Department of Biological Sciences, University
of Calgary, Calgary, Alberta T2N 1N4, Canada
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TEXT |
Rhizobia are soil bacteria that are
able to induce nitrogen-fixing nodules on the roots of leguminous
plants. The formation and maintenance of a nitrogen-fixing nodule are
determined by the expression and regulation of plant and bacterial
genes (16, 28). Many of the bacterial genes necessary for an
effective symbiotic association are plasmid borne (1, 2, 3, 6, 13,
19, 24, 26, 31, 39, 40).
Sinorhizobium meliloti typically contains two megaplasmids
of approximately 1,400 and 1,600 kb (2, 3, 8, 9, 19, 24, 26,
37). In strain RCR2011 these plasmids have been termed
pRme2011a and pRme2011b, respectively (also referred to as
pRmeSU47a and pRmeSU47b or as pSymA and pSymB). Collectively, these
plasmids comprise approximately 40% of the genome of this strain
(23, 37). Although this represents a substantial proportion of this bacterial genome, relatively few traits have been ascribed to
these replicons. The larger of these megaplasmids, pRme2011b, has been
shown to carry determinants for exopolysaccharide synthesis, thiamine biosynthesis, high-affinity phosphate transport, and dicarboxylic acid transport (4, 12, 19, 22, 26, 39, 40). The
construction of a genetic map facilitated the genetic characterization
of this replicon, leading to the identification and localization of
several catabolic loci (11, 12, 14).
In contrast, pRme2011a is not as well characterized. The phenotypes
ascribed to this replicon are restricted to a region comprising less
than one-third of the plasmid and are primarily involved in nodulation
and nitrogen fixation (3, 5, 6, 7, 15, 30, 31, 32). The
majority of pRme2011a is still considered cryptic. In an effort to
characterize this replicon genetically, we have attempted to create
large deletions by using a positive selection strategy which has been
previously used successfully on the smaller plasmids of Rhizobium
leguminosarum (24, 25). Here we present data which show
that repetitive rounds of Tn5B12-S mutagenesis with
selection for deletion can be used successfully to cure the
nod-nif megaplasmid, pRme2011a, of strain Rm2011. Physical
analysis using pulsed-field gel electrophoresis and Southern blot
analysis of the putatively cured strain are consistent with the
hypothesis that the nod-nif plasmid has been cured.
Deletion and curing of pRme2011a.
Creation of a derivative of
Rm2011 which has been cured of pRme2011a was achieved in two steps.
Rm2011-14 carrying Tn5B12-S insert 14, which was previously
demonstrated to be in pRme2011a (25), was
plated onto TY (tryptone-yeast extract) agar containing 5%
sucrose. Resultant colonies were screened by Eckhardt gel
electrophoresis (18, 24, 25) for putative deletions. The
colony which carried the largest deletion was designated SmA146. From
Eckhardt gels, using VF39SM as a size standard, it was estimated that
this deletion reduced pRme2011a to approximately 600 kb (Fig.
1). This deletion was designated 
14-6,
and the replicon carrying this deletion was designated
pRme2011a14-6.
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FIG. 1.
Eckhardt gel showing plasmid profiles of S. meliloti strains with deletions in pRme2011a. Lanes: A, R. leguminosarum LRS39501 (24) (size standard); B, Rm2011;
C, SmA818; D, SmA146; E, Rm2011. All lanes are from the same gel, but
some intervening lanes showing derivatives with smaller deletions have
been removed for clarity.
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To reduce the size of the residual replicon in SmA146, the strain was
subjected to a further round of transposon mutagenesis, utilizing
Tn5B12-S. Single colonies were conjugally mated with Agrobacterium tumefaciens UBAPF2 to identify those
colonies which carried a Tn5B12-S insertion on
pRme2011a14-6. Eckhardt gel electrophoresis analysis was
carried out on putative transconjugants to ensure that the proper
plasmid had been transferred. S. meliloti colonies which had a Tn5B12-S insertion on pRme2011a14-6 were
grown overnight in TY broth and plated onto TY agar containing
5% sucrose. Three colonies of 300 were found to be neomycin sensitive
and no longer sensitive to sucrose. Of these, one appeared to be
missing pRm2011a14-6 (Fig. 1). This strain was designated SmA818.
