New insights into the role of ppGpp
and DksA through their effect on
transcriptional regulation of
housekeeping and colonization related
genes of Escherichia coli
Anna Åberg
Department of Molecular Biology
Umeå University
Umeå 2008
Department of Molecular Biology
Umeå University
SE-901 87 Umeå
Sweden
Copyright © 2008 by Anna Åberg
ISSN 0346-6612
ISBN 978-91-7264-536-3
Printed by Print & Media, Umeå University, Umeå
Cover pictures: Electronmicropgraph pictures of Escherchia coli expressing type 1 fimbriae (left)
and flagella (right) structures (courtesy of Juan David Cabrer and Jorge Fernández). Pictures were
taken at Servei de microscòpia electrònica de transmissió, Serveis centificotècnics, Universitat de
Barcelona.
2
Till pappa
Nothing in life is to be feared. It is only to be understood
Marie Curie (1867-1934) Polish Scientist
Success is going from failure to failure without a loss of enthusiasm
Winston Churchill (1874-1965) English Statesman
3
Abbreviations used:
DNA
Deoxyribonucleic acid
iNTP
Initiator NTP
mRNA
Messenger RNA
ncRNA
Non-coding RNA
NTP
Nucleoside triphosphates
ORF
Open-reading frame, coding sequence of a gene
ppGpp
Guanosine tetra- and pentaphosphate
RelA
ppGpp synthetase I
RNA
Ribonucleic acid
RNAP
RNA polymerase (α2ββ’ω-subunits)
rrn
Genes encoding the rRNA
rRNA
Ribosomal RNA
Rsh
RelA and SpoT homologues
SpoT
ppGpp synthetase II and ppGpp hydrolase
stable RNA
rRNA and tRNA
TF
Transcription factor
tRNA
Transfer RNA
UPEC
Uropathogenic E. coli
Science is a wonderful thing if one does not have
to earn one's living at it
Albert Einstein (1879-1955) German Physicist
4
Table of contents
ABSTRACT…………………………………………………………………………………….7
PAPERS IN THIS THESIS………………………………………………………………..8
INTRODUCTION…………………………………………………………………………….9
1.
Sensing the environment……………………………………………………………………. 9
2.
2.1
2.2
2.2.1
2.2.2
2.3
2.3.1
2.3.2
Transcription……………………………………………………………………………………..10
The players in transcription…………………………………………………………….. 12
Transcription step-by-step…………………………………………………………………13
Promoter binding and initiation of transcription………………………………14
Elongation and termination……………………………………………………………….15
Regulation of transcription………………………………………………………………. 16
Transcription factors………………………………………………………………………… 17
Non-proteinaceous regulatory molecules…………………………………………. 18
3.
3.1
3.2
3.2.1
3.2.2
3.3
3.3.1
3.3.2
ppGpp – a stress alarmone………………………………………………………………… 19
Stringent response……………………………………………………………………………. 20
DksA – a possible co-regulator…………………………………………………………..22
Gre-factors………………………………………………………………………………………… 23
Regulation through the secondary channel……………………………………… 23
Mechanism of regulation by ppGpp and/or DksA………………………………25
Direct negative regulation of transcription……………………………………… 25
Direct and indirect positive regulation……………………………………………. 26
4.
4.1
4.1.1
4.2
4.2.1
4.2.2
4.2.3
Escherichia coli - a model system……………………………………………………. 27
Strains of E. coli………………………………………………………………………………..28
Uropathogenic E. coli………………………………………………………………………..28
Type 1 fimbriae………………………………………………………………………………….29
General characteristics………………………………………………………………………30
Genetic organization and regulation………………………………………………… 30
Biofilm formation……………………………………………………………………………….33
AIMS OF THIS THESIS………………………………………………………………….35
RESULT AND DISUCSSION…………………………………………………………… 36
5.
5.1
5.2
Global effect of ppGpp and/or DksA on gene expression in E. coli… 36
Effect of ppGpp and DksA on bacterial cell physiology…………………… 37
Effect of ppGpp and DksA on transcriptional
stimulation and repression………………………………………………………………. 37
5
6.
6.1
6.2
Involvement of ppGpp in biofilm formation by commensal
and pathogenic E. coli……………………………………………………………………….38
ppGpp regulate expression of type 1 fimbriae at the phase
variation level…………………………………………………………………………………… 39
ppGpp regulation of factors important for biofilm formation………… 40
7.
7.1
Divergent effects of ppGpp and DksA on gene expression in vivo….. 41
Effect of DksA-deficiency on type 1 fimbriae and flagella
expression…………………………………………………………………………………………. 42
8.
Dissecting the mechanism of regulation of fimB transcription
by ppGpp and DksA…………………………………………………………………………… 43
Direct effects of ppGpp and DksA on transcription from
the fimB P2 promoter………………………………………………………………………. 43
Co-dependent versus independent effects of ppGpp and DksA
on fimB transcription………………………………………………………………………… 44
8.1
8.2
9.
Involvement of Gre-factors in differential regulation by
ppGpp and DksA in vivo……………………………………………………………………. 45
9.1
9.2
9.3
Gre-factors stimulate fimB transcription in vivo and in vitro………… 45
Gre-factors are important for FliC expression in vivo……………………..46
Competition for the secondary channel of RNAP……………………………..47
CONCLUDING REMARKS……………………………………………………………… 48
ACKNOWLEDGMENTS………………………………………………………………….49
REFERENCES……………………………………………………………………………….51
Research is what I'm doing when I don't know what I'm doing
Wernher von Braun (1912-1977) German Scientist
6
Abstract
Bacteria have the ability to sense different environmental signals. When an
environmental stress is detected, bacteria rapidly adjust their gene expression
profile to be able to survive and thrive. The transduction of such environmental
signals often requires the coordinated involvement of several factors that
constitute complex regulatory networks. Hence, depending on the combination of
signals, a unique gene expression profile required to adapt to the specific stress
conditions is generated. Proteins are the best-known regulatory factors. However,
non-proteinaceous molecules are also important in signal-responsive control of
bacterial gene expression. Alarmones are low molecular weight non-proteinaceous
regulatory factors which can characteristically be rapidly turned-over to mediate
instant changes in gene expression. One such alarmone is the modified nucleotide
ppGpp, which directly binds to RNA polymerase to alter its activity. The levels of
this alarmone are expected to rapidly increase in response to any environmental
stress that result in slow proliferation. DksA, a putative ppGpp co-regulator that
likewise directly targets RNA polymerase, has been suggested to be required for
both the positive and negative regulation mediated by ppGpp in Escherichia coli.
This thesis describes dissection of the role of ppGpp and DksA on transcriptional
regulation, primarily using the fim genetic determinant that encodes for the type
1 fimbriae. Type 1 fimbriae are involved in adhesion to abiotic surface and initial
adhesion/invasion of bladder cells, as well as in biofilm formation. We found that
ppGpp regulates phase variation by increasing the sub-population of cells that
express the fimbriae. The effect of ppGpp was ultimately traced to its role in
transcription of the fimB gene that encodes a recombinase involved in the phase
variation process (paper 1). In contrast, we unexpectedly found that lack of DksA
causes an increase, rather than a decrease, in transcription from the fimB P2
promoter in vivo. However, in vitro transcription studies demonstrated that
ppGpp and DksA, both independently and co-dependently, stimulate transcription
from the fimB P2 promoter. These seemingly contradictory results from the in
vivo and in vitro transcriptional studies were shown to be, at least in part, a
consequence of the increased association of Gre-factors with RNA polymerase that
can occur in the absence of DksA in vivo (paper 2).
The results outlined above have implications for the role of ppGpp and/or DksA in
global gene expression. Using gene expression profile (microarray analysis) during
the transition from logarithmic to stationary phase of E. coli, we found that while
most of the genes regulated by ppGpp and DksA are regulated in the same
direction by the two factors, many were not. In addition to the fim genes, genes
involved in flagella functioning, taxis responses, and a few genes encoding
different transport systems are also differentially regulated in ppGpp- and DksAdeficient strains in vivo. Our results clearly indicate that the effect of deficiencies
in ppGpp and DksA is far more complex than phenotypic similarity of the
corresponding mutants anticipated by the proposed concerted action of ppGpp
and DksA on gene expression (paper 2 & 3).
7
Papers in this thesis
The results and discussion of this thesis is based on the following
papers and manuscript labelled with their roman letters (I-III).
Paper I
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. Micro.
60:1520-1533.
Paper II
Åberg, A., Shingler, V. and Balsalobre, C. (2008). Regulation of
the fimB promoter: a case of differential regulation by ppGpp
and DksA in vivo. Mol. Micro. 67:1223-1241.
Paper III
Åberg, A., Fernández, J., Sánchez, A. and Balsalobre, C. (2008).
Global gene regulation by ppGpp and DksA in Escherichia coli – A
transcriptomic approach. Manuscript.
8
Introduction
1.
Sensing the environment
Organisms from all domains of life have the potential to sense their
surroundings which provides them with the fundamental information required
for development, adaptation and/or survival. Bacteria are arguably the most
adaptive life forms, with an enormous diversity of species that are able to
colonise the most adverse environments. Even though bacteria harbour very
condensed genomes, they have developed an array of strategies for sensing,
responding and adapting to different environmental conditions. The complex
regulatory networks that bacteria have evolved allow them to modify their
gene expression patterns in accordance with the environmental conditions
encountered. These changes occurs at the molecular, physiological and
cellular levels to generate a phenotypic changes in entire clonal populations
(201).
However, having a population of bacteria that all express the same
genes/proteins is not always beneficial for survival. To promote phenotypic
heterogeneity in an otherwise homogeneous genetic background, bacteria
have developed another level of regulation known as phase variation. Phase
variation results in differential expression for a specific genetic determinant
in the clonal population to generate two sub-populations: one lacking or
having decreased level of expression of the gene(s) under phase variation
control and the other sub-population expressing the gene(s) fully. The key
feature of phase variation is to switch from the “ON” to “OFF” phenotype and
vice versa. Although a cell of the “OFF” phenotype does not express a given
determinant, it has not lost the ability to switch to the “ON” orientation. The
frequency of phase variation varies extensively in different genetic system,
ranging from one cell in 10 per generation to one in 10,000. This gives rise to
a heterogenic and dynamic changing population. It is believed that the phase
variation event is random, since it is impossible to predict which cell in a
population will undergo the switch. However, phase variation mechanisms
have been shown to be very tightly regulated processes controlled by complex
regulatory networks that can integrate several environmental signals. The
frequency of the phase variation event can be modulated by involvement of
accessory proteins or by changing the amount/activity of the proteins
mediating the phase variation event (192, 193).
Phase variation control of gene systems has been extensively studied in
pathogenic bacteria. Several surface structures like fimbriae, surface
proteins, capsule and LPS are regulated by phase variation in bacteria species
that live in symbiosis with other organisms. It is believed that phase variation
represent a powerful adaptative strategy and that might promote the
possibilities to evade the host immune response. Nevertheless, phase
9
variation has been described in non-pathogenic species and in bacteria not
associated with other organisms (192, 193). Hence, it is possible that phase
variation mediated control of gene expression is much more broadly dispersed
than what has been documented until now. Studies of the mechanisms of
phase variation will help to understand the importance of heterogeneity in a
bacterial population during environmental survival.
2.
Transcription
Transcription is defined as the DNA-dependent synthesis of RNA which is
catalyzed by an enzyme complex known as RNA polymerase (RNAP) (40).
DNA (deoxyribonucleic acid) is a polymer of repeated deoxyribonucleotides
and the universal molecule that contains the genetic information of most
organisms. Each monomer (deoxyribonucleotide) consists of a deoxyribose
sugar and a nitrogenous base. There are four different nitrogenous bases:
adenine (A), cytosine (C), guanine (G) and thymine (T), defining the four
different nucleotides present in the DNA. The sugars of the monomers are
joined together by phosphodiester bonds forming long polymers. RNA is very
similar in chemical structure to the DNA. The RNA (ribonucleic acid) is a
polymer of ribonucleotides, which are formed by a ribose sugar and a
nitrogenous base. In the case of the RNA, there are also four different bases,
but in contrast to DNA, the uracil (U) nitrogenous base is used instead of the
thymine. Generally, the RNA is a single-stranded molecule produced as a copy
of one of the DNA strands (template strand) during transcription.
