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Journal of Bacteriology, May 2001, p. 3134-3141, Vol. 183, No. 10
Unité de Glycobiologie Structurale et
Fonctionnelle, UMR USTL-CNRS 8576, Université des Sciences et
Technologies de Lille, 59655 Villeneuve d'Ascq
Cedex,1 and Unité de Microbiologie
et Génétique, INSA, ERS INSA-UCB-CNRS 2009, INSA, 69621 Villeurbanne Cedex,2 France
Received 5 December 2000/Accepted 6 March 2001
Erwinia chrysanthemi is a phytopathogenic
enterobacterium causing soft rot disease in a wide range of plants.
Osmoregulated periplasmic glucans (OPGs) are intrinsic components of
the gram-negative bacterial envelope. We cloned the opgGH
operon of E. chrysanthemi, encoding proteins involved in
the glucose backbone synthesis of OPGs, by complementation of the
homologous locus mdoGH of Escherichia coli.
OpgG and OpgH show a high level of similarity with MdoG and MdoH,
respectively, and mutations in the opgG or opgH
gene abolish OPG synthesis. The opg mutants exhibit a
pleiotropic phenotype, including overproduction of exopolysaccharides,
reduced motility, bile salt hypersensitivity, reduced protease,
cellulase, and pectate lyase production, and complete loss of
virulence. Coinoculation experiments support the conclusion that OPGs
present in the periplasmic space of the bacteria are necessary for
growth in the plant host.
Osmoregulated periplasmic glucans
(OPGs) are a family of oligosaccharides found in the periplasmic space
of gram-negative bacteria. Their two common features are the presence
of glucose as the sole constituent sugar and their increased level in
media of low osmolarity (5).
Members of the family Enterobacteriaceae and related
bacteria synthesize a family of linear and branched OPGs that are
variously substituted. The linear backbone is constituted by glucose
units joined by Many factors are involved in the virulence of pathogenic bacteria, and
OPGs appear to be among them. In P. syringae pv. syringae, the causal agent of brown spot disease of the common bean
(Phaseolus vulgaris), the hrpM mutant, obtained
after transposon mutagenesis, was isolated because it failed to incite
disease on the host plant and to cause the hypersensitive reaction on a
non-host plant such as tobacco (29). The hrpM
mutant does not synthesize OPGs, and the hrpM locus
complements the OPG synthesis defect of the
mdoH200::Tn10 mutant of E. coli. The amino acid sequences of HrpM and MdoH are 75.5%
identical and 87.5% similar (25). More recently, a
transposon insertion in a gene similar to hrpM/mdoH was
isolated because it severely reduces the virulence in
Pseudomonas aeruginosa PA14, an opportunistic pathogen
for human, mice, and the worm Caenorhabditis elegans
(26).
The pectinolytic bacterium E. chrysanthemi, a member of the
Enterobacteriaceae family, causes soft rot disease in a wide
range of plant species. The virulence of this bacterium is strongly associated with the synthesis and secretion of a set of enzymes that
degrade plant cell wall components, such as pectinases, cellulases, and
proteases. The action of these enzymes, particularly the pectinases, leads to the extension of the disease, causes maceration of the plant
tissues, and supplies the bacteria with carbon sources derived from the
degraded polymers. The combination of pectin lyase, polygalacturonase, pectate lyase, pectin acetyl esterase, and pectin methyl esterase activities is needed for the degradation of pectin, reflecting the
structural complexity of this polymer (2, 36). Among these
proteins, endo pectate lyases play the most important role in the
maceration process.
The variability of the plant cell wall is reflected by the great number
of pectinase genes and correlated with the great number of stimuli
affecting the differential expression of the pel genes (15). These genes seem to be, at least in part, the reason
for the broad host range of this bacterium (3). E. chrysanthemi 3937 synthesizes five major endo pectate lyases,
encoded by pelA to pelE (39), and a
set of minor ones. Transcription of the pel genes is induced
by pectin catabolic products, the stationary growth phase, host plant
metabolites (7), and iron depletion (28).
