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Pathogenecity-associated
genes modulate Escherichia coli
adhesion and motility
Annika Sjöström

Department of Molecular Biology

Umeå University Sweden 2009
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New series Nr. 1266
ISSN 0346-6612
ISBN: 978-91-7264-789-3
Editor: The Dean of the Faculty of Medicine

Pathogenecity-associated
genes modulate
Escherichia coli adhesion
and motility

Annika E. Sjöström

Department of Molecular Biology

Umeå University Sweden 2009
Department of Molecular Biology
Umeå University
SE-901 87 Umeå, Sweden

Copyright © 2009 by Annika E. Sjöström

Printed by Print & Media

Under cover picture:

"Bakterier" ritade av Ossian 7.5år, Erik 10.5år, Anton snart
12år, Vera 5år, Holger 3.5år, Maja 12år och Hugo snart 7år.

The drawing shows: One bacterium — different magnifications (Ossian), a

pathogen (Erik), view in a microscope (Anton), a plant pathogen and an
animal pathogen (Vera), capsulated bacteria (Holger), a commensal
(Maja), and antigenic variation (Hugo).
Table of contents

ABSTRACT....................................................................................1

PAPERS IN THIS THESIS.............................................................. 2

BACKGROUND.............................................................................. 3

1. INTRODUCTION....................................................................... 4

1.1. Escherichia coli................................................................. 4

1.1.1. Flagella...................................................................... 6
1.2. Pathogenicity islands (PAIs)............................................. 8
1.3. Fimbriae or pili................................................................. 9
1.3.1. Receptor specificity and tissue tropism...................... 10
1.3.2. Biogenesis of the fimbriae......................................... 11
1.3.3. Visualization and measuring of fimbriae bio-
mechanical properties............................................... 13
1.3.4. Regulation of fimbriae expression............................. 15
1.3.4.1. Type 1 fimbriae (core chromosome) ........................ 16
1.3.4.2. PAI-encoded fimbriae............................................ 17
1.3.4.2.1. Regulatory crosstalk among fimbrial operons..... 18
1.3.5. Other regulatory factors............................................ 18
1.3.5.1. The H-NS protein................................................... 18
1.3.5.2. The RpoS, σs, stress sigma factor............................. 18
1.3.5.3. The cyclic-di-GMP signal molecule............................ 19
1.3.5.4. The Hfq protein..................................................... 20
1.4. The X and Y genes............................................................ 20

2. AIMS OF THIS THESIS.............................................................. 21

3. RESULTS AND DISCUSSION......................................................21

3.1. Paper I............................................................................. 22

3.1.1. Strategy.....................................................................22
3.1.2. Effects of sfaXII mutations in the IHE3034 strain....... 23
3.1.3. Effects of complementation and overexpression of
the sfaXII gene on motility......................................... 23
3.1.4. Effects of complementation and overexpression of
the sfaXII gene on type 1 fimbriae expression........... 24
3.1.5. Effects of expression of the sfaXII gene on
pathogenicity-associated genes.................................26
3.1.6. The SfaXII protein...................................................... 27
3.1.7. Conclusion................................................................. 29
3.2. Paper II................................................................................ 29
3.2.1. Strategy.....................................................................29
3.2.2. S fimbriae in comparison to other fimbriae................ 30
3.2.3. Effect of sfaX mutation.............................................. 31
3.2.4. Clinical isolate vs. ectopic fimbriae expression.......... 31
3.2.5. Conclusion................................................................. 32
3.3. Paper III.......................................................................... 32
3.3.1. Strategy.....................................................................32
3.3.2. Expression profile of sfaXII........................................ 33
3.3.3. sfaXII is included in the sfaII fimbrial transcriptional
unit........................................................................... 34
3.3.4. Discovery of an additional gene, sfaYII, in the
sfaII fimbrial operon.................................................. 35
3.3.5. Conclusion................................................................. 36
3.3. Paper IV.......................................................................... 37
3.4.1. Strategy.................................................................... 37
3.4.2. Effect of RpoS on sfaXII expression............................ 37
3.4.3. Effect of Hfq on sfaXII expression.............................. 38
3.4.4. Effect of combined hfq and sfaXII expression on fim
expression................................................................ 39
3.4.5. Random mariner transposon mutagenesis................. 39
3.4.6. Conclusion................................................................. 40

4. CONCLUDING REMARKS........................................................... 40

ACKNOWLEDGEMENTS................................................................. 42

REFERENCES ............................................................................... 44
ABSTRACT
Escherichia coli strains typical of UPEC (uropathogenic E. coli) and NBM
(newborn meningitis) isolates carry chromosomally located PAIs
(pathogenicity islands) that are absent in non-pathogenic E. coli strains.
The PAIs include genes for virulence factors such as toxins and genes
coding for specific adhesins and pili/fimbriae formation. Commonly, the
gene clusters for such fimbriae in E. coli consist of a set of genes for
biogenesis of the actual fimbriae organelles: a chaperone, an usher, the
fimbrial subunits, and an adhesin, as well as some regulatory genes.
Genetic studies of the fimbrial gene clusters in PAIs containing the pap
genes, the prs genes, or the sfa genes led to the discovery of nearby open
reading frames coding for putative cytoplasmic 17 kDa proteins — the X
genes. Molecular genetic studies of the sfaXII locus in the clinical NMEC
isolate IHE3034 have been performed. The results suggested that
expression of the sfaXII gene had regulatory functions affecting both type
1 fimbriae expression and the flagella-mediated motility.
Type 1 fimbriae expression was found to be affected at the level of fim
operon transcription and a major reason was SfaXII-mediated modulation
of expression from the fimB and fimE recombinase genes. Quantification
of SfaII-fimbriated bacteria in a comparison between wild type and SfaXII
mutant strains gave no indication that the sfaXII gene product also would
be affecting expression and/or biogenesis of SfaII fimbriae.
Biomechanical properties of the SfaII fimbriae produced by wild type and
the sfaXII mutant IHE3034 were studied using force measuring optical
tweezers (FMOT) and compared to other PAI-encoded fimbriae as well as
to the type 1 fimbriae encoded on the core chromosome. The FMOT
methodology assesses unfolding and refolding properties and we found
that S fimbriae had weaker layer-to-layer interactions than both P and
type 1 fimbriae, however the unfolding kinetics was slightly faster.
The expression profile and regulation of the sfaXII gene were determined
by use of reporter gene fusions and it was found that expression was
affected by environmental cues such as pH, osmolarity and temperature.
It was also discovered that the nucleoid structuring protein H-NS and the
sigma factor RpoS had strong direct or indirect repressive effects on sfaXII
gene expression.
Further genomic analysis of the PAI fimbrial operons revealed that in
some cases an additional ORF was found between the X genes and the
fimbrial adhesion genes. Examination of the sfaII operon in IHE3034
indicated that this new gene, denoted sfaYII, coded for a protein that had
the EAL domain motif and thereby could be considered a putative
phosphodiesterase involved in controlling the level of cyclic-di-GMP in the
bacterial cells.

1
PAPERS IN THIS THESIS

Paper I
The SfaXII Protein from Newborn Meningitis E. coli is Involved in
Regulation of Motility and Type 1 Fimbriae Expression
Annika E. Sjöström, Carlos Balsalobre, Levente Emödy, Benita
Westerlund-Wikström, Jörg Hacker, and Bernt Eric Uhlin.
Microbial Pathogenesis 46 (2009) p. 243–252

Paper II
Unfolding and Refolding Properties of S Pili on Extraintestinal
Pathogenic Escherichia coli
Mickaël Castelain, Annika E. Sjöström, Erik Fällman, Bernt Eric Uhlin, and
Magnus Andersson.
Manuscript.

Paper III
Analysis of the sfaXII Locus in the Escherichia coli Meningitis
Isolate IHE3034 Reveals Two Novel Regulatory Genes within the
Promoter-distal Region of the Main S-fimbrial Operon.
Annika E. Sjöström, Berit Sondén, Claudia Müller, Anna Rydström, Ulrich
Dobrindt, Sun Nyunt Wai, and Bernt Eric Uhlin.
Microbial Pathogenesis 46 (2009) p. 150–158

Paper IV
Growth Phase Regulated Expression of sfaXII is Negatively
Influenced by the RpoS Sigma Factor and the Hfq RNA Chaperone
Annika E. Sjöström and Bernt Eric Uhlin.
Manuscript.

2
BACKGROUND

Escherichia coli is one of the most well-studied bacterial species found in

the intestinal tracts of humans. Despite the common species name,
different E. coli strains affect the human host in quite diverse ways,
ranging from helpers to non-affecters to killers. On one hand, there are
the commensal E. coli strains present in the gut of the human that live in
symbiosis with their host, helping to prevent colonization of pathogenic
bacteria that might enter the gastrointestinal tract through contaminated
food or water. On the other hand, there are the pathogenic E. coli strains
that cause severe or even deadly diseases in humans e.g., bloody
diarrhea, meningitis, and urinary tract infections (UTI) that can develop
into pyelonephritis.
These pathogenic strains have developed special "weapons" and tools to
disguise themselves from the human immune response, using properties
that are lacking in the commensal strains e.g., toxins, capsules (shields),
and several kinds of fimbriae for adhesion (anchoring devices). An
important factor for the survival and persistence of the pathogenic
bacterium in the body is how to control the function of these equipment -
to be able to "realize" when it is suitable to use one type of device and not
another, and to quickly respond to changes in the surrounding
environment.
During my Ph.D. period, my research has been focused on the regulatory
mechanisms involved in adhesion, in particular the role of one of these
"controllers" — regulators — the SfaXII protein. This kind of protein (X-
protein) is only found in pathogenic E. coli strains and my aim was to
figure out what function/s it controls and under what circumstances
(temperature, availability of nutrition, pH, etc.) the bacterium needs it.

3
1. INTRODUCTION
1.1. Escherichia coli
Broadly, the E. coli strains can be divided into three different pathotypes
— the commensal E. coli, the intestinal pathogenic E. coli, and the
extraintestinal pathogenic E. coli (Russo and Johnson, 2000) (Fig. 1).

Commensal strains
"Harmless" commensal E. coli

Intestinal pathogenic strains (IPEC)

EPEC — enteropathogenic E. coli
EHEC — enterohemorrhagic E. coli
Causing diarrhea
EIEC — enteroinvasive E. coli
ETEC — enterotoxogenic E. coli

Extraintestinal pathogenic strains (ExPEC)

NMEC — meningitis E. coli, causing newborn meningitis (NBM)
UPEC — uropathogenic E. coli, causing UTI and pyelonephritis
SEPEC — sepsis associated E. coli

Fig. 1 A few classes of different Escherichia coli strains — "pathotypes".

