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Methods in
Molecular Biology 2741

Véronique Arluison
Claudio Valverde Editors

Bacterial
Regulatory RNA
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
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advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
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 Bacterial Regulatory RNA

Methods and Protocols

Second Edition

Edited by

Véronique Arluison
Laboratoire Léon Brillouin LLB, CEA, CNRS UMR 12, CEA Saclay, Gif-sur-Yvette, France;
Université Paris Cité, Paris, France

Claudio Valverde
Laboratorio de Fisiología y Genética de Bacterias Beneficiosas para Plantas (LFGBBP), Centro de
Bioquímica y Microbiología del Suelo (CBMS), Departamento de Ciencia y Tecnología,
Universidad Nacional de Quilmes – CONICET, Bernal, Buenos Aires, Argentina
Editors
Véronique Arluison Claudio Valverde
Laboratoire Léon Brillouin LLB, Laboratorio de Fisiologı́a y Genética de Bacterias Beneficiosas
CEA, CNRS UMR 12, CEA Saclay para Plantas (LFGBBP), Centro de Bioquı́mica y Microbiologı́a
Gif-sur-Yvette, France del Suelo (CBMS), Departamento de Ciencia y Tecnologı́a,
Universidad Nacional de Quilmes – CONICET
Université Paris Cité
Bernal, Buenos Aires, Argentina
Paris, France

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-3564-3 ISBN 978-1-0716-3565-0 (eBook)
https://doi.org/10.1007/978-1-0716-3565-0
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Preface/Summary

Regulatory small non-coding RNAs (sRNAs) are ubiquitous key regulators of gene expres-
sion in prokaryotes, operating mostly at the post-transcriptional level to influence the fate of
mRNA translation and/or stability, in most cases with the complicity of RNA binding
proteins. Although significant progress has been made in the past 25 years to understand
the function of individual sRNAs, high-throughput RNomics has recently revealed the
enormous wealth of non-coding RNA species existing in a wide variety of prokaryotes,
thus significantly expanding the implications of this class of regulatory molecules and
boosting this new field of research of yet undimensioned relevance.
The understanding of many of the fundamental processes underlying the evolution,
expression, structure, subcellular location, dynamics, and function of sRNA requires the
availability of solid experimental approaches that may be applied either singly or in combina-
tions to explore key aspects of sRNA biology. This volume collects many of the most
important methods that have been recently set up for studying prokaryotic non-coding
RNAs and their protein accomplices and complete the first edition published in 2018. These
methods cover different aspects in the biology of the field presented in different sections:
Discovery of sRNAs, their functional analysis, characterization of sRNA interactomes, and
structural studies. Each method includes a section with advice and tips from the authors.
This volume aims to provide a guidebook to scientists, which we hope will lead to new tools
and procedures for further development in the field of sRNA biology.

Gif-sur-Yvette, France Véronique Arluison

Bernal, Buenos Aires, Argentina Claudio Valverde

v
Contents

Preface/Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I SRNA DISCOVERY

1 RNA Extraction from Gram-Positive Bacteria Membrane
Vesicles Using a Polymer-Based Precipitation Method . . . . . . . . . . . . . . . . . . . . . . . 3
Paul Briaud and Ronan K. Carroll
2 Extraction and Purification of Outer Membrane Vesicles
and Their Associated RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Anaı̈s Blache and Wafa Achouak
3 Analysis of Phage Regulatory RNAs: Sequencing Library
Construction from the Fraction of Small Prokaryotic RNAs
Less Than 50 Nucleotides in Length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Sylwia Bloch, Natalia Lewandowska, Wojciech Wesołowski,
Aleksandra Łukasiak, Paulina Mach, Bożena Nejman-Faleńczyk,
and Grzegorz We˛grzyn
4 Discovering Novel Bacterial Small RNA by RNA-seq Analysis
Toolkit ANNOgesic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Chin-Hsien Tai, Deborah Hinton, and Sung-Huan Yu

PART II SRNA FUNCTIONS

5 Ribosome Profiling Methods Adapted to the Study

of RNA-Dependent Translation Regulation in Staphylococcus aureus. . . . . . . . . . . 73
Maximilian P. Kohl, Béatrice Chane-Woon-Ming,
Roberto Bahena-Ceron, Jose Jaramillo-Ponce, Laura Antoine, Lucas
Herrgott, Pascale Romby, and Stefano Marzi
6 CRISPR Interference-Based Functional Small RNA Genomics . . . . . . . . . . . . . . . 101
Gianluca Prezza and Alexander J. Westermann
7 Investigation of sRNA-mRNA Interactions in Bacillus subtilis In Vivo . . . . . . . . . 117
Inam Ul Haq, Peter Müller, and Sabine Brantl
8 In Vitro Methods for the Investigation of sRNA-mRNA Interactions
in Bacillus subtilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Inam Ul Haq, Peter Müller, and Sabine Brantl
9 RNA Double-Helix Hybridization Measured by Fluorescence
Correlation Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Arne Werner
10 New Perspectives on Crosstalks Between Bacterial Regulatory
RNAs from Outer Membrane Vesicles and Eukaryotic Cells . . . . . . . . . . . . . . . . . . 183
Moumita Roy Chowdhury and Eric Massé

vii
viii Contents

11 Experimental Validation of RNA–RNA Interactions by Electrophoretic

Mobility Shift Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Eva Maria Sternkopf Lillebæk and Birgitte Haahr Kallipolitis
12 Dynamics and Function of sRNA/mRNAs Under the Scrutiny
of Computational Simulation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Agustı́n Ormazábal, Juliana Palma, and Gustavo Pierdominici-Sottile
13 Analysis of sRNAs and Their mRNA Targets in Sinorhizobium meliloti:
Focus on Half-Life Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Robina Scheuer, Jennifer Kothe, Jan W€ a hling,
and Elena Evguenieva-Hackenberg
14 Evaluation of 5′-End Phosphorylation for Small RNA Stability
and Target Regulation In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Alexandra Schilder, Yvonne Göpel, Muna Ayesha Khan,
and Boris Görke
15 In-Gel Cyanoethylation for Pseudouridines Mass Spectrometry
Detection of Bacterial Regulatory RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Antony Lechner and Philippe Wolff

PART III SRNA INTERACTOME

16 Directed Screening for sRNA Targets in E. coli Using a Plasmid Library . . . . . . . 291
Xing Luo and Nadim Majdalani
17 Defining Bacterial RNA-RNA Interactomes Using CLASH . . . . . . . . . . . . . . . . . . 307
Sofia Esteban-Serna, Liang-Cui Chu, Mehak Chauhan,
Pujitha Raja, and Sander Granneman
18 Global Identification of RNA-Binding Proteins in Bacteria. . . . . . . . . . . . . . . . . . . 347
Thomas Søndergaard Stenum and Erik Holmqvist
19 An Integrated Affinity Chromatography-Based Approach to Unravel
the sRNA Interactome in Nitrogen-Fixing Rhizobia . . . . . . . . . . . . . . . . . . . . . . . . 363
Natalia Isabel Garcı́a-Tomsig, Antonio Lagares Jr., Anke Becker,
Claudio Valverde, and José Ignacio Jiménez-Zurdo

PART IV SRNA STRUCTURE

20 sRNA Structural Modeling Based on NMR Data . . . . . . . . . . . . . . . . . . . . . . . . . . . 383

Pengzhi Wu and Lingna Yang
21 Circular and Linear Dichroism for the Analysis of Small Noncoding
RNA Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Florian Turbant, Kevin Mosca, Florent Busi, Véronique Arluison,
and Frank Wien

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
Contributors

WAFA ACHOUAK • CEA, CNRS, Aix Marseille University Lab of Microbial Ecology of the
Rhizosphere (LEMiRE), UMR7265 BIAM, Saint-Paul-lez-Durance, France
LAURA ANTOINE • Architecture et Réactivité de l’ARN, CNRS 9002, Université de
Strasbourg, Strasbourg, France
VÉRONIQUE ARLUISON • Laboratoire Léon Brillouin LLB, CEA, CNRS UMR 12, CEA
Saclay, Gif-sur-Yvette, France; Université Paris Cité, Paris, France
ROBERTO BAHENA-CERON • Architecture et Réactivité de l’ARN, CNRS 9002, Université de
Strasbourg, Strasbourg, France
ANKE BECKER • Center for Synthetic Microbiology (SYNMIKRO), University of Marburg,
Marburg, Germany
ANAÏS BLACHE • CEA, CNRS, Aix Marseille University Lab of Microbial Ecology of the
Rhizosphere (LEMiRE), UMR7265 BIAM, Saint-Paul-lez-Durance, France
SYLWIA BLOCH • Department of Molecular Biology, Faculty of Biology, University of Gdansk,
Gdansk, Poland
SABINE BRANTL • Matthias-Schleiden-Institut für Genetik, Bioinformatik und Molekulare
Botanik, AG Bakteriengenetik, Friedrich-Schiller-Universit€ at Jena, Jena, Germany
PAUL BRIAUD • Department of Biological Sciences, Ohio University, Athens, OH, USA
FLORENT BUSI • Université Paris Cité, Paris, France; BFA, UMR 8251, Université Paris cité,
CNRS, Paris, France
RONAN K. CARROLL • Department of Biological Sciences, Ohio University, Athens, OH, USA;
Molecular and Cellular Biology Program, Ohio University, Athens, OH, USA; Infectious
and Tropical Disease Institute, Ohio University, Athens, OH, USA
BÉATRICE CHANE-WOON-MING • Architecture et Réactivité de l’ARN, CNRS 9002,
Université de Strasbourg, Strasbourg, France
MEHAK CHAUHAN • Centre for Engineering Biology, School of Biological Sciences, University
of Edinburgh, Edinburgh, UK
MOUMITA ROY CHOWDHURY • Department of Biochemistry, RNA Group, Université de
Sherbrooke, Sherbrooke, QC, Canada
LIANG-CUI CHU • Centre for Engineering Biology, School of Biological Sciences, University of
Edinburgh, Edinburgh, UK
SOFIA ESTEBAN-SERNA • Centre for Engineering Biology, School of Biological Sciences,
University of Edinburgh, Edinburgh, UK
ELENA EVGUENIEVA-HACKENBERG • Institute of Microbiology and Molecular Biology,
University of Giessen, Giessen, Germany
NATALIA ISABEL GARCÍA-TOMSIG • Structure, Dynamics and Function of Rhizobacterial
Genomes (RhizoRNA Lab), Estacion Experimental del Zaidı́n, Consejo Superior de
Investigaciones Cientı́ficas (CSIC), Granada, Spain
YVONNE GÖPEL • Department of Microbiology, Immunobiology and Genetics, Max Perutz
Labs, University of Vienna, Vienna Biocenter (VBC), Vienna, Austria; Lexogen, Campus
Vienna Biocenter 5, Vienna, Austria
BORIS GÖRKE • Department of Microbiology, Immunobiology and Genetics, Max Perutz Labs,
University of Vienna, Vienna Biocenter (VBC), Vienna, Austria

ix
x Contributors

SANDER GRANNEMAN • Centre for Engineering Biology, School of Biological Sciences,

University of Edinburgh, Edinburgh, UK
INAM UL HAQ • Matthias-Schleiden-Institut für Genetik, Bioinformatik und Molekulare
Botanik, AG Bakteriengenetik, Friedrich-Schiller-Universit€at Jena, Jena, Germany
LUCAS HERRGOTT • Architecture et Réactivité de l’ARN, CNRS 9002, Université de
Strasbourg, Strasbourg, France
DEBORAH HINTON • Gene Expression and Regulation Section, Laboratory of Biochemistry
and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, MD, USA
ERIK HOLMQVIST • Department of Cell and Molecular Biology, Biomedical Centre, Uppsala
University, Uppsala, Sweden
JOSE JARAMILLO-PONCE • Architecture et Réactivité de l’ARN, CNRS 9002, Université de
Strasbourg, Strasbourg, France
JOSÉ IGNACIO JIMÉNEZ-ZURDO • Structure, Dynamics and Function of Rhizobacterial
Genomes (RhizoRNA Lab), Estacion Experimental del Zaidı́n, Consejo Superior de
Investigaciones Cientı́ficas (CSIC), Granada, Spain
BIRGITTE HAAHR KALLIPOLITIS • Department of Biochemistry and Molecular Biology,
University of Southern Denmark, Odense, Denmark
MUNA AYESHA KHAN • Department of Microbiology, Immunobiology and Genetics, Max
Perutz Labs, University of Vienna, Vienna Biocenter (VBC), Vienna, Austria; Lexogen,
Campus Vienna Biocenter 5, Vienna, Austria
MAXIMILIAN P. KOHL • Architecture et Réactivité de l’ARN, CNRS 9002, Université de
Strasbourg, Strasbourg, France
JENNIFER KOTHE • Institute of Microbiology and Molecular Biology, University of Giessen,
Giessen, Germany
ANTONIO LAGARES JR • Center for Synthetic Microbiology (SYNMIKRO), University of
Marburg, Marburg, Germany; Laboratorio de Fisiologı́a y Genética de Bacterias
Beneficiosas para Plantas (LFGBBP), Centro de Bioquı́mica y Microbiologı́a del Suelo,
Departamento de Ciencia y Tecnologı́a, Universidad Nacional de Quilmes – CONICET,
Bernal, Argentina
ANTONY LECHNER • Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN,
UPR 9002, Strasbourg, France
NATALIA LEWANDOWSKA • Department of Molecular Biology, Faculty of Biology, University of
Gdansk, Gdansk, Poland
EVA MARIA STERNKOPF LILLEBÆK • Department of Biochemistry and Molecular Biology,
University of Southern Denmark, Odense, Denmark
ALEKSANDRA ŁUKASIAK • Department of Molecular Biology, Faculty of Biology, University of
Gdansk, Gdansk, Poland
XING LUO • Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD,
USA
PAULINA MACH • Department of Molecular Biology, Faculty of Biology, University of Gdansk,
Gdansk, Poland
NADIM MAJDALANI • Laboratory of Molecular Biology, National Cancer Institute, Bethesda,
MD, USA
STEFANO MARZI • Architecture et Réactivité de l’ARN, CNRS 9002, Université de
Strasbourg, Strasbourg, France
ERIC MASSÉ • Department of Biochemistry, RNA Group, Université de Sherbrooke,
Sherbrooke, QC, Canada
 Contributors xi