Transcription of these genes is repressed by nitrogen starvation, high
temperature, and high osmolarity and is under catabolic repression
(16). Several proteases and the cellulase EGZ, encoded by
the celZ gene, are secreted by E. chrysanthemi 3937, but they seem to play a minor role in pathogenesis. The cellulase
and most of the pectinases are secreted in the medium by the specific
Out secretion system encoded by the 15 out genes (34). Synthesis and secretion of pectinases are tightly
associated. Thus, expression of the out genes is regulated
in the same way and by the same factors as the pel genes
(11). Because of iron depletion found in plant fluids, two
iron-scavenging systems are needed for full virulence
(27).
In this paper, we describe the molecular analysis of the
opgGH operon involved in OPG backbone synthesis in E. chrysanthemi. opgG and opgH mutants were obtained by
reverse genetic manipulation and analyzed for the synthesis and
secretion of plant cell wall-degrading enzymes and for virulence.
Bacterial strains and plasmids.
E. chrysanthemi
and E. coli K-12 strains are listed in Table
1. Plasmids used for subcloning
experiments were pYZ4 (kanamycin resistance) and pUC18 (ampicillin
resistance) (25).
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3134-3141.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Osmoregulated Periplasmic Glucan Synthesis Is
Required for Erwinia chrysanthemi Pathogenicity
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
,1-2 linkages, and the branches are made of one
glucose unit linked to the main chain by a ,1-6 linkage. In
Escherichia coli, the backbone, containing 7 to 13 glucose
units, is substituted with phosphoglycerol, phosphoethanolamine, and
succinyl residues (19). In Erwinia
chrysanthemi, the backbone contains 5 to 12 glucose units
substituted with succinyl and acetyl residues (9), and in
Pseudomonas syringae, the backbone, consisting of 6 to 13 glucose units, is not substituted (38). In E. coli, the OPG backbone is synthesized by the products of the
mdoGH operon located in the vicinity of pyrC, a
gene involved in the biosynthesis of uracil (6). In this
bacterium, the defect in OPG synthesis does not confer an easily
selectable phenotype in laboratory conditions. Thus, the
mdoGH locus was cloned using the linked selectable genetic marker pyrC (23).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains.
Media and growth conditions. Bacteria were grown at 30°C (E. chrysanthemi) or 37°C (E. coli) in Luria-Bertani broth (LB) or in minimal medium M63 supplemented with the required metabolites and a carbon source added at a concentration of 2 g/liter (29). Polygalacturonate was added at a concentration of 4 g/liter. Potato extract was added at a concentrations of 4 or 8% (vol/vol). When low-osmolarity medium was required, LB without NaCl or low-osmolarity medium (LOS) was used (23). Solid media were obtained by adding agar at 15 g/liter.
Potato extract was obtained as follows. Potato tubers were crushed in a domestic mixer, and the resulting suspension was passed through a French pressure cell at 20,000 lb/in2 (1.4 × 107 Pa) and filter sterilized (0.22-µm; Millipore). Motility tests were made on LB plates containing agar at 0.4 g/liter. The MIC of bile salts (no. 3; Difco Laboratories) was determined on LB plates. The solid media used to test the pectinase and cellulase activities have been described previously (16, 28), and protease activity was detected by adding 1% milk to LB solid medium. Antibiotics were used in media at the following concentrations: streptomycin, 100 µg/ml; ampicillin and kanamycin, 50 or 10 µg/ml; and tetracycline, 25 and 5 µg/ml for E. coli and E. chrysanthemi, respectively. X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) was used in media at a concentration of 20 µg/ml.
Transduction and transformation.
Transduction of E. chrysanthemi with phage 
EC2 was carried out according to
Resibois et al. (32).
Transfer of RP4 derivative plasmids by mating. Plasmid pULB110, a kanamycin-sensitive RP4::mini-Mu derivative (41), was used to generate R-prime derivatives containing bacterial DNA. Matings between recipient and donor strains carrying plasmids were performed by spreading 0.2 ml of overnight cultures of the strains on 63 minimal medium plates and incubating for 5 h at 30°C. Bacteria were resuspended in 1 ml of 63 minimal medium and spread on the appropriate selective media.
Recombinant DNA techniques. Standard procedures were performed for genomic and plasmid DNA extraction (35). Restriction enzymes (Eurogentec) and T4 DNA ligase (Gibco-BRL) were used according to the manufacturer's recommendations. DNA sequences were determined with the Sequenase version 2.0 kit (U.S. Biochemical Corporation). For DNA hybridization, 10 µg of DNA was digested with the appropriate restriction enzyme(s) and treated as previously described (35). DNA fragments were transferred and fixed onto a Biotrans nylon membrane as recommended by the manufacturer (ICN). The digoxigenin DNA-labeling and detection kit was used for labeling of probes, hybridization, and detection of hybrids according to the manufacturer's recommendations (Boehringer Mannheim).
The DNA sequences and deduced amino acid sequences were analyzed by using computer programs made available from Infobiogen (http://www.infobiogen.fr/).Construction of opgG::uidA-kan
and opgH::uidA-kan insertions and
marker exchange recombination.
The uidA-kan cassette
was liberated by SphI digestion of plasmid pUIDK11
(1). In order to inactivate the opgG or
opgH gene, this SphI DNA fragment was then
introduced into pNFW32, containing the opgGH operon,
partially digested by SphI, either in the SphI site located in opgG or in the first of the two
SphI sites located in opgH (Fig.
1). The resulting plasmids were
introduced into E. chrysanthemi cells by electroporation.
The insertions were then integrated into the E. chrysanthemi
chromosome by marker exchange recombination after successive cultures
in low-phosphate medium in the presence of kanamycin (33).
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OPG analysis. To measure OPG synthesis in E. chrysanthemi, cells were grown overnight in LB without NaCl (50 ml). Bacteria were collected by centrifugation at 4°C for 15 min at 8,000 × g. Cell pellets were resuspended in 1 ml of distilled water and extracted with 5% trichloroacetic acid (TCA). The TCA extracts were neutralized with ammonium hydroxide 10%, desalted on a Sephadex G15 column (Pharmacia), and concentrated by rotary evaporation. The resulting material (2 ml) was then fractionated by gel filtration on a Bio-Gel P-4 (Bio-Rad). The column (55 by 1.6 cm) was equilibrated with 0.5% acetic acid. The column was eluted in the same buffer at a flow rate of 15 ml/h, and fractions of 2.5 ml were collected. Sugar content in each fraction was determined colorimetrically by the anthrone reagent procedure (37).
To measure OPG synthesis in E. coli, cultures (5 ml) were made in LOS containing 0.45 mM [2-3H]-glycerol (296 MBq/mmol; New England Nuclear), and labeled OPGs were extracted by the charcoal adsorption procedure; 0.2 ml from the 2 ml of pyridine extract obtained by this procedure was used for counting (23). When necessary, 1.5 ml of pyridine extract was chromatographed on a Sephadex G25 medium column (Pharmacia). The column (2 by 45 cm) was equilibrated with 0.15 M ammonium acetate in 7% (vol/vol) aqueous propanol. The column was eluted with the same buffer at a rate of 17 ml/h. Fractions (1.5 ml) were collected, and radioactivity was counted.Determination of enzyme activities.
Enzymatic assays were
performed on toluene-treated cells. Pectate lyase activity was
determined by monitoring spectrophotometrically the formation of
unsaturated products from polygalacturonate at 230 nm. The assay
mixture consisted of 0.1 M Tris-HCl (pH 8.5), 0.1 mM CaCl2,
and polygalacturonate at 0.5 mg/ml. One unit of activity was defined as
the amount of enzyme required to produce 1 µM unsaturated product per
min (30). -Galactosidase activity was determined in Z
buffer (29).
Pathogenicity test.