Different types of RNA products are produced by the RNAP during
transcription. The messenger RNA (mRNA) is the molecule that is used as
template to synthesize proteins during the process called translation that is
catalyzed by the ribosomes (40). Two other types of RNA are produced by
transcription that are not translated to protein but are involved in the
translation of mRNA to protein: ribosomal RNAs (rRNA) that are components of
the ribosome and transfer RNAs (tRNA) that are carriers of specific amino
acids to the ribosome. In more recent years, it has been shown that other
small non-coding RNAs (ncRNA) play additional different roles in gene
regulation (174).
DNA is compacted into chromosomes by the help of proteins and RNA. The
number of chromosomes varies considerably between different organisms. In
most cases, bacteria have one circular chromosome, although many
exceptions have been described. In addition, extra chromosomal DNA
elements, called plasmids, can be present in the bacterial cells. The segments
of DNA that carry the genetic information for proteins – the building blocks of
all living organisms - are called genes (Fig. 1).
10
Fig. 1. Illustration of the gene organization in Bacteria. A. Presentation of the different parts of a
gene; the promoter which controls the expression of the gene, and coding and non-coding
sequences. The coding sequence is the part of the gene that encodes a protein and the noncoding sequence can be involved in regulating the expression of the gene. During transcription
RNA is produced from the promoter to the terminator, containing both non-coding and coding
sequences of the gene. During translation the ribosome will bind to the Shine-Dalgarno sequence
and only the coding sequence will be translated into a protein. The coding sequences (ORFs) are
organized in operons containing only one ORF called monocistronic operon (A) or several ORFs
called polycistronic operon (B).
Genes contain all the information to produce proteins (via mRNA), or non
coding RNAs (tRNA, rRNA, ncRNA) (Fig. 1A). A gene is delineated by a region
called promoter - where RNAP binds to initiate transcription - and a
downstream sequence called a transcription terminator - where transcription
ends. The sequence between the promoter and the terminator is transcribed
to RNA. In the case of genes that encodes for proteins, the sequence of the
DNA that specify the amino acid sequence of the protein is denoted the
coding sequence or open-reading frame (ORF). All coding sequences are
preceded by a Shine-Dalgarno sequence that is recognized by the ribosomes to
start the translation of the RNA to a protein. In bacteria, the genetic
information in the genomes is found in a specialized organization, and the
expression unit is defined as operon. Different operons are termed
monocistronic when it contains a single ORF (Fig. 1A), bicistronic when
encoding two ORFs and polycistronic operon when more than two ORFs are
under the control of a single promoter (Fig. 1B).
11
2.1
The players in transcription
Transcription is the first step in the process of gene expression and is
catalyzed by the enzymatic complex RNAP in all organisms (Eukarya, Archaea
and Bacteria). The activity of the RNAP is a major target in signal-responsive
regulation of gene expression. Bacteria, in contrast to other divisions, only
have one RNAP for the production of all RNA products. The bacterial catalytic
core RNAP enzyme, with a molecular mass of ~400 kDa, is build up by five
subunits (α2, β, β’ and ω, see Fig. 2) and is evolutionary conserved in terms of
its primary sequence, tertiary structure and function (210). The β- and β’subunits play a role in binding DNA and NTP (nucleoside triphosphates) and
catalyze the RNA synthesis at the active site. These two subunits form the two
pincers of the “crab-claw” shape of the RNAP (210). The catalytic activity of
the RNAP is dependent on a two-metal-ion mechanism and the stabilization of
binding of two coordinated Mg2+ ions in the active site of RNAP is crucial for
its activity (166). The two identical α-subunits have two functional domains
(see below, Fig. 2), the carboxyl terminal domain (α-CTD) that binds to the
UP element of the extended promoter and the amino terminal domain (αNTD)
that interact with the β- or β’-subunits of the RNAP (63). The ω-subunit has a
role in both the assembly of the RNAP core complex (114), and in subsequent
sensitivity of RNAP to the regulatory effects of small molecules (206). For
correct positioning, RNAP needs to recognize a promoter sequence. This
process is mediated by the association of an additional subunit, namely the
sigma factor (σ), that ensures promoter specificity of the resulting RNAP
holoenzyme (29, 84, 188).
Fig. 2. An illustration of the different subunits forming the RNAP holoenzyme (α2, β, β’, ω and σ)
and its interaction with different elements of a promoter. The consensus sequence of the -35 and
-10 promoter elements recognized by the σ70-factor are shown below. The box next to -10
element indicates the extended -10 element. Adapted from (26).
Having several σ-factors provides powerful means to redirect the expression
of sets of genes by a process known as promoter recognition which is
determined by the σ-factor. Most bacteria genomes encode more than one σfactor. There seem to be an approximate correlation between the number of
σ-factors and the physiological diversity of the bacteria. For example, the soil
bacterium Streptomyces coelicolor harbour the largest number of genes
12
encoding σ-factors, a total of 63 genes (66). In Escherichia coli there are
seven σ-factors (reviewed in (84)) that are classified into two families, σ70like and σ54-like (203). The most extensively studied and most abundant σfactor is σ70, also called σD, which is involved in the expression of most
housekeeping genes. σ38 (σS) is essential for some genes expressed upon entry
into stationary phase. σ32 (σH) is involved in expression of heat shock proteins
and σ24 (σE) in expression of genes whose products deal with misfolded
proteins in the periplasm. σ28 (σF) controls the expression of flagella and
chemotaxis genes and σFecI in expression of the fec operon. These σ-factors all
belong to the σ70-like family. σ54 (σN) is involved in expression of some stress
response genes and genes involved in nitrogen scavenging. The σ54-factors
have no primary sequence similarity with σ70 and have a different domain
organization (28). In this thesis only the σ70-like family of σ-factors will be
reflected upon.
Five DNA sequence elements of the promoter have been identified to be
important for the promoter recognition by the RNAP holoenzyme (Fig. 2). The
two major determinants are the -35 and the -10 elements, which are located
approximately at 35 and 10 base pairs (bp) upstream of the transcriptional
start site (+1 site), respectively. Consensus hexameric sequences of the -35
and -10 elements have been established and these regions are separated by a
spacer consisting of 17 ± 1 bp (31, 44, 121). Other elements that can also be
important for the expression of some genes are the UP element, the extended
-10 element and the discriminator located between -10 and +1. The UP
element is a DNA sequence of about 20 bp, located upstream of the -35
element of some promoters, which is bound by the αCTD of the RNAP (153).
The extended -10 element is a 3-4 bp motif, which has been suggested to be
important for a subset of promoters that lack or have poor consensus -35 or 10 hexamers (78, 115). The discriminator refers to a G+C-rich region between
the positions -10 to +1 present in some promoters (186).
2.2
Transcription step-by-step
As illustrated in Fig. 3, transcription is a cyclic process and can roughly be
divided into three major steps: promoter binding and initiation, elongation
and termination. In brief, the first step in the cycle requires the presence of
RNAP core enzyme with a promoter specific σ-factor (84). The RNAP
holoenzyme slides along the DNA until it encounters an appropriate promoter
sequence where it binds, opens the two DNA strands around the
transcriptional start site to form the transcription bubble required for RNA
synthesis to start (155). The RNAP first enters an abortive initiation cycle with
the production of short RNA products. Once the RNA products exceed ~12 to
14 nucleotides in length, transition to elongation takes place and RNAP leaves
the promoter (promoter escape) and the σ-factor is released. Transcription
elongation will continue until the RNAP reaches a termination signal, at which
13
point RNAP is releases from the DNA and so becomes available for association
with another σ-factor to restart the cycle once again (20).
Fig. 3. The transcription cycle consist of four main steps; 1 – Promoter recognition, 2 – Initiation,
3 – Elongation and 4 – Termination. Upon transcriptional termination, RNAP is released (5) and
can bind to another σ-factor (6), thus the cycle continues.
2.2.1
Promoter binding and initiation of transcription
Transcription initiation is a multi-step process consisting of three main steps;
promoter recognition, initiation and promoter escape (Fig. 4). Transcription
initiation involves the interaction of the RNAP to the promoter element. As
already mentioned in the preceding sections, this requires that the RNAP is
associated with a specific σ-factor. The σ-factor has three main functions;
identification of the promoter sequence, positioning of the RNAP holoenzyme
and aiding the unwinding of the two DNA strands (203).
When the RNAP initially binds to the promoter, a RNAP-DNA complex is
formed called the closed-complex (RPc). In this status the DNA is doublestranded and the RNAP is covering a region from ~-55 to +1 relative to the
transcriptional start site (38, 107). The complex undergoes several
conformational changes in both RNAP and DNA known as isomerization steps.
Eventually, the open-complex (RPo) develops as the DNA strands get separated
in the region ~-11 to +3, allowing a base on the template strand to pair with
the initiating NTP (iNTP) (38, 100, 146). After the formation of the opencomplex, incorporation of NTPs drives transcription forward. However,
initially the RNAP synthesize short abortive products from most promoters (27 nucleotides long) before starting elongation (79). During this process, the
RNAP maintains contact with the -35 element and moves back and forth by a
process also known as “scrunching”. The energy that is stored in these
14
intermediates has been suggested to allow the RNAP to begin the transition
into the elongating complex (92, 150).
Fig. 4. Different steps in promoter recognition and transcription initiation. RNAP holoenzyme (R)
binds to the promoter (P) and forms the closed-complex (RPc) with the DNA double-strand. After a
series of isomerization steps, the DNA double-strand is opened forming the open-complex (RPo).
As the initiating NTP (iNTP) pairs with the +1 (transcriptional start) this forms the initiating
complex (RPinit). Several rounds of abortive initiation occur, producing short RNA products before
the elongating complex (EC) leaves the promoter and the σ-factor disassociate from the RNAP.
Adapted from (146).
2.2.2
Elongation and termination
When the RNAP leaves the promoter (promoter escape) and transcription
elongation starts it has been assumed that the σ-factor is always released
from the RNAP. However, more recently it has been shown that the release is
not obligatory for escaping from the promoter and that σ-factor release
occurs stochastically during the early stages of elongation (149). The
elongating complex (EC) is much stable and processive as compared to the
initiation complex. During elongation, the RNAP-DNA complex can change
between an active (elongating competent) and inactive (backtracked/paused)
state (67, 124). Pauses occurring during elongation determine the overall
elongation rate. Unlike during initiation, the elongation complex does not
disassociate from the DNA when these pausing events occurs. There are two
mechanistically distinct classes of pause signals; hairpin-dependent (e.g. his
leader pause) and hairpin–independent pauses (e.g. ops pause) (5, 11). During
pausing of RNAP, regulatory interactions, such as binding of transcription
elongation factors and the proper positioning of the ribosome take place (6,
151). The exact position and time that the RNAP spend in the paused state
can determine the effect of RNAP pausing on gene expression. Furthermore,
pausing also plays a role in folding of the mRNA (132, 133) and in the
termination process (68).
Elongation continues until the RNAP encounters a termination signal. There
are two different classes of signals known: the Rho-independent, also known
as intrinsic terminator, and the Rho-dependent ((74) and references therein).
The intrinsic terminator is composed of a G+C-rich stem-loop followed by a
15
series of U residues. The Rho-dependent termination requires the binding of
the Rho-factor to a rut (Rho utilization) site on the nascent transcript
followed by the interaction with the RNAP. Thus like initiation, elongation and
termination are dynamic processes controlled at different levels.
2.3
Regulation of transcription
The activity of RNAP can be regulated throughout the entire transcription
cycle. Although it has been anticipated that most transcription regulation
occurs at the level of initiation, there are also numerous factors influencing
the elongation and termination steps of transcription. In this thesis, effectors
that regulate the first steps of transcription (initiation and elongation) are
primarily discussed.
When studying bacterial gene regulation, it is important to bear in mind that
the intracellular levels of RNAP are limiting. During active growth, most of the
RNAP in the cell is associated with the genes encoding stable RNAs (rRNA and
tRNA) that are very highly expressed since they are crucial components of the
translational machinery. Therefore, there is a limited number of RNAP
available to transcribe the 4-5,000 genes present in the cell (84). We might
consider that inside the cell there is a competition between the nearly 2,000
different promoter sequences for the accessible RNAP (156). Several factors
might affect the affinity of the RNAP to a specific promoter: promoter
sequence, σ-factor composition, small ligands, transcription factors and
structure of the chromosome. Each of these factors potentially promotes
variations in the level of transcription and the impact of each of them varies
from gene (or sets of genes) to gene.
