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Journal of Bacteriology, September 1999, p. 5160-5166, Vol. 181, No. 17
Department of Biology, University of
Richmond, Richmond, Virginia 23173
Received 5 March 1999/Accepted 10 June 1999
A combined genetic and physical map of the Agrobacterium
tumefaciens A348 (derivative of C58) genome was constructed to
address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two
nonhomologous chromosomes. The combined map confirms the two-chromosome
genomic structure and the correspondence of the initial genetic maps to
the circular chromosome. The linear chromosome is almost devoid of
auxotrophic markers, which probably explains why it was missed by
genetic mapping studies.
Most of the work on
Agrobacterium tumefaciens, since its identification as the
causal agent in crown gall disease of dicotyledonous plants at the turn
of the century, has rightfully focused on the mechanism of tumor
induction (52; for recent reviews of all aspects of
the disease, see references 2, 10, 23, 39, and
58). The virulence mechanism turns out to be unique
among interactions between prokaryotic pathogens and eukaryotic hosts. Since most of the virulence genes lie on the Ti plasmid, the
chromosomal complement of A. tumefaciens has been relatively understudied.
Initial chromosomal maps for A. tumefaciens, based on
chromosome mobilization and recombination of genetic markers, suggested a single circular chromosome (11, 29, 44, 47, 48). However, recent physical mapping data strongly suggests that A. tumefaciens has two chromosomes, one circular chromosome of ~3
Mbp and one linear chromosome of ~2.1 Mbp (1, 31). This
chromosome organization appears to be a conserved trait throughout the
genus (32). While multiple chromosomes have been found in
some other eubacteria (7-9, 43, 54, 57), we were interested
in the discrepancy between the initial genetic maps and the more recent
physical mapping data. We hypothesized that the original genetic
mapping techniques somehow missed the linear chromosome. To test this hypothesis, we constructed a combined genetic and physical map of the
A. tumefaciens genome by collecting a large number of
transposon-mediated auxotrophic mutations, using a transposon carrying
rare restriction sites, and then physically mapping the transposon
insertions by using pulsed-field gel electrophoresis (PFGE). Our
results confirm the two-chromosome genome organization, and we found
that almost all the auxotrophic markers lie on the circular chromosome.
We put forward an explanation for the discrepancy between the initial genetic maps and the physical mapping data and suggest some hypotheses for the gene organization in this bacterial species.
(Partial results of this work were presented at the 19th Annual Crown
Gall Meeting [24] and at the Microbial Genomes III Conference [25].)
Strains, plasmids, and growth conditions.
The A. tumefaciens strain and plasmids used in this study are described
in Table 1. A. tumefaciens
cultures were grown in a modified Luria-Bertani (LB) medium (only
5 g of NaCl/liter) at 30°C. Screens for A. tumefaciens auxotrophic mutants were carried out in M9 minimal
medium with sucrose as a carbon source (45). The antibiotics
carbenicillin, kanamycin, rifampin, and tetracycline were used as
needed at 50, 50, 20, and 10 µg/ml, respectively.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Combined Genetic and Physical Map of the Complex
Genome of Agrobacterium tumefaciens
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
Matings. Donor (either Escherichia coli or A. tumefaciens carrying a plasmid) and recipient (A. tumefaciens) strains were mixed by streaking on a modified LB agar plate and incubated at 30°C for 48 h. When necessary (when the donor plasmid was a mobilizable pRK290 derivative), a third strain, E. coli carrying pRK2013, was included in the mating. The cell mixture was scraped off, resuspended in M9 minimal medium without a carbon source, diluted, and plated on the appropriate selective medium.
Mutant isolation and characterization. A. tumefaciens A348 was mated with E. coli S17-1/pUT::Tn5(pfm). A. tumefaciens carrying Tn5(pfm) insertions were selected on modified LB medium containing kanamycin [selective for the presence of Tn5(pfm)] and rifampin (selective for A. tumefaciens). Single colonies were screened for auxotrophy by plating on M9 and modified LB medium. Potential Tn5(pfm)-induced auxotrophs were tested on M9 plates with various nutrient pools and later supplemented with specific pathway intermediates (13).