KEVIN MOSCA • Laboratoire Léon Brillouin LLB, CEA, CNRS UMR 12, CEA Saclay,
Gif-sur-Yvette, France; Synchrotron SOLEIL, L’Orme des Merisiers Saint Aubin, Gif-sur-
Yvette, France; SANOFI, Marcy-l’Etoile, France
PETER MÜLLER • Matthias-Schleiden-Institut für Genetik, Bioinformatik und Molekulare
Botanik, AG Bakteriengenetik, Friedrich-Schiller-Universit€at Jena, Jena, Germany
BOŻENA NEJMAN-FALEŃCZYK • Department of Molecular Biology, Faculty of Biology,
University of Gdansk, Gdansk, Poland
AGUSTÍN ORMAZÁBAL • Departamento de Ciencia y Tecnologı́a, Universidad Nacional de
Quilmes, Bernal, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Cientı́ficas
y Técnicas, CONICET, CABA, Buenos Aires, Argentina
JULIANA PALMA • Departamento de Ciencia y Tecnologı́a, Universidad Nacional de Quilmes,
Bernal, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Cientı́ficas y Té
cnicas, CONICET, CABA, Buenos Aires, Argentina
GUSTAVO PIERDOMINICI-SOTTILE • Departamento de Ciencia y Tecnologı́a, Universidad
Nacional de Quilmes, Bernal, Buenos Aires, Argentina; Consejo Nacional de
Investigaciones Cientı́ficas y Técnicas, CONICET, CABA, Buenos Aires, Argentina
GIANLUCA PREZZA • Helmholtz Institute for RNA-based Infection Research (HIRI),
Helmholtz Centre for Infection Research (HZI), Würzburg, Germany
PUJITHA RAJA • Division of Pathway and Infection Medicine, University of Edinburgh,
Edinburgh, UK
PASCALE ROMBY • Architecture et Réactivité de l’ARN, CNRS 9002, Université de
Strasbourg, Strasbourg, France
ROBINA SCHEUER • Institute of Microbiology and Molecular Biology, University of Giessen,
Giessen, Germany
ALEXANDRA SCHILDER • Department of Microbiology, Immunobiology and Genetics, Max
Perutz Labs, University of Vienna, Vienna Biocenter (VBC), Vienna, Austria; Doctoral
School in Microbiology and Environmental Science, University of Vienna, Vienna, Austria
THOMAS SØNDERGAARD STENUM • Department of Cell and Molecular Biology, Biomedical
Centre, Uppsala University, Uppsala, Sweden
CHIN-HSIEN TAI • Laboratory of Molecular Biology, Center for Cancer Research, National
Cancer Institute, National Institutes of Health, Bethesda, MD, USA
FLORIAN TURBANT • Laboratoire Léon Brillouin LLB, CEA, CNRS UMR 12, CEA Saclay,
Gif-sur-Yvette, France; Synchrotron SOLEIL, L’Orme des Merisiers Saint Aubin, Gif-sur-
Yvette, France
CLAUDIO VALVERDE • Laboratorio de Fisiologı́a y Genética de Bacterias Beneficiosas para
Plantas (LFGBBP), Centro de Bioquı́mica y Microbiologı́a del Suelo, Departamento de
Ciencia y Tecnologı́a, Universidad Nacional de Quilmes – CONICET, Bernal, Provincia
de Buenos Aires, Argentina
JAN WA€ HLING • Institute of Microbiology and Molecular Biology, University of Giessen,
Giessen, Germany
GRZEGORZ WE˛GRZYN • Department of Molecular Biology, Faculty of Biology, University of
Gdansk, Gdansk, Poland
ARNE WERNER • Institute for Biochemistry and Molecular Biology, Department of Chemistry,
Faculty of Mathematics, Computer Science and Natural Science, Hamburg University,
Hamburg, Germany; Institute for Microbiology, Faculty for Mathematics and Natural
Science, University Kiel, Kiel, Germany
WOJCIECH WESOŁOWSKI • Department of Molecular Biology, Faculty of Biology, University of
Gdansk, Gdansk, Poland
xii Contributors

ALEXANDER J. WESTERMANN • Helmholtz Institute for RNA-based Infection Research

(HIRI), Helmholtz Centre for Infection Research (HZI), Würzburg, Germany; Institute of
Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany
FRANK WIEN • Laboratoire Léon Brillouin LLB, CEA, CNRS UMR 12, CEA Saclay, Gif-
sur-Yvette, France
PHILIPPE WOLFF • Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN,
UPR 9002, Strasbourg, France; Plateforme protéomique Strasbourg Esplanade FRC1589
du CNRS, Université de Strasbourg, Strasbourg, France
PENGZHI WU • Hefei National Laboratory for Physical Sciences at the Microscale, School of
Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of
China, Hefei, Anhui, People’s Republic of China; Department of Biology, Institute of
Biochemistry, ETH Zürich, Zürich, Switzerland
LINGNA YANG • Hefei National Laboratory for Physical Sciences at the Microscale, School of
Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of
China, Hefei, Anhui, People’s Republic of China
SUNG-HUAN YU • Institute of Precision Medicine, National Sun Yat-sen University,
Kaohsiung, Taiwan; School of Medicine, College of Medicine, National Sun Yat-sen
University, Kaohsiung, Taiwan
 Part I

sRNA Discovery
 Chapter 1

RNA Extraction from Gram-Positive Bacteria Membrane

Vesicles Using a Polymer-Based Precipitation Method
Paul Briaud and Ronan K. Carroll

Abstract
Investigations into the biological role and composition of bacterial extracellular vesicles have grown in
popularity in recent years. Vesicles perform a variety of functions during interactions with eukaryotic host
cells, ranging from antibiotic resistance to immune modulation. It is necessary to isolate vesicles in order to
understand their biological functions. Here we describe a polymer-based precipitation method allowing
high-yield isolation of extracellular vesicles and their cargo RNA from the Gram-positive bacterium
Staphylococcus aureus.

Key words Membrane vesicle, RNA, Gram-positive, Staphylococcus, Extraction, MV, OMV

1 Introduction

Extracellular vesicles (EVs) are membrane-derived lipid spheres

produced by all domains of life. EVs have different names depend-
ing on the organism they originate from and the subcellular loca-
tion of production. In Gram-negative bacteria, EVs are typically
called outer-membrane vesicles (OMVs), as they are pinched off
from the outer-membrane [1]. For decades, EVs from Gram-
positive bacteria (also called MVs for membrane vesicles) were
neglected due to the presence of a thick cell wall at the surface,
which was thought to hinder the production of MVs [2, 3]. In the
late 2000s, the first studies showed that Gram-positive bacteria
could produce MVs with the same features as described for Gram-
negative bacteria: (i) MVs are made of a lipid bilayer, which origi-
nates from the bacterial membrane; (ii) the cargo is composed of
nucleic acids (DNA and RNA) and proteins, which appear to be
actively sorted into vesicles; (iii) MVs can deliver their content into
surrounding cells and trigger a wide array of responses depending
on the cargo packaged [2, 4–9]. Similar to OMVs, the precise
mechanism of MV production remains elusive and species-specific

Véronique Arluison and Claudio Valverde (eds.), Bacterial Regulatory RNA: Methods and Protocols, Methods in Molecular Biology,
vol. 2741, https://doi.org/10.1007/978-1-0716-3565-0_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

3
4 Paul Briaud and Ronan K. Carroll

genetic determinants have been implicated [10]. MV functions are

closely correlated to the cargo packaged as toxins, proteases, and
other virulence factors can be released in vesicles from pathogenic
bacteria [2, 8, 11]. Interestingly, nucleic acid content such as RNA
has been shown to play a role in the host cell response.
OMV-associated RNA can trigger pro-inflammatory response via
Rig-1-like receptor (RLR) recognition [12]. Also, OMVs from
Pseudomonas aeruginosa carry a small RNA that is predicted to
interact with eukaryotic mRNA involved in pro-inflammatory cyto-
kine production [13]. More studies are needed to address the role
of MV-associated RNAs in interactions with host cells. Isolation/
separation of vesicles from secreted proteins in the supernatant is
essential to studying their composition and their interactions with
other cells. Different methods have been described to isolate vesi-
cles for eukaryotic cells and Gram-negative bacteria, such as diafil-
tration coupled with ultracentrifugation [14]. Diafiltration
removes particles smaller than the filter size used and concentrates
the remaining solution. Then, ultracentrifugation uses centrifugal
force to pellet vesicles while less dense particles and molecules
remain in the upper phases. Such methods require working with
large starting volumes of cultures and rely on expensive equipment
such as tangential flow filtration devices and ultracentrifuges.
Polymer-based precipitation methods, on the other hand, use
volume-excluding polymers to decrease the solubility of vesicles
and allow isolation by low-speed centrifugation. Precipitation-
based protocols have become increasingly popular over the past
few years as they can isolate and purify vesicles from culture super-
natants or body fluids faster and more efficiently than previous
methods and use common laboratory equipment, reducing the
cost and allowing for higher throughput assays than traditional
ultracentrifugation technics. Here, we describe a polymer-based
precipitation method to isolate Gram-positive bacterial MVs using
the ExoQuick-TC buffer and the subsequent steps to isolate MV
RNA. We use Staphylococcus aureus as the working model.

2 Materials

Prepare all solutions with purified deionized water and sterilize by

autoclaving if needed. All solutions can be stored at room
temperature.
1. Tryptic Soy Broth (TSB), autoclaved.
2. 250 mL sterilized glass flask
3. 1.5 mL microcentrifuge tubes
4. 50 mL conical tubes
5. 37 °C shaking incubator
 RNA Extraction from Extracellular Vesicles 5

6. Vortex.
7. Pierce™ BCA Protein Assay kit (Thermo Scientific, 23225).
8. β-Mercaptoethanol (Sigma, M6250),
9. Macrosep Advance 100 kDa concentrators (Pall,
MAP100C37).
10. 0.22 μm filter membranes
11. Ethanol 80%.
12. Isopropanol.
13. 37 °C water bath.
14. 50 mL syringes.
15. ExoQuick-TC buffer (System Bioscience, ExoTC10A-1).
16. Phosphate-buffered saline (PBS 1×): 137 mM NaCl, 2.7 mM
KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, autoclaved.
17. miRNeasy kit (Qiagen, 217604).
18. RLT buffer from miRNeasy kit.
19. AL buffer from miRNeasy kit.
20. gDNA column eliminator from miRNeasy kit.
21. RNeasy column from miRNeasy kit.
22. RWT buffer from miRNeasy kit.
23. RPE buffer from miRNeasy kit.
24. Turbo DNAse kit (Invitrogen AM1907).
25. Deactivation reagent from Turbo DNAse kit.
26. RNAse-free water, autoclaved.
27. FM™ 4–64 Dye (Invitrogen™, T3166).
28. Refrigerated centrifuge with swinging buckets.
29. Refrigerated tabletop centrifuge.
30. Optional: 2100 Agilent Bioanalyzer and Agilent RNA 6000
Pico Kit (Agilent, 5067–1513).

3 Methods

3.1 Concentration of Our lab and others [4, 15] were successful in isolating MVs from
MVs from Culture S. aureus using a precipitation method. If working with a different
Supernatants bacterial species, we recommend adapting growth conditions to
your bacterium of interest.
1. Grow bacterial cultures (25 mL TSB in a 250 mL flask) with
shaking (250 rpm) until mid-exponential (see Note 1).
2. Centrifuge the 25 mL culture in a 50 mL conical tube at
3000 × g for 15 min at 4 °C.
6 Paul Briaud and Ronan K. Carroll

3. Filter sterilize the supernatant through a 0.22 μm filter with a

50 mL syringe.
4. Transfer the cell-free supernatant (~ 15 mL) to the 100 kDa
concentrators (see Note 2).
5. Centrifuge at 3000 × g for 60 min at room temperature in a
swing bucket rotor (see Note 3).

3.2 Precipitation 1. Transfer the concentrated supernatants into a 1.5 mL micro-

of MVs centrifuge tube, and estimate the volume recovered with a
micropipette.
2. Add ExoQuick-TC buffer to the concentrated supernatants for
a final ratio of 1:5 (ExoQuick-TC buffer:supernatant) (e.g., if
200 μL of concentrated supernatant was recovered, add 50 μL
of ExoQuick-TC buffer).
3. Mix well by vortexing for ~10 s, and incubate on ice at 4 °C
overnight.
4. Centrifuge the mixture at 1500 × g for 30 min at 4 °C. A
white/beige pellet should be visible.
5. Carefully remove the supernatant by pipetting.
6. Centrifuge again for a few seconds at 1500 × g, and pipette off
the remaining supernatant.
7. Resuspend the MV pellet in a suitable volume of 1× PBS (ice
cold) (see Note 4).
8. Use the BCA assay kit to estimate the number of vesicles
isolated (see Note 5).

3.3 Isolation of RNA We usually perform the RNA extraction directly from the MV pellet
from MVs with the from step 6 in Subheading 3.2. Phosphate-buffered saline used to
miRNeasy Kit resuspend MVs could reduce/inhibit the lysis step.
1. Add 260 μL of RLT buffer containing β-mercaptoethanol.
2. Vortex for 1 min.
3. Add 80 μL of buffer AL and mix thoroughly with a pipet.
4. Incubate at room temperature for 3 min.
5. Transfer the lysate to a gDNA eliminator column and spin
down at 8000 × g for 30 s.
6. Add one volume (340 μL) of isopropanol to the flow through
and pipet up and down 8 times.
7. Transfer the mixture onto an RNeasy column placed on a
collection tube.
8. Centrifuge at 8000 × g for 15 s.
9. Pipet 700 μL of RWT buffer and spin down at 8000 × g for
15 s.
 RNA Extraction from Extracellular Vesicles 7

10. Wash the column with 500 μL of buffer RPE and centrifuge at
8000 × g for 15 s.
11. Add 500 μL of EtOH 80% and centrifuge at 8000 × g for 15 s.
12. Place the RNeasy column onto a new collection tube and dry
the column at full speed for 1 min.
13. Place the RNeasy column onto a clean 1.5 mL microcentrifuge
tube, and add 35 μL of RNAse-free water.
14. Incubate for 1 min at room temperature.
15. Elute MV RNA at full speed for 1 min.
16. Add 4 μL of 10x TurboDNase buffer and 1 μL of TurboDNase
enzyme to remove DNA.
17. Incubate at 37 °C for 30 min.
18. Add 4 μL of deactivation reagent and mix by pipetting up
and down.
19. Centrifuge for 2.5 min at 10,000 × g.
20. Transfer the supernatant (~ 30 μL) into a fresh 1.5 mL
microcentrifuge tube.
21. Determine the concentration and size of MV RNA with Agi-
lent 2100 Bioanalyzer (see Note 6).

4 Notes

1. MVs can be formed from active budding at the bacterial mem-

brane or passively, with auto-assembly of bacterial membrane
fragments upon cell death. It is preferred to work with bacteria
during mid-exponential phase as the cell lysis is minimal and
the amount of MVs from cell lysis is reduced. If working at a
later time point, samples may contain both active and passive
MV forms.
2. Be careful to load the same volume of cell-free supernatant,
particularly when working with supernatants from different
conditions (e.g., WT vs. mutant). This allows a direct compari-
son of the MV yield between conditions.
3. If using a swing bucket rotor, the position of the concentrator
membrane with respect to the rotor axis is not important.
However, if using a fixed rotor, the concentrator membrane
needs to be perpendicular to the rotor axis.
4. The volume of PBS to add will depend on the amount of MV
produced by the bacteria. We typically resuspend MVs in
500 μL of 1× PBS. After resuspending the MV pellet, it is
also possible to increase the purity of the sample by performing
a density gradient purification (e.g., Optiprep).
8 Paul Briaud and Ronan K. Carroll

5. Characterization of MV content should be performed after

each isolation to estimate the concentration of vesicles isolated.
As MVs are composed of proteins and lipids, we routinely use
the Pierce BCA protein assay kit for protein concentration
estimation and the fluorescent FM4-64 dye for lipid detection.
We then normalize MVs by their protein and/or lipid concen-
tration when using them for subsequent assays. We highly
recommend performing, at least once, a nanoparticle tracking
analysis (e.g., LM10 NanoSight) and/or transmission elec-
tronic microscope analysis to visualize MVs directly and ensure
that isolation occurred correctly (Fig. 1).
6. The profile and concentration of MV RNA obtained can differ
depending on the bacterial species studied. For S. aureus, we
routinely get concentrations in the nanograms / μL range. The
RNA profile shows an abundance of short-sized RNAs (Fig. 2).
The RIN (RNA integrity number) cannot be computed as no
defined rRNA bands can be determined. RNA isolated can be
used for sequencing.