There are several potential explanations for how one species can have
such seemingly opposing roles in contact with humans. One factor is
where the bacteria are found in the body. Commensal E. coli strains
residing in the gut are the most frequent species in the fecal flora and
usually do no harm to the human host even if they spread to other
locations in the body, unless the host is immunocompromised. Also,
ExPEC strains normally reside in the intestinal tract where they can be
considered part of the commensal flora. But, in contrast to the commensal
strains, ExPEC strains outside the intestinal tract cause severe harm even
in otherwise completely healthy hosts (Russo and Johnson, 2000). In year

4
2000, it was proposed that the term ExPEC should be used instead of
UPEC, NMEC, and SEPEC, because of these strains’ ability to cause disease
at multiple anatomical sites (Russo and Johnson, 2000) (Johnson and
Russo, 2002). Of the diseases caused by ExPEC, the most common is
urinary tract infection (UTI). Furthermore, 70–95% of the UTI cases are
caused by ExPEC. More than 10% (350 000) of Swedish women >18
years old need antibiotic treatment for at least one occurrence of UTI/yr.
Among those, 30-40% need treatment for recurrent UTI the following year
(Läkemedelsverket 2009, http://www.lakemedelsverket.se).
The main reason commensal E. coli and ExPEC strains, localized in
extraintestinal areas, elicit such different host responses is because of
dissimilarities in gene composition. The genomic sizes of E. coli strains
range from 4.64 Mb (K-12 MG1655, NC_000913) to 5.57 Mb (O157:H7
EC4115, NC_011353), thus almost 1 Mb difference. Comparisons of all the
hitherto sequenced E. coli strains reveal that they all carry a common but
slightly flexible ~4 Mb core chromosome coding for ~2800 open reading
frames (ORFs) (Dobrindt et al., 2004) (Dobrindt, 2005). The genes found
on this core chromosome are the housekeeping genes, essential for
cellular functions, and genes that are beneficial under special conditions
(e.g., antibiotic resistance genes). Two other gene systems that are of
importance for my research, namely the type 1 fimbrial operon
(adherence) and all genes needed to express functional flagella (motility),
are also encoded on the core chromosome. Type 1 fimbriae will be
described in the section "Fimbriae or pili" but I want to highlight that
these fimbriae also will be referred to as the core chromosome-encoded
fimbriae.

5
1.1.1. Flagella
Most E. coli strains are motile, both non-pathogenic and pathogenic
strains. An effective means for movement is provided by flagella (Fig. 2).
These are long, rope-like helical filaments that by rotating create force
resulting in movement of the bacterium.

1 μm

Fig. 2 Atomic force microscopy (AFM) image of the flagellated E. coli strain M15/pQE30.
(Image courtesy of Monica Persson).

Repeated switching from counter clockwise (CCW) flagellar rotation to

clockwise (CW) flagellar rotation and back again keeps the bacterium
moving. The synchronized CCW rotation will push the bacterium forward
in a directed movement, while a non-synchronized CW rotation leads to
movement without direction — a tumble (Larsen et al., 1974). Depending
on the availability of nutrients, the switching frequency varies, resulting in
directed movements — chemotaxis.
The biosynthesis of flagella relies on a complex regulatory system divided
into three levels — the early, middle, and late genes. This beautifully
organized flagellar transcriptional hierarchy and assembly of the subunits
is reviewed by Chilcott and Hughes (Chilcott and Hughes, 2000) and
Macnab (Macnab, 2003) (Fig. 3). The major regulators (early genes)
respond to several different environmental cues as well as to global
regulators within the bacterium. Upon activation, these early genes are
expressed, building heteromeric double complexes that activate the

6
middle genes. The middle genes code for proteins building up the hook-
basal body (which anchors the flagellum to the cell membrane but also
functions as a secretion apparatus). One of the middle genes codes for a
sigma factor needed for expression of the late genes. This sigma factor
(σ28) is inactive until the basal-hook body is complete, whereupon the
active σ28 mediates transcription of the genes coding for the flagellum, the
motor, and the motor regulators. As stated before, all the genes needed
to express functional flagella are located on the core chromosome.

Global regulatory Completed flagellum

signals
Middle Genes
flgAMN
flgBCDEFGHIJKL
Early Genes flhBAE
flhC flhD fliAZY
fliDST
fliE Late Genes
fliFGHIJK flgMN
fliMNOPQR flgKL
fliC
fliDST
Hook-Basal Body fljBA
motAB
cheAW
tar
cheRBYZ
tsr
aer
Inactive Active Figure modified
28 from Chilcott and
σ σ28 Hughes (Chilcott
and Hughes, 2000)

Fig. 3 Schematic drawing of the flagellar transcriptional hierarchy leading to assembly of

the flagella. Expression of the middle genes is activated by the FlhDC complex (yellow
oval and white circle) that is formed as a response to environmental signals or global
regulators. The proteins of the middle genes build up the hook-basal body (black parts),
that also acts as a secretion machinery. FlgM (purple arc) and σ28 (FliA, red oval) makes
an inactive complex, but as soon as the secretion machinery is functional the FlgM/σ28
complex dissociates and FlgM is transported out of the cell and the active sigma factor
can bind to the RNA polymerase and direct transcription to the late genes. Expression
products of these late genes build up the actual flagella and the motor (black parts).

7
1.2. Pathogenicity islands (PAIs)
As mentioned above, the E. coli core chromosome has a size of
approximately 4 Mb that is greatly expanded in pathogenic strains,
thereby reaching 5 to 6 Mb. This additional DNA is scattered throughout
the entire chromosome and consists of bacteriophages, plasmids, insertion
sequence (IS) elements, transposons and integrons, and blocks of DNA in
the size range of 30-200 kb (probably acquired by horizontal gene
transfer). These DNA blocks are termed pathogenicity islands (PAIs) and
are defined as genomic regions present in a pathogenic strain, but are less
common in closely related non-pathogenic bacteria. PAIs are also, by
definition, often unstable and contain mobility genes that encode
integrases or transposases. Further, they have a G+C content that differs
from that of the rest of the chromosome, are frequently associated with
tRNA genes, carry one or several virulence-associated genes, and are
often flanked by repeat sequences (Hacker et al., 1997). Examples of
virulence genes present on PAIs are genes coding for toxin production,
capsule formation, iron acquisition, and genes coding for specific adhesins
involved in fimbriae/pili formation (Fig. 4).

Pathogenicity island
PAI

Core
chromosome

capsule toxin fimbrial determinant

Fig. 4 Schematic drawing of a pathogenic E. coli chromosome. In the magnified PAI,

virulence-associated genes are symbolized by rectangles flanked by repeat sequences
(black triangles).

8
1.3. Fimbriae or pili
To aid in persistence and colonization of a bacterial population in the
human body, a number of adhesive systems have evolved. The strength
of the adhesive properties of these systems varies depending on the
nature of the bacterium-tissue interaction and its binding specificity. One
adhesive strategy is development of fimbriae or pili (the term fimbriae will
be used in the rest of this text). Fimbriae are composed of many (100–
1000) small protein subunits building up a long, hair-like, filamentous
appendage that is attached to the outer cell membrane on the bacterial
surface (Fig. 5).

1 μm

Fig. 5 Atomic force microscopy (AFM) image of the fimbriated E. coli strain
HB101/pAZZ50 expressing S fimbriae. (Image courtesy of Monica Persson).

In this work, the focus is on the SI and SII fimbriae, encoded on the sfaI
and sfaII operons, and the type 1 fimbriae encoded on the fim operon.
Another fimbrial system that will be discussed is the P fimbriae encoded
on the pap operon (Fig. 6).

9
 sfaI and sfaII fimbriae operons 1 kb

C B A D E F G S H Y X

pap fimbriae operon

I B A H C D J K E F G Y X

fim operon

B E A I C D F G H

regulation chaperone minor subunits regulation?

major subunit usher adhesin

Fig. 6 Schematic overview of the gene organization of different E. coli fimbrial operons.
sfaI is from the UPEC 536 strain, sfaII from the NMEC IHE3034 strain, pap from the UPEC
J96 strain, and fim from the K-12 MG1655 strain. The adhesins are colored in orange and
different patterns of black to pinpoint their differences in receptor specificity.

The type 1 encoding genes, the fim operon, are present in many members
of the Enterobacteriaceae family and in virtually all E. coli strains
(Buchanan et al., 1985). In E. coli, all the fim genes are present on the
core chromosome in contrast to the genes encoding for the SI, SII and P
fimbriae that are situated on PAIs. This implies that these fimbriae are
only expressed by pathogenic E. coli, whereas type 1 fimbriae can be
expressed by both pathogenic and non-pathogenic strains. Consequently,
SI, SII, and P fimbriae will be referred to as the PAI-encoded fimbriae in a
similar manner as referring to type 1 fimbriae as the core chromosome
encoded fimbriae.

1.3.1. Receptor specificity and tissue tropism

An E. coli bacterium that carries several different fimbrial operons in its
genome, and thereby has the potential to express different kinds of
fimbriae, usually never expresses more than one type of fimbriae at a
time (reviewed by Holden et al.) (Holden and Gally, 2004). Although the
genetic organization and functions are similar among the S, P, and type 1

10
fimbrial systems, there are also some fundamental dissimilarities. The
choice of fimbriae expressed might very well reflect one of these
differences — the fact that each type of fimbriae binds to specific
receptors. These receptors are expressed on host cells and tissue, and
consist of glycolipids with different sugar moieties. Due to the specificity
of the fimbrial adhesins and the occurrence of its receptors on certain
tissue, fimbriae have been associated with specific diseases (Table 1).

Table 1. E. coli fimbriae, their receptors, tissue type localization of receptors, and
associated diseases.

Tissue/Associated
Fimbrium Receptor Reference
disease
kidney/urinary tract
(Parkkinen et al.,
SI infection
α-sialyl-2,3- 1986) (Prasadarao
and brain endothelial
β-galactose et al., 1993) (Virkola
SII cells/newborn
et al., 1988)
meningitis
(Leffler and
Svanborg-Edén,
erythrocytes,
Gal(α1-4) 1981) (Virkola et al.,
P kidney/pyelonephritis,
βGal 1988) (Striker et al.,
cystitis
1995) (Johanson et
al., 1992)
α-D- erythrocytes, (Old, 1972) (Virkola
Type 1
mannoside bladder/cystitis et al., 1988)

1.3.2 Biogenesis of the fimbriae

Biogenesis of these fimbriae — S, P, and type 1 fimbriae — occurs via the
chaperone-usher pathway (Fig. 7). From the bacterial cytosol via the
general secretory pathway, the different fimbrial subunits and the adhesin
are transported across the inner cell membrane. In the periplasm the
chaperone forms a complex with each subunit and stabilizes it by
"donating" a beta strand to complete an immunoglobulin-like fold (Sauer

11
et al., 1999). The chaperone-subunit complex is then transported to the
usher site, where it dissociates. The N-terminal strand of the incoming
subunit binds in the gap left in the last assembled subunit (completing its
immunoglobulin-like fold), thereby adding to the growing helical filament
— the donor-strand exchange (Sauer et al., 2002).

Outer membrane D H C
C
C A
C G
C H C C
G
A A
C
A Misfolding/Degradation
C
A Periplasm

C Inner membrane
Figure modified from
Sauer et al. 2004
(Sauer et al., 2004)

Fig. 7 Schematic figure of fimbriae assembly by the chaperone-usher pathway

exemplified by type 1 fimbriae. Subunit proteins enter the periplasm via the Sec system
(purple ovals). In case no chaperone-subunit complex is formed, the subunit misfolds,
aggregates, and is subsequently proteolytically degraded. If a chaperone-subunit
complex is formed, the chaperone (blue shape) folds, stabilizes, and caps the subunit
(red, orange and beige shapes). The complex docks to the pore-forming usher (white
shape) where it dissociates and the subunit is incorporated into the growing fimbrium via
donor-strand exchange.

12
1.3.3. Visualization and measuring of fimbriae biomechanical
properties
A visual comparison of the fimbriae in this work using atomic force
microscopy (AFM) reveals no major differences in appearance. All of them
look more or less like the fimbriae that are shown in Fig. 5, i.e., with a
diameter of approximately 5–7 nm and a length of approximately 2–5 µm.
To be able to appreciate possible differences, a more sophisticated
method, force measuring optical tweezers (FMOT), can be used
(Andersson et al., 2006b). Under certain conditions, highly focused laser
light can trap micrometer-sized objects (inorganic as well as biological
objects) by transfer of light momentum. The FMOT technique can be used
to initiate contact and measure forces between biological objects and
allows control and precise manipulation of living bacteria by light. The
possibility to apply and measure dynamic and static forces and the high
force resolution, <0.5 pN, can be used to obtain information at a
molecular level. An illustration of an optical tweezer constructed around
an inverted microscope is shown in Fig. 8.

