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Applied and Environmental Microbiology, January 2000, p. 87-91, Vol. 66, No. 1
0099-2240/0/$04.00+0
Importance of pfkA for Rapid Growth of
Enterobacter cloacae during Colonization of Crop Seeds
D. P.
Roberts,1,*
P. D.
Dery,1
I.
Yucel,1,
and
J.
S.
Buyer2
Biocontrol of Plant Diseases
Laboratory1 and Soil Microbial Systems
Laboratory,2 USDA Agricultural Research
Service, Beltsville, Maryland 20705
Received 21 June 1999/Accepted 19 October 1999
 |
ABSTRACT |
Enterobacter cloacae A-11 is a prototrophic, glycolytic
mutant of strain 501R3 with a single transposon insertion in
pfkA. The populations of strain A-11 on cucumber and radish
seeds were smaller than the populations of strain 501R3 in natural
soil, but the populations of these two strains on pea, soybean,
sunflower, and sweet corn seeds were similar (D. P. Roberts,
P. D. Dery, I. Yucel, J. Buyer, M. A. Holtman, and D. Y. Kobayashi, Appl. Environ. Microbiol. 65:2513-2519, 1999).
The net effect of the mutation in pfkA in vitro was a shift
from rapid growth on certain carbohydrates detected in seed exudates to
much slower growth on other carbohydrates, amino acids, and organic
acids. The impact of the mutation in pfkA was greatest on
the growth rate of E. cloacae on the seeds that released
the smallest quantities of fructose, other carbohydrates, and amino
acids. Corn, pea, soybean, and sunflower seeds released total amounts
of carbohydrates and amino acids at rates that were approximately 10- to 100-fold greater than the rates observed with cucumber and radish
seeds for the first 24 h after inhibition began. The growth rate of
strain A-11 was significantly less (50% less) than the growth rate of
strain 501R3 on radish seeds, and the growth rate of strain A-11 was too low to estimate on cucumber seeds in sterile sand for the first
24 h after inhibition began. The growth rate of strain A-11 was
also significantly lower on soybean seeds, but it was only 17% lower
than the growth rate of strain 501R3. The growth rates of strains 501R3
and A-11 were similar on pea, sunflower, and corn seeds in sterile sand
for the first 30 h after imbibition began. Large reductions in the
growth rates of strain A-11 on seeds were correlated with subsequent
decreased levels of colonization of seeds compared to the levels of
colonization of strain 501R3. The strain A-11 populations were
significantly smaller than the strain 501R3 populations only on radish
and cucumber seeds. The mutation in pfkA appears to
decrease the level of colonization by E. cloacae for seeds
that release small quantities of reduced carbon compounds by decreasing
the size of the pool of compounds that support rapid growth by this bacterium.
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INTRODUCTION |
Colonization of the subterranean
portions of plants by bacteria can be an essential process when these
organisms are used for beneficial purposes, such as plant growth
promotion, plant disease control, and bioremediation (2, 3,
5). Several traits, including motility, chemotaxis, salt
tolerance, binding to roots, and the production of the O-antigenic side
chain of lipopolysaccharide, have been correlated with the colonization of plant surfaces (1, 6, 7, 9, 10, 21). It has also been
established that microbial growth is an essential process for
colonization (20, 22-26, 28, 29). The complex mixtures of
carbohydrates, amino acids, organic acids, and other nutrients (4) released from seeds and roots are thought to support the growth of beneficial bacteria in the spermosphere and rhizosphere. We
have used a mutational approach to study the role of the bacterial genes and catabolic pathways and the nutrients supplied by the host
plant during growth and colonization of seeds by the potential biocontrol bacterium Enterobacter cloacae 501R3 (17,
18, 22-26).
E. cloacae A-11 is a prototrophic, glycolytic mutant of
strain 501R3 with a single mini Tn5-Km transposon insertion
in pfkA (22, 26). Roberts et al. found that the
populations of strain A-11 on cucumber and radish seeds in natural soil
were smaller than the populations of strain 501R3 but the populations
of these two strains on pea, soybean, sunflower, and sweet corn seeds
were similar (26). It is reasonable to speculate that the
mutation in pfkA and the resulting block in glycolysis
decrease the populations of E. cloacae A-11 on cucumber and
radish seeds by limiting the number of available compounds that support
rapid growth by this bacterium. The following findings support this
hypothesis. (i) E. cloacae 501R3 was capable of rapid in
vitro growth on a large number of carbohydrates found in seed exudates,
while strain A-11, which has a mutation in pfkA, had a
dramatically lower in vitro growth rate or exhibited no in vitro growth
on all of the carbohydrates detected in seed exudates that support
rapid growth except fructose (22, 25, 26). Wild-type growth
of strain A-11 on fructose was expected since this sugar enters
glycolysis after the metabolic block in pfkA
(27). And (ii) strains A-11 and 501R3 exhibited comparable
levels of colonization of seeds that released relatively large
quantities of fructose. Cucumber and radish seeds released substantially less fructose than pea, sunflower, soybean, and sweet
corn seeds (26).
We studied the impact of the mutation in pfkA on the growth
rate during colonization of seeds by E. cloacae in a sterile
sand system by using germinating seeds as continuous sources of reduced carbon compounds. Other methods, such as growth in batch culture or in
chemostats containing defined mixtures of reduced carbon compounds, do
not approximate spermosphere nutritional conditions (8, 11-13,
16). We used the sterile sand system to demonstrate that the
mutation in pfkA decreases the growth rate of E. cloacae during colonization of the spermospheres of certain seeds.
The impact of this mutation on the growth rate during colonization by
E. cloacae was greatest for the seeds that released the
smallest quantities of fructose, other carbohydrates, and amino acids.
(Portions of this work have been published previously
[25].)
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MATERIALS AND METHODS |
Analysis of aqueous seed extracts.
Extracts of cucumber,
pea, radish, soybean, sunflower, and sweet corn seeds were prepared
essentially as described previously (17, 22, 25) after
various incubation periods in sterile distilled water (SDW). The total
carbohydrate contents of samples were estimated by using the anthrone
assay (15); glucose was used as the standard. Individual
carbohydrates were identified and quantified as trifluoracetyl
derivatives by using gas chromatography as described previously
(25, 31). The total amino acid contents of samples were
estimated by using the ninhydrin assay (30); L-leucine was used as the standard.
Estimation of growth rates on seeds.
E. cloacae
strains were grown overnight at 35°C and 250 rpm in Luria-Bertani
broth (14) supplemented with 100 µg of rifampin per ml for
strain 501R3 and 100 µg of rifampin per ml and 50 µg of kanamycin
per ml for strain A-11. Overnight cultures were washed, resuspended,
and applied to single cucumber (Cucumis sativum cv. Marketmore 76), radish (Raphinus sativus cv. Cherry Bomb),
pea (Pisum sativum cv. Sugarsnap), soybean (Glycine
max cv. Chesapeake), sunflower (Helianthus giganteus),
and sweet corn (Zea mays cv. Stowells Evergreen) seeds, and
the seeds were each buried in 4 g of washed, sterile sand
containing 4 ml of SDW in a 14-ml sterile snap cap tube; the resulting
preparations were incubated at 22°C for 30 h. The seeds were
surface disinfested with 5% bleach and washed with SDW before the
bacterial suspensions were applied. The numbers of CFU were determined
periodically by spiral plating (Spiral Systems, Cincinnati, Ohio) the
entire contents of a tube onto Luria-Bertani agar containing 100 µg
of cycloheximide per ml and the appropriate antibiotics for each
bacterial strain. Experiments were performed with surface-disinfested
seeds and sterile sand in order to decrease the effects arising from
competition with indigenous microbes for nutrients released from the
seeds. In all of the experiments rifampin-resistant, E. cloacae-like colonies represented 90 to 99% of the bacteria
detected in the samples.
Nonlinear regression techniques were used to obtain a logistic growth
curve for each of the experiments in which we compared growth of
strains 501R3 and A-11 on individual plant species. Each experiment was
performed with seeds from one plant species and was performed six times
with four replicate seeds at each of five sampling times (6, 12, 18, 24, 30 h). The results obtained in all experiments performed with
all six plant species were combined prior to analysis. The growth rate
and final population size were estimated from each growth curve and
were analyzed by using mixed-model analysis of variance techniques (SAS
Institute, Cary, N.C.) to determine the fixed effects of the seed type
and the bacterial strain and their interactions. Our interpretation of
the analyses was based on strain-seed interactions and on pairwise mean
comparisons of strains for each seed type.
In vitro growth assays.