Potato tubers were inoculated as
previously described (24). Sterile pipette tips containing
a bacterial suspension of 107 cells in 5 µl were inserted
into the tuber parenchyma. After 24 to 72 h of incubation in a dew
chamber, tubers were sliced vertically through the inoculation point,
and the weight of decayed tissue was taken as the characteristic of
disease severity. Moreover, in coinoculation tests containing both
Kanr (Opg
) and Kans
(Opg+) bacteria, all the macerated tissues (ranging from
0.3 to 2.0 g) were collected, weighed, and diluted to a
concentration of 1 g of macerated tissue in 2 ml of sterile
physiological distilled water. The numbers of bacteria originating from
each of the two strains were determined by spreading appropriate
dilutions of the cell suspensions onto LB plates with or without
kanamycin. Chicory leaves were slightly wounded prior to inoculation
(106 bacteria). After 24 h of incubation in a dew
chamber, the length of rotted tissue was measured to estimate disease
severity. These tests were repeated in at least three independent experiments.
Nucleotide sequence accession number. Sequence data will appear in the EMBL/GenBank/DDBJ nucleotide sequence data libraries under accession number AJ294718.
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RESULTS |
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Materials and Methods
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Discussion
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Complementation of the mdoH200::Tn10 mutation of E. coli with an RP4 derivative of E. chrysanthemi. Since plasmids harboring the P. syringae hrpM gene complemented the E. coli mdoH200::Tn10 mutation, we postulated that the genes involved in OPG backbone synthesis in E. chrysanthemi may also complement such mutations. The absence of a selectable phenotype in mdoGH mutants of E. coli prevented direct selection for Opg+ complementation. We decided to use the selectable marker pyrC, located 13 kb downstream from the mdoGH locus in E. coli. Despite some important genomic rearrangements, the genetic organization of several genomic regions is quite similar in E. coli and E. chrysanthemi (17).
Plasmid pULB110, a kanamycin-sensitive RP4::mini-Mu derivative (41), was used to generate R-prime derivatives containing an insert of bacterial DNA from E. chrysanthemi complementing the pyrC mutation of E. coli strain NFB223 (pyrC mdoH200::Tn10). Ura+ (Pyr+) transconjugants were isolated on M63 medium plates supplemented with streptomycin. The plasmid contents of 12 Pyr+ transconjugants were analyzed. Four plasmids with a DNA insert of 15 to 44 kb were tested for the Opg phenotype that they conferred. One of them, pR24, complemented the mdoH mutation and conferred an Opg+ phenotype. Moreover, the amount of OPGs produced by the mdoH200::Tn10 strain harboring pR24 was 1.5 times higher than in the wild-type E. coli strain.Cloning of opgGH locus of E. chrysanthemi. Plasmid pR24 was digested with EcoRI, and the resulting DNA fragments were ligated into the EcoRI site of pYZ4. Plasmid pNFW3, containing a 9.5-kb DNA insert, was Opg+ (Fig. 1) and thus complemented the mdoH200::Tn10 mutation of NFB216, but only at the wild-type level. The sequences of both ends of the insert were established. The region adjacent to the lac promoter present in the vector was highly similar to the beginning of the open reading frame of the E. coli mdoG gene (starting at amino acid number 72). Then, a truncated promoterless version of the E. chrysanthemi locus has been cloned, and complementation of the mdoH200::Tn10 mutation was most probably the result of transcription from the lac promoter. A similar observation was made during the cloning of the mdoGH operon (23).
In order to restore a complete version of the operon, a 3.3-kb SalI DNA fragment of pR24 overlapping the pNFW3 insert was detected by Southern blot using the 1.1-kb EcoRI-BamHI fragment of the pNFW3 insert as a probe (Fig. 1) and cloned into pUC18. The resulting plasmid, pNFW27, introduced into NFB702 complemented the mdoG202::neo mutation. The reconstitution of the whole operon was done as follows. pNFW3 was digested with BamHI and partially digested with EcoRV to obtain a 4.1-kb BamHI-EcoRV DNA fragment (Fig. 1). This DNA fragment was ligated into pNFW27 digested with BamHI and SmaI (using a site originating from the vector polylinker) to give pNFW32 containing a 6.4-kb DNA insert. Complementation of mdoG202::neo and mdoH200::Tn10 was observed when pNFW32 was introduced into strains NFB702 and NFB216, respectively. Moreover, strains harboring pNFW32 contained an OPG level twice that of the wild type. The same increasing effect was observed when E. coli mdoGH was cloned into a multicopy plasmid (23). These results clearly indicated that the pNFW32 insert contains the entire E. chrysanthemi locus necessary for complementation of the mdoGH operon of E. coli.Analysis of nucleotide sequence of opgGH genes of
