong et al. J Biol Chem, 281: 7568–7577, 2006.197D. S. Touw, D. R. Patel, and B. Van Den Berg. PLoS One, 5: e15016, 2010.120NNNNCOOHHOOCx4?Figure 5.1: The fully assembled structure of the C. crescentus LPS. Included is the lipid A,the core oligosaccharide, five repeats of the OPS, and the longer of the two possibleend groups. The red question mark denotes the one currently undetermined linkage,the bond between the OPS and the core.121cannot be the only way forward. The halides, specifically iodine, were the best candidates toconsider at the beginning and we indeed got some very enticing early data from them. It seemedthat small optimizations might be satisfactory to complete the project. We got drawn in by thesesuccesses and it delayed us trying new strategies.Our troubles with phasing are at least in part due to a dearth of useful residues in the proteinto couple with heavy atoms. For instance, there are too few methionines for seleomethionineincorporation (if that was possible in C. crescentus) and there are no cysteines which limitsthe use of many heavy metals.225 We can modify RsaA to contain any residues we want, atleast at selected positions within RsaA; this is the strategy we have already begun with ourGSCC cassette insert (see section 2.2.1). Cysteine-rich and methionine-rich inserts at positionsknown to tolerate heterologous insertions (e.g. AA 644, 690, 723, 784, 805, and 944) are obviousapproaches to be tried. As of this writing, cloning has begun to insert various peptides into RsaAfor the purposes of improving our phasing. We do have to consider the possibility that the insert-strategy is inherently flawed. We saw that our GSCC inserted RsaA did not crystallize well.The cause for that is not clear and it is possible that there is a structural instability introduced byexogenous peptides. Less common methods, like molecular replacement from a low resolutioncryo-electron microscopy structure,226 are also possibilities that should be considered.Once we have figured out our difficulties with the RsaA structural analysis, we can startto assess the current peptide display strategies in the context of a protein structure. Rationaldesign choices can then be used to improve the display products. Currently display peptidesare inserted into rsaA at sites that were first found by trial-and-error and evaluated by manyyears of use.157 Despite their consistent efficacy, we do not know how these display sites fit intothe structure of RsaA. Locating sites on RsaA that are less buried in the protein may improvepeptide accessibility to the environment. Choosing display sites with increased flexibility mayallow for more complex tertiary structures for the display peptides to adopt.225S. A. Islam et al. Acta Crystallgr D, 54: 1199–1206, 1998.226M. M. Jore et al. Nature Struc & Biol, 18: 529–536, 2011.157W. H. Bingle, J. F. Nomellini, and J. Smit. J Bacteriol, 179: 601–611, 1997.122Designing the insert peptides with respect to how they fit into the larger three-dimensionalstructure of the S-layer may open up new directions of innovation. For example, we cancurrently co-display two-separately engineered versions of RsaA on the surface of a singleC. crescentus cell. But with more fine-detail information about RsaA’s structure, we could imag-ine displaying two types of RsaA each containing a specifically arranged half of a functionaldimer that assembles in the S-layer. Another example that would be of immediate interest toour laboratory would be to improve the targeting of HIV. C. crescentus and RsaA constructshave been created in our laboratory that inhibit HIV entry into T-cells.59,60 The three copiesof RsaA in the center of three-fold symmetry in the S-layer (see fig. 1.5) could each display aGP120-binding peptide, rationally spaced to match the trimer structure of GP120.The LPS of C. crescentus is presented in chapter 3 but the study of Caulobacter’s outer mem-brane polysaccharides is not complete. Specifically for the OPS, there is one glycosidic bondthat was not abundantly clear in our data. The linkage between the OPS and the core oligosac-charide is still unknown (see fig. 5.1). This single bond is particularly difficult to determinebecause it is present in nuclear magnetic resonance spectroscopy (NMR) spectra alongside thehighly amplified data coming from the repeating OPS structure. A project is underway to doubleour efforts to identify that one missing bond by NMR. That bond could possibly be determinedthrough genetic knockouts. There are six sugars in the core oligosaccharide that could be thebase of the OPS, but only three sugars if we assume it is on the C-5 branch from the Kdo (as iscommon in LPS). If we knockout the glycosyltransferase responsible for adding the base sugarthat supports the OPS, then we would see a loss of smooth-LPS. But that points out anothergap in our understanding: the genetics of the C. crescentus LPS are still an open question. Thework of Awram et al. (2001) did the first survey of OPS biosynthetic genes,147 but