Confirmation of the linkage between Tn5(pfm) insertion and auxotrophic mutation. A. tumefaciens auxotrophic mutant strains were grown overnight at 30°C in 2-ml cultures. Total genomic DNA was isolated from each strain and resuspended at ~0.4 to 0.5 µg/ml (16). Approximately 4 to 5 µg (10 µl) of each sample was electroporated into competent wild-type A. tumefaciens A348 cells, and the transformed cells were plated on modified LB medium containing kanamycin (5). Three days later, the few resulting colonies were picked from each transformation and streaked onto modified LB plates containing kanamycin, M9 plates containing kanamycin, and M9 plates containing kanamycin and the specific nutrient required by the original A. tumefaciens auxotrophic mutant.
PFGE of intact and digested DNAs. Wild-type and mutant A. tumefaciens strains used in physical mapping were grown for 48 h at 30°C in 2-ml cultures. Cells were pelleted, suspended in 2% agarose plugs, digested overnight with pronase E (2 mg/ml) at 50°C and washed (53). Restriction enzyme digest of genomic DNA in the agarose plugs by PacI and SwaI (New England Biolabs) were carried out at 25°C for 24 h. PFGE was carried out in a contour-clamped homogeneous electric field apparatus (Bio-Rad), in 0.5× TBE buffer (45 mM Tris, 45 mM borate, 1.25 mM EDTA [pH 8.3]). Restriction enzyme digests of genomic DNA were electrophoresed through 1% agarose gels with a ramp of 40 to 90 s for 22 h at 180 V. Lambda ladder (Bio-Rad) served as size markers.
Plasmid-mediated mobilization of chromosomal markers. R68.45, a conjugable plasmid used in several of the original genetic mapping experiments, was mobilized into several A. tumefaciens Tn5(pfm)-induced auxotrophic mutants in independent overnight matings with E. coli harboring R68.45. The presence of R68.45 in the A. tumefaciens strains was selected by growth on modified LB medium containing kanamycin [selective for the presence of Tn5(pfm)], carbenicillin (selective for the presence of R68.45), and rifampin (selective for A. tumefaciens). A. tumefaciens strains carrying R68.45 were used as donors in overnight matings on modified LB medium with recipient A. tumefaciens strains harboring different auxotrophic mutations. Transconjugants in which the auxotrophic marker of the recipient strain had been replaced by the wild-type counterpart from the donor chromosome were selected by plating dilutions of a mating mixture onto M9-sucrose medium.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of a combined genetic and physical map for A. tumefaciens A348.
To build on the results of
Allerdet-Servent et al. (1), we first repeated their
experiments with A. tumefaciens A348. We obtained identical
results for strain A348 (data not shown), which differs from strain C58
only by having a chromosomal rifampin resistance mutation and a
different Ti plasmid (22). Next, we devised a strategy for
physical localization of genetic markers with digestions by
PacI and SwaI, the same enzymes used in the initial physical mapping experiments (1).
Tn5(pfm), a minitransposon carrying selectable markers and
several rare restriction sites, was introduced by mating into A. tumefaciens A348 (60). From 30 independent matings,
approximately 11,000 kanamycin-resistant colonies were replicated onto
LB and M9-sucrose minimal media. Of these, 103 were identified as
Tn5(pfm)-induced auxotrophs. A total of 56 independently
isolated auxotrophs were chosen for further analysis, with 45 eventually being characterized down to the affected biosynthetic
pathway and the remainder having unknown requirements (Tables
2 and 3).
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Essential features of the map. The results of the mapping were entirely consistent with the findings of earlier studies indicating two independent chromosomes, a 3.0-Mb circle and a 2.1-Mb linear structure (1). Furthermore, the restriction enzyme digestions of the genomic DNA of the various Tn5(pfm) mutants allowed us to order the PacI and SwaI fragments on each of the chromosomes and to localize a large number of the transposon insertions (Fig. 2). Insertions were found on both chromosomes. None of the transposon insertions localized to the small SwaI fragments J, K (doublet), L, M, and N. All but one of these small fragments had been assigned to chromosomes earlier by Southern hybridization (1), but we were unable to assign specific map positions for them.