SmA818 has deletions of all known genetic markers.
It has been
shown previously that the deletion 14-6 did not carry the
nodPQ region (35). To determine the extent of the deletion of 14-6 in SmA146 and to verify that SmA818 did not contain
sequences associated with pRme2011a, Southern blot analysis was carried
out on genomic DNA from these strains. Probes used corresponded to
regions associated with pRme2011a (6, 15, 27, 33, 34).
The results of these analyses showed that strains SmA146 and SmA818 did
not contain sequences homologous to nifHDK,
fixLJ, fixG, or syrB (Table
1). SmA146 did, however, contain
sequences that hybridized to the fixN probe (Table 1). In
S. meliloti strain Rm2011, fixN is reiterated on
pRme2011a (31). Further analysis revealed that the
hybridizing fragment in SmA146 corresponded to the fixN'
region, which is located close to a locus necessary for trigonelline
catabolism (7). Since SmA146 did not contain nifHDK sequences but did contain fixN' sequences,
this suggests that one end point of the pRme201114-6 deletion
was between these two regions. As genes encoding trigonelline
catabolism (trc) are found between these two markers, it was
of interest to determine whether SmA146 could utilize trigonelline as a
sole carbon source. The results showed that SmA146 could not use this
compound as a sole carbon source (Table
2). Together these data suggest that one
end point of the 14-6 deletion is between the trc and
fixN' loci and that it extends approximately 750 kb (Fig.
2).
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FIG. 2.
Schematic representation of pRme2011a showing relative
positions of known genetic markers. The approximate position of
syrB::Tn5 in strain MB101
(5) was determined by conjugal-transfer experiments similar
to those which were described for pRmeSU47b (11). The
oriT used for these experiments was that of
 30::Tn5-11 from Rm5420 (which is linked to
nifH) (18). The direction of transfer was
determined to be clockwise, using
fixJ2.3::Tn5 from strain GMI 5704 as a
marker for conjugal transfer (15). The arc shown represents
the approximate position of 14-6. The end points of this deletion
are undefined. The large arrows indicate the positions of
PmeI restriction sites (23). PmeI
fragment 5 extends between sites 1 and 2, fragment 6 extends between
sites 1 and 3, and fragment 7 extends between sites 2 and 3. All three
fragments are missing in strain SmA818.
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In an effort to confirm that pRme201114-6 had indeed been cured from
SmA818, plasmid DNA from SmA146 carrying pRme201114-6 was isolated
from a preparative Eckhardt gel, labeled, and used to probe Southern
blots containing DNA from Rm2011, SmA146, and SmA818. Consistent with
the hypothesis that pRme2011a was cured in SmA818, the results showed a
large reduction in the number, and in most cases the intensity, of
hybridizing fragments when Rm2011 and SmA818 were compared (Fig.
3). We note that it has previously been
shown that a great deal of reiteration exists in the S. meliloti and other rhizobial genomes (20). Moreover, it
has also been shown that Rm2011 contains at least five different insertion elements, four of which are highly reiterated (as many as 11 or 12 copies) within the genome (36). It has also been shown
that 18% of the sequence of the nodulation plasmid of
Sinorhizobium strain NGR234 shows homology to insertion
elements (21). The high proportion of reiterated sequences
in the rhizobia may explain the presence of the few remaining
hybridizing bands seen in SmA818 (Fig. 3). It is also theoretically
possible that small segments of plasmid pRme2011a do remain in strain
SmA818, integrated into either the chromosome or plasmid pRme2011b.
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FIG. 3.
Southern blot analysis of SmA818. Equal amounts of
EcoRI-restricted DNA from Rm2011 and SmA818 were
electrophoresed, blotted to a nylon membrane, and probed with
pRme201114-6 DNA which was isolated from a preparative Eckhardt gel.
Lanes: A, Rm2011; B, SmA818.
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Genomic analysis of SmA146 and SmA818 using pulsed-field gel
electrophoresis.
