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Broek, G. V. Bloemberg and B. Lugtenberg
Environmental Microbiology (2005) 7(11), 1686–1697
doi:10.1111/j.1462-2920.2005.00912.x
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The role of phenotypic variation in rhizosphere
Pseudomonas bacteria
Daan van den Broek,*† Guido V. Bloemberg and
Ben Lugtenberg
Leiden University, Institute of Biology, Clusius Laboratory,
Wassenaarseweg 64, 2333 AL Leiden, the Netherlands.
Summary
Colony phase variation is a regulatory mechanism at
the DNA level which usually results in high frequency,
reversible switches between colonies with a different
phenotype. A number of molecular mechanisms
underlying phase variation are known: slipped-strand
mispairing, genomic rearrangements, spontaneous
mutations and epigenetic mechanisms such as differential methylation. Most examples of phenotypic variation or phase variation have been described in the
context of host–pathogen interactions as mechanisms allowing pathogens to evade host immune
responses. Recent reports indicate that phase variation is also relevant in competitive root colonization
and biological control of phytopathogens. Many
rhizospere Pseudomonas species show phenotypic
variation, based on spontaneous mutation of the
gacA and gacS genes. These morphological variants
do not express secondary metabolites and have
improved growth characteristics. The latter could contribute to efficient root colonization and success in
competition, especially since (as shown for one
strain) these variants were observed to revert to their
wild-type form. The observation that these variants
are present in rhizosphere-competent Pseudomonas
bacteria suggests the existence of a conserved strategy to increase their success in the rhizosphere.
Introduction
Phenotypic variation or phase variation has been defined
by as a process of reversible, high-frequency phenotypic
switching that is mediated by DNA mutations, reorganization or modification (Saunders et al., 2003). Phase variaReceived 15 March, 2005; accepted 25 July, 2005. *For correspondence. E-mail [email protected]; Tel. +31 338504076; Fax
+31 338502035. †Present address: Meander Medisch Centrum, Utrechtseweg 160, 3818 ES Amersfoort, the Netherlands.
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd
tion is used by several bacterial species to generate
population diversity that increases bacterial fitness under
certain circumstances and is important in niche adaptation, including immune evasion. Phase variation occurs at
a high frequency of > 10−5 switches per cell per generation
(Henderson et al., 1999) and can result in reversible ON
or OFF switching of traits or in the variation of surface
phenotypes. Phase variation is one of the mechanisms
enabling pathogens to survive in the host by escaping the
immune response (Craig and Scherf, 2003). This is illustrated by the fact that phase variation poses a problem in
vaccine production due to the high frequency of variation
in epitopes exposed by the pathogen (Maskell et al., 1993;
Pedersen et al., 2004). Although phase variation, or
antigenic variation, has primarily been associated with
host–pathogen interactions, a number of reports describe
phase and phenotypic variation in a broader context.
These reports show that phenotypic variation is also
involved in the production of exo-enzymes, production of
secondary metabolites and affects colonization behaviour
and biocontrol activity of rhizosphere bacteria (Chabeaud
et al., 2001; Chancey et al., 2002; Sanchez-Contreras
et al., 2002; van den Broek et al., 2003). This indicates
that phase variation can have a much broader impact on
the ecology of bacteria, affecting a high number of traits
and processes, and therefore phase variation is not only
relevant in host–pathogen interactions but also in more
ecological and industrial processes.
Genetic mechanisms of phase variation
Phase variation is a phenomenon encompassing a variety
of genetic mechanisms. These can be divided into programmed and unprogrammed variation (Borst, 2003).
Programmed variation is characterized by two properties, (i) a family of genes encoding proteins with the same
or similar function, which is combined with (ii) the ability
to express only one of the gene family members at a time
and alter the expression of these members from time to
time (Borst, 2003). Programmed variation entails regulated DNA conversions as the result of slipped-strand
mispairing (slipped-strand mispairing is, despite the fact
that the variation is based on errors during DNA replication, considered to be programmed due to the requirement
Phenotypic variation in rhizosphere Pseudomonas bacteria 1687
of a specific repeat tract) or genomic rearrangements
(including inversions, deletions, recombinational events
and gene conversions) but can also be epigenetic when
based on differential methylation. Unprogrammed phase
variation is based on DNA alterations through the accumulation of errors during DNA replication, imperfect DNA
repair, the recombination between non-identical genes, or
reassortment of gene segments if the genome is not
present in one molecule (Borst, 2003).