Variations in the promoter sequence offers one way of regulating transcription
by promoting the recruitment of the RNAP. However, this can hardly be
considered as a mechanism of modulation of gene expression in the short
term, since an adjustment would involve alterations in the DNA sequence.
Nevertheless, accumulation of mutations through generations might provide a
more appropriately controlled promoter sequence for bacterial adaptation to
certain conditions. Thus promoter sequence changes might play an important
role in the control of gene expression on an evolutionary time scale.
As mentioned earlier, most of the bacteria can express more than one σfactor simultaneously leading to competition for binding of the different σfactors to the RNAP (84). Depending on the specific σ-factor that binds to the
core RNAP, expression of a specific subset of genes will be favored.
Therefore, depending on the global composition of σ-factors present in the
cell, a specific global gene expression profile would be generated (reviewed
in (84, 203)). The global transcriptional capacity is also modulated by the
16
activity of anti-sigma factors that bind and sequester specific σ-factor so that
they can not associate with RNAP (82).
The bacterial DNA in the cell is negatively supercoiled and its conformation
changes according to environmental conditions and/or by the action of the socalled nucleoid-associated proteins (e.g. H-NS, Fis, IHF and HU). Most of these
factors are very abundant and bind DNA non-specifically (8, 185). The
conformation of DNA can affect both the accessibility of the RNAP to a
promoter as well as the activity of the RNAP during transcription (185).
The role of the transcription factors and small ligands in transcription will be
extensively discussed in the following sections.
2.3.1
Transcription factors
Classically, transcription factors (TFs) have been considered as proteins that
bind to DNA. However, it is now evident that not all TFs directly bind DNA.
For example, their action can be mediated by modifying the binding or
changing the activity of other TFs or by interacting directly with the RNAP.
Moreover, in the last years, studies have shown that many factors directly
involved in regulating transcription are non-proteinaceous, like RNA products
and small molecules.
In E. coli there are more than 300 predicted genes that encode for proteins
that bind to DNA and either up- or down-regulate transcription (138, 181).
The function of about half of those proteins have been verified
experimentally. Most of them bind to sequence-specific regions on the DNA
and control the expression of either a large number of genes (global
regulator) or just one or few genes (specific regulator). In E. coli, about 50 %
of all genes have been suggested to be regulated by seven global regulators:
cyclic AMP receptor protein (CRP), fumarate reductase and nitrite reductase
(FNR), integration host factor (IHF), factor for inversion stimulation (Fis),
anaerobic respiratory control (Arc), nitrite regulation (Nar) and leucine
regulatory protein (Lrp) (111). The TFs regulate gene expression in response
to environmental changes. Sensing changes in environmental parameters
might alter the amount, the availability and/or the activity of TFs and
consequently induce important alterations in the expression profile of the
bacterial cell. Certain conditions might induce the levels of TFs in the cell
with consequent effects on gene expression. In some cases the TFs might
either interact or not with other cellular factors which determine whether it
is active or not. The availability of active TFs can also be altered as a
consequence of environmental signals, following an alteration of the gene
expression pattern. The alteration of the activity of the TFs can be mediated
by binding to small ligands, like cAMP for the CRP protein (71), by covalent
modifications, like phoshorylation by a sensor kinase in two-component
systems, etc (172).
17
As the TFs bind to the DNA they can either increase (i.e. activator) or
decrease (i.e. repressor) transcription. Different mechanisms have been
described to promote activation or repression of transcription by studying how
TFs affect specific genetic systems. Hence, the classical activation of
transcription by improving the recruitment of the RNAP to the promoter can
be achieved by several specific mechanisms: binding to the UP element (e.g.
cAMP-CRP), binding to the -35 element (e.g. λ CI protein) or by changing the
conformation of the promoter by binding to the -35 to -10 region (e.g. MerR
family). Correspondingly, repression of transcription can occur by different
and diverse mechanisms: steric hindrance for the RNAP to bind to the
promoter (e.g. Lac repressor), by DNA looping that shuts transcription
initiation (e.g. GalR), by modulation of an activator (e.g. CytR), etc (26). In
addition to the enormous mechanistic diversity that exists, when analyzing
how regulation occurs in the cell, is important to have in mind that frequently
the activity of a promoter is influenced by several environmental cues and
regulated by more than one TF simultaneously. Moreover, it is common that
global regulators and specific regulators work co-ordinately to regulate the
expression of a genetic locus (111, 112). Furthermore, some TFs are
regulating the expression and activity of other TFs, forming very complex
regulatory networks.
2.3.2
Non- proteinaceous regulatory molecules
There are several non-proteinaceous molecules in bacteria that can regulate
transcription and gene expression, both directly and/or indirectly, in different
ways. Examples are small regulatory RNAs, modified nucleotides (cAMP,
ppGpp and cyclic di-GMP) and quorum sensing molecules (AHLs and
oligopeptides).
Small RNAs (sRNA) are non-coding RNAs of 40 to 400 nucleotides in length,
which have the ability to regulate gene expression. In E. coli at least 50 sRNAs
has been described so far, although just a few of them have been
characterized. The expression of most of sRNA studied to date is induced in
response to changes in environmental conditions. They regulate gene
expression by using different mechanistic strategies (reviewed in (69, 113,
173, 175)), e.g.:
- by base-pairing with target mRNAs (both in cis and trans) affecting
transcription termination, mRNA degradation and/or inhibiting
translation.
- by interacting with proteins and thereby inhibiting or changing its activity.
- by directly interacting with the RNAP. For instance, the 6S RNA interacts
with the RNAP changing its promoter preference. The 6S RNA is abundant
in E. coli and other species, and its synthesis is induced upon entry in
18
stationary phase of growth. 6S RNA represses expression of several σ70RNAP promoters and induces expression of several σS-RNAP promoters. Its
interaction with the active site of the RNAP mediates both the inhibition
(by DNA blockage) and the activation of transcription (by template RNA
synthesis) (195).
There are several low molecular mass molecules that play a role in bacterial
gene regulation. The quorum sensing autoinducers (e.g. AHLs, AI-2, PQS and
oligopeptides) modulate gene expression in response to population density.
These molecules can regulate gene expression by interacting either with a
transcription factor or with a response-regulator of a two-component system
(197).
Several cytoplasmic second messengers are modified nucleotides. The cyclic
dinucleotide 3’5’-cyclic diguanylic acid (c-di-GMP) is synthesized from GTP by
proteins having diguanylate cyclase activity and degraded by proteins having
phosphodiesterase activity. Intracellular c-di-GMP levels are altered by
external signals (environmental changes) causing concomitant effects on gene
expression. So far, the exact mechanism of how c-di-GMP alter gene
expression is not known (152). Both quorum sensing and c-di-GMP coordinately regulate processes such as biofilm formation and virulence.
Therefore, it has been proposed that the two regulatory pathways are linked
(30). Cyclic-adenosine 3’5’ monophosphate (cAMP) is synthesized from ATP by
adenylate cyclases. cAMP binds to CRP (catabolite repressor protein) and
activates the dimeric form of this global regulator. cAMP-CRP complex binds
to the DNA, commonly at the promoter region, exerting its control of the gene
expression (71). Another modified nucleotide involved in gene control is the
guanosine-3’,5’-bispyrophosphate (ppGpp). ppGpp is synthesized in response
to different kinds of stress conditions and interacts directly with the RNAP
(33, 35). This will be discussed more extensively below.
3.
ppGpp - a stress alarmone
Guanosine tetra- and penta-phosphate, collectively called ppGpp or “magic
spot”, are modified nucleotides that act as alarmones or stress signals in
bacteria. ppGpp is very rapidly synthesized from GTP (or GDP) and ATP in
response to any stress condition that will result in growth arrest (33). As early
as 1969, Cashel and Gallant described that stimulation of intracellular level of
ppGpp in E. coli in response to nutrient starvation has important
consequences in the pattern of gene expression, termed the stringent
response (32). Since then, it has been illustrated that ppGpp is also
synthesized during many other stress conditions. ppGpp is not restricted to
Gram-negative bacteria, it is also found in Gram-positive bacteria (85) and in
the chloroplasts of plant cells where it also functions in stress related
processes (179).
19
3.1
Stringent response
The hallmark of the stringent response in E. coli is the down-regulation of
stable RNA synthesis (rRNA and tRNA) and ribosome production that occurs
upon amino acid starvation (33, 157, 170). This is mediated by the small
regulatory molecule, ppGpp (32). In E. coli, the synthesis of ppGpp occurs by
two related proteins: RelA and SpoT (33, 208). The RelA protein mainly
synthesize ppGpp in response to amino acid starvation by recognizing and
binding to stalled ribosomes, that have an uncharged tRNA bound in their Asite (33, 200). SpoT is a bifunctional protein that synthesize ppGpp in
response to other stress conditions than amino acid starvation (208). The
mechanism leading to SpoT activation is not known (61, 200). Recently it was
shown that SpoT is binding to CgtAE, a ribosome-associated protein in E. coli,
but whether this interaction has any effect on the activity of the SpoT protein
remains to be elucidated (204). Elevated levels of ppGpp during a long period
time are deleterious for the cell physiology and therefore it is essential to
hydrolyse it. In E. coli, the SpoT protein is responsible for the hydrolysis (33,
61). An E. coli strain that lacks both RelA and SpoT proteins do not synthesize
ppGpp under any growth conditions and is denoted ppGpp0. These strains are
unable to grow in minimal media as a consequence of extensive amino acid
requirement (auxotrophy) (208).
There are several bacteria species that only harbour one copy of a RelA-SpoT
homologue with both synthetase and hydrolase activity (116). These
homologues are called Rsh (RelA-SpoT homologue) proteins and such
bifunctional enzymes modulate the ppGpp levels through two distinct active
sites (21). It has been shown that in some Gram-positive bacteria, the Rsh
proteins do not require interaction with the ribosome to trigger their ppGpp
synthetase activity. However, in the presence of stalled ribosomes the
activity of the Rsh proteins increases (85). Rsh proteins have also been found
in chloroplasts of plants cells and are responsible for accumulation of ppGpp
due to stress (62, 179, 191). Notably, it has been found that there are some
microorganisms that do not produce ppGpp, including some parasitic bacteria
(Treponema pallidum, Chlamydia species, Rickettsia prowazekii) and some
Achaea species (116).
ppGpp binds to the β- and β’-subunits of the RNAP core enzyme (34, 184). It
has been shown that mutations within the genes rpoB, rpoC and rpoD that
encodes for the RNAP subunits β , β’ and σ70 respectively, can render a RNAP
that is unresponsive to the levels of ppGpp in the cell (33, 122). Cross-linking
and co-crystallization studies of the RNAP of Thermus thermophilus and
ppGpp has suggested that ppGpp interacts with the β- and β’-subunits in close
proximity to the active site, coordinating the position of the Mg2+-ions (7, 34).
The co-crystallization studies suggested that ppGpp can bind to the RNAP in
two different orientations. The significance of these two binding positions for
the mechanism of transcriptional regulation by ppGpp has not been
determined (7). Recently, it has been suggested that the predicted binding
20
site for ppGpp within the RNAP of T. thermophilus may not be accurate for
the E. coli RNAP. Substitutions of the predicted interacting amino acids
residues in the core enzyme did not result in loss of ppGpp-mediated
regulatory effects (205). Further studies are needed to elucidate the exact
binding position of ppGpp within the RNAP. This knowledge will facilitate the
understanding of the regulatory mechanism of ppGpp on transcription.
The interaction of ppGpp with the RNAP has pleiotropic effects on gene
expression. Numerous genes are both repressed and induced in response to
increasing concentration of ppGpp (see Fig. 5). Although the decrease of
protein synthesis, as a consequence of repression of stable RNA, is perhaps
the most predominant effect during the stringent response, ppGpp also
regulates DNA replication, fatty acid synthesis and cell wall component
expression, among others. There is also induction of amino acid biosynthesis,
stress response related proteins, and alternative sigma factor such as RpoS,
RpoE and RpoH (σS, σE and σH) (33, 50).
