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Annu. Rev. Phytopathol. 2001. 39:461–90
c 2001 by Annual Reviews. All rights reserved
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MOLECULAR DETERMINANTS OF RHIZOSPHERE
COLONIZATION BY PSEUDOMONAS
Ben J. J. Lugtenberg, Linda Dekkers, and
Guido V. Bloemberg
Leiden University, Institute of Molecular Plant Sciences, Clusius Laboratory
Wassenaarseweg 64, 2333 AL Leiden, The Netherlands;
e-mail: [email protected]
Key Words Pseudomonas, root, rhizosphere competence, biocontrol,
pathogenic fungi
■ Abstract Rhizosphere colonization is one of the first steps in the pathogenesis
of soilborne microorganisms. It can also be crucial for the action of microbial inoculants used as biofertilizers, biopesticides, phytostimulators, and bioremediators.
Pseudomonas, one of the best root colonizers, is therefore used as a model root colonizer. This review focuses on (a) the temporal-spatial description of root-colonizing
bacteria as visualized by confocal laser scanning microscopal analysis of autofluorescent microorganisms, and (b) bacterial genes and traits involved in root colonization.
The results show a strong parallel between traits used for the colonization of roots
and of animal tissues, indicating the general importance of such a study. Finally, we
identify several noteworthy areas for future research.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ADVANTAGES OF A GNOTOBIOTIC SYSTEM FOR
STUDYING RHIZOSPHERE COLONIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . .
SIMULTANEOUS IMAGING OF DIFFERENT POPULATIONS
OF MICROBES IN THE RHIZOSPHERE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ISOLATION OF (COMPETITIVE) ROOT
TIP COLONIZATION MUTANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
FLAGELLA, MOTILITY, AND CHEMOTAXIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PILI AND TWITCHING MOTILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LIPOPOLYSACCHARIDE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OTHER CELL SURFACE POLYSACCHARIDES . . . . . . . . . . . . . . . . . . . . . . . . . . .
PROTOTROPHY FOR VITAMINS AND BUILDING BLOCKS . . . . . . . . . . . . . . . .
NUTRITIONAL BASIS OF RHIZOSPHERE COLONIZATION . . . . . . . . . . . . . . . .
INTERACTION OF P. FLUORESCENS WCS365 WITH
PUTRESCINE IN EXUDATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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PROTEIN SECRETION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SITE-SPECIFIC RECOMBINATION
AND COLONY PHASE VARIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OUTER MEMBRANE PERMEABILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ROLE OF NADH DEHYDROGENASES IN COMPETITIVE
ROOT TIP COLONIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ROLE OF RHIZOSPHERE COLONIZATION
IN BIOCONTROL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
QUORUM SENSING AND MICROCOLONIES . . . . . . . . . . . . . . . . . . . . . . . . . . . .
COLONIZATION AND BIOFILM FORMATION . . . . . . . . . . . . . . . . . . . . . . . . . . .
OTHER COLONIZATION MUTANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PROMOTORS INDUCED BY EXUDATE
AND IN THE RHIZOSPHERE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
COMPARISON OF GENES AND TRAITS INVOLVED IN
COLONIZATION OF ROOTS AND OF ANIMAL TISSUES . . . . . . . . . . . . . . . . .
CAN COLONIZATION BE IMPROVED? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
COLONIZATION AND FUNCTIONAL GENOMICS . . . . . . . . . . . . . . . . . . . . . . . .
FUTURE PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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INTRODUCTION
Rhizosphere bacteria can have a profound effect on plant health. Rhizosphere
colonization is important not only as the first step in pathogenesis of soilborne
microorganisms, but also is crucial in the application of microorganisms for beneficial purposes. Most significant among these applications are biofertilization,
phytostimulation, biocontrol, and phytoremediation (75). Colonizing microorganisms can be detected attached to the root, as free organisms in the rhizosphere
(e.g., attracted to the root environment by nutrients present in exudate), or as endophytes. Endophytes, usually experimentally defined as not accessible to mild
root surface sterilization, are not considered in this review. It has long been assumed that many microorganisms are attracted by nutrients exuded by plant roots.
Indeed, this “rhizosphere effect” was first described in 1904 by Hiltner (58) who
observed increased numbers and activity of microorganisms in the vicinity of plant
roots.
The methods chosen to study root colonization depend to a considerable degree
on the particular problem to be studied. For example, determining which microorganisms enter the rhizosphere from the soil requires an experimental approach
different from that needed to follow the fate of an inoculant bacterium after application on the seed for commercial purposes. For details on these strategies the
reader is referred to a review by Kloepper & Beauchamp (65). Classical electron
microscopy studies as well as the use of marked strains (33, 35, 118) have shown a
nonuniform distribution of bacteria on the root: Some areas, including the extreme
tip of the root, are practically free from bacteria whereas other areas can be heavily
populated (10, 43, 45, 46, 87, 90, 110). With Pseudomonas the heavily populated
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areas are usually found at junctions between epidermal root cells, indented parts of
the epidermal surface, or sites of side root appearance (4, 21), all presumed sites of
exudation. Rhizobium bacteria also attach to root hair tips (121, 122), presumably
because of the presence of specific receptors (129). The observation that strain
GB03 of Bacillus subtilis did not colonize the root (46) is consistent with results in
our group showing that Bacillus is a poor colonizer (B. Lugtenberg, L. Dekkers &
G.V. Bloemberg, unpublished data).
Initially bacteria were not detected on the root because the root surface is
covered by a distinct matrix, the mucigel (62). When this layer was disrupted
during preparation for electron microscopy (42, 44) rhizosphere bacteria became
visible.
Using a modified method, Chin-A-Woeng et al (21) were able to make this
layer semitransparant and visualize the bacteria underneath the mucigel layer
by scanning electron microscopy (SEM). The mucigel layer does not prevent
visualization of green fluorescent protein-(GFP) labeled bacteria using confocal laser scanning microscopy (CLSM) (4, 5, 27) or visualization of lacZ marked
strains (8).
Root colonization has been most intensively studied in Pseudomonas, the most
effective root-colonizing bacterium. This review therefore mainly focuses on rootcolonization genes and traits of Pseudomonas.
ADVANTAGES OF A GNOTOBIOTIC SYSTEM FOR
STUDYING RHIZOSPHERE COLONIZATION
The interactions between plants and microorganisms are immensely complex and
very little is known about the sum of factors that lead to reliable biocontrol application (66). As one of our main purposes is to understand the mechanism of
microbiological control of plant diseases caused by fungi at the molecular level,
we have developed a less complex system devoid of field-soil variables, which
therefore yields more reproducible results (120). In this system sterile germinated
seedlings are grown after bacterization by incubating them in a suspension containing 107 to 108 CFUs/ml for 15 min. At 108 CFUs/ml the number of bacteria
attaching to the seed is close to saturation (T.F.C. Chin-A-Woeng, unpublished
data). After inoculation, the seedlings are aseptically placed 5 mm below the surface of a 10-cm long quartz sand column moisturized with a plant nutrient solution
(PNS) without added carbon source. After growth for seven days the plant, including the sand particles adhering to the root, is removed. The root is divided into
segments that are then shaken in phosphate buffered saline (PBS) to remove bacteria. The number of colony forming units (CFUs) is determined after plating (120).
