Laboratoire de Pathologie Végétale, UMR INRA/INA-PG, 16 rue Claude Bernard, 75231 Paris cedex 05, France1
Author for correspondence: Corine Enard. Tel: +33 1 44 08 17 06. Fax: +33 1 44 08 16 31. e-mail: enard{at}inapg.inra.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: iron transport, siderophore, achromobactin, chrysobactin, pathogenicity
Abbreviations: EDDHA, ethylenediamine-N-N'-bis(2-hydroxyphenylacetic acid)
The GenBank accession number for the sequence reported in this paper is Y15888.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In response to iron starvation, Er. chrysanthemi 3937 synthesizes the catechol-type siderophore chrysobactin (Persmark et al., 1989 ). Iron acquisition mediated by chrysobactin is essential for the bacterium to disseminate throughout its host plant (Enard et al., 1988
; Masclaux & Expert, 1995
). Mutants affected in chrysobactin-mediated iron transport cannot use EDDHA [ethylenediamine-N-N'-bis(2-hydroxyphenylacetic acid)]-chelated iron as an iron source but retain the ability to grow on a medium containing 2,2'-dipyridyl (Enard et al., 1988
). Growth on 2,2'-dipyridyl is dependent on a second iron-acquisition system based on the production of achromobactin, a siderophore which is neither a catechol nor a hydroxamate (Mahé et al., 1995
) but a citrate derivative (Münzinger et al., 2000
). For instance, chrysobactin-deficient mutants lacking a functional ferriachromobactin permease encoded by the cbrABCD operon failed to grow in the presence of 2,2'-dipyridyl (Mahé et al., 1995
). These two siderophore-dependent iron-transport pathways are differentially regulated by iron: derepression of the chrysobactin system requires more severe iron deficiency than that of the achromobactin system. Repression by iron is mediated by the ferric uptake regulatory protein, Fur, that was recently characterized in Er. chrysanthemi 3937. The Fur repressor plays a key role in the coordinate regulation of genes encoding iron-transport proteins and pectinases (Franza et al., 1999
). In addition, Er. chrysanthemi 3937 is able to use the two xenosiderophores, enterobactin and ferrichrome, produced by enterobacteria and the fungal genus Ustilago.
Here, we describe a TonB- mutant isolated by insertional mutagenesis of a chrysobactin-deficient strain. In Escherichia coli, the function of the TonB protein has been thoroughly studied (for reviews see Braun, 1995 ; Moeck & Coulton, 1998
). The passage of the ferrisiderophore through the outer membrane requires active transport via a receptor which functions as a pore energized by the cytoplasmic-membrane-generated protonmotive force transduced by the TonB protein and auxiliary proteins ExbB and ExbD forming the Ton complex (Higgs et al., 1998
; Larsen et al., 1999
; Postle, 1999
). The Er. chrysanthemi TonB- mutant studied displays a leaky phenotype regarding utilization of the ferric complex of achromobactin.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Cross-feeding assay.
Utilization of ferriachromobactin, ferrichrysobactin, ferrienterobactin and ferrichrome by the tested strains was determined in a bioassay under low-iron conditions. Plates were poured with 25 ml EDDHA-L-agar medium seeded with an overnight L-broth culture of each strain at a final concentration of 104 c.f.u. ml-1. Ferriachromobactin, ferrichrysobactin and ferrienterobactin were provided as culture supernatants of strains 3937 cbsE1, 3937 acs-59 and MM272-60, respectively. Sterile disks of 6 mm diameter were placed on the agar surface and 60 µl of each filter-sterilized fluid was added. The radii of zones of growth of the tested strains were measured after 48 h.
Mating, transduction and transformation methods.
These were as described by Franza et al. (1991) .
DNA methods.
The cosmid library of Er. chrysanthemi was constructed in vector pLA2917 linearized with BglII using genomic DNA partially digested with Sau3A. The ligated DNA was packaged in vitro using the Gold Kit packaging extracts as recommended by the supplier (Stratagene). The packaged extracts were used to transduce E. coli ED8767. Tcr clones were selected and screened for kanamycin sensitivity. Other DNA methods were as previously described (Franza & Expert, 1991 ).
