Download Some Types of Bacterial Phase Variation Are Epigenetic

Some Types of Bacterial Phase
Variation Are Epigenetic
Methylating DNA sites can control phase variation, enabling bacteria
to inherit a gene expression state that remains reversible
Marjan W. van der Woude
n so many bacterial experiments, assuming that all the individual cells of Escherichia coli or other bacterial species in a
particular flask are identical in terms of
genotype and phenotype is reasonable
and usually works well. Indeed, accepting this
premise led several generations of microbiologists to study many microbiological properties
and to uncover important biological principles,
including metabolic pathways, the basics of nucleic acids and genes, and many mechanisms
underlying their regulation and DNA replication.
However, individual cells can vary phenotypically, even within a clonal population. Indeed,
I
Summary
• Individual bacterial cells can vary phenotypically, even within a clonal population.
• Phase variation provides a means for regulating
specific genes between an expressed, or “on”
state, and a nonexpressed, or “off” state; these
two expression states are heritable and reversible.
• The mechanisms that lead to phase variation are
either genetic or epigenetic; if epigenetic, they
entail no changes in DNA sequence.
• More generally, phase variation apparently provides a means for generating diversity and
thereby increasing the chances of survival amid
changing environmental conditions.
• Understanding phase variation may allow us to
manipulate bacterial populations to provide
health benefits, including better diagnostic tests
and vaccines.
F. W. Andrewes of St. Bartholomew’s Hospital
in London, England, observed morphology variation between colonies of a Salmonella strain as
early as 1922. Those differences later proved to
be due to antigenic variation of the flagellum.
The biological significance and mechanisms underlying this and other heterogeneities within
particular bacterial populations are important
features that microbiologists continue to study.
Population Heterogeneity
and Phase Variation
The phenomenon described by Andrewes consists of variation in the antigenic properties of a
particular protein. Although all cells in a population will express the protein, in some of
those cells it has a different antigenic form.
A second, related phenomenon that also
generates heterogeneous phenotypes within
a population is known as “phase variation.”
This term describes a means for regulating
genes that can switch a specific gene between an expressed, or “on” state (blue in
Fig 1), and a nonexpressed, or “off” state
(white in Fig 1). The expression state is
heritable, yet reversible.
Thus, cells in the “on” phase give rise to
progeny that predominantly express that
particular gene with only a minority that are
switched “off” and do not express that
gene, and vice versa. Therefore, phase variation results in a population of clonal cells
that are not identical, yet can revert to each
other’s phenotype (Fig. 1). The frequency of
this switch can be as high as 1 per 10 cells
per generation or as low as 1 per 10,000
cells. The latter frequency is low enough
Marjan W. van der
Woude is Senior
Lecturer in Microbiology in the Department of Biology
and the Hull York
Medical School,
University of York,
United Kingdom.
Volume 3, Number 1, 2008 / Microbe Y 21
FIGURE 1
that multiple generations are required to see
changes in phenotype in the absence of selective pressures. However, this frequency is
still more than 100 times higher than spontaneous mutation rates.
The higher frequency and, equally importantly, the reversible nature of phase variation distinguish this hereditary change in
gene expression from classical mutations.
Furthermore, the molecular mechanisms
leading to phase variation are distinct from
those causing mutations.
Molecular Mechanisms of Phase
Variation May Be Genetic or Epigenetic
Phase variation is a heritable yet reversible form of regulation and results in a
heterogenous population. In the absence of selective pressures the two phenotypes will coexist in a population. (A) What can happen after the expression state
of one of two daughter cells of an “off “cell has switched, and both grow to a
colony. “On” cells are depicted blue and “off” cells white. (B) Colonies from a
streak of a single white colony of a strain in which the agn43 phase-varying
promoter drives the lacZ reporter fusion. Colonies with predominantly “on” cells
are blue, and colonies with predominantly “off” cells are white on this medium
containing the substrate Xgal for LacZ activity. (C) Typical result of streaking an
“on” (blue) colony and an “off” (white) colony for single colonies. The plates
clearly illustrate the bias in progeny phenotype as a result of parental phenotype.
The number of generations during which this bias exists depends on the frequency
of switching.
22 Y Microbe / Volume 3, Number 1, 2008
The mechanisms that lead to phase variation
are classified either as genetic or epigenetic. If
genetic, changes in DNA sequence account
for differences in expression state between
cells in the “on” and “off” phase. Such genetic changes may involve different molecular mechanisms (Table 1), including changes
in reading frame of the coding sequence
through slipped-strand mispairing or a flip in
orientation of a promoter relative to the transcribed gene through site-specific recombination. In each case the sequence change is small
and, importantly, reversible.
Meanwhile, epigenetic phase variation involves no change in DNA sequence but is
also reversible. Accounting for how a change
can be heritable but reversible in the absence
of DNA sequence changes has occupied several research labs for a number of years.