Fig. 5. Synthesis of ppGpp in E. coli and consequential global effects on gene expression. ppGpp
is synthesized from GTP/GDP and ATP by two parallel pathways depending on the stress condition
encountered (33), the RelA- or the SpoT-dependent pathway. ppGpp is very rapidly hydrolysed by
the SpoT protein. ppGpp binds to RNAP and redirects gene expression to survive the stress
conditions. Adapted from (109).
21
Furthermore ppGpp has been shown to play an important role in regulating
genes involved in virulence and survival under host/environmental induced
stress (21, 85). Hence, ppGpp regulates long-term persistence of
Mycobacterium tuberculosis (41), quorum sensing and virulence in
Pseudomonas aeruginosa (52), biofilm formation in E. coli and Listeria
monocytogenes (1, 180), and has an effect on virulence in Vibrio cholerae,
Salmonella, Legionella, L. monocytogenes, Borrelia burgdorferi and
Campylobacter jejuni (60, 70, 119, 140, 165, 180).
3.2
DksA – a possible co-regulator
DksA is a 151 amino acid long (17 kDa) polypeptide encoded by a non-essential
gene found in E. coli and related bacteria. DksA was originally found as a
multicopy suppressor of the temperature-sensitive phenotype of dnaKJ
mutants in E. coli (91). Studies of dksA over-expression and dksA deletion
strains suggest a role for DksA in a variety of cellular processes, including cell
division, stringent response, quorum sensing and virulence in E. coli, P.
aeruginosa, Salmonella and Shigella flexneri (76, 89, 117, 187, 198). Several
studies suggested that DksA acts as a co-regulator for the ppGpp-dependent
regulation of genes in E. coli. A dksA mutant strain shows an auxotrophy
phenotype that can be suppressed by a rpoB mutation (βT563P) which also
suppresses the auxotrophy of a ppGpp0 strain (122). Moreover, the effect of a
dksA mutation on rpoS induction is similar to that observed in the absence of
ppGpp (76). Furthermore, it has been shown that DksA enhances the effect of
ppGpp on both the negatively (134) and positively (135) regulated genes, both
in vivo and in vitro. However, it is notable that DksA is not as widely
distributed among bacterial species as ppGpp (136, 137).
Conversely, when studying the phenotypes of a dksA and ppGpp0 mutant
strains some differential phenotypes have been observed (2, 25, 110). The
amino acid requirements are not exactly the same for the ppGpp0 and the
dksA mutant strains (25) and the auxotrophic phenotype of the ppGpp0 strain
can not be restored by over-expressing DksA in all E. coli K-12 strain
backgrounds (110). Moreover, several genes in E. coli are differentially
affected in ppGpp0 and dksA mutant strains in vivo, including genes for
adhesion, chemotaxis and motility. It has been shown that DksA and ppGpp
may exert independent effects on transcription both in vivo and in vitro (2,
110, 143). These conclusions will be further discussed in the Result and
Discussion section of this thesis.
The crystal structure of DksA reveals a prominent coiled-coil domain in the Nterminus and a conserved Zn-finger motif in the C-terminus (137, 196). DksA is
structurally very similar to the transcription elongation factor GreA, although
they do not share any sequence homology (Fig. 6, (169)). DksA co-purifies
with the RNAP. Although no co-crystallization structure between DksA and
RNAP has yet been reported, it has been suggested that DksA interacts with
22
RNAP directly by protruding the coiled-coil N-terminus into the secondary
channel, thereby stabilizing the interaction between RNAP and ppGpp (137).
3.2.1
Gre-factors
The Gre-factors are proteins that interact with the RNAP during transcription
and suppress RNAP arrest or pausing by inducing cleavage of the RNA
transcript within the active site of the enzyme (55, 123). In E. coli there are
two genes coding for the Gre-factors, greA and greB. Although, neither greA
nor greB are essential for viability, several phenotypes has been associated
with gre mutant strains, including sensitivity to divalent metal ions, salt and
temperature (171, 176). In Pseudomonas, a greA mutant strain is unable to
grow in minimal media (108). The Gre-factors are evolutionary conserved and
found in more than 60 bacterial species. Members of the Gre-family, which
includes DksA and Gfh1, share a conserved two-domain structure, the globular
C-terminal domain and an extended coiled-coil N-terminal domain (105, 137,
169, 178).
The Gre-factors bind to the RNAP with the C-terminal domain near the
secondary channel and the N-terminal coiled-coil domain protrudes into the
channel so that its tip come in close proximity to the active site (106, 130,
167). The acidic residues, at the tip of the coiled-coil domain of the Grefactors help to coordinate the Mg2+ ions in the catalytic center of the RNAP,
thereby inducing the endonucleolytic cleavage reaction (Fig. 6). The induced
cleavage reactivates the stalled elongation complex formed as a result of
backtracking or arrest of the RNAP (106, 167). The Gre-factors are also
involved in earlier steps of the transcription, since they facilitate promoter
escape and entrance to the elongation phase by decreasing the production of
abortive RNAs (54, 80, 176). A recent microarray study in E. coli has shown
the importance of GreA in gene regulation in vivo. The stimulatory effect of
GreA on gene expression occurs by facilitating the promoter escape event that
depends on the ability to stimulate transcript cleavage. On the other hand,
GreA-mediated negative regulation appears to be indirect through some
unknown factors (171). The cleavage reaction catalyzed by the Gre-factors
removes miss-incorporated nucleotides and, therefore, the Gre-factors also
contribute to the proofreading and fidelity of transcription (55, 209).
3.2.2
Regulation through the secondary channel
The secondary channel of the RNAP links the external milieu to the active
site, acting as an entry port for NTPs and regulatory factors (123). The Grefactors are members of a family of secondary channel regulators that share a
common tertiary structure but have different effects on transcription
regulation (45, 123). DksA influences ppGpp-mediated regulation by
destabilizing open-complex formation (134, 135). GreA and GreB, as already
23
mentioned, facilitate promoter escape and rescue of stalled/arrested RNAP
elongation complexes by inducing endonucleolytic cleavage (55). Another
member, Gfh1 from the Thermus genus, does not stimulate endonucleolytic
cleavage of RNAP but instead inhibits the GreA-mediated effect on RNA
cleavage and RNA synthesis (104, 178). When the crystal structure of GreA
and Gfh1 were compared, a striking difference in the orientation of the Nterminal and C-terminal domain was revealed. In Gfh1, the N-terminal domain
is flipped ~170° as compared to GreA (178). Interestingly, the Gfh1 can adopt
two different conformations (inactive and active) with the switch to the
active conformation being dependent on low pH (105). Superimposition of the
structures of DksA and GreA illustrated that the position of the acidic residues
of the coiled-coil domain of DksA is likely oriented differently as compared to
GreA (137).
Fig. 6. Structure of the RNAP core enzyme with the main channel containing the DNA (solid line)
and the secondary channel marked in white. The dotted line shows the synthesized RNA exiting
through the RNA exit channel and the two Mg2+ ions in the active site are marked by circles. To
the right are shown factors predicted to enter (NTP and ppGpp) or bind to the secondary channel
(DksA and GreA/GreB) in E. coli. The β-subunit is shown in dark grey and the β’-subunit in light
grey. Adapted from (123).
It has been suggested that all these regulators by interacting with the
secondary channel might have overlapping or antagonistic effects on gene
expression. Potrykus et al showed that GreA and DksA have antagonistic
effects on rrnB transcription in vitro and in vivo (143). On the other hand,
Rutherford et al showed that while GreA had only modest effects on
transcription, GreB could fulfill some of the regulatory roles of DksA on rrnB
transcription in vitro (154). The presence of multiple secondary channel
regulators in the cell implies that competition for binding to the secondary
channel could potentially occur. Such competition would be influenced by
24
both the intracellular concentration of the various factors as well as their
intrinsic binding affinities towards RNAP.
It has been shown in E. coli that the intracellular concentration of DksA, GreA
and GreB are relatively constant through growth, with DksA being more
abundant than either GreA and GreB (154). DksA and GreB were shown to
have similar affinities for the binding to the RNAP, which apparently are much
higher than the affinity shown by GreA. Due to the differences in relative
concentration, it is assumed that a substantial proportion of the RNAP would
be preferentially occupied with DksA. Whether that is valid for all growth
phases and conditions remains to be elucidated. The knowledge that the
structure of Gfh1 changes from an active to an inactive form upon pH
alterations suggests that under certain conditions conformational changes of
factors that interact with the secondary channel might alter their specific
affinity for RNAP and, therefore, make important adjustments in the amount
of RNAP interacting with each specific factor. Another possibility that has not
been considered in the literature is that the affinity of RNAP for the different
secondary channel TFs might be altered when RNAP binds a specific promoter
sequence or interacts with a particular σ-factor. These interactions could
potentially change the conformation of the secondary channel of RNAP
holoenzyme and thereby interactions of regulatory factors through this part of
the enzyme.
3.3
Mechanism of regulation by ppGpp and/or
DksA
ppGpp and DksA can be classified as global transcriptional regulators because
transcriptomic and proteomic studies have shown that they influence the
expression of numerous bacterial genes. They activate and repress
transcription from a number of promoters, both directly and indirectly (50,
110, 139, 161, 182). In most cases, the two factors seem to regulate
transcription co-dependently. However, there are cases where apparently
they affect transcription by independent mechanisms (2, 110).
3.3.1
Direct negative regulation of transcription
Negative regulation mediated by ppGpp and DksA on rRNA production has
been extensively studied over the last years (136). Moreover, direct inhibitory
effects of ppGpp have also been reported for other promoters (13, 33, 142).
Several mechanisms have been suggested to explain the direct negative
effects of ppGpp on transcription.
- The rrn promoters form intrinsically unstable open-complexes and are
very sensitive for further destabilization (13, 134, 136). Direct binding of
25
ppGpp and/or DksA to RNAP further destabilizes their already intrinsically
unstable open-complexes leading to down-regulation.
- ppGpp also has direct negative effects on promoters with stable opencomplexes. In these cases it has been proposed that ppGpp disturbs the
promoter escape process to exert its negative effect (95, 142).
- the amount of iNTP have shown to be important for some promoters
regulated by ppGpp, suggesting that competition between ppGpp and
iNTPs for the active site could account for negative ppGpp-dependent
effects (88, 208).
- ppGpp has also been demonstrated to induce pausing during the
elongation step of transcription (88, 189).
These different mechanisms are not exclusive and could
simultaneously in the fine regulation of different genetic systems.
3.3.2
function
Direct and indirect positive regulation
ppGpp has been shown to be a positive regulator of several genes in vivo (33).
The mechanism for the direct positive regulation by ppGpp has not been as
extensively studied as those of direct negative regulation. For many years, no
clear experimental evidences of a direct positive regulation of transcription
by ppGpp could be obtained. Therefore, it was thought that the positive
regulation by ppGpp occurred only by an indirect mechanism. However, when
it was discovered that the DksA protein promotes ppGpp-mediated effect on
transcription, direct positive effects of ppGpp has been revealed in vitro in
the presence of DksA. These studies were first done with the amino acid
biosynthesis genes (135). Direct effects of ppGpp on the phage promoter λPaQ
has also been shown (141).
Promoters that are positively regulated by ppGpp and/or DksA often form very
stable open-complexes. The destabilization mediated by ppGpp and DksA of
the open-complex formation might actually help promoter escape and thereby
transcription initiation from these promoters (14). Alternatively, it has been
proposed that ppGpp/DksA lower the transition state energy required for
formation of a rate-limiting intermediate in the pathway to open-complex
formation thereby stimulating promoter output (136).
Superimposed on direct effects, it has been suggested that ppGpp could
indirectly activate gene expression by altering the availability of the RNAP in
the cell. Since ppGpp down-regulates transcription of the rrn genes, in the
presence of ppGpp more σ70-RNAP holoenzyme would be accessible for other
promoters (12, 211).