Transport experiments.
Bacterial culture and transport experiments were performed as previously described (Mahé et al., 1995 ) with the following modifications: radiolabelled iron (1 µM) was supplied as 59Fe-achromobactin (metal:ligand ratio of approximately 1:4). The source of achromobactin was a filter-sterilized supernatant of a culture of strain 3937 cbsE1tonB60 grown for 8 h in Tris medium. Formation of the ferric complex of achromobactin was estimated using the CAS assay. Filters were washed with 20 ml supernatant fluid containing a 10-fold excess of unlabelled Fe-achromobactin. Experiments were performed in duplicate.
Determination of pectate lyase activity in bacterial culture.
Cultures were grown as previously described (Franza et al., 1999 ). Pectate lyase specific activity is expressed as µmol degradation products liberated min-1 (mg dry weight of bacteria)-1. Each experiment was performed three times.
Pathogenicity assay.
Pathogenicity was tested on potted African violets (cv. Blue Rhapsody), as reported by Expert & Toussaint (1985) with modifications: the inoculum was 100 µl of a bacterial culture grown in M63 medium for 18 h diluted in the same medium to give an OD600 of 0·3. Twelve plants were inoculated with each strain. Progression of the symptoms was scored for 5 weeks.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate the level of ferriachromobactin transport in more detail, we carried out uptake experiments using 59Fe-achromobactin (Fig. 1). The strain harbouring the tonB60 mutation, like the receptor mutant, failed to incorporate radioactive iron. The parental strain incorporated 40 pmol ferric iron (a quarter of the labelled iron present in the assay) within 20 min. Thus, the mutation affects the transport of ferriachromobactin. We also checked the ability of the mutant to utilize chrysobactin, enterobactin and ferrichrome as iron sources. No halo of growth was observed in cross-feeding assays for any of these three siderophores. Therefore, the transport step affected by the tonB60 mutation is required for the utilization of all siderophores taken up by the bacterium.
|
DNA sequence of the Er. chrysanthemi tonB gene and organization of the tonB region
Plasmid pCE1 was used for sequencing (GenBank accession no. Y15888). The 1122 bp sequenced in the genomic insert included an ORF of 759 bp (nucleotide positions 3021060) encoding a putative protein of 252 amino acids with a predicted molecular mass of 27600 Da. The deduced translation product is 58% identical and 72% similar to the Serratia marcescens TonB protein. The amino acid sequence is rich in proline (19%), with nearly half the residues from amino acid positions 72 to 118 being proline. The region includes a (Lys-Pro)3-(Glu-Pro)-(Lys-Pro)2 repeat. This Pro-rich region is typical of TonB proteins and confers a rod-like structure to the protein such that it can reach the outer membrane from its anchor in the inner membrane (Hannavy et al., 1990 ; Larsen et al., 1993
). This anchor is located at the N-terminal end of the predicted protein (amino acids 1535, Karlsson et al., 1993a
, b
; Postle & Skare, 1988
) and harbours the highly conserved motif SHLS (Koebnik, 1993
; Koebnik et al., 1993
) known to be essential in E. coli for interaction with the auxiliary protein ExbB of the Ton complex (Braun et al., 1996
; Traub et al., 1993
). The central part of the predicted protein contains the highly conserved motif PXYP, which is thought to be required for the protein to adopt the conformation necessary to allow interaction with the outer membrane receptors of the ferrisiderophores (Larsen et al., 1997
). The C-terminal end of the predicted protein exhibited a putative amphipatic helix (amino acids 211226) believed to facilitate interaction between TonB and the outer membrane (Larsen et al., 1997
). A highly hydrophobic C-terminal end similar to those of the E. coli and Salmonella typhimurim TonB proteins was identified. The role of this segment in the E. coli and S. typhimurium TonB proteins is unknown (Anton & Heller, 1991
; Bruske et al., 1993
; Larsen et al., 1997
). Pairwise comparisons of the Er. chrysanthemi predicted TonB protein (TonBEch) with tonB gene products of the other bacteria showed that TonBEch shares at least 50% identity with TonB proteins from enterobacteria and 2040% identity with TonB proteins from other bacteria.
A putative ribosome-binding site and a 30 bp promoter/operator potential region were identified upstream from the translation start codon (Fig. 2a). This putative promoter is nearly identical to the tonB promoter shared by 6 of the 20 known tonB genes, including the -35 and -10 sites and a Fur box (see Fig. 2a
). In Er. chrysanthemi, only 28 of the 30 bp are conserved (Fig. 2a
). The differences are both in the potential Fur operator sequence, although this promoter/operator region is perfectly conserved among the six other enterobacteria. The conservation of this promoter suggests a common origin of those tonB genes. CLUSTAL W phylogenic tree analysis (Thompson et al., 1994
) indicates that TonBEch is closer to enterobacterial than the other TonB proteins (data not shown). In addition, the N-terminal Prodom domain 10309, only common to the enterobacteria, is also present in TonBEch (amino acids 165249). Upstream from the tonB gene there is a segment highly similar to the E. coli region that spans the bacteriophage
80 attachment site (70% DNA sequence identity on one strand; Fig. 2b
). Nevertheless, strain 3937 is not sensitive to
80.
|
The location of the prophage insertion in the tonB gene was determined using pCE2. The insertion is 48 bp downstream from the translation start. The position of this insertion excluded the possibility of translation of a functional truncated TonB protein that could explain the residual growth of the mutant observed in cross-feeding assays.
Genetic mapping of the tonB60 mutation
To locate the tonB gene on the linkage map of Er. chrysanthemi 3937, we used the kanamycin-sensitive conjugative plasmid pULB110, derived from RP4:miniMu. The cotransfer of the trp marker and the kanamycin marker of the mutation was 96%, suggesting possible cotransduction of the two markers with the phage ECII. An average of 45% cotransduction was obtained. Therefore, the tonB gene maps very close to the trp marker, less than 62 kb away (the size of the phage genome). For a map of strain 3937 see Hugouvieux-Cotte-Pattat et al. (1996)
.
Pathogenicity of the TonB- mutant
The pathogenicity of the TonB- mutant was assessed using potted African violets. Unlike the parental strain, which gave rise to a systemic infection on a quarter of the inoculated plants, the mutant strain had no systemic effects. However, the mutant was able to macerate the inoculated leaf (two-thirds of the inoculated plants), as has been observed for mutants affected in chrysobactin biosynthesis (Enard et al., 1988 ). It is surprising that a strain deficient in all high-affinity iron-uptake pathways was not less agressive than a mutant affected in the chrysobactin system only. No revertants were recovered from inoculated leaves (data not shown) and thus the ability of the TonB- mutant to invade the leaf did not result from the reversion of the tonB mutation. We determined the total pectate lyase activity of the tonB mutant, and for comparison, the wild-type strain. Both strains were grown under various conditions of iron availability, i.e. in Tris medium with or without EDDHA, a strong ferric iron chelator. The carbon source was either glycerol or polygalacturonate, the latter being the inducer of genes encoding pectinases. The tonB mutant grew slightly slower than the wild-type in Tris medium (Fig. 3a
). Therefore, although poor in iron because of the low phosphate concentration, Tris medium contains traces of iron allowing the tonB mutant to grow. However, in the presence of EDDHA, the growth of the mutant strain was almost abolished (data not shown). In the presence of glycerol no pectate lyase activity was detected. In the presence of the pectic inducer polygalacturonate, the pectate lyase activity produced by the tonB mutant was twice as high as that of the wild-type strain and the activity of the mutant appeared earlier (Fig. 3b
). Increase in this enzymic activity may explain why the virulence of the mutant was not as severely impaired as expected.
|
The apparent discrepancy in the behaviour of the TonB- mutant in the cross-feeding assays and the uptake experiments is interesting. In a cross-feeding assay, bacterial growth is recorded after 24 h incubation. Possibly, the relatively long period of time allows expression of a TonB-independent transport mechanism that cannot be detected during the uptake experiment. Similarly, TonB-independent transport of ferrioxamines B and E has been described in Salmonella enterica. This transport was only revealed by cross-feeding assays and its level has not been determined (Kingsley et al., 1999 ).