Programmed and unprogrammed phase variation are
subject to regulation. Especially in a host–pathogen situation the regulation of phase variation is important to allow
expression under relevant conditions. Phase variation
itself is therefore often regulated by environmental factors.
These environmental factors include temperature
(Schwan et al., 1992; Gally et al., 1993; White-Ziegler
et al., 2002), medium composition (Struve and Krogfelt,
1999; White-Ziegler et al., 2000) and stress conditions
(Nicholson and Low, 2000; Hengge-Aronis, 2002; Yildiz
et al., 2004). One of the effects of growth limitation or
stress conditions is an increase in stationary-phase mutations (Moxon et al., 1994; Kivisaar, 2003; Lombardo et al.,
2004). This is due to a downregulation of MMR (mismatch
repair) (Tsui et al., 1997; Richardson and Stojiljkovic,
2001), the spontaneous mutations of MMR components
(Horst et al., 1999; Shaver et al., 2002; Shaver and Sniegowski, 2003) and, possibly, the activity of error-prone
polymerases like DinB (Strauss et al., 2000; McKenzie
and Rosenberg, 2001).
In Pseudomonas, the occurrence of phenotypic variants
is clearly correlated to stress conditions. Culture conditions, medium composition and the scale of the cultures
strongly influence the percentage of gac mutants in
Pseudomonas fluorescens CHAO. The genetic stability
and therefore the occurrence of gac mutants in this strain
were correlated with stress conditions such as high electrolyte concentrations and certain mineral amendments
(Duffy and Défago, 1995; 2000). A central role for stress
in phenotypic variation of Pseudomonas is genetically
supported by the relationship between the frequency of
spontaneous mutation of gac and expression of the general stress response sigma factor RpoS (van den Broek
et al., 2003; 2005a). Constitutive expression of the rpoS
gene resulted in a 10-fold increase in the frequency of gac
mutants in Pseudomonas sp. PCL1171 whereas mutation
of rpoS reduced this frequency 20-fold (van den Broek
et al., 2005a).
A number of mechanisms of phase variation are known
to play a role in specific host–pathogen interactions. Most
of these species are human pathogens. We will shortly
discuss the four well-known mechanisms of phase variation: slipped-strand mispairing, genomic rearrangements,
differential methylation and unprogrammed phase variation via spontaneous mutation.
Slipped-strand mispairing
Slipped-strand mispairing uses short sequence repeats to
regulate gene expression at the translational or transcriptional level. These repetitive DNA sequences are increasingly being identified in prokaryotes (Tomb et al., 1997;
van Belkum et al., 1998; Aras et al., 2003) and can consist
of homopolymeric repeat tracts or multimeric, heterogeneous repeats (Levinson and Gutman, 1987; Henderson
et al., 1999). The stability of these repeat tracts is dependent on MMR and the sequence characteristics of the
repeat tract (Levinson and Gutman, 1987; Lovett and Feschenko, 1996; Bayliss et al., 2002; Bayliss et al., 2004a,b;
Fernandez-Lopez et al., 2004). Repeats associated with
a single locus, present in the promoter region or within the
coding region, can alter gene expression as a result of
changes in the number of repeats (Fig. 1A). The number
of repeats is varied via a RecA-independent mechanism
through the formation of heteroduplex DNA (H-DNA),
which is induced by superhelical coiling (Belland, 1991;
Dybvig, 1993; Lovett and Feschenko, 1996). This H-DNA
consists of a triple-stranded region, based on the formation of triple residue bonds within the repeat region, with
as a result a single-stranded region, which will stimulate
slipped-strand mispairing (Belland, 1991; Henderson
et al., 1999). Altering the number of repeats will result in
an incomplete gene product due to a shift in reading frame
(Fig. 1A). For example, the regulation of expression of the
Fig. 1. Model for phase variation via slipped-strand mispairing.
A. Model for ON and OFF switching of traits via slipped-strand mispairing. Variations in the number of repeats () within the coding region
of the gene results in a shift of reading frame in or out of frame. A
shift out of frame will introduce premature stop codons (*).
B. Model for volume control via slipped-strand mispairing. Variations
in the number of repeats within the promoter region of the gene will
vary promoter −10 and −35 spacing, thereby increasing (ON++) or
decreasing (ON or OFF) promoter efficiency.