Colonization is evaluated by counting the numbers of released bacteria after shaking root segments for 20 min. in PBS. The high bacterial numbers at the root base
(107 CFU/cm) rapidly decrease (to 103 to 104 CFU/cm at the root tip). The total
number of wild-type P. fluorescens WCS365 bacteria on the root increases 16- to
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32-fold in 7 days. However, this does not necessarily mean that all applied bacteria
make 4 to 5 divisions. The adhering sand contains few bacteria, and the shaking
procedure removes practically all bacteria from the root (21). Since Loper et al
(74) showed a log normal distribution of rhizosphere bacteria, the data are log10
(CFU + 1)/cm transformed before any statistical treatment.
Most commercial inoculants are coated to the seed or are applied in the furrow
where the bacteria can reach the young seedling. However, to increase reproducibility, bacterized seedlings are often used instead of seeds, because this allows
the choice of a homogeneous set of seedlings at the start of the experiment. For
potato, sterile stem cuttings are used after these have been allowed to root (31, 36).
This gnotobiotic system functions well for tomato, radish, potato, cucumber,
wheat, and grass. The advantages of the gnotobiotic system over the use of field
soil are (a) better reproducibility due to the use of the plant growth medium; (b)
higher numbers of the test bacterium on the root in the absence of competition from
indigenous soil bacteria. For an efficient colonizer such as P. fluorescens WCS365
cell numbers are tenfold lower when quartz sand is replaced by potting soil; (c)
simple testing of the effect of individual biotic factors, such as other bacteria (27),
fungi (70), or protozoae; and (d ) easier screening of mutants for their colonizing
ability (29, 119).
Colonization is studied mainly in the context of biocontrol of tomato foot and
root rot caused by Fusarium oxysporum f. sp. radicis lycopersici. Because this
fungus can reside deep in the soil, the deeper root parts must also be protected by
the biocontrol agent. Colonization is therefore defined as the ability of bacteria
applied on the seed(ling) to reach the growing root tip.
Using gfp-labeled microorganisms (see Figure 1), Lagopodi et al observed
that the biocontrol bacteria P. fluorescens WCS365 and P. chlororaphis PCL1391
applied to the seedlings, and the pathogenic fungus Fusarium oxysporum f. sp.
radicis lycopersici applied in the soil, occupy the same sites on the tomato root.
The observation that the biocontrol bacterium occupies these sites faster than the
phytopathogenic fungus presumably underlies a crucial aspect of the bacterium’s
biocontrol ability (70).
In the gnotobiotic system, all six tested single strains of wild-type pseudomonads, in the absence of competing organisms, reached the root tip. However, after
inoculation of seedlings with a 1:1 mixture of two different wild-type strains, some
strains (e.g. WCS315 and WCS358) performed poorly in competitive root tip colonization whereas WCS365 outcompeted the other five tested strains (120). That
WCS365 was also the best strain of an inoculation mixture of 11 Pseudomonas
strains in the colonization of a 100-cm long potato root system growing in soil (6)
further supports the relevance of the gnotobiotic system to practical conditions.
Additional support for choosing this strain as a model came from the observation
that mutants, established to be defective in colonization in soil, also appeared to
be defective in colonization in the gnotobiotic system (120).
When wheat plants were grown gnotobiotically after inoculation with one of
150 single fluorescent Pseudomonas isolates from the wheat rhizosphere, 40% of
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the strains inhibited plant growth, 20% had no effect, and 40% stimulated plant
growth (9). Note that had this test been carried out in soil, the number of strains
that affect plant growth would certainly have been smaller since soil contains
high numbers of indigenous bacteria that would have competed with the inoculant
strains for sites and nutrients from the rhizosphere. Consistent with this notion
are the observations that barley plants grown under gnotobiotic conditions have
increased susceptibility to two powdery mildew fungi (114) and that biocontrol
Pseudomonas bacteria perform much better in suppressing tomato foot and root
rot in a gnotobiotic system than in soil (A.L. Lagopodi, B. Lugtenberg & G.
Bloemberg, unpublished).
We have observed in gnotobiotic root colonization studies that the root tips of
the monocots grass and wheat are colonized with ten times more Pseudomonas
bacteria than are those of dicots such as potato, radish, and tomato. This observation
correlates with a better growth yield in root exudates of monocots.
SIMULTANEOUS IMAGING OF DIFFERENT POPULATIONS
OF MICROBES IN THE RHIZOSPHERE
Green fluorescent protein (GFP) (133) has been used as a marker for noninvasive
observation of the fate of individual cells, and its gene has been used as a reporter
for the activity of specific promoters (2) and to show that more than one rhizobial
species can be present in the same nodule (128). The problem of GFP being too
stable to study real-time gene expression has been overcome by constructing g fp
mutants with a short half-life (107). This technology allows detailed studies of
the behavior of individual cells in the rhizosphere (4, 144), e.g., with respect to
ribosomal content and synthesis rates (40).
WCS365 derivatives of P. fluorescens harboring genes encoding enhanced cyan,
green, and yellow variants of GFP and encoding the red fluorescent protein (DsRed)
were constructed (47) in the rhizosphere-stable cloning vector pME6010 (56).
After seedling inoculation and gnotobiotic plant growth, the roots were isolated,
rinsed briefly in PBS to remove most sand particles, then put on microscopic
slides in PBS to prevent them from drying during microscopy. Using confocal
laser microscopy, it was confirmed that Pseudomonas cells on the tomato root are
present mainly as elongated stretches on indented areas, such as junctions between
epidermal cells and the deeper parts of the epidermis on the root surface (4, 5),
and on root hairs (27). Up to three differently labeled bacteria could be visualized
simultaneously. Since the vector has a broad host range for Gram-negative bacteria,
the constructed plasmids will be of great value in analyzing microbial communities
in processes such as colonization, biofilm formation, competition for niches, and
gene expression in the rhizosphere (5).
Mixed inoculation of tomato seedlings with differently labeled WCS365 derivatives showed that mixed microcolonies are present predominantly on the upper part
of the root whereas on the lower part of the root microcolonies consist of only one
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population to which cells from another population are sometimes attached. This
observation strongly suggests that (a) only infrequently does a single bacterium
start forming a microcolony and (b) such a growing homogeneous microcolony
is only infrequently reached by other cells (27). A low frequency of microcolony
formation also explains the strong variation found among plants with respect to
ratios of two strains found at the root tip after mixed inoculation.