Collectively, those researchers learned that
differential methylation of only a few target
DNA sequences accounts for this phenomenon.
Specifically, deoxyadenosine methyltransferase (Dam) mediates phase variation to
control expression of some outer surface
structures in Escherichia coli. More generally, Dam is the main DNA methyltransferase of E. coli, and this enzyme modifies the
adenine residue of the 4-bp recognition sequence GATC. A few of the GATC sequences in the E. coli genome control phase
variation.
David Low, now at the University of California at Santa Barbara, uncovered DNA
methylation-dependent phase variation while
studying the pilus operon (pap) in E. coli. He
and his lab members determined that Dam
and the global regulator Lrp are key players
in regulating pap phase variation, along with
several other proteins. Other fimbriae are
regulated by homologous systems.
A selection of bacterial phase variation mechanisms and some
examples of species in which these occur
Phase variation
classification
Molecular Mechanism
Genetic
Epigenetic Phase Variation:
DNA Methylation-Dependent
Ag43 Phase Variation
Another example of Dam-dependent phase
variation is Ag43, an outer-membrane protein that is involved in biofilm formation.
Ag43 from E. coli K12 is being further studied to address questions regarding methylationdependent epigenetic regulation, including stochastic events.
Borge Diderichsen at CERIA in Belgium and
his collaborators recognized phase variation in
the Ag43-encoding gene in 1980, naming the
locus flu for “fluffing” because changes in Ag43
expression triggered cells to aggregate. They
also noted that flu appeared to be “metastable.”
Nearly two decades later, Ian Henderson, working with Peter Owen at Trinity College in Dublin, Ireland, and now at the University of Birmingham in England, renamed the gene agn43
and showed that this phase variation requires
Dam and the regulatory protein OxyR.
A 76-bp stretch in the regulatory region of
agn43 contains three sequence elements that are
essential for Ag43 phase variation (Fig. 2A).
Ag43 phase variation is a result of regulated
transcription, and the first essential element is
the promoter, which is ␴70-dependent and has a
sequence that is very similar to a consensus
promoter region. Second, this 76-bp stretch contains three Dam target sequences, each consisting of 4 bp, GATC. Dam methylates the adenine
residue at these GATC sequences (Fig. 2). Third,
the 76-bp stretch contains a site to which the
protein OxyR binds; this global regulator
mainly coordinates expression of genes in response to oxidative stress. At agn43 the OxyR
binding site overlaps the three GATC sequences
as well as the -10 sequence of the promoter (Fig.
2); when OxyR binds, transcription is repressed.
The conservation of these three sequence components in an otherwise variable stretch of DNA
at agn43 genes of different E. coli isolates is
striking and suggests that phase variation is a
conserved feature.
Slipped-strand mispairing
Bacterial species
Haemophilus influenzae, Neisseria
meningitidis, Helicobacter pylori
Site specific recombination Escherichia coli, Bacteroides fragilis
Epigenetic
Recombination
Streptococcus pneumoniae,
Neisseria meningitidis
DNA methylation
Escherichia coli, Salmonella enterica
Phase variation occurs when OxyR binds
agn43 or if Dam gains access and methylates its
three unbound target GATC sequences within
this site (Fig 2B, top). In the absence of OxyR
binding, Dam methylates its target sequences,
and this methylation blocks subsequent OxyR
binding. OxyR can bind to unmethylated sites,
however, in turn blocking Dam access. Since
OxyR represses transcription of this gene,
agn43 expression is off when OxyR is bound. In
contrast, when the GATC sequences are methylated, that gene is expressed and such cells
produce Ag43.
After cells divide, daughter cells are biased to
the parental Ag43 expression state, reflecting
the inherited methylation state of the agn43
GATC sequences. Because switching appears to
be mainly stochastic, determining whether other
specific cellular events contribute to this infrequent event remains a challenge. E. coli cells
contain no enzyme to remove methyl groups
from their DNA. Thus, switching from “on” to
“off” depends on DNA synthesis during replication to generate unmethylated GATC sequences
(Fig. 2B). Dam acts on hemimethylated as well
as nonmethylated DNA, and hemimethylated
DNA in cells tends to become fully methylated.
However, OxyR can also bind to hemimethylated agn43 GATC sequences, blocking Dam
access, and would confer an “off” phenotype on
such cells (Fig 2B).
Changes in relative concentrations of Dam
and OxyR can bias the switch, according to
genetic experiments. Specifically, increasing
Dam or decreasing OxyR levels biases cells to
the “on” phase, and vice versa. Further, mutating the first GATC sequence, rendering this site
unmethylated, also biases cells to the “off”
Volume 3, Number 1, 2008 / Microbe Y 23
the “on” phase. The OxyR regulon mediates
a specific protective response to oxidative
stress, changing the oxidation state of OxyR
and its DNA binding recognition sequence.