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1686–1697
1688 D. van den Broek, G. V. Bloemberg and B. Lugtenberg
opa (opacity) genes in Neisseria meningitides and Neisseria gonorhoeae species, switching loci ON and OFF is
based on changes in the number of pentameric repeat
elements (5′-CTTCT-3′) with which the expression state
of the opa gene(s) changes. For example, 6, 9 or 12
repeats are equivalent to an Opa+ phenotype in which the
gene is in frame (Fig. 1A). All other numbers (e.g. 7, 8 or
13) shift the gene out of frame, resulting in incomplete
gene products and an Opa− phenotype (Stern et al., 1986)
(Fig. 1A).
An alternative slipped-strand mispairing mechanism
regulates gene expression at the level of transcription
(Sarkari et al., 1994). This regulation is mediated by the
presence of repeats upstream of the encoding gene,
which upon variation of the number of repeats, results in
an increase or a decrease in expression by varying the
promoter spacing. The expression of the opc promoter in
N. meningitidis is attenuated by a polyC tract. A tract of
12 or 13 bases increases the expression of opc 10-fold
(Opc++ phenotype) when compared with a promoter with
a polyC tract of 11 or 14 bases (Opc+ phenotype)
(Fig. 1B). When the number of repeats exceeds 15 or
becomes less than 10 no expression of opc is detected
anymore (Sarkari et al., 1994) (Fig. 1B). Slipped-strand
mispairing as a regulatory mechanism is present in a wide
range of bacteria regulating various traits. Examples of
traits regulated via slipped-strand mispairing are presented in Table 1.
Genomic rearrangements
Genomic rearrangements combine a wide range of processes involved in phase variation. These include inversions, deletions, gene duplication and gene transfer using
silent copies (recombinational deletion) (Borst, 2003).
Control of expression of, for example, type 1 fimbriae in
Escherichia coli is based on the presence of inverted
repeats and the action of site-specific recombinases
(Fig. 2). The presence of inverted repeats within the promoter region facilitates the inversion of the promoter
switching expression ON or OFF (Abraham et al., 1985;
McClain et al., 1991; 1993). Alternatively, when the promoter itself is flanked by inverted repeats, as described
for H1 and H2 flagellin genes of Salmonella typhimurium,
different sets of genes can be expressed. One orientation
of the promoter will result in the expression of h2 and the
repressor Rh1 of the h1 promoter. Upon inversion both h2
and rh1 are no longer expressed, lifting the repression of
h1 by rh1 (Zieg et al., 1977).
A second form of variation based on genomic rearrangements, for example, regulating variation of type IV
pili in N. gonorrhoeae (Jonsson et al., 1992; Seifert, 1996)
and the expression of surface proteins in Borrelia spp.
(Barbour, 2003), uses deletions, gene duplications and
gene transfer to create a large number of potential proteins to express. Although in many systems recA mutants
are not yet available, those mutants analysed show that
these rearrangements are dependent on the recA gene,
and based on the deletion of one allele present in an
active locus, which is subsequently replaced by transcriptionally inactive alleles present elsewhere on the genome
(Koomey et al., 1987; Dybvig, 1993; Henderson et al.,
1999; Meyer and Hill, 2003). This is often combined with
the presence of highly variable and semivariable regions
within these alleles, thereby increasing the variation
potential of the gene product (Haas and Meyer, 1986;
Meyer and Hill, 2003). This can enable bacteria to produce
up to 107 variant proteins (Haas et al., 1992). Examples
of traits regulated via genomic rearrangements are presented in Table 1.
Differential methylation
Phase variation of pap fimbriae and expression of antigen
43 in E. coli are dependent on a differential DNA methylation pattern and therefore represents an epigenetic
mechanism of phase variation (van der Woude et al.,
1996; Henderson et al., 1999). Methylation of GATC sites
in the genome is dependent on deoxyadenosine methylase (dam), which binds to the GATC site and methylates
adenosine at the N6 position (Palmer and Marinus, 1994).
Normally, methylation provides the organism with a regulatory mechanism for DNA repair, protection from restriction endonucleases, and timing and targeting of cellular
events (Marinus, 1996). Methylation of GATC sites within
regions involved in gene regulation can inhibit or facilitate
the binding of regulatory proteins at specific sites, and
thus alter gene expression (Nou et al., 1995; van der
Woude et al., 1996). A paradigm for regulation via differential methylation is presented in Fig. 3. Examples of
genes controlled via differential methylation are presented
in Table 1.
