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Previous Article | Next Article 
Applied and Environmental Microbiology, June 1999, p. 2513-2519, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Role of pfkA and General Carbohydrate
Catabolism in Seed Colonization by Enterobacter
cloacae
D. P.
Roberts,1,*
P. D.
Dery,1
I.
Yucel,1,
J.
Buyer,2
M. A.
Holtman,3 and
D.
Y.
Kobayashi3
Biocontrol of Plant Diseases
Laboratory1 and Soil Microbial Systems
Laboratory,2 USDA Agricultural Research
Service, Beltsville, Maryland, and Department of Plant
Pathology, Rutgers University, New Brunswick, New
Jersey3
Received 4 December 1998/Accepted 19 March 1999
 |
ABSTRACT |
Enterobacter cloacae A-11 is a transposon mutant of
strain 501R3 that was deficient in cucumber spermosphere colonization and in the utilization of certain carbohydrates (D. P. Roberts, C. J. Sheets, and J. S. Hartung, Can. J. Microbiol.
38:1128-1134, 1992). In vitro growth of strain A-11 was reduced or
deficient on most carbohydrates that supported growth of strain 501R3
but was unaffected on fructose, glycerol, and all amino acids and organic acids tested. Colonization by strain A-11 was significantly reduced (P 
0.05) for cucumber and radish seeds
compared to that of strain 501R3, but colonization of pea, soybean,
sunflower, and sweet corn seeds was not reduced. Pea seeds released
several orders of magnitude more total carbohydrates and amino acids
than cucumber and radish seeds and approximately 4,000-fold more
fructose. Fructose was the only carbohydrate detected in the seed
exudates which supported wild-type levels of in vitro growth of strain A-11. Soybean, sunflower, and sweet corn seeds also released
significantly greater amounts of fructose and total carbohydrates and
amino acids than cucumber or radish seeds. The exogenous addition of fructose to cucumber and radish seeds at quantities similar to the
total quantity of carbohydrates released from pea seeds over 96 h
increased the populations of strain A-11 to levels comparable to those
of strain 501R3 in sterile sand. Molecular characterization of strain
A-11 indicated that the mini-Tn5 kanamycin transposon was
inserted in a region of the genome with significant homology to
pfkA, which encodes phosphofructo kinase. A comparison of
strain A-11 with Escherichia coli DF456, a known
pfkA mutant, indicated that the nutritional loss phenotypes
were identical. Furthermore, the pfkA homolog cloned from
E. cloacae 501R3 complemented the nutritional loss
phenotypes of both E. coli DF456 and E. cloacae A-11 and restored colonization by strain A-11 to near wild-type levels.
These genetic and biochemical restoration experiments provide strong
evidence that the quantities of reduced carbon sources found in seed
exudates and the ability of microbes to use these compounds play
important roles in the colonization of the spermosphere.
| |
INTRODUCTION |
Colonization of subterranean
portions of plants can be an essential process for a number of
beneficial microbial activities, including plant growth promotion,
plant disease control, and bioremediation (2, 6). Much
research has been conducted in attempts to understand the processes by
which microbes colonize plants, and several bacterial traits have been
correlated with the colonization of seeds and roots in specific systems
(e.g., references 3, 4, 7-9, 12, 14, 18, 26, and
41). One trait, microbial growth, has been
established as an essential process for colonization, and recent
studies have been published concerning the nutritional requirements for
microbial growth on subterranean portions of plants (24, 29-32,
35, 36). However, the contributing roles played by the catabolic
pathways of beneficial microbes, and the nutrients supplied by the host
plant, in growth and colonization are still unclear.