We find it surprising that growing microcolonies consist of one population for
so long; rather we would expect many bacteria to use chemotaxis to reach a new
site of exudation. The results suggest that a single bacterium rarely swims toward
exudate. We propose that most bacteria are somehow contained, e.g., bound to
the plant surface, caught under the mucigel layer, or aggregated to other bacteria.
Aggregation brought about by PspB (12) may play an important role in this process.
Mixed inoculation of tomato seedlings with two biocontrol pseudomonads,
strain PCL1391 (acting through production of PCN) and strain WCS365 (acting
through ISR) in equal numbers, showed that at the older part of the root the two
cell types were present in larger microcolonies of either one or both cell types.
Small microcolonies, usually of one cell type, and single cells were present at the
middle part of the root. At the root tip, single cells or small groups of two to four
cells, predominantly of one cell type, were observed.
The observation that mixed microcolonies exist in the rhizosphere implies that
two populations can influence each other extensively, through either direct contact
or signal exchange. Direct contact could explain the observation (132, 140) that
the frequency of recombination in the rhizosphere is high. Exchange of signal
molecules by bacteria in the wheat rhizosphere has been reported by Pierson et al
(98).
Mixed inoculation with equal numbers of WCS365 and PCL1391 cells indicated
that the two bacteria show some preference for different sites. On the middle part
of the root, WCS365 cells were approximately five times more abundant, whereas
root hairs were colonized almost exclusively by PCL1391 cells. Equal numbers of
both cells were found at the root tip (27).
Inoculation by a mixture of WCS365 and PCL1391 cells tended to improve
biocontrol in comparison with cells of a single strain inoculation (27). We interpret
this to be the result of a synergistic effect due to the use of two different mechanisms.
This positive synergistic effect, however, is likely to be reduced by the twofold
dilution of each strain (27).
ISOLATION OF (COMPETITIVE) ROOT
TIP COLONIZATION MUTANTS
Two approaches have been used to isolate colonization mutants. In the targeted
approach, mutants in traits possibly involved in colonization are tested against the
parental strain for colonization ability. This approach showed the importance for
colonization of the following traits: motility (36), production of the O-antigen of
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lipopolysaccharide (LPS) (31), of cellulose (83), of thiamine (120), of amino acids
(119), and of biotin (125), as well as the synthesis of an isoflavonoid-inducible
efflux pump (93). In the second approach, based on the method developed by
Lam et al (71), two mutants (labeled with Tn5 and Tn5lacZ, which allows their
discrimination on X-gal plates) were screened in competition with each other on
one tomato plant (29, 119), using the gnotobiotic system of Simons et al (120).
After plant growth, bacteria were removed from the root tip and transfered to King’s
medium B plates supplemented with X-gal. Putative colonization mutants were
tested for motility and auxotrophy, already known as traits for colonization. After a
second screening for competitive colonization on four plants, this time against the
wild-type strain P. fluorescens WCS365 or its lacZ-marked derivative PCL1500,
the remaining mutants were tested for their ability to synthesize the O-antigen of
LPS. The remaining mutants were tested for competitive growth in liquid King’s
medium B to avoid collecting mutants that are colonization defective because of
aspecific poor growth. Mutants without any growth defect were considered as
novel colonization mutants. The nature of their mutations is discussed later in this
review.
Starting with 1300 mutants, 15 appeared to be nonmotile, 13 auxotrophic for
amino acids, 6 did not produce the O-antigen of LPS, 1 produced a shortened
O-antigen side chain, and 11 mutants, including the 6 O-antigen deficient ones,
had a 5% to 50% growth rate defect. Eight mutants remained and were analyzed
to identify the novel genes and traits involved in colonization. When tested alone
in the gnotobiotic system, these mutants all colonized the root system as well as
their parental strain, although they were severely defective in competitive colonization, both in the gnotobiotic system (29) and in potting soil (28, 29; C. Mulders
& B. Lugtenberg, unpublished). Furthermore, these mutants were all defective
in competitive colonization on several crops, including the monocot wheat, indicating that they are impaired in colonization traits covering a broad host range
(29).
The temporal spatial competitive colonization behavior of wild-type WCS365
and colonization mutant PCL1233 was analyzed after inoculation of potato cuttings
with a 1:1 mixture of these bacteria. The percentage of mutant cells gradually
decreased along the root, and the percentage at the root tip decreased with time,
i.e., with increasing root length (28).
FLAGELLA, MOTILITY, AND CHEMOTAXIS
Pseudomonas bacteria produce up to nine polar flagella per cell. Nonmotile mutants are easy to screen for since, unlike wild type, they do not swarm on medium
containing 0.3% agar. The role of flagella in colonization has been disputed
in the 1980s. Howie et al (60) and Scher et al (115) reported that nonmotile
mutants of Pseudomonas are not impaired in root colonization of wheat and
soybean, respectively. On the other hand, de Weger et al (36) found that
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several different nonmotile mutants of P. fluorescens strain WCS374 were severely
impaired in colonization of potato roots, with the defect largest at sites most distant from the site of inoculation. In later studies, using different parental Pseudomonas strains, soil systems, and plants (29), we observed that nonmotile mutants
belong to the most defective competitive class of colonization mutants (19, 29,
120).
To test whether the role of flagella in root colonization is based on random
motility or on chemotaxis (e.g., to root exudate components), H. Vermeiren,
J. Vanderleyden & R. de Mot, unpublished) isolated mutants in the general chemotaxis gene cheA of biocontrol strains WCS365 (27, 49), OE 28. 3 (30), SBW 25
(105), and F113 (117) of the European P. fluorescens. All mutants were as severely
(100- to 1000-fold) defective in competitive tomato root tip colonization as nonmotile strains (C. Mulders, S. de Weert & B. Lugtenberg, unpublished), indicating
that chemotaxis rather than random motility is involved in root colonization. Surprisingly, when nonmotile mutants or cheA mutants are tested alone in the gnotobiotic system, the ability to colonize the root tip is not significantly different from
that of the wild type (S. de Weert & B. Lugtenberg, unpublished). This implies
that other factors are also involved in transportation of bacteria to the root tip. Root
growth is an obvious candidate.
The realization that chemotaxis is important for colonization has been used to
select bacterial strains that are good cereal root colonizers (67).
PILI AND TWITCHING MOTILITY
Pili or fimbriae are cell surface appendages involved mainly in attachment. The
presence of pili has been described extensively for pathogens for which they are
considered to be virulence factors (55, 127). In P. aeruginosa, type 4 pili mediate the initial contact between the bacteria and the epithelial cell surface (55, 97),
are involved in a unique type of locomotion known as twitching motility (24), and
have also been implicated in attaching to abiotic surfaces and forming biofilms
(92). Roles for type 4 pili have also been described for plant pathogens (91, 109).
Furthermore, type 4 pili are involved in colonization of both plant and fungal hosts
of Azoarcus, an associative nitrogen-fixing bacterium (37).
Although the WCS series of biocontrol pseudomonads had been isolated after
thorough washing of the roots (49), and therefore could be assumed to be tightly
bound to the plant root, several attempts by our group to visualize pili were unsuccessful. More recently, Camacho Carvajal (8) was able to visualize, after growth
in vitro, an average of one to two type 4 pili per WCS365 cell, no pili on cells of
a pilA mutant, and three to four pili on the surface of pilT mutant cells. pilA is the
gene encoding prepilin, whereas pilT is involved in pilus retraction and is required
for twitching motility (148). Furthermore, she has shown expression of the pilA
promoter in the tomato rhizosphere and defects in competitive root tip colonization
for the pilA mutant PCL1092 and for the pilT mutant PCL1093 (8). We conclude
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that type 4 pili play a role in competitive tomato root tip colonization, presumably
through twitching motility. We suggest that rhizosphere conditions could enhance
the production and activity of type 4 pili. However, preliminary attempts to test the
influence of exudate on pilA expression were not positive (M. Camacho Carvajal,
G. Bloemberg & B. Lugtenberg, unpublished).
LIPOPOLYSACCHARIDE
Lipopolysaccharide (LPS) of Pseudomonas and of most other Gram-negative bacteria consists of lipidA, core, and O-antigen. The latter can consist of many repeating units. LPS of a pure culture is heterogenous with respect to the number
of repeating units in its O-antigen. The result is that upon sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) a ladder pattern forms in
which every band represents a different LPS molecule, with the O-antigen having
a distinct number of repeating units. Short molecules run fastest (52, 94). Different Pseudomonas strains produce LPSs that differ in ladder pattern obtained after
SDS-PAGE (34). Since LPS acts as the receptor for many bacteriophages, mutants in LPS structure can simply be obtained by isolating clones resistant to such
phages (31). Mutants devoid of the LPS ladder pattern, and therefore defective
in the O-antigenic side chain, appear to be impaired in potato root colonization,
as judged from tests in clay soil (31) and in the gnotobiotic quartz sand system
(120). As expected from these results, screening of large numbers of mutants for
competitive root tip colonization in the gnotobiotic system also yielded several
mutants with a defective O-antigen (29).
Originally the interpretation of defects in root colonization by mutants missing
their O-antigen was difficult because these mutants had serious growth rate defects
in both laboratory medium (29) and in root exudate (C. Phoelich, L.C. Dekkers &
B. Lugtenberg, unpublished). However, from the screening of 1300 mutants on
competitive root colonization defects, the colonization mutant strain PCL1205 appeared to have a shortened LPS ladder, next to six mutants that were completely
missing the ladder (29). In contrast to these six mutants, strain PCL1205 had a
normal growth rate in laboratory medium and in exudate despite its colonization defect. Therefore, the simplest explanation of these results is that part of the
O-antigen is required for competitive colonization but not for maximal growth rate.
The mechanism by which this part of the O-antigen plays a role in colonization is
not known.
Mutant PCL1216 appeared to have a mutation in a gene homologous to htrB
from Haemophilus influenzae (72). The LPS of both mutants migrates faster than
that of their corresponding wild-type strain (29, 72). HtrB of Escherichia coli
encodes a lauroyl transferase that uses (KDO)2 lipid IVA as the laurate receptor
(22). The assumption that lipid A is underacylated explains the faster migration
of the LPSs and also why PCL1216 cells lose competition, because of a defective
outer membrane structure, in root exudate (77).
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OTHER CELL SURFACE POLYSACCHARIDES
Matthysse & McMahan used a root colonization assay that involved dipping
roots in a suspension of Agrobacterium tumefaciens cells, followed by growth
for 10 days. Bacterial numbers on the tomato root increased 10,000-fold during
this period. A cellulose-minus mutant (84) colonized the tomato root 10,000-fold
less and the Arabidopsis root 10- to 100-fold less (83).
De Weger et al described a polysaccharide of P. putida WCS358 that shares
characteristics with K-antigens of E. coli. A mutant lacking this polysaccharide,
isolated using magnetic monoclonal antibodies, had wild-type colonizing ability
on potato root (32).
PROTOTROPHY FOR VITAMINS AND BUILDING BLOCKS
As early as in 1961 (112), biotin was reported to be present in exudates of all ten
plants tested in amounts sufficient to influence the growth of rhizosphere microorganisms (112). Indeed, Streit et al (125, 126) reported that 4 nM biotin promotes
growth of strain 1021 of Rhizobium meliloti in laboratory medium. These authors
reported that growth of the same strain in the rhizosphere is supported by the watersoluble vitamins biotin, thiamine, and riboflavin (96, 126). Mutants of R. meliloti
unable to synthesize biotin are poor competitors in the alfalfa rhizosphere (126).
Similarly, a thiamine auxotroph of P. fluorescens WCS365 appeared to be a poor
competitor in the tomato rhizosphere (120). Apparently, the ability to synthesize
these vitamins is an important colonization trait.
The presence of amino acids in root exudates has been reported many times
(48, 64, 96, 102, 134–139). In an analysis of tomato root exudate, we found that
aspartic acid, glutamic acid, isoleucine, leucine, and lysine are the major amino
acid components. To test whether the amino acid level in exudate is sufficient to
support growth of auxotrophs, five different mutants of strain WCS365 unable
to synthesize leucine, arginine, histidine, valine + isoleucine, and tryptophan,
respectively, were isolated. None of these mutants was able to colonize the tomato
root tip in the gnotobiotic system, either alone or after coinoculation with the wildtype strain. However, addition of the appropriate amino acid to the system restored
colonization by the mutants, usually to wild-type levels (119). Glandorf (51) had
reported previously that amino acid auxotrophs of WCS365 are impaired in potato
root colonization in field soil. Apparently the ability to synthesize amino acids is
also an important colonization trait.
The competitive colonization mutant PCL1218 of P. fluorescens WCS365,
which also loses competition in exudate, has a transposon insertion in two overlapping genes with different orientations, namely wbpN and tyrB. Gene wbpN plays
an unknown role in lipooligosaccharide synthesis. Gene tyrB encodes an aromatic
amino acid amino transferase. Since the addition of tyrosine, phenylalanine, aspartic acid, and leucine restores both competitive colonization and competitive
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growth in exudate, it was concluded that the colonization defect is caused by the
tyrB mutation (77).
The mutant PCL1202 of P. fluorescens WCS365 is impaired in competitive
tomato root tip colonization as well as in competitive growth in tomato root exudate. The transposon Tn5lacZ is inserted in the pyrR gene, which encodes a
regulatory protein of the de novo pyrimidine biosynthetic pathway. Downstream
of pyrR the pyrimidine biosynthetic genes pyrB and pyrC were identified. An independently constructed pyrR mutant also appeared to be defective in competitive
colonization ability. Addition of exogenous uracil restored both competitive colonization as well as competitive growth on exudate, despite the observation that
PCL1202 is not auxotrophic. Expression studies showed that PyrR has a positive
effect on the transcription of the pyrB promoter. These results strongly suggest that
this enhanced transcription is required for optimal competition in the rhizosphere.