26
ppGpp increases pausing of RNAP at all promoters tested. The ppGpp-induced
pausing increases the time that the RNAP occupies the promoter. This
increased residence time could facilitate productive interaction and binding
of regulatory factors that could be important for stimulating transcription of
some genes (22, 101).
ppGpp and/or DksA are also required for transcription from promoters
recognized by alternative σ-factors (e.g. σ32, σS, σ54 and σE) which might be
explained by three possible mechanisms (17, 39, 64, 65, 86, 103):
1. ppGpp/DksA directly affects the interaction of the σ-factor with the
RNAP core enzyme thereby affecting the assembly of the alternative
holoenzymes.
2. The promoters that are regulated by alternative σ-factors have kinetic
properties which make them sensitive to the presence of ppGpp and
DksA.
3. The repression of rrn transcription in the presence of ppGpp and DksA
increases the availability of pool of free core RNAP in the cell, thereby
facilitating holoenzyme formation with alternative σ-factors.
As with negative regulation by ppGpp, these mechanisms are not mutually
exclusive and more than one can act simultaneously.
4.
Escherichia coli - a model organism
E. coli is a rod-shaped Gram-negative bacterium that was first discovered by
Theodor Escherich in 1885. E. coli is classified as a γ-proteobacteria and part
of the Enterobacteriaceae family. It is about 2 micrometer long and 0.5
micrometer wide. E. coli is commonly found in the lower intestine of warmblooded animals and it is part of the normal flora of the gut. Most of the
strains are harmless and provide benefits to the host by producing vitamin K2
(16) or by preventing the colonization of pathogenic bacteria within the
intestine, among other mechanisms (81). E. coli is a model organism
commonly used in microbiology and genetics to study bacterial physiology,
metabolism, gene regulation, signal transduction, and cell wall structure and
function.
Wild-type E. coli has meager growth requirements and can synthesize all the
macromolecular components of the cell from glucose. The bacterium can
grow both in the presence (aerobic) and absence (anaerobic) of oxygen. Like
other bacteria, E. coli has the ability to respond to environmental signals such
as chemicals, pH, temperature, osmolarity, etc.
27
4.1
Strains of E. coli
E. coli strains are divided into several different serotypes based on their O(somatic), H- (flagella) and K- (capsule) antigens. Practically, E. coli can
roughly be divided into three sub-groups of strains: commensal, pathogenic
and laboratory strains.
The commensal strains colonize the infant gastrointestinal tract within 40
hours after birth and may persist for life time. E. coli is the predominant
facultative organism in the human gastrointestinal tract but represent only 1
% of the total bacterial mass (183). Commonly, the commensal strains are
considered as non-pathogenic.
The pathogenic E. coli strains can cause diverse diseases. Although they are
mostly known as the causative agents of gastroenteritis, urinary tract
infection and neonatal meningitis, they can also cause peritonitis, mastitis,
septicemia and pneumonia (183). Based on their serological characteristics
and virulence properties, pathogenic E. coli strains are classified into
different virotypes. The virotype causing urinary tract infections are
uropathogenic E. coli. The differences between commensal and pathogenic E.
coli strains are evident when comparing their genome sequences. Pathogenic
E. coli have acquired additional virulence genes by horizontal gene transfer.
The “extra” DNA detected in the genomes of pathogenic strain is often
located in discrete regions of the genome that are called pathogenicity
islands. Interestingly, regulation of these virulence factors often involves
transcription factors that are encoded by the backbone chromosome.
The laboratory strains are either commensal or virulent/pathogenic isolates
that have been used as model organisms in the research laboratories for many
years. After cultivation under the laboratory conditions, most strains loose
some of their original phenotypes. The most commonly used laboratory strains
of E. coli belong to the K-12 serotype.
During my thesis work I used laboratory K-12 strains and uropathogenic
isolates of E. coli. The characteristic phenotypes of the former are described
in the following section.
4.1.1
Uropathogenic E. coli – UPEC
Uropathogenic E. coli (UPEC) strains can colonize the urethra (urethritis), the
bladder (cystitis) and/or kidneys (phyleonephritis) and are the causal agent of
about 90 % of all the urinary tract infections each year. UPEC express a
number of different virulence determinants such as adhesins, iron uptake
systems and cytotoxins (87) that promote colonization of the host tissues and
successful establishment of infections (Fig. 7).
28
Fig. 7. Illustration of the different virulence and colonization factors that UPEC strains may
express and that are important for colonization and establishment of infection. Adapted from
(87).
4.2
Type 1 fimbriae
During the infection process, the adhesins expressed by UPEC aid colonization
of different tissues and organs of the urinary tract by recognizing different
surface receptors on the endothelial cells. The ability of individual UPEC
isolates to colonize a specific tissue within the urinary tract depends on which
kind of adhesins can be expressed (120).
Fimbriae, also called pili, are proteinaceous hair-like appendages found on
the surface of the bacterial cell. The word “fimbriae” and “pili” comes from
the Latin word for “thread” or “hair” (23). They range from 3-10 nanometers
in diameter and can be several micrometers in length. They are built up by
several different subunit types and have a specific adhesin protein at their
tip. Bacteria use fimbriae to attach to each other, to biotic or abiotic surfaces
and to host cells. In general, most Gram-negative bacteria produce fimbrial
structures and similar systems have been described for some Gram-positive
bacteria. The structure and assembly of the fimbrial subunits vary between
different fimbriae systems and bacterial species. Different strains of E. coli
have the genes and the ability to express several types of fimbriae structures.
The studies presented in this thesis have been focused on better
understanding of how the type 1 fimbriae are regulated in UPEC strains.
29
4.2.1
General characteristics
Type 1 fimbriae are the most common fimbriae in species of the
Enterobacteriace family. About 70 to 80 % of all wild-type E. coli strains have
the genes to express these fimbriae. Type 1 fimbriae are involved in
attachment to biotic and abiotic surfaces (144) and are important for the
initial steps of biofilm formation (158, 159). Moreover, they are a crucial
virulence factor of UPEC because they mediate initial adhesion and invasion of
bladder cells by binding to mannose-containing receptors on the
uroepithelium (83, 120, 162). UPEC can evade the human host responses by
invading the epithelial cells of the bladder where they form intracellular
bacterial communities (90). In addition, type 1 fimbria are required for
maturation of such intracellular bacterial communities formed within
epithelial cells that provides a protective mechanism against the host immune
response (207).
Type 1 fimbriae are about 7 nanometers in diameter and 0.2 to 2 micrometers
in length. The assembly of the fimbriae structures occurs by the
“chaperone/usher-pathway” (118). The fimbriae consist of several noncovalently linked subunits, a major rod (FimA), a tip fimbrillum (FimF and
FimG) and an adhesin (FimH) (Fig. 8). The adhesin recognize mannosecontaining receptors and the binding of the fimbriae can be blocked by Dmannose or α-methylmannosides (120).
4.2.2
Genetic organization and regulation
The genes encoding for the synthesis of type 1 fimbriae are clustered in the
fim determinant, which consist of a polycistronic operon containing the
structural genes (fimAICDFGH) and two monocistronic operons (fimB, fimE)
encoding for the recombinases involved in the regulation of the expression of
the structural genes (see Fig. 8).
Transcription of the polycistronic fim operon is regulated by phase variation
and by promoter activity. The phase variation is mediated by inversion of a
314 bp DNA fragment that contains the promoter for the polycistronic fim
operon located just upstream of the major subunit gene, fimA (Fig. 9 and (56,
99)). Depending on the orientation of this invertible DNA fragment, the
promoter is positioned to direct transcription of the structural fim genes
(“ON” orientation) or not (“OFF” orientation). The “switching-mechanism” is
mediated by the site-specific recombinases FimB and FimE (Fig. 9). FimB
mainly mediates the switch from OFF-to-ON, and FimE the switch from ON-toOFF (58, 98). In an E. coli K-12 population, grown in standard laboratory
conditions, only about 10 % of the cells have the main fim promoter in the
ON-orientation, therefore, most cells in the population are afimbriated under
these conditions.
30
Fig. 8. The genetic organization of the fim determinant of E. coli. The genes encoding the
different subunits of the fimbriae are color-coded to match the fimbriae illustration shown to the
right of the figure.
The phase variation event is further affected by DNA-binding factors as H-NS
(heat-stable nucleoid-structuring protein), IHF (Integration host-factor) and
Lrp (Leucine-responsive protein) (18, 51, 57, 59, 94, 125, 129). The expression
of type 1 fimbriae is regulated in response to environmental stress conditions
such as high osmolarity, pH and temperature, and expression is induced upon
entry into stationary phase (49, 57, 160). The environmental-responsive
regulation is a consequence of both regulation of the expression of the
recombinases and by directly influencing the phase variation event (Fig. 9).
As previously indicated, fimB and fimE are transcribed by their own separate
promoters and several factors regulate the expression of these factors (Fig.
9). Interestingly, the number of factors described to affect expression of fimB
is much higher than the numbers of factors affecting fimE. Transcription of
fimB is regulated by the alarmone ppGpp, the alternative σ-factor RpoS, the
heat-stable nucleoid-structuring protein H-NS, the sialic acid (Neu5Ac)
regulator NanR and the GlcNAc-6P-responsive regulator NagC (1, 47, 49, 163,
164). The expression of fimE is orientation dependent, meaning that it is
expressed when the fim switch is in the ON-orientation (102). In addition,
fimE is also negatively regulated by the global regulator H-NS (128, 129).
31
32
Fig. 9. Summary of the factors involved in regulating the expression of type 1 fimbriae. The expression of type 1 fimbriae is regulated at two
levels, promoter activity (main promoter located upstream of the fim operon) or by phase variation (invertible region between arrowheads). The
phase variation event is mediated by the recombinases FimB and FimE and further fine-tuned by the binding of accessory factors like Lrp and
IHF. The expression of FimB and FimE is affected by several TFs and this in turn will have an effect on phase variation. There are several
environmental conditions that have been suggested to influence the expression of the fimbriae.
4.2.3
Biofilm formation
A biofilm is an organized community of microorganisms (e.g. bacteria,
archaea, protozoa, fungi and algae) covered by a protective and adhesive
matrix. Biofilms are usually found on solid substrates but can also form
floating mats on liquid surfaces. Biofilms are commonly found on rocks and
pebbles at the bottom of most water-streams where they serve as an
important component in the food-chains. In recent years, it has been shown
that biofilms causes serious troubles in industry by clogging pipes and causing
corrosion, in food industry and in water distribution systems. From a medical
perspective, biofilms formed on catheters and implants might be the origin of
severe diseases (75, 199, 212). Another relevant feature of the biofilms is the
observed increased resistance to: antibiotics, environmental stress and host
defenses (3, 48, 77). Bacteria living in a biofilm usually have different
properties from free-floating (planktonic) bacteria of the same species, as has
been shown by different approaches, including microarray analysis studies
(46, 158).
Fig. 10. The different steps and factors involved in biofilm formation of E. coli. Adapted from
(168, 194).
In E. coli, biofilm formation proceeds in a biological circle consisting of five
steps (see Fig. 10):
1- Initial adhesion by planktonic (free-swimming) cells to a surface.
2- Transition to a irreversible state known as surface attachment.
3- Microcolony formation, early development of the biofilm consisting of
a few layers of cells.
4- Maturation of the biofilm with the characteristic pedestal formations.
5- Dispersion of the cells into the surrounding environment i.e. return to
the planktonic state.
33
The initiation of a biofilm is regulated by environmental signals as nutrients,
temperature, osmolarity, pH, iron and oxygen (126, 168). Nutrient availability
also influences the thickness of the biofilm and dispersion (168). Therefore, it
can be proposed that the starvation response pathway plays an overall role in
the control of biofilm formation.
Several surface structures of E. coli contribute in biofilm formation ((194) and
Fig 10). Flagella and motility have been shown to promote cell-to-surface
contact and promote the bacterial spread along surfaces and in dispersal from
the biofilm (144). Type 1 fimbriae are critical for the initial stable cell-tosurface attachment in a range of different media and biofilm growing systems
(15, 144, 148, 158). Ag43, a self-recognizing adhesin mediating autoaggregation, induces microcolony formation (42, 97). Both type 1 fimbriae and
Ag43 has been shown to play a role in the intracellular bacterial communities
formed inside the uroepithelium during urinary tract infection (4, 207).