Fig. 1 Nanoparticle tracking analysis (NTA) of MVs isolated from S. aureus. Most
of the vesicles concentrate around a particle size of ~82 nm, which is the usual
size for vesicles isolated from Gram-positive bacteria
 RNA Extraction from Extracellular Vesicles 9

Fig. 2 RNA profile of MV RNA from S. aureus grown at 37 °C for 3 h. A high abundance of short-sized RNAs is
observed. The Agilent RNA 6000 Pico Kit was used

References
1. Roier S, Zingl FG, Cakar F, Schild S (2016) 7. Lee J, Lee E-Y, Kim S-H, Kim D-K, Park K-S,
Bacterial outer membrane vesicle biogenesis: a Kim KP, Kim Y-K, Roh T-Y, Gho YS (2013)
new mechanism and its implications. 6. Microb Staphylococcus aureus extracellular vesicles
Cell 3:257–259 carry biologically active β-lactamase. 6. Antimi-
2. Briaud P, Carroll RK (2020) Extracellular- crob Agents Chemother 57:2589–2595
vesicle (EV) biogenesis and functions in 8. Bitto NJ, Cheng L, Johnston EL, Pathirana R,
Gram-positive bacteria. Infect Immun. Phan TK, Poon IKH, O’Brien-Simpson NM,
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3. Brown L, Wolf JM, Prados-Rosales R, Casade- (2021) Staphylococcus aureus membrane vesi-
vall A (2015) Through the wall: extracellular cles contain immunostimulatory DNA, RNA
vesicles in Gram-positive bacteria, mycobac- and peptidoglycan that activate innate immune
teria and fungi. 10. Nat Rev Microbiol 13: receptors and induce autophagy. J Extracell
620–630 Vesicles 10:e12080
4. Briaud P, Frey A, Frey A, Marino EC, Bastock 9. Dean SN, Rimmer MA, Turner KB, Phillips
RA, Zielinski RE, Wiemels RE, Keogh RA, DA, Caruana JC, Hervey WJ, Leary DH, Wal-
Murphy ER, Shaw LN, Shaw LN, Carroll RK per SA (2020) Lactobacillus acidophilus mem-
(2021) Temperature influences the composi- brane vesicles as a vehicle of Bacteriocin
tion and cytotoxicity of extracellular vesicles delivery. Front Microbiol 11:710
in Staphylococcus aureus. https://doi.org/ 10. Lee E-Y, Choi D-Y, Kim D-K, Kim J-W, Park
10.1128/msphere.00676-21 JO, Kim S, Kim S-H, Desiderio DM, Kim Y-K,
5. Choi E-J, Lee HG, Bae I-H, Kim W, Park J, Kim K-P, Gho YS (2009) Gram-positive bacte-
Lee TR, Cho E-G (2018) Propionibacterium ria produce membrane vesicles: proteomics-
acnes-derived extracellular vesicles promote based characterization of Staphylococcus
acne-like phenotypes in human epidermis. 6. J aureus-derived membrane vesicles. 24. Proteo-
Invest Dermatol 138:1371–1379 mics 9:5425–5436
6. Hong S-W, Choi E-B, Min T-K, Kim J-H, Kim 11. Choi JH, Moon CM, Shin T-S, Kim EK,
M-H, Jeon SG, Lee B-J, Gho YS, Jee Y-K, McDowell A, Jo M-K, Joo YH, Kim S-E,
Pyun B-Y, Kim Y-K (2014) An important role Jung H-K, Shim K-N, Jung S-A, Kim Y-K
of α-hemolysin in extracellular vesicles on the (2020) Lactobacillus paracasei-derived extra-
development of atopic dermatitis induced by cellular vesicles attenuate the intestinal inflam-
Staphylococcus aureus. 7. PLoS One 9: matory response by augmenting the
e100499 endoplasmic reticulum stress pathway. Exp
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Mol Med. https://doi.org/10.1038/s12276- 14. Brennan K, Martin K, FitzGerald SP,

019-0359-3 O’Sullivan J, Wu Y, Blanco A, Richardson C,
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Immune modulation by bacterial outer mem- for the isolation and separation of extracellular
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Scharfe M, Gerber SA, Mielcarz DW, Demers Lebtig M, Fehrenbacher B, Schaller M,
EG, Dolben EL, Hammond JH, Hogan DA, Ebner P, Nega M, Otto M, Kretschmer D,
Stanton BA (2016) A novel mechanism of Peschel A (2018) The mechanism behind bac-
host-pathogen interaction through sRNA in terial lipoprotein release: phenol-soluble mod-
bacterial outer membrane vesicles. 6. PLoS ulins mediate toll-like receptor 2 activation via
Pathog 12:e1005672 extracellular vesicle release from Staphylococ-
cus aureus. 6. MBio 9:e01851–e01818
 Chapter 2

Extraction and Purification of Outer Membrane Vesicles

and Their Associated RNAs
Anaı̈s Blache and Wafa Achouak

Abstract
Outer membrane vesicles (OMVs), produced by Gram negative-bacteria and sRNAs, are key players in cell-
to-cell communication and interactions of bacteria with the environment. OMVs act as information carriers
and encapsulate various molecules such as proteins, lipids, metabolites, and RNAs. OMVs and sRNAs play a
broad range of functions from pathogenesis to stress resistance, to biofilm formation and both mediate
interkingdom signaling. Various studies indicate that there is a mechanism of intercellular communication
mediated by OMV-derived bacterial RNAs that is conserved among certain bacterial species. Here we
describe methods for the extraction and purification of vesicles produced by Gram-negative bacteria, such
as Pseudomonas brassicacearum and Escherichia coli, and address methods for the extraction of OMVs-
derived sRNA and techniques for the analysis of sRNAs.

Key words OMVs, sRNA, Gram-negative bacteria, Extraction methods, Purification methods,
OMVs-derived RNA extraction

1 Introduction

Gram-negative bacteria produce outer membrane vesicles (OMVs)

by pinching off portions of the outer membrane. OMVs, key
players in cell-to-cell communication, carry various molecules like
proteins, RNAs, secondary metabolites, and lipids [1, 2]. OMVs act
as information carriers. A lipid bilayer protects the cargo from
environmental stress. For some species, OMVs can activate quorum
sensing, immune regulation, and biofilm formation [3, 4]. Secretion
of OMVs allows rapid adaptation to environmental changes by
transporting signaling molecules, misfolded proteins, toxins, and
virulence factors [1, 5–10].
Analysis of the OMV profile of human pathogenic bacteria
reveals the presence of sRNAs that have been shown to be inter-
nalized by human cells [11, 12]. Pseudomonas aeruginosa OMVs
carrying sRNAs have been shown to transfer their sRNAs to human

Véronique Arluison and Claudio Valverde (eds.), Bacterial Regulatory RNA: Methods and Protocols, Methods in Molecular Biology,
vol. 2741, https://doi.org/10.1007/978-1-0716-3565-0_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

11
12 Anaı̈s Blache and Wafa Achouak

airway epithelial cells and in the mouse lung, attenuating their

immune response [13].
sRNAs are key players in cell-to-cell communication, including
plant–pathogen interactions [14]. A 115 nt sRNA from the plant
pathogen Xanthomonas has been reported to regulate 63 genes
associated with signaling and virulence functions in pepper
[15]. In the absence of any evidence at present, we can expect
bacterial sRNAs to be transferred from OMVs into plant cells.
Functional studies of OMVs and identification of their cargo
are challenging because of the slow extraction process and low
purification yield. Most experiments require purified vesicles, for
example, in the case of host–pathogen interactions. The most
commonly used extraction method is ultracentrifugation. Objects
larger than 0.45 μm are eliminated by filtration, but heavy objects
like membrane pieces, pili, and flagella remain. During ultracentri-
fugation, these objects will be pelleted and disturb the purity of the
OMVs. According to the purpose of the study, a purification step is
mandatory. The volume of OMVs required will influence the choice
of extraction and purification method. The ultracentrifugation pro-
cess requires less equipment than ultrafiltration, but the volume of
the supernatant is limiting. Small-scale extraction and purification
involve the ultracentrifugation process and density gradient purifi-
cation, or the use of a commercial extraction and purification kit.
Ultrafiltration allows to process culture supernatant without vol-
ume boundaries and can be used to enhance the extraction yield by
concentrating a large volume of supernatant. This method has been
used for OMVs produced by Escherichia coli with a 100 kDa mem-
brane [16, 17] and 70 kDa [18]. Ultrafiltration and gel filtration
columns compose the large-scale process. Once the OMVs have
been purified, their RNAs can be extracted, quantified, and quality
checked, and then used for RT-PCR/qRT-PCR, Northern blot, or
RNA-Seq analysis. The methods described here are efficient with
Gram-negative bacteria such as Pseudomonas brassicacearum and
Escherichia coli.

2 Materials

2.1 Bacterial Growth 1. Tryptic Soy Broth (TSB) diluted tenfold or LB Broth.
2. Glass culture flasks.
3. Incubator.

2.2 OMVs Extraction 1. Centrifuge.

2.2.1 Ultracentrifugation 2. Fixed angle rotor 5Jla 9 1000 or Jla 16 250.
3. Sterile 1 L or 200 mL bottles able to spin at 10,000 × g.
4. PES Filtration units 0.45 μm or 0.22 μm.
 Gram-Negative Outer Membrane Vesicles and Associated RNAs 13

5. Vacuum pump.
6. Ultracentrifuge.
7. Fixed angle rotor (i.e., 50.2. Ti).
8. Ultracentrifuge bottles (26.3 mL) capable of spinning at
150,000 × g.
9. Phosphate-buffered saline (PBS), pH 7.4.
10. DPBSS (Dulbecco’s phosphate buffered supplemented in salt):
0.901 mM CaCl2, 2.682 mM KCl, 0.4918 mM MgCl2 hexa-
hydrate, 136.8 mM NaCl2 and 15.21 mM Na3PO4 dibasic).

2.2.2 Ultrafiltration 1. Ultrafiltration cassette: Pellicon Biomax 30 kDa 0.57 m2 D

screen.
2. Pellicon cassette acrylic holder and assembly.
3. 5 L of 0.1 M NaOH.
4. 10 L of ultrapure H2O.
5. Sterile bottle (5 L).
6. Peristaltic pump.
7. Autoclavable tubing adaptable to the peristaltic pump.
8. Perforated caps.
9. 2 clamps for tubing.
10. pH paper.

2.2.3 Ion-Exchange- 1. ExoBacteria™ OMV Isolation Kit for Gram-negative bacteria.

Based Columns 2. Cold room.
3. Shaker.

2.3 OMVs 1. Optiprep density gradient medium.

Purification 2. Ultracentrifuge.
3. Fixed angle rotor (e.g., 50.2. Ti).
4. 8 mL ultracentrifugation tubes capable of spinning at
150,000 × g.
5. Sterile 1 mL syringes.
6. Needles: Sterican 0.70 × 40 mm 22G × 11/2.
7. Ultracentrifuge.
8. Rotor MLA-55.
9. Ultracentrifuge tubes (26.3 mL) capable of spinning at
200,000 × g.

2.4 OMVs Analysis 1. 5 μg/mL lipophilic fluorescent dye FM1–43.

2. Multimode microplate reader.
14 Anaı̈s Blache and Wafa Achouak

3. Zetasizer.
4. 50–2000 μL UVette 220–1600 nm.
5. Nanoparticle Tracker Analyzer (NTA).
6. Transmission electron microscope (TEM).
7. 0.1% Phosphotungstic acid.
8. Electrophoresis cell.
9. Generator.
10. Imperial Protein Stain.
11. SDS 20x MES Running Buffer.
12. NUPAGE 10% Bis-tris gel.
13. LDS sample buffer.
14. Protein all blue standard.
15. Bradford protein estimation kit.

2.5 RNA Extraction 1. TRIzol Reagent.

and Analysis 2. Chloroform.
2.5.1 RNA Extraction 3. Isopropanol.
Methods 4. Ethanol 75%.
5. RNAse-free water.
6. Centrifuge.
7. Vortex.
8. MiVac DNA Concentrator.
9. RNAse A/T1.
10. Nanovue spectrophotometer.
11. GIGAPUR RNAse-DEKONTA OFF.

2.5.2 Quality Control 1. Qubit 4 fluorometer.

2. Qubit RNA IQ Assay Kit.
3. Qubit RNA BR Assay Kit.

2.5.3 RNA Analysis 1. TURBO DNA-free Kit.

2. Transcriptor First Strand cDNA Synthesis Kit.
3. Q-PCR LightCycler 480 SYBR Green I Master kit.
4. Light Cycler 480 or other real-time PCR system.
5. Thermocycler.
 Gram-Negative Outer Membrane Vesicles and Associated RNAs 15

3 Methods

3.1 OMVs Extraction 1. Start an overnight culture from an isolated clone in 10 mL of

tenfold diluted Tryptic Soy Broth (TSB 1/10) or LB Broth.
3.1.1 Bacterial Growth
2. Prepare sterile flasks of 1/10 TSB or LB. The volume should be
adjusted according to the extraction method. Choose 80%
aeration of the volume.
• Ultracentrifugation: 600 mL in a 3 L flask (see Note 1).
• Ultrafiltration: 7 × 600 mL in 3 L flasks.
• Exobacteria OMVs isolation kit: 100 mL in a 500 mL flask.
• Centrifugation steps: 600 mL in 3 L flask.
3. Grow the cultures at the appropriate temperature and at
140 rpm for 24 h (see Note 2).
4. Dispense the culture into 200 mL or 1 L sterile bottles. To
separate vesicles and cells, centrifuge the cultures twice at
10, 000 × g for 30 min. Use a new sterile bottle for each
centrifugation step, and discard the bacterial pellet.
5. Filter the supernatant through 0.45 μm PES filters. If the
OMVs are smaller than 200 nm, filter the supernatant through
a 0.22 μm PES filter unit.
6. After this step, a cell-free supernatant is obtained (see Notes 3
and 4).

3.1.2 Ultracentrifugation The ultracentrifugation process is the most common method for
extracting OMVs. This technique is convenient and repeatable but
restricts the volume of supernatant used.
1. Transfer the cell-free supernatant into sterile 26.3 mL tubes for
ultracentrifugation. The vials may be sterilized with 15% H2O2
and washed twice with sterile water (see Note 5).
2. Centrifuge the supernatant at 150,000 × g for 2 h at 4 °C (see
Note 6).
3. Filter the phosphate-buffered saline (PBS at pH 7.4) through
0.22 μm filter units.
4. After ultracentrifugation, the OMVs are concentrated in the
pellet. Discard the supernatant and resuspend the pellets with
the correct volume of sterile PBS (see Note 7).