The plant-beneficial bacterium Enterobacter cloacae
suppresses Pythium ultimum damping-off of cucumber and other
crops Pythium ultimum by (22) and colonizes the
spermospheres and rhizospheres of a number of plant species
(13, 16, 17, 22, 28-31, 39). It is believed that seeds and
roots support growth by bacteria such as E. cloacae through
the release of complex mixtures of carbohydrates, amino acids,
water-soluble and -insoluble organic acids, and other nutrients
(5). The growth of strain A-11, a nutritional mutant of
E. cloacae 501R3 (29), was reduced or deficient
on almost all carbohydrates released by seeds and roots that supported
the growth of strain 501R3 in vitro (29, 32). The
colonization of seeds of a cucumber (but not of a pea) cultivar by
strain A-11 was significantly reduced (P 0.05)
relative to that of strain 501R3 in studies conducted in natural soil
and in sterile sand (29, 32). We have characterized the
mutation in strain A-11 at the molecular level in an attempt to
understand the colonization behavior of this strain. We report here
that glycolysis in E. cloacae A-11 is blocked by a mutation
in pfkA and that this gene is most important for
colonization of seeds that release limited quantities of reduced carbon
sources. Portions of this work have been published previously
(32).
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
Descriptions of the
bacterial strains and plasmids are in Table
1. Unless otherwise indicated, E. cloacae and Escherichia coli strains were grown to the
stationary phase at 35°C and 250 rpm. The following media were used:
Luria-Bertani (LB) broth or agar (19), M9 basal salts broth
or agar (19), and M56 basal salts broth or agar
(23) supplemented as previously described. The antibiotic
levels used to maintain or select strains and plasmids were 100 µg/ml
for rifampin (RIF) and streptomycin (STR), 50 µg/ml for kanamycin
(KAN), and 12.5 µg/ml for tetracycline (TET).
In vitro growth on reduced carbon and nitrogen sources.
Growth of bacterial strains on various reduced carbon compounds was
compared spectrophotometrically in basal salts broth or on basal salts
agar amended with 0.2% carbohydrate or with 0.5% L-amino
acid or 0.5% organic acid as previously described (19, 29,
30). The carbohydrates used were arabinose, cellobiose, fructose,
galactose, glucose, N-acetylglucosamine, lactose, maltose, mannitol, mannose, raffinose, rhamnose, ribose, salicin, stachyose, sucrose, trehalose, and xylose. The L-amino acids and
organic acids tested were alanine, asparagine, proline, glutamate,
glutamine, serine, pyruvate, and malate. The growth of E. cloacae 501R3 and A-11 on alanine, asparagine, cysteine,
glutamate, glutamine, proline, and serine as sources of nitrogen were
measured as previously described (30).
Seed colonization assays.
E. cloacae strains were
grown, washed, resuspended, and applied to single cucumber
(Cucumis sativum cv. Marketmore 76), radish (Raphinus
sativus cv. Cherry Bomb), pea (Pisum sativum cv. Sugar snap), soybean (Glycine max cv. Chesapeake), sunflower
(Helianthus giganteus), or sweet corn (Zea mays
cv. Stowells Evergreen) seeds at approximately 104 CFU per
seed as previously described (29). Seeds were buried in
4 g of a natural Galestown gravelly loamy sand soil (77.8% sand,
12.6% silt, 9.6% clay, and 0.6% organic matter) that had been
previously equilibrated to 
75 kPa or in 4 g of washed, sterile sand moistened with 4 ml of sterile distilled water (SDW) in 14-ml sterile snap cap tubes and incubated at 22°C. CFU were determined by
spiral plating (Spiral Systems, Cincinnati, Ohio) the contents of the
tubes onto LB agar containing 100 µg of cycloheximide/ml and the
appropriate antibiotics for each bacterial strain.
Experiments were performed three times each with three replicates for
each seed-strain combination at each of three sampling times (24, 45, and 96 h) for experiments comparing colonization of seeds by
strains A-11 and 501R3. The mean log10 CFU per seed were
determined and compared for strains 501R3 and A-11 at each time for
each seed type by using Student's t test (SAS Institute, Cary, N.C.). Data from all three experiments were combined prior to
analysis. There was a significant experiment effect (P 0.05); however, there was no significant experiment×treatment
effect for experiments conducted in natural soil and in sterile sand.
