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Applied and Environmental Microbiology, December 1999, p. 5357-5363, Vol. 65, No. 12
0099-2240/99/$04.00+0
Utilization of Heterologous Siderophores Enhances
Levels of Iron Available to Pseudomonas putida in the
Rhizosphere
Joyce E.
Loper* and
Marcella D.
Henkels
Agricultural Research Service, U.S.
Department of Agriculture, and Department of Botany and Plant
Pathology, Oregon State University, Corvallis, Oregon
Received 7 April 1999/Accepted 1 October 1999
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ABSTRACT |
Pseudomonas spp. have the capacity to utilize
siderophores produced by diverse species of bacteria and fungi, and the
present study was initiated to determine if siderophores produced by
rhizosphere microorganisms enhance the levels of iron available to a
strain of Pseudomonas putida in this natural habitat. We
used a previously described transcriptional fusion
(pvd-inaZ) between an iron-regulated promoter
(pvd) and the ice nucleation reporter gene
(inaZ) to detect alterations in iron availability to
P. putida. Ice nucleation activity (INA) expressed from the
pvd-inaZ fusion by P. putida N1R or
N1R Pvd
, a derivative deficient in the production of a
pyoverdine siderophore, was inversely related to the concentration of
ferric citrate in a culture medium. In culture, INA expressed by N1R
Pvd (pvd-inaZ) was reduced in the
presence of the ferric complex of pseudobactin-358, a pyoverdine
siderophore produced by P. putida WCS358 that can be
utilized as a source of iron by N1R Pvd. In the
rhizosphere of cucumbers grown in sterilized soil, N1R Pvd (pvd-inaZ) expressed INA,
indicating that iron availability was sufficiently low in that habitat
to allow transcription of the iron-regulated pvd promoter.
Coinoculation with WCS358 or N1R significantly decreased INA expressed
by N1R Pvd (pvd-inaZ) in the
rhizosphere, whereas coinoculation with a pyoverdine-deficient mutant
of WCS358 did not reduce INA expressed by N1R Pvd
(pvd-inaZ). These results indicate that iron
availability to N1R Pvd
(pvd-inaZ) in the rhizosphere was enhanced by
the presence of another strain of P. putida that produces a
pyoverdine that N1R Pvd
(pvd-inaZ) was able to utilize as a source of
iron. In culture, strain N1R Pvd also utilized ferric
complexes of the siderophores enterobactin and aerobactin as sources of
iron. In the rhizosphere of cucumbers grown in sterilized soil, INA
expressed by N1R Pvd (pvd-inaZ)
was reduced in the presence of strains of Enterobacter cloacae that produced enterobactin, aerobactin, or both
siderophores, but INA expressed by N1R Pvd
(pvd-inaZ) was not altered in the presence of a
mutant of E. cloacae deficient in both enterobactin and
aerobactin production. Therefore, the iron status of P. putida was altered by siderophores produced by an unrelated
bacterium coinhabiting the rhizosphere. Finally, we demonstrated that
INA expressed by N1R containing pvd-inaZ in the
rhizosphere differed between plants grown in sterilized versus
nonsterilized field soil. The results of this study demonstrate that
(i) P. putida expresses genes for pyoverdine production and uptake in the rhizosphere, but the level of gene expression is influenced by other bacteria that coexist with P. putida in
this habitat, and (ii) diverse groups of microorganisms can alter the availability of chemical resources in microbial habitats on root surfaces.
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INTRODUCTION |
In environments in which iron is
limited, fluorescent pseudomonads produce pyoverdines, a class of
siderophores comprised of a dihydroxyquinoline chromophore linked to a
peptide of variable length and composition (1). Pyoverdines
are secreted from the bacterial cell and can chelate ferric iron in the
environment via the hydroxamate and hydroxyacid groups present within
the peptide moiety of the molecule (1). Strains of
Pseudomonas spp. have outer membrane receptor proteins that
transport ferric iron complexed to their cognate pyoverdines into the
bacterial cell (3), where the iron becomes available for
metabolic processes. Certain strains can also utilize ferric complexes
of pyoverdines produced by other strains of Pseudomonas spp.
due to the presence of multiple outer membrane receptors that recognize
heterologous pyoverdines (16). Furthermore,
Pseudomonas spp. can utilize iron complexes of a variety of
different siderophores produced by fungi and bacteria (references
12 and 25 and references therein), including the catechol enterobactin (29) and the
hydroxamate aerobactin, which are produced by members of the
Enterobacteriaceae.