However, expression is controlled so that co-expression is likely to be
mutually exclusive (159). Curli are heteropolymetric proteinaceous
filamentous appendages that influence adherence properties of biofilmforming E. coli (127). Over-expression of the curli subunit, CsgA, resulted in a
constitutive biofilm forming phenotype of the cells (202) with the
development of a mature biofilm with mushroom shaped pillars and medium
filled channels (147). The extracellular polysaccharide produced by E. coli
necessary for the establishment of the complex three dimensional structure
and depth of the biofilm (43).
34
Aims of this thesis
The main aim of my thesis work was to elucidate the role of
small regulatory molecules in transcriptional regulation of genes
in E. coli, in particular in control of genes involved in
colonization and survival of uropathogenic E. coli strains.
Specific aims:
• To determine whether ppGpp regulatory networks are
involved in biofilm formation and type 1 fimbriae expression
(Paper I).
• To establish the regulatory mechanism of ppGpp-mediated
regulation of type 1 fimbriae (Paper I).
• To resolve the molecular mechanism of how ppGpp
stimulates the expression of the fimB gene (Paper II).
• To investigate the participation of DksA in the ppGppmediated regulation of type 1 fimbriation (Paper II).
• To clarify the discrepancy observed between the ppGppand DksA-mediated regulation of fimB (Paper II).
• To elucidate similarities and differences in the global
transcription pattern of ppGpp- and DksA-deficient strains of
E. coli (Paper II & III).
Science never solves a problem without creating ten more
George Bernard Shaw (1856-1950) Irish Dramatist
35
Result and Discussion
In this section I present and discuss the main results obtained during my thesis
work. Data presented in the Papers of the thesis are referred to by their
roman letters (I-III) and corresponding figure numbers.
5.
Global effect of ppGpp and/or DksA on gene
expression in E. coli
A commonly used strategy to elucidate whether a specific gene is under the
control of a particular regulatory factor is to determine the expression profile
in cells deficient for that regulatory factor (mutants). In order to analyze
redistribution of the global gene expression patterns in E. coli mutants’
deficient for the global transcriptional regulators ppGpp and DksA, we used a
transcriptomic approach. The previously described identical and opposing
effects of ppGpp and DksA on gene expression (2, 110, 135) primed us to
analyze and compare the difference in gene expression pattern in single
ppGpp0 and ΔdksA mutant strains as well as in the double ppGpp0 ΔdksA
mutant strain. Transcriptomic analyses of the effects of ppGpp on gene
expression in E. coli and other species have been performed previously, but
most of those studies were performed under growth conditions where the
levels of ppGpp were induced by amino acid starvation (RelA-dependent
induction) (24, 50, 53, 145). Our work presumably represents the first study of
E. coli K-12 gene expression in a ppGpp0 compared to a Wt strain under
conditions commonly used to study general effects by ppGpp, i.e. entry into
stationary phase after growth in LB-media. Moreover, the gene expression
profile of DksA- and ppGpp/DksA-deficient strains was also analyzed.
We could observe an extensive rearrangement of gene expression in all three
mutant strains (ppGpp0, ΔdksA and ppGpp0 ΔdksA) as compared to the Wt
strain. About 20 % of all ORFs represented in the microarray were found to be
significantly altered in the different mutants. The analysis focused on ORFs
whose mRNA levels were altered more than 2-fold in the mutant strains as
compared to the Wt. Among these, about 7 % of the total numbers of ORFs
were affected in the single mutant strains and about 13 % in the double
mutant strain (Paper III, Fig. 1A-B). This is consistent with previous work by
others obtained from transcriptomic studies analyzing the effect of ppGpp and
DksA on gene expression in E. coli and Salmonella (50, 139, 161, 182).
Interestingly, considering the known co-dependent effect of ppGpp and DksA
on gene expression very few ORFs were found to be altered in all three
mutant strains as can readily be visualized when the array data was plotted in
Venn diagrams (Paper III, Fig. 1A-B, Fig. 3).
36
5.1
Effect of ppGpp and DksA on bacterial cell
physiology
In order to get an overview of the role of ppGpp and DksA in bacterial cell
physiology, the ORFs with >2-fold altered expression were organized into
categories according to the function of the protein that they encode. From
this analysis, it was evident that both ppGpp and DksA repress expression of
ORFs that encode proteins involved in transcription, purine and pyrimidine
synthesis, protein synthesis, protein fate and cell envelope biogenesis, while
stimulating expression of ORFs that encode proteins involved in energy
metabolism, transport/binding, central intermediary metabolism and amino
acid biosynthesis (Paper III, Fig. 1C and Table 2). All these alterations in gene
expression will result in a decrease of the rate of translation and change in
energy metabolism, thereby preparing the cell for adaptation to the slower
growth condition, and are in full agreement with previous reports describing
the general effect of ppGpp and DksA on bacterial physiology (33, 50, 139,
161, 182).
Interestingly, in most of the functional groups, the number of ORFs affected
in the double mutant strain (ppGpp0 ΔdksA) was higher that the number of
ORFs altered in each single mutant strain (Paper III, Fig. 1C). To rationalize
these observations, two explanations might be proposed. Firstly, some ORFs
are slightly affected in the single mutant strains (less than 2-fold) but are
synergistically affected by the absence of both factors. Secondly, ORFs that
are independently regulated by ppGpp or DksA, by one but not the other,
would also be found among the genes affected in the double mutant strain.
When analyzing the expression levels of the different ORFs altered in the
double mutant strain, we found ORFs whose expression pattern exemplifies
both of these proposed explanations (Paper III, Table 3 and S1).
5.2
Effect of ppGpp and DksA on transcriptional
repression and stimulation
To our surprise the diversity of expression patterns when looking at the
different ORFs was large, suggesting a remarkable complexity of the effect of
ppGpp and DksA on transcription. This was further exemplified when analyzing
the number of up- versus down-regulated ORFs in the different mutant strains
(Paper III, Fig. 3A-B). The result showed that; i) a larger number of genes
were affected in the double mutant strain than the single mutants; ii) the
number of ORFs affected in all three mutant backgrounds was very limited;
and iii) an surprising number of ORFs were only affected in either the ppGppor DksA-deficient strains. A significantly higher number of ORFs were found to
be up-regulated in the DksA-deficient strain as compared to the number of
down-regulated ORFs. These results might suggest that DksA play a more
prominent role in repression than in stimulation of gene expression. As will be
37
discussed later, up-regulation of gene expression in the DksA-deficient strain
does not always imply that DksA has a repressing effect on transcription. It
could also be explained by an increased availability of the secondary channel
of RNAP due to the physical lack of DksA (Paper II, Fig. 8 and Paper III, Fig. 7).
The differences in the numbers of up- and down-regulated ORFs and
differential regulation of individual ORFs suggested that the mechanism of
repression and stimulation might be diverse and that the requirement for
ppGpp and DksA in these processes might be dissimilar.
6.
Involvement of ppGpp in biofilm formation
by commensal and pathogenic E. coli
The establishment of biofilm communities is a multi-factor process in which
the commitment to grow as a biofilm can be instigated in response to several
environmental inputs and physiological stresses (48, 93, 168). However, which
regulatory factors are involved in transduction of these signals is not fully
understood. Recently it was shown that uropathogenic E. coli form complex
bacterial communities with biofilm-like traits during intracellular growth in
umbrella cells of the bladder, and therefore that biofilm formation is likely a
key feature during its pathogenesis (4, 90). The involvement of ppGpp in
regulating biofilm formation and survival has previously been suggested for E.
coli K-12 strains (10).
To study the involvement of ppGpp in biofilm formation, mutations of the two
ppGpp synthetases, RelA and SpoT, were introduced into two uropathogenic
E. coli isolates, J96 and 536. We found that the biofilm-forming abilities of
J96 and 536 were severally affected in the absence of ppGpp (Paper I, Fig.
1B). Very little biofilm formation was observed in the ppGpp0 derivatives. A
similar result was observed when using a non-pathogenic K-12 strain (Paper I,
Fig. 1C).
The next obvious question was at what level does the regulation occur? There
are several factors important for the biofilm formation in E. coli (Fig. 10). By
blocking type 1 fimbriae mediated adhesion by addition of mannosides, the
biofilm formation of the UPEC strains and K-12 strain were diminished,
illustrating the importance of the type 1 fimbriae on the biofilm formation of
these strains (Fig. 10 and Paper I, Fig. 1B-C). In the presence of mannosides,
no difference on biofilm formation was observed between the Wt and ppGpp0
derivatives, suggesting that ppGpp regulates the expression of the type 1
fimbriae. This was corroborated by analyzing a fimH mutant strain (lacking
type 1 adhesin), again no difference between the Wt and the ppGpp0
derivatives were observed (Paper I, Fig. 1C). Furthermore, when
phenotypically analyzing the expression of type 1 fimbriae in both UPEC and
K-12 strains by mannose-sensitive yeast agglutination (MSYA), a distinct
38
difference was observed, illustrating that the ppGpp0 derivatives did not
express any fimbriae on their surface.
6.1
ppGpp regulates the expression of type 1 fimbriae
at the phase variation level
It was of interest to investigate at what level the type 1 fimbriae system was
regulated by ppGpp. Transcription of the polycistronic fim operon, encoding
for the synthesis of type 1 fimbriae, is regulated by both phase variation and
by promoter activity (Figs 8-9). The phase variation is catalyzed by two sitespecific recombinases, FimB and FimE. Type 1 fimbriae synthesis is regulated
in response to environmental stress conditions such as high osmolarity, pH and
temperature, and expression is induced upon entry into stationary phase (49,
57, 160). The environmental regulation is consequence of both regulation of
the expression of the recombinases and by directly influencing the phase
variation event (Fig. 9).
The effect of ppGpp on fim expression was determined by transcriptional
studies using reporter fusions and mutants for ppGpp synthesis. A PCR-based
method was used to determine the phase variation state of the fim promoter
region. The transcriptional profile of fimA-lacZ (major subunit of the
structural genes) showed maximal expression during entry into stationary
phase (Paper I, Fig. 2A), where it has been reported that levels of ppGpp are
highest during growth in LB-media (9, 185). Expression of the fimA-lacZ fusion
was severely down-regulated in the ppGpp-deficient strain (Paper I, Fig. 2A).
Dissecting the mechanism for ppGpp-regulation on expression of type 1
fimbriae revealed that it operates exclusively through modification of the
phase variation state of the fim promoter region (Paper I, Fig. 3). First, in the
absence of phase variation, ppGpp has very little effect on transcription from
the fim promoter per se, but a phase variable strain that lacked ppGpp had
>8-fold reduced expression of a fimA-lacZ reporter (Paper I, Fig. 2 and Table
1). Second, a phase variable strain lacking ppGpp had a reduced population of
cells with the fim promoter in the ON orientation (ON-cell) as measured by a
PCR based approach (Paper I, Fig. 3). Third, a concomitant increase in the
number of ON-cells was observed by artificially increasing the ppGpp levels in
the cell (Paper I, Fig. 4F).
We could trace the effect of ppGpp on phase variation to the expression of
the fimB gene that encodes the OFF-to-ON FimB recombinase. The expression
of fimB was >3-fold higher in ppGpp proficient strains as compared to ppGppdeficient strains whereas the ON-to-OFF FimE recombinase was only slightly
affected (Paper I, Table 2). Furthermore, increasing the intracellular levels of
ppGpp had an immediate effect on fimB expression. In further support,
artificial elevation of FimB levels bypasses the need for ppGpp in type 1
fimbriae expression and biofilm formation (Paper I, Fig. 4G and data not
shown).
39
6.2
ppGpp regulation of factors important for biofilm
formation
Type 1 fimbria is not the only surface structure that is important for biofilm
formation in E. coli. Flagella, Ag43 and curli are others envelope structures
that are important at different steps in the biofilm formation process ((194)
and Fig. 10).
Ag43 is a self-recognizing adhesin that causes bacterial cells aggregation in
liquid culture when highly expressed (96, 131). Magnusson et al showed that
the auto-aggregation phenotype of E. coli was decreased in ppGpp0 strains
and furthermore, the Ag43 protein level was also reduced in this strain (110).
Similarly, an effect of ppGpp on ag43 expression was observed in our
transcriptomic study (data not shown), with a clear decrease of ag43
transcript in the ppGpp0 strain as compared to the Wt. Ag43, as for type 1
fimbriae, are regulated both by phase variation and at the promoter activity
level by several regulators and environmental conditions (73, 190). Results in
our research group indicate that phase variation of Ag43 is strongly affected
by ppGpp (J.D. Cabrer et al, unpublished data).
