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Journal of Bacteriology, October 2000, p. 5351-5358, Vol. 182, No. 19
Max-Planck-Institut für Zellbiologie,
Rosenhof, D-68526 Ladenburg, Germany
Received 18 February 2000/Accepted 26 June 2000
Sucrose is an important storage and transport sugar of plants and
an energy source for many phytopathogenic bacteria. To analyze regulation and biochemistry of sucrose metabolism of the fire blight
pathogen Erwinia amylovora, a chromosomal fragment which enabled Escherichia coli to utilize sucrose as sole carbon
source was cloned. By transposon mutagenesis, the scr
regulon of E. amylovora was tagged, and its nucleotide
sequence was determined. Five open reading frames, with the genes
scrK, scrY, scrA, scrB,
and scrR, had high homology to genes of the scr
regulons from Klebsiella pneumoniae and plasmid pUR400.
scrB and scrR of E. amylovora were fused to a histidine tag and to the maltose-binding protein (MalE) of
E. coli, respectively. ScrB (53 kDa) catalyzed the
hydrolysis of sucrose with a Km of 125 mM.
Binding of a MalE-ScrR fusion protein to an scrYAB promoter
fragment was shown by gel mobility shifts. This complex dissociated in
the presence of fructose but not after addition of sucrose. Expression
of the scr regulon was studied with an scrYAB
promoter-green fluorescent protein gene fusion and measured by flow
cytometry and spectrofluorometry. The operon was affected by catabolite
repression and induced by sucrose or fructose. The level of gene
induction correlated to the sucrose concentration in plant tissue, as
shown by flow cytometry. Sucrose mutants created by site-directed
mutagenesis did not produce significant fire blight symptoms on apple
seedlings, indicating the importance of sucrose metabolism for
colonization of host plants by E. amylovora.
The gram-negative bacterium
Erwinia amylovora causes fire blight of apple, pear, and
other rosaceous plants. Pathogenecity depends on the ability to produce
the exopolysaccharide amylovoran (10, 13), to elicit a
hypersensitive response on non-host plants (6, 8), and to
metabolize sorbitol of the host plants (1). Rosaceous plants
contain sorbitol and sucrose as storage and transport carbohydrates.
The distribution of these carbohydrates is dependent on environmental
conditions, species, and plant tissue (28, 52). The highest
concentration of sucrose was found in the nectaries of host plants
(14), which are assumed to be the main entry site for the
pathogen when the pathogen is distributed by insects.
Sucrose is utilized by some but not all bacteria extracellularily or
intracellularily. E. amylovora can metabolize sucrose via
the secreted levansucrase, which polymerizes the homopolysaccharide levan and releases glucose from sucrose (27, 29), but also by uptake and intracellular metabolism.
The sucrose-utilizing system of enteric bacteria has been studied in
Klebsiella pneumoniae and in some isolates of
Escherichia coli and Salmonella spp. (45,
49). In E. coli and Salmonella spp., the
conjugative plasmid pUR400 confers the ability to utilize sucrose
(54), whereas the scr regulon of K. pneumoniae is located on the chromosome (42, 49). In
these bacteria, the uptake of sucrose is mediated via the
phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system
(PTS), yielding sucrose 6-phosphate, which is cleaved by an
intracellular hydrolase into glucose 6-phosphate and fructose (45,
49). The scr regulons of K. pneumoniae and
pUR400 consist of four structural genes: scrK codes for an
ATP-dependent fructokinase (5), scrY codes for a
sucrose-specific porin of the outer membrane (30),
scrA codes for enzyme IIscr of the PTS, and
scrB codes for an intracellular
In this work, we cloned, sequenced, and characterized the
scr regulon of E. amylovora. The regulation of
sucrose metabolism was studied by gel shift assays and promoter-green
fluorescent protein gene (gfp) fusions analyzed by flow
cytometry also with bacteria extracted from plant tissue. Mutants
carrying mutations in the scr genes were nonvirulent.
(A preliminary report has been published as a proceedings contribution
[15].)
Strains, plasmids, and oligonucleotides.
Table
1 lists the strains, plasmids, and
oligonucleotides used in the experiments.
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Molecular Analysis of Sucrose Metabolism of
Erwinia amylovora and Influence on Bacterial
Virulence
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-fructofuranoside fructohydrolase (EC 3.2.1.26), which
cleaves sucrose 6-phosphate into -D-fructose and
-D-glucose 6-phosphate (51). The regulon is
controlled by the negative regulator ScrR (34) and is
induced in medium containing sucrose, fructose, or raffinose (45,
46).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Stains, plasmids, oligonucleotides, and phage used
Mutagenesis and screening of scr mutants.