Pseudomonas putida and Pseudomonas fluorescens
are common rhizosphere and soil inhabitants, and certain strains
promote plant growth and health by suppressing diseases caused by
soilborne pathogens. The capacity of these strains to produce
pyoverdines is linked, in some cases, with disease suppression; mutants
deficient in pyoverdine production can be less effective than parental
strains in biological control (17). Pyoverdines produced in
situ by Pseudomonas spp. are thought to chelate iron in a
form that is unavailable to pathogens, thereby preventing the
pathogens' access to the already limited pool of soluble iron in the
rhizosphere. We previously described an iron sensor
(pvd-inaZ) that is useful in assessing levels of
biologically available iron in the rhizosphere, in soil, and on leaf
surfaces (18, 20). The iron sensor is comprised of an
iron-regulated promoter of a pyoverdine production and uptake
(pvd) region from Pseudomonas syringae fused to
an ice nucleation reporter gene (inaZ). In culture and in
natural habitats, ice nucleation activity (INA) expressed by
Pseudomonas spp. containing pvd-inaZ
is inversely related to iron availability (18, 20). Our
previous results indicated that INA expressed by P. fluorescens containing pvd-inaZ was not
static on root surfaces but instead varied over time (18).
Numerous factors, such as the solubilization of iron by organic
acids exuded from plant roots (23) or the presence
of phytosiderophores (13) or microbial siderophores
(4), are likely to influence concentrations of iron
available to Pseudomonas spp. on root surfaces, and these could contribute to temporal variations in INA expressed in situ by
Pseudomonas spp. containing pvd-inaZ.
The present study was initiated to determine if levels of iron
available to P. putida are altered by siderophores produced by its microbial coinhabitants in the rhizosphere. The capacity to
utilize heterologous siderophores produced by other members of the
rhizosphere microflora is a proposed fitness factor, conferring a
selective advantage on strains of Pseudomonas spp. (2,
12, 30). In support of this hypothesis, Bakker et al.
(2) demonstrated that the rhizosphere population
size of a mutant of P. putida WCS358 deficient in
pyoverdine production (Pvd) is enhanced in the
presence of the parental strain or another strain that produces a
pyoverdine that WCS358 can utilize. Furthermore, a strain of P. fluorescens that cannot utilize the ferric complex of
pseudobactin-358 is suppressed by WCS358 in the rhizosphere, but this
suppression is eliminated by introduction of the pupA gene,
which encodes the outer membrane receptor for the ferric complex
of pseudobactin-358 (30). These experiments indicate that the capacity to utilize heterologous siderophores can alter the
outcome of interactions among strains of Pseudomonas spp. in
the rhizosphere, as evaluated by the relative population sizes of
coinoculated strains. Because bacterial population size can be altered
by many factors other than iron availability, we initiated this study
to determine if the presence of heterologous siderophores can alter the
iron status of a strain of P. putida in the rhizosphere. We
demonstrated that the rhizosphere bacterium P. putida N1R
can utilize ferric complexes of pseudobactin-358, enterobactin, and aerobactin as sources of iron. Furthermore, INA expressed by N1R containing pvd-inaZ was reduced in the presence
of strains producing these siderophores in the rhizosphere, indicating
that microbial coinhabitants alter iron availability to N1R in this
natural habitat. Finally, we demonstrated that INA expressed by N1R
containing pvd-inaZ in the rhizosphere differs
between plants grown in sterilized versus nonsterilized field soil. The
results of these experiments indicate that the iron status of
P. putida is altered by its coinhabitants in the rhizosphere.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
Bacterial strains are listed
in Table 1. The
pvd-inaZ construct consists of a stable plasmid
containing an iron-regulated promoter, obtained from a cluster of
pyoverdine biosynthesis and uptake genes from P. syringae,
fused to a promoterless ice nucleation reporter gene (20).