To verify that genomic rearrangements resulting
in insertion of portions of pRme2011a into another replicon had not
occurred during the deletion process that generated strains SmA146 and SmA818, the genomes of these strains were analyzed by pulsed-field gel
electrophoresis using restriction enzymes that cut the S. meliloti genome infrequently (23, 37). Using the
enzymes I-CeuI, PacI, PmeI, and
SwaI, we ascertained that the chromosomal fragments in
SmA818 and SmA146 had mobilities indistinguishable from those of the
wild type, Rm2011. Furthermore, there were no detectable changes in the
sizes and restriction patterns of bands known to correspond to
pRme2011b. Pulsed-field analysis using PacI and SwaI, both of which cut pRme2011a once, revealed the absence
of a 1,400-kb band in SmA818 (Fig. 4).
Moreover, analysis using PmeI, which yields seven
PmeI fragments in the wild-type strain, resulted in only
four discernible fragments in SmA818; bands 5, 6, and 7, which
comprise pRme2011a (23), were absent (data not shown). In strain SmA146, only a single 600-kb PmeI fragment
remained, suggesting that a single PmeI site [labeled
PmeI (3) in Fig. 2] remained in the form
of the pRme2011a replicon with the deletion. The presence of a
single PmeI site in pRme2011a14-6 is
consistent with the mapping of the deletion end point between
fixN' and trc, both of which are found on
PmeI fragment 6 to the left of PmeI fragment 5 (Fig. 2), and with the Southern analysis, which shows that
fixN' remains in SmA146. Together these data strongly
suggest that plasmid pRme2011a is missing completely in strain SmA818.
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FIG. 4.
Pulsed-field gel profiles of PacI-digested
S. meliloti strains. Lanes: A, SmA818; B, SmA146; C, Rm2011;
D, concatemers, molecular size markers.
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Phenotypic characterization of SmA818.
Growth experiments were
performed to characterize SmA818. These experiments demonstrated that
SmA818 and Rm2011 have identical doubling times when grown either in
Vincent's minimal medium (38) containing succinate (15 mM)
or glucose (15 mM) as a sole carbon source or in complex media (TY).
It has been shown previously that unidentified dehydrogenase activity
was encoded by pRme2011a (14). To provide corroborating evidence that pRme2011a was indeed cured, cell extracts of Rm2011, SmA818, and A. tumefaciens UBAPF2(pRme2011a) were
prepared, separated on native polyacrylamide gel electrophoresis gels,
and stained for unidentified dehydrogenase (14). The data
show that SmA818 did not have this activity, whereas Rm2011 and
UBAPF2(pRme2011a) both had an unidentified dehydrogenase band of
activity (Fig. 5).
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FIG. 5.
Unidentified dehydrogenase (Udh) and SOD activities in
S. meliloti. Cell extracts of Rm2011 and SmA818 were run on
nondenaturing polyacrylamide gels. The gel was stained as previously
described (8). Lanes: A, Rm2011; B, SmA818.
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When native gels containing Rm2011 and SmA818 extracts were stained for
prolonged periods, an achromatic band(s) was often observed. Native
gels stained for superoxide dismutase (SOD) or tetrazolium
oxidase activities yielded a negative band on a dark background
(17). Interestingly, a negative staining band also appeared
to be missing in SmA818 (Fig. 5). Under our growth conditions, we were
able to resolve three bands of SOD activity in the wild-type strain
(data not shown). SmA818 was found to be missing one of these
bands. Transfer of pRme2011a to A. tumefaciens resulted in
the appearance of a band of SOD activity with an
Rf similar to that of the band missing in SmA818
(data not shown). This suggests that pRme2011a carries a determinant(s)
that influences the expression of SOD activity. We note that
plasmid-borne SOD activity has previously been reported for R. leguminosarum (1).
To reveal possible catabolic defects present in a strain lacking
pRme2011a, SmA818 was screened for potential phenotypes using Biolog
plates, which enabled screening for utilization of 96 carbon sources
simultaneously. Putative phenotypes were confirmed by streaking SmA818,
SmA146, and Rm2011 on defined media containing the carbon source being
tested (Table 2). This analysis shows that SmA818 was unable to
catabolize inosine, 
-aminobutyric acid (GABA), gluconate, glycine,
and serine as sole carbon sources (Table 2). Further analysis of the
glycine and serine phenotypes suggested that both of these phenotypes
map to one locus and that one of the genes involved may be a transport
protein responsible for the uptake of these amino acids. Moreover,
cosmