1 Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, 53706, USA
2 Room 256 Biochemistry, 420 Henry Mall and Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI, 53706, USA
Correspondence
Michael G. Thomas
thomas{at}bact.wisc.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In silico identification of secondary metabolite biosynthetic potential is facilitated by the observation that bacteria use conserved protein machinery for production of many of these metabolites. In particular, the synthesis of nonribosomal peptides and polyketides uses conserved enzymic machinery to generate the backbone of the corresponding metabolite (Walsh, 2003). Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are the multiprotein complexes that produce nonribosomal peptides and polyketides, respectively. Importantly for in silico analyses, NRPSs and PKSs have conserved protein motifs; thus searching a genome with a known NRPS or PKS protein sequence can identify NRPS- and PKS-encoding genes within a genome. Bioinformatic analysis of the adjacent genes can then identify putative gene clusters that might encode production of novel secondary metabolites. Biochemical and genetic analyses can then be used to identify the metabolite produced.
The genome of the plant pathogen Agrobacterium tumefaciens C58 was recently sequenced by two groups (Goodner et al., 2001; Wood et al., 2001
). We investigated whether this bacterium has the genetic potential to produce secondary metabolites by taking an in silico approach. Using standard BLAST algorithms (Altschul et al., 1990
, 1997
), we identified a gene cluster in A. tumefaciens C58 that could encode the production of a secondary metabolite. Bioinformatic analysis of the gene cluster showed that the encoded proteins have the most significant amino acid similarity with proteins encoded by a putative secondary metabolite biosynthetic gene cluster from the filamentous cyanobacterium Nostoc sp. PCC7120 (Kaneko et al., 2001
), rather than with proteins involved in the production of any known secondary metabolite. Analysis of mutant strains of A. tumefaciens C58 disrupted in several genes in this cluster suggested that the cluster encodes the enzymic machinery for the production of a siderophore. The genes in this cluster do not show the strongest similarity with known siderophore biosynthetic genes, indicating that the biosynthesis and structure of this siderophore may be novel.
Siderophores are small molecules that bind iron with high affinity, and are typically produced by prokaryotes, fungi and some plants in response to iron deprivation (reviewed by Crosa & Walsh, 2002; Neilands, 1995
; Winkelmann, 2002
). Siderophores are generally categorized by the main functional group involved in chelating iron; this may be a catecholate, hydroxamate or hydroxycarboxylate group (Crosa & Walsh, 2002
; Winkelmann, 2002
). In Gram-negative bacteria, siderophores are secreted by the cell, bind iron extracellularly, and then are taken up by the cell by means of a dedicated TonB-dependent membrane transport system (reviewed by Faraldo-Gomez & Sansom, 2003
; Visca et al., 2002
). Many siderophores are peptidic molecules that are synthesized by NRPS systems. These often contain salicylate or dihydroxybenzoic acid as the N-terminal starter molecule, which requires a dedicated NRPS module that recognizes these aryl acids (Crosa & Walsh, 2002
; Quadri, 2000
). Siderophores demonstrate a high degree of structural diversity, with hundreds of structures known so far (Winkelmann, 1991
).
A. tumefaciens is known to synthesize a number of siderophores, but production of these molecules appears to be strain specific. For example, strain B6 produces the siderophore agrobactin (Ong et al., 1979), and strain MAFF301001 produces a siderophore whose structure has not been determined (Sonoda et al., 2002
). Conflicting reports have been published concerning the ability of strain C58 to synthesize siderophores. Leong & Neilands (1982)
found that C58 synthesized a siderophore, but Penyalver et al. (2001)
later reported that no detectable siderophores were produced by this strain. As we detail in this report, detection of siderophore production by A. tumefaciens C58 is medium dependent and seems to require addition of an iron chelator to the medium, suggesting that this strain is highly efficient in iron scavenging.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Construction of A. tumefaciens C58 mutant strains.