Introduction of pvd-inaZ confers iron-regulated INA on Pseudomonas spp. (18, 20). N1R, N1R
Pvd, WCS358 Pvd, and B10 do not produce
detectable ice nuclei without an introduced ice nucleation gene.
pvd-inaZ was introduced into these strains by
conjugation, as described previously (20).
Transcriptional activity of the pvd promoter in
culture.
The effect of iron on INA expressed by
Pseudomonas spp. containing pvd-inaZ
was evaluated in 5-ml shake cultures of modified RSM medium
(5). For 1 liter of medium, 0.75 g of
Ca(NO3)2 · 4H2O, 0.25 g
of MgSO4 · 7H2O, 18.22 g of ACES
[N-(2-acetamido)-2-aminoethanesulfonic acid], and
2.00 g of NaOH were dissolved in 879 ml of deionized water, and
the pH was adjusted to 7.0. After autoclaving, the following sterile
stock solutions were added: 1 ml of 1 M KH2PO4 (pH 7.0), 20 ml of 50% glycerol, and 100 ml of 10% (wt/vol) Casamino Acids. The medium was supplemented with different concentrations of
ferric citrate
(Fe3C6H5O7). To
equalize treatments for citrate, citric acid trisodium salt dihydrate
(C6H5O7Na3 · 2H2O) was added to give a total citrate concentration of
103 M. After 24 h of shaking at 25°C, samples were
evaluated for INA and numbers of culturable bacteria (CFU). The number
of ice nuclei was determined by the droplet freezing assay
(21), and the number of CFU was determined by spreading
diluted samples on King's medium B (KMB) agar (14). INA,
expressed as log10 (ice nuclei/CFU), was calculated as
previously described (21). Reported values are the means of
three replicate cultures. The results from duplicate experiments were
similar, and results of a representative experiment are presented.
Evaluation of siderophore production and utilization by P. putida N1R.
Siderophore production was detected by the
formation of orange halos surrounding bacterial colonies on CAS agar
(32) after 48 h of incubation at 27°C. The capacity
of N1R Pvd to utilize siderophores produced by test
strains of P. putida or Enterobacter cloacae was
detected in a cross-feeding assay. KMB agar was autoclaved, cooled to
50°C, amended with 600 µg of 2,2'-dipyridyl per ml, and seeded with
105 CFU of N1R Pvd per ml. After
solidification, test strains were inoculated by spotting 5-µl drops
on the surface of the seeded agar. Plates were incubated for 48 h,
and growth of N1R Pvd surrounding colonies of test
strains was considered indicative of cross-feeding. The experiment
was done three times with the same results.
Effect of the ferric complex of pseudobactin-358 on INA expressed
by Pseudomonas spp. containing
pvd-inaZ in culture.
Pseudobactin-358
complexed with iron at 80% (to avoid free iron in the system) was
kindly provided by Peter A. H. M. Bakker (Utrecht University,
Utrecht, The Netherlands). The effects of different concentrations of
the ferric complex of pseudobactin-358 on INA of N1R Pvd
(pvd-inaZ), WCS358 Pvd
(pvd-inaZ), and B10
(pvd-inaZ) were evaluated in 5-ml shake cultures containing modified RSM medium amended with 106 M ferric
citrate. After 24 h of incubation at 25°C, samples were evaluated for INA (21) and CFU. Reported values are the
means of three replicate cultures. The results from duplicate
experiments were similar, and results of a representative experiment
are presented.
Cross-feeding of N1R Pvd
(pvd-inaZ) in the rhizosphere.