Finally, the TonB- mutant was not strongly impaired in its ability to invade leaf tissues following inoculation. The total pectate lyase activity produced by bacterial cells grown under iron-deficient conditions was twice as high in the mutant strain as in the wild-type. Previous work (Franza et al., 1999 ) demonstrating the existence of coordinate regulation, by iron via the Fur sensory and regulatory protein, between iron transport functions and synthesis of several pectate lyases may explain this effect. Depletion of the intracellular iron pool, as probably occurs in the TonB- mutant, could result in up-regulation of iron-controlled genes encoding pectate lyase.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anton, M. & Heller, K. J. (1991). Functional analysis of a C-terminal altered TonB protein of Escherichia coli.Gene 105, 23-29.[Medline]
Bachmann, B. J. (1987). Derivations and Genotypes of Some Mutant Derivatives of Escherichia coli K12. Washington, DC: American Society for Microbiology.
Barras, F., Van Gijsegem, F. & Chatterjee, A. K. (1994). Extracellular enzymes and pathogenesis of soft-rot Erwinia.Annu Rev Phytopathol 32, 201-234.
Braun, V. (1995). Energy-coupled transport and signal transduction through the Gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins.FEMS Microbiol Rev 16, 295-307.[Medline]
Braun, V., Gaisser, S., Herrmann, C., Kampfenkel, K., Killmann, H. & Traub, I. (1996). Energy-coupled transport across the outer membrane of Escherichia coli: ExbB binds ExbD and TonB in vitro, and leucine 132 in the periplasmic region and aspartate 25 in the transmembrane region are important for ExbD activity.J Bacteriol 178, 2836-2845.[Abstract]
Bruske, A. K., Anton, M. & Heller, K. J. (1993). Cloning and sequencing of the Klebsiella pneunoniae tonB gene and characterization of Escherichia coliK. pneunoniae TonB hybrid proteins.Gene 131, 9-16.[Medline]
Castilho, B. A., Olfson, P. & Casadaban, M. J. (1984). Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposons.J Bacteriol 158, 488-495.[Medline]
Enard, C., Diolez, A. & Expert, D. (1988). Systemic virulence of Erwinia chrysanthemi 3937 requires a functional iron assimilation system. J Bacteriol 170, 2419-2426.[Medline]
Expert, D. & Toussaint, A. (1985). Bacteriocin-resistant mutants of Erwinia chrysanthemi: possible involvement of iron acquisition in phytopathogenicity.J Bacteriol 163, 221-227.[Medline]
Expert, D., Sauvage, C. & Neilands, J. B. (1992). Negative transcriptional control of iron transport in Erwinia chrysanthemi involves an iron-responsive two factor system.Mol Microbiol 6, 2009-2017.[Medline]
Expert, D., Enard, C. & Masclaux, C. (1996). The role of iron in plant hostpathogen interactions.Trends Microbiol 4, 232-237.[Medline]
Faelen, M. (1987). In Phage Mu, pp. 309316. Edited by N. Symonds, A. Toussaint, P. Van de Putte & M. M. Howe. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Figurski, D. H. & Helinski, D. R. (1979). Replication of an origin containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.Proc Natl Acad Sci U S A 76, 1648-1652.[Abstract]
Franza, T. & Expert, D. (1991). The virulence-associated chrysobactin iron uptake system of Erwinia chrysanthemi 3937 involves an operon encoding transport and biosynthetic functions.J Bacteriol 173, 6874-6881.[Medline]
Franza, T., Enard, C., Van Gijsegem, F. & Expert, D. (1991). Genetic analysis of the Erwinia chrysanthemi 3937 chrysobactin iron-transport system: characterisation of a gene cluster involved in uptake and biosynthetic pathway.Mol Microbiol 5, 1319-1329.[Medline]