E. cloacae 501R3 was
grown overnight in M56 salts broth (19) containing 0.2%
glycerol at 200 rpm and 32°C. The overnight cultures were
centrifuged, washed with 10 mM magnesium sulfate, and suspended in 10 mM magnesium sulfate to an optical density at 540 nm of 1.00. Test
tubes containing 5 ml of M56 salts broth supplemented with a
carbohydrate at a concentration of 0.2%, an L amino acid
at a concentration of 0.5%, or an organic acid at a concentration of
0.2% were inoculated with 100-µl portions of this suspension. The
cultures were incubated at 32°C and 200 rpm, and the optical
densities at 540 nm were determined periodically. Generation times were
calculated as described by Miller (14).
| |
RESULTS AND DISCUSSION |
Growth rates on seeds and seed colonization.
Previously, it
was shown that glycolysis in E. cloacae A-11 was blocked by
a mutation in pfkA and that this gene was most important for
colonization of seeds that released limited quantities of reduced
carbon compounds. The populations of strain A-11 were significantly
smaller than the populations of strain 501R3 on cucumber and radish
seeds in sterile sand and in natural soil but not on pea, soybean,
sunflower, and sweet corn seeds. Roberts et al. described the strain
A-11 phenotype in detail but did not explore the underlying reasons for
the smaller strain A-11 populations on cucumber and radish seeds
(26).
Data obtained in this current study indicated that the lower levels of
colonization of cucumber and radish seeds by strain
A-11 were due to
dramatically lower strain A-11 growth rates on
these seeds. A
comparison of the growth rates of strains A-11
and 501R3, derived from
a logistic model of seed colonization
by these strains, showed that the
growth rate of strain A-11 was
one-half the growth rate of strain
501R3 on radish seeds (significantly
different at
P 
0.003) and that the strain A-11 growth rate on
cucumber seeds
was so low that it could not be estimated (Table
1). The growth rate of strain A-11 was
only 17% lower on soybean
seeds (significantly different at
P 0.001) than the growth rate
of strain 501R3 and
was similar to the growth rate of strain 501R3
on pea, sunflower, or
sweet corn seeds (Table
1). As expected
with dramatic reductions in the
growth rate, the strain A-11 populations
were significantly smaller
(
P 0.05) than the strain 501R3 populations
on radish
seeds 18 h after application and on cucumber seeds 12,
18, 24, and
30 h after application, as determined in these experiments
(Fig.
1). The sizes of the strain A-11 and
501R3 populations were
similar at all sampling times on pea, soybean,
sunflower, and
sweet corn seeds, on which the growth rates of strain
A-11 were
essentially similar to the growth rates of strain 501R3.
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TABLE 1.
Growth rates and final population sizes of E. cloacae strains during colonization of various seeds, as estimated
with a logistic model
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FIG. 1.
Population dynamics of E. cloacae A-11 ( )
and 501R3 ( ) on various types of seeds in sterile sand during the
first 30 h after imbibition began. The error bars represent 1 standard deviation from the mean.
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In vitro growth on compounds in seed exudates.
The generation
times of E. cloacae 501R3 were determined by performing in
vitro growth assays in minimal medium containing individual
carbohydrates detected in seed exudates. The generation times of strain
501R3 were 69.6 ± 13.1 min on arabinose, 51.4 ± 5.5 min on
cellobiose, 60.8 ± 0.1 min on fructose, 58.5 ± 3.1 min on
galactose, 51.3 ± 5.9 min on glucose, 70.8 ± 21.2 min on maltose, 55.6 ± 4.5 min on mannitol, 62.3 ± 6.8 min on
raffinose, 69.2 ± 1.2 min on ribose, 51.0 ± 2.3 min on
sucrose, 55.4 ± 2.2 min on trehalose, and 85.1 ± 11.7 min
on xylose. We also determined the generation times of strain 501R3 on
individual L amino acids and organic acids that have been
reported to be present in seed exudates that support growth of E. cloacae. The generation times of strain 501R3 were 127.8 ± 26.7 min on alanine, 136.7 ± 11.5 min on asparagine, 198.6 ± 30.9 min on proline, 130.8 ± 2.5 min on glutamate, 200.1 ± 51.6 min on glutamine, 376.2 ± 79.8 min on serine, 100.9 ± 7.4 min on pyruvate, and 94.1 ± 2.7 min on malate. The mean
generation times during in vitro growth of E. cloacae 501R3
on the carbohydrates, amino acids, and organic acids tested were
61.7 ± 9.9, 195.0 ± 86.5, and 97.5 ± 3.4 min, respectively.