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Correspondence of the physical-genetic map to previous genetic maps. It seemed reasonable to suggest that the circular chromosome recognized by physical mapping and further characterized in this study is the same as the circular chromosome from earlier genetic mapping studies. To further anchor our physical and genetic map in comparison to previous genetic maps, we tested whether a methionine biosynthetic gene used in previous genetic mapping studies was the same as any of the methionine biosynthetic genes identified by Tn5(pfm) mutagenesis. The chemically induced auxotrophic mutation met6 had previously been shown to map very close to both the att gene cluster, required for initial binding of A. tumefaciens to plant cells, and the cel gene cluster, encoding a cellulose biosynthesis pathway (48). Both the att and cel gene clusters have been separately isolated from genomic cosmid libraries (40, 41). To determine if the cosmid clones carrying the att and cel gene clusters also harbored the wild-type met6 gene and whether that gene would complement any of our Tn5(pfm)-induced methionine auxotrophs, the att gene cluster cosmid pG644, the cel gene cluster cosmid pCP13.101, and pRK290, the parent plasmid on which the cosmids are based (17, 21, 36), were independently mobilized into A. tumefaciens A348 strains carrying the auxotrophic mutations met-101::Tn5C22, met-102::Tn5C23, and met-102::Tn5C24. The parent plasmid pRK290 and the att gene cluster cosmid pG644 failed to complement any of the mutations. However, the cel gene cluster cosmid pCP13.101 complemented the met-102::Tn5C23 and met-102::Tn5C24 mutations, which have transposon insertions at the same location.
Ability of the circular and linear chromosomes to be
mobilized.
One possible explanation for the failure of the
original genetic mapping experiments to detect both chromosomes may be
a reduced ability of the linear chromosome to be mobilized. We used
R68.45, the same conjugable plasmid used in many of the original
genetic mapping experiments, in experiments to determine if the
circular and linear chromosomes each could be mobilized
(27). The basic strategy was to mate donor and recipient
strains carrying different Tn5(pfm) insertions. The donor
strain also harbored R68.45. Chromosome mobilization was determined by
selection for recipients in which the Tn5(pfm) insertion
site of the recipient strain had been replaced by its wild-type
counterpart from the donor strain (Table
5). In a control experiment where the
donor (cys-101::Tn5C9) and recipient (cys-101::Tn5C9) strains carried the
exact same transposon insertion, no wild-type transconjugants were
found, as expected. This also showed that Tn5(pfm)
insertions do not revert at a measurable frequency, which makes sense
since this minitransposon lacks a transposase gene (60). In
experiments where the donor and recipient strains carried different
Tn5(pfm) insertions, the circular and linear chromosomes
were mobilized at comparable frequencies. The only exceptions were
cases in which the auxotrophic markers in the donor and recipient
strains were near each other on the same chromosome
(trp-101::Tn5C33 × cys-101::Tn5C9,
cys-101::Tn5C9 × trp-101::Tn5C33, and
aux-108::Tn5L7 × thr-101::Tn5L5).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We were able to confirm the two-chromosome genome organization in
A. tumefaciens by constructing a combined genetic and
physical map of the circular and linear chromosomes (Fig. 2). A strong case can be made that the circular chromosome is the chromosome on
which previous genetic maps are based. The previous genetic maps are
congruent with one another and are consistent with a circular
chromosome (11, 29, 44, 47, 48). Furthermore, the
chvAB genes, encoding enzymes involved in extracellular
-glucan production, had been placed on one of the genetic maps and
were later shown in the initial physical mapping to hybridize to
PacI fragment A and SwaI fragment A of the
circular chromosome (1, 11, 18). Finally, we were able to
prove that the met6 gene, located on one of the genetic
maps, is the same as one of the methionine biosynthetic genes we
physically localized to the circular chromosome (48).
We can rule out some explanations of why the genetic mapping approaches missed the linear chromosome. First, the chromosome mobilization experiment shows that genetic markers on the linear chromosome can be mobilized by R68.45 at frequencies comparable to those for markers on the circular chromosomes (Table 5). This shows that the linear chromosome is not recalcitrant to conjugation-based mobilization due to its topology. Second, we found prototrophic Tn5(pfm) insertions at similar frequencies for both chromosomes. This discounts the possibility of transpositional bias between the chromosomes. Therefore, we are left with a simple but intriguing hypothesis. Since the genetic mapping approaches mainly used auxotrophic markers and we found that virtually all such markers (~86%) lie on the circular chromosome, it is possible that the small collections of auxotrophic strains used for the genetic maps do not contain any examples of mutations on the linear chromosome. We believe that this hypothesis is robust based on the large number of independent auxotrophic markers, both characterized and uncharacterized, that we physically mapped. The fact that some biosynthetic pathways (arginine, lysine, phenylalanine, proline, and tyrosine) are not represented in our collection is not worrisome, since it is possible to find such auxotrophic markers for these pathways in A. tumefaciens and since several of these markers were mapped to the circular chromosome by purely genetic approaches (11, 29, 44, 47, 48).