Standard molecular biology techniques were used in all plasmid constructions (Ausubel et al., 1994). Primers were designed from the A. tumefaciens C58 genome sequence in GenBank (GenBank accession no. AE008689; NCBI NC_003305). PCR amplification was used to generate fragments of atu3683 and atu3673 that were approximately 2 kb in size. Amplification primers were designed containing PstI restriction enzyme recognition sites at the ends, and they were digested with PstI and cloned into pJQ200mp18 (Quandt & Hynes, 1993
) digested with PstI, generating pMTA1 and pMTA2 respectively. pCRS487 (Reeve et al., 1999
) was digested with NotI, and the gusAnptII cartridge from this plasmid was cloned directly into the NotI site in pMTA1 or was blunt-ended with Klenow enzyme and cloned into the blunted XhoI site in pMTA2. pMTA5 was generated by digesting pHP45
(Prentki & Krisch, 1984
) with SmaI and cloning the spectinomycin resistance cassette into blunted NotI-digested pMTA1.
Conjugation was used to introduce mutations into the A. tumefaciens C58 chromosome by standard techniques (Simon et al., 1983, 1986
). Briefly, plasmids to be introduced into A. tumefaciens C58 were transformed into E. coli strain S17-1, which contains the necessary functions for conjugal transfer of pJQ200mp18 derivatives (Simon et al., 1983
). E. coli and A. tumefaciens C58 cells were grown to late exponential phase, washed in 10 mM MgSO4, and mixed together in a 1 : 1 ratio. Aliquots of 10 µl were spotted onto a TY agar plate and incubated at 28 °C overnight. Cells were then scraped from the plate and resuspended in 1 ml 10 mM MgSO4. Samples were plated onto selective medium, selecting gentamicin resistance (for integration of the plasmid) and counter-selecting with ampicillin or nalidixic acid. Exconjugants were then grown in TY medium containing kanamycin or spectinomycin, and aliquots of these cultures were plated onto TY medium containing kanamycin or spectinomycin plus 5 % (w/v) sucrose. Colonies that arose on media containing sucrose were then tested for loss of gentamicin resistance. Putative mutants were confirmed by PCR analysis and restriction digestion of the resulting PCR products, using the original amplification primers.
Transposon mutagenesis.
Plasmid pCRS538 (Reeve et al., 1999) was used to mutagenize strain MTA100. The plasmid was delivered by conjugation from E. coli S17-1, and exconjugants were selected using gentamicin and kanamycin. Colonies were then patched to TY plus the
-glucuronidase (GUS) indicator 5-bromo-4-chloro-3-indoyl
-D-glucopyranoside, to identify colonies with altered gene expression.
Quantitative GUS assays.
GUS assays were performed as described previously (Bittinger & Handelsman, 2000), except that a colorimetric substrate, p-nitrophenyl
-D-glucuronide, was used. Values are expressed as nmol p-nitrophenol produced min1 (mg total cell protein)1. The BCA protein kit (Pierce) was used to measure protein concentration. Strains were grown in YEM for 24 h and then used to inoculate YEM, YEMFe and YEM containing 2,2'-dipyridyl (DIP) (YEM+DIP) at a 1 : 200 dilution. Cultures were grown for an additional 24 h before assays were done. Assays were done in duplicate, and the values presented are the means of four independent assays.
Quantitative growth curves.
Growth was measured on 5 ml cultures growing in 18 mm test tubes. OD590 was measured using a Spectronic 20 spectrophotometer. Cultures were grown at 28 °C in YEMFe supplemented with either 0·15 or 0·2 mM DIP.
In vitro siderophore assay.
Siderophore production was measured using the chrome azurol S (CAS) liquid assay as described by Schwyn & Neilands (1987). Solid CAS medium was made by adding 10 ml CAS stock (Schwyn & Neilands, 1987
) to 100 ml YEM medium containing 1·5 % agar.
Siderophore purification and HPLC analysis.
Wild-type A. tumefaciens C58 was grown in Tris-minimal medium (100 ml) for 4 days. The cells were removed by centrifugation and the pH of the supernatant was adjusted to 2·5 with HCl. The supernatant was passed through a Dowex 50X2-100 (H+ form) column previously equilibrated with ddH2O. The column was washed with ddH2O and the bound metabolites were eluted with a step gradient of 0·5, 1·0, 2·0, 3·0 and 4·0 M NH4OH. The fractions were collected and lyophilized. Each of the lyophilized fractions was resuspended in ddH2O and the pH neutralized with HCl. The fractions containing the siderophore were identified using the in vitro siderophore assay described above. CAS-positive fractions were pooled and subsequently analysed and purified by HPLC. For HPLC analysis and purification, a Vydac C18 small-pore column at a flow rate of 1 ml min1 was used. The HPLC elution used the following solvents: solvent A, ddH2O and 0·1 % trifluoroacetic acid (TFA); solvent B, acetonitrile and 0·1 % TFA. The HPLC profile for separation was 5 min isocratic development at 100 %A/0 %B; 15 min linear gradient of 100 %A/0 %B to 0 %A/100 %B; 5 min isocratic development at 0 %A/100 %B. The elution of metabolites was monitored at 254 nm.