E. chrysanthemi.
The 5.0 kb starting from the first
EcoRV (Fig. 1) and encompassing the opgGH locus
were sequenced in both strands. The analysis of this sequence confirmed
the genetic analysis, and two open reading frames were detected. The
first open reading frame, opgG, encoding a 522-amino-acid
polypeptide, begins with a putative start codon (GTG) and
ends at a TAA codon. The GTG start codon represents 11.5%
(versus 86.5% for ATG) of the start codons found in the 88 sequences of E. chrysanthemi available in the EMBL sequence database (release 62, March 2000). The second open reading frame, opgH, encoding an 850-amino-acid polypeptide, begins with a
putative start codon (ATG) and ends at a TGA codon. Moreover,
the end of the opgG sequence overlaps the start of
opgH (ATGAATAA). An identical sequence was observed in E. coli, leading to the same
overlapping between mdoG and mdoH
(25). The putative ribosome-binding site is located 8 nucleotides upstream from the GTG start codon of opgG.
The 
10 and 35 regions of the putative promoter are separated by 18 nucleotides, and the GTG start codon is located 32 nucleotides downstream from the putative transcriptional start site. In
E. coli, the same promoter sequence was found except
for one nucleotide in the 10 region. Thus, the two genes most
probably form an operon functionally homologous to the
mdoGH operon.
Construction of opgG and opgH mutants and localization of opgGH locus on chromosome of E. chrysanthemi. Both gene disruptions were obtained by insertion of a uidA-kan cassette into pNFW32 followed by transfer into the chromosome (see Materials and Methods) to give NFB3500 (opgG500::uidA-kan) and NFB3501 (opgH501::uidA-kan). Recombination of these constructions with the homologous locus into the chromosome was confirmed by Southern blot hybridization using the 3.3-kb SalI DNA fragment of pNFW27 (data not shown).
OPG synthesis was measured in both mutant strains. OPGs were extracted from overnight cultures grown in LB without NaCl, and the extracts were loaded onto a Bio-gel P-4 column (see Materials and Methods). The peak containing OPGs, normally observed in extracts of the wild-type strain 3937 (9), was absent in both extracts, indicating that these mutants are affected in OPG backbone synthesis (data not shown). Chromosomal mobilization mediated by plasmid pULB110 was used for conjugation with various polyauxotrophic recipients to localize the opgGH locus on the E. chrysanthemi chromosome. Using the opgG500::uidA-kan fusion, the Kanr marker cotransferred at 4% with ade-377, 10% with ogl, and 96% with ura-2. Thus, the opgGH locus is located very near the ura-2 marker on the E. chrysanthemi chromosome (17). In addition, the auxotrophy of the ura-2 mutant is complemented by the four pyrC+ R-prime plasmids isolated above, strongly suggesting that ura-2 is a mutation affecting the pyrC gene of E. chrysanthemi. Moreover, in a generalized transduction by phageEC2, the Kanr resistance of the
opgG500::uidA mutant was cotransduced at 80% with ura-2.
Pleiotropic phenotypes of opg mutants of E. chrysanthemi.
Colonies of the opg mutants have a
mucoid aspect on minimal medium agar plates, a typical sign of
increased exopolysaccharide (EPS) biosynthesis. We analyzed the effect
of the opgG mutation on the transcription of the EPS
biosynthetic operon of E. chrysanthemi using the
epsG::lacZ fusion present in the
eps-29 mutant (A2575). The -galactosidase activity was
increased about threefold in the
opgG500::uidA-kan derivative (data not shown)
grown with either glycerol or glucose as the sole carbon source.
Induction of transcription of the EPS genes was previously observed in
opg mutants of E. coli (12) and
Agrobacterium tumefaciens (8).