now we couldgo back and start identifying these genes’ functions based on changes in polysaccharide struc-ture. Only one true LPS synthesis protein from C. crescentus has been characterized, LpxI17959J. F. Nomellini et al. PLoS One, 5: e10366, 2010.60C. Farr et al. PLoS One, 8: e65965, 2013.179L. E. Metzger IV et al. Nat Struct Mol Biol, 19: 1132–1138, 2012.123(this study misidentified the C. crescentus lipid A precursors to be glucosamines instead ofdiaminoglucoses); there are many more to be studied.Even before that last bond is determined, progress can continue to use the LPS to improvethe Caulobacter peptide-display technology and move S-layers onto abiotic surfaces. NMRanalysis demonstrated that we can liberate free-OPS by mild-acid hydrolysis and that thereare carboxyl groups present on the Kdo that can be chemically linked to an amine.170 Weshould redouble our efforts to chemically produce OPS functionalized surfaces, regardless ofour previous negative results. Additionally, working with the Cullis Lab in the Departmentof Biochemistry and Molecular Biology at UBC, lipid nanoparticles227 have been created thatare in part composed of C. crescentus LPS. The ultimate goal is to produce nanoparticles thatdisplay therapeutic targeting molecules. The work is preliminary but they have already beenproduced in various sizes and have been loaded with siRNA or fluorescent dyes. Efforts todemonstrate successful formation of S-layers of recombinant RsaA onto the nanoparticles areunderway. Thus nanoparticles may be potent platforms to support an S-layer peptide-displaytechnology.A further area of future study is the new rhamnan polysaccharide. I am particularly inter-ested in what role this rhamnan polysaccharide plays in the cells’ lifestyle. There are questionsto be asked related to how it affects sedimentation rates, surface adhesion, and phage resistance.Discovering a new polysaccharide, rhamnan polysaccharide, like our discovery that OmpWacts as a outer membrane channel, begs the question of what else lies in the outer membraneand cell envelope waiting to be discovered next?170E. S. Jauho et al. J Immuno Meth, 242: 133–143, 2000.227A. K. K. Leung et al. J Phys Chem B, , 2015.124Bibliography[1] M. D. JONES, E. VINOGRADOV, J. F. 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(see p. 124)153AppendixMAYTTAQLVTAYTNANLGKAPDAATTLTLDAYATQTQTGGLSDAAALTNTLKLVNSTTAV -60AIQTYQFFTGVAPSAAGLDFLVDSTTNTNDLNDAYYSKFAQENRFINFSINLATGAGAGA -120TAFAAAYTGVSYAQTVATAYDKIIGNAVATAAGVDVAAAVAFLSRQANIDYLTAFVRANT -180PFTAAADIDLAVKAALIGTILNAATVSGIGGYATATAAMINDLSDGALSTDNAAGVNLFT -240AYPSSGVSGSTLSLTTGTDTLTGTANNDTFVAGEVAGAATLTVGDTLSGGAGTDVLNWVQ -300AAAVTALPTGVTISGIETMNVTSGAAITLNTSSGVTGLTALNTNTSGAAQTVTAGAGQNL -360TATTAAQAANNVAVDGGANVTVASTGVTSGTTTVGANSAASGTVSVSVANSSTTTTGAIA -420VTGGTAVTVAQTAGNAVNTTLTQADVTVTGNSSTTAVTVTQTAAATAGATVAGRVNGAVT -480ITDSAAASATTAGKIATVTLGSFGAATIDSSALTTVNLSGTGTSLGIGRGALTATPTANT -540LTLNVNGLTTTGAITDSEAAADDGFTTINIAGSTASSTIASLVAADATTLNISGDARVTI -600TSHTAAALTGITVTNSVGATLGAELATGLVFTGGAGADSILLGATTKAIVMGAGDDTVTV -660SSATLGAGGSVNGGDGTDVLVANVNGSSFSADPAFGGFETLRVAGAAAQGSHNANGFTAL -720QLGATAGATTFTNVAVNVGLTVLAAPTGTTTVTLANATGTSDVFNLTLSSSAALAAGTVA -780LAGVETVNIAATDTNTTAHVDTLTLQATSAKSIVVTGNAGLNLTNTGNTAVTSFDASAVT -840GTGSAVTFVSANTTVGEVVTIRGGAGADSLTGSATANDTIIGGAGADTLVYTGGTDTFTG -900GTGADIFDINAIGTSTAFVTITDAAVGDKLDLVGISTNGAIADGAFGAAVTLGAAATLAQ -960YLDAAAAGDGSGTSVAKWFQFGGDTYVVVDSSAGATFVSGADAVIKLTGLVTLTTSAFAT -1020EVLTLA -1026Figure A.1: The amino acid sequence of the S-layer protein, RsaA from C. crescentusNA1000. GeneID: CCNA_01059. Ensembl Accession number: ACL94524. Theencoding gene has the coordinates of 1 159 693 bp–1 162 773 bp on the forwardstrand of the C. crescentus chromosome. The underlined sequence corresponds tothe amino acids 1–222, the section of the protein that was removed to generate acrystallizable C-terminal fragment. The bolded sequences are the RTX motifs inRsaA. For the crystallization of RsaA, see chapter 2 page 31.154MKKLALSLVAFGALAAGAAQAQDFTPNAKGDLIVHARLTQVAPAKDAAILTAAGANSGLK -60AHVGNDIKPTLGFTYFLTDKVAVEAILGTTEHNIRAQGPGTDVLVHKTWVLPPVVTLQYH -120PLPASQVSPYVGAGLNYMLFYSGKNKNGFTVKVDDGVGYALQAGVNIKMKNSWLVNADVK -180KVYFSTDAKINGGALKAKVDLDPVVASIGLSRKF -214Figure A.2: The amino acid sequence of the outer membrane channel, OmpW fromC. crescentus NA1000. GeneID: CCNA_01475. Ensembl Accession number:ACL94940. The encoding gene has the coordinates of 1 582 599 bp–1 583 243bp on the forward strand of the C. crescentus chromosome. The bolded ‘K’ is thelocation of the putative hydrophobic gate in E. coli and P. aeruginosa, where thatresidue encodes for a tryptophan; in C. crescentus that residue is a lysine. For ourinvestigations into OmpW, see chapter 4 on page 91155
Thesis/Dissertation
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Microbiology and Immunology
Vancouver : University of British Columbia Library
Graduate
Studies on the structure and composition of the outer membrane of Caulobacter crescentus
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