Cross-feeding of N1R Pvd (pvd-inaZ)
in the rhizosphere of cucumbers was evaluated in enclosed soil
chambers. Cucumber seeds (Cucumis sativus L. cv. Marketmore)
were surface sterilized in a 95% ethanol solution (30 s) followed by a
1% hypochlorite solution (10 min). Seeds were rinsed thoroughly in
sterile deionized water and placed in covered sterile beakers
containing moist filter papers for 2 days at 27°C. Chambers consisted
of centrifuge tubes (Beckman Polyallomer [38 by 102 mm]; Beckman
Instruments Inc., Palo Alto, Calif.) containing 80 ml of Warden sandy
silt loam (pH 7.4; 6.0 mg of Fe per kg) at a matric potential of 
0.03
MPa. Chambers were capped with 50-ml glass beakers and, except where noted, autoclaved for 60 min. The bacterial inoculum was prepared by
growing strains for 24 h at 27°C in 5-ml shake cultures
containing either SM medium (20) amended with
104 M ferric citrate for Pseudomonas spp. or
Luria-Bertani medium (31) for E. cloacae. Cells
were harvested by centrifugation and resuspended in sterile deionized
water to an optical density of 0.1 at 600 nm, which corresponded to
approximately 108 CFU/ml. The inoculum consisted of these
suspensions (to achieve approximately 106 CFU/root system),
100-fold dilutions of these suspensions (to achieve approximately
104 CFU/root system), or 1:1 (vol:vol) mixtures of
suspensions from two bacterial strains. Ten minutes before planting in
the chambers, seedlings were dipped in bacterial suspensions. One
seedling was planted in each chamber. Following planting, capped
chambers were incubated at 25°C with a photoperiod of 12 h. At
various times after planting, root systems were retrieved from each of
five replicate tubes. Root systems were removed, gently shaken to
remove loose soil, and placed in culture tubes containing wash buffer (22). Tubes containing root systems were placed in a
bath-style sonicator for 5 min prior to serial dilution of root
washings. Samples from each dilution series were evaluated by the
droplet freezing assay to estimate INA (21) and spread on
KMB agar supplemented with antibiotics to estimate CFU. Antibiotics
were rifampin at 100 µg/ml for strains of E. cloacae,
P. putida N1R, and P. putida WCS358 and rifampin
at 100 µg/ml and kanamycin at 50 µg/ml for N1R Pvd,
N1R (pvd-inaZ), N1R Pvd
(pvd-inaZ), WCS358 Pvd, and WCS358
Pvd (pvd-inaZ). Petri plates were
incubated at 27°C for 48 h before colonies were counted.
Colonies of Pvd+ and Pvd strains of P. putida could be distinguished by the presence or absence of
fluorescence under UV light (
= 366 nm). Nonamended KMB was
used in all experiments for total bacterial counts and to detect any
contaminants in the rhizosphere of plants grown from untreated control
seed in sterilized soil. All experiments were done twice, with similar
results. Results of a representative experiment of each are presented.
Statistical analysis.
The SAS (Statistical Analysis Systems
Institute, Cary, N.C.) General Linear Models procedure was used for
statistical analysis of INA data. Mean bacterial population sizes in
the rhizosphere were calculated by averaging the logarithm (base 10) of
values obtained for five individual root systems, each of which served as a replicate for statistical analysis. Where statistical differences among treatments are specified, Fisher's protected least significant difference at P of 0.05 was used to separate mean values.
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RESULTS |
P. putida N1R Pvd utilizes ferric
complexes of siderophores produced by other strains of rhizosphere
bacteria.
A Pvd mutant of N1R (N1R
Pvd) did not produce a zone on CAS agar, indicating a
lack of siderophore production on this medium. N1R Pvd
grew very poorly on KMB containing 600 µg of the iron chelator 2,2'-dipyridyl per ml, except within zones surrounding colonies of
bacteria that produced siderophores that N1R Pvd
could utilize. Zones of growth of N1R Pvd were observed
surrounding colonies of strains N1R and WCS358, but no zone was
observed surrounding colonies of WCS358 Pvd (Table
2). Zones of growth of N1R
Pvd also were observed surrounding colonies of
E. cloacae JL1157, which produces the siderophores
enterobactin and aerobactin; LA122, a mutant that produces only
enterobactin; and LA266, a mutant that produces only aerobactin. No
zones of N1R Pvd growth were observed surrounding
colonies of LA235, a mutant of E. cloacae JL1157 that
produces neither aerobactin nor enterobactin.
INA expressed by Pseudomonas spp. containing
pvd-inaZ is inversely related to iron
availability in culture.
INA expressed by P. putida N1R
(pvd-inaZ), P. putida N1R
Pvd (pvd-inaZ), P. putida WCS358 Pvd (pvd-inaZ),
and P. fluorescens B10 (pvd-inaZ)
decreased by 5 to 8 orders of magnitude as the concentration of
ferric citrate in RSM medium was increased from 107 M to
103 M (Fig. 1). INA
expressed by N1R did not differ significantly from that expressed
by N1R Pvd, indicating that the mutation in the
Pvd derivative did not alter INA expressed from the
pvd-inaZ fusion.