3.1.3 Ultrafiltration The ultrafiltration technique is easy and effective but requires the
use of rather expensive equipment such as the acrylic ultrafiltration
cassette holder and the ultrafiltration membrane.
1. Place the Pellicon membrane into the holder, and screw the
holder nuts strongly with a wrench to avoid any leakage (see
Note 8).
16 Anaı̈s Blache and Wafa Achouak

1. Flushing 2. Concentrating cell-free supernatant

2. Concentrating cell-free supernatant 3. Washing

Created with BioRender.com

Fig. 1 Ultrafiltration technique: (1) Flushing step with H2O, (2) Concentrating supernatant, (3) Cleaning step
with NaOH

2. Connect the inlet of the peristaltic pump with a 10 L bottle of

ultrapure H2O and the retentate and permeate to trash bottles
(Fig. 1). Start the pump at a low flow rate. Check the system for
leaks (see Note 9).
3. The first step is to flush the membrane with ultrapure H2O to
avoid any residual storage solution (0.1 M NaOH). Determine
the pH of the permeate and retentate before flushing. Start the
pump at a maximum flow rate (120 rpm) to flush the mem-
brane with 3 L of ultrapure H2O.
4. Close the retentate with a clamp, the entire flow must pass
through the membrane and flush with 2 L of ultrapure
H2O. Check the pH of the permeate and retentate; if it is
equivalent to pH of H2O, stop flushing. Otherwise, keep flush-
ing the membrane.
5. Connect the peristaltic pump and the retentate to the superna-
tant bottle, and the permeate to the trash bottle (Fig. 1).
6. Close the retentate to concentrate the supernatant. When the
desired volume of supernatant is achieved, open the retentate
and close the permeate. Reverse the peristaltic pump direction
to collect all the concentrated supernatant.
 Gram-Negative Outer Membrane Vesicles and Associated RNAs 17

7. Connect the pump to a 0.1 M NaOH bottle, the retentate and

permeate go into trash bottles (Fig. 1). Close the retentate and
clean the membrane with 3 L of NaOH. Open the retentate
and finish the cleaning with 2 L of 0.1 M NaOH. Check the
permeate and retentate pH to make sure that the NaOH passes
through the membrane.
8. Store the Pellicon cassette at 4 °C in 0.1 M NaOH.
9. Re-filter the concentrated supernatant with 0.45 μm or
0.22 μm PES filters.
10. Ultracentrifuge the supernatant at 150,000 × g for 2 h at 4 °C
and resuspend the pellet with the appropriate volume of sterile
PBS (depending on the pellet thickness).

3.1.4 Centrifugation This method allows direct OMV purification with a long and high-
Steps speed centrifugation step [19]. Supernatant volumes can be higher
than those used with ExoBacteria™ OMV Isolation Kit (System
Biosciences).
1. Sterilize DPBSS using 0.22 μm PES filter units.
2. Centrifuge the supernatant at 38,400 × g for 2 h and collect the
pellet in 25 mL of sterile Dulbecco’s phosphate-buffered saline
with added magnesium and calcium [19].
3. Ultracentrifuge OMVs at 100,000 × g for 1 h (6), and resus-
pend the pellet in the appropriate volume of sterile DBPS
or PBS.
After extraction by ultracentrifugation or ultrafiltration meth-
ods, OMVs are cell-free but not pure. The sample may contain
membrane debris, flagella, and pili. To study the vesiculation pro-
cess or to compare vesicle production between strains, the extrac-
tion steps are sufficient. For other investigations as host–OMVs
interactions, OMVs need to be purified. Flagellin is a microbe-
associated molecular pattern capable of activating the plant immune
response [20]. The most commonly used technique for purification
is density gradient purification. For large-scale extraction, gel
extraction columns can be employed. Some like Collins et al.
(2022) [21] use size exclusion chromatography.

3.2 OMVs Density gradient purification is the most commonly used method
Purification for OMV purification [17, 18, 22, 23]. This method provides high
purity but causes low purification yield and poor reproducibility.
3.2.1 Density Gradient
Purification 1. Pool the three 100–200 μL OMVs samples in a 26.3 mL sterile
ultracentrifugation tube. Centrifuge the sample at 150,000 × g
for 2 h at 4 °C. Discard the supernatant and resuspend the
pellet in 410 μL of sterile PBS.
18 Anaı̈s Blache and Wafa Achouak

Gradient density
fractionation
Optiprep Gradient

Collect fractions Lipid content of

20% each fraction
30% 150,000 x g for 20 h (FM1-43)
at 4°C 8E-05

Relative fluorescence
35% 6E-05

per CFU
4E-05
40%
2E-05
45% (OMVs) 0E+00
1 2 3

Fig. 2 Density gradient purification

2. From the Optiprep 60% density gradient medium, prepare

1.5 mL of diluted solutions: 45%, 40%, 35%, 30%, 25%, and
20%. The solutions are diluted with sterile PBS except 45%
solution which is already diluted with 410 μL OMVs sample.
2.5 mL of 20% solution is needed (see Note 10).
3. Sterilize an 8 mL tube with 15% H2O2 and wash twice with
sterile H2O.
4. Carefully and slowly lay 1 mL of each Optiprep solution as
described in Fig. 2 into the sterilized tube, beginning with the
45% solution, in descending order (from 45% to 20%) (see Note
11).
5. Centrifuge the gradient at 150,000 × g for at least 20 h at 4 °C
(see Note 12).
6. After centrifugation, extract each 1 mL fraction with a 1 mL
sterile syringe and needle. Fix the tube and begin extraction
from the top of the gradient (see Note 13).
7. Use the lipophilic fluorescent dye FM1-43 (Thermo Scientific)
to quantify the OMVs in each fraction. Run the assay as
described in the OMVs analysis section. OMVs should be
found in the third and fourth extraction fraction, in the case
of P. brassicacearum.
8. Sterilize a 26.3 mL ultracentrifugation tube with 15% H2O2
and wash twice with sterile H2O.
9. Pool the OMVs-containing fractions into the sterile 26.3 mL
centrifugation tube. Fill the tube with approximately 15 mL of
sterile PBS and equilibrate the tube (±0.05 g).
10. Centrifuge the tube at 200,000 × g for 2 h at 4 °C. Discard the
supernatant and resuspend the pellet with the appropriate
volume of sterile PBS.
 Gram-Negative Outer Membrane Vesicles and Associated RNAs 19

3.3 OMVs Analysis The production of OMVs can be estimated with 5 μg/mL of the
lipophilic fluorescent dye FM1-43 in a 96-well microplate. As there
3.3.1 OMVs
is no calibration curve available, this assay is only an estimate and
Concentration
does not provide the concentration of OMVs. This method is a
quick and efficient way to compare the production of OMVs (see
FM1-43 Dosage
Note 14).
1. Fill each well. Three blanks are required (see Note 15).
2. Incubate for 5 min in the dark.
3. Use a multimode microplate reader. The excitation and emis-
sion wavelengths of FM1-43 are 479 nm and 600 nm,
respectively.

ZetaSizer OMV samples may be analyzed with a ZetaSizer instrument to

determine the size distribution of OMVs. This method confirms
the presence of OMVs in the sample. It allows the size distribution
of nanosized particles and the presence of aggregates to be deter-
mined. The ZetaSizer analysis does not provide the concentration
of OMVs.
1. Put 50 μL of your OMVs sample into a UVette (Eppendorf),
and insert it into the Zetasizer instrument.
2. Use the ZetaSizer software with automatic measurement dura-
tion and three measurements per sample.

Nanoparticles Tracker The Nanoparticle Tracker Analyzer (NTA) is capable of measuring

Analyzer (NTA) the concentration of OMVs directly from the sample. This method
is rapid, easy, precise, and reproducible, but the NTA equipment is
quite expensive [23, 24].
1. Clean and calibrate the NTA machine with ultrapure H2O.
2. Dilute the sample 103-fold with PBS.
3. Triplicate each measure.

Bradford Assay The Bradford assay evaluates the concentration of proteins in the
sample. The sample must be purified to correlate the protein con-
centration with the OMV concentration. Follow the Bradford pro-
tein estimation kit protocol [25].

3.3.2 OMVs Observations Direct observation of OMVs is a good method to confirm sample
purity and OMVs integrity and to determine the size of OMVs.
Transmission Electron Transmission electron microscopy (TEM) is the only tool available
Microscopy (TEM) to observe OMVs accurately. TEM also brings out the presence of
flagella and pili, an efficient tool to ensure sample purity. For TEM
observations, OMVs should be negatively stained, such as with
0.1% phosphotungstic acid.
20 Anaı̈s Blache and Wafa Achouak

SDS-PAGE Electrophoresis Protein electrophoresis is used to observe the protein composition

of OMVs, to compare the production of OMVs, and to prove their
purity. The protein sizes of flagella and pili are known and can be
identified on SDS-PAGE gel. Anti-flagellum or pili protein anti-
bodies can be used to ensure the absence of these contaminants in
the samples.
1. Mix 15 μL of OMVs sample with 5 μL of 4x LDS sample buffer.
2. Heat samples at 95 °C for 10 min.
3. Load 20 μL of OMVs sample onto a 10% Bis-Tris
NUPAGE gel.
4. Run at 130 V for 1 h.
5. Incubate the SDS-PAGE gel for 1 h in imperial protein stain for
staining.
6. Bleach the gel overnight in H2O.
7. Observe the protein profile.

3.4 Extraction of sRNAs are an important part of the OMVs cargo and key players in
RNAs cell-to-cell communication. To identify the cargo whole of RNAs
or to study the encapsulation of specific sRNAs, sRNAs must be
extracted from OMVs samples.
To distinguish sRNA extracted from the OMVs cargo and
sRNAs located at the membrane surface, incubate the samples in
the presence and absence of RNAse. Add 1 μL of RNAse A/T1
(Thermoscientific) to the samples and incubate at 37 °C for
1 H. Use 0.5 mL 30 kDa Amicon to eliminate RNAse after incuba-
tion. Measure sRNA concentrations and compare data from sam-
ples with and without RNAse.
RNAs are very sensitive to high temperatures. To avoid RNA
degradation, always manipulate RNA samples on ice (see Note 16).

3.4.1 RNA Extraction 1. Trizol allows the extraction of small RNAs from
Methods low-concentration samples [23]. Use the TRIzol Reagent and
follow the User Guide from “Lysis” to “Isolate RNA” sections
(see Note 17).
2. The RNAeasy extraction Kit is efficient on OMVs samples
[13]. This Kit allows extraction without the use of chemical
hood and provides rapid and reproductible results. Follow the
manufacturer’s user guide instructions.
3. RNAzol RT Kit (Sigma-Aldrich) is recommended for the
extraction of small RNAs (<200 bp) [26]. Follow the instruc-
tions in the user guide.
4. RNA can also be extracted and purified as described by Busi
et al. [27].
 Gram-Negative Outer Membrane Vesicles and Associated RNAs 21

3.4.2 Quality Control Nanovue is a good tool to quickly evaluate RNA concentration and
protein contamination after extraction. However, to obtain the
RNA concentration accurately, use the Qubit RNA BR Assay Kit.
To evaluate sample integrity, use Qubit RNA IQ Assay Kit.

3.4.3 RNA Analysis 1. Add DNAse to the samples to avoid biasing the results by DNA
contamination. The TURBO DNA-free Kit works on sRNA
samples extracted from OMVs. Follow the instructions in the
user guide for routine DNAse treatment.
After this step, RNA-seq analysis can be performed on the
RNA samples.
For RT-PCR or qRT-PCR, the steps are described below.
2. The Transcriptor First Strand cDNA synthesis kit works on
sRNAs from OMV samples. Follow the supplier’s instructions
(see Note 18). The results of the RT-PCR can be observed by
cDNA electrophoresis.
qRT-PCR allows quantitative comparison of sRNA pro-
duction between samples. For qRT-PCR experiments, design
specific primers to amplify the sRNAs under study and conven-
tional reagents. Use the real LightCycler 480 SYBR Green I
q-PCR Master kit [28].
3. Test the specific primers and use a concentrated sample to
evaluate PCR efficiency.
4. Run the PCR according to the instructions in the user guide.

4 Notes

1. One ultracentrifugation run requires 300 mL of culture super-

natant. With three 600 mL cultures, you can perform three
ultracentrifugations in one day.
2. Higher aeration enhances vesicle production but should be
chosen carefully to avoid excessive stress conditions.
3. All manipulations of OMVs should be done under sterile con-
ditions where possible to avoid bacterial contamination. The
culture supernatant and OMVs samples should be handled at
4 °C to avoid degradation of protein RNAs and metabolites.
4. To ensure OMVs sterility, spread 2–5 μL of each OMVs sample
on a 1/10 TSB or LB agar plate.
5. Balance the tubes before centrifugation with a balance
(±0.05 g).
6. Each ultracentrifugation run requires 300 mL of cell-free
supernatant.
7. For long-term storage, use freezers at – 45 °C or – 80 °C.
22 Anaı̈s Blache and Wafa Achouak

8. Depending on the size of the holder, two ultrafiltration mem-

branes can be used: 30 kDa or 100 kDa. A 100 kDa membrane
is recommended to avoid concentrating supernatant proteins.
9. For ultrafiltration, if the tubing doesn’t go to the bottom of the
bottle, use a cut glass pipette Pasteur to reach the bottom of the
vial and aspirate all the liquid.
10. Lay the needed volume of 20% solution (between 1.5 and
2.5 mL) to completely fill the tube, and avoid crushing the
tube during ultracentrifugation.
11. Each layer of the Optiprep must be visible and must not be
mixed with the others. Manipulate the tube very carefully.
12. Equilibrate the tubes before centrifugation with a balance
(±0.05 g).
13. Before extraction, to avoid any contamination, wash the ultra-
centrifugation tubes with ethanol 100%.
14. To calculate the amount of vesicle production per cell, for each
method, determine the number of colony-forming units
(CFU) at the end of the bacterial growth.
15. If the strain is known to produce a high number of vesicles, the
sample can be diluted fourfold.
16. Before manipulating RNAs, use gloves and clean the bench
with GIGAPUR RNAse-DEKONTA OFF (Shimitek).
17. For the transfer of the aqueous phase (Step 7, “Lysis” section),
carefully collect the upper phase and leave a small volume of
this phase to avoid pipetting the interphase. During RNA
precipitation (Step 1.d., Isolate RNA section), note the pellet
position before centrifugation because it will not be easily
visible. Avoid the last incubation step (Step 3.b, RNA Isolation
section).
18. The denaturation step is optional but highly recommended to
improve efficiency as it allows denaturation of secondary
structures.

Acknowledgments

This work was supported by a PhD program grant from CEA. This
work is part of the COMBAT project (FR. ECCOREV).