Fig. 8 Schematic illustration of the force-measuring optical tweezers setup. The Nd:YVO4
laser beam (blue) traps a target in a fixed position by use of lenses and mirrors.
Deviation from this position is detected by a position-sensitive detector (PSD) that senses
light deflections created by the target from the probe laser (red) (Andersson, 2007).

13
By this method, the biomechanical properties of fimbriae can be assessed
at an individual level. Measuring the biomechanical properties of fimbriae
is performed by first fixing a mounting bead (9 µm), covered with poly-L-
lysine, to a cover slide. A bacterium is trapped by laser light and moved
and mounted on the mounting bead. Thereafter, a force transducer bead
(3 µm) is trapped by the tweezers and its behavior in the trap, i.e., the
trap stiffness, is determined by a statistical calibration. As shown in Fig. 9,
when a bead held in the optical trap is subjected to an external force, its
position will be shifted. The length of this shift is a direct and immediate
measure of the force applied to the bead. When a fimbrium is allowed to
adhere to the trapped bead, it is therefore possible to determine the
forces and kinetic properties of the fimbrium by monitoring the position of
the bead in the trap as the attached fimbrium is slowly dragged away
(thus creating a force between the bead and the fimbrium under study).

Fig. 9 Schematic illustration of a trapped bead (force transducer bead, 3 µm) used to
measure force probed by a laser light source (solid red). The light is monitored on a PSD
that through a calibration procedure converts the deflection into force. A bacterium is
mounted on 9 µm bead that is fixed to a cover slide. The mounted bacterium is
positioned to allow binding to the 3 µm bead. A. The probe light focused onto the
detector by a bead in its equilibrium position. No external force is applied. B. The
bacterium is pulled away, thereby moving the trapped bead, causing a deflection of the
light. The magnitude of deflection is converted to a corresponding force (Andersson,
2007).

The adhesion force in this type of measurement is given by the maximum

deflection of the bead in the trap before the binding between the bead and
the bacterium breaks. In addition to adhesion force measurements, it is
also possible to continuously monitor the force as a function of elongation

14
and elongation speed. In this way, various types of elastic as well as
dynamic elongation and retraction properties of the object under study
can be assessed and provide information about the internal structure of
the molecule.
Using the FMOT method has brought understanding to the structural
arrangement of a fimbrium, and the measurements have verified
mathematical models to explain mechanical behaviors (Andersson et al.,
2006a) (Andersson et al., 2006b) (Andersson et al., 2007). In addition,
the measurements can be used to discriminate between different types of
fimbriae since all types of fimbriae analyzed thus far have unique values
(Paper II).

1.3.4. Regulation of fimbriae expression

Expression of fimbriae is an energetically costly process and needs
therefore to be thoroughly regulated. In E. coli, two regulatory steps
maintain control of fimbrial expression. In the first step, it is determined
whether or not fimbriae will be produced at all, which illustrates how one
genetically identical population can have two different phenotypes. This
mechanism is called phase variation and allows the possibility for a pure
bacterial population — genetically identical — to include both bacteria
expressing fimbriae (ON) and bacteria not expressing fimbriae (OFF) —
different phenotypes. In the second regulatory step, the degree of
fimbriation of an ON-bacterium is determined and this depends on the
promoter activity of the fimbrial biogenesis operon. Both the phase
variation and promoter activity are tightly regulated and respond to
environmental signals such as osmolarity, temperature and aeration (Gally
et al., 1993) (Schwan et al., 2002) (White-Ziegler et al., 2000) (Schmoll
et al., 1990). Although the fimbrial systems commonly found in ExPEC
strains respond to similar cues, there are differences in the ways these
systems are regulated. PAI-encoded fimbriae expression is regulated by
binding of several proteins (activators and repressors) to the promoter

15
region, whereas type 1 fimbriae expression relies in addition on a
recombination event of an invertible DNA fragment.

1.3.4.1 Type 1 fimbriae (core chromosome)

Type 1 fimbriae expression is dependent on the orientation of an invertible
DNA fragment containing the promoter for the structural fim genes
(Abraham et al., 1985) (Fig. 10). In this case, the expressed
recombinases encoded upstream of the promoter region are the main
proteins responsible for phase switching in combination with a number of
intrinsic global regulators (Gally et al., 1996) (McClain et al., 1991)
(Dorman and Higgins, 1987) (Blomfield et al., 1993) (Blomfield et al.,
1997) (Schembri et al., 1998) (O'Gara J and Dorman, 2000) (Åberg et al.,
2006) (Müller et al., 2009) When the DNA fragment is in the ON-direction
(Fig. 10A), the promoter activity determines the degree of fimbriation of
the bacterium. When the fragment is in the OFF-direction (Fig. 10B), no
fimbriae are produced.

B E A
Off On

FimB

FimE
B

Off On
B E A

Fig. 10 Regulation of the fim operon. A. The recombinase FimB can mediate a switch of
the invertible DNA fragment containing the promoter region to an ON-state, i.e., fimbriae
are produced. B. The recombinase FimE mediates a switch of the invertible DNA
fragment containing the promoter region to an OFF-state, i.e., no fimbriae are produced.

16
1.3.4.2 PAI-encoded fimbriae
Regulation of PAI-encoded fimbriae does not involve an invertible DNA
element. Instead, the phase variation of these fimbriae relies entirely on
expression of activators and enzymes that in turn respond to
environmental and global regulatory signals. The two genes flanking the
promoter region encode for proteins that have regulatory functions.
Expression of fimbriae depends on binding of these proteins to the
promoter region (Fig. 11).

A PI CRP Lrp PB

4 5 6 1 2 3
papI papB

site 1 site 2 site 3

GATC-I GATC-II
DAM-methylase sites
B PI PB

Lrp
papI papB

methylated
unmethylated

C PI PB
PapI/Lrp
CRP
papI papB

methylated
unmethylated

Fig. 11 Regulation of a PAI-encoded fimbria exemplified by the pap operon. A. Protein

and methylation binding sites in the promoter regions: grey line — PapB binding site,
purple circle — Lrp binding site, green circle — CRP binding site, and light green circle
DAM methylase site. B. When the GATC-II site is unmethylated (light green circle), Lrp
(purple oval) binds to sites 1, 2 and 3 thereby preventing transcription of the pap
structural genes, i.e., no fimbriae produced. C. In complex with PapI (grey shape), the
Lrp protein binds to sites 4, 5 and 6 and the GATC-II site becomes methylated (red
circle). Upon binding of CRP (purple oval), transcription of the pap structural genes is
initiated, i.e., fimbriae are produced.

17
1.3.4.2.1 Regulatory crosstalk among fimbrial operons
The genes flanking the promoter region of a PAI-encoded fimbriae operon
show high homology to corresponding genes in other ExPEC fimbrial
operons and it is shown that expression of regulators from one operon can
affect expression of fimbriae of other operons e.g., prf regulators affect
expression of S fimbriae (Morschhäuser et al., 1994) and pap and foc
regulators affect type 1 fimbriae (Lindberg et al., 2008) (Holden et al.,
2001), (Xia et al., 2000). Lindberg et al. showed in addition that
heteromers of different B regulators have an impact on fimbriae
expression (Lindberg et al., 2008).

1.3.5 Other regulatory factors

1.3.5.1. The H-NS protein

H-NS, a nucleoid-associated protein extensively reviewed by Dorman
(Dorman, 2004), is known to bind to curved DNA. Since curved DNA
commonly is found in promoter regions, the binding of H-NS will thereby
affect expression of numerous genes throughout the entire bacterial
genome. DNA binding of this protein affects the gene transcription,
predominantly by repressing expression.

1.3.5.2. The RpoS, σs, stress sigma factor

Transcription of DNA is dependent on unique sequences in the promoter
region referred to as the -10 and -35 boxes. For transcription to occur the
RNA polymerase must bind to these regions. This binding is mediated by
so-called sigma factors (in E. coli there are seven different) that bring
specificity to the polymerase. When the σs factor binds to RNA
polymerase, this complex will specifically recognize -10 and -35 boxes
upstream of genes coding for proteins needed in the transition of
vegetative-to-stationary phase growth or other stress conditions.

18
1.3.5.3. The cyclic-di-GMP signal molecule
c-di-GMP acts as an intracellular signal molecule found in many different
bacterial species. The level of this molecule affects expression of several
classes of genes e.g., genes important for cellular morphology and genes
with regulatory functions (Mendez-Ortiz et al., 2006). The levels of c-di-
GMP depend on expression of two classes of enzymes — one raising the
levels of c-di-GMP (diguanylate cyclases containing their specific GGDEF
domain) (Stins et al., 1994) (Ryjenkov et al., 2005) and another lowering
the levels (phosphodiesterases containing their specific EAL domain)
(Simm et al., 2004) (Schmidt et al., 2005). In Fig. 12, the schematic
drawing shows known effects of c-di-GMP in E.coli.

pGpG
2 GTP

Phophodiesterases
Diguanylate
cyclases

2 GMP

Biofilm
Motility

Fig. 12 Schematic drawing of c-di-GMP synthesis and hydrolysis (dotted arrows) and the
effects on E. coli (black arrows).

19
1.3.5.4. The Hfq protein
In E. coli, Hfq is highly abundant with an estimated 50,000 to 60,000
copies (approximately 10,000 hexamers) per cell where 80-90% are found
in the cytoplasmic fraction in association with ribosomes (Vassilieva and
Garber, 2002). The Hfq protein is usually described as an RNA chaperone
that binds to sRNA, thereby facilitating base-pairing of target mRNAs
(Valentin-Hansen et al., 2004). By that means the complexes regulate
translation and stability, in most cases negatively. The Hfq protein
displays two RNA binding sites: one site binding sRNA and mRNA; and
another site binding poly(A) tails or RNA sites containing a short, single-
stranded stretch of uridines and adenosines, which are either preceded or
followed by a stem-loop structure (Valentin-Hansen et al., 2004). It has
also been shown to interact with PAP I, PNP and RNase E, proteins that
are involved in mRNA decay (Brennan and Link, 2007).

1.4. The X and Y genes

Through genomic comparison of fimbrial determinants — PAI-associated
and chromosomal — new, interesting differences between the operons
were found. The PAI-encoded fimbriae operons, in contrast to the type 1
fimbrial operon, were shown to include additional genes downstream of
the adhesin-coding genes (Figs. 6 and 13). Computer analysis and
homology searches predicted these genes, designated X and Y, to possess
regulatory properties. The genes and their corresponding gene products
are the main subjects in the following pages of this thesis.

adhesin

Y X

Fig. 13 Schematic figure of a PAI-encoded fimbrial operon.

20
2. AIMS OF THIS THESIS
To characterize the genes in the promoter distal region of PAI-encoded S
fimbrial operons and to elucidate the roles and the modes of action of the
sfaXII and the sfaYII gene products in the newborn meningitis Escherichia
coli clinical isolate IHE3034.