To identify the fractions containing iron-chelating activity, the fractions were collected, dried, resuspended in ddH2O, and a portion of the fraction was subsequently tested using the CAS assay. Repeated injections and collections of the CAS-reactive fraction allowed for collection of sufficient quantities of siderophore for in vivo studies. The purified siderophore from 100 ml of culture was resuspended in 500 µl ddH2O for use in in vivo siderophore assays.
The purification protocol and HPLC analysis outlined above were followed for the supernatant from a culture of strain MTA100; however, since MTA100 did not produce a CAS-reactive metabolite, the fractions from the Dowex 50X2-100 column that were comparable to the CAS-reactive samples from the wild-type were collected and analysed by HPLC.
In vivo siderophore assay.
Growth of strains in the presence or absence of purified siderophore was tested as follows. Fresh cultures of wild-type or MTA100 cells grown in YEM were used (10 µl) to inoculate 1 ml YEMFe containing 0·00, 0·15 or 0·20 mM DIP, with or without the addition of 10 µl purified siderophore. The cultures were grown for 2 days at 28 °C and the final OD600 was determined using an ultraviolet/visible spectrophotometer (Beckman DU640).
Plant assays.
The assay for tumour formation on carrot slices was performed as described by Jen & Chilton (1986). Infection of tomato plants was performed as described by Palumbo et al. (1998)
, with modifications as follows. Two-week-old seedlings of tomato plants (Lysopersicon esculentum cultivar Wisconsin 55 or cultivar Husky Gold Hybrid) were inoculated by stabbing the stem with a needle that had been dipped into a colony of the appropriate A. tumefaciens strain grown on YEM agar. Plants were grown in a growth chamber with a 12 h light period and a constant temperature of 24 °C, and were watered on alternate days (from the bottom, to minimize contamination) with distilled water or Hoagland's solution (Baker, 1981
). Plants were scored 3 weeks after inoculation.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Siderophore purification
To demonstrate that the wild-type strain produces a siderophore that is absent from an ATS mutant strain, we compared the metabolite profiles of wild-type and MTA100 (atu3683 : : gusAnptII) strains. For this analysis, the wild-type and MT100 strains were grown in Tris-minimal medium for 4 days, after which the cells were removed by centrifugation and the supernatant was used for subsequent siderophore purification. Cation-exchange chromatography of the supernatant, followed by in vitro CAS assay analysis of the eluted fractions, identified fractions that contained a CAS-reactive metabolite. These fractions were pooled and analysed by reverse-phase HPLC (Fig. 4). CAS assays of eluted fractions from the HPLC identified a fraction, corresponding to an absorption peak at 16·4 min (Fig. 4
), which was positive for iron chelation. This metabolite was not observed in the equivalent cation-exchange chromatography fractions from the supernatant of strain MTA100 (Fig. 4
).
|
To test whether the purified metabolite (eluting at 16·4 min) could enhance the growth of a strain disrupted in the ATS gene cluster when grown under iron-limiting conditions, MTA100 (atu3683 : : gusAnptII) and the wild-type strain were grown in YEMFe medium with varying concentrations of DIP, with or without the addition of the purified siderophore. Measurement of the final OD600 of the cultures after 48 h growth showed that the purified metabolite was able to restore the growth of MTA100 to near wild-type levels in the presence of DIP (Table 3).
|
|
Structure of the ATS gene cluster
We identified transposon insertion mutations that affected expression of the ATS gene cluster. Among these mutations was a set that abolished transcription of the gusA fusion in MTA100 (not shown). Analysis of the site of insertion of these transposons indicated that they were located in atu3682, atu3681, atu3677 and atu3676, consistent with the idea that these genes are organized into one transcriptional unit (see Fig. 1). Additionally, there is significant translational overlap between many of the ORFs in the cluster, and no ORFs are separated by more than 42 bp; these findings support the idea that the ORFs are co-transcribed. The ATS gene cluster appears to be arranged into two divergently transcribed operons. Our data are consistent with the hypothesis that the products of both operons are required for synthesis of one siderophore; thus we consider both to be part of the ATS gene cluster.