Unprogrammed variation
Random unprogrammed variation is based on the introduction of mutations due to imperfect replication and the
subsequent removal of these mutations, coinciding with a
switch back to the wild-type situation. One of the drawbacks of a mechanism stimulating diversification based on
imperfect replication is a high mutational load. Higher
organisms had to evolve a mechanism of mutation while
controlling the mutation rate using mechanisms like mismatch repair pathways (Borst, 2003; Schofield and Hsieh,
2003), or, although still under discussion, specific errorprone DNA polymerases transcribing specific genomic
regions (Moxon et al., 1994; McKenzie and Rosenberg,
2001; Kivisaar, 2003; Tegova et al., 2004). Most
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1686–1697
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1686–1697
Motility/flagella/biofilm formation
Root colonization/flagella/exo-enzymes
Root colonization
Root colonization/motility/biofilm formation
Motility and assimilation of certain sugars
Virulence factors
Secondary metabolism and exo-enzymes
Bordetella bronchiseptica
Pseudomonas sp. PCL1171
Pseudomonas aeruginosa
Pseudomonas brassicacearum
Pseudomonas fluorescens WCS365
P. fluorescens F113
Asospirillum lipoferum
Capsule production
Secondary metabolism/morphology
Autoaggregation
Pilus expression
Fimbrial expression
Surface lipoproteins
Surface proteins
Fimbrial expression
Variable surface glycoproteins
Flagellar expression
Type IV fimbriae
Fimbrial expression
Major outer membrane protein
Surface lipoprotein antigens
DNA restriction and modification properties
Adhesion/invasion
LPS antigenicity, Lewis Y antigen
LPS antigenicity
Fimbrial expression
Capsular polysaccharides
Flagellum export
Adhesion/invasion/neutrophil interaction
Two component sensing
Antigenicity of LOS
ABC transporter
ToxR regulon
Hemoglobin binding outer membrane proteins
Hemoglobin receptors
Property affected
Streptococcus pneumoniae
Pseudomonas tolaasii
E. coli
E. coli
Salmonella typhimurium
Salmonella spp.
Moraxella lacunata
LPS, lipopoly saccharide; LOS, lipo-oligo saccharide; ABC, ATP-binding cassette.
Mechanism unknown
vir locus
gacA/S (AY236957)
Spontaneous
mutations ON↔OFF
vsp (AF396970 and AH008162)
vsp/vlp (AF049852)
cap3, cap8, tts (Z12159,
AJ239004, AJ131985)
pheN (U95300)
pilE (AF043652)
vsg genes
Recombinational
deletion ON↔OFF
Spontaneous
duplications
ON↔OFF
M. bovis
Borrelia spp.
hin (V01370)
piv (M34367)
ONa/OFFb↔ONb/OFFa
agn43 (U24429)
pap (X03391)
pef (AB041905)
N. gonorrhoeae/N. meningitides
Trypanosome spp.
fimA (Z37500)
omp1 (U02462)
vspA (L81118)
hsd1 (AF003541)
Site-specific
inversion ON↔OFF
Differential
methylation
ON↔OFF
N. meningitides
opc (A44611)
Volume control
OFF→ON+/ON++→OFF
Escherichia coli
Dichelobacter nodusus
Moraxella bovis
Mycoplasma pulmonis
Helicobacter pylori
Haemophilus influenza
Neisseria gonorhoeae/N. meningitides
N. meningitides
Pseudomonas putida
N. gonorhoeae/N. meningitides
Bordetella spp.
Haemophilus somnus
Mycoplasma fermentans
Vibrio cholerae
N. gonorrhoeae
N. meningitidis
fucT2 (AF076779)
lic1A,2A,3A (M37912-14)
pilC (Z49120)
siaD (M95053)
flhB (AF031418)
opa (P11296)
bvgS (M25401)
lob1 (U94833)
p78 (AF100324)
tcpH (X74730)
hpuA (AF031495)
hmbR, hpuAB (AF105339, U73112)
Slipped-strand
mispairing
ON↔OFF
Species
Locus
Mechanism
Table 1. Examples of phase variable traits.