3. RESULTS AND DISCUSSION

While studying the genome sequences downstream of the genes required
for fimbrial biogenesis in PAI-associated fimbrial operons, an open reading
frame (ORF) was detected (Dobrindt et al., 2001) (Vetter, 1995)
(Marklund et al., 1992). Initially these ORFs were called the "17kDa
genes", the name reflecting the predicted protein sizes. They were found
downstream of the adhesin-coding gene of the fimbrial biogenesis operons
and showed high homology to each other (80-99%). BLAST searches in
databases were performed and the results suggested that these putative
cytoplasmic proteins (the 17kDa family), grouped into the MarR-like
regulatory family, should have DNA binding properties and act as
regulators. However, a hint of what these proteins might specifically
regulate could not be determined, as this MarR-like family affects quite
diverse functions, e.g., the EmrR (MprA) protein which represses a multi-
drug resistance pump (Lomovskaya et al., 1995) or the SlyA protein,
which acts as an activator of a cryptic hemolysin (Oscarsson et al., 1996).
To be able to distinguish different members of the 17kDa family, the
genes were given names in accordance with the associated fimbrial
operon, e.g., the 17kDa gene close to the pap operon got the name papX.

21
3.1. Paper I

3.1.1. Strategy
To find a role for the 17kDa gene, two approaches were taken: i) mutating
the gene and comparing the behavior of the wt and the mutant bacteria,
and ii) creating an overexpression system and observing how the
increased levels of gene product affected the bacterium.
Pathogenic E. coli strains usually carry a number of different PAIs and
thereby have the potential to carry several different fimbriae operons. In
order to obtain a defined single locus X mutant, an E. coli strain with only
one PAI-encoded fimbriae operon was needed. The chosen strain was the
clinical NMEC strain, IHE3034, which was isolated in the middle of the
seventies from a 6-month-old baby boy who died from the infection
(Korhonen et al., 1985). This strain has the potential to express three
different types of fimbriae — type 1, MatB, and SII fimbriae — of these
only the SII fimbriae is encoded on a PAI (Selander et al., 1986) (Pouttu et
al., 2001) (Korhonen et al., 1985).
The X gene associated with the sfaII fimbrial operon is called sfaXII. To
accomplish proper analysis, several sfaXII mutants of the strain IHE3034
and plasmid overexpression systems were created. The most frequently
used mutants were a Km-cassette insertion mutant, strain AES1, and a
deletion mutant lacking more than 70% of the sfaXII coding sequence,
strain AES4. The most often used plasmid constructs were a pBR322
(medium copy number vector) clone that contains the sfaXII gene under
its own promoter, pAES1, and a pBAD30 (medium copy number vector)
clone that contains the sfaXII gene under the control of an arabinose-
inducible ParaBAD promoter, pASS5.
To investigate the effect of sfaXII expression on type 1 fimbriae
expression, different fim gene lacZ operon fusion strains and one
fimA::lacZ fusion plasmid were used.

22
3.1.2. Effects of sfaXII mutations in the IHE3034 strain
The initial work, trying to detect effects of the sfaXII mutation in the
IHE3034 strain, gave very subtle or no results under the growth
conditions tested. No effects on growth rate could be detected when the
strains were grown under different conditions, such as rich or minimal
media, different temperatures, various carbon sources, and anaerobic
conditions. As the sfaXII gene was predicted to be a regulator and
furthermore is situated in close proximity to a fimbrial operon, we
speculated that it might have regulatory functions, acting on fimbriae
expression, either on the nearby sfaII operon or on the other fimbrial
operons (fim or matB) through crosstalk. Agglutination tests using
different types of blood (horse, sheep, goat, human, and bovine) and
yeast gave an indication that type 1 fimbriae expression was enhanced in
the sfaXII mutant.
A tendency of the sfaXII mutation to have a positive effect on motility was
also observed when the strains (wt and mutants) were grown on low
concentration agar plates (motility plates). In repeat experiments it was
observed that the mutants were equally good, or in some cases even
better, at spreading.
Both of these phenotypes indicated that the sfaXII gene, encoded on a
PAI, could affect expression of genes on the core chromosome. These
weak but still significant phenotype effects of the sfaXII mutations were
further explored when complementation studies were performed.

3.1.3. Effects of complementation and overexpression of the

sfaXII gene on motility
Normally, the IHE3034 strain and its derivative strains, AES1 and AES4
(sfaXII-), are quite motile, but upon introduction of pAES1 (sfaXII+) into
the strains they became very slow-spreading when grown on low
concentration agar plates for motility screening. Expression of sfaXII
seemed to prevent synthesis of flagella as judged by AFM images, where
no flagella at all could be detected in the trans-complemented strains.

23
RNA isolated from shaken cultures and protein extracts taken from the
same cultures verified the lack of flagella at both the transcriptional (fliC
RNA, Northern) and translational (FliC protein, Western) levels. As
mentioned in the "Introduction", flagellar synthesis is dependent on a
complicated regulation cascade involving more than 50 genes (Fig. 3). The
effect of sfaXII expression, lower levels of fliC transcript, can obviously be
due to repression of genes at several steps in the flagellar transcription
hierarchy. As the highly homologous PapX from the UPEC isolate CFT073
recently was shown to bind the flhD promoter DNA and thereby suggested
repressed expression of the master regulatory complex (Simms and
Mobley, 2008), it is likely that SfaXII also could have the same impact.
Such repression would then, as a consequence, affect all downstream
genes in the flagellar synthesis cascade (including the fliC gene) as was
indicated in the PapX case (Simms and Mobley, 2008). However, it has
not been ruled out that SfaXII might act at some other level. Several
attempts to detect differences in RNA transcript levels from early (flhD)
and middle genes (flgN) between the wt/vector, sfaXII mutant/vector, and
sfaXII mutant/complemented strains were unfruitful and gave inconclusive
results.

3.1.4. Effects of complementation and overexpression of the

sfaXII gene on type 1 fimbriae expression
Similar to the motility case, agglutination tests revealed that the slightly
enhanced expression of type 1 fimbriae in sfaXII-mutated IHE3034 strains
was reduced to wt levels, or even lower, when trans-complemented with
the pAES1 plasmid. Co-introduction of pAES1 and a transcriptional
fimA::lacZ reporter fusion plasmid into the lac- derivative of AES1 (sfaXII-)
was done. Measurement of β-galactosidase activity from the fimA fusion
verified that sfaXII expression repressed type 1 expression.
Expression of type 1 fimbriae, like other fimbriae, is regulated at both
phase variation (fim-switching) and promoter activity levels. To
investigate the mechanism of this repressive effect of SfaXII, a number of

24
lacZ reporter fusion strains were used. All of these reporter constructs are
in non-pathogenic E. coli, so the initial experiment was performed to see if
ectopically expressed sfaXII had any impact on type 1 expression in a
strain allowing both fim-switching and promoter regulation (Fig. 14A). It
was found that the observed effect in the clinical isolate indeed occurred
also in the non-pathogenic E. coli K-12 strain. fim expression was reduced
to half in the strain ectopically expressing sfaXII (pAES1) compared to the
vector control. Thus, SfaXII does not need unique IHE3034 co-factors to
repress type 1 expression. After introduction of pAES1 into the fim locked-
ON strain (Fig. 14B), which reports only promoter activity, we concluded
that the effect of sfaXII expression to a minor extent affected promoter
activity and that the major impact instead was on phase switching.

B E A’ lacZ

B
B E A’ lacZ

Fig. 14 Schematic drawings of the fimA::lacZ fusions used to measure the impact of
sfaXII expression on type 1 expression. A. fimA::lacZ fusion allowing measurement of
expression due to promoter activity and fim-switching. B. fimA::lacZ fusion allowing
measurement of expression dependent only on promoter activity, since both
recombinases are functionally mutated and the invertible DNA fragment is locked in the
ON-orientation.

Two possible scenarios could explain down-regulation of type 1 fimbriae

due to phase variation: i) sfaXII expression repressed OFF-to-ON
switching, or ii) sfaXII expression activated ON-to-OFF switching. Two
recombinases are responsible for these switching events, FimE and FimB.
FimE is described to only mediate ON-to-OFF switching, whereas FimB is
able to promote recombination in both directions (McClain et al., 1991)
(Gally et al., 1996). When measuring the activity of the fimE- and fimB-
lacZ fusions, the expected results would be either an up-regulation of fimE
expression or an effect on fimB expression. After measuring β-
galactosidase in the recombinase fusion strains carrying pAES1, it was

25
obvious that fimB expression was down-regulated. Thus, the results
suggested that sfaXII expression repressed the FimB-mediated OFF-to-ON
switching. This was furthermore also demonstrated in a test where
bacterial cultures in the OFF-state remained OFF in the presence of SfaXII,
whereas the OFF-state culture started to switch ON in the absence of
SfaXII.

3.1.5. Effects of expression of the sfaXII gene on pathogenicity-

associated genes
The effects of the sfaXII gene product so far described, were in my studies
found on systems considered as part of the E. coli core chromosome and
present also in non-pathogenic strains. To look for potential effects on
genes associated with pathogenicity, a so-called pathoarray (described in
Paper III below) was used for analysis of changes at the transcriptional
level. This pathoarray consists of 456 probes specific for typical virulence-
associated genes of ExPEC, IPEC, and Shigella, providing information
about the transcriptome of PAIs (Dobrindt et al., 2003). cDNA from the
IHE3034 wt and sfaXII mutant strains containing the pAES1 (sfaXII+) or
the vector control were used for hybridization to the arrays. The resulting
data from the experiment revealed an intriguing role of SfaXII in the
regulatory system for iron uptake encoded in the high pathogenicity island
(HPI) element (Carniel et al., 1996) (Bach et al., 2000). Both the irp and
the ybt genes were down-regulated in the sfaXII mutant strain, whereas
complementation and overexpression of sfaXII caused up-regulation of
these iron uptake systems. It will be of interest to reveal the actual
mechanism(s) of the observed SfaXII regulatory effects on the irp and the
ybt genes.

26
3.1.6. The SfaXII protein
As the SfaXII protein is postulated to have DNA binding properties, I
initiated trials to purify the protein for gel retardation studies
(unpublished). Several attempts using different types of affinity tags (e.g.,
6-His-SfaXII and GST-SfaXII) were done, but all of these trials had a
common result — a massive formation of inclusion bodies. Solubilizing
inclusion bodies is known to affect activity and since it is vital to have
active proteins to perform DNA binding experiments, it was necessary to
use big culture volumes to get small volumes of soluble SfaXII protein.
These X-family members are presumed to have DNA binding properties
but no consensus DNA binding sites are known, so another dilemma was
how to choose DNA fragments to carry out the binding assays. The first
approach was to use the His-tagged SfaXII protein and DNA fragments
from the fimB and fimA promoters (since type 1 fimbriae expression was
affected by sfaXII expression). As a negative control, a DNA fragment in
the fimA gene coding region was used. Unfortunately, no DNA binding was
detected to any of the different DNA fragments, so we speculated that the
His-tag might interfere and thereby prevent binding. Constructions of an
SfaXII fusion with a removable GST tag was followed by new gel shift
experiments. The same effect for all of the different DNA fragments was
detected, but with completely opposite results — the protein bound to all
of them with equal strength. One possibility was that some DNA-binding
protein(s) co-purified with the GST-tagged SfaXII construct.
Examining the amino acid sequence of the SfaXII protein reveals six
histidine residues (His) that are positioned in an intriguing pattern within
the polypeptide (Fig. 15).

MNNTDTLEKIIRHQKNKDPVYPFREHLLMQLCIRANKRMQDNISEFLGAYGINHSVYMVL
TTLFTAESHCLSPSEISQKLQFTRTNITRITDFLEKTGYVKRTDSREDRRAKKISLTSEGMFF
IQRLTLAQSMYLKEIWGYLTHDEQELFEVINKKLLAHLDDVSS

Fig. 15 Amino acid sequence of the SfaXII protein. His residues are highlighted in yellow.