Bioinformatic analysis of the ATS gene cluster yields limited information regarding the structure of the siderophore
Together, the entire cluster encodes five NRPSs, two PKSs, and one acyl-CoA ligase protein associated with an acyl carrier protein, as well as several putative modifying enzymes that may act on the NRPS/PKS-synthesized core molecule. The predicted functions of the ORFs are shown in Table 2. Based on the NRPS and PKS components, the metabolite produced by this gene cluster is likely to contain seven amino acids, two carboxylic acids and one initiating acyl moiety.
In some cases it is possible to predict the structure of a NRPS/PKS-synthesized molecule based on the predicted substrates of the biosynthetic proteins and on the arrangement of ORFs in the gene cluster. An application of these principles can be seen in the analysis of the recently completed genome of the model actinomycete Streptomyces coelicolor. Eighteen novel gene clusters found in the genome were analysed, and molecules produced by seven of these gene clusters were proposed, including a novel siderophore, coelichelin (Bentley et al., 2002; Challis & Ravel, 2000
).
Analysis of the predicted substrate specificity of the NRPS components of the ATS gene cluster was of limited value in predicting the amino acids incorporated into the siderophore, since little similarity to known substrate specificity determinants was found. The online version of this paper (http://mic.sgmjournals.org) contains a supplementary table showing the substrate-specificity determinations for each NRPS component. The most informative substrate-specificity prediction came from Atu3682, where cysteine was one amino acid predicted to be incorporated by this protein. The association of a cyclization and an oxidase domain with this putative cysteine-activating protein suggests that the final product will include a thiazole ring. Several siderophores contain thiazole rings as a means of chelating iron, so this prediction is consistent with the molecule's proposed function as a siderophore (Crosa & Walsh, 2002).
Identification of a similar gene cluster in the Nostoc sp. PCC7120 genome
While analysing the ATS gene cluster via BLASTP and PSI-BLAST algorithms (Altschul et al., 1990, 1997
), we noted that the proteins showing the highest level of amino acid similarity were consistently part of a gene cluster in the filamentous cyanobacterium Nostoc sp. PCC7120 (formerly Anabaena sp. PCC7120). A comparison of the predicted functions of the ORFs in both gene clusters is shown in the supplementary data section of the online version of this paper, as is a comparison of the gene arrangements. Of the 15 ORFs in the A. tumefaciens C58 gene cluster, 11 have as their most similar protein in the database an ORF from the corresponding gene cluster from Nostoc sp. PCC7120. Of particular interest is the finding that the substrate specificity predictions from the Nostoc sp. PCC7120 NRPS proteins were nearly identical to those found in A. tumefaciens C58, suggesting that the proteins incorporate similar precursor molecules. This extensive similarity suggests that the A. tumefaciens C58 and Nostoc sp. PCC7120 gene clusters generate metabolites of related structure and function. Since we predict that the molecule made by A. tumefaciens C58 is a siderophore, it is likely that the cyanobacterial molecule also functions as a siderophore.