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 Chapter 3

Analysis of Phage Regulatory RNAs: Sequencing Library

Construction from the Fraction of Small Prokaryotic RNAs
Less Than 50 Nucleotides in Length
Sylwia Bloch , Natalia Lewandowska , Wojciech Wesołowski ,
Aleksandra Łukasiak , Paulina Mach, Bożena Nejman-Faleńczyk ,
and Grzegorz We˛grzyn

Abstract
So far, bacterial regulatory sRNAs of length less than 50 nucleotides have been poorly understood, and a
low number of such molecules has been identified. The first microRNA-size functional ribonucleic acid
occurring in a bacterial cell has been described only recently, and it was found to be encoded by a
bacteriophage. One of the reasons for such a scarcity in this field is the lack of procedures intended for
the isolation and selection of molecules of this size from bacterial cells. To meet these difficulties, we
describe here the few-step procedure of isolation, purification, selection, and sequencing library preparation
that is dedicated to the fraction of very small, bacterial RNA molecules.

Key words Sequencing library, microRNA-size RNAs, Bacteriophages, Bacteria

1 Introduction

The most frequently reported small regulatory RNAs (sRNAs)

occurring in prokaryotic cells vary in size between 50 and
500 nucleotides (nt) in length. They show high structural diversity,
exhibit different molecular mechanisms of action, and act by base
pairing sharing either perfect (cis-acting sRNAs) or more limited
(trans-acting sRNAs) complementarity with their target transcripts
[1, 2]. Small, non-coding, prokaryotic RNAs have been identified
in a wide range of bacteria, including pathogenic strains of Escher-
ichia, Salmonella, and Yersinia, and found to play an important role

Sylwia Bloch and Natalia Lewandowska contributed equally to this work as co-first authors, Bożena Nejman-
Faleńczyk and Grzegorz We˛grzyn are also equal contributors to this work and designated as co-corresponding
authors.

Véronique Arluison and Claudio Valverde (eds.), Bacterial Regulatory RNA: Methods and Protocols, Methods in Molecular Biology,
vol. 2741, https://doi.org/10.1007/978-1-0716-3565-0_3,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

25
26 Sylwia Bloch et al.

in the regulation of many processes such as carbon metabolism,

virulence, motility, quorum sensing, biofilm formation, bacterial
adaptation to changing conditions, and response to stresses [3–
7]. Importantly, among sRNAs, there are also molecules of phage
origin that play important roles in the development of phages and
their bacterial hosts [8, 9].
Surprisingly, although very small, noncoding RNA molecules
less than 50 nt in length have been found broadly in multicellular
organisms (humans, animals, and plants), their occurrence in pro-
karyotic cells is not entirely clear. Up to now, only a low number of
such sRNAs (15–36 nt) have been reported in prokaryotic cells.
These RNAs have been named in the literature as ‘microRNA-size
molecules’ and were found in Streptococcus mutans in 2011 [10]
and in E. coli in 2013 [11], although their functions remain to be
determined. Another small RNA molecule of bacterial origin has
been identified in Mycobacterium marinum, a fish pathogen able to
produce a unique mycolactone toxin, mycolactone F [12]. Small
RNAs less than 50 nt in length have also been identified in peri-
odontal pathogens, including Aggregatibacter actinomycetemcomi-
tans; Porphyromonas gingivalis, Treponema denticola bacteria [13],
and a key player in the colonization of the human oral cavity,
Streptococcus sanguinis [14]. A little is also known about the very
small, shorter than 50 nt, RNA molecules derived from phage
genomes. One reported example is the 24B_1 small RNA molecule
(20-nt long) that has been identified after the induction of Shiga
toxin-converting (Stx) phage Φ24B in E. coli [15].
One of the reasons why bacterial regulatory sRNAs shorter
than 50 nt are still poorly understood is the scarcity of described
procedures aimed at their selection and detection among the excess
of larger molecules. Even though the recent development of high-
throughput RNA sequencing technologies has facilitated the iden-
tification and characterization of sRNAs and their targets in differ-
ent bacterial species, the identification attempts undertaken by
scientists concerned mostly sRNA molecules longer than 50 nt
[16, 17]. The key stage in the RNA-Seq analysis is the preparation
of the cDNA library which includes various steps. The study of
sRNAs usually involves a size-fractionation step (either on a gel or
gel-free), from which molecules are extracted using a suitable kit. A
main drawback of this method is the possible exclusion of very small
RNAs, less than 50 nt, which can be lost not only at the stage of size
selection of the library but even during the initial RNA isolation.
To overcome the above-described difficulties, we present here
the procedure of next-generation sequencing (NGS) library con-
struction, focusing on the phage and bacterial sRNAs shorter than
50 nt and encompassing all important steps allowing for their
isolation, purification, concentration, selection, and library prepa-
ration and validation. From the outset and at every stage, this
procedure is aimed at obtaining only the smallest RNA particles
(<50 nt). Importantly, unlike most protocols dedicated to
 Analysis of Phage Regulatory RNAs: Sequencing Library Construction. . . 27

Fig. 1 Concentrations, electropherograms, and sequencing results for cDNA libraries generated from the
fraction of bacterial small RNAs (less than 50 nt) according to the procedure described in this section. Panel (a)
presents a table with sample description and obtained library concentrations (ng/μL). Libraries were prepared
with a fraction of RNA-containing molecules shorter than 50 nt. RNA was isolated from three independent
bacterial strains, two E. coli strains, MG1655(Φ24B) and EDL933, after prophage induction, and one E. coli
O157:H7 strain after infection with phage vB_Eco4-M7. Concentration was measured using the Qubit™
dsDNA HS Assay Kit. The gel image and electropherograms from the Agilent 2100 Bioanalyzer, presented in
Panel (b), were generated from the prepared libraries using the Agilent High Sensitivity DNA kit. Peaks labeled
with green and purple correspond to DNA reference markers included in each analysis. Small peaks observed
in the 143–153 bp size range correspond to adapter dimers, and all peaks above this size correspond to the
size of processed sRNAs. The peak at about 175 bp corresponds to the fraction of microRNA-size RNA
molecules. Interestingly, although the concentration of the library produced from RNA isolated from sample
No. 3 was the lowest (11 ng/μL), it yielded a major and the highest peak at 175 bp. The other two library
profiles (of samples 1 and 2) show more and higher peaks above 175 bp, which was reflected in the
sequencing results presented in Panel (c). Results of reads length distribution show a higher number of
sequences shorter than 50 nt identified in samples 1 and 2 in comparison with sample No. 3. Besides, only a
small percentage of sequences of length in the range of 50–99 nt and 100–149 nt were identified in all
investigated samples 1–3 (less than 0.3% and 0.04% respectively), whereas sequences larger than 150 nt
were not observed at all

eukaryotic microRNAs and larger bacterial sRNAs, we propose to

carry out the stage of size selection of sRNA molecules less than
50 nt before cDNA synthesis, rather than after this step. As pre-
sented in Fig. 1a–c, this procedure allowed us to obtain cDNA
libraries encompassing mainly very small molecules, shorter than
50 nt, and identify molecules of these sizes in the sequencing
results. Importantly, one of the found molecules was 24B_1, the
regulatory microRNA-size RNA of phage origin, which was inves-
tigated by us previously [15]. This proves that the proposed proce-
dure works well and according to its purpose.
28 Sylwia Bloch et al.

2 Materials

2.1 Reagents to Be Always use proper microbiological aseptic techniques when work-
Supplied by User ing with RNA. Wear a suitable lab coat and disposable gloves to
prevent accidental contamination. Store all reagents at consistent
temperatures according to the manufacturer’s instructions. For
more information, consult the appropriate safety data sheets
(SDSs), available from the product supplier. To avoid RNA degra-
dation, prepare all solutions using ultrapure, diethylpyrocarbonate
(DEPC)-treated water.
2.1. Acrylamide: ready-to-use 40% solution (w/v) of 19:1 acryl -
bis-acryl ratio.
2.2. Agilent High Sensitivity DNA kit (Agilent Technologies).
2.3. Ammonium persulfate (APS): 10% solution of APS in DEPC-
treated H2O. Prepare this mixture fresh each time.
2.4. Dialysis membrane with 3.5 kDa molecular weight cut off
(MWCO).
2.5. Denaturing acrylamide gel: for 15% denaturing gel, mix
0.75 mL 10 × TBE buffer, 3.15 g urea, 2.8 mL 40% acrylam-
ide, and DEPC-treated H2O up to 7.5 mL. Stir at room
temperature until the urea is completely dissolved, then add
37.5 μL 10% APS and 7.5 μL TEMED. Pour the gel into a
prepared mold immediately.
2.6. 10 × bacterial cells killing buffer: 200 mM sodium azide,
50 mM MgCl2, 200 mM M Tris-HCl, pH 8.0.
2.7. Chloroform.
2.8. Diethyl pyrocarbonate (DEPC)-treated water: add 1 mL of
freshly prepared 0.1% DEPC to 1000 mL of distilled
H2O. Mix well to disperse DEPC through H2O. Incubate
the mixture at 37 °C for at least 12 h, and then autoclave it
(20 min, 121 °C) to inactivate the remaining DEPC. Let the
mixture cool to room temperature prior to use.
2.9. 100% ethanol.
2.10. 96% ethanol.
2.11. 70% ethanol.
2.12. Ethidium bromide solution (EtBr): 10 mg/mL solution in
H2O.
2.13. Ethylenediaminetetraacetic acid (EDTA), pH 8.0: for 0.5 M
solution, dissolve 73.061 g of EDTA (m.w. 292.244 g/mol)
in 200 mL DEPC-treated H2O using a magnetic stirrer.
Adjust the pH to 8.0 by adding 10 M NaOH. Transfer the
solution to a 500 mL measuring cylinder and add DEPC-
treated H2O to bring the final volume to 500 mL. Autoclave
for 20 min at 121 °C on liquid cycle.
Analysis of Phage Regulatory RNAs: Sequencing Library Construction. . . 29

2.14. 2 × gel loading buffer II (Invitrogen): ready-to-use mixture

of 95% formamide, 18 mM EDTA, 0.025% SDS, 0.025%
xylene cyanol, and 0.025% bromophenol blue.
2.15. Magnesium chloride: for 1 M solution, dissolve 4.761 g of
MgCl2 (m.w. 95.211 g/mol) in 40 mL of DEPC-treated
H2O. Adjust the volume to 50 mL with DEPC-treated
H2O. Autoclave for 20 min at 121 °C on liquid cycle.
2.16. microRNA Marker (New England BioLabs): ready-to-load
RNA ladder that contains three synthetic, single-stranded
RNA oligonucleotides 17, 21, and 25 residues long.
2.17. N, N, N ′, N ′ - Tetramethyl-ethylenediamine (TEMED):
ready-to-use.
2.18. NucleoSpin® Gel and PCR Clean-Up kit (Macherey-Nagel).
2.19. PureLink™ miRNA Isolation Kit (Invitrogen).
2.20. Qubit™ dsDNA HS Assay Kit (Invitrogen).
2.21. Qubit™ microRNA Assay Kit (Invitrogen),
2.22. RNA Marker 1 RTU (A&A Biotechnology, Poland): ready-
to-load RNA ladder that contains RNA fragments in the
range of 100–1000 nt.
2.23. SMARTer smRNA-Seq kit for Illumina (Clontech
Laboratories).
2.24. Sodium acetate: for 3 M solution, dissolve 24.610 g of
C2H3NaO2 (82.034 g/mol) in 70 mL of DEPC-treated
H2O using a magnetic stirrer. Adjust the pH to 5.3 by adding
glacial acetic acid. Transfer the solution to a 100 mL measur-
ing cylinder and add DEPC-treated H2O to bring the final
volume to 100 mL. Sterilize by filtration using a 0.22-μm-
pore size SFCA membrane and then store at room
temperature.
2.25. Sodium azide: for 1 M solution, dissolve 0.650 g of NaN3
(m.w. 65.010 g/mol) in 8 mL of DEPC-treated
H2O. Adjust the volume to 10 mL with DEPC-treated
H2O. Sterilize by filtration using a 0.22-μm-pore size
SFCA membrane and then store at room temperature.
2.26. 10 × Tris–Borate–EDTA electrophoresis buffer (10x TBE):
for 10 × TBE buffer, dissolve 108 g Tris and 55 g boric acid
in 800 mL DEPC-treated H2O using a magnetic stirrer. Add
40 mL 0.5 M EDTA, pH 8.0. Adjust volume to 1000 mL.
Store 10 × TBE buffer at room temperature.
2.27. 1 × Tris–Borate–EDTA electrophoresis buffer (1 × TBE):
dilute the 10 × TBE stock solution by 10 × in DEPC-treated
H2O.
Exploring the Variety of Random
Documents with Different Content
such common occurrence in those days, that no further notice was
ever taken of an accident of the kind than by giving the injured
person all the assistance that could be administered at the time.

However, it may well be supposed that Sir Osborne Maurice felt

no ordinary interest in the sight before him. By an extraordinary
coincidence, overthrown by his hand, though without intention, and
apparently nearly killed, lay the persevering enemy who had
swallowed up the fortunes of his house, and had sought so
unceasingly to sweep it for ever from the face of the earth; and
while he lay there, prostrate at his feet, with the ashy hue of his
cheek paler than ever, and his dark eye closed as if in death, Sir
Osborne still thought he could see the same determined malignity of
aspect with which he had declared that he would found his title to
the lordship of Chilham Castle on the death of its heir.

Still holding the lance in his hand, the knight bent over the bow of
his saddle, and through the bars of his volant-piece contemplated
the face of his fallen adversary, till he began to unclose his eyes and
look around him; when Sir Thomas Neville, thinking that the
stranger was animated merely by feelings of humanity, turned to
him, saying that Sir Payan had only been a little stunned, and would
do very well now.

"Gentlemen," continued he, addressing the king and Sir Osborne,

"we must, according to promise, let you pass away unquestioned;
but I will say, that two more valiant and skilful knights never graced
a field, nor is it possible to say which outdoes the other; but ye are
worthy companions and true knights both, and so fare ye well."

The king did not reply, lest he should be recognised by his voice;
but bending low, in token of his thanks, rode out of the lists,
accompanied by Sir Osborne and followed by Longpole.

"Now, by my fay, sir knight!" cried Henry, when they had once
more reached the cover of the wood, "you have far exceeded my
expectations; and I thank you heartily--good faith, I do!--for your
aid. But I must have you stay with me. Our poor court will be much
graced by the addition of such a knight. What say you? ha!"

"To serve your grace," replied Sir Osborne, "is my first wish; to
merit your praise my highest ambition. It is but little to say that you
may command me when you command all; but if my zeal to obey
those commands may be counted for merit, I will deserve some
applause."

"Wisely spoken," answered the king; "we retain you for ours from
this moment; and that you may be ever near our person, we shall
bid our chamberlain find your apartments in the palace. How say
you, sir knight? are you therewith contented?"

"Your grace's bounty outstrips even the swift wings of Hope,"

replied Sir Osborne; "but I will try to fly Gratitude against it; and
though, perhaps, she may not be able to overtop, she shall, at least,
soar an equal pitch."

The knight's allusion to the royal sport of falconry was well

adapted to the ears that heard it. Every one must have remarked,
that whatever impressions are intended to be produced on the mind
of man are always best received when addressed to his heart
through its most common associations. Whether we wish to explain,
to convince, to touch, or to engage, we must refer to something that
is habitual and pleasing; and, therefore, the use of figures in
eloquence is not so much to enrich and to deck, as to find admission
to the soul of the hearer, by all the paths which its own habits have
rendered most easy of access.