27
This encouraged me to test whether it was possible to take advantage of
these histidines for binding to nickel-coated beads. Maybe the SfaXII
protein naturally folded in a manner exposing the histidines present, and
this exposure could be sufficient for binding to nickel beads and allow
purification (thereby eliminating problems with a tag). After introduction
of the inducible plasmid pASS5 into an hns mutant strain (to rule out co-
purification of the DNA binding H-NS protein) the protein purification
procedure was performed as before but with use of less stringent elution
conditions. Using this method, a surprisingly pure SfaXII protein could be
obtained (Fig. 16).

70

55

43

34
SfaXII dimer

26

17 SfaXII monomer

Elution with 50 mM
Imidazole buffer
Fig. 16 Purification of native SfaXII (unpublished data). Two liters of AAEC198Ahns-
/pASS5 culture was induced with 0.004% arabinose at OD600 = 0.5 grown at 25 °C. The
cells were harvested at OD600 = 1.25, lysed, and the lysate was added to Ni-agarose
resin. Monomeric and dimeric forms of SfaXII were eluted with 50 mM imidazole.
Molecular weights (kDa) are shown.

Several attempts to verify DNA binding properties of this protein have

been performed, but so far no clear binding effects have been recorded.
However, these negative results do not rule out that the SfaXII protein
regulates gene expression through DNA binding; it could just be a matter
of finding the right DNA targets. Another possibility is that the protein
needs additional co-factor(s) not present in the in vitro experiments to be
able to bind target DNA.

28
The strong repressive effect of the sfaXII mutant phenotypes (enhanced
type 1 expression and higher motility) when trans complemented might
reflect that very low levels of SfaXII are needed to affect the target genes.
Therefore the use of medium copy number plasmids might result in
protein levels far too high compared to levels in the wt. This theory is
supported by the fact that the SfaXII protein never has been detected in
IHE3034 (wt) whole cell protein extracts collected for Western blot
analysis. The reasons might be that optimal conditions for expression
have not been found, or that the SfaXII protein is transiently expressed
and rapidly degraded. In Paper III, the expression profile of the SfaXII
protein is investigated.

3.1.7. Conclusion
The data in Paper I demonstrated that sfaXII expression affected motility
and that overexpression of SfaXII resulted in reduced flagellar synthesis.
Furthermore, I showed that sfaXII expression repressed the FimB-
mediated OFF-to-ON switching but also to a lesser degree the promoter
activity of the fim operon.

3.2. Paper II

3.2.1. Strategy
The biomechanical properties of the type 1 and P fimbriae have recently
been investigated using force measuring optical tweezers, FMOT, and
dynamic force spectroscopy, DFS (Andersson et al., 2006b) (Andersson et
al., 2006a) (Andersson et al., 2007). Differences in their properties were
observed that correlate with the respective niches in which these fimbriae
are mainly thought to act. These findings inspired us to investigate
whether other ExPEC fimbriae could be distinguished from one other on
similar grounds.

29
The same methodology as earlier was used, now focusing on the two
different types of S fimbriae: the SI fimbriae encoded on the sfaI operon
and the SII fimbriae encoded on the sfaII operon. These operons encode
rod subunits that show 80% homology at the amino acid level. The sfaI
fimbriae operon cloned from the UPEC strain 536 (pANN108-13) and the
sfaII fimbriae operon cloned from the NMEC strain IHE3034 (pAZZ50)
were introduced into a laboratory strain (HB101) that does not produce its
own fimbriae. In addition, plasmids pAES7 and pAES8 containing a cloned
SII fimbriae operon with sfaXII mutations were introduced into HB101. The
physical and biomechanical properties of these fimbriae were also
measured and included in the comparative studies.
Finally, measurements were performed to investigate if a plasmid-encoded
fimbrium had different properties compared to the corresponding
chromosome-encoded fimbrium.

3.2.2. S fimbriae in comparison to other fimbriae

The previously performed comparisons of the core chromosome encoded
type 1 fimbriae and the PAI-encoded P fimbriae have revealed several
differences in the physical and biomechanical behaviors e.g., that type 1
fimbriae possess catch bonds and P fimbriae slip bonds. Catch bonds
respond by becoming longer-lived with tensile mechanical forces (Nilsson
et al., 2006) (Thomas et al., 2006) (Yakovenko et al., 2008) in contrast to
slip bonds which respond by becoming shorter lived (Björnham et al.,
2009) (Dembo et al., 1988). The kinetic properties are also different
between type 1 and P fimbriae, where the type 1 fimbriae is much slower
— the time it takes for a completely stretched out type 1 fimbrium to
retract is more than 30 times longer than for a P fimbrium.
When measurements of the two different S fimbriae were conducted, it
was obvious that S fimbriae showed more similarities to P fimbriae than to
type 1 fimbriae, a phenomenon that might reflect the fact that all of these
types of fimbriae — P, SI, and SII fimbriae — are encoded on PAIs, in
contrast to the type 1 fimbriae. Comparisons of SI and SII fimbriae

30
properties revealed differences in the kinetics — SI fimbriae was seven
times faster than the SII fimbriae. Furthermore, the force needed to break
the layer-to-layer interactions was higher in the SII fimbrium rod.

3.2.3. Effect of sfaX mutation

The 20% difference in amino acid sequences of the SI and SII fimbriae
major subunits might very well explain the differences detected in the
biomechanical behavior of those fimbriae. Nevertheless, when analyzing
the sequences of the plasmids carrying the cloned operons, it was found
that the SI fimbrial operon was not complete. Approximately 60% of the
last gene (the sfaXI gene) of the operon was missing. To exclude that the
truncated X gene was responsible for the detected differences in physical
and biomechanical properties of the two types of S fimbriae rods, X gene
mutations in the SII fimbriae expression plasmid pAZZ50 were created.
Measurements of the resulting fimbriae were performed, but it seemed
that those sfaXII mutations only had negligible effects on the physical and
biomechanical properties compared to a wt SII fimbrium. Thus, the
observed differences between the SI and SII fimbriae are probably not due
to altered levels of sfaX expression.

3.2.4. Clinical isolate vs. ectopic fimbriae expression

The physical and biomechanical properties have so far been unique for
each type of fimbriae investigated. With this information, it was
interesting to see if it was possible to measure and identify fimbriae
expressed from a natural system, i.e., fimbriae expressed by a clinical
isolate. Moreover, if that was the case, it was interesting to see if those
fimbriae had the same properties as ectopically expressed fimbriae from
plasmids in laboratory strains. The NMEC isolate IHE3034 has the
potential to express both type 1 and SII fimbriae. Using this strain as a
model organism, it was possible to distinguish and identify different types
of fimbriae. It was also verified that the properties of respective fimbriae
remain the same irrespective of the expression system chosen.

31
3.2.5. Conclusion
In paper II we demonstrated that the PAI-encoded SI and SII fimbriae
show more resemblance, with respect to the biomechanical properties, to
P fimbriae than to the core chromosome encoded type 1 fimbriae. It was
also shown that the X proteins do not contribute to the physical and
biomechanical properties of the fimbrium. Furthermore, these behaviors
are not changed if the fimbrial operons are moved from their natural
expression system (clinical isolate) and introduced for ectopic expression
in other expression systems (plasmid expression in laboratory strain).
Finally, we concluded that FMOT measurements can be used as an
immediate and precise technique to recognize and identify different
fimbriae since the fimbriae investigated to date show unique parameter
values.

3.3. Paper III

3.3.1. Strategy
To quantify SfaXII protein expression, reporter transcriptional fusions to
the sfaXII gene were created. In this way, it was possible to check under
what circumstances the gene was expressed. Two different types of
systems were constructed, a luxAB and a lacZ fusion. The two reporter
fusions were constructed in suicide plasmids that later were allowed to
recombine into the chromosome of the clinical isolate IHE3034 (Forsberg
et al., 1994) (Kalogeraki and Winans, 1997). All transcripts that contain
the sfaXII gene promoter region, plus 200 bp of the gene, can be
measured using these chromosomal constructs (strains AES5
(sfaXII::luxAB) and AES13 (sfaXII::lacZ)). An additional lacZ operon fusion
construction that measures the transcript of only the sfaXII gene was
made in a K-12 strain, AES24 (sfaXII::lacZ).
Transcriptional analysis to examine and map the sfaXII promoter region
was performed when suspicions arose that the AES5 and AES13 strains

32
measured activity from more than one transcript. Both primer extension
and reverse transcript (RT) PCR techniques were used.
During these examinations, an additional ORF, between the sfaHII gene
(coding for the adhesin) and sfaXII, caught my interest. This gene, sfaYII,
was cloned into several different plasmids. The most often used constructs
were a pMMB66EH (low copy number vector) clone that contains the sfaYII
gene under the control of an IPTG inducible Ptac promoter, pAES22, and a
pBAD30 (medium copy number vector) clone that contains the sfaYII gene
under the control of an arabinose-inducible ParaBAD promoter, pAES25.
Phenotypic changes were analyzed after introduction and overexpression
of the sfaYII gene in different strains.

3.3.2. Expression profile of sfaXII

Examination of the sfaXII gene expression profile was started using the
sfaXII fusion strains, AES5 and AES13. The luxAB reporter system (in
AES5) has the advantage that it can be rapidly analyzed in a few steps.
On the other hand, this system has the disadvantages that it is
temperature-sensitive and less reliable when applied to old cultures (late
stationary phases), maybe due to the fact that capsule formation prevents
the substrate (decanal) from entering the cells. The lacZ reporter system
(in AES13) is reliable in any growth phase, but has the disadvantage that
it is time-consuming and requires multiple steps. Both of these constructs
are, as mentioned, transcriptional fusions. This implies that even if high
measurement values are obtained, there are no assurances that high
levels of proteins will be detected because of post-transcriptional events
e.g., low mRNA stability or translational inhibition.
The reporter strains, AES5 and AES13, were subjected to different
environmental conditions and expression of the sfaXII transcripts was
monitored during growth. A summary of the results is shown in Fig. 17,
where a typical expression profile during growth in LB at 37°C also is
shown.

33
 Lux activity/OD600
pH 7 is optimal for expression AU OD600
40 10
37°C is optimal for expression
35
expression is repressed at high 30
osmolarity (0.4 M NaCl) 1
25
expression is repressed at anaerobic 20
growth
15
0.1
the expression peak height is 6-fold
increased in hns mutant background 10 OD
5 expression
the expression peak height is 2-fold
increased when papB or papI is over- 0 0.01
expressed 0 100 200 300 400 500 600 700
Time (min)

Fig. 17 Summary of growth conditions affecting expression of sfaXII transcripts. The

diagram shows a typical expression profile (red) and growth curve (black) for a AES5
(sfaXII::luxAB) culture shaken at 37°C in LB.

When comparing these findings to data obtained from studies looking for
optimal conditions for S fimbriae expression, it was found that many of
the parameter values coincided (Schmoll et al., 1990). This made us
consider the possibility that the AES5 and AES13 fusion strains detected
expression not only from a monocistronic sfaXII transcript but also from
the complete sfaII operon.