For biochemical restoration of seed colonization, E. cloacae
strains were grown, washed, and resuspended in SDW or SDW containing 6% fructose. Suspensions (40 µl) were applied to individual cucumber or radish seeds in sterile sand, and populations were determined after
18 h as described above. The quantities of fructose added to the
cucumber and radish seeds were similar to the quantity of carbohydrates
exuded from pea seeds over the initial 96 h after the onset of
imbibition. Experiments were performed twice with six replicates for
each treatment. For genetic restoration of seed colonization, E. cloacae strains were added to cucumber seeds in sterile sand as
described above and sampled at 18 h. Experiments were performed
three times with three replicates for each treatment. Means were
determined and compared by least significant differences (SAS) for both
the biochemical and the genetic restoration experiments. Results from
both sets of experiments were combined prior to analysis.
Analysis of aqueous seed extracts.
Seed extracts were made
essentially as previously described (21, 29). Seeds (25 g)
in a 250-ml Erlenmeyer flask were surface disinfested in 10% Clorox
for 20 min followed by two 20-min rinses in SDW. Surface-disinfested
seeds (2.5 g) were added to 10 ml of SDW and incubated at room
temperature in the dark. At the sampling times (24, 45, and 96 h)
the aqueous contents of the flasks were decanted and checked for
microbial contamination by spotting 10-µl aliquots onto nutrient
agar. SDW (10 ml) was subsequently added to the flasks, and the flasks
were incubated as described above until the next sampling time. All
noncontaminated samples from each sampling time for each seed type were
pooled, frozen, and lyophilized to dryness.
Total carbohydrates in the samples were estimated by the anthrone assay
(20) with glucose as the standard. Individual carbohydrates were identified and quantified as trifluoroacetyl derivatives by using
gas chromatography in 24-, 45-, and 96-h samples as previously described (32, 38). Total amino acids in samples were
estimated by the ninhydrin assay (37) with
L-leucine as the standard.
Molecular techniques and bacterial matings.
DNA isolations,
transformations, restriction digests, electrophoresis, ligations, and
Southern blot hybridizations were performed as previously described
(34). Complementation of strain A-11 was performed by
mobilizing a total genomic DNA cosmid library of E. cloacae
501R3 in the cosmid vector pLAFR3 (constructed by J. Loper,
Agricultural Research Service, Corvallis, Oreg.) into strain A-11 by
triparental mating (10) and selecting directly for
transconjugants which grew on M56 basal salts agar containing 0.2%
N-acetylglucosamine, RIF, and TET. All other transconjugants were selected by growth on LB agar supplemented with RIF and TET, except matings into E. coli DF456, which were selected by
growth on LB agar supplemented with STR and TET.
Nucleotide sequence was determined by the dideoxy chain termination
method by fluorescence labelling with Amplitaq (Applied Biosystems Inc.
[ABI]) run on an ABI model 373 automated sequencer. Overlapping
sequences were generated by using a series of nested deletions of
fragments subcloned into pGEM7Z(+) generated by digestion with
ExoIII (15) or by the use of selected primers.
Sequences were analyzed using the DNA analysis programs of DNAStar
(Lasergene, Inc.). BLAST searches of databases (1) were
conducted with translated proteins by using the Blastp program
available on the National Center for Biotechnology Information web page
(20a).
Nucleotide sequence accession number.
The nucleotide
sequence and the sequences of the translated proteins have been
deposited in GenBank under accession no. AF098509.
| |
RESULTS AND DISCUSSION |
Seed colonization.
E. cloacae A-11 increased slightly
(17-fold) from 104 CFU per seed to approximately
105 CFU per seed over the initial 24 h after
application to cucumber seeds in natural soil. Populations of strain
A-11 remained at this level 45 and 96 h after application (Table
2). Populations of strain 501R3 increased
continuously (200-fold) and more rapidly than those of strain A-11 over
this 96-h period to approximately 106 CFU per seed.