The flagella system consists of numerous operons harbouring several openreading frames. The operons are expressed in a temporal order and are
divided in early, middle and late operons (36). The expression of the middle
ORFs are regulated by the FlhDC regulators and σ70-RNAP holoenzyme while
the late ORFs are regulated by FlgM anti-sigma factor and σF(σFliA)-RNAP
holoenzyme. Contradictory results of the effect of ppGpp on flagella
expression have been published (50, 110). Magnusson et al also showed that
the ppGpp-deficient strain was deficient in motility on plates (110).
Conversely, Dufree et al showed that the flagella and chemotaxis genes were
down-regulated in presence of high levels of ppGpp (50). They also suggested
that ppGpp/DksA directly regulate expression of FlhDC (master regulators of
middle genes). There are some major differences in the experimental set up’s
that could in part be the explanation for the differences in the results
obtained.
Our experimental results showed that ppGpp act as a positive regulator of the
transcription of the genes involved in flagella biosynthesis and chemotaxis
(Paper III, Fig. 5A). Furthermore, we could observe a clear down-regulation of
FliC (major subunit) and FliA (σF, regulating the late ORFs) and several other
flagella ORFs in the ppGpp0 strains as compared to the Wt (Paper III, Figs 5-6).
Our data indicated that ppGpp primarily regulates the early and middle
operons (Paper III, Fig. 5), i.e. ORFs that are transcribed by σ70-RNAP
holoenzyme. The studies performed by Dufree et al were performed during
amino acid starvation conditions in mid-log phase of growth. Furthermore,
Durfee et al did not use a ppGpp-deficient strain and the studies followed the
expression during 30 minutes after inducing high levels of ppGpp by amino
40
acid starvation (50). It might be that under these conditions, some additional
regulator is expressed resulting in the differential effects observed. It remains
to be elucidated whether the effect we observe on flagella expression in the
ppGpp-deficient strain is direct or indirect, and at what level it occurs.
Metabolic stresses and many environmental conditions that reduce bacterial
growth are cues for rapid induction of the intracellular levels of ppGpp (33).
During maturation of the intracellular bacterial communities of uropathogenic
E. coli, it was described that after an initial fast growing phase, a significant
decrease in the growth rate occurs with the concomitant appearance of
biofilm traits (90). Our results provide a possible regulatory mechanism for
the changes in the expression pattern during maturation of the intracellular E.
coli communities. Under this scenario, reduced growth rate would presumably
induce elevated levels of ppGpp, which in turn would stimulate the expression
of type 1 fimbriae, Ag43 and flagella, and trigger the successful establishment
of an intracellular biofilm-like community.
Nevertheless, knowing that all factors can not be expressed at the same time
shows the importance of additional level of regulation of expression. Both the
fim operon and ag43 are regulated by phase variation while flagella operons
are temporally regulated. A reciprocal regulation of motility, adhesion and
aggregation is logical and cross-regulation between the fimbrial gene products
and both flagella genes and ag43 has been shown (27, 37, 72). This suggests
that very rarely will there be a bacterial cell in a population expressing all
three factors simultaneously. This illustrates one example where having a
population of bacteria expressing the same genes/proteins is not always
beneficial for the survival.
7.
Divergent effects by ppGpp and DksA on
type 1 fimbriae and flagella in vivo
During the analysis of the array data, a number of the ORFs that were
differentially altered in at least two of the three mutant strains as compared
to the Wt were identified (i.e. up-regulated in one and down-regulated in the
other). About 10-20 % of the ORFs affected in the three mutant strains fall
into this group that are differentially by ppGpp and DksA in vivo. These ORFs
were grouped into different classes depending on their pattern of expression
in the different mutant strains (Paper III, Fig. 4). There were very few ORFs
that were repressed by ppGpp (i.e. up-regulated in the ppGpp0 strain) and
stimulated by DksA (i.e. down-regulated in the ΔdksA strain). Most of the
differentially affected ORFs were activated by ppGpp (i.e. down-regulated in
the ppGpp0 strain) and repressed by DksA (i.e. up-regulated in the ΔdksA
strain) (Paper III, Fig. 4). These results suggest that the concerted action by
ppGpp and DksA seems to depend on whether a promoter is positively or
negatively regulated by ppGpp. Many of the ORFs differentially regulated by
41
ppGpp and DksA (Paper III, Fig. 4B) encode proteins involved in type 1
fimbriae expression, flagella biosynthesis and chemotaxis. The differential
effect of ppGpp and DksA on these genetic determinants was further
analyzed.
7.1
Effect of DksA-deficiency on type 1 fimbriae and
flagella expression
The observed effect of ppGpp on biofilm formation and the discovery of DksA
as a possible co-regulator of ppGpp-mediated regulation prompt us to analyze
the involvement of DksA in biofilm formation. In contrast to the ppGpp0
strain, the ΔdksA strain showed an increase in biofilm formation that could
effectively be abolished by the addition of mannosides (Paper II, Fig. 3C). This
suggests that the increase in biofilm formation was due to elevated levels of
type 1 fimbriae in the ΔdksA strain (Paper II, Fig. 3C). This was further
corroborated by analyzing the MSYA phenotype of the mutant strain which
showed elevated levels of type 1 fimbriae on the surface of the ΔdksA
mutant. The findings were also corroborated by studying the fimA-lacZ
reporter fusion. We concluded that in the ΔdksA strain there is an increase in
type 1 fimbriae expression by increasing the percentage of ON-cells (Paper II,
Fig. 3A-B). When dissecting the mechanism of hyperfimbriation in the DksAdeficient strains it was revealed that the observed effect of DksA on the phase
variation occurred by enhanced transcription of the fimB gene (Paper II, Fig.
3). Recently, the negative effect of DksA on type 1 fimbriation in E. coli was
also evidenced by MSYA and electron microscopy by Magnusson et al (110).
Similar to the type 1 fimbriae expression, genes of the flagella operons were
also shown to be differentially regulated by ppGpp and DksA in the array data
(Paper III, Fig. 5). The effect of DksA was confirmed by analyzing the protein
levels of FliC (major subunit) and FliA (σF, regulator). A clear up-regulation of
both proteins could be observed in the DksA-deficient strain (Paper III, Fig. 6).
Furthermore, many of the ORFs important for the chemotaxis response were
also up-regulated in the DksA-deficient strain. We tested the chemotactic
response of the three mutant strains to L-serine, maltose and glucose, as
compared to the Wt strain (Paper III, Fig. 5B). The DksA-deficient strains
clearly showed an increased chemotactic response as compared to the Wt,
whereas the ppGpp0 strain did not respond at all. At what level ppGpp and
DksA affect the expression of the flagella and chemotaxis operons is still not
known.
42
8.
Dissecting the mechanism of regulation of
fimB transcription by ppGpp and DksA
In order to elucidate the molecular mechanism behind the differential
regulation of ppGpp and DksA on the transcription of fimB, we dissected the
independent and co-dependent effects that these factors have on fimB
expression. It has been suggested that fimB transcription is controlled by
three promoters and that the expression is influenced by several regulatory
factors (Fig. 9), i.e. the alternative σ-factor RpoS, the heat-stable nucleoidstructuring protein H-NS, the sialic acid (Neu5Ac) regulator NanR and the
GlcNAc-6P-responsive regulator NagC (47, 49, 163, 164). Our results illustrate
that fimB is mainly transcribed from the P2 promoter (Paper I, Fig. S1) and
primer extension analysis showed that both ppGpp and DksA affect
transcription from the predominant P2 promoter both in the commensal and
pathogenic strain (Paper I, Fig. 4D-E and Paper II, Fig. 5C). Additionally, the
effect of ppGpp and DksA was determined in mutants of the previously known
regulators. However, none of those factors were found to be involved in the
ppGpp- or DksA-mediated regulation of fimB expression (Paper I, Table 2 and
Paper II, Table 2). Together, these results suggested that the effect by ppGpp
and DksA could occur either directly at the fimB promoter or through a so far
unknown factor.
8.1
Direct effects of ppGpp and DksA on transcription
from the fimB P2 promoter
To investigate the possible direct effects of ppGpp and DksA on fimB
transcription, DNA templates spanning the fimB P2 promoter were tested in a
reconstituted in vitro transcription system. In vitro transcription is an
approach used to determine direct effects of regulatory factors on
transcription. In this assay, transcription is quantified after combining the
specific template DNA with RNAP holoenzyme and the regulators to be tested.
By varying the concentration of the components and the timing or the order of
addition of the different components it is possible to determine regulation at
different steps.
To elucidate whether ppGpp and DksA have a direct effect on fimB
transcription, multiple round in vitro transcription assays were performed in
the presence of σ70-RNAP holoenzyme and increasing concentrations of ppGpp
or DksA. Surprisingly, transcription from the fimB P2 promoter was stimulated
by the presence of either factor separately (Paper II, Fig. 6A-B) and an
additive effect on fimB P2 transcription could be observed in the presence of
both factors (Paper II, Fig. 6C). These results contrast the observed negative
effect of DksA in vivo and the proposed concerted action of ppGpp and DksA
on transcription (Paper II, Fig. 1C).
43
To further test the idea that ppGpp can stimulate fimB transcription
independently of DksA in vivo, ppGpp levels were increased by inducing a
plasmid expressing a truncated version of RelA. This allele do not require the
association to a stalled ribosome to induce the synthesis of ppGpp (177). The
expression of fimB was increased both in presence and absence of DksA as
measured by the expression of a chromosomal fimB-lacZ allele in a ppGpp0
strain (Paper II, Fig. 8C). In agreement with the in vitro data, the increase
was not as extensive in the absence of DksA, indicating that for full expression
of fimB both ppGpp and DksA are required, although ppGpp can
independently stimulate transcription both in vivo and in vitro. The in vitro
and in vivo data suggested that ppGpp and DksA have both independent and
co-dependent effects on fimB transcription.
8.2
Co-dependent versus independent
ppGpp and DksA on fimB transcription
effects
of
The apparent co-dependent and independent effects of ppGpp and DksA on
transcription of fimB in vivo and in vitro prompted us to try and dissect these
different effects in vitro. This involved analysis of the effects of ppGpp and
DksA on the association of RNAP to the promoter DNA, on open-complex
stability, and on the rate of formation of full-length transcript from the fimB
P2 promoter.
DksA has been shown to facilitate the binding of RNAP to promoter DNA in
vitro (139, 143). To explore if such a mechanism could explain the stimulatory
effect of DksA on fimB transcription, we performed gel-shift assays with a
DNA template containing the fimB P2 promoter, a constant amount of RNAP
and increasing concentrations of DksA (Paper II, Fig. 7A). DksA dramatically
enhanced the ability of RNAP to bind to the fimB P2 promoter. On the other
hand, ppGpp did not show any effect on the binding of RNAP to the promoter
DNA (Paper II, Fig. S1).
The molecular mechanism(s) by which ppGpp and DksA have their codependent effects on transcription from σ70-promoters are still not very well
known. One feature commonly studied is the concerted action of ppGpp and
DksA on destabilizing open-complexes promoters (134, 135). For promoters
with very unstable open-complexes (e.g. rrn) this destabilization will result in
repression of transcription, while for promoters with very stable opencomplexes this destabilization might help RNAP escape the promoter and
consequently promote productive transcription (14). The fimB P2 promoter
forms very stable complexes (Paper II, Fig. 7B) and, ppGpp and DksA did not
show any effect independently on the open-complex stability. However,
together they decrease the half-life from >200 min to ~30 min (Paper II, Fig.
7B).
44
The rate of formation of full-length transcripts was tested in an in vitro
transcription assay, since it has been postulated that ppGpp might have an
effect on abortive initiation, promoter escape and transcriptional pausing (14,
22, 101). Interestingly, ppGpp showed a repressive effect on the rate of
formation of full-length transcripts both in presence and absence of DksA
(Paper II, Fig. 7C-D, 8D-E). Despite the repression of the rate of formation of
full-length transcripts, the maximal amount of transcript from the fimB P2
promoter was increased in the presence of ppGpp and even further increased
when both regulatory molecules were provided simultaneously.