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FIG. 1.
INA [log10 (ice nuclei/cell)] expressed by
Pseudomonas spp. containing pvd-inaZ
in RSM medium amended with ferric citrate. P. putida N1R
(pvd-inaZ) ( ) and the pyoverdine-deficient
mutant N1R Pvd (pvd-inaZ) ( )
(A), P. putida WCS358 Pvd
(pvd-inaZ) ( ) (B), and P. fluorescens B10 (pvd-inaZ) ( ) (C) were
grown for 24 h in RSM medium amended with 103 to
107 M ferric citrate. INA values are means of three
replicate cultures. Error bars represent standard errors of the means;
most error bars are obscured by graph symbols.
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INA expressed by N1R Pvd
(pvd-inaZ) in culture is diminished in the
presence of ferric pseudobactin-358.
In RSM medium containing
106 M ferric citrate, INA was expressed by virtually
every cell of N1R Pvd (pvd-inaZ),
WCS358 Pvd (pvd-inaZ), or B10
(pvd-inaZ) (i.e., approximately 0 log10 [ice nuclei/cell]) (Fig.
2). When the medium was amended with
104 M of the ferric complex of pseudobactin-358, the
pyoverdine produced by WCS358, INA expressed by N1R Pvd
(pvd-inaZ) decreased by 2 orders of magnitude
(Fig. 2). INA expressed by WCS358 Pvd
(pvd-inaZ) decreased by almost 5 orders of magnitude in the presence of 104 M ferric
pseudobactin-358 (Fig. 2). In contrast, INA expressed by B10
(pvd-inaZ) did not change in the presence of
ferric pseudobactin-358 (Fig. 2), a siderophore that B10 could not
utilize as a source of iron (data not shown). Therefore, exogenous
sources of ferric pseudobactin-358 decreased the INA of only those
strains that could utilize the siderophore as a source of iron.
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FIG. 2.
Influence of ferric pseudobactin-358 on INA
[log10 (ice nuclei/cell)] expressed by
Pseudomonas spp. containing pvd-inaZ.
P. putida N1R Pvd
(pvd-inaZ) (A), P. putida WCS358
Pvd (pvd-inaZ) (B), and
P. fluorescens B10 (pvd-inaZ) (C)
were grown for 24 h in a medium amended with 104,
105, or 106 M ferric pseudobactin-358 or a
nonamended medium. INA values are means of three replicate cultures.
Error bars represent standard errors of the means.
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INA expressed by N1R Pvd
(pvd-inaZ) in the rhizosphere of cucumbers
decreases in the presence of WCS358 or N1R.
One day after
inoculation of roots, N1R Pvd
(pvd-inaZ) expressed 1.4 log10 (ice
nuclei/cell) in the rhizosphere of cucumbers (Fig. 3A and
C). When coinoculated with
106 CFU of WCS358 or the wild-type strain N1R per
root, both of which produce pyoverdines that N1R Pvd
could utilize, INA expressed by N1R Pvd
(pvd-inaZ) was only ca. 2.6 log10
(ice nuclei/cell) (Fig. 3A). In contrast, coinoculation with WCS358
Pvd did not decrease INA expressed by N1R
Pvd (pvd-inaZ). A similar pattern
of INA expression by N1R Pvd
(pvd-inaZ) was observed at 6 days following
its inoculation onto cucumber roots (Fig. 3B), although INA at 6 days
was less than that at 1 day for all treatments. When coinoculated with
104 CFU of WCS358 or N1R per root, INA expressed by N1R
Pvd (pvd-inaZ) was decreased
significantly at 6 days following inoculation (Fig. 3D).
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FIG. 3.