Genes located adjacent to the ATS gene cluster are consistent with siderophore production
We noticed that directly adjacent to the proposed siderophore biosynthetic gene cluster are a number of predicted genes also potentially involved in iron metabolism. These are not shown in Fig. 1, but are located 3' to atu3685. atu3686 encodes a putative protein similar to FhuF, which has been proposed to be involved in mobilization of iron from ironsiderophore complexes (Müller et al., 1998
). atu3687 encodes a putative TonB-dependent outer membrane receptor similar to those that bind siderophores (reviewed by Braun & Killmann, 1999
; Faraldo-Gomez & Sansom, 2003
). atu3688, atu3689, atu3690 and atu3691 code for proteins with similarity to ABC-dependent transporters involved in ironsiderophore complex transport (reviewed by Andrews et al., 2003
), including a periplasmic binding protein, cytoplasmic membrane-spanning proteins, and an inner-membrane-associated ATPase. atu3692 encodes a putative sigma factor that shows the most similarity with sigma factors involved in iron-dependent regulation of siderophore biosynthesis in Pseudomonas spp., including PbrA from P. fluorescens M114 (Venturi et al., 1995
), PfrI from P. putida WCS358 (Sexton et al., 1995
), and PvdS from P. aeruginosa (Ochsner et al., 1995
). Finally, atu3693 encodes a protein with similarity to FecR, also involved in iron-mediated gene regulation (Braun et al., 2003
; Ochs et al., 1995
). The presence of these genes adjacent to the ATS gene cluster supports our hypothesis that this gene cluster encodes the biosynthesis of a siderophore. Regulatory and transport genes would be needed to control siderophore biosynthesis and to transport the molecule into the cell, and genes encoding these functions are often found associated with siderophore biosynthetic pathways in other organisms. However, direct testing is needed to confirm that these other genes are involved in iron metabolism and that they function in collaboration with the siderophore produced by the ATS gene cluster.
Secondary metabolite production in Agrobacterium spp.
As with many other bacteria, secondary metabolite production in A. tumefaciens, including siderophore production, seems to be highly strain specific. Ong et al. (1979) found that A. tumefaciens strain B6 produced a siderophore called agrobactin. Agrobactin is composed of threonine, spermidine, and three molecules of 2,3-dihydroxybenzoic acid. The genes for agrobactin biosynthesis have not been identified in strain B6. However, a recent report of siderophore production in strain MAFF301001 identified a gene cluster, called agb, that might be responsible for production of agrobactin or an agrobactin-like molecule (Sonoda et al., 2002
). In that study, Southern hybridization studies revealed the lack of similarity between the agb genes and any locus in A. tumefaciens C58, suggesting that C58 does not make agrobactin. Penyalver et al. (2001)
reported the production of a siderophore in the related Agrobacterium rhizogenes K84, a strain that is used for biological control of crown gall. Those authors identified mutants that did not produce the siderophore, and they localized the mutations to a putative NRPS-encoding gene. They did not detect production of this siderophore by strain C58, and DNA hybridizing to DNA from the siderophore biosynthetic locus in strain K84 was not found in the A. tumefaciens C58 genome, suggesting that the siderophore produced by K84 is distinct. There are also several reports of marine Agrobacterium spp. that produce cytotoxic agents, namely agrochelin (Cañedo et al., 1999
), and sesbanamide A and C (Acebal et al., 1998
). None of these compounds have structures that are consistent with the predicted functions of the proteins encoded by the ATS gene cluster. Thus, it is likely that the siderophore produced by C58 is not a molecule previously isolated from Agrobacterium spp.
Conclusions
Siderophores are defined as ferric ion chelating molecules synthesized by microbes growing under low-iron stress (Neilands, 1995). Our results show that A. tumefaciens C58 contains a biosynthetic pathway for production of a novel siderophore. We detected this compound only under conditions of iron stress, and mutants affected in the biosynthetic pathway were defective in growth and production of CAS-reactive material under iron-limiting conditions. Further, we showed that expression of the gene cluster is increased when cells are grown in low-iron medium.
In this study we have provided a function for a large secondary metabolite gene cluster in A. tumefaciens C58. The function for this gene cluster is to produce a large array of enzymes involved in the biosynthesis of a siderophore required for A. tumefaciens C58 growth under iron-limiting conditions. Significantly, the proteins encoded by the ATS gene cluster do not show the most similarity with other known siderophore biosynthetic enzymes, but rather show the most similarity to enzymes encoded by a gene cluster in Nostoc sp. PCC7120. This lack of similarity with other known siderophore biosynthetic enzymes is intriguing and suggests that a novel siderophore is produced by A. tumefaciens C58, and that Nostoc sp. PCC7120 is likely to produce a structural analogue. Current studies are under way to determine the structure of the purified siderophore to give further insights into the novel enzymology and biology associated with A. tumefaciens C58 siderophore biosynthesis.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403410.[CrossRef][Medline]
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 33893402.