Franza, T., Sauvage, C. & Expert, D. (1999). Iron regulation and pathogenicity in Erwinia chrysanthemi strain 3937: role of the Fur repressor protein.Mol PlantMicrobe Interact 12, 119-128.[Medline]
Gaisser, S. & Braun, V. (1991). The tonB gene of Serratia marcescens: sequence, activity and partial complementation of Escherichia coli tonB mutants. Mol Microbiol 5, 2777-2787.[Medline]
Hannavy, K., Barr, G. C., Dorman, C. J. & 7 other authors (1990). TonB protein of Salmonella typhimurium. A model for signaling transduction between membranes. J Mol Biol 216, 897910.[Medline]
Hantke, K. & Braun, V. (1978). Functional interaction of the tonA/tonB receptor system in Escherichia coli.J Bacteriol 135, 190-197.[Medline]
Higgs, P. I., Myers, P. S. & Postle, K. (1998). Interactions in the TonB-dependent energy transduction complex: ExbB and ExbD form homomultimers. J Bacteriol 180, 6031-6038.
Howe, M. M. (1973). Prophage deletion mapping of bacteriophage Mu-1.Virology 54, 93-101.[Medline]
Hugouvieux-Cotte-Pattat, N. & Robert-Baudouy, J. (1985). Lactose metabolism in Erwinia chrysanthemi.J Bacteriol 162, 248-255.[Medline]
Hugouvieux-Cotte-Pattat, N., Condemine, G., Nasser, W. & Reverchon, S. (1996). Regulation of pectinolysis in Erwinia chrysanthemi.Annu Rev Microbiol 50, 213-257.[Medline]
Karlsson, M., Hannavy, K. & Higgins, C. F. (1993a). A sequence-specific function for the N-terminal signal-like sequence of the TonB protein. Mol Microbiol 8, 379-388.[Medline]
Karlsson, M., Hannavy, K. & Higgins, C. F. (1993b). ExbB acts as a chaperone-like protein to stabilize TonB in the cytoplasm. Mol Microbiol 8, 389-396.[Medline]
Kingsley, R. A., Reissbrodt, R., Rabsch, W. & 7 other authors (1999). Ferrioxamine-mediated iron(III) utilization by Salmonella enterica. Appl Environ Microbiol 65, 16101618.
Koebnik, R. (1993). The molecular interaction between components of the TonB-ExbBD-dependent and of the TolQRA-dependent uptake systems.Mol Microbiol 9, 219.[Medline]
Koebnik, R., Bäumler, A. J., Heesemann, J., Braun, V. & Hantke, K. (1993). The TonB protein of Yersinia enterocolitica and its interactions with TonB-box proteins.Mol Gen Genet 237, 152-160.[Medline]
Kotoujansky, A., Lemattre, M. & Boitard, P. (1982). Utilization of a thermosensitive episome bearing transposon Tn10 to isolate Hfr donor strains of Erwinia carotovora subsp. chrysanthemi. J Bacteriol 150, 122-131.[Medline]
Larsen, R. A., Wood, G. E. & Postle, K. (1993). The conserved proline-rich motif is not essential for energy transduction by Escherichia coli TonB protein.Mol Microbiol 10, 943-953.[Medline]
Larsen, R. A., Foster-Hartnett, D., McIntosh, M. A. & Postle, K. (1997). Regions of Escherichia coli TonB and FepA proteins essential for in vivo physical interactions.J Bacteriol 179, 3213-3221.[Abstract]
Larsen, R. A., Thomas, M. G. & Postle, K. (1999). Protonmotive force, ExbB and ligand-bound FepA drive conformational changes in TonB.Mol Microbiol 31, 1809-1824.[Medline]
Lojkowska, E., Masclaux, C., Boccara, M., Robert-Baudouy, J. & Hugouvieux-Cotte-Pattat, N. (1995). Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937.Mol Microbiol 16, 1183-1195.[Medline]
Mahé, B., Masclaux, C., Rauscher, L., Enard, C. & Expert, D. (1995). Differential expression of two siderophore-dependent iron-acquisition pathways in Erwinia chrysanthemi 3937: characterization of a novel ferrisiderophore permease of the ABC transporter family.Mol Microbiol 18, 33-43.[Medline]
Masclaux, C. & Expert, D. (1995). Signalling potential of iron in plantmicrobe interactions: the pathogenic switch of iron transport in Erwinia chrysanthemi.Plant J 7, 121-128.