Influence of coinoculated strains of P. putida on INA [log10 (ice nuclei/cell)] expressed by
P. putida N1R Pvd
(pvd-inaZ) in the rhizosphere of cucumber grown
in sterilized soil. Surface-sterilized cucumber roots were inoculated
with 104 CFU of N1R Pvd
(pvd-inaZ) per root system alone (Control) or in
combination with 106 CFU (A and B) or 104 CFU
(C and D) of a coinoculated strain (WCS358, WCS358
Pvd, or N1R) per root system and planted in sterilized
soil. INA expressed by P. putida N1R Pvd
(pvd-inaZ) was assessed at 1 day (A and C) or 6 days (B and D) after planting. INA values are means of five replicate
root systems. Error bars represent standard errors of the means.
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When inoculated at 10
6 CFU/root, all of the
strains (WCS358, WCS358 Pvd
, and N1R) decreased the
population size of N1R Pvd
(
pvd-
inaZ) to similar degrees (Table
3). When inoculated at
10
4
CFU/root, none of the strains consistently decreased the population
size of N1R Pvd
(
pvd-
inaZ) in two
replicated experiments.
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TABLE 3.
Influence of P. putida strains differing in
pyoverdine production on population size of P. putida N1R
Pvd (pvd-inaZ) in the
rhizosphere of cucumber
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INA expressed by N1R Pvd
(pvd-inaZ) in the rhizosphere of cucumbers
decreases in the presence of E. cloacae JL1157.
When inoculated at 104 CFU/root, E. cloacae JL1157 decreased INA expressed by coinoculated cells
of N1R Pvd (pvd-inaZ) at 1 day
after planting (Fig. 4A). In contrast,
mutants of JL1157 deficient in aerobactin production (LA122),
enterobactin production (LA266), or production of both siderophores
(LA235) did not decrease significantly INA expressed by coinoculated
cells of N1R Pvd (pvd-inaZ). Five
days following its inoculation onto cucumber roots, N1R
Pvd (pvd-inaZ) expressed less INA
in rhizospheres coinoculated with JL1157 or the aerobactin-producing
derivative LA266 than in the rhizosphere of control plants (Fig. 4B).
INA expressed by N1R Pvd (pvd-inaZ)
was greater in rhizospheres coinoculated with LA235, which produces
neither enterobactin nor aerobactin, than in the rhizosphere of control
plants. Coinoculation with LA235 also decreased the population size
of N1R Pvd (pvd-inaZ) in one
experiment (Table 4), but none of the
strains of E. cloacae had a consistent influence on the
population size of N1R Pvd
(pvd-inaZ) in both replicated experiments.
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FIG. 4.
Influence of coinoculated strains of E. cloacae on INA [log10 (ice nuclei/cell)] expressed
by P. putida N1R Pvd
(pvd-inaZ) in the rhizosphere of cucumber grown
in sterilized soil. Surface-sterilized cucumber roots were inoculated
with 104 CFU N1R Pvd
(pvd-inaZ) per root system alone (Control) or in
combination with 104 CFU of a strain of E. cloacae (JL1157 [Ent+ Iuc+], LA122
[Ent+ Iuc], LA266 [Ent
Iuc+], or LA235 [Ent Iuc])
per root system and planted in sterilized soil. INA expressed by
P. putida N1R Pvd
(pvd-inaZ) was assessed at 1 day (A) or 5 days
(B) after planting. INA values are means of five replicate root
systems. Error bars represent standard errors of the means.
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TABLE 4.
Influence of E. cloacae strains differing in
enterobactin and aerobactin production on population size of P. putida N1R Pvd (pvd-inaZ)
in the rhizosphere of cucumber
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INA expressed by N1R Pvd
(pvd-inaZ) and N1R
(pvd-inaZ) in the rhizosphere of cucumbers
grown in field soil.
Because the results described above indicated
that siderophores produced by specific bacteria could alter iron
availability to N1R Pvd (pvd-inaZ)
in the rhizosphere, we evaluated the influence of the native microflora
on INA expressed by N1R Pvd
(pvd-inaZ) and N1R (pvd-inaZ).
One day after inoculation of cucumber roots grown in sterilized soil,
N1R Pvd (pvd-inaZ) expressed
34-fold-greater INA than that expressed by N1R
(pvd-inaZ) (Fig.