The mutation in
pfkA in
E. cloacae A-11 reduced
the number of carbohydrates in seed exudates that supported wild-type
in vitro
growth of this bacterium. Of the carbohydrates detected in
seed
exudates, strain A-11 grew rapidly in vitro only on fructose.
The
growth of strain A-11 and the growth of strain 501R3 on amino
acids and
organic acids were similar (
26). When we extrapolated
from
the results of in vitro growth experiments performed with
strain 501R3,
the net effect of the mutation in
pfkA in strain
A-11 on
growth on compounds released in seed exudates was a shift
from rapid
growth on carbohydrates to slower growth on amino acids
and organic
acids.
Seed exudation and growth rate.
The mutation in
pfkA in strain A-11 appears to decrease the growth rate and
the level of colonization by E. cloacae on seeds that
release small quantities of reduced carbon compounds by limiting the
quantity of the compounds that support rapid growth of this bacterium.
Three lines of evidence support this hypothesis. First, the decreases
in the growth rates of strain A-11 compared to the growth rates of
strain 501R3 were substantially greater on cucumber and radish seeds
than on the other four types of seeds tested (Table 1). The quantities
of compounds released by cucumber and radish seeds that supported rapid
growth of E. cloacae were substantially less than the
quantities of compounds released by the other four types of seeds
tested (Fig. 2). Corn, pea, soybean, and
sunflower seeds released 40- to 1,500-fold more carbohydrates (as
detected by gas chromatography) that supported rapid in vitro growth of E. cloacae than cucumber or radish seeds released. The
carbohydrates that supported rapid growth (defined as in vitro
generation times of 65 min) were cellobiose, fructose, galactose,
glucose, mannitol, raffinose, sucrose, and trehalose. In addition,
corn, pea, and soybean seeds released approximately 50- to 100-fold
more total carbohydrate than cucumber and radish seeds released during
the first 24 h after imbibition began. Sunflower seeds released
approximately 10-fold more total carbohydrate than cucumber and radish
seeds released (Fig. 2). In general, carbohydrates support much more rapid in vitro growth than amino acids or organic acids support. Second, several sugars were detected, but strain A-11 could grow rapidly only on fructose. Pea, soybean, sunflower, and sweet corn seeds
released 56- to 2,500-fold more fructose than cucumber and radish seeds
released. In fact, the quantities of fructose released by pea, soybean,
and sweet corn seeds during the first 24 h after imbibition began
were greater than the combined total quantities of carbohydrates and
amino acids released by cucumber and radish seeds. Third, the
quantities of sugars available that supported rapid growth of E. cloacae were significantly greater with strain 501R3 than with
strain A-11 on cucumber and radish seeds; on these seeds the quantities
of sugars that supported rapid growth were very small, the growth rate
of strain A-11 was significantly lower than the growth rate of strain
501R3, and the populations of strain A-11 were significantly smaller
than the populations of strain 501R3. The concentrations of
high-growth-rate sugars available to strains 501R3 and A-11 were
0.13 ± 0.09 and 0.02 ± 0.01 µg per seed, respectively, on
cucumber seeds and 0.09 ± 0.05 and 0.03 ± 0.01 µg per
seed on radish seeds, respectively.
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FIG. 2.
Cumulative quantities of fructose, high-growth-rate
sugars, total carbohydrate, and total amino acid released from sweet
corn ( ), pea ( ), cucumber ( ), soybean (), radish ( ), and
sunflower () seeds during the first 24 h after imbibition
began. The high-growth-rate sugars resulted in in vitro generation
times of 65 min for strain 501R3. The high-growth-rate sugars are
cellobiose, fructose, galactose, glucose, mannitol, raffinose, sucrose,
and trehalose. The error bars represent 1 standard deviation from the
mean.
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The colonization data presented here indicate that the spermospheres of
all of the seeds tested were not carbon limited. The
estimated final
population sizes of strains A-11 and 501R3 were
similar despite the
inability of strain A-11 to use substantial
quantities of reduced
carbon that were available to strain 501R3
(Table
1 and Fig.