Populations of strain 501R3 were significantly greater (P 0.02) than those of strain A-11 at 96 h on cucumber seeds in
natural soil. Similar results were obtained for experiments performed
in radish spermospheres in natural soil. Populations of both strains
increased over this 96-h period. However, populations of strain A-11
were significantly lower than those of strain 501R3 at 45 h
(P 0.001) and at 96 h (P 0.01) after application. Similar results were also obtained when
seed colonization studies were performed in sterile sand. Populations
of strain A-11 grew more slowly and were significantly smaller
(P 0.005) than those of strain 501R3 at 24 h in
both cucumber and radish spermospheres (Table 2).
There was evidence of strain A-11 achieving slightly lower populations
than strain 501R3 on pea, soybean, sunflower, and sweet corn seeds at
45 and 96 h after application. However, populations of strains
A-11 and 501R3 increased significantly and were statistically similar
(P 0.05) in pea, soybean, sunflower, and sweet corn spermospheres at 24, 45, and 96 h after application in both
natural soil and sterile sand (Table 2). This suggests that compounds other than reduced carbon compounds are limiting to colonization in the
spermospheres of these pea, soybean, sunflower, and sweet corn seed cultivars.
Analysis of seed exudate.
The cultivars of pea, soybean, and
sweet corn seeds tested released approximately 1,000-fold more glucose
equivalents, as determined by the anthrone assay, than radish or
cucumber seeds, while sunflower seeds released approximately 20-fold
more glucose equivalents (Table 3). This
trend was confirmed by gas chromatography (Fig.
1). Fructose comprised a major portion of
the carbohydrates released by each seed for all six seed cultivars over
96 h. However, there were approximately 2 to 3 orders of magnitude
more fructose released by the cultivars of the sunflower, pea, soybean,
and sweet corn seeds tested than by those of the cucumber and radish seeds. Other prevalent carbohydrates detected were glucose, galactose, sucrose, and stachyose. Significantly more total amino acids, as
determined by the ninhydrin assay, were also released by pea, soybean,
sunflower, and sweet corn seeds than by the cultivars of cucumber or
radish seeds tested over this 96-h period (Table 3).
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FIG. 1.
Individual carbohydrates released by cucumber, radish,
pea, soybean, sunflower, and sweet corn seeds. Quantities represented
are summed from 0 to 24 h, 24 to 45, and 45 to 96 h samples.
Error bars represent one standard deviation from the mean. Note that
quantities are micrograms/seed for pea, soybean, and sweet corn seeds
and nanograms/seed for cucumber, radish, and sunflower seeds.
|
|
Characterization of E. cloacae A-11.
Strain 501R3
grew on 13 of 16 carbohydrates detected by gas chromatography in the
seed exudates (Fig. 1) when supplied as sole sources of reduced carbon.
Strain 501R3 did not grow on D-lactose, L-rhamnose, or stachyose. In contrast, strain A-11 showed
significant growth on only fructose. Growth on fructose by strain A-11
was similar to that by strain 501R3 (data not shown). Strains 501R3 and
A-11 had similar growth on the L-amino acids alanine,
asparagine, glutamine, glutamate, proline, and serine and on pyruvate
and malate when supplied as sole sources of reduced carbon. Strains 501R3 and A-11 also had similar growth on glycerol supplied as a
reduced carbon source and on the L-amino acids alanine,
asparagine, cysteine, glutamate, glutamine, proline, and serine when
supplied as sole sources of nitrogen (data not shown). This nutritional utilization profile is consistent with that of pfkA mutants
of the closely related bacterium E. coli (33).
Cosmid p82, which was identified from a genomic library of strain
501R3, restored the ability of strain A-11 to grow on M56 basal salts
agar amended with either 2% N-acetylglucosamine or 2%
salicin. Plasmid pSubB, containing an 8.5-kb subcloned fragment from
p82 (Fig. 2), restored the growth of
strain A-11 on M56 minimal salts medium amended individually with
salicin, N-acetylglucosamine and all 12 other carbohydrates
tested. The 8.5-kb DNA fragment from pSubB was used as a probe in
Southern hybridizations to genomic DNA from strains A-11 and 501R3
digested with EcoRI. Analysis of the blot (data not shown)
indicated that transposon mini-Tn5 KAN was inserted within a
1.5-kb EcoRI fragment located within pSubB (Fig. 2). A
2.5-kb BamHI fragment which contained this EcoRI fragment was subsequently subcloned from pSubB in both orientations into pRK415, resulting in plasmids pBB1415a and pBB1415b. Both plasmids
restored the growth of strain A-11 on salicin,
N-acetylglucosamine, and all 12 other carbohydrates tested,
indicating that this 2.5-kb BamHI fragment contained the
gene of interest in its entirety. In contrast, pSubB-3, which consisted
of a 2-kb BglII deletion of pSubB, did not restore the
growth of strain A-11 on either salicin or
N-acetylglucosamine (Fig. 2).