5A). In sterilized soil, INA expressed by
N1R (pvd-inaZ) was not altered significantly by
coinoculation with WCS358 or WCS358 Pvd, whereas INA
expressed by N1R Pvd (pvd-inaZ) was
decreased in the presence of WCS358. INA expressed by both
strains was lower at 6 days than at 1 day following inoculation, but
the influence of WCS358 on INA expressed by N1R Pvd
(pvd-inaZ) was statistically significant on both
days.
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FIG. 5.
Influence of soil sterilization on INA
[log10 (ice nuclei/cell)] expressed by P. putida N1R (pvd-inaZ) or N1R
Pvd (pvd-inaZ) in the rhizosphere
of cucumbers. Surface-sterilized cucumber roots were inoculated with
104 CFU of N1R (pvd-inaZ) or N1R
Pvd (pvd-inaZ) per root system
alone (Control) or in combination with 104 CFU of a second
strain (WCS358 or WCS358 Pvd) per root system and planted
in sterilized soil (A and B) or nonsterilized soil (C and D). INA
expressed by P. putida N1R Pvd
(pvd-inaZ) and N1R (pvd-inaZ) was
assessed at 1 day (A and C) or 6 days (B and D) after planting. INA
values are means of five replicate root systems. Error bars represent
standard errors of the means.
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One day after inoculation, INA expressed by N1R
(
pvd-
inaZ) and N1R Pvd
(
pvd-
inaZ) was lower in the rhizosphere of plants
grown in nonsterilized
field soil than in sterilized field soil (Fig.
5). INA expressed
by the strains did not decrease markedly over time in
the rhizosphere
of plants grown in nonsterilized field soil.
Furthermore, WCS358
did not reduce INA expressed by N1R
Pvd
(
pvd-
inaZ) significantly in the
rhizosphere of cucumbers grown
in nonsterilized field
soil.
Populations of all strains were approximately 100-fold larger in the
rhizosphere of plants grown in sterilized soil than in
nonsterilized
field soil (Table
5). In one experiment,
the population
size of N1R Pvd
(
pvd-
inaZ) in the rhizosphere of plants grown in
sterilized soil
was decreased when coinoculated with WCS358
Pvd
(Table
5). In this experiment, coinoculation with
strain WCS358
altered the population size of N1R
(
pvd-
inaZ) but not of N1R Pvd
(
pvd-
inaZ) in the rhizosphere of plants grown
in sterilized soil.
These effects were not observed in the rhizosphere
of plants grown
in sterilized soil in a second experiment (data not
shown) or
in nonsterilized field soil in either of the two experiments.
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TABLE 5.
Influence of P. putida strains differing in
pyoverdine production on population size of P. putida N1R
Pvd (pvd-inaZ) or P. putida N1R (pvd-inaZ) in the rhizosphere of
cucumber grown in sterilized or nonsterilized soil
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| |
DISCUSSION |
The results of this study demonstrate that the iron status of
P. putida in the rhizosphere is altered by the presence
of siderophores produced by other bacterial strains
occupying that habitat. These results complement those of a previous
study demonstrating that the rhizosphere population size of
P. putida is influenced by its capacity to utilize
pyoverdines produced by coinoculated strains (2, 30). The
ice nucleation reporter gene system described here provides a
complementary approach to the assessment of population size for studies
evaluating the importance of siderophore utilization patterns in
microbial interactions in the rhizosphere. In this study, certain
bacterial strains influenced INA expressed by N1R Pvd (pvd-inaZ) while having minimal
effects on the population size of N1R Pvd
(pvd-inaZ). We speculate that INA may be
sensitive to alterations in iron availability that are too small to
have immediate effects on population size. Because iron availability
affects many metabolic processes of bacteria, alterations in iron
availability that are not associated with immediate and detectable
changes in population size are nonetheless likely to have ultimate
effects on the fitness of a bacterial strain. The
pvd-inaZ construct provides a tool for assessing
specific effects of bacteria on the physiology of their microbial
coinhabitants in natural habitats.
Pseudomonas spp. are known to have the capacity to utilize
siderophores produced by diverse species of microorganisms (12, 16, 25, 29). In this study, we provide evidence that
E. cloacae produces enterobactin and aerobactin in the
rhizosphere in concentrations that are recognized as iron sources by
a coinoculated strain of P. putida. Strains of
E. cloacae that produced aerobactin, even in the absence of
enterobactin production, were particularly effective in enhancing iron
availability to P. putida. Although the affinity of
aerobactin for iron is lower than that of enterobactin (10,
11), aerobactin is more stable and water soluble than enterobactin and is active over a range of pH values (28).