Deziel et al. (2001)
Chabeaud et al. (2001)
Dekkers et al. (1998)
Sanchez-Contreras et al. (2002)
Vial et al. (2004)
Monack et al. (1989)
van den Broek (2005)
Waite et al. (2003)
Han et al. (1997)
Owen et al. (1996)
Blyn et al. (1990)
Nicholson and Low (2000)
Haas and Meyer (1986)
Agur et al. (1989);
Barry and McCulloch (2003)
Lysnyansky et al. (1999)
Barbour (2003)
Zieg et al. (1977)
Marrs et al. (1990);
Heinrich and Glasgow (1997)
Abraham et al. (1985)
Moses et al. (1995)
Lysnyansky et al. (1996)
Dybvig and Yu (1994)
Sarkari et al. (1994)
Wang et al. (1999)
Hood and Moxon (2003)
Jonsson et al. (1991)
Hammerschmidt et al. (1996)
Segura et al. (2004)
Stern et al. (1986)
Stibitz et al. (1989)
Inzana et al. (1997)
Theiss and Wise (1997)
Carroll et al. (1997)
Chen et al. (1998)
Lewis et al. (1999)
Reference
Phenotypic variation in rhizosphere Pseudomonas bacteria 1689
1690 D. van den Broek, G. V. Bloemberg and B. Lugtenberg
these regions can consist of small deletions (50–500 bp),
mismatches and duplications (Monack et al., 1989; Han
et al., 1997; Waite et al., 2003; van den Broek et al.,
2005b). Still unclear is the exact mechanism by which the
capsule locus is switched ON again, which factors determine the switch OFF and the relevance of this mechanism
in disease. Examples of spontaneous mutations in phase
variation, switching genes ON and OFF are presented in
Table 1.
Phenotypic variation and Pseudomonas
Fig. 2. Model for phase variation via a 314 bp invertible element.
Inversion of a 314 bp promoter fragment will switch expression of fimA
ON or OFF. The inversion is facilitated by two site-specific recombinases FimE and FimB, recognizing the two 9 bp inverted repeats (IR,
the orientation is indicated with an arrow). FimE promotes the switch
from ON to OFF, while FimB can invert the fragment in both directions.
An Integration Host Factor (IHF) is required for efficient expression.
As mutation of one of the subunits of the IHF locks the expression of
fimA either in an ON or in the OFF configuration, the IHF is also
involved in the inversion of the fimA promoter (Dorman and Higgins,
1987). Histone-like protein (H-NS) is directly involved in suppression
of the fimB gene, suppressing the inversion from OFF to ON (Donato
et al., 1997; O’Gara and Dorman, 2000). The Leucine Responsive
protein (Lrp) stimulates expression of fimB and slightly decreases
expression of fimE, stimulating inversion in both directions as shown
by a decrease in the frequencies of inversion, upon mutation of lrp
(Blomfield et al., 1993).
organisms use this strategy to create diversity, for example, in antibody genes (Gearhart, 2002), but this mechanism has also been suggested to play a role in adaptive
evolution in microorganisms (Moxon et al., 1994). In the
context of phase variation, the mutations accumulating in
Phase variation in Pseudomonas bacteria is still a relatively unexplored phenomenon, but an increasing number
of interesting examples of phase variation and switching
genes have been described. These examples can be
divided into (i) mechanisms, of which the molecular basis
is often unknown, affecting a specific trait such as variation of epitopes, expression of exo-enzymes, or flagella,
and (ii) mechanism based on spontaneous mutation of
gacA/S, which affects the expression of the secondary
metabolism. Both mechanisms of phenotypic variation
often present themselves in the form of morphological
variants. These morphological variants have been
described as thick, small and opaque versus thin translucent colonies (Han et al., 1997; Bull et al., 2001; Chabeaud et al., 2001; Deziel et al., 2001; Chancey et al.,
2002; Sanchez-Contreras et al., 2002; van den Broek
et al., 2003). The determinants of these differences in
colony morphology in Pseudomonas are not known. In
addition to colony morphology, a number of other traits
are affected by this phenotypic variation. Pseudomonas
aeruginosa species regulates, in a temperaturedependent manner, the expression of the phosphocholine
Fig. 3. Paradigm for phase variation of pap via
differential methylation in E. coli. Differential
methylation of two GATC sites, GATC1028 and
GATC1130, regulates expression of papBA.
Methylation of GATC1130 will inhibit papBA
expression. The regulation is based on competition for binding sites as Lrp and methylation
sites overlap. Binding of PapI to Lrp will reduce
the affinity for binding to sites overlapping with
GATC1130. The Lrp–PapI complex will preferentially bind to the sites overlapping GATC1028,
probably after replication, and thus facilitate
methylation of GATC1130 to enable papBA
expression. Binding of CAP and PapB will lift HNS suppression (Forsman et al., 1992) and
enable transcription of papI. In addition, binding
of PapB upstream of the papI promoter will
stimulate papI transcription (van der Woude
et al., 1996).