3.3.3. sfaXII is included in the sfaII fimbrial transcriptional unit

If the AES5 and AES13 fusion strains produced sfaII operon transcripts
detectable with the sfaXII reporter fusion, it would support the theory that
also sfaXII belonged to the sfaII fimbrial operon. To check this, plasmids
carrying the P fimbrial activators were introduced into the AES5 and
AES13 fusion strains. In parallel, the plasmids were introduced also into
the K-12 derivative AES24 (sfaXII::lacZ) which only detects expression of
the monocistronic sfaXII transcript. The P fimbrial activators, PapB and
PapI, both activate P fimbriae expression but other fimbrial operon
promoters are known to be activated as well (Morschhäuser et al., 1994)

34
(Lindberg et al., 2008). Two-fold increases in the operon fusion strains
were found, whereas no activation of the monocistronic transcript could be
detected. In other words, the increased expression from the sfaXII
reporter fusions came from a transcript sensing the fimbrial activators,
most likely a transcript from the fimbrial biogenesis promoter.
As the transcript sfaBI - sfaHI already has been described (Balsalobre et
al., 2003), the region sfaHII - sfaXII was now further analyzed. Isolated
RNA of the IHE3034 strain was used to examine transcripts of this region.
RT-PCR analysis revealed the presence of a transcript covering the entire
sfaHII - sfaXII region. Taken together, these two transcriptional
assessments implied a complete sfaBII - sfaXII transcript. Furthermore,
primer extension analysis verified that the sfaXII gene also was
transcribed from its own promoter, resulting in a monocistronic sfaXII
transcript.

3.3.4. Discovery of an additional gene, sfaYII, in the sfaII fimbrial

operon
Meanwhile, examination of an ORF found within the sfaHII - sfaXII region,
later denoted sfaYII, was initiated. Homology searches in genomic
databases stated that its deduced amino acid sequence coded for a
protein containing an EAL domain. Proteins with such domains usually
possess phosphodiesterase activity, which hydrolyzes c-di-GMP (Simm et
al., 2004) (Schmidt et al., 2005). The same approach to finding a role for
the sfaYII gene as for the sfaXII was used — construction of sfaYII mutants
and overexpression systems. Several attempts to clone the gene were
carried out. Seemingly correct clones (sequence analysis) never produced
any detectable SfaYII protein, however, as judged by Coomassie staining
(no antibody against the protein was available). As weak biofilm formation
and high motility are known phenotypes of low c-di-GMP levels, it was
speculated that introduction of plasmids carrying sfaYII might give
detectable effects (as low c-di-GMP levels is a response to high levels of
phosphodiesterase). The IHE3034 strain forms very poor biofilm, therefore

35
a Vibrio cholerae strain, C6706, was chosen. This strain, as many of the V.
cholerae strains, forms good liquid-air biofilm pellicles (Tischler and
Camilli, 2004) (Waters et al., 2008). sfaYII was ectopically expressed in
the C6706 strain and an almost total absence of biofilm suggested that
even though the SfaYII protein could not be detected, it had a role in
biofilm formation. When expressed, it also brought a smooth phenotype to
the otherwise rugose V. cholerae colonies (Rashid et al., 2003).
Surprisingly, overexpression of sfaYII in IHE3034 seemed to result in less
motile bacteria, in contrast to what would be expected. This might be due
to the fact that wt IHE3034 bacteria produce more S fimbriae than sfaYII
mutants as preliminary results from immunofluorescence analysis
indicate. A simultaneous expression of flagella to swim and fimbriae to
adhere seems a waste of energy as it promotes two completely divergent
activities.

3.3.5. Conclusion
In Paper III, I showed that sfaXII gene expression was affected by
environmental factors such as temperature, pH, and osmolarity and
effects were most pronounced under conditions typically found in a human
host. I also demonstrated that the sfaXII gene was transcribed from two
transcripts — one monocistronic transcript containing only the sfaXII gene
and one transcript covering the entire sfaII operon (sfaBII - sfaXII).
sfaYII, a new gene here shown to also belong to the sfaII operon, was
found to affect biofilm formation, possibly through lowered c-di-GMP levels
caused by SfaYII phoshpdiesterase activity. In addition, preliminary data
suggests that motility and SII fimbriae expression is affected by sfaYII,
expression, however these results need further exploration.

36
3.4. Paper IV

3.4.1. Strategy
Experiments that successfully detected sfaXII expression using the
reporter gene fusion derivatives of IHE3034 revealed an expression
pattern suggesting a growth-dependent behavior. This pattern resembled
that of many operons under the control of the RpoS stationary phase
sigma factor. To investigate this further, the plasmid pMMkatF2, encoding
a wt rpoS allele, was introduced into the AES5 (sfaXII::luxAB) and AES13
(sfaXII::lacZ) strains, since the parent strain (IHE3034) is known to carry
a defective rpoS gene (Wang and Kim, 2000). Furthermore, a Tn10-
inactivated rpoS gene was transduced into the rpoS locus of the K-12
sfaXII::lacZ fusion strain (AES24). These strains, AES24 and its rpoS
mutant derivative AES25, enabled a comparison of the monocistronic
sfaXII transcript expression with and without functional σS factor.
To investigate if Hfq might be involved in facilitating the regulatory effects
of SfaXII, a knock-out of the hfq gene in the AES24 strain was first
constructed to verify that sfaXII is expressed in an hfq mutant. Then the
hfq mutant allele was introduced into the K-12 fimA::lacZ fusion strains
(AAEC198A and AAEC374A) together with the plasmids pBR322 and
pAES1 (sfaXII+).
To initiate a search for additional genes that potentially could affect the
sfaXII expression, random mutagenesis by mariner transposon insertion
into AES13 (sfaXII::lacZ) was performed.

3.4.2. Effect of RpoS on sfaXII expression

As mentioned above, the expression profiles of the sfaXII gene encouraged
investigation into whether rpoS somehow could be involved. As both
IHE3034-derived reporter fusion strains, AES5 and AES13, only express
negligible levels of σS (due to a point mutation in the rpoS gene (Wang
and Kim, 2000)), a functional rpoS gene was provided to the strains to
monitor effects on sfaXII expression. The response to this trans-expressed
rpoS was an almost total abolishment of activity from the sfaXII reporter

37
fusions. Earlier experiments (Paper III) have shown that these fusions
measure activity from two transcripts, the transcript covering the entire
sfaII operon and the monocistronic sfaXII transcript, implying that the
direct or indirect rpoS repressive effect could be achieved from two
different promoters. However, the long transcript did not seem to be
repressed by the expressed σS factor. When analyzing SfaA protein levels
by Western blot in the same samples used for Lux activity measurements,
it was found that the trans-complemented AES5 strain (rpoS+) expressed
slightly more SII fimbriae compared to the vector control (rpoS-). In the
case of the K-12 sfaXII::lacZ strain, AES24, measurements of β-
galactosidase activity were made only from the monocistronic transcript in
an rpoS-positive background. To be able to test how expression of the
single sfaXII transcript was affected by σS, the wt rpoS allele was
exchanged for a mutated allele in the AES24 strain. Analysis of the
different strains supported the results that σS had a direct or indirect
repressive effect on sfaXII expression.

3.4.3. Effect of Hfq on sfaXII expression

The SfaXII protein is proposed to have DNA binding properties, but
attempts to verify this statement have been unfruitful, maybe due to lack
of co-factor(s) needed to facilitate this binding. To investigate if a small
RNA-Hfq complex could be involved, an hfq mutant strain was needed to
abolish hfq expression. It was also crucial to verify that sfaXII was
expressed in an hfq mutant strain background. Therefore, an hfq mutant
allele was constructed in AES24 (sfaXII::lacZ). Due to the fact that rpoS
expression is dependent on Hfq, the expected result was that the sfaXII
expression profile should mimic the profile obtained from the σS mutant.
Those expectations proved to be right in the sense that sfaXII expression
was enhanced in the hfq mutant strain, but it was also found that the
expression was further enhanced compared to the σS mutant strain. Thus,
Hfq seemed to directly or indirectly repress sfaXII expression.

38
3.4.4. Effect of combined hfq and sfaXII expression on fim
expression
When it was verified that SfaXII could be expressed in hfq mutant strains,
the hfq mutant allele was introduced into the fim::lacZ fusion strains
together with the pAES1 or pBR322 plasmids. After the β-galactosidase
measurements had been conducted, the first striking finding was that type
1 fimbriae expression was greatly affected by the hfq mutation. The
expression of fimA was two times higher in the wt strain compared to the
hfq mutant strain (strains allowing fim switching). fimA promoter activity
was also affected by the hfq mutation, but only significantly in log phase
cultures. Thus, Hfq seemed to primarily affect type 1 fimbrial phase
variation. Regarding the question about an SfaXII effect without Hfq, no
significant differences in fimA expression between sfaXII-expressing and
control strains of the hfq mutants could be found. Whether this is due to
SfaXII needing Hfq to repress type 1 fimbriae expression or the hfq
mutation overriding the effect of SfaXII remains to be elucidated.

3.4.5. Random mariner transposon mutagenesis

To find genes that could influence sfaXII expression, a random transposon
mutagenesis project was initiated. Ninety-three mariner transposon
mutants in the AES13 (sfaXII::lacZ) fusion strain were isolated. As the
screening was performed visually by blue-white screening on X-gal plates,
two groups were identified. One group contained the darker blue colonies
compared to the wt, i.e., colonies expressing more sfaXII and thereby
carrying the transposon in a potential repressor. The other group
contained the lighter blue colonies, expressing less sfaXII and thereby
carrying transposons in potential activators. Only four of these mutations
have been localized in this initial study, two of which were in the fusion
construction. The remaining two genes code for a putative hybrid
polyketide-non-ribosomal peptide and a hypothetical protein of unknown
function. Continuous screening of the transposon insertions would be

39
expected to render hits in the hns and hfq genes and also hopefully some
genes revealing the reason for the growth-dependent expression pattern.

3.4.6. Conclusion
In paper IV I showed that expression of sfaXII was repressed, presumably
indirectly, by σS and most likely also by Hfq. One potential co-factor for
SfaXII-mediated repression of fim expression could be the Hfq protein,
with or without accompanying sRNA. I also showed that type 1 fimbriae
expression was dependent on hfq expression, most likely by affecting
some component(s) involved in regulating the phase variation.

4. CONCLUDING REMARKS
A short summary of the findings:
• Expression of the sfaXII gene affects type 1 fimbriae expression,
mainly by repression of the fimB-mediated OFF-to-ON switching
• Expression of the sfaXII gene affects motility, presumably by
reduced biogenesis of flagella
• The SfaXII protein does not contribute to physical or biomechanical
properties of the fimbrium
• The sfaXII gene is part of the main sfaII operon
• The sfaXII gene is transcribed from two promoters
• The sfaXII gene expression is affected by environmental factors
• The sfaXII gene expression is repressed by H-NS, RpoS and Hfq
• The sfaYII gene is part of the main sfaII operon
• Expression of the sfaYII gene affects biofilm formation, motility, and
SII fimbriae expression

The results of my aims to characterize the genes (sfaXII and sfaYII) of the
PAI-encoded SII fimbrial operon and to elucidate the roles and modes of
action of their gene products in IHE3034 have shown that these genes
encode for regulators affecting virulence-associated genes located both in
the core chromosome of E. coli and within PAIs (e.g., HPI). It should also

40
be emphasized that the sfaXII and sfaYII loci are representatives of gene
families present in several different gene clusters found in ExPEC isolates.
Intriguing questions arise regarding strains that carry several fimbrial
operons, strains that therefore have the ability to express more than one
of the X and/or Y family members (Fig. 18).

sfaI & sfaII Y X

(536 (SI) & IHE3034 (SII))

prf (536) X

foc (CFT073) X

prs (CFT073) X

pap (J96) Y X
adhesin

Fig. 18 Schematic drawing of the distal genes in some ExPEC fimbrial operons. The
fimbrial operons are marked in italics and the strains in which they are found are in
parenthesis. The last part of the truncated adhesin genes is colored in orange.