Aerobactin's stability and other chemical properties contribute to its
importance as a virulence factor in Escherichia coli
(28) and also are likely to be important factors in
determining its role in ecological interactions of bacteria in the
rhizosphere. Other factors, such as the relative levels of
aerobactin and enterobactin produced in situ by E. cloacae, could also account for the differential contribution of
these siderophores to the iron status of P. putida. Irrespective of the relative importance of enterobactin and aerobactin in the rhizosphere, the contribution of both siderophores to the iron
nutrition of P. putida suggests that diverse groups of
microorganisms can alter the chemical composition of microbial habitats
on root surfaces.
Previously, we demonstrated that the iron-regulated promoter evaluated
in this study is not expressed uniformly over time by P. putida in the rhizosphere or the spermosphere. Instead, transcriptional activity reached a maximum within approximately 24 h after P. putida was inoculated onto root or seed surfaces and decreased thereafter. One possible explanation for this decrease in
transcription was that iron complexed by microbial siderophores became
more available to P. putida in the rhizosphere or
spermosphere over the course of an experiment. The results of the
present study argue that siderophores produced by other rhizosphere
bacteria enhance iron availability to P. putida.
Nevertheless, siderophores produced by indigenous soil microorganisms
could not account for the temporal patterns of pvd gene
expression by P. putida in the rhizosphere of plants grown
in nonsterilized field soil. INA expressed by N1R
(pvd-inaZ) and N1R Pvd
(pvd-inaZ) was stable in nonsterilized field soil
relative to sterilized soil, a result opposite that which would be
expected if the decrease in pvd gene expression were caused
by siderophores produced by the native soil microflora. The population
sizes of N1R and N1R Pvd increased to much higher levels
in the rhizosphere of plants grown in sterilized than in nonsterilized
field soil, and the relative physiological statuses of the bacteria in
the two environments provide one possible explanation of the temporal
patterns of pvd gene expression. In the field soil,
competition for resources other than iron could have limited the
population size of P. putida such that the pvd
promoter was transcribed at a stable, moderate level throughout the
experiment. In contrast, sterilized soil may be transiently replete in
carbon, because the organic matter content of soil is enhanced
(8) and few microbial competitors are present immediately
after autoclaving. With adequate carbon, iron may have been the
limiting resource to P. putida immediately after it was
inoculated onto the root surfaces of plants grown in sterilized soil,
but carbon or other resources could then become limiting as they were
utilized by the inoculated bacterium.
The production of pyoverdines by Pseudomonas spp. in the
rhizosphere is one mechanism by which these bacteria suppress soilborne bacteria and fungi that cause plant disease (17). The
results of this study confirm that P. putida expresses genes
for pyoverdine production and uptake in the rhizosphere, but the level
of gene expression is influenced by other bacteria that coexist with
P. putida in this habitat. Furthermore,
siderophore-mediated interactions between microorganisms can be
altered by the indigenous microflora, as evidenced by the differential
effects of WCS358 on INA expressed by N1R Pvd
(pvd-inaZ) in sterilized and nonsterilized field
soils. These data provide an example of how the expression of genes
involved in biological control can be altered by biotic components of
the environment and point to microbial community context as
a factor influencing the in situ activities of
biological control agents.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge the helpful reviews of this manuscript
that were provided by Mary Hagen, Steve Lindow, and Virginia Stockwell.
We are also grateful to Peter Bakker for the generous gift of
P. putida WCS358 and WCS358 Pvd and for
the authentic sample of pseudobactin-358.
This research was supported in part by interagency agreement
DWI2935653-01-2 between the biotechnology program of the U.S. Environmental Protection Agency and the U.S. Department of Agriculture.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Horticultural
Crops Research Laboratory, Agricultural Research Service, U.S.
Department of Agriculture, 3420 N.W. Orchard Ave., Corvallis, OR 97330. Phone: (541) 750-8771. Fax: (541) 750-8764. E-mail:
loperj{at}bcc.orst.edu.
| |
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Abstract
Introduction