Transposon insertions in scr genes were detected after
infection of Escherichia coli S17-1(pSCR100) with phage
::Tn5seq as white colonies on MacConkey agar
(Gibco-BRL) with sucrose in the presence of kanamycin. Plasmids
pSCR100-1 and pSCR100-3 were transferred into E. amylovora
as described by Bernhard et al. (13). Plasmid pPH1JI, which
is incompatible with pSCR100-1 and pSCR100-3, was then conjugated into
a mutant, and cells were selected for resistance to chloramphenicol,
kanamycin, and streptomycin to screen for bacteria with a transposon
insertion in the chromosome.
Enzyme assays. Sucrose hydrolase activity was determined in 1 ml of 100 mM phosphate buffer (pH 7.0) with 200 mM sucrose incubated at 28°C for 30 min. The reaction was stopped by boiling for 1 min. After removal of denatured protein, the glucose was determined with 10-µl aliquots added to 1 ml of 10 mM phosphate buffer-0.05% sodium azide with peroxidase (1 U), glucose oxidase (10 U), and 1 mg of ABTS (2,2-azino-di-3-ethyl-benzthiazolinsulfonate) (Boehringer, Mannheim, Germany). The reaction was complete after 30 min at 28°C, and the absorption was determined at 436 nm.
Protein purification and analysis. (His)6 tag fusions were expressed in E. coli strain GI698 after growth at 37°C to an optical density at 600 nm (OD600) of 0.5. The gene was induced with 1 mM IPTG (isopropylthiogalactopyranoside) for 4 h at 28°C. The fused histidine residues of recombinant proteins were bound to an Ni-nitrilotriacetic acid matrix (Qiagen). The native protein (H-ScrB) was eluted with 250 mM imidazole, whereas the denaturated repressor (H-ScrR) was eluted with buffer D (8 M urea, 0.1 M NaH2PO4 [pH 5.9], 0.01 M Tris-HCl). The purification of the proteins was done with the QIAexpress system according to the protocol of the manufacturer.
For expression and purification of maltose-binding protein (MBP)-ScrR, E. coli strain DH5(pMalscrR) was grown in Luria-Bertani (LB) medium with 0.2% glucose to an OD600 of 0.5. Expression of the malE-scrR fusion was induced with 0.5 mM
IPTG for 3 h at 37°C. Cells were harvested, washed, and
resuspended in column buffer (20 mM Tris-HCl [pH 7.4], 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol) and sonicated. The cell debris was
pelleted by centrifugation (13,000 × g), and the
supernatant was mixed with the amylose resin (1:1) and incubated at
4°C for 30 min. After extensive washing with column buffer, the bound
protein was eluted with column buffer supplemented with 10 mM maltose.
Protein concentrations were determined by the method of Lowry
(37) with bovine serum albumin as the standard. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed in 10% polyacrylamide gels (36).
Sequencing. Sequencing was done with the automated laser fluorescent DNA sequencer (A.L.F. Express; Pharmacia Biotec). Initial sequences were obtained with fluorescent universal primers. Nucleotide sequences were determined by primer walking using unlabeled primers synthesized with an oligonucleotide synthesizer (Beckman), after brief incorporation of fluorescein-labeled dATP followed by a chase with unlabeled dATP. Nucleotide sequences were determined with plasmids pSCR107, pSCR109, and pSCR141 in both strands of the inserts. Computer analysis was done with databases for similarities to DNA and protein sequences using the programs BLAST (3; http://www3.ncbi.nlm.nih.gov /Entrez/), IDENTIFY (41; http://dna.stanford.edu/identify/), and additional programs from the BCM-Launcher (47; http://www.hgsc.bcm.tmc.edu/SearchLauncher/).
Media and virulence assays. MM2 medium has been described (9, 11, 18). Sorbitol (1%) was replaced with other carbohydrates when indicated. DNA manipulations followed standard procedures (44).