Exoenzyme synthesis and secretion in opgG mutant of E. chrysanthemi. Pectate lyase activity was first estimated on plates containing polygalacturonate and a second carbon source. No halo derived from polygalacturonate degradation was observed when the opg mutants were grown in the presence of glycerol, glucose, or sucrose. A reduced halo, compared with that of the wild-type strain, was observed when succinate or galacturonate was added or in the absence of a second carbon source. Thus, pectate lyase activity appeared to be reduced at various levels in the opg mutants, depending on the carbon source used by the bacteria (data not shown).
Assay of the global pectate lyase activity demonstrated that production of these enzymes decreased by twofold in the presence of the opgG mutation in either the presence or absence of an inducer (Table 2). A similar decrease was observed when pelC::lacZ or pelE::lacZ transcriptional fusions were used to analyze the expression of these genes (Table 2).
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Pathogenicity of opg mutants of E. chrysanthemi.
Inoculation with the opgG mutant
incited no tissue maceration on potato tubers after 24 h, while an
outS mutant produced reduced symptoms (Fig.
3A). Identical results were obtained when
the opgG and opgH mutants were used to inoculate
chicory leaves (Fig. 3B). Moreover, after a 10-day incubation, plants
inoculated with opg mutants did not exhibit any symptoms.
The opg mutants were thus completely nonvirulent.
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bacteria, while the inoculum contained 9 × 106 Opg bacteria. Since the population would
have increased only by a factor of 1.3 (in the range of error), one can
consider that no growth of the mutant strain occurred in the plant
tissues. During the same time, the wild-type population increased by a
factor of 4,580. Thus, intercellular complementation did not occur
between the opgG mutant and wild-type strains, indicating
that the loss of virulence of opgG mutants did not result
simply from motility or exoenzyme deficiencies. After plant infection,
the opg mutant stopped its growth but remained viable.
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DISCUSSION |
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Materials and Methods
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Discussion
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This paper describes the molecular organization of the opgGH operon from E. chrysanthemi, the phenotypes observed when these genes are disrupted, and their implication in its pathogenicity.
As in E. coli, the two genes necessary for OPG backbone synthesis in E. chrysanthemi are organized in an operon, opgGH, in the vicinity of the pyrC gene. In both organisms, yceE, a gene of unknown function, is found downstream from mdoH or opgH. In E. coli, this gene is transcribed with msyB, which is only known to encode a suppressor of a secY thermosensitive mutation. Nevertheless, in E. chrysanthemi, msyB is not present at this locus (Fig. 1). The DNA sequences of the two organisms are conserved over the entire sequences of the opgGH and mdoGH operons, including the promoter region. In E. coli, the mdoC gene, involved in OPG succinylation, is located just upstream from the mdoGH operon (21). Despite the fact that the OPGs of E. chrysanthemi are succinylated (9), no gene homologous to mdoC was found upstream of the opgGH operon. The 13-bp direct repeats found upstream from opgGH could be the limit of a chromosomal rearrangement mediated by a transposable element. Moreover, the gene encoding the OPG succinylation activity could differ strongly from mdoC, since several attempts to obtain PCR amplification of that gene failed (data not shown).
The opg mutants exhibit a pleiotropic phenotype. Colonies of opg mutants are mucoid, indicating increased biosynthesis of EPS. This correlates with a threefold increase in the expression of the epsG::lacZ fusion in an opgG mutant background. This increase could be the result of a direct action of OPGs on eps gene transcription via a regulatory protein(s). Actually, in E. coli, the cps genes, involved in colanic acid capsular polysaccharide biosynthesis, are regulated by a two-component sensor-regulator system, RcsB/RcsC. In an mdoH mutant, cps gene transcription is increased but remains regulated in an RcsB/RcsC-dependent fashion. This suggests that RcsC, the sensor of the two-component system, could sense the level of OPG in the periplasm in a still unknown way (12). In Erwinia amylovora and Erwinia stewartii, the RcsB and RcsC proteins are involved in the regulation of EPS biosynthesis and show strong similarities with the RcsB and RscC proteins of E. coli, indicating the existence of a family of related capsule activator proteins (4). Thus, in E. chrysanthemi, OPGs could also participate in EPS biosynthesis regulation by acting via a similar two-component regulatory system. Alternatively, this increase could be one of the responses resulting from a disorganization of the cell envelope induced by the lack of OPGs. Disorganization of the cell envelope is suggested by the other phenotypes of the opg mutants, particularly bile salt hypersensivity and defective chemotaxis. Lack of OPGs could result in abnormal assembly of several envelope components due to an unknown structural function of the glucans, or OPG concentration could be detected and used to regulate the synthesis of several envelope components, especially those which are subjected to osmotic regulation, like the flagellum apparatus (14) and colanic acid synthesis (12) in E. coli.