This finding seems consistent with Ag43
phase variation helping cells to withstand
oxidative stress through inducing Ag43-dependent aggregation.
However, both oxidized and reduced OxyR
bind to the agn43 region in a DNA methylation-dependent manner, repressing agn43
transcription. Furthermore, applying oxidative stress to a culture does not increase the
percentage of Ag43 “on” cells. This absence
of environmental signaling from the OxyRdependent mechanism could mean that OxyR
was “hijacked” with Dam to generate constant phenotypic diversity.
Besides Dam and OxyR, no other transcription factors are required for regulating
agn43, while no environmental signaling alters agn43 phase variation. Thus, a random
event—phase variation— dictates whether individual cells produce Ag43.
FIGURE 2
ON
Special Significance of
Epigenetic Phase Variation
(A) agn43 regulatory region showing the sequence elements that are essential for
phase variation and their organization (not to scale). (B) Events during and after
DNA replication that contribute to a change or maintenance of the agn43 “on”
expression state. Competition between OxyR and Dam for free hemimethylated
DNA is a key event. Newly synthesized DNA that is unmethylated initially is
depicted in red. The black cross indicates that methylation by Dam or OxyR binding
can not occur. Other symbols are explained in the Key box.
phase, while mutating the second or third
GATC sequence locks cells in the “off” phase.
These data support the notion that Dam and
OxyR compete for the agn43 regulatory region
and that their relative concentrations are important for controlling phase variation. However,
no natural conditions seem to change the Dam
or OxyR concentration.
One way in which E. coli cells cope with
environmental stress, including oxidative stress,
is to form aggregates. Extensive clumping occurs in stressed cultures whose cells are fully in
24 Y Microbe / Volume 3, Number 1, 2008
To determine whether phase variation regulatory mechanisms confer survival advantages, it would behoove us to know whether
phase variation itself is advantageous, an
issue that remains problematic. Phase-varying genes mostly encode proteins that directly or indirectly affect bacterial surface
structures, which, for pathogens, are exposed to host immune systems.
When genes encoding such structures are
switched off, the bacteria can more effectively “hide” from adaptive immune responses than when those same genes are on
and producing those telltale surface proteins.
Hence, some investigators consider phase variation a mechanism for evading host immune responses. Furthermore, “off” settings for phasevariable proteins might also help pathogens to
evade innate immune responses, which some of
those surface border proteins can trigger.
However, this “immune evasion” hypothesis
regarding phase variation tends to focus too
narrowly on pathogens. Phase variation instead
might be a general means for generating diversity and thereby increasing the chances of sur-
vival amid changing environmental condiFIGURE 3
tions, according to some investigators such
as Richard Moxon at the University of Oxford in England, who considers such genes as
belonging to “contingency loci.” Of course,
both the “off” and “on” phases might enhance survival, depending on the situation
and role played by a particular phase-variable protein.
It proves difficult to determine whether
phase variation affects virulence. For example, in studying cystitis and pyelonephritis in
mice, Harry Mobley and colleagues at the
University of Michigan in Ann Arbor concluded that, although type I E. coli fimbriae
are a virulence factor, phase variation of
those fimbriae is not. However, phase variation allows several Salmonella serovars with
The same level of gene expression in a population can underlie different gene
expression patterns in individual cells. This illustrates a drawback of methods that
multiple fimbrial genes in common to coexist
average the expression level from a population including microarrays. Here the
in an individual host animal, which those
shades of grey indicate the level of expression of a gene: (A) A completely
serovars could not do if they all expressed
homogenous population as a result of uniform gene expression is depicted (B) a
heterogeneous population with a gradient in the level of gene expression, for
every fimbrial type, according to Andreas
example as a result of stochastic variation in the concentration of a regulatory
Baümler and colleagues, now at the Univerprotein; (C) heterogeneity with “on” and “off” cells due to phase variation.
sity of California, Davis.
Thus, biological context affects how phase
variation contributes to virulence or bacteare mutually exclusive. However, predicting
rial survival. Nevertheless, one can speculate
DNA sites where regulatory proteins bind is still
that different environmental conditions gave
a challenge, as is predicting whether or what
rise to very different mechanisms for regulating
residue changes block such binding.
phase variation. Although both the epigenetic
An additional complication is that the DNA
system of pap and site-specific recombination
binding of some regulatory proteins is not alsystem of type 1 fimbriae are well suited for
tered by methylation, even if binding blocks
responding to environmental signals, slipped
Dam access. For instance, when the repressor of
strand mispairing is not.
the glucitol utilizing operon, GutR, binds to that
segment of DNA, its footprint includes an unHow Common Is Methylationmethylated target sequence. Its binding, howDependent Phase Variation?
ever, does not correlate with DNA methylationdependent regulation of any kind. Indeed, most
Dam in conjunction with either the global reguof the several dozen unmethylated GATC selator Lrp or OxyR mediates epigenetic phase
quences among the total of 18,000 in the E. coli
variation in E. coli and Salmonella, as elucidated
chromosome are not associated with methylafor pap and other fimbriae and for agn43, retion-dependent regulation.
spectively. Meanwhile, genomes of species beFinally, there is no reason for thinking that
longing to at least five orders of the ␥-proteobacDNA-methylation-dependent
phase variation
teria also encode a Dam homolog, and several
can
be
conferred
only
by
Dam,
and
not by other
also encode OxyR and Lrp.
DNA
methyltransferases.
The
only
known preConceivably, Dam-dependent phase variation
requisite
is
that
they
may
not
be
part
of a funcsystems similar to those controlling pap and
tioning
restriction
modification
system
because
agn43 occur in these species. Furthermore,
an
unmethylated
target
sequence
would
render
Dam-dependent phase variation could involve
the
cell
unviable
in
the
presence
of
the
cognate
regulatory proteins other than OxyR and Lrp, as
restriction enzyme. Thus, the extent of methyllong as binding of these proteins to DNA and
ation-dependent phase variation is not known.
methylation at the specific phase variation sites
Volume 3, Number 1, 2008 / Microbe Y 25
Phase Variation May Affect Research
Results as well as Diagnostics and
Vaccines Being Developed
No matter how it arises, phenotypic heterogeneity can dramatically affect the outcome of microbiological experiments and how they are interpreted. Phase variation is no exception (Fig.
3). If gene expression in a particular cell population is determined, for example, with an enzymatic assay of a reporter gene or a microarray, a
population average is measured. Although the
data would indicate the same level of expression
in all three cases, individual cells within the
population would be registering very different
gene expression patterns in each case. Thus,
single-cell analyses might be needed to identify
this sort of heterogeneity.
Understanding phase variation may allow us
to manipulate bacterial populations to provide
human health benefits. Moreover, phase variation should be taken into account when devel-
oping diagnostic tests and vaccines. For instance, diagnostic tests that include serotyping
may be sensitive to biochemical changes on the
bacterial cell surface that reflect phase variation.
Meanwhile, phase variation that leads to immune evasion might undermine the efficacy of
some candidate vaccines or therapeutic agents.
Analyzing the molecular mechanisms underlying phase variation can yield insights and
could help us to better understand the role of
how stochastic events influence the dynamics
of heterogeneity in cell populations. On a larger
scale, we are learning to appreciate how bacteria thrive in diverse environments, sometimes
relying on minor variants within the greater
natural population. Thus, identifying additional, heterogeneously expressed genes and understanding how such heterogeneity is generated and how it can prove beneficial will deepen
our understanding of how microbial populations behave.
ACKNOWLEDGMENTS
I thank my lab members and Dr. Thomas, Chong, and Pohlschroder for critical reading of this article. Work in my lab has been
supported by grants from the National Science Foundation, a Marie Curie International Re-integration grant, the Biotechnology and Biological Sciences Research Council, and the Wellcome Trust.
SUGGESTED READING
Casadesus, J., and D. Low. 2006. Epigenetic gene regulation in the bacterial world. Microbiol. Mol. Biol. Rev. 70:830 – 856.
Hernday, A., M. Krabbe, B. Braaten, and D. Low. 2002. Self-perpetuating epigenetic pili switches in bacteria. Proc. Natl.
Acad. Sci. USA 99:16470 –16476.
Hernday, A. D., B. A. Braaten, G. Broitman-Maduro, P. Engelberts, and D. A. Low. 2004. Regulation of the pap epigenetic
switch by CpxAR: phosphorylated CpxR inhibits transition to the phase ON state by competition with Lrp. Mol.
Cell.16:537–547.
Lim, H. N., and A. van Oudenaarden. 2007. A multistep epigenetic switch enables the stable inheritance of DNA methylation
states. Nature Genet. 39:269 –275.
Moxon, R., C. Bayliss, and D. Hood. 2006. Bacterial contingency loci: the role of simple sequence DNA repeats in bacterial
adaptation. Annu. Rev. Genet. 40:307–333.
van der Woude, M. W., and A. Baumler. 2004. Phase variation and antigenic variation in bacteria. Clin. Microbiol. Rev.
17:581– 611.
van der Woude, M. W. 2006. Re-examining the role and random nature of phase variation. FEMS Microbiol. Lett
254:190 –197.
Wallecha, A., V. Munster, J. Correnti, T. Chan, and M. W. van der Woude. 2002. Dam- and OxyR-dependent phase variation
of agn43: essential elements and evidence for a new role of DNA methylation. J. Bacteriol. 184:3338 –3347.
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