Leaf tips of young apple seedlings (cv. Golden Delicious) were cut with scissors and inoculated with a toothpick dipped into an overnight culture of a gfp-labeled E. amylovora strain. Migration of bacteria was evaluated in a fluorescence microscope (Zeiss Axiovert, type 135) under fluorescein isothiocyanate conditions after 5 days of incubation in a growth chamber. Virulence on pears was visually determined from ooze formation at 1 week after inoculation of 0.5-cm-thick slices in a petri dish. The assays were done with at least three plant specimens for each strain.Cytometric and fluorometric analyses. For analysis by flow cytometry and spectrofluorometry, bacteria were grown for 16 h in LB medium with 0.5% carbohydrate, centrifuged, and washed in phosphate-buffered saline (PBS) (44). The bacterial pellet was suspended in 0.5 ml of PBS for determination of fluorescence. Flow cytometry was carried out in a FACScan system (Becton Dickinson). Illumination was done with a 15-mW, 488-nm argon laser, and emission light was detected by a 530 ± 30-nm band pass filter. Photomultiplier voltages were kept constant in a given series of experiments. The fluorescence detector was set at a photomultiplier tube voltage of 546 V. Forward scatter was collected by a photomultiplier tube set at 100, and sideward scatter was collected at a photomultiplier tube set at 400 V. Data were collected for 104 particles per sample and analyzed with the program WinMDI 2.7 (http://facs.scripps.edu /software.html). Bacteria and other particles were separated by their different light-scattering properties (32), which are a complex function of their size, shape, and refractive indices (2, 40, 43). The region R1 (see Fig. 4) was defined to measure bacterial fluorescence on the light-scattering plots. LB cultures of Ea1/79(pSU18-Y1gfpR), LB medium, and extracts from uninfected plant tissue were compared to define an area where more than 95% of the particles were recovered as bacterial signals. For evaluation of R1, at least 1,000 particles were measured. An SPF-500 spectrofluorometer (American Instrument Company) was used to measure bacterial fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 510 nm.
DNA mobility shift assay.
The scrYAB promoter
fragment was amplified by PCR using primers 3341 and 3342. About 0.5 pmol of the promoter fragment was incubated with protein MBP-ScrR (37 to 50 pmol) for 30 min at 30°C in 20 µl of DNA-protein binding
buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 10 mM dithiothreitol,
0.1 mM EDTA, 0.05 µg of DNA per µl, and 0.5 µg of bovine
serum albumin per µl). Before incubation, 10 mM sugar was added if
indicated. Protein binding to DNA was assayed by electrophoresis in a
native 5% polyacrylamide gel. After electrophoresis, the gel was
stained with a 1:104 dilution of SYBR Green I (Biozym) in
Tris-Borate-EDTA buffer for 30 min. The gel was analyzed with a Fluor-S
Multi-Imager from Bio-Rad.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Identification and analysis of the scr regulon from
E. amylovora.
Cosmid pSCR100 (Fig.
1) was isolated from a genomic library of
E. amylovora (10) obtained after
HindIII digestion. Cells of E. coli strain
S17-1 with the library were screened for the ability to utilize sucrose
as a carbon source on MacConkey agar plates with sucrose and
tetracycline and to grow on minimal medium with 1% sucrose. Cosmid
pSCR100 contained a 20-kb HindIII fragment, and the
scr regulon of E. amylovora was localized by
transposon mutagenesis of S17-1(pSCR100) using phage
::Tn5seq and screening on 1% sucrose,
tetracycline, and kanamycin. From 1,100 transposon mutants, 2 colonies
with an scr-negative phenotype were found, and cosmids
pSCR100-1 and pSCR100-3 were isolated from white colonies with
different insertions of Tn5seq in the scr regulon
of pSCR100. By subcloning after digestion with various restriction
endonucleases, the transposon insertions were localized in a 10-kb
region of pSCR100.
|
. P,
putative promoter; B, BamHI; E, EcoRI; EV,
EcoRV; H, HindIII; M, MluI; Ps,
PstI; S, SalI; Sm, SmaI; Sp,
SphI.
-D-fructofuranoside fructohydrolase (EC 3.2.1.26)
encoded by pUR400 (46, 51). A consensus sequence of the
catalytic active domain (H-x(2)-P-x(4)-[LIVM]-N-D-P-N-G) of
invertases was found in the N terminus of the protein. ScrR of E. amylovora shows 83% similarity to the regulator protein of the
scr regulon of pUR400. The proteins share a well defined N-terminal helix-turn-helix DNA-binding motif, characteristic of many
repressors of the LacI/GalR family (53).