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1686–1697
Phenotypic variation in rhizosphere Pseudomonas bacteria 1691
Fig. 4. Work model for the genetic regulation of spontaneous mutations accumulating in gac (van den Broek, 2005). A schematic representation
of the regulatory roles of GacA/S, RpoS and MutS in phase variation of PCL1171. Phase I cells harbour intact gacA and gacS genes that are
required for the expression of rpoS, which, in combination with additional factors in stationary phase negatively regulates mutS expression (van
den Broek et al., 2005a). Inefficient repair of mutations due to downregulation of mutS results in a decrease in the repair of spontaneous mutations,
which in turn will result in the accumulation of mutations in gacA and gacS (indicated with an asterisk). As a result the cells will switch to the
phase II phenotype. Mutation of gacA/S decreases rpoS expression and the subsequent increase in mutS expression, thus limiting the mutation
rate. In addition, the mutation of rpoS is hypothesized to affect both the introduction and the repair of mutations in gacA/S (van den Broek, 2005).
epitope of a 43 kDa protein through phase variation,
which is hypothesized to play a role in its pathogenicity
(Weiser et al., 1998). In addition, phase variation in this
bacterium is described to regulate the expression of type
IV pili, which affects swimming, swarming and twitching
motility and, as a result biofilm formation in P. aeruginosa.
This variation is hypothesized to be aimed at the diversification of cultures resulting in a prior presence of phenotypic forms well adapted to initiate the formation of biofilm
as soon as environmental conditions are favourable for
biform formation (Deziel et al., 2001).
Recently phenotypic variants were shown to play an
important role in competitive root colonization. An effect
of phase variation on root colonization was suggested by
the observation of a reduced competitive tomato root tip
colonization upon mutation of sss encoding a site-specific
recombinase in P. fluorescens WCS365. It was hypothesized that due to the mutation of sss, a fraction of the cells
become locked in a configuration less fit for the rhizosphere (Dekkers et al., 1998). The link between phase
variation, root colonization and sss was also studied in P.
fluorescens F113. During root colonization of alfalfa by P.
fluorescens F113, phenotypic variants were isolated. It
was shown that the sss gene is responsible for the majority of the phenotypic variation, which is combined with a
phenotypic selection for gac mutations. Three morphologically different variants, which showed a difference in
colonization pattern, and in the production of cyanide,
exo-protease and siderophores, were isolated (SanchezContreras et al., 2002; Martinez-Granero et al., 2005).
The effect of phenotypic variation on root colonization has
been described in more detail for the colonization of
Arabidopsis thaliana by Pseudomonas brassicacearum
NFM421. Pseudomonas brasicacearum shows two distinct morphological variants, designated phase I and
phase II. Phase II cells of P. brassicacearum show an
overproduction of flagellin by phase II bacteria, which
results in a higher ability to swim and swarm compared
with phase I bacteria. In root colonization experiments of
P. brasicacearum, these phase II bacteria are found at the
tip of the main root and on secondary roots, while the
phase I bacteria are primarily localized at the basal parts
of the root. Similarly, in Pseudomonas putida DOT-T1E the
expression of the flhB gene, encoding a protein involved
in flagellin export, is controlled in response to environmental changes via slipped-strand mispairing (Segura et al.,
2004). Based on the role of these variants, phenotypic
variation is suggested to be a strategy, increasing the
colonization ability of P. brassicacearum (Achouak et al.,
2004). In addition, in P. brasicacearum the expression of
an extracellular alkaline protease, a serine protease
homologue, and of a lipase is only expressed in a phase
I morphology. Although the genes coding for the protease
and lipase are organized in a single operon, the mechanism responsible for the ON and OFF switching of this
operon has not yet been described (Chabeaud et al.,
2001).