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FIG. 2.
Physical maps of plasmid pSubB and pSubB-derived
subclones. Abbreviations: B, BamHI; Bg, BglII; E,
EcoRI; H, HindIII; S, SalI. Arrows
indicate the direction of transcription. The thick line indicates the
sequenced portion of pSubB. E. cloacae A-11 harboring the
indicated plasmid was capable (+) or incapable () of in vitro growth
on M56 minimal medium plus 0.2% salicin or
N-acetylglucosamine.
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|
Nucleotide sequence analysis of the 2.5-kb BamHI fragment
from pBB1415 indicated the presence of two complete open reading frames
(ORFs) and part of a third ORF, all with the same transcriptional orientation (Fig. 2). Each of these ORFs contained a predicted ATG
start codon that was preceded within 6 to 10 bases by sequences resembling ribosome binding sites. The predicted start codon of ORF1
was located 212 bases from the BamHI site and encoded a
predicted protein product of 320 amino acids. Two DNA inverted repeat
sequences, representing potential transcriptional termination sites,
were identified immediately downstream of the stop codon. The first repeat was located 12 bases from the stop codon and consisted of the
7-base inverted repeat sequence of
GCCCGGT-N12-ACCGGGC. The second repeat was
located 20 bases downstream from the first repeat and consisted of the
sequence GCCGGGT-N12-ACCCGGC. Database searches
revealed that the predicted protein for this ORF had an amino acid
sequence identity of 88% to the pfkA gene product of
E. coli (GenBank accession no. P06998). pfkA
encodes phosphofructo kinase, a key enzyme in glycolysis catalyzing the
conversion of fructose-6-phosphate to fructose-1,6-phosphate
(33). To verify that the mutation in strain A-11 was located
within pfkA, a 1.5-kb BamHI-SalI
fragment containing the entire pfkA ORF and only the 5' end
of the sbp ORF was subcloned into pRK415. The resultant plasmid, pBS1415 (Fig. 2), restored the growth of strain A-11 on all 14 carbohydrates tested. In addition, pBS1415 was mobilized into E. coli DF456, a pfkA-deficient strain, restoring growth on N-acetylglucosamine.
The second ORF (ORF2) within the 2.5-kb BamHI fragment was
positioned 1,384 bp from the BamHI site, beginning 202 bases
from the termination codon of the pfkA homolog, and
consisted of a predicted protein product of 329 amino acids. Database
searches indicated 83% sequence identity to the sulfur binding protein of E. coli encoded by sbp (GenBank accession no.
S40860). Analysis of the nucleotide sequence extending an additional
3.25 kb downstream from the 2.5-kb BamHI fragment indicated
that pfkA and sbp are physically linked to a gene
with 76% sequence identity to cdh of E. coli,
which encodes CDP-diglyceride hydrolase (GenBank accession no. P06282)
and an ORF (ORF4) of unknown function. Database searches for this ORF
did not identify any matches of significance at the nucleotide or
predicted amino acid sequence levels. A fifth ORF (ORF5) encodes a
protein with 91% identity to triose phosphate isomerase of E. coli (GenBank accession no. P04790) (Fig. 2).
Genetic and biochemical restoration of seed colonization by strain
A-11.