Do all X genes regulate the same sets of genes, thereby assuring that the
regulatory effect of an X gene is always maintained, irrespective of which
PAI-encoded fimbriae is expressed?
Computerized structure predictions of the SfaXII protein suggest that this
putative DNA binding protein is a helix-turn-helix protein. Those types of
proteins usually form dimers when active. If several different X proteins
are expressed in a strain — can those proteins form heteromers? And in
that case, will the heteromers function differently than the homomers?
Expression of X is down-regulated in the presence of σS — what impact
does that have on the action and role of X genes in the rpoS-negative
strain IHE3034 compared to the rpoS-positive strain 536?
Regarding the Y genes, the sfaYII gene seems to affect SII fimbriae
expression — does the papY homolog affect P fimbriae expression?
Is there some kind of counterselection for the Y genes, since not all
fimbrial operons carrying an X gene carry a Y gene?

41
ACKNOWLEDGEMENTS
Since I have been in the molecular biology building "for ages", I think it is
impossible to remember to thank all of U as much as I really should...I
promise...it is not my intention to leave someone out!!! So initially I would
like to thank everyone (no-one mentioned, no-one forgotten ☺) that has
made my years here a memorable time. Then, of course... there are some
people that have been more closely involved in the "all-in-a-days-work"
that deserve to be highlighted:
Bernt Eric — I'm grateful that U gave me the chance to complete this Ph.
D. study and I hope that U are a little bit proud of me anyway!...but...I
would lie if I said that I always have appreciated your priority skills! ☺
Carlos — This is all your "fault"!...I would never begun these studies if it
wasn't for U...Your help has been invaluable and blablablablabla for 14
pages more!! ☺ Monica. — U are the solid rock...the shoulder to lean
on...our comforter!!! Stina — We are a nice couple, eh? The complaining
twins! ☺ But...what will happen now when I loose my private training
coach?? Connie — Please...never change!!! Hyun-Sook and Ryoma —
U give politeness a face!! That is needed in the group! Ting — U make me
happy! Berit — U're the best…NEVER forget that!!! On top of that...my
mental support! Jörgen — U always have time for my questions and
seem to cope with my teasing! ☺ Strong guy!! Sun — My Vibrio guru!
Barbro — "the unpredictable woman"...I like your style!!! Tianyan — I
am convinced that your Swedish soon will be better than my English!
(Envy!! ☺) Takahiko — The silent worker! Pramod — Always with a
smile in your face! Wonderful! Edmund, Karolis, Jonas, Theresa and
Sakura — What would U say if I claimed that the world would be a better
place without the sRNAs??? ☺...and Edmund...I am jealous of your self
confidence!...and....Karolis...I still havn't lost my hope to get the photos
from Japan...should I reconsider? Anna — Always giving a helping
hand...always listening...U are a GOOD friend!!! Magnus and Mick — May
the Force be with U!!

42
And now to the "past-people"...and I must emphasize that I mention U all
in random order ☺...Claudia — I have NEVER met anyone so genuinely
positive in my whole life...and so generous! Emma — Always nice to meet
U...and your delicious chocolate cakes!!! ☺ Jurate — I admire your ability
to always "land on your feet". Marija — I dream of visiting U some
day!...So much talk I would expose U to!!! ☺ Christer, Pär och Marie —
Sven's group must be quite quiet now!!! Betty — THANK U!! Marie W — I
took over the "sleeping-at-seminar-role" when U left...oooooops! ☺ Janne
— There are too few "word-joke-creators" in the world....could U do
something to it?!
Slutligen några ord till mina nära och kära: Per — Oljan på mitt upprörda
hav, fenderten på min otympliga båt och stjärnan på min nattsvarta
himmel!!! Anton — Du är snäll, stark och rättvis och jag tycker om att
vara din mamma!! Mamma och pappa — Ni har gett/ger mig så otroligt
mycket och alltid trott på mig...vilka beslut jag än tagit. Joakim och
Sylvia — Jag är stolt över att vara er storasyster! Carina och Stefan —
Ta väl hand om mina syskon! Det är de verkligen värda!! Maja, Erik,
Ossian och Holger — Jag har världens bästa syskonbarn!!! Eva och
Lennart — Mina "reservföräldrar"...så hjälpsamma...så generösa...så
förstående!!! Lars och Camilla — Även om jag verkar "borta" ibland är ni
ALLTID välkomna! Hugo och Vera — Jag har världens bästa Per-
syskonbarn!!! Bosse — Kussin!!!! ☺

This work was carried out in the frame of the European Virtual Institute
for Functional Genomics of Bacterial Pathogens (CEE LSHB-CT-2005-
512061) and the ERA-NET project "Deciphering the intersection of
commensal and extraintestinal pathogenic E. coli" and was supported by
grants from the Swedish Research Council, the Swedish Foundation for
International Cooperation in Research and Higher Education (STINT), the
Faculty of Medicine, Umeå University, and it was performed within the
Umeå Centre for Microbial Research (UCMR).

43
REFERENCES

Abraham, J.M., Freitag, C.S., Clements, J.R., and Eisenstein, B.I. (1985)
An invertible element of DNA controls phase variation of type 1
fimbriae of Escherichia coli. Proc Natl Acad Sci U S A 82: 5724-
5727.
Andersson, M., Fällman, E., Uhlin, B.E., and Axner, O. (2006a) A sticky
chain model of the elongation and unfolding of Escherichia coli P pili
under stress. Biophys J 90: 1521-1534.
Andersson, M., Fällman, E., Uhlin, B.E., and Axner, O. (2006b) Dynamic
force spectroscopy of E. coli P pili. Biophys J 91: 2717-2725.
Andersson, M. (2007) Construction of force measuring optical tweezers
instrumentation and investigations of biophysical properties of
bacterial adhesion organelles. In Faculty of Science and Technology,
Physics. Doctor of Philosophy Umeå: Umeå University. ISBN: 978-
91-7264-435-9.
Andersson, M., Uhlin, B.E., and Fällman, E. (2007) The biomechanical
properties of E. coli pili for urinary tract attachment reflect the host
environment. Biophys J 93: 3008-3014.
Bach, S., de Almeida, A., and Carniel, E. (2000) The Yersinia high-
pathogenicity island is present in different members of the family
Enterobacteriaceae. FEMS Microbiol Lett 183: 289-294.
Balsalobre, C., Morschhäuser, J., Jass, J., Hacker, J., and Uhlin, B.E.
(2003) Transcriptional analysis of the sfa determinant revealing
mmRNA processing events in the biogenesis of S fimbriae in
pathogenic Escherichia coli. J Bacteriol 185: 620-629.
Björnham, O., Nilsson, H., Andersson, M., and Schedin, S. (2009) Physical
properties of the specific PapG-galabiose binding in E. coli P pili-
mediated adhesion. Eur Biophys J 38: 245-254.
Blomfield, I.C., Calie, P.J., Eberhardt, K.J., McClain, M.S., and Eisenstein,
B.I. (1993) Lrp stimulates phase variation of type 1 fimbriation in
Escherichia coli K-12. J Bacteriol 175: 27-36.
Blomfield, I.C., Kulasekara, D.H., and Eisenstein, B.I. (1997) Integration
host factor stimulates both FimB- and FimE-mediated site-specific
DNA inversion that controls phase variation of type 1 fimbriae
expression in Escherichia coli. Mol Microbiol 23: 705-717.
Brennan, R.G., and Link, T.M. (2007) Hfq structure, function and ligand
binding. Curr Opin Microbiol 10: 125-133.
Buchanan, K., Falkow, S., Hull, R.A., and Hull, S.I. (1985) Frequency
among Enterobacteriaceae of the DNA sequences encoding type 1
pili. J Bacteriol 162: 799-803.
Carniel, E., Guilvout, I., and Prentice, M. (1996) Characterization of a
large chromosomal "high-pathogenicity island" in biotype 1B
Yersinia enterocolitica. J Bacteriol 178: 6743-6751.

44
Chilcott, G.S., and Hughes, K.T. (2000) Coupling of flagellar gene
expression to flagellar assembly in Salmonella enterica serovar
typhimurium and Escherichia coli. Microbiol Mol Biol Rev 64: 694-
708.
Dembo, M., Torney, D.C., Saxman, K., and Hammer, D. (1988) The
reaction-limited kinetics of membrane-to-surface adhesion and
detachment. Proc R Soc Lond B Biol Sci 234: 55-83.
Dobrindt, U., Blum-Oehler, G., Hartsch, T., Gottschalk, G., Ron, E.Z.,
Fünfstück, R., and Hacker, J. (2001) S-Fimbria-encoding
determinant sfa(I) is located on pathogenicity island III(536) of
uropathogenic Escherichia coli strain 536. Infect Immun 69: 4248-
4256.
Dobrindt, U., Agerer, F., Michaelis, K., Janka, A., Buchrieser, C.,
Samuelson, M., Svanborg, C., Gottschalk, G., Karch, H., and
Hacker, J. (2003) Analysis of genome plasticity in pathogenic and
commensal Escherichia coli isolates by use of DNA arrays. J
Bacteriol 185: 1831-1840.
Dobrindt, U., Hochhut, B., Hentschel, U., and Hacker, J. (2004) Genomic
islands in pathogenic and environmental microorganisms. Nat Rev
Microbiol 2: 414-424.
Dobrindt, U. (2005) (Patho-)Genomics of Escherichia coli. Int J Med
Microbiol 295: 357-371.
Dorman, C.J., and Higgins, C.F. (1987) Fimbrial phase variation in
Escherichia coli: dependence on integration host factor and
homologies with other site-specific recombinases. J Bacteriol 169:
3840-3843.
Dorman, C.J. (2004) H-NS: a universal regulator for a dynamic genome.
Nat Rev Microbiol 2: 391-400.
Forsberg, Å.J., Pavitt, G.D., and Higgins, C.F. (1994) Use of transcriptional
fusions to monitor gene expression: a cautionary tale. J Bacteriol
176: 2128-2132.
Gally, D.L., Bogan, J.A., Eisenstein, B.I., and Blomfield, I.C. (1993)
Environmental regulation of the fim switch controlling type 1 fimbrial
phase variation in Escherichia coli K-12: effects of temperature and
media. J Bacteriol 175: 6186-6193.
Gally, D.L., Leathart, J., and Blomfield, I.C. (1996) Interaction of FimB
and FimE with the fim switch that controls the phase variation of
type 1 fimbriae in Escherichia coli K-12. Mol Microbiol 21: 725-738.
Hacker, J., Blum-Oehler, G., Mühldorfer, I., and Tschäpe, H. (1997)
Pathogenicity islands of virulent bacteria: structure, function and
impact on microbial evolution. Mol Microbiol 23: 1089-1097.
Holden, N.J., Uhlin, B.E., and Gally, D.L. (2001) PapB paralogues and their
effect on the phase variation of type 1 fimbriae in Escherichia coli.
Mol Microbiol 42: 319-330.
Holden, N.J., and Gally, D.L. (2004) Switches, cross-talk and memory in
Escherichia coli adherence. J Med Microbiol 53: 585-593.