Phenotypic variation can regulate a diversity of traits in
Pseudomonas species. These variations can increase the
population diversity and could have a positive effect on
the success of a population. Similar effects are described
for the second class of mechanisms responsible for phenotypic variation in Pseudomonas: spontaneous mutation
of gacA/gacS. The gacA/gacS two-component regulatory
system consists of a sensor kinase GacS and a response
regulator GacA belonging to the FixJ family of transcrip-
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1686–1697
1692 D. van den Broek, G. V. Bloemberg and B. Lugtenberg
tional regulators (Laville et al., 1992). This two-component
system regulates secondary metabolism and the production of exo-enzymes in Pseudomonas (Laville et al., 1992;
Rich et al., 1994; Kitten et al., 1998; Blumer et al., 1999;
van den Broek et al., 2003). Phenotypic variants as a
result of spontaneous mutation of the gacA or gacS gene
are known to occur in Pseudomonas species known for
their biocontrol activity of phytopathogens. As a result
spontaneous gac mutants are often isolated from the
rhizosphere (Rich et al., 1994; Duffy and Défago, 1995;
2000; Bull et al., 2001; Chancey et al., 2002; SanchezContreras et al., 2002; van den Broek et al., 2003;
Schmidt-Eisenlohr et al., 2003). Spontaneous gac
mutants have been reported for Pseudomonas chlororaphis (Schmidt-Eisenlohr et al., 2003), Pseudomonas
tolaasii (Han et al., 1997), P. fluorescens (Duffy and Défago, 2000; Bull et al., 2001), Pseudomonas aureofaciens
30–84 (Chancey et al., 2002) and Pseudomonas sp.
PCL1171 (van den Broek et al., 2003; 2005b). In these
mutants the growth characteristics (Schmidt-Eisenlohr
et al., 2003; van den Broek et al., 2005a,b), the expression of secondary metabolites (Duffy and Defago, 2000;
Chancey et al., 2002; van den Broek et al., 2003) and
pathogenicity (Han et al., 1997) are affected. The presence of these mutants is therefore an important factor, for
example, for the efficient biocontrol activity of these
strains.
Mechanisms influencing phase variation via gac
Molecular characterization of these mutations showed
that in both the gacA and the gacS gene mutations, which
are random in nature as well as in distribution, accumulated. These mutations include base mismatches, small
insertions (1 bp) and deletions (up to 12 bp) but also large
rearrangements (Bull et al., 2001; van den Broek et al.,
2005b). The observation that spontaneous mutations form
the basis for phenotypic variation in Pseudomonas is supported by the possibility to complement these mutants
using wild-type gacA or gacS genes and by the central
role of MMR (van den Broek et al., 2003; 2005a). In
Pseudomonas sp. PCL1171 the frequency of variation
was directly correlated to the expression of MutS-dependent mismatch repair mechanisms. As the expression of
mutS is regulated by RpoS in this strain, it was suggested
that gac mutants are the result of stress-induced inefficient repair of replication-related mismatches (Fig. 4) (van
den Broek et al., 2005a). The reason for the relatively high
frequencies at which these mutants occur has been attributed to a positive selection for mutation of gac (Bull et al.,
2001) or to the growth advantage of these mutants over
wild-type bacteria (Schmidt-Eisenlohr et al., 2003; van
den Broek et al., 2005a,b). The latter is observed as a
decrease in the length of the lag-phase after reinoculation
into fresh medium in combination with a slight increase in
growth rate. This increase could be an effect of a reduced
metabolic load of a gac mutant (Schmidt-Eisenlohr et al.,
2003; van den Broek et al., 2005a,b). The effect on the
lag-phase is the result of the need for intact gacA/S genes
to fully express the rpoS gene (Schmidt-Eisenlohr et al.,
2003) (Fig. 4). The combination of these growth characteristics is suggested to enable these derivatives to reinitiate growth more readily and to grow faster than the wild
type, thereby providing a mixed population with a competitive advantage (Schmidt-Eisenlohr et al., 2003; van den
Broek et al., 2005a) (Fig. 4).
An additional effect of the spontaneous mutation of gac
is the loss of secondary metabolism. Gac-negative
subpopulations do not produce antimicrobial compounds
such as hydrogen cyanide, protease, lipase, 2,4diacetylphloroglucinol, pyoluteorin or pyrrolnitrin (Rich
et al., 1994; Han et al., 1997; Duffy and Defago, 2000; van
den Broek et al., 2003; Schmidt-Eisenlohr et al., 2003)
and are also limited in their cell-to-cell communication
and biofilm formation (Schmidt-Eisenlohr et al., 2003).