Although no function for PyrR of P. fluorescens was apparent under laboratory conditions, the tests under rhizosphere conditions described here revealed a function
for this protein (8).
NUTRITIONAL BASIS OF RHIZOSPHERE COLONIZATION
Root exudates are important carbon and energy sources for colonizing microorganisms. Thirty percent of the total net photosynthesis in wheat seedlings is used
for rhizosphere respiration by roots and associated microorganisms. Sixty percent of this amount was attributed to microbial respiration of exudates (14, 111).
The presence of bacteria can alter the profile of exuded metabolites (145, 151).
Extracellular metabolites of various microorganisms, such as Pseudomonas and
Fusarium oxysporum, increase carbon exudation significantly (87).
Generally, amino acids, monosaccharides, and organic acids are considered as
the major exudate compounds, but other components can also be present (96). We
have determined the amino acid (119), sugar (78), and organic acid (79) composition of tomato root exudate and used mutants to evaluate the importance of these
components for proliferation and root colonization.
Glucose, xylose, and fructose are the major monosaccharides in tomato root
exudate (78). A mutant was isolated that was impaired in growth on the sugars
glucose, fructose, sucrose, and xylose but which grew normally on organic acids
such as succinate. The mutation in this mutant, strain PCL1083, was localized
in zwf, which encodes glucose-6-phosphate dehydrogenase. The mutant was able
to compete with the wild type for tomato root tip colonization, indicating that
utilization of exudate sugars is not important for rhizosphere competence (78).
The major organic acids in seedling exudate are citric, oxalic, pyruvic, and lactic
acids, whereas citric, malic, lactic, and succinic acids are the major organic acids
of root exudate. The total amount of organic acids in root exudate is five times
higher than the amount of sugars (79). Mutant PCL1085 of P. fluorescens strain
WCS365 was isolated and found to grow poorly in vitro on the organic acids malic
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and succinic acid and moderately on citric acid. Consistent with this result, the
mutation was found just upstream of the single gene mdh operon, which encodes
malate dehydrogenase. Mutant PCL1085 appeared to be such a poor competitive
colonizer on tomato root tips that it was completely outcompeted by its parental
strain. These results are consistent with the composition of the tomato root exudate
and also show that utilization of exudate organic acids is the nutritional basis
of tomato root colonization by Pseudomonas. These data open opportunities for
choosing sets of beneficial microbe-plant pairs based on exudate composition and
high bacterial growth rate on the major exudate carbon sources (79).
INTERACTION OF P. FLUORESCENS WCS365
WITH PUTRESCINE IN EXUDATE
The competitive colonization mutant PCL1206 has a mutation immediately upstream of the pot operon, which encodes the putrescine uptake system and consists
of two potF genes (potF encodes a putative putrescine binding protein), followed
by potG, potH, and potI. This observation suggested the presence of the polyamine
putrescine in exudate. Indeed, putrescine, but not the polyamines cadaverine, spermine, and spermidine, is present in tomato root exudate, its concentration being
higher in exudate of 21-day-old plants than in that of 8-day-old plants. The mutant
shows impaired growth on putrescine as the sole N-source. The obvious interpretation, namely that putrescine is an important N-source for the root colonizing strain
WCS365, was unlikely since the plant nutrient solution present in the gnotobiotic
system contains sufficient nitrate (5 mM). A new mutant, strain PCL1270, was
constructed within the first potF gene. This strain is not impaired in colonization.
Subsequent experiments on the effect of putrescine on growth, viability, and on the
rate of 14C-putrescine uptake resulted in the following interpretation. Wild-type
WCS365 takes up putrescine to a certain intracellular level that is determined by
a regulator protein which can prevent transcription of the pot operon. In strain
PCL1206 the binding site for this regulator protein is mutated. This causes excessive putrescine uptake in strain PCL1206, which results in transient bacteriostasis
but does not cause cell death. This phenomenon is likely to be the cause of its colonization defect. The other mutant, strain PCL1270, is unable to take up putrescine
owing to its potF mutation. It uses nitrate as the N-source in the rhizosphere when
studied in the gnotobiotic system.
In retrospect, mutant PCL1206 did not contribute to our knowledge of the
colonization process per se. However, analysis of this mutant strain indicated that
(a) putrescine is present in exudate; (b) Pseudomonas is able to protect itself
against levels of putrescine that otherwise would cause slower growth; and (c)
putrescine concentrations in the rhizosphere are bacteriostatic unless the uptake of
this polyamine is carefully regulated (68).
Interestingly, the pot operon of Agrobacterium tumefaciens has been implicated
in attachment of bacterial cells to carrot suspension culture cells. The ability of
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mutants to bind to host cells was restored by the addition of conditioned medium
(85). The relationship of this finding with P. fluorescens mutant PCL1206 is not
clear.
PROTEIN SECRETION
Mutant PCL1268 has a mutation in the single gene operon secB but no growth
rate defect (I. Kuiper, G. Bloemberg & B. Lugtenberg, unpublished). In E. coli
this gene encodes a chaperonin involved in secretion of proteins over the cytoplasmic membrane, such as periplasmic proteins, outer membrane proteins, and
extracellular proteins. Since no extracellular proteins are known to be secreted by
WCS365, we assume that the colonization defect caused by secB affects one or
more periplasmic and/or outer membrane proteins involved in colonization.
Rainey (104) described many genes of the plant-growth-promoting SBW25
strain of P. fluorescens that are specifically induced during rhizosphere colonization. About one third of these genes showed significant homology to sequences
in GenBank that are involved in nutrient acquisition, stress response, or secretion.
Interestingly, one gene is a homologue of hrcC, a type III secretion pathway, not
previously described in nonparasitic bacteria (104). In a more recent study, it was
shown that hrcC resides in a 20-kb gene cluster that encodes a functional secretion
pathway capable of delivering ArrB from P. syringae to plant cells. hrp genes
appeared to be widespread among Pseudomonas spp, suggesting that type III secretion has functional significance in both pathogens and plant growth promoting
rhizobacteria (101).
SITE-SPECIFIC RECOMBINATION
AND COLONY PHASE VARIATION
Mutant PCL1233 is at least 50-fold impaired in competitive root tip colonization
in the gnotobiotic system on tomato, potato, and wheat. It is also colonization
defective on tomato when tested in potting soil. The transposon in this mutant
resides in the orf 235/233 homologue of a partly sequenced operon consisting of
lppL, lysA, dapF, orf 235/233, xerC/sss, and the largely incomplete orf 238. Further
studies have revealed that the xerC/sss homologue is crucial for colonization (28).
xerC in E. coli and sss in P. aeruginosa belong to the λ-integrase family of
site-specific recombinases, which play a role in phase variation caused by DNA
rearrangements. These enzymes promote conservative reciprocal recombination,
which does not require DNA synthesis, between two small (approximately 15-bp)
homologous DNA sequences. The presence and orientation of two of these sequences will lead to inversion or excision of the DNA fragment situated between
these small recognition sites (113).