Thus, Sir Osborne, without knowing it, drew his metaphor from a
sport in which the king delighted; and, more convinced of his zeal by
these few words than if the young knight had spoken for an hour,
the king replied, "I doubt ye not; 'faith, I doubt ye not. But this night
we give a mummery unto our lady queen, when I will bring you to
her knowledge: 'tis a lady full of graciousness, and though 'tis I who
say it, one that will love well all that I love. But now let us haste, for
the day wears; and as you shall be my masking peer, we must think
of some quaint disguise: Darby shall be another; and being all light
of foot, we will tread a measure with the fair ladies. You are a proper
man, and may, perchance, steal some hearts, wherein you shall have
our favour, if 'tis for your good advancement. But turn we down this
other path; in that I see some strangers. Quick! Mary Mother! I
would not be discovered for another kingdom!"

CHAPTER XX.

Not rain she finds the charmful task,

In pageant quaint, in motley mask.--Collins.

During this expedition of Henry and Sir Osborne, Lord Darby had
acted with more prudence than might have been expected from one
so light and volatile as himself. But, with all the levity of youth, he
had a great fund of shrewdness and good sense, which enabled him
keenly to perceive all the weaknesses of the king's character, and
adapt his own behaviour exactly to the circumstance, whenever he
was brought particularly in contact with the monarch.

In the present instance, seeing that the spirit of mystery had

seized upon Henry, he consented to forego all more active
amusement; so that, when the king and his young companion
returned, they found the earl seated in the saloon wherein Sir
Osborne had been armed, never having quitted it during their
absence.
 Henry was in high spirits. All had gone well with him: his
expedition had been both successful and secret, and he was not a
little pleased to find that the earl had not joined any of the gay
parties of the court while he had been away.

"Ha, my lord!" cried he, as he entered; "still here! You have done
well; you have done well. 'Tis a treasure you have brought me, this
good knight. Snell, unlace my casque; I must thank you for him as a
gift, for he is now mine own. He outdoes all expectation; nay, say
not against it, Sir Osborne; I should be able to judge of these
matters: I have broken spears enow, and I pronounce you equal to
any knight at this court. Call some one to undo these trappings. But,
Darby, you must not quit the court to-night. Dine here; 'tis time,
i'faith; near one o' the clock! and take Sir Osborne Maurice with you.
Make him known to the best of the court: say the king holds him
highly. But stay," he added, "I had forgot;" and sending for the sub-
controller of the household, he gave commands that the young
knight should be furnished with apartments in the palace from that
moment, and receive the appointment of a gentleman of the privy
chamber. "The number is complete," he continued, turning to Sir
Osborne; "but, nevertheless, you shall be rated as such, and yourself
and men provided in the palace. See it be done, Sir John Harvey.
Darby, return hither privately with your friend, at nine to-night. We
have a masque and revel afoot; but take no heed to send to London
for disguise; we will be your furnishers."

"I hope, sir," said the sub-controller, as the knight and his friend
followed him from the presence, "you are aware that only three
servants are allowed to a gentleman of the privy chamber."

"Three will be as much as I shall have occasion for," answered the

knight; "the other shall remain in London."

"If you will follow me, then," said the officer, "I will show you to
the apartment. Ho! send me a yeoman usher there," he continued,
speaking to a servant who passed. "This way, sir, we shall find the
rooms."

"What!" cried Lord Darby, after they had ascended a good many
steps in one of the wings of the building; "are you going to put my
friend in a third story? Think, Sir John Harvey, may not the king find
it strange when he hears that a knight he honours with his regard
has been so lodged?"

"I can assure you, my lord," answered the controller, "they are
absolutely the only ones in the palace vacant which are at all equal
to the knight's quality; and in truth, were it not for the height, are
among the best in the place. They are large and spacious; exactly
the same size as those which were appointed yesterday, by the
queen's command, for Lady Constance de Grey, and which are
immediately underneath."

"I was going to offer Sir Osborne the use of mine," said Lord
Darby, with a laughing glance towards the knight, "till you could find
him better; but if they are so very good as you say, maybe he will
prefer having his own at once. Ha! Sir Osborne?"

The controller looked solemn, seeing there was some joke, and
not understanding it; but, however, he was joined in a moment after
by a yeoman usher, bearing a bunch of keys, from which he selected
one, and opened the door at which they had been standing while
the earl spoke. A little ante-chamber conducted into three others
beyond, all very well furnished according to the fashion of the day,
with a beautiful view of the wild park from the windows of some of
the rooms, and of the river from the others; on which advantage the
worthy sub-controller descanted with much the tone and manner of
a lodging-house keeper at a watering-place; little knowing that one
word regarding the proximity of Constance de Grey would have been
a higher recommendation to the young knight than all the prospects
in the world, though he loved the beautiful and varied face of earth
as much as any one.
 "Go to the wardrobe of beds, usher," said the officer, when he had
promenaded the knight and Lord Darby through the apartment; "go
to the wardrobe of beds, and tell the undermaster to come hither
and garnish this apartment with all speed. As I do not know the
honourable knight's face," continued he, "it is probable that he is
new to this court, and is not aware of the regulations, which,
therefore, I will make bold to tell him. Dinner and supper are served
at the board of estate, every day, at noon and at nightfall. No rere-
suppers are given, nunchions, beverages, or breakfast; but to each
gentleman of the privy-chamber his grace commands a livery every
night."

"A livery!" said Sir Osborne; "pray, Sir John, what is that?"

"Its value, sir," said the controller, "depends upon the station of
the person to whom it is given. I have known it cost as much as ten
pounds; such was sent every night to the gentlemen who came to
seek the Princess Mary for the French king; but the livery given by
his grace the king to the gentlemen of the privy-chamber, and others
bearing the same rank, is a cast of fine manchet bread, two pots of
white or red wine at choice, one pound weight of sugar, four white
lights, and four yellow lights of wax, and one large staff torch, which
is delivered every evening at seven of the clock."

Without proceeding further with such discourse, we shall merely

say that the arrangement of Sir Osborne's apartment was soon
completed, himself unarmed, his servants furnished with what
modern lacqueys would call dog-holes, and with truckle-beds; and
having, by intercession with a gentleman wearing black velvet and a
gold chain, and calling himself the chief cook, obtained some dinner,
for the board of estate had long been cleared, Lord Darby and Sir
Osborne sauntered forth on the parade, where the young gallants of
the court were beginning to show themselves; some taking, as it
were, a furtive walk across, afraid to be seen there before the
moment of fashion sanctioned their appearance, and some, who,
from either ignorance or boldness, heeded no mode but their own
convenience. Fashions are nine times out of ten affectations;
affectations in those who lead and in those who follow; and as it is
now, so was it in the days of Henry the Eighth.

The presence of Lord Darby, however, who gradually gathered

round him a little multitude as he walked, soon rendered the parade
more populous. Sir Osborne was introduced to all who were worthy
of his acquaintance; and the same persons who three days before
might hardly have given him a courteous answer, if he had asked
them a question, were now mortified at not being numbered with his
acquaintance. The knight himself, however, was absent and
inattentive, his eye continually seeking Lady Constance de Grey
through the crowd, and his mind sometimes occupied with pleasing
dreams of love, and hope, and happiness to come, and sometimes
pondering over his unexpected encounter with Sir Payan Wileton,
and its probable results.

So strange is the world, that this very abstractness of manner and

carelessness in regard to those about him had its grace in the eyes
of the court. They seemed to think that he who cared so little about
anybody, must be somebody of consequence himself; and when,
after a prolonged saunter, the two friends re-entered the palace, Sir
Osborne's name had acquired a degree of éclât which the most
attentive politeness would scarcely have obtained. Still no Constance
de Grey had he seen, and he sat down in the apartments of Lord
Darby, not peculiarly satisfied with their walk.

The young earl himself had also suffered a similar

disappointment, for in the midst of all the nonchalant gaiety which
he had displayed to the crowd, his eye had not failed to scan every
group of ladies that they met for the form of Lady Katrine Bulmer,
and he felt a good deal mortified at not having seen her. But very
different was the manner in which his feelings acted, from the
deeper and more ardent love of Darnley. He laughed, he sung, he
jested his companion upon his gravity, and in the end consoled him,
by assuring him that they should meet with both their lady-loves
that night at the queen's, so that if he were not in a very expiring
state, he might hope to live to see her once more.

The hours quickly flew, and a little before nine the knight and his
companion presented themselves at the door of the king's private
apartments, where they were admitted by a page. When they
entered Henry was reading, and pursued the object of his study
without taking any notice of their approach by word or sign. Nothing
remained to be done but to stand profoundly still before him, waiting
his good pleasure, which remained full a quarter of an hour
unmanifested.

"Well, gentlemen both," cried the king at last, starting up and

laying down the book; "I have kept ye long--ha? But now, to make
amends, I will lead ye to the fair ladies. Oh, the disguises! the
disguises! Bring the disguises, Minton; the three I chose but now.
You, Darby, shall be a Muscovian; you, Maurice, a Polacco; and I an
Almaine. Say, Darby, did you see my good lord cardinal this morning
ere you came? Holds he his mind of going to York, as he stated
yesterday?"

"I did not see the very reverend lord this morning," replied Lord
Darby, who was Wolsey's ward, as well as the chief lord of his
household. "But his master of the horse informed me that he still
proposed going at ten this morning. Your grace knows that he never
delays when business calls him; and in the present case he thinks
that his presence may quell the murmurers of Yorkshire, as well as
Lord Howard has put down the Rochester fools."

"Ah, 'twas a shrewd business that of Rochester," said the king.

"Now would I give a thousand marks to know who 'twas that set
that stone a-rolling. Be you sure, Darby, that the brute shipwrights
would ne'er have dreamed such a thing themselves. They were set
on! They were set on, man! Ha, the disguises! Quick! come into this
closet, and we will robe us. 'Tis late, and our lady has promised to
give, as well as to receive, a mask."
 So saying, Henry led the way to a cabinet at the side of the
saloon in which they were; and here the two young lords offered to
assist in dressing him, but of this he would not permit, bidding them
haste with their own robes, or he would be ready first. The disguise
assigned to Sir Osborne was a splendid suit of gold brocade trimmed
with fur, intended to represent the dress of a Pole; having a sort of
pelisse with sleeves of rich gold damask and sables thrown over the
back, and held by a baldrick, crossing from the right shoulder under
the left arm. His head was covered with a square bonnet of cloth of
gold, like his dress, with an edge of fur; and his face concealed by a
satin mask with a beard of golden threads.

The dress of Lord Darby was not very dissimilar, with only this
difference, that in place of the pelisse, he was furnished with a robe
with short sleeves, and wore on his head a sort of turban, or toque,
with a high feather. In a very different style was the king's disguise,
being simply a splendid German dress of cloth of gold, trimmed with
crimson velvet, but certainly not so unlike his usual garments as to
afford any great degree of concealment. All being masked and
prepared, Henry sent the page to see if the torchbearers were ready,
and issuing out of the palace the three maskers, preceded by half-a-
dozen attendants, crossed the greater quadrangle, passed out at the
gate, and making a circuit round the building, came immediately
under the windows of the queen's great hall, from each of which a
broad blaze of light flashed forth upon the night, and cast a line of
twinkling splendour across the river, that otherwise flowed on, dark
and indistinct, under a clouded and moonless sky.

"Sir Osborne," said Henry, in a low voice, as they entered the

open doors, and turned into a suite of apartments anterior to the
room where the queen held her assembly--"Sir Osborne, your voice
being unknown, you shall be our orator, and in your fine wit seek a
fair compliment for our introduction."

Had his face been uncovered, perhaps the young knight might
have sought to excuse himself; but there is wonderful assurance in a
mask; and feeling a boldness in his disguise, which perhaps the eye
of Constance de Grey might have robbed him of, had he not been
concealed from its glance, he at once undertook the task, saying
that he would do his best.

As he spoke, a couple of hautboys, by which Henry was preceded,

paused at the entrance of the great hall, and placing themselves on
each side, began a light duet, to announce that some masks were
coming. The doors were thrown open, and a splendid scene burst on
the view of Sir Osborne, full of bright and glittering figures, fleeting
about in the blaze of innumerable lights, like the gay phastasms of a
brilliant dream. The knight instinctively paused, but Henry urged him
on.

"Quick! quick!" whispered he; "to the lady, to the lady; you forget
your task."

Sir Osborne instantly recollected himself, and seeing a lady, who,

standing unmasked at the farther end of the hall, bore about her
that air of royalty, and that majestic beauty, scarcely touched by
time, for which the noble Catherine was famous, he advanced
directly towards her, and bent one knee to the ground. Nature had
given him somewhat of a poet's inspiration, which came now happily
to his aid, and if his verses were not very good, they were at least
ready.

"Lady of beauty, queen of grace,

Strangers three have come to thee,
To gaze on thine unclouded face,
Where so many maskers be.
Oh! never shade that brow so high
With the mummers' painted wile.
Sure you keep that lip and eye,
Welcome on your slaves to smile."
 "I thank you, fair sir; I thank you," replied the queen, with a
pleased and gracious smile: "be most welcome, you and your
company. I should know you, and yet I do not. But will you not
dance? Choose your fair ladies; and, chamberlain, bid the music
sound."

Sir Osborne passed on, and the king and Lord Darby followed.

"Excellent well, my knight! excellent well!" whispered Henry. "Now

show your wit in choice of a fair dame. I'faith, one must be keen in
these same masks to tell the foul from the fair. However, let us
disperse and find the jewels, though they be hid in such strange
rinds."

At the word the three maskers took different paths amongst the
various figures with which the hall was now nearly filled; Lord Darby
and the knight, each in search of the object of his love; while Henry,
as yet unrecognised, glided through the apartment, it might be in
quest of some fair one also.

For some time Sir Osborne sought in vain, bewildered amongst

the crowd of quaint disguises with which he was surrounded. Now
he thought he beheld the form of Lady Constance here, and after
following it for a moment was called away by the sight of one that
resembled her more. That again he gave up, convinced by some
turn or some gesture that it was some other. Another presented
itself, which perhaps he might have mistaken, but the gay flutter of
her manner at once showed that it was not the person he sought.
He saw that already Lord Darby had found his partner; the tuning of
the musical instruments was over, and mentally cursing his own
stupidity, or his own ill-fortune, he was proceeding once more
towards the part of the room where stood the queen, with his heart
beating between eagerness and vexation, when he beheld a lady,
dressed in silver brocade, with a plain satin mask, glide into the hall,
and passing by several who spoke to her, approach that spot, as if to
take a seat which stood near. Sir Osborne darted forward. He felt
that it was her; and, eager to prevent any one intercepting him,
almost startled her with the suddenness of his address.

"Fair mask," said the knight, in a voice that trembled with delight
and hope, "will you tread a measure with a stranger, for courtesy's
sake?"

"I should know your voice," said the lady, in a low tone; "but I
can scarce believe I see you here. But one word, to tell me who you
are?"

"My motto," replied the knight, "is Constanc-y; my crest, a lady's

glove."

The lady instantly put her hand into his. "Darnley!" said she, in a
voice so low as to be inaudible to any one but himself, who, bending
his head over her, trembled to catch every accent.

"Ah! Constance," he replied, in the same subdued tone, "what is it

I have dared to say to you? what is it I have dared to hope?
Friendless and fortuneless as I am, can you ever pardon my
boldness?"