The paucity of auxotrophic markers on the linear chromosome brings up the question of the origin of the two-chromosome state in this genus (32). If the current chromosomes resulted from a splitting of a single ancestral chromosome, one might expect those two chromosomes to have similar densities of auxotrophic markers. To see if this is true for single chromosome genomes, we looked at the distribution of putative auxotrophic markers (i.e., genes involved in amino acid, cofactor or vitamin, and nucleotide biosynthesis that, when mutated, would lead to auxotrophy) in the published genomic sequences of several members of the Eubacteria and Archaea (3, 4, 14, 20, 33, 35, 37, 51, 56). In the genomes analyzed, auxotrophic markers are rarely separated by more than 100 kbp, with the largest gap being less than 300 kbp. An even better comparison is the recent low-resolution sequencing of approximately one-third of the ~0.9-Mb chromosome II of Rhodobacter sphaeroides 2.4.1T (8, 9, 54). Putative auxotrophic markers were found at a density slightly lower than but comparable to that for the single-chromosome genomes. In contrast, we found only six auxotrophic markers on the 2.1-Mbp linear chromosome of A. tumefaciens, with approximately one-third of the linear chromosome being devoid of such markers. As detailed genetic maps or complete genomic sequences become available for other species with multiple non-homologous chromosomes, such as Brucella melitensis (31, 43), Burkolderia (formerly Pseudomonas) cepacia (7), and Vibrio cholera (57), it will be interesting to see if they show asymmetry in the distribution of auxotrophic markers between their chromosomes.
One hypothesis to explain the lack of auxotrophic markers on one-third of the linear chromosome is a bias against Tn5(pfm) jumping into this region due to a different base composition. The only data we obtained that can address this idea is the distribution of randomly chosen prototrophic Tn5(pfm) insertions. Of 15 such insertions on the linear chromosome, 4 mapped to the region lacking auxotrophic markers (Table 4). This number closely matches that expected for random insertion of the transposon. While this small data set cannot disprove the hypothesis, it is highly suggestive that transpositional bias is not the cause of the asymmetrical distribution of auxotrophic markers on the linear chromosome.
An alternative hypothesis is the acquisition or evolution of a large cluster of nonessential genes either on the ancestral chromosome before the split into two chromosomes or on the linear chromosome after the split. For example, the linear chromosome may contain a large gene cluster specifically involved in the interaction of A. tumefaciens with plant tissue. This is seen in several animal pathogens, where many virulence genes are clustered into "pathogenicity islands" (28, 38). Of the known A. tumefaciens chromosomal virulence genes, only the chvAB operon, the att gene cluster, the cel gene cluster, and the ros gene have been mapped, and all are located on the circular chromosome (1, 11, 48). To further test this hypothesis, the other known chromosomal virulence genes and nonvirulence genes implicated in the plant-microbe interaction need to be localized on the physical map (6, 26, 30, 34, 42, 46, 49, 50, 55).
Ultimately, a fuller understanding of the genetic structure and role of the two chromosomes in the ecology of A. tumefaciens will require genomic sequencing. We have initiated such an effort for the ~710-kbp PacI fragment D of the linear chromosome. We hope to verify the real size of the auxotrophic marker gap on the linear chromosome. Other benefits will include the identification of (i) additional genes that can be used for structure-function, evolutionary, and comparative genomic studies, (ii) a bacterial telomere, and (iii) genes involved in the interaction of A. tumefaciens with plant tissues.
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ACKNOWLEDGMENTS |
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Research support was provided by a University of Richmond Faculty Research Grant to B.W.G.; University of Richmond Undergraduate Research Grants to M.C.F., B.P.M., J.L.R., and L.M.H.; and University of Richmond Summer Undergraduate Research Fellowships to B.P.M. and B.A.S.
We are grateful to Jeff Elhai, Todd Steck, Ann Matthysse, Kwong Kwok Wong, and Michael McClelland for the gifts of strains and plasmids and for many helpful suggestions. We also thank the students of the BIOL315 course at University of Richmond for help in the initial auxotrophic mutant screens, Bill Shanabruch for helpful instruction on PFGE, two anonymous reviewers for their comments, and Dahlia Doughty and Charlaine Scott for help with the experiments needed to address the reviewers' comments.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, Science Center E-105, University of Richmond, Richmond, VA 23173. Phone: (804) 289-8661. Fax: (804) 289-8233. E-mail: bgoodner{at}richmond.edu.
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Journal of Bacteriology, September 1999, p. 5160-5166, Vol. 181, No. 17
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