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Most DNA rearrangements involved in phenotypic switching regulate expression of phase-variable surface antigens, such as fimbriae, flagella, LPS, and lipoprotein. These events have been studied in detail in animal pathogens for which it
makes sense to change the cell surface when another niche has to be occupied
(e.g., within host cells the presence of flagella is probably a handicap) or to escape
the animal’s (immune) defense system. For the biocontrol bacterium P. fluorescens
WCS365, we favor Dybvig’s hypothesis (39) that subpopulations generated by
DNA rearrangements enable a bacterial population to respond adequately at all
times to environmental changes. According to this notion, WCS365 is present in
at least two subpopulations and its sss mutant PCL1233 is locked in a genetic
configuration that is not optimally rhizosphere competent (28).
DNA rearrangements are often associated with colony sector formation. Indeed,
it was found that 5-day-old colonies of P. fluorescens strain WCS365 contain
morphologically distinct sectors. Sectoring was observed less frequently in sss
mutant strain PCL1233 (28).
Höfte et al (59) have described a sss mutant of P. aeruginosa. The major defect
in this sss mutant was its inability to produce the siderophore pyoverdin in a certain
medium. Furthermore, the mutant had slight defects in log phase growth and in colonization. This mutant, strain 7NSK2, is therefore different from PCL1233, which
is unaffected in growth and siderophore synthesis but has a serious colonization
defect (28).
To identify genes encoding traits that are targets of sss, an sss mutant of strain
PCL1391 of P. chlororaphis, which has many traits relevant for biocontrol (20, 21),
was tested. The sss derivative PCL1126 appeared not to be altered in the production
of PCN, chitinase, protease, and HCN. The observation that PCL1126 is defective
in competitive tomato root tip colonization and biocontrol (19) shows that targets
of sss other than the intact traits already noted are involved in colonization and
biocontrol.
After the discovery that colony phase variation plays a role in colonization, we
investigated a series of 43 Pseudomonas strains from which colony phase variation
was obvious from segmented colonies (usually opaque and translucent). From this
work it appeared that production of antifungal activity (as judged from antagonism
on plates), biosurfactant and chitinase are correlated with colony phase variation,
whereas the production of lipase, protease, and siderophore is often, but not always,
correlated with colony phase variation (D. van den Broek, T. F. C. Chin-A-Woeng,
G. Bloemberg & B. Lugtenberg, unpublished).
Vesper (143) described two colony types for the PCA (phenazine-1-carboxylic
acid) producing biocontrol strain 2-79 of P. fluorescens (131). He observed that, in
contrast to cells of mucoid colonies, those of the nonmucoid colonies were highly
piliated and showed hydrophobic attachment to polystyrene, attachment to corn
roots, and were active in hemagglutination (143).
Recently, a new Pseudomonas species, Pseudomonas brassicacearum, was described as a root colonizer of Arabidopsis thaliana and Brassica napus (1). After
growth for prolonged periods on rich media a new colony type appeared that, unlike the original “phase I” isolate, showed fluorescent pigmentation when grown
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in iron-poor medium. Furthermore, this “phase II” variant differs from the original isolates with respect to nitrate reduction and does not hydrolyze gelatin and
Tween 20 (1), indicative of extracellular protease and lipase, respectively. Using
a DNA probe containing the alkaline protease gene aprA from P. aeruginosa,
Chabeaud et al (12) identified hybridizing signals with DNA fragments of identical size in digests of both phases of P. brassicacearum. Further cloning resulted
in the identification of a fragment containing a single operon containing seven
ORFs of which the predicted proteins are homologous to alkaline protease (aprA),
protease inhibitor (aprI), and the ABC transporter (aprDEF) of P. aeruginosa, and
to the putative serine protease (pspB) and the lipase (lipA) of P. fluorescens. Since
the operon was only expressed in phase I cells, its expression must be controlled
by phase variation (12). No serine protease activity was detected when the serine
protease gene was expressed in E. coli (12). Since many members of the autotransporter family function as adhesins (57), the notion that PspB might be involved
in the adhesion of bacteria to plant roots was examined. E. coli cells harboring
the pspB containing fragments did indeed form large aggregates (12). We suggest that autoaggregation could be very relevant for colonization and biocontrol,
e.g., it could play a role in microcolony formation, which is likely to be important
for biocontrol (21).
OUTER MEMBRANE PERMEABILITY
Mutant PCL1210 of P. fluorescens WCS365 is impaired in the two-component
sensor/response regulator system colR/colS (25) downstream of which a putative
orf222-inaA/wapQ operon was found (26). Since wapQ encodes a putative heptose
kinase and since two-component systems often regulate a nearby operon, we tested
whether the integrity of the mutant’s outer membrane was impaired because of a
defective LPS. Indeed, mutant PCL1210 is more resistant to various chemically
unrelated antibiotics and grows more slowly on some nutrients. The only single
factor to explain these results is a less permeable outer membrane. Further evidence for an impaired outer membrane came from the finding that the mutant is
more sensitive toward the antibiotic polymixin B, which attacks the outer membrane from the outside (76). We conclude that mutant PCL1210 has a less permeable outer membrane, presumably a disadvantage in the competitive uptake of
exudate components on the root (26).
ROLE OF NADH DEHYDROGENASES IN COMPETITIVE
ROOT TIP COLONIZATION
Dekkers et al (29) found that a mutant of P. fluorescens WCS365 in the nuoD
gene is impaired in competitive root colonization. Two membrane-bound NADH
dehydrogenases have been identified in E. coli: NADH: ubiquinone oxidoreductase or NADH dehydrogenase I (NDH-I), which is encoded by the 14-gene nuo
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operon (147), and NDH-II, which is encoded by ndh (149, 150). Dekkers et al (29)
observed that a competitive root colonization defective mutant of P. fluorescens
WCS365 has its mutation in a nuoD homologue, suggesting a role for NDH-I in
colonization (29). By constructing a second nuoD mutant and biochemically characterizing the enzymes, Camacho Caraval (8) demonstrated that the colonization
defect is indeed due to the defective NDH-I. They showed further the presence
of NDH-II encoded by ndh in P. fluorescens and also that a mutation in this gene
has no effect on competitive colonization. Both the nuo and ndh promoters are
expressed in the rhizosphere (8). NDH-I is involved in the generation of the proton
motive force, which can be used for uptake of various nutrients, generation of ATP,
and ATP-dependent rotation of flagella. The finding that NDH-I plays a role in
colonization can be understood in this context.