Populations of strain 501R3 increased from approximately
104 CFU per seed to greater than 107 CFU per
seed in 18-h assays on surface-disinfested cucumber seeds in sterile
sand (Table 4). Populations of strains
A-11 and A-11(pRK415) showed no substantial increases after 18 h
when approximately 104 CFU per seed were added to the
cucumber seeds. Populations of strains A-11 and A-11(pRK415) were
substantially smaller than those of strain 501R3 at this time. Plasmids
pBB1415 and pBS1415 (Fig. 2) restored the ability of A-11 to grow on
cucumber seeds to near the levels associated with the wild-type strain,
strain 501R3 (Table 4).
Addition of fructose, a carbohydrate capable of supporting wild-type
growth of strain A-11, to treatments containing strain A-11, also
restored the ability of strain A-11 to grow on both cucumber and radish
seeds to levels similar to those of strain 501R3 (Table
5). The quantity of fructose added to
cucumber and radish seeds in these treatments was similar to the
quantity of total carbohydrates exuded from pea seeds over the initial
96 h after the onset of imbibition.
Genetic and biochemical data presented here strongly suggest that the
strain A-11 colonization phenotype is solely due to inactivation of
pfkA rather than to the loss of other physically linked
genes that appear to be involved in general carbohydrate catabolism
(Fig. 2). A DNA fragment containing only pfkA restored the
strain 501R3 phenotype to strain A-11 with regard to in vitro growth
and seed colonization. Also, the presence of an inverted repeat
indicative of a transcriptional termination signal downstream from
pfkA suggests this gene is expressed independently of other genes. Finally, the exogenous addition of fructose in colonization experiments circumvented the impact of the mutation in pfkA
on colonization by supplying strain A-11 with a reduced carbon source utilizable for growth. Fructose is not expected to biochemically complement any of the genes physically linked to pfkA. These
observations link the loss of pfkA function, the loss of
carbohydrate catabolic capabilities, and decreased colonization of
seeds that release relatively small quantities of reduced carbon such
as the cucumber and radish cultivars tested here.
Conclusion.
Nutrient-rich plant exudates are expected to
support an abundance of microbial growth while nutrient-poor exudates
are not. Since it has been established that growth is an essential
component of microbial colonization processes (24, 29-31,
35), it can be assumed that the catabolic capabilities of a
bacterium, with regard to specific reduced carbon compounds found in
exudates, contribute directly to its ability to colonize a given host
plant. Under nutrient-poor conditions a greater ability to catabolize compounds in exudates is expected to improve colonization. Although these assumptions are obvious and accepted by many, there is little evidence to support them. Our studies with E. cloacae 501R3
and the near-isogenic strain A-11 directly support these assumptions for microbial colonization of the spermosphere. Strain A-11 had decreased catabolic capabilities with regard to specific carbohydrates in seed exudates relative to those of strain 501R3. The colonization of
cucumber and radish seeds by strain A-11 was also reduced relative to
that by strain 501R3, but there were no differences between the two
strains relative to the colonization of pea, soybean, sunflower, and
sweet corn seeds. The exudates from these cultivars of pea, soybean,
sunflower, and sweet corn seeds contained, on average, several orders
of magnitude more carbohydrates and amino acids than the exudate from
cucumber or radish seeds.
Work with additional carbohydrate utilization mutants of E. cloacae and other plant-beneficial bacteria needs to be performed to substantiate the findings presented here. In addition, mutants affected in the utilization of other nutrients such as nitrogen need to
be analyzed. The acquisition of reduced carbon compounds and other
nutrients is fundamentally important for growth and other desired
microbial activities such as biocontrol in the spermosphere and
rhizosphere (25). An understanding of the nutritional
requirements of beneficial bacteria colonizing subterranean portions of
plants and the impact of available nutrients on beneficial activities such as colonization and biocontrol is required if the behavior of
beneficial bacteria in specific environments is to be accurately predicted.
| |
ACKNOWLEDGMENTS |
We acknowledge Kim Brandon, Ricky Brathwaite, and Sean Wu for
excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biocontrol of
Plant Diseases Laboratory, Bldg. 011A, Rm. 275, 10300 Baltimore Ave., 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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Abstract
Introduction