Cloning of scrB and purification and enzymatic activity of the sucrose hydrolase from E. amylovora. The gene scrB of E. amylovora was amplified from plasmid pSCR100 by PCR with primers 3584 and 3523, creating SphI and HindIII sites at the ends of the 1.5-kb PCR fragment. The amplified fragment with scrB was digested and inserted into pQE31 to create pQE31scrB by fusion of scrB with the (His)6 tag sequence of the vector. The plasmid was transformed into E. coli strain GI698. The protein was expressed and purified as described in Materials and Methods. After elution of the native gene product from an Ni column, a major band of 53 kDa was detected by SDS-PAGE, corresponding in size to ScrB as deduced from the encoding ORF.
The purified sucrose hydrolase cleaved sucrose with an optimal enzymatic activity of 18,300 U/mg of homogeneous enzyme in 100 mM phosphate buffer (pH 7.0) and 200 mM sucrose at 28°C. The Michaelis-Menten constant of the sucrose hydrolase for cleavage of sucrose was 125 mM (data not shown). After freezing the enzyme in 10% glycerol at
80°C, no significant (<1%) loss of enzymatic activity
was detected after 1 month. Enzymatic activity decreased at ionic
strengths higher than 200 mM or lower than 50 mM sodium phosphate. The
temperature optimum of the hydrolase was between 18 and 28°C. The
maximum enzymatic activity was recovered at pH 7.0, whereas a pH shift
to 5.6 or 8.6 caused loss of the activity.
Gene cloning, purification, and characterization of the repressor ScrR from E. amylovora. The gene scrR of E. amylovora was amplified by PCR with primers 3583 and 3506 as a 1-kb fragment. The amplified scrR was digested with BamHI and HindIII and inserted into pQE31 to create pQE31scrR, expressed in strain GI698. Purification of the gene product was done under denaturing conditions due to low solubility of the fusion protein. The purified probe was analyzed by SDS-PAGE and showed a dominant band of 38 kDa, corresponding to the size deduced from the ORF. Several attempts to renature the protein after purification were unsuccessful.
Insolubility after overexpression in E. coli has been described for other ScrR repressors (33). However, a fusion of the repressor to E. coli MBP could yield a soluble protein. Therefore, the 1-kb BamHI-HindIII fragment from pQE31scrR was cloned into pMa1-c2 to create pMalscrR. After expression in E. coli, the MBP-ScrR protein was purified by its affinity to an amylose resin. In order to show that scrR codes for a regulatory protein which binds to the scrYAB promoter region, a gel mobility shift assay with the 278-bp promoter fragment (PscrYAB) of pSU18-Y1 was carried out. The promoter fragment was shifted after addition of MBP-ScrR but was not affected by the presence of DNA (Fig.
2). To identify the intracellular
inducer, fructose, sucrose, and glucose were tested for their ability
to release bound ScrR from the promoter fragment. Fructose but not
sucrose or glucose dissociated the repressor-promoter complex (Fig. 2),
suggesting that fructose is the intracellular inducer of the
scr operon.
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Regulation of scrYAB in E. amylovora.
To
study transcriptional regulation of the scrYAB operon of
E. amylovora, the putative promoter-operator region
preceding the operon was amplified as a 278-bp PCR fragment with
primers 3342 and 3341. The fragment was inserted into vector pSU18 via the restriction sites SalI and XbaI. In the
resulting plasmid (pSU18-Y1), the orientation of the putative promoter
PscrYAB is directed against the lac
promoter of the vector. To fuse the promoterless gfp gene
with PscrYAB, a 700-bp
XbaI-SalI fragment from pFG30 was subcloned into
pSU18-Y1, yielding pSU18-Y1gfp. Constitutive expression of the reporter
gene in Ea1/79(pSU18-Y1gfp) without the repressor gene cloned on the
plasmid was assayed by fluorometry and flow cytometry in LB medium with
and without sucrose. In order to increase the intracellular level of
the repressor, a 1.2-kb fragment containing scrR was
amplified with primers 3366 and 3367 by PCR. The primers introduced
EcoRV and Asp718 restriction sites into
pSU18-Y1gfp. The plasmid created, pSU18-Y1gfpR, was transferred into
Ea1/79, and expression of the gfp gene in LB cultures with
various carbohydrates was determined (Fig.