ROLE OF RHIZOSPHERE COLONIZATION
IN BIOCONTROL
The ability of P. putida to utilize heterologous siderophores can contribute to the
bacterium’s competence in the rhizosphere (103). The production of phenazine antibiotics contributes to ecological fitness by competing with the resident microflora
(86).
Biocontrol mechanisms of bacteria, such as certain Pseudomonas strains, are
usually based on secreted bioactive factors that attack the pathogen, e.g., antibiotics, exo-enzymes, or HCN (reviewed in 131). Colonization of large parts of the
root system will obviously facilitate biocontrol since colonization can be expected
to function as the delivery system for bacterial cells that act as factories of antifungal metabolites. Indeed, Schippers et al (116) showed that inadequate colonization
leads to decreased biocontrol activity, and Bull et al (7) reported an inverse relation between the numbers of bacteria present on the wheat root and the number of
take-all lesions seen on the plant. On the other hand, Roberts et al (108) claimed
that colonization is not important for biocontrol.
The availability of a series of different colonization genes made it possible to test
thoroughly whether colonization is required for biocontrol (19). As a test system
we used tomato root and foot rot caused by the fungus Fusarium oxysporum f. sp.
radicis lycopersici. Strain PCL1391 of P. chlororaphis controls this disease through
the production of the antifungal metabolite phenazine-1-carboxamide (PCN) (19).
Different colonization-defective mutants of this strain were constructed that were
nonmotile, auxotrophic for phenylalanine, or contained a tetracyclin cassette in the
sss gene. The mutants appeared not to be altered in the production of the extracellular metabolites PCN, HCN, chitinase, and protease and were still antagonistic
against the fungus in a plate test. All three mutants were impaired in colonization
in the gnotobiotic system as well as in potting soil. In contrast to the parental strain,
none of the mutants showed biocontrol activity. Therefore, these results showed for
the first time that root colonization plays a crucial role in biocontrol, presumably
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by functioning as the delivery system for cells producing the antifungal metabolite
PCN (19).
One biocontrol mechanism is not based on direct attack of the pathogen by
the biocontrol microbe but on induction of systemic resistance of the plant by
the microbe (reviewed in 141). Biocontrol by P. fluorescens WCS365 has been
described to act by induced systemic resistance (ISR) (50), in which the mechanism
is different from that of systemically acquired resistance (SAR) in that salicylic
acid and pathogenesis-related (PR) proteins do not play a role in ISR. Mutations in
various colonization genes of WCS365 did not consistently decrease the biocontrol
activity of the strain (27), in contrast to observations for similar mutations in P.
chlororaphis PCL1391. The results suggest that colonization plays a less important
role, if any, in ISR-induced biocontrol. We propose that a temporary or local
presence of ISR-inducing cells is sufficient to cause long-lasting resistance of the
whole root system (27).
QUORUM SENSING AND MICROCOLONIES
Quorum sensing-dependent processes such as conjugation (99) and PCN production (16) require a minimal critical bacterial cell density. The mechanism of quorum
sensing is based on the intracellular requirement of membrane-permeable N-acyl
homoserine lactones (N-AHLs) as autoinducers and transcription factors (47).
Pseudomonas biocontrol bacteria form microcolonies on plant roots (4, 21, 27, 47).
We hypothesize that microcolonies are perfect sites for quorum sensing to take
place because the bacterial cell density is high and the mucigel layer covering the
bacteria could delay diffusion of N-AHLs. Also the observation by Van Elsas et al
(140), that conjugation between pseudomonads is significantly and unexpectedly
stimulated in the rhizosphere, has been attributed to quorum-sensing stimulated
conjugation in microcolonies (21).
COLONIZATION AND BIOFILM FORMATION
In most natural, clinical, and industrial settings, bacteria are predominantly present
in biofilms and not as planktonic cells such as those grown under laboratory conditions (23, 100). O’Toole & Kolter (92) have isolated mutants of strain WCS365 of
P. fluorescens impaired in the initiation of biofilm formation, i.e., in the attachment
to the abiotic polyvinylchloride (PVC) surface of microtiter dishes. They observed
that protein synthesis is required for the initiation of biofilm formation and that the
ClpP protein, a component of the cytoplasmic Clp protease, participates in biofilm
formation. Biofilm formation is strongly influenced by environmental signals: The
presence of Fe3+ and casamino-acids increased biofilm formation whereas high osmolarity decreased it (92). Among the surface attachment defective (sad ) mutants
were many mutants in genes of unknown function (92).
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Since microcolony formation on plant root surfaces can be considered as a form
of biofilm formation, it was interesting to see that strains impaired in flagellar
synthesis were found among sad mutants (92).
OTHER COLONIZATION MUTANTS
The Tn5lacZ competitive colonization mutant PCL1207 (29) spontaneously forms
white colonies on the root after a single inoculation. The colonization defect is
apparently caused by the fact that the transposon is unstable in this mutant, resulting in an altered ratio of blue and white colonies. The mutation of the competitive
colonization mutant PCL1204 (29) is located in moxR, which encodes a regulatory gene of methanol dehydrogenase (L. Dekkers, S. de Weert, C. Mulders &
B. Lugtenberg, unpublished). The mutations in PCL1208 and PCL1217 (29) are
in genes with no significant homology to known genes.
Espinoza-Urgel et al (40) have isolated KT2440 mutants of P. putida impaired
in adhesion to seeds. Three of the eight sequences obtained show no obvious
similarities with known genes, and therefore a novel function has been attributed
to them. One gene showed limited similarities to surface proteins whereas the
other four correspond to putative surface and membrane proteins: a calciumbinding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux
pump (8).
PROMOTORS INDUCED BY EXUDATE
AND IN THE RHIZOSPHERE
Corn root exudates strongly induce put genes of P. putida that are involved in
proline uptake and utilization (13).
Van Overbeek & Van Elsas (142) described a Tn5lacZ R2f mutant of P. fluorescens in which the reporter gene was induced by wheat exudate and by proline.
The induction is specific since root exudates of maize and grass were less effective,
whereas clover root exudate was inactive.
Rainey (104) identified promoters that are active in the rhizosphere but not in
laboratory growth medium.