"Hush!" she said, "for pity's sake speak not in that way. Now I
know you love me, that is enough. Friendless you are not, and
fortuneless you cannot he, when all that is Constance's is yours. But
see! they are going to dance; afterwards we will speak more. Do not
think me bold, Darnley, or too easily won; but were I to affect that
reserve which still perhaps might be right, we are so circumstanced
that we might be ruined before we understood each other."

The knight poured forth a thousand thanks, and strove to explain

to Lady Constance how deeply grateful he felt for that generous
candour which is ever the companion of the truest modesty; and,
the music now beginning, he led her through the dance with calm
and graceful ease. As soon as the measure was ended, the queen's
chamberlain pronounced, with a loud voice, that in the other halls
the knights and ladies who had danced would find cool air and shady
bowers; and, gladly taking advantage of this information, Sir
Osborne led his partner into the chamber beyond, which by the
queen's device had been divided into a thousand little arbours,
where artificial trees and shrubs, mingled with real ones, and often
ornamented with gilt fruit or flowers, formed a sort of enchanted
garden, for the dancers to repose themselves; not very exquisite in
its taste, indeed, but very much to the taste of the day.

Singling out the farthest of all the arbours, and the one which
permitted its occupants most easily to observe the approach of any
other party, Darnley led Lady Constance to one of the seats which it
contained, and placing himself by her side, paused for a moment in
silence, to enjoy the new delights that came thrilling upon his heart.
"Oh, Constance!" said he at length, looking up to the sweet hazel
eyes that gazed upon him through the meaningless mask; "never,
never did I think to know such happiness on earth! Could I have
dreamed of this when I left you for Flanders?"

"I do not know," replied Constance; "I have done nothing but
think ever since--ever since you took my glove; and I have fancied
that my dear father foresaw this, and wished it, as you tell me he
was aware who you were; for never, even at that age, was I
permitted to know, and converse with, and see intimately, any young
cavalier but yourself. And then, do not you remember, when you
used to teach me to shoot with the bow, how he would stand by and
praise your shooting? Oh! I can call to mind a thousand things to
make me think so."

"Could I but believe it," said Darnley, "I should be even happier
than I am. But still, dear Constance, I hope, I trust, that in the end I
may be enabled to seek your hand, not as an outcast wanderer. Your
good cousin, Lord Darby, has brought me to the knowledge of the
king, whose favour I have been happy enough to gain. He has
retained me as one of the gentlemen of his privy chamber, appointed
me apartments in the palace, which are just above your own; and I
hope so far to win his regard by this opportunity, that he may be
induced to hear my cause against the villain who has seized our
inheritance, and do justice to us at last. And then, Constance, with
rank, and fortune, and favour, all restored, Darnley may hope."

"And what if not restored, Darnley?" said Lady Constance. "Do

you think that rank, or fortune, or favour, will make any difference in
the regard of Constance de Grey? No, Darnley: if--but I won't say if--
-you love me, the cardinal may do what he will, but I will never wed
another. He may find means, as they hint, to forfeit my English
lands, yet he cannot take my French ones; and even if he did, I
would rather be beggar and free than married to a man I do not
love. Not that I do not love Darby as my cousin; he is kind, and
generous, and frank; but oh!! it is very, very different. But you say
that he introduced you to the king; I did not know you were even
acquainted."

"It is a long story, dear Constance," replied the knight; "I will give
it you some other time; but now tell me, while we are yet
uninterrupted, how may I see you? To watch for you, even to catch
a word during the day, certainly were delight; but still 'tis hard,
situated as we are, not to be able to communicate together more
freely. May not I come to see you?"

"Certainly," replied Lady Constance; "but you know that I can

hardly have any private conversation with you even when you do;
for good Dr. Wilbraham is with me the greater part of the morning,
and one of my women always." She paused for a moment in
thought, and, raising her eyes to his, "Darnley," she said, "I never
could love a man in whose honour I could not entirely confide;
therefore I do not think it shows me either weak or wrong when I
say that I will be entirely guided by you. We are not situated as
people in general, and therefore we cannot act as people in general
do. Tell me, then, what you think right, and I will do it. But here are
two of the maskers coming directly towards us. Say what must I
do?"
 "It is necessary, Constance," said the knight quickly, "absolutely
necessary, that I should sometimes be allowed half-an-hour's
conversation alone, especially at the present moment. I will come to-
morrow early, very early, if it can be then. May I?"

"Yes," said Lady Constance, "I will see. But who are these? They
are coming to us."

"It is Lord Darby," said the knight, "and, if I mistake not, Lady
Katrine Bulmer."

"Dear Polacco!" cried Lord Darby, approaching with a lady, who,

to use an old writer's description, was wondrous gay in her apparel,
with a marvellous strange and rich tire on her head: "dear Polacco, I
am but now aware of how much I have to thank you for. What! you
were near tilting at the Rochester host, and broaching me half-a-
dozen plank-shavers on your spear in defence of a fair lady, and also
took my part even before you knew me? Now, will I guess who is
this silver fair one by your side? she's blushing through her mask as
if I were going to pronounce her name with the voice of a trumpet.
Well, sweet cousin! will you own that you have a wild and rattle-
pated relation in the good town of Westminster? and if so, though
you cannot love him, will you love a very loveable creature for his
sake?"

"Hush, mad-cap! let me speak!" said the voice of Lady Katrine

Bulmer. "Lady," she continued, placing herself by the side of Lady
Constance, "will you hate one that would fain love you very much,
and have your love again?"

"Heaven forbid!" replied Lady Constance. "'Tis so sweet to be

loved ourselves, that feeling it, we can scarce refuse it again to
those that love us: with a reservation, though," she added.

"Granted the reservation, that there is still a one must be loved

best," said Lady Katrine; "we all four know it," and she glanced her
merry eyes round the circle. "Oh, what a happy thing is a mask!
Here one may confess one's love, or laugh at one's friends, or abuse
one's relations, without a blush; and surely, if they were worn
always, they would save a world of false smiles and a world of false
tears. Oh, strange economy! What an ocean of grimaces might be
spared if man were but to wear a pasteboard face!"

"I am afraid that he does so more than you think, lady," replied
Sir Osborne. "You will own that his countenance is hollow, and that
its smiles are painted: in short, that it is all a picture, though a
moving one."

"Listen to him!" cried Lady Katrine, raising her look to Lord Darby;
"think of his having the impudence to moralise in the presence of
two women! Would you have believed it?"

"Nay, fair lady! it was you who led the way," replied Sir Osborne.
"But what means that trumpet in these peaceful halls?"

"'Tis either a sound to supper," replied Lord Darby, "or the

entrance of one of those pageants of which our gracious king is so
fond. At all events, let us go and see."

Thus speaking, he led away Lady Katrine gaily to the door,

towards which all the other parties from the enchanted garden were
now proceeding. Sir Osborne and Lady Constance followed more
slowly. "Darnley," said the fair girl, as she leaned on his arm, "I know
not what sort of presentiment led me hither to-night, for I have been
so vexed and so distressed with much that has happened since my
arrival in London, that I can hardly call myself well. I am now much
fatigued, and if I can escape, I will hie me to my bed. When you
come to-morrow, you shall answer me a thousand questions that I
have to ask. Oh! I see I can pass round by that other door. Farewell
for this night!"

"Oh, that I dared hope it had been a happy one to you, as it has
been to me!" said the knight, still holding her hand with a fond and
lingering pressure.
 "It has, Darnley; it has!" replied Lady Constance; "it has been one
that I shall never forget. Farewell!" and turning away, she passed
out of the door at the side, which led to the apartments in that wing
of the building: not, however, without one look more into the room
where her lover stood gazing still, to catch the last glance of that
graceful figure ere it left his sight.

When she was gone, the young knight, with a high-beating heart,
turned to the door of the great hall, and entered with some of the
last lingerers, who were now changing their slowness into speed, in
order to get a place before the pageant entered. The thoughts of Sir
Osborne, however, were employed on so much more engrossing
subjects, that he took no pains to hasten his steps till he was fairly
within the chamber, when, seeing the whole of the guests arranged
on the farther side of the hall, with the queen in the centre, under
her canopy or cloth of estate, he felt the impropriety of standing
there alone, and hastened to seek a place.

At that moment he observed Henry, who, still disguised, was

seated amongst the rest, and who made him a sign to take a place
beside him. Notwithstanding his mask, however, it was very evident
that the king was known; for, on his sign to Sir Osborne, all around
made way for the young knight to approach the monarch. Scarcely
had he taken his seat when, through the great doors of the hall, a
huge machine was rolled in, before which extended a double cloth of
arras, so arranged as to hide every part of the gewgaw within, only
leaving a twinkling light here and there, seen through the crevices,
like the lamps that, through the cracks of the last scene in a
pantomime, announce the brilliant change that is soon to take place
to the temple of Love or Venus, or some other such sweet power,
that deals in pasteboard and spangles.

But such a thing can never be so well described as in the words of

those who saw it, and whose old stiff style harmonises admirably
well with the quaint and graceless show that they detail. We shall
therefore only so far modify the account which Hall, the chronicler,
gives of this very pageant, as to render him generally intelligible.

"Then," says he, "there was a device or pageant brought in, out
of which pageant issued a gentleman richly apparelled, that showed
how, in a garden of pleasure, there was an arbour of gold, wherein
were lords and ladies, much desirous to show pleasure and pastime
to the queen and ladies, if they might be licensed so to do; who was
answered by the queen, how sire and all other there were very
desirous to see them and their pastime, when a great cloth of arras,
that did hang before the same pageant, was taken away, and the
pageant brought more near. It was curiously made and pleasant to
behold; it was solemn and rich, for every post or pillar thereof was
covered with frieze gold. Therein were trees of hawthorn, eglantines,
roses, vines, and other pleasant flowers of divers colours, with
gillofers and other herbs, all made of satin, damask, silk, silver and
gold, accordingly as the natural trees, herbs, or flowers ought to be.
In which arbour were six ladies, all apparelled in white satin and
green, set and embroidered full of H. and K. of gold, knit together
with laces of gold of damask, and all their garments were
replenished with glittering spangles gilt over; and on their heads
were bonnets all opened at the four quarters, overfriezed with flat
gold of damask. In this garden also were six lords, apparelled in
garments of purple satin, all of cuts with H. and K. Every edge
garnished with friezed gold, and every garment full of posies, made
in letters of fine gold in bullion, as thick as might be; and every
person had his name in like letters of massy gold. The first, Cœur
Loyal; the second, Bonne Volure; the third, Bon Espoir; the fourth,
Valiant Désire; the fifth, Bonne Foi; the sixth, Amour Loyal. Their
hose, caps, and coats, were full of posies and H. K.'s of fine gold in
bullion, so the ground could scarce appear, and yet in every void
place were spangles of gold. When time was come, the said pageant
was brought forward into presence, and then descended a lord and
lady by couples, and then the minstrels, which were disguised, also
danced, and the lords and ladies danced, that it was a pleasure to
behold."
 Such is old Hall's description of the pageant which now entered:
and it may easily be imagined that Sir Osborne, accustomed to a
less luxurious court, was somewhat astonished at the splendour of
the scene, if he was not much gratified by the good taste of the
device.

When the eye of Henry, pampered with such gaudy food from day
to day, had taken in enough of the pageant, he rose from his seat,
and waving his hand for the musicians to cease, "Thanks, gentle
lords and ladies; thanks!" he cried; and taking off his own mask,
added, "Let us ease our faces of their vizards."

As he spoke, every one rose and unmasked; and Henry, taking Sir
Osborne by the hand, led him forward to the queen, while all eyes
naturally fixed upon him.

"Fair lady mine," said the king, "I bring you a good knight, Sir
Osborne Maurice, who, as you see, has wit at will, and who, I can
vouch, is as keen a champion in the saddle as he is a graceful
dancer in the hall. In short, he is a very gentle perfect knight, whom
you must cherish and receive for my love."

While Sir Osborne knelt and kissed the hand that she extended to
him, Katherine replied, "Indeed, my lord, you have brought me one
that I have longed to see. This is the good knight who, on his
journey towards London, took charge of my giddy girl and
namesake, Katrine Bulmer, and defended her from the Rochester
rioters. Come hither, Kate, and in our presence thank the knight for
all the trouble I am sure he had with thee upon the road."

"Nay, your grace," said Lady Katrine, advancing, "I have thanked
him once already, and men are all too saucy and conceited to thank
them twice."

"'Tis thou art saucy, my fair mistress," said the king, laughing;
and then bending down his head to the queen, who was still seated,
he whispered something to her which made her smile and raise her
eyes to the knight and Lady Katrine. "A handsome pair, indeed!" said
she, in reply to what the king had whispered. "But the banquet is
ready."

"Lords and ladies," said Henry, raising his voice, "our royal
mistress will not let us part without our supper. All, then, come in
pairs, for in the White Hall is prepared a banquet. Sir Osborne, lead
in Lady Katrine there; you shall be coupled for an hour at least."

Sir Osborne glanced his eye to Lord Darby; but the earl was
perfectly master of his countenance, and looking as indifferent as if
nothing had happened, led in some other lady, while the knight
endeavoured to entertain Lady Katrine as well as he might, labouring
under the comfortable assurance that she would very much have
preferred another by her side.

CHAPTER XXI.

Would I a house for happiness erect,

Nature alone should be the architect.--Cowley.

Light hath no tongue, but is all eye;

If it could speak as well as spy,
This were the worst that it could say,
That being well I fain would stay.--Donne.

We must now pass over a brief space of time with but little
commemoration.

It was a bright and beautiful morning in the beginning of the

month of May, when the sky was of that soft, tender blue which it
possesses in the early year, ere the ardent rays of summer have
dyed it with a deeper tint; and yet there was nothing of that misty
faintness of hue which foretels that the blue eye of heaven may be
filled with tears before nightfall. It was clear, though it was soft; and
the light white clouds that, winged by the breeze, sped quickly over
the wide expanse, gave to the earth no trace of their passing, except
the fleeting shadows that followed them, which, hurrying rapidly
over the distant fields and woods, made each spot as they left it look
brighter than before. Every object that met the eye spoke of spring.
The bright green of the trees, and the fields, and the woods, clearly
told that they had not known the burning touch of summer, which,
like manhood and the world's experience, coming o'er the fresh
dreams of youth, withers while it ripens, and with its very first
approach steals somewhat of the refreshing hue of early nature. The
wild singing of the birds, rejoicing in the return of brightness to the
earth, and making the whole air vocal with the bursting happiness of
their renewed enjoyment; the busy hum of animated being rising up
from hill, and dale, and wood, and joining with their song upon the
breeze; all spoke of refreshed existence. Flowers painted the fields,
and blossoms hung upon the trees, and perfume shook its light
wings in the morning air and sprinkled it with balm.