Taken together, these in vitro experiments illustrated that ppGpp and DksA
have independent and co-dependent effects on multiple steps of transcription
from the fimB promoter, namely i) a ppGpp-independent effect of DksA on
RNAP-DNA complex formation, ii) co-dependent effects on open-complex
stability and iii) a DksA-independent effect of ppGpp on the rate of
accumulation of full-length transcripts, which might be consequence of
alterations in promoter escape, abortive initiation or elongation.
Notably, in no instance was DksA found to have a negative effect on fimB
transcription in vitro. These seemingly contradictory results for the role of
DksA on fimB transcription in vitro and in vivo could readily be explained if
another factor(s) are operational in absence of DksA in vivo.
9.
Involvement of Gre-factors in differential
regulation by ppGpp and DksA in vivo
A possible explanation for the paradoxical in vitro versus in vivo discrepancy
of the role of DksA in transcription from the fimB promoter might be that in
the absence of DksA, the regulatory activity of some additional factor(s) is
more efficient. DksA is a structural homologue to GreA and GreB, and all
three proteins exert their regulatory effects through the secondary channel of
the RNAP (19, 130, 137, 169, 178). The Gre-factors can reduce abortive
initiation, facilitate promoter escape, suppress elongation pausing and arrest
(19, 54, 80). We reasoned that the lack of DksA may facilitate interaction of
the Gre-factors with the RNAP, which in turn may specifically affect the
expression of some genes.
9.1
Gre-factors stimulate fimB transcription in vivo
and in vitro
In order to determine if the Gre-factors could be responsible for the increased
expression of fimB in the absence of DksA in vivo, we analyzed mutants for
the Gre-factors in E. coli, over-expressed the Gre-proteins in vivo and
performed in vitro transcription assays with purified GreA and GreB-His
proteins.
45
Mutations of the greA and greB were introduced into ppGpp- and/or DksAdeficient strains harbouring a fimB-lacZ reporter. Loss of GreA and GreB had
no effect in the DksA-proficient strains, however, in the absence of DksA the
loss of the Gre-factors counter-balance the observed increase of fimB
expression (Paper II, Fig. 8A). Similarly, when over-expressing the Gre-factors
in presence and absence of DksA a significant increase on fimB expression
could be observed in the absence of DksA, independently of the presence or
absence of ppGpp (Paper II, Fig. 8B).
The in vivo data suggest that the Gre-factors can stimulate the expression of
fimB transcription in the absence of DksA. Therefore, we tested if the Grefactor could relieve the ppGpp-mediated delay of the production of fulllength transcripts, since the Gre-factors have been suggested to have an
effect on abortive initiation, promoter escape, elongation pausing and arrest
(19, 54, 80). Addition of the Gre-factors to the in vitro transcription reaction
relieved the ppGpp-mediated delay, both in the presence and absence of DksA
suggesting that the Gre-factors regulate fimB transcription directly by
affecting the later stages of transcription initiation and/or elongation (Paper
II, Fig. 8D). The Gre-factors additionally increased the maximal amount of
full-length transcript produced from the fimB P2 promoter. The absolute
maximal levels of transcripts were observed in the presence of all four
factors, i.e. ppGpp, DksA, GreA and GreB (Paper II, Fig. 8E). This net effect
on transcription is most likely a combination of all their independent and codependent effects on the different steps of transcription initiation (Fig. 11).
9.2
Gre-factors are important for FliC expression in
vivo
To analyze if the expression profiles of the flagella and chemotaxis operons
observed in the DksA-deficient strain might, similarly to the fim operon, be a
consequence of the increased association of Gre-factors with the RNAP, the
effect of gre mutations on the expression of the flagella expression was
studied. The amount of FliC (flagella major subunit) was determined by
Western blot analysis of the different mutant strains (Paper III, Fig. 7).
Intriguingly, in the ΔgreA ΔgreB and the ΔdksA ΔgreA strains the amount of
FliC was severely down-regulated as compared to the Wt and ΔdksA strains,
suggesting the Gre-factors play an important role in the FliC expression even
in the presence of DksA in vivo. Whether this effect occurs directly at the fliC
promoter or indirectly by regulating the expression of another factor are
speculations that will require further experimentation to be corroborated.
The effect of the Gre-factors has also been determined for the argI
transcription (data not shown), a gene that is positively regulated by both
ppGpp and DksA (Paper II, Table 1). In this case, no effect of the Gre-factors
on the expression of this gene was observed either in the presence or in the
46
absence of DksA. This suggests that the Gre-factors do not always alter the
expression of promoters that are under control of DksA.
9.3
Competition for the secondary channel of RNAP
Notably, our studies suggest that the Gre-factors might have different role in
regulation of ppGpp and/or DksA dependent genes in vivo depending on the
promoter. For fimB transcription, the Gre-factors only have an effect in vivo
in the absence of DksA (Fig. 11 and Paper II), for FliC the Gre-factors affect
the expression even in the presence of DksA (Paper III), while for argI the Grefactors are dispensable (Paper II, data not shown).
The intracellular concentrations of DksA, GreA and GreB, and their predicted
affinities for the RNAP has suggest that most RNAP would be occupied by DksA
rather than GreA and GreB (154). However, whether this scenario is true for
all growth conditions remain to be examined. Data has been presented
indicating that Gfh1 (belonging to the Gre-family) exhibits a pH-dependent
switch between an active and inactive conformation (105). If this is also the
case for the other Gre-family proteins needs to be further studied. I consider
that our data suggest that there can be conditions where the affinity of the
secondary channel factors could change to override their relative abundance.
It is also plausible that interactions of the RNAP to a specific promoter
sequence or with a particular σ-factor might alter the conformation of the
secondary channel of the RNAP thereby changing the affinity of the RNAP for
the different secondary channel TFs.
Fig. 11. Regulatory network controlling transcriptional regulation of the fimB promoter.
Schematic illustration of the fimB regulatory region is shown with DNA-binding TFs H-NS, NanR
and NagC depicted below the DNA and non-DNA-binding regulatory molecules that directly target
RNAP (αββ’ωσ) depicted above the DNA; ppGpp as dots, DksA as red circles, GreA as orange
diamonds and GreB as red diamonds. Stimulatory effects on transcription from the fimB promoter
are exemplified as arrow-heads whereas repressive effects are indicated by dash-ended lines.
Note, while DksA stimulates transcription, its absence would allow greater access of the Grefactors to RNAP, which would also have stimulatory effects. Adapted from (Paper II, Fig. 9).
47
Concluding Remarks
The data presented in this thesis highlights new insights into gene regulation
by ppGpp and DksA. Early studies have mostly implicated ppGpp in regulating
house-keeping genes that will slow-down the translation machinery during
amino acid starvation conditions (stringent response). A growing number of
studies have showed that intracellular levels of ppGpp are likely to be induced
in bacteria in response to any environmental stress condition that results in
slow proliferation. Furthermore, it is now established that ppGpp also plays a
central role in regulating genes involved in virulence and survival in the face
of host induced stress.
For many years it was a struggle to corroborate direct effects of ppGpp on
gene regulation in vitro. Although, genetic, cross-linking, and cocrystallization studies suggested that ppGpp interacts directly with the β- and
β’-subunits of the RNAP within the active site cleft, very little or no effect of
ppGpp could be observed in vitro compared to the major effects observed in
vivo. It was suggested that DksA was the missing co-factor for ppGppdependent regulation. However, as shown in this thesis as well as by others,
there are a number of genes regulated only by one of these factors, strongly
suggesting that concerted action by ppGpp and DksA is not true for all genes
regulated by ppGpp.
The discovery of the involvement of the Gre-factors in the ppGpp-mediated
regulation introduces a new perspective to the importance of the secondary
channel of the RNAP in gene regulation. It also illustrates a new dynamic view
of the RNAP conformation that might change by association to different
transcription factors consequently altering expression from certain susceptible
promoters. It is still not known if the interaction of one factor with RNAP can
affect the binding of another, or for how long one factor remains associated
with RNAP. Nevertheless, it is plausible to speculate that there might be an
active exchange of RNAP binding factors through out the transcription
process.
I propose that we are moving towards a new model of ppGpp-mediated
regulation, which is far from as fully elucidated as one might hope after
almost 40 years of research.
Ju mer man tänker, ju mer inser man att det inte finns något enkelt svar
Nalle Puh/ A.A. Milne
48
Acknowledgements
First, making sure that I will not forget anyone, I would like to say thanks to
everyone that have supported and helped me during these 6 years of PhD
studies at the Department of Molecular Biology, both within and outside of
work.
Enormous gratitude goes to my “never-ending” smiling supervisor Carlos (the
man with the voice), without you I would never have managed to get this far,
your huge support and sometimes crazy ideas have made working with you a
pleasure, I also want to thank you for being a good friend also outside work.
To the people in Microbiology department in Barcelona; Juanda, Jorge (the
two Spanish guys that have survived long visits in Umeå, thank you for the EM
pictures that made the cover of my thesis look beautiful), Núria, Aitziber,
Christina (thanks to you 3 for all the help with the microarray analysis), Mari
(most happy and crazy person I have ever met) and the others; big thanks to
you for giving me a nice time during my trips to Spain.
In the department I would like to start to say thanks to Vicky for your
tremendous help and support, for always trying your best to answer all of my
questions and teaching me so much, thanks to you I finally understood what
all of my result meant. Big thanks also go to your group members who have
taken me in as a “member”; to Linda and Pelle (kommer aldrig glömma våra
champagne-kvällar), Lisandro (for being such a good friend), Ellinor and the
others. I want to thank past and present members of the Friday journal club
and people whom I have shared lab with during these years; Bernt Eric (my
second supervisor) and Sun, thank you for allowing me to participate in many
activities and the nice gatherings at your house; to Annika (for your
wonderful book-reviews and stories of your night-dreams and for being who
you are), Stina (for your smile), Jurate (for the nice dinners and “pyssel” at
your place), Claudia (for following me to learn Salsa, among other things) and
Monica, thank you for the trips and all fun stuff that we have been doing
inside and outside the lab, you all mean a lot to me; Emma (for being my
training partner and a good friend), Anna R (for bringing a breath of fresh air
to the lab), Connie, Hyun-Sook, Barbro, Karolis, Tianyan, Takahiko, Soni,
Pramod, Berit (for still sharing office with me), Jörgen, Edmund (maybe one
day you will hear me sing), Jonas, Jonny, Teresa, Sakura etc. To the people
in SvB group; Sven, Christer, Pär, Marie, Marija, Yngve, Ingela, Lelle, Betty,
etc., for good times on trips to Germany and Japan, and for all the fun things
that would never had happened at the department without you. I want thank
the people of the “bunker/beer corner”; Jenny, Maria N, Petra E, Sofia, Sara
C, Micke, Stefan, Jocke, Giovanni, Sara R, etc; and to the secretaries
Marita, Berit, Helena and Ethel, to Jonny, to Siv, to Media, and to
Marianella, without you the department would not function properly.
49
Jag vill också tacka mina släktingar och mina vänner som stöttat mig och
försökt förstå vad det egentligen är jag har pysslat med på dagarna under
dessa år; Petra och Helen med familjer, vi har hållt ihop länge nu och det
kommer blir många år till, TACK för att ni finns. Alla er i Sävar med familjer;
Jesper, Anette och Björn, Susanne och Hans, Sussie, vi har haft mycket
roligt ihop under åren. Kusin Maria, du är underbar, allt stöd du har gett
under det senaste året är helt otroligt, jag lovar att det kommer vara mera
glada smilisar hädanefter ☺ ☺ ☺.
Dessan, min vän, du kommer för alltid stå mig otroligt nära, ditt stöd och din
vänskap har hjälp mig otroligt mycket genom åren, mer än jag kan beskriva i
ord. TACK och åter TACK för att du finns, ÄLSKAR DIG, och din underbara
familj, Maja (vi ska äta popcorn snart), Filip (längtar att få se dig gå) och
Anders.
Jag vill avsluta med att tacka min familj så otroligt mycket, Mamma, ”Storebror” Robert och Pappa (saknar dig, hoppas du har det bra där uppe), ni
betyder allt för mig, utan er skulle jag inte vara någonting, ÄLSKAR ER.
50
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