3). A high induction of the
transcriptional fusion was found for 0.1% sucrose, with less effect of
lower or higher concentrations (Fig. 3A). Fructose also induced the
reporter fusion to some extent (Fig. 3B). Catabolite repression was
observed for growth with glucose in addition to sucrose. For the
levan-deficient mutant Ea7/74-LS7(pSU18-Y1gfpR), expression of the
reporter gene was increased twofold compared to expression in strain
Ea1/79(pSU18-Y1gfpR). Crude protein extracts of Ea7/74-LS7 cultures
grown in LB with 0.5% sucrose, fructose, glucose, glycerol, or sucrose
plus glucose were tested for sucrose hydrolase activity. The induction
of enzyme activities by the different carbohydrates correlated with the induction of the gfp gene measured by flow cytometry and
spectrofluorometry with Ea7/74-LS7(pSU18-Y1gfpR) or
Ea1/79(pSU18-Y1gfpR) (Fig. 3).
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Site-directed mutagenesis of the scr regulon in E. amylovora. Plasmid pSCR100-1 was transferred into E. amylovora strain Ea7/74 and the levan-deficient strain PD494, and pSCR100-3 was transformed into strain Ea1/79. Homolog recombination events were screened after conjugation of plasmid pPH1JI, which is incompatible with RP4-derived plasmids. Mutants with site-specific recombination of the transposon were white on MacConkey plates with sucrose and unable to grow on solidified MM2 medium with sucrose. The mutants with transposon insertions in scrY (pSCR100-1) and scrA (pSCR100-3) were named Ea7/74-S1, PD494-S1, and Ea1/79-S3, accordingly. Complementation of the mutants was verified with pSCR100 on MacConkey agar plates with sucrose. Their level of levan synthesis was the same as for the parent strains.
Growth features of scr mutants in culture.
The
growth properties of the scr mutants on various carbon
sources were compared with those of their parent strains. No difference in the growth kinetics was observed in LB medium or minimal medium with
glucose or sorbitol. In contrast to the parent strains, there was no
growth of the scr mutants in minimal medium with 1% sucrose after 24 h. In LB medium with sucrose (5 to 40%, wt/wt), the
growth of the mutants was reduced fourfold compared to the wild type, which is able to use sucrose as a carbon source, whereas the mutants must rely on the ingredients of the LB medium. For sucrose
concentrations higher than 40%, the mutants and the wild-type strains
were unable to grow, as shown for the levan-deficient strain PD494Sm
and corresponding sucrose mutant PD494-S1 (Fig.
5), limiting E. amylovora to
propagation in extreme sugar concentrations independent of their
genetic features.
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Virulence of E. amylovora scr mutants. In virulence assays on slices of immature pears, the E. amylovora wild-type strains and the scr mutants produced similar amounts of ooze. The low content of sucrose in those pears (G. Geier and K. Geider, unpublished) is probably compensated for by other carbon sources, such as sorbitol, fructose, and glucose, or organic acids.
When inoculated into leaves of apple seedlings, wild-type strains of E. amylovora caused necrosis and sometimes even wilt. In contrast, the sucrose mutants Ea7/74-S1, PD494-S1, and Ea1/79-S3 showed strongly reduced symptoms in these assays. To confirm the inability of the mutants to colonize leaf tissue, the strains were labeled with plasmid pfdC1Z'-gfp in order to trace them via the fluorescence of the GFP (16). The parent strains labeled with gfp showed the expected migration in the veins of apple leaves at day 5 after inoculation. Conversely, the labeled mutant strains did not move from the narrow zone of inoculation at the leaf tip. Sucrose, which cannot be utilized by scr mutants, is thus an important energy source for E. amylovora to colonize the tissue of the fire blight host plants.| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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For colonization of plants by E. amylovora, the causative agent of fire blight, not only the sorbitol (1) but also the sucrose metabolism of the bacteria is important. These carbohydrates vary among species and within parts of a plant (14, 28, 50). Sucrose is high in the nectaries of flowers (14), a main entry site for the pathogen. High sucrose concentrations usually inhibit bacterial growth, but E. amylovora can grow even in 40% sucrose (38). Its inability to grow in 50% sucrose restricts propagation of the pathogen in an environment with extreme sugar concentrations.
The genes involved in sucrose metabolism of E. amylovora showed significant homology to the scr genes of Salmonella enterica serovar Typhimurium with pUR400 and of K. pneumoniae (45, 49). Conserved amino acid motifs in the N terminus of scr proteins of E. amylovora indicated biological function related to the enzymes from pUR400, K. pneumoniae, and other proteins in the family. The overlapping stop codon of scrA and start codon of scrB (ATGA) support transcriptional and translational coupling of the two genes, as described for the scr genes of other species (42, 51).