45
Johanson, I., Lindstedt, R., and Svanborg, C. (1992) Roles of the pap- and
prs-encoded adhesins in Escherichia coli adherence to human
uroepithelial cells. Infect Immun 60: 3416-3422.
Johnson, J.R., and Russo, T.A. (2002) Extraintestinal pathogenic
Escherichia coli: "the other bad E coli". J Lab Clin Med 139: 155-
162.
Kalogeraki, V.S., and Winans, S.C. (1997) Suicide plasmids containing
promoterless reporter genes can simultaneously disrupt and create
fusions to target genes of diverse bacteria. Gene 188: 69-75.
Korhonen, T.K., Valtonen, M.V., Parkkinen, J., Väisänen-Rhen, V., Finne,
J., Ørskov, F., Ørskov, I., Svenson, S.B., and Mäkelä, P.H. (1985)
Serotypes, hemolysin production, and receptor recognition of
Escherichia coli strains associated with neonatal sepsis and
meningitis. Infect Immun 48: 486-491.
Larsen, S.H., Reader, R.W., Kort, E.N., Tso, W.W., and Adler, J. (1974)
Change in direction of flagellar rotation is the basis of the
chemotactic response in Escherichia coli. Nature 249: 74-77.
Leffler, H., and Svanborg-Edén, C. (1981) Glycolipid receptors for
uropathogenic Escherichia coli on human erythrocytes and
uroepithelial cells. Infect Immun 34: 920-929.
Lindberg, S., Xia, Y., Sondén, B., Göransson, M., Hacker, J., and Uhlin,
B.E. (2008) Regulatory Interactions among adhesin gene systems of
uropathogenic Escherichia coli. Infect Immun 76: 771-780.
Lomovskaya, O., Lewis, K., and Matin, A. (1995) EmrR is a negative
regulator of the Escherichia coli multidrug resistance pump EmrAB. J
Bacteriol 177: 2328-2334.
Macnab, R.M. (2003) How bacteria assemble flagella. Annu Rev Microbiol
57: 77-100.
Marklund, B.I., Tennent, J.M., Garcia, E., Hamers, A., Båga, M., Lindberg,
F., Gaastra, W., and Normark, S. (1992) Horizontal gene transfer of
the Escherichia coli pap and prs pili operons as a mechanism for the
development of tissue-specific adhesive properties. Mol Microbiol 6:
2225-2242.
McClain, M.S., Blomfield, I.C., and Eisenstein, B.I. (1991) Roles of fimB
and fimE in site-specific DNA inversion associated with phase
variation of type 1 fimbriae in Escherichia coli. J Bacteriol 173:
5308-5314.
Mendez-Ortiz, M.M., Hyodo, M., Hayakawa, Y., and Membrillo-Hernandez,
J. (2006) Genome-wide transcriptional profile of Escherichia coli in
response to high levels of the second messenger 3',5'-cyclic
diguanylic acid. J Biol Chem 281: 8090-8099.
Morschhäuser, J., Vetter, V., Emödy, L., and Hacker, J. (1994) Adhesin
regulatory genes within large, unstable DNA regions of pathogenic
Escherichia coli: cross-talk between different adhesin gene clusters.
Mol Microbiol 11: 555-566.

46
Müller, C.M., Åberg, A., Straseviciene, J., Emödy, L., Uhlin, B.E., and
Balsalobre, C. (2009) Type 1 fimbriae, a colonization factor of
uropathogenic Escherichia coli, are controlled by the metabolic
sensor CRP-cAMP. PLoS Pathog 5: e1000303.
Nilsson, L.M., Thomas, W.E., Trintchina, E., Vogel, V., and Sokurenko,
E.V. (2006) Catch bond-mediated adhesion without a shear
threshold: trimannose versus monomannose interactions with the
FimH adhesin of Escherichia coli. J Biol Chem 281: 16656-16663.
O'Gara J, P., and Dorman, C.J. (2000) Effects of local transcription and H-
NS on inversion of the fim switch of Escherichia coli. Mol Microbiol
36: 457-466.
Old, D.C. (1972) Inhibition of the interaction between fimbrial
haemagglutinins and erythrocytes by D-mannose and other
carbohydrates. J Gen Microbiol 71: 149-157.
Oscarsson, J., Mizunoe, Y., Uhlin, B.E., and Haydon, D.J. (1996) Induction
of haemolytic activity in Escherichia coli by the slyA gene product.
Mol Microbiol 20: 191-199.
Parkkinen, J., Rogers, G.N., Korhonen, T., Dahr, W., and Finne, J. (1986)
Identification of the O-linked sialyloligosaccharides of glycophorin A
as the erythrocyte receptors for S-fimbriated Escherichia coli. Infect
Immun 54: 37-42.
Pouttu, R., Westerlund-Wikström, B., Lång, H., Alsti, K., Virkola, R.,
Saarela, U., Siitonen, A., Kalkkinen, N., and Korhonen, T.K. (2001)
matB, a common fimbrillin gene of Escherichia coli, expressed in a
genetically conserved, virulent clonal group. J Bacteriol 183: 4727-
4736.
Prasadarao, N.V., Wass, C.A., Hacker, J., Jann, K., and Kim, K.S. (1993)
Adhesion of S-fimbriated Escherichia coli to brain glycolipids
mediated by sfaA gene-encoded protein of S-fimbriae. J Biol Chem
268: 10356-10363.
Rashid, M.H., Rajanna, C., Ali, A., and Karaolis, D.K. (2003) Identification
of genes involved in the switch between the smooth and rugose
phenotypes of Vibrio cholerae. FEMS Microbiol Lett 227: 113-119.
Russo, T.A., and Johnson, J.R. (2000) Proposal for a new inclusive
designation for extraintestinal pathogenic isolates of Escherichia
coli: ExPEC. J Infect Dis 181: 1753-1754.
Ryjenkov, D.A., Tarutina, M., Moskvin, O.V., and Gomelsky, M. (2005)
Cyclic diguanylate is a ubiquitous signaling molecule in bacteria:
insights into biochemistry of the GGDEF protein domain. J Bacteriol
187: 1792-1798.
Sauer, F.G., Futterer, K., Pinkner, J.S., Dodson, K.W., Hultgren, S.J., and
Waksman, G. (1999) Structural basis of chaperone function and
pilus biogenesis. Science 285: 1058-1061.
Sauer, F.G., Pinkner, J.S., Waksman, G., and Hultgren, S.J. (2002)
Chaperone priming of pilus subunits facilitates a topological
transition that drives fiber formation. Cell 111: 543-551.

47
Sauer, F.G., Remaut, H., Hultgren, S.J., and Waksman, G. (2004) Fiber
assembly by the chaperone-usher pathway. Biochim Biophys Acta
1694: 259-267.
Schembri, M.A., Olsen, P.B., and Klemm, P. (1998) Orientation-dependent
enhancement by H-NS of the activity of the type 1 fimbrial phase
switch promoter in Escherichia coli. Mol Gen Genet 259: 336-344.
Schmidt, A.J., Ryjenkov, D.A., and Gomelsky, M. (2005) The ubiquitous
protein domain EAL is a cyclic diguanylate-specific
phosphodiesterase: enzymatically active and inactive EAL domains.
J Bacteriol 187: 4774-4781.
Schmoll, T., Ott, M., Oudega, B., and Hacker, J. (1990) Use of a wild-type
gene fusion to determine the influence of environmental conditions
on expression of the S fimbrial adhesin in an Escherichia coli
pathogen. J Bacteriol 172: 5103-5111.
Schwan, W.R., Lee, J.L., Lenard, F.A., Matthews, B.T., and Beck, M.T.
(2002) Osmolarity and pH growth conditions regulate fim gene
transcription and type 1 pilus expression in uropathogenic
Escherichia coli. Infect Immun 70: 1391-1402.
Selander, R.K., Korhonen, T.K., Väisänen-Rhen, V., Williams, P.H.,
Pattison, P.E., and Caugant, D.A. (1986) Genetic relationships and
clonal structure of strains of Escherichia coli causing neonatal
septicemia and meningitis. Infect Immun 52: 213-222.
Simm, R., Morr, M., Kader, A., Nimtz, M., and Römling, U. (2004) GGDEF
and EAL domains inversely regulate cyclic di-GMP levels and
transition from sessility to motility. Mol Microbiol 53: 1123-1134.
Simms, A.N., and Mobley, H.L. (2008) PapX, a P fimbrial operon-encoded
inhibitor of motility in uropathogenic Escherichia coli. Infect Immun
76: 4833-4841.
Stins, M.F., Prasadarao, N.V., Ibric, L., Wass, C.A., Luckett, P., and Kim,
K.S. (1994) Binding characteristics of S fimbriated Escherichia coli to
isolated brain microvascular endothelial cells. Am J Pathol 145:
1228-1236.
Striker, R., Nilsson, U., Stonecipher, A., Magnusson, G., and Hultgren, S.J.
(1995) Structural requirements for the glycolipid receptor of human
uropathogenic Escherichia coli. Mol Microbiol 16: 1021-1029.
Thomas, W., Forero, M., Yakovenko, O., Nilsson, L., Vicini, P., Sokurenko,
E., and Vogel, V. (2006) Catch-bond model derived from allostery
explains force-activated bacterial adhesion. Biophys J 90: 753-764.
Tischler, A.D., and Camilli, A. (2004) Cyclic diguanylate (c-di-GMP)
regulates Vibrio cholerae biofilm formation. Mol Microbiol 53: 857-
869.
Valentin-Hansen, P., Eriksen, M., and Udesen, C. (2004) The bacterial Sm-
like protein Hfq: a key player in RNA transactions. Mol Microbiol 51:
1525-1533.
Wang, Y., and Kim, K.S. (2000) Effect of rpoS mutations on stress-
resistance and invasion of brain microvascular endothelial cells in
Escherichia coli K1. FEMS Microbiol Lett 182: 241-247.

48
 Vassilieva, I.M., and Garber, M.B. (2002) The regulatory role of the Hfq
protein in bacterial cells. Mol Biol (Mosk) 36: 970-977.
Waters, C.M., Lu, W., Rabinowitz, J.D., and Bassler, B.L. (2008) Quorum
sensing controls biofilm formation in Vibrio cholerae through
modulation of cyclic di-GMP levels and repression of vpsT. J
Bacteriol 190: 2527-2536.
Vetter, V. (1995) Analysis of the S-fimbriae-minor-subunit-proteins
Mediating Adherence of Pathogenic Escherichia coli. In Institut für
Molikulare Infektionsbiologie Würzburg: Bayerischen Julius-
Maximilians-Universität, pp. 167.
White-Ziegler, C.A., Villapakkam, A., Ronaszeki, K., and Young, S. (2000)
H-NS controls pap and daa fimbrial transcription in Escherichia coli
in response to multiple environmental cues J Bacteriol 182: 6391-
6400.
Virkola, R., Westerlund, B., Holthöfer, H., Parkkinen, J., Kekomäki, M.,
and Korhonen, T.K. (1988) Binding characteristics of Escherichia coli
adhesins in human urinary bladder. Infect Immun 56: 2615-2622.
Xia, Y., Gally, D., Forsman-Semb, K., and Uhlin, B.E. (2000) Regulatory
cross-talk between adhesin operons in Escherichia coli: inhibition of
type 1 fimbriae expression by the PapB protein. EMBO J 19: 1450-
1457.
Yakovenko, O., Sharma, S., Forero, M., Tchesnokova, V., Aprikian, P.,
Kidd, B., Mach, A., Vogel, V., Sokurenko, E., and Thomas, W.E.
(2008) FimH forms catch bonds that are enhanced by mechanical
force due to allosteric regulation. J Biol Chem 283: 11596-11605.
Åberg, A., Shingler, V., and Balsalobre, C. (2006) (p)ppGpp regulates type
1 fimbriation of Escherichia coli by modulating the expression of the
site-specific recombinase FimB. Mol Microbiol 60: 1520-1533.

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