Miller, J. F. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Moeck, G. S. & Coulton, W. (1998). TonB-dependent iron acquisition: mecanisms of siderophore-mediated active transport.Mol Microbiol 28, 675-681.[Medline]
Münzinger, M., Budzikiewicz, H., Expert, D., Enard, C. & Meyer, J.-M. (2000). Achromobactin, a new citrate siderophore of Erwinia chrysanthemi. Z Naturforsch 55C, 328-332.
Murray, N. E., Brammar, W. J. & Murray, K. (1977). Lambdoid phages that simplify the recovery of in vitro recombinants.Mol Gen Genet 150, 53-61.[Medline]
Persmark, M., Expert, D. & Neilands, J. B. (1989). Isolation, characterisation, and synthesis of chrysobactin, a compound with siderophore activity from Erwinia chrysanthemi. J Biol Chem 264, 3187-3193.
Pettis, G. S., Brickman, T. J. & McIntosh, M. A. (1988). Transcriptional mapping and nucleotide sequence of the Escherichia coli fepAfes enterobactin region.J Biol Chem 263, 18857-18863.
Postle, K. (1999). Active transport by customized ß-barrels.Nature Struct Biol 6, 3-6.[Medline]
Postle, K. & Good, R. F. (1985). A bidirectional Rho-independent transcription terminator between the E. coli tonB gene and an opposing gene.Cell 41, 577-585.[Medline]
Postle, K. & Skare, J. T. (1988). Escherichia coli TonB is exported from the cytoplasm without proteolytic cleavage of its amino terminus.J Biol Chem 263, 11000-11007.
Pugsley, A. P. & Reeves, P. (1976). Characterization of group B colicin-resitant mutants of Escherichia coli K-12: colicin resistance and the role of enterochelin.J Bacteriol 127, 218-228.[Medline]
Résibois, A., Colet, M., Faelen, M., Schoonejans, E. & Toussaint, A. (1984). PhiEC-2, a new generalized transducing phage of Erwinia chrysanthemi.Virology 137, 102-112.
Rogers, H. J. (1973). Iron-binding catechols and virulence in Escherichia coli.Infect Immun 7, 445-456.
Schwyn, B. & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160, 47-56.[Medline]
Shevchik, V. E., Robert-Baudouy, J. & Hugouvieux-Cotte-Pattat, N. (1997). Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family. J Bacteriol 179, 7321-7330.[Abstract]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalities and weight matrix choice.Nucleic Acids Res 22, 4673-4680.[Abstract]
Traub, I., Gaisser, S. & Braun, V. (1993). Activity domains of the TonB protein. Mol Microbiol 8, 409-423.[Medline]
Van Gijsegem, F. & Toussaint, A. (1982). Chromosome transfer and R-prime formation by an RP4:mini-Mu derivative in Escherichia coli, Salmonella typhimurium, Klebsiella pnemoniae and Proteus mirabilis.Plasmid 7, 30-44.[Medline]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.Gene 33, 103-119.[Medline]
Received 25 October 1999;
revised 28 February 2000;
accepted 15 May 2000.