2). The
final population sizes of strain A-11
on cucumber seeds could not be
estimated with the model due to
the low growth rate. In another study,
the population sizes of
strains A-11 and 501R3 were similar 96 h
after application for
all six seed types in seed colonization
experiments performed
in sterile sand (
26). This suggests
that required nutrients
other than reduced carbon compounds were
limiting in the spermospheres
of the
seeds.
The growth rates of strains A-11 and 501R3 were not strictly correlated
with the reduced carbon compounds detected in seed
exudates. For
example, the estimated growth rate of strain 501R3
on corn seeds was
much lower than the growth rate of strain 501R3
on sunflower seeds.
Corn seeds release substantially greater quantities
of individual
sugars that support rapid growth of
E. cloacae,
total
carbohydrates, and total amino acids (Table
1 and Fig.
2). A regression
analysis indicated that there was no correlation
between the growth
rates on seeds of the plant species tested
and the quantities of total
carbohydrate, total amino acid, fructose,
or high-growth-rate sugars
released or the rates of release of
these compounds (data not shown).
High-level correlations between
growth rate and exudation of reduced
carbon compounds by the seed
types tested were not expected under
non-carbon-limiting
conditions.
Conclusions.
It has been established that microbial growth is
an essential process for colonization of plant surfaces (20,
22-26, 28, 29). It can be assumed that the ability of a
microorganism to grow on reduced carbon compounds in exudates
contributes directly to the ability of the organism to colonize a
particular host plant. Hence, the qualitative nature of reduced carbon
compounds in exudates with regard to supporting rapid growth should
influence colonization. Rapid growth on seeds and roots should be
advantageous when it comes to utilizing limiting resources, such as
nutrients and space in competitive environments like the plant
spermosphere and rhizosphere. It is thought that introduced beneficial
bacteria must preempt indigenous bacteria to become established
(32).
Studies of the growth of bacteria in batch cultures have shown that
different reduced carbon compounds influence microbial
growth rates. In
these studies bacteria were grown on highly concentrated
inorganic
minimal media supplemented with reduced carbon compounds
at high
concentrations (
12). However, microbes growing in
environmental
situations are typically exposed to complex mixtures of
substrates
which are present at relatively low concentrations
(
16). Other
studies of microbial growth have been performed
with low concentrations
of defined mixtures of reduced carbon sources
in chemostats (
8,
11,
13). Unfortunately, the exact
qualitative and quantitative
compositions of seed exudates and the
dynamics of the release
of the exudates are not known, which makes
extrapolating the results
of chemostat studies to the spermosphere and
rhizosphere environments
difficult.
Studies in which we used
E. cloacae 501R3, the near-isogenic
strain A-11, seeds, and our sand system provided evidence that
directly
supports the hypothesis that the ability of a microorganism
to rapidly
grow on reduced carbon compounds in exudates contributes
to its ability
to grow on and colonize a particular host plant.
Compared with strain
A-11, strain 501R3 could grow rapidly on
a larger number of compounds
present in seed extracts. Strain
A-11, with a mutation in
pfkA, was not able to grow rapidly on
carbohydrates that
enter the Embden-Meyeroff-Parnass pathway after
fructose 6-phosphate
(
26,
27). When the quantities of compounds
that support
rapid growth of
E. cloacae were limited, as they
are in the
cucumber and radish spermospheres, the ability of strain
501R3 to grow
on a more diverse collection of sugars resulted
in a strain 501R3
growth rate that was dramatically greater than
the growth rate of
strain A-11. The growth rates of strains A-11
and 501R3 were
essentially very similar in pea, soybean, sunflower,
and sweet corn
spermospheres, in which substantially more sugars
that supported rapid
growth of both
E. cloacae strains were detected.
Colonization of cucumber and radish seeds by strain A-11 was reduced,
and the growth rate was dramatically affected, while colonization
of
pea, soybean, sunflower, and sweet corn seeds by strain A-11
was not
affected.
| |
ACKNOWLEDGMENTS |
We thank Kim Brandon and Stanley Tesch for providing excellent
technical assistance, Larry Douglas for performing the statistical analysis, and Don Kobayashi and Mark Wilson for reviewing the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biocontrol of
Plant Diseases Laboratory, USDA-ARS, Beltsville, MD 20705. Phone: (301) 504-5680. Fax: (301) 504-5968. E-mail:
DROBERTS{at}asrr.arsusda.gov.
Present address: U.S. Patent Office, Arlington, VA 22202.
| |
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