COMPARISON OF GENES AND TRAITS INVOLVED IN
COLONIZATION OF ROOTS AND OF ANIMAL TISSUES
Several of the genes and traits important for root colonization have also been
implicated in colonization of animal tissues by pathogenic bacteria. Flagella and
motility were shown to be virulence factors in burn wound sepsis by P. aeruginosa
(11). Motility plays a role in colonization of the host light organ by Vibrio fischeri
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(53). LPS plays a role in colonization of the large intestine by S. typhimurium
in mouse (89). The htrB gene, which is impaired in the competitive rhizosphere
colonization mutant PCL1216 of P. fluorescens (29), encoding lauroyl transferase
involved in the biosynthesis of the lipid A part of LPS (22), plays a role in colonizing
the organs of the lymphatic system by S. typhimurium in mouse (63). LPS of
the human pathogens Vibrio cholera, Klebsiella pneumoniae (95), Salmonella
typhimurium, and Shiga toxin-producing E. coli (81) is essential to enable the
pathogen to colonize animal tissues. Found among mutants of Vibrio cholerae
impaired in colonization of the mouse intestine were auxotrophic mutants as well
as mutants in the biosynthesis of biotin and LPS (15). Colonization mutants of
a strain of E. coli that lost the capacity to colonize the GI tract of infant rats
included mutants affected in LPS biosynthesis, tip adhesin on type 1 pili, and
methionine biosynthesis (82). Interestingly, strain PAO-1 of the human pathogen
P. aeruginosa appears to be a biocontrol strain since it is able to protect wheat and
cucumber from the fungal pathogens Gaeumannomyces graminis and Pythium
ultimum, respectively (see 132). Furthermore, the two established prerequisites
for biocontrol of strain PCL1391, colonization (19) and PCN biosynthesis (20),
are the same as the traits found to be important for pathogenesis by strain PA14 of
the human opportunistic pathogen P. aeruginosa (130).
CAN COLONIZATION BE IMPROVED?
It seems reasonable to assume that colonization plays an important role in agroindustrial applications of microbial inoculants such as biocontrol (19, 27), biofertilization (124), phytostimulation, and phytoremediation (69). Three ways to improve
colonization have been identified. First, introduction of multiple copies of an ssscontaining fragment from strain WCS365 into the poor colonizer P. fluorescens
WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold, and
8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic
goal (27). Note that introduction of the same fragment into WCS365 tended to
decrease competitive root colonization, albeit not significantly. We interpret the
results by assuming that multiple copies of sss result in an equilibrium between
the various phases. According to this interpretation, the majority of the wild-type
WCS365 cells would be in a colonization-competent state and multiple sss copies
would lead to a decrease in colonization. In contrast, most cells of strains F113
and WCS307 would be in a colonization-incompetent state and multiple copies of
sss would lead to a higher percentage of colonization-competent cells (27). Second, we have isolated enhanced colonizers by enrichment. To our knowledge, this
classical microbiological approach had never been used to isolate plant-beneficial
bacteria. In this particular case, the goal was to combine the abilities to colonize
plant roots and to degrade the xenobiotic naphtalene. A suspension of bacteria
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from the rhizosphere of a plant grown in naphtalene-contaminated soil was used
to inoculate seedlings. After growth, bacteria that had reached the root tip were
isolated, grown as a mixture on naphtalene as the sole carbon source, and harvested. The procedure was repeated twice. This approach resulted in the isolation
of a bacterium, P. putida PCL1444, which is a 10-fold and 100-fold better root
tip colonizer than strain WCS365 in the absence and presence, respectively, of
naphtalene. We predict that this approach will also be applicable to isolating more
effective biopesticides, biofertilizers, and phytostimulators owing to enhanced colonization properties (69). A third way to enhance colonization was found to be Tn5
mutagenesis of P. fluorescens WCS365 and subsequent enrichment for enhanced
root tip colonizers, as described previously. This resulted in a variety of different mutants that are currently being characterized. Preliminary results indicate
that one mutation is located in a homologue of the ferrisiderophore receptor gene
(L. Dekkers, S. de Weert & B. Lugtenberg, unpublished). Consistent with this
observation is the finding that the ability to utilize ferric pseudobactin M114 does
not improve the ecological fitness of Pseudomonas sp. B24 in the rhizosphere
(88).
Webster et al (146) reported that endophytic colonization of wheat roots by
Azorhizobium caulinodans can be improved by the presence of the flavonoid naringenin. Consistent with this result is the finding by Batinic et al (3) that biocontrol of
Pythium ultimum Trow on cucumber by strain W24 of P. fluorescens can be significantly increased by various flavonoids. We did not find a significant effect of the
presence of 10-µM naringenin in the gnotobiotic system on root tip colonization
by P. fluorescens WCS365 (S. de Weert & B. Lugtenberg, unpublished).
COLONIZATION AND FUNCTIONAL GENOMICS
The complete nucleotide sequence of P. aeruginosa PAO-1 has been determined
(123). Interestingly, this strain is an excellent rhizosphere colonizer of wheat (132).
Once the DNA corresponding with open reading frames has been spotted on
microarrays, many interesting questions can be answered:
1. Which genes are expressed during seed coating, on the seed, and in the
rhizosphere?
2. Which global and specific regulatory genes next to phzI, phzR, gacS (17),
gacA (106), and lexA (18) are involved in the synthesis of the antifungal
metabolite PCN?
3. Through which genes and processes do environmental factors such as glycerol, oxygen limitation, and Fe3+ (54) exert their regulatory action on PCN
expression?
4. What are the target genes of the regulatory genes colR/colS (25), moxR, and
sss (28) involved in rhizosphere colonization?
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481
5. Which novel genes are regulated by quorum sensing (17)?
6. Which genes are influenced by the presence of fungi?
FUTURE PERSPECTIVES
Two developments should dramatically influence our knowledge of rhizosphere
microbiology. First, visualization technology, using CLSM in combination with
various forms of GFP, will allow detailed studies on interactions between various
organisms as well as temporal-spatial aspects of gene expression. Second, functional genomics (including proteomics and metabolomics) will allow us to identify
all genes expressed in the rhizosphere.
The use of promoters that are specifically expressed in the rhizosphere will allow
the engineering of microorganisms for beneficial purposes with minimal loss of
energy. Finally, the interactions between bacteria and fungi in the rhizosphere (41,
73) is an exciting new field of research.
ACKNOWLEDGMENTS
The research described here was supported by numerous grants from the Netherlands Organization for Scientific Research, either directly for Crop Protection and
in cooperation with the Russian Federation or through the Foundations for Earth
and Life Sciences, Chemical Sciences and Technological Sciences. Moreover,
grants were obtained from the European Community through the programs
BIOTECH (BIO2-CT93.0053, BIO2-CT93.0196, BIO4-CT96.0027, BIO4-CT96.
0181, and BIO4-CT98.0254), and Marie Curie fellowships (ERBFMBI-CT982930 and HPMF-CT-1999-00377).
We thank the following colleagues for sending us relevant preprints and reprints:
Dieter Haas, Thierry Heulin, Ann Matthijsse, Ole Nybrö, Jos Raaymakers, Paul
Rainey, Juan Ramos, Jan Sørensen, Linda Tomashow, Jan Tommassen, and David
Weller. We also thank Ludmila Frankova, Janneke van Gaal, Martijn E. Lugtenberg,
and Martha Rodrigues Gomes for typing and literature searches.
Visit the Annual Reviews home page at www.AnnualReviews.org
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Figure 1 Confocal laser scanning microscopical analysis of a tomato root surface
colonized by Pseudomonas fluorescens WCS365 expressing ds-red and Fusarium
oxysporum f. sp. radicis lycopersici expressing g f p (image taken by A. Lagopodi).