A transcriptional fusion of the promoter PscrYAB with gfp in the presence of scrR (pSU18-Y1gfpR) and expression studies of ScrB indicated regulation of the promoter activity from a region preceding scrYAB. Computer analysis of PscrYAB predicted binding domains for the regulator ScrR and a cyclic AMP-cyclic AMP receptor protein complex. The promoter was not only regulated by ScrR, but also induced by sucrose and fructose and suppressed by glucose. Gel mobility shift assays with the 300-bp DNA fragment preceding scrY and the repressor showed that ScrR binds to this region and that fructose and not sucrose is the molecular inducer of scrYAB transcription, as in pUR400 and K. pneumoniae. The scr repressors of pUR400 and K. pneumoniae interact with a helix-turn-helix with the operator palindrome TAAACC/GGTTTA preceding scrY and scrK and bind to fructose or fructose 1-phosphate but not to sucrose (34). A part of this sequence (AACC/GGTT) was found in front of scrY and is possibly the operator in the scr regulon of E. amylovora. Based on a possible leucine zipper motif at the C terminus of ScrR and the observation that the palindrome is present twice in the PscrYAB region of K. pneumoniae, Jahreis and Lengeler (34) proposed binding of ScrR as a tetramer to the operator. This is not supported by E. amylovora, with one palindrome in this region and without a leucine zipper motif in ScrR.
An influence of host cells on the promoter activity of a pathogen has been measured in Mycobacterium smegmatis (23), Bartonella henselae (22), Bacillus cereus (24), and Listeria monocytogenes (17) by the combination of flow cytometry and the gfp reporter gene as a marker. gfp expression in E. amylovora was also analyzed by flow cytometry. Fluorescence of bacteria from stem sections was increased twofold compared to bacteria from young leaves of apple seedlings. This increased fluorescence can be explained by the relatively low sucrose concentration in young leaves and the high concentration in stem tissue (28). The highest in vitro induction was found for sucrose concentrations between 0.01 and 0.8%, comparable to concentrations in the xylem of apple plants. Other parts of the plants can contain much higher concentrations of the sugar (14, 25, 31). In LB medium with sucrose concentrations higher than 0.8%, the activity of PscrYAB was reduced. Low fluorescence of the bacteria from cotoneaster flowers (unpublished data) can be explained by the high sucrose concentration in the nectaries, which also caused reduced fluorescence in LB medium.
Reduced virulence of sorbitol (1) and sucrose mutants could be due to a low level of nutrients in xylem vessels, requiring access to sucrose and sorbitol for colonization of the host plant by E. amylovora. The scr mutants Ea7/74-S1, PD494-S1, and Ea1/79-S3 did not grow in minimal medium with sucrose as the sole carbon source, independent of levan synthesis.
Levan may provide fast protection of E. amylovora against
plant defense mechanisms (27, 29). Levansucrase could reduce the induction of the scr regulon by decreasing the sucrose
concentration and providing glucose for catabolite repression shown for
the levan mutant Ea7/74-LS7(pSU18-Y1gfpR) with increased fluorescence in the presence of sucrose compared with the wild type. Intracellular sucrose metabolism and extracellular levan formation can depend on a
single enzyme, as in SacB of Bacillus subtilis
(19). At high sucrose concentrations (>30 mM), sucrose is
cleaved by extracellular SacB to form levan, and at low sucrose
concentrations (
1 mM) it is hydrolyzed in the cells. The
sacB gene is regulated by sucrose via an antitermination
process (21). Expression of the levansucrase gene of
E. amylovora is not induced by sucrose (27), but
is regulated by LsrA, encoded in the hrp region
(56). Unlike the nitrogen-fixing bacterium Acetobacter
diazotrophicus, which depends on an extracellular levansucrase to
use sucrose as a carbon source (4), E. amylovora
thus encodes two enzymes to metabolize sucrose (ScrB and Lsc). The
external release of glucose did not substitute for a deficiency in
sucrose metabolism, since the defect in scr mutants of
E. amylovora cannot be suppressed by secreted levansucrase. Sucrose metabolism via the scr regulon of E. amylovora is thus strictly required for pathogenicity.
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FOOTNOTES |
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* Corresponding author. Mailing address: Max-Planck-Institut für Zellbiologie, Rosenhof, D-68526 Ladenburg, Germany. Phone: 49-6203-106-117 or 120. Fax: 49-6203-106-122. E-mail: kgeider{at}zellbio.mpg.de.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, October 2000, p. 5351-5358, Vol. 182, No. 19
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