It was one of those mornings when the heart opens, and when
every vein thrills with glad existence; when we feel, as it were, the
Deity on the morning's breath; when we hear Him in the voice of
creation; when we worship Him in his works, and adore Him in the
temple He himself has raised. The scene, too, was lovely. It was in a
wide open park, where the rich thick grass spread like velvet over
every slope and lawn; so rich, so thick, its elasticity almost raised the
foot that trod it. On its luxuriant bosom the wide old trees, scattered
in clumps, or gathered together in broad sweeping woods, cast a
deep shadow, defined and clear, making the glossy softness and the
vivid green shine out more strongly for the contrast. It was the elm
and the oak that principally tenanted that park, though occasionally
a hawthorn or a beech would interpose; and wherever they
congregated in a wood there was to be found every sort of shrub
and brushwood clinging round their roots. Many a glade, however,
appeared, and many a lawn between; and where the trees broke
away, there a wide extended view presented itself, showing a rich
and fertile country beyond, full of green hedgerows and fields,
broken and diversified by the lines of hamlets and villages, mingling
an air of wealth, prosperity, and living gladness, with the bright
sweetness of the morning and the calm tranquillity of the park itself.

At the foot, then, of one of the old oaks in Richmond Park sat
Lady Constance de Grey, while her woman Margaret stood at a little
distance with a page, and Sir Osborne Maurice leaned by her side.
They had met by chance--really by chance--at that early hour in that
remote part of the park; though it is more than probable that the
same thoughts, acting on hearts so nearly allied, had led them both
forth to meditate on their fate. And even after they had met, the
stillness of the scene seemed to have found its way to their souls,
for they remained almost in silence watching the clouds and gazing
at the view, content to feel that they enjoyed together the same
sweet morning and the same lovely scene.

It may be as well, however, before proceeding further, to give

some slight sketch of what had occurred since the close of the last
chapter; though were we to account for every day, it would be but
detail of just after just, tourney after tourney, revel upon revel,
wearisome from their repetition, and sickening from their vain
splendour. Suffice it that Sir Osborne still maintained his place in the
king's favour. His lance was always held by the judges of the field as
next to the king's: his grace in the hall, or at the court, his dexterity
in martial exercises, his clerkly learning, and his lighter
accomplishments, won him much admiration; while a sort of
unassumingness, which seemed to hold his own high qualities as
light, silenced much envy. In short, it became the fashion to praise
him; and it is so easy for courtiers to applaud or to decry, as the
veering breath of favour changes, that to believe the outward
semblance, Sir Osborne Maurice, next to the king himself, and
Charles Brandon Duke of Suffolk, was the god of the court's idolatry.
 There was, however, many a curious whisper of--Who was he?
Whence did he come? What was his family? And some of the knights
who had served abroad, and had been with the king at Terouenne
and Tournay, conferred together, and shook the wise head; but still it
was remarked that they were amongst those who most praised and
sought the young knight. Sir Osborne marked with a keen and
observing eye all that passed about him; and seeing that he was
recognised by more than one, he felt that he must hasten to prevent
his secret being communicated to the king by any lips but his own;
and now high in favour, he only waited a fitting opportunity to
hazard all by the avowal of his name and rank.

Wolsey had been absent for nearly a month in his diocese at York,
and, removed from the influence of his presence, Lord Darby and
Lady Katrine Bulmer, Sir Osborne and Constance de Grey, seemed to
have forgot his stern authority, and given course to the feelings of
their hearts. The knight had seen Lady Constance almost every day;
and good Mistress Margaret, her woman, with whom Sir Osborne
was no small favourite, took care not to exercise towards him that
strict etiquette which she practised upon all other visitors, leaving
them full opportunity to say all that the heart sought to
communicate, as she very well perceived what feelings were busy in
their breasts.

Thus everything between them was explained, everything was

known: there was no coldness, there was no reserve, there was
none of that idle and base coquetry which delights in teasing a heart
that loves. Constance de Grey loved sincerely, openly, and she had
too high an esteem for the man she had chosen, to suppose that the
acknowledgment of that love could make it less worthy in his eyes.
Happy indeed it was for them both that the most perfect confidence
did exist between them, for Henry had conceived the project of
marrying the young knight to Lady Katrine; and though the queen,
with the instinctive perception of a woman in those matters, soon
saw that such a plan would very ill accord with the feelings of either
party, and quickly discouraged it, yet Henry, giving way to all his own
impetuosity, hurried it on with precipitation, took every occasion to
force them together, and declared that he would have them married
as soon as the court returned from the meeting with the French king
at Guisnes.

The situation of Sir Osborne was not a little embarrassing, the

more especially as Lady Katrine, in her merry malice, often seemed
to give in entirely to the king's schemes, having a threefold object in
so doing, if object can be attributed to such heedless gaiety; namely,
to coquet a little with Sir Osborne, which she did not dislike with
anybody, to enjoy his embarrassment, and, at the same time, to
tease Lord Darby.

With these three laudable motives she might have contrived to

make Sir Osborne and Lady Constance unhappy, had not that mutual
confidence existed between them which set all doubts at defiance.
Nor, indeed, was it Lady Katrine's wish to do harm: whimsical, gay,
and thoughtless, she gave way to the impulse of the moment. If she
was in good humour, she was all liveliness and spirit, running as
close to the borders of direct flirtation as possible with whomsoever
happened to be near; but, on the contrary, if anything went wrong
with her, she would be petulant and irritable, showing forth a
thousand little airs of affected dignity and reserve which were not
natural to her. No one's good regard did she seek more than that of
Lady Constance de Grey; and yet she seemed to take every way to
lose it. But Constance, though so different herself, understood her
character, appreciated the good, made allowance for the faults, and
secure in Darnley's affection, forgave her little coquetry with her
lover.

In regard to Lord Darby, he knew Lady Katrine too; and if ever he

gave himself a moment's uneasiness about her waywardness, he did
not let it appear. If she flirted, he flirted too; if she was gay, he took
care not to be a whit behind; if she was affectionate, he was gentle;
and if she was cross, he laughed at her. She never could put him out
of humour, though, to do her all manner of justice, she tried hard;
and thus finding her attempts to tease ineffectual, she gradually
relaxed in the endeavour.

In the mean time, the days of Sir Osborne and Lady Constance
flew by in a sweet calm, that had something ominous in its
tranquillity. He had almost forgotten Sir Payan Wileton; and in the
mild flow of her happiness, Constance scarcely remembered the
schemes with which the avaricious and haughty Wolsey threatened
to trouble the stream of her existence. But, nevertheless, it was to
be expected that if the dispensation had not yet arrived from Rome,
it could not be delayed more than a few days; and that, at the
return of the minister from York, the command would be renewed
for her to bestow her hand upon Lord Darby. Such thoughts would
sometimes come across Constance's mind with a painful sensation of
dread; and then, with a spirit which so fair and tender an exterior
hardly seemed to announce, she would revolve in her mind a plan
for baffling the imperious prelate at all risks, and yet not implicate
her lover at the very moment that his "fortunes were a-making."

Then, again, she would often hope that the extraordinary

preparations that were going forward for the speedy meeting of the
two courts of France and England, all the ceremonies that were to
be arranged, and the many important questions that were to be
discussed, would divert the mind of the cardinal from herself, at
least till after that meeting had taken place; during which interval
chance might produce many circumstances more favourable to her
hopes. At all events, her resolution was taken: she felt, too, that no
power on earth was adequate to combat that determination; and
thus, with fixed purpose, she turned her mind from the
contemplation of future dangers to the enjoyment of her present
happiness.

The scene in Richmond Park, to which the court had now

removed from Greenwich, as well as the bright gentleness of the
May morning in which she met Sir Osborne there, was well
calculated to nurse the most pleasing children of hope; and yet there
was something melancholy even in the magnificent aspect of the
day. I know not how, but often in those grand shining mornings the
soul seems to swell too powerfully for the body; the spirit to feel
galled, as it were, by the chain that binds it to mortality. Whatever
be the cause, there is still, in such a scene, a pensiveness that steals
upon the heart; a solemnity that makes itself felt in those innermost
recesses of the mind where thought and sensation blend so
intimately as to be hardly separable from each other. Constance and
Darnley both felt it; but still it was not sorrow that it produced; for,
mingling with their fervent love and their youthful hope, it gave their
feelings something of divine.

"This is very, very lovely, Darnley," said Lady Constance, after

they had long gazed in silence. "Oh, why are not all days like this!
Why must we have the storm, and the tempest, and the cloud!"

"Perhaps," replied the knight, "if all days were so fair, we might
not esteem them so much: we should be like those, Constance, who
in the world have gone on in a long course of uninterrupted
prosperity, and who have enjoyed so much that they can no longer
enjoy."

"Oh, no, no!" cried she; "there are some pleasures that never
cloy, and amongst them are those that we derive from
contemplating the loveliness of nature. I cannot think that I should
ever weary of scenes like these. No! let me have a fairy sky, where
the sunshine scarcely knows a cloud, and where the air is always
soft and sweet like this."

At this moment Mistress Margaret approached, with some

consternation in her aspect. "Good now, lady!" cried she; "look! who
is that coming? Such a strange-looking little man, no bigger than an
atomy! Oh! I am glad the knight is with us; for it is something
singular, I am sure."
 "You are very right, Mistress Margaret," said Sir Osborne; "this is,
indeed, a most singular being that approaches. Constance, you have
heard the queen and her ladies speak of Sir Cesar, the famous
alchymist and astrologer. He is well known to good Dr. Wilbraham,
and seems, for some reason, to take a strange interest in all my
proceedings. Depend on it, he comes to warn us of something that
is about to happen, and his warning must not be slighted; for, from
wheresoever his knowledge comes, it is very strange."

Lady Constance and the knight watched the old man as he came
slowly over the green towards them, showing little of that vivacity of
demeanour by which he was generally characterised. On
approaching near, he bowed to Lady Constance with courtly ease,
saluted the knight in a manner which might be called affectionate;
and, without apology for his intrusion, seated himself at the lady's
feet, and began a gay and easy conversation upon the justs of the
day before.

"There is no court in the world," said he, after a little--"and there

are few courts I have not seen--where such sports are carried to the
height of luxury that they are here. I never saw the tournaments,
the justs, the pageants of Henry the Eighth, King of England,
excelled but once."

"And when was that, may I ask?" demanded Lady Constance,

whose feelings towards the old man were strangely mingled of awe
and curiosity, so much had she heard of him and his strange powers
during her residence at the court.

"It was in Germany," replied Sir Cesar, "at the city of Ratisbon;
and it was conducted as all such displays should ever be conducted.
Each knight wore over his armour a motley suit, and on his casque a
cap and bells; the hilt of his sword was ornamented with a bauble,
and as they made procession to the lists, the court fools of all the
electors in the empire followed behind the knights, and whipped
them on with blown bladders."
 "Nay, nay, you are a satirist," said Lady Constance; "such a thing,
surely, could never happen in reality."

"In truth it did, lady," answered Sir Cesar; "it was called the
Tournament of Fools, though I wot not to distinguish it from other
tournaments, which are all foolish enough. Osborne," he continued,
turning abruptly to the young knight, "you will ride no more at this
court."

"How mean you?" demanded Sir Osborne: "why should I not?"

"I mean," replied the old man, "that I come to forewarn you of
approaching evil. Perhaps you may turn it aside, but there is much
that threatens you. Are you not losing time? The king's regard is
gained; wherefore, then, do you delay? While Wolsey is absent--
mark me! while Wolsey is absent--or you are lost for the moment."

"Oh! say not so," cried Lady Constance, clasping her hands; "oh!
say not so, for I hear that he returns to-morrow."

"Fear not, lady," said Sir Cesar, who had now risen; "the danger
will last but for a time, and then pass away. So that, whatever
happens to either of you, let not your hearts sink; but be firm,
steadfast, and true. All the advice I can give you is but the advice of
an ordinary mortal like yourselves. Men judge rashly when they think
that even those who see clearest can yet see clear. All that I know,
all that I behold, is but a dim shadowing forth of what will be, like
the indistinct memory of long gone years; a circumstance without a
form. I see in both your fates an evil and a sorrowful hour
approaching, and yet I cannot tell you how to avoid it; but I can
descry that 'twill be but for a while, and that must console you."

"Good Sir Cesar," said the young knight, "I will ask you no
questions, for I have now learned that you were a dear friend of my
father, and I feel sure that you will give all knowledge that may be
useful to me; and if you will tell me what is good to do in this
conjuncture, I will follow it."
 "Good, now!" said Sir Cesar, with a gratified look: "good! I see
you are overcoming your old fault, though you have been a long
while about it. Three thousand years! three thousand years to my
remembrance."

Constance turned an inquiring look to her lover, who, however,

was not capable of giving her any explanation. "Think you,"
demanded he, addressing Sir Cesar, "that it would be best to inform
his grace of everything at once?"

"I think it would," said the old man; "I think it would, but I
scarcely dare advise you. Osborne, there is a conviction pressing on
my mind, which I have perhaps learned too late. Can it be that those
who are permitted to read certain facts in the book of fate are
blinded to the right interpretation of that which they discover?
Perhaps it may be--I have reason to believe it. Nought that I have
ever calculated has proved false; but often, often it has been verified
in a sense so opposite to my expectations, yet so evident when it did
appear, that it seems as if heaven held the search presumptuous,
and baffled the searcher even with the knowledge he acquired.
Never more will I presume to expound aught that I may learn. The
fact I tell you: an evil and a bitter hour is coming for you both, but it
shall not last, and then you shall be happy--when I am no more."
And turning away without other farewell, he left them, and took the
way to the palace.

Lady Constance gazed on the face of her lover with a look of

apprehensive tenderness that banished all thought of himself. "Oh,
my Constance!" said he, "to think of your having to undergo so
much for me is too, too painful! But fear not, dear Constance; we
are still in a land where laws are above all power, and they cannot,
they dare not ill-treat you!"

"For myself, Darnley," replied Constance, "I have no fear. They

may threaten, they may wrong me, they may do what they will, but
they can never make me marry another. It is for you I fear. However,
he said that we should be happy at last, though he hinted that you
would be driven from the court. Oh, Darnley! if that be the case--if
you find there be the least danger--fly without loss of time----"

"And leave behind me," said Darnley, "all I love in the world! Oh,
Constance! would not the block and axe itself be preferable? It
would, it would, a thousand times preferable to leaving you for
ever!"

"It might," said Constance; "I myself feel it might, if you feel as I
feel. But, Darnley, I tell you at once I boldly promise to follow."

"But still, Constance, dear, excellent girl!" said the knight, "would
it be right, would it be honourable, in me to accept such a sacrifice?"

"Darnley," said Lady Constance, firmly, "my happiness is in your

hands, and what is right and honourable is not to throw that
happiness away. Now that my love is yours, now that my hand is
promised to you, you have no right to think of rank, or fortune, or
aught else. If I were obliged to fly, would you not follow me? and
wheresoever you go, there will I find means to join you. All I ask, all
I pray in return is, that if there be the least danger, you will instantly
fly. Will you promise me? If you love me you will."

"I will," said Sir Osborne. "What would I not do to prove that love!
But I trust, dear Constance, there may be no need of hasty flight. All
they can do will be to banish me the court, for I have committed no
crime but coming here under a feigned name."

"I know not; I know not," said the lady; "'tis easy, where no crime
is, to forge an accusation; and, if report speak truth, such has been
Wolsey's frequent policy, when any one became loved of our
gracious king; so that even the favour you have gained may prove
your ruin. But you have promised to fly upon the first threatening of
danger, and I hold as a part of that promise that you will stay for no
leave-taking."