Among the various effects observed in opg mutants, the most important seems to be the reduction in pectate lyase production and secretion, because they are known to play a crucial role in virulence. opg mutations result in a twofold decrease in pectate lyase production associated with a similar decrease in pel gene transcription. OPGs could modulate pel gene expression via the two-component sensor-regulator system PecM/PecS (15) in a way which remains to be elucidated, but the existence of new regulatory factors cannot be excluded. The reduction in Pel protein synthesis is associated with a reduction in their secretion through the cell envelope. This defect in the Out secretory apparatus could be explained in part by a decrease in its synthesis and in part by an alteration in its assembly and/or functioning resulting from the cell envelope disorganization.
The opg mutants are completely nonvirulent when inoculated into potato tubers or chicory leaves. This phenotype has already been observed in planta with the hrpM mutant of P. syringae pv. syringae (28) and for the hrpM mutant of P. aeruginosa PA14 (26). The major consequence of pel and out mutations is the severely reduced virulence of strains harboring these mutations. However, the complete loss of virulence of the opg mutants cannot be explained only by a defect in pectate lyase secretion, since the outS mutant (severely impaired in pectate lyase secretion) induces reduced symptoms, while the opg mutant, which secretes more pectate lyases than the outS mutant, induces no symptoms.
The outS (altered secretion) and msrA (altered motility and peptide methionine sulfoxide reductase) mutant cells could be sustained by coinoculation with no more than 1% of the wild-type strain in potato tubers. These mutant strains grew poorly in planta and produced few macerated tissues by themselves, but they are able to use the macerated tissues produced by the wild-type strain for their growth. This clearly suggests that only the plant-bacterium interaction is disrupted in these mutants. On the other hand, the opg mutant cells cannot grow when coinoculated in potato tubers with as much as 90% wild-type cells, while growth remains unaffected when the coinoculation occurs in laboratory conditions. However, the opg mutants which are unable to use macerated tissues for their growth can survive in these conditions. These results indicate that OPGs are necessary not only for plant colonization, but also for bacterial multiplication in macerated tissues. When E. chrysanthemi is grown in a liquid medium, a large fraction of the OPGs produced can be recovered in the medium, at least under certain circumstances (9). We have no indication whether this is true when bacteria grow in planta. However, our experiments clearly show that if OPGs are released in the plant tissues during co-inoculation with wild-type cells, these external OPGs cannot complement the growth defect of the opg mutants. Moreover, OPG-defective mutants of A. tumefaciens remain nonvirulent when inoculated in planta with purified OPGs (8). This strongly suggests that OPGs need to be inside the periplasmic space and not in the external environment during the infection process.
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ACKNOWLEDGMENTS |
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We are grateful to F. Barras and to our colleagues at Unité de Microbiologie Génétique for providing us with bacterial strains.
This research was supported by grants from the Centre National de la Recherche Scientifique and the Ministère de l'Education Nationale, de la Recherche et de la Technologie.
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FOOTNOTES |
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* Corresponding author. Mailing address: U.S.T.L., Bât. C9, 59655 Villeneuve d'Ascq Cedex, France. Phone: 34 (0)3 20 43 65 92. Fax: 34 (0)3 20 43 65 55. E-mail: Jean-Pierre.Bohin{at}univ-lille1.fr.
Present address: Departomento de Microbiologia, Facultad de
Ciencias Bioquimicos Formacenticas, Universidad Nacional de Rosario UNR
Suipocha 531, 2000 Rosario Santa Fe, Argentina.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, May 2001, p. 3134-3141, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3134-3141.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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