Andrews, S. C., Robinson, A. K. & Rodríguez-Quiñones, F. (2003). Bacterial iron homeostasis. FEMS Microbiol Rev 27, 215237.[CrossRef][Medline]
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1994). Current Protocols in Molecular Biology. New York: Wiley.
Baker, A. (1981). Accumulators and excluders strategies in the response of plants to heavy metals. J Plant Nutr 3, 643654.
Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M. & 40 other authors (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141147.[CrossRef][Medline]
Beringer, J. E. (1974). R-factor transfer in Rhizobium leguminosarum biovar phaseoli. J Gen Microbiol 84, 188198.[Medline]
Bittinger, M. A. & Handelsman, J. (2000). Identification of genes in the RosR regulon of Rhizobium etli. J Bacteriol 182, 17061713.
Braun, V. & Killmann, H. (1999). Bacterial solutions to the iron-supply problem. Trends Biochem Sci 24, 104109.[CrossRef][Medline]
Braun, V., Mahren, S. & Ogierman, M. (2003). Regulation of the FecI-type ECF sigma factors by transmembrane signalling. Curr Opin Microbiol 6, 173180.[CrossRef][Medline]
Cañedo, L. M., de la Fuente, J. A., Gesto, C., Ferreiro, M. J., Jiménez, C. & Riguera, R. (1999). Agrochelin, a new cytotoxic alkaloid from the marine bacteria Agrobacterium sp. Tetrahedron Lett 40, 68416844.[CrossRef]
Challis, G. L. & Ravel, J. (2000). Coelichelin, a new peptide siderophore encoded by the Streptomyces coelicolor genome: structure prediction from the sequence of its non-ribosomal peptide synthetase. FEMS Microbiol Lett 187, 111114.[CrossRef][Medline]
Cornelis, P. & Matthijs, S. (2002). Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines. Environ Microbiol 4, 787798.[CrossRef][Medline]
Crosa, J. H. & Walsh, C. T. (2002). Genetic assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev 66, 223249.
Faraldo-Gomez, J. D. & Sansom, M. S. (2003). Aquisition of siderophores in Gram-negative bacteria. Nat Rev Mol Cell Biol 4, 105116.[CrossRef][Medline]
Goodner, B., Hinkle, G., Gattung, S. & 28 other authors (2001). Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science 294, 23232328.
Guerinot, M. L. (1991). Iron uptake and metabolism in the rhizobia/legume symbiosis. Plant Soil 130, 199209.
Hori, K., Yamamoto, Y., Minetoki, T., Kurotsu, T., Kanda, M., Miura, S., Okamura, K., Furuyama, J. & Saito, Y. (1989). Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene. J Biochem 106, 639645.[Abstract]
Jen, G. C. & Chilton, M.-D. (1986). Activity of T-DNA borders in plant cell transformation by mini-T plasmids. J Bacteriol 166, 491499.[Medline]
Kaneko, T., Nakamura, Y., Wolk, C. P. & 19 other authors (2001). Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res 8, 205213.[Medline]
Krätzschmar, J., Krause, M. & Marahiel, M. A. (1989). Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases. J Bacteriol 171, 54225429.[Medline]
Leong, S. A. & Neilands, J. B. (1981). Relationship of siderophore-mediated iron assimilation to virulence in crown gall disease. J Bacteriol 147, 482491.[Medline]
Leong, S. A. & Neilands, J. B. (1982). Siderophore production by phytopathogenic microbial species. Arch Biochem Biophys 218, 351359.[Medline]
May, J. J., Wendrich, T. M. & Marahiel, M. A. (2001). the dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin. J Biol Chem 2001, 72097217.[CrossRef]
Müller, K., Matzanke, B. F., Schünemann, V., Trautwein, A. X. & Hantke, K. (1998). FhuF, an iron-regulated protein of Escherichia coli with a new type of [2Fe2S] center. Eur J Biochem 258, 10011008.[Abstract]
Neilands, J. B. (1995). Siderophores: structure and function of microbial iron transport compounds. J Biol Chem 270, 2672326726.