The presence of large populations of spontaneous gac
mutants, as a result of the high frequency of switching or
of inoculation with wild-type phase II cells, reduced the
biocontrol efficiency of Pseudomonas sp. PCL1171 (van
den Broek et al., 2003). This suggests that the presence
of phenotypic variants could explain the variability of biocontrol efficiency in the field but only when it reduces the
presence of wild-type bacteria on the root (van den Broek
et al., 2003). The effect of a subpopulation of spontaneous
gac mutants can therefore increase the competitive success as a result of more efficient growth and improved
colonization abilities. However, these mutants lack secondary metabolites, which are often the basis for their
biocontrol activity. The effect of this decrease in secondary
metabolism on the biocontrol activity will usually be limited
as this would require a significant fraction of bacteria to
harbour a gac mutation. The presence of such significant
populations of gac mutants in the rhizosphere was shown,
especially in natural soils, not to be the result of the
increased growth characteristics (Chancey et al., 2002;
Schmidt-Eisenlohr et al., 2003). Under normal circumstances the presence of subpopulations of gac mutants
can therefore be beneficial in the competitive environment
of the rhizosphere and could in addition explain the relative success of Pseudomonas species in the rhizosphere.
Reversibility of gac mutations
Recent publications put the spontaneous mutation of gac
in a new perspective. Pseudomonas sp. PCL1171 was
described to switch reversibly between gac mutants and
the wild type. This phase variation occurs at a frequency
of 6.4 × 10−5 switches per cell per generation from phase
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1686–1697
Phenotypic variation in rhizosphere Pseudomonas bacteria 1693
I to phase II and 8.2 × 10−2 switches per cell per generation for phase II to phase I (van den Broek et al., 2005a,b).
This ability to restore the wild-type gac genes was also
shown both on agar plates and at molecular level (van den
Broek et al., 2005b). A similar observation was made for
the pheN gene in P. tolaasii. This homologue of gacS was
found to harbour a 661 bp reversible duplication (Han
et al., 1997). Variation based on the introduction and subsequent removal of mutations is not dependent on specific
DNA features, and spontaneous duplications and deletions have been described to control, for example, capsule
genes in Streptococcus pneumonia (Waite et al., 2003),
the pathogenicity and colony morphology in P. tolaasii and
Ralstonia solanacearum (Han et al., 1997; Poussier et al.,
2003), and virulence of Bordetella bronchiseptica
(Monack et al., 1989). Presently the mechanism responsible for the exact restoration of the wild-type sequence is
unknown for any of these cases.
Interestingly, differences in the stability of phase II bacteria were observed, ranging from relatively instable phase
II cells to a stable mutation in the form of a 307 bp deletion
(van den Broek et al., 2005b). This suggests that phase II
colonies growing on agar plates only represent the stable
derivates. Although for none of the other Pseudomonas
species reversion of the gac mutations has been reported,
there is a possibility that this reversible phase variation via
spontaneous mutation is a conserved strategy of rhizosphere Pseudomonas species, which improves their success in the heterogeneous and challenging environment
of the rhizosphere (Dekkers et al., 1998; 2000; SchmidtEisenlohr et al., 2003; Achouak et al., 2004; van den
Broek, 2005). The observation that gac mutations can be
reversible (Han et al., 1997; van den Broek, 2005) makes
the suggestion of a competitive advantage for mixed population more plausible. It can explain why gac mutants
cannot out-compete their wild-type population despite
their growth advantage and do not pose, under normal
conditions, a threat to biocontrol efficacy (Chancey et al.,
2002; Schmidt-Eisenlohr et al., 2003). Furthermore, it
enables a gac mutant under less limiting conditions to
switch back to its wild-type form to express its secondary
metabolism and, as a result, become a biocontrol agent
for plant pathogens, for example.
Conclusions
Recent work has shown that phenotypic variation is a
phenomenon not only relevant in host–pathogen interactions. In contrast, research on Pseudomonas species
has shown that phenotypic variation is a more broadly
active phenomenon affecting phenomena such as root
colonization, biocontrol activity, and the expression of
exo-enzymes and secondary metabolites. The most
important known mechanism responsible for the variation
in Pseudomonas is spontaneous mutation of gacA/S,
affecting the production of exo-enzymes and secondary
metabolites.
Overall, the different forms of phenotypic variation are
relevant for rhizosphere bacteria to introduce a certain
amount of diversity into a population by allowing the formation of specific subpopulations. These subpopulations
can react rapidly to environmental changes or opportunities. The observation that these mechanisms are active in
rhizosphere competent Pseudomonas bacteria suggests
the existence of a conserved strategy to increase their
success in the rhizosphere. Whether the variation via
spontaneous mutation of gac is reversible for all these
species remains to be seen, but this would substantially
increase the importance of such mechanisms as it allows
bacteria to switch between wild-type and mutant stages.
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