Ochs, M., Veitinger, S., Kim, I., Welz, D., Angerer, A. & Braun, V. (1995). Regulation of citrate-dependent iron transport of Escherichia coli: FecR is required for transcription activation by FecI. Mol Microbiol 15, 119132.[Medline]
Ochsner, U. A., Vasil, A. I. & Vasil, M. L. (1995). Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters. J Bacteriol 177, 71947201.[Abstract]
Ong, S. A., Peterson, T. & Neilands, J. B. (1979). Agrobactin, a siderophore from Agrobacterium tumefaciens. J Biol Chem 254, 18601865.[Abstract]
Palumbo, J. D., Phillips, D. A. & Kado, C. I. (1998). Characterization of a new Agrobacterium tumefaciens strain from alfalfa (Medicago sativa L.). Arch Microbiol 169, 381386.[CrossRef][Medline]
Penyalver, R., Oger, P., Lopez, M. M. & Farrand, S. K. (2001). Iron-binding compounds from Agrobacterium spp. biological control strain Agrobacterium rhizogenes K84 produces a hydroxamate siderophore. Appl Environ Microbiol 67, 654664.
Prentki, P. & Krisch, H. M. (1984). In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303313.[CrossRef][Medline]
Quadri, L. E. N. (2000). Assembly of aryl-capped siderophores by modular peptide synthetases and polyketide synthases. Mol Microbiol 37, 112.[CrossRef][Medline]
Quandt, J. & Hynes, M. F. (1993). Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene 127, 1521.[CrossRef][Medline]
Ravel, J. & Cornelis, P. (2003). Genomics of pyoverdine-mediated iron uptake in pseudomonads. Trends Microbiol 11, 195200.[Medline]
Reeve, W. G., Tiwari, R. P., Worsley, P. S., Dilworth, M. J., Glenn, A. R. & Howieson, J. G. (1999). Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria. Microbiology 145, 13071316.[Medline]
Rosen, R., Matthysse, A. G., Becher, D., Biran, D., Yura, T., Hecker, M. & Ron, E. Z. (2003). Proteome analysis of plant-induced proteins of Agrobacterium tumefaciens. FEMS Microbiol Ecol 44, 355360.[CrossRef]
Schwyn, B. & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160, 4756.[Medline]
Sexton, R., Gill, P. R., Callanan, M. J., O'Sullivan, D. J., Dowling, D. N. & O'Gara, F. (1995). Iron-responsive gene expression in Pseudomonas fluorescens M114 cloning and characterization of a transcription activating factor, PbrA. Mol Microbiol 15, 297306.[Medline]
Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1, 784791.
Simon, R., O'Connell, M., Labes, M. & Pühler, A. (1986). Plasmid vectors for the genetic analysis and manipulation of rhizobia and other Gram-negative bacteria. Methods Enzymol 118, 640659.[Medline]
Sonoda, H., Suzuki, K. & Yoshida, K. (2002). Gene cluster for ferric iron uptake in Agrobacterium tumefaciens MAFF301001. Genes Genet Syst 77, 137146.[CrossRef][Medline]
Todd, J., Wexler, M., Sawers, G., Yeoman, K. H., Poole, P. S. & Johnston, A. W. B. (2002). RirA, an iron-responsive regulator in the symbiotic bacterium Rhizobium leguminosarum. Microbiology 148, 40594071.[Medline]
Venturi, V., Ottevanger, C., Bracke, M. & Weisbeek, P. (1995). Iron regulation of siderophore biosynthesis and transport in Pseudomonas putida WCS358 involvement of a transcriptional activator and of the Fur protein. Mol Microbiol 15, 10811093.[Medline]
Vincent, J. M. (1970). A Manual for the Practical Study of Root Nodule Bacteria. Oxford, UK: Blackwell Scientific Publications.
Visca, P., Leoni, L., Wilson, M. J. & Lamont, I. L. (2002). Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas. Mol Microbiol 45, 11771190.[CrossRef][Medline]
Walsh, C. (2003). Antibiotics. Washington, DC: American Society for Microbiology.
Winkelmann, G. (1991). CRC Handbook of Microbial Iron Chelates. Boca Raton, FL: CRC Press.
Winkelmann, G. (2002). Microbial siderophore-mediated transport. Biochem Soc Transact 30, 691696.[Medline]
Wood, D. W., Setubal, J. C., Kaul, R. & 48 other authors (2001). The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294, 23172323.
Received 10 May 2004;
revised 21 June 2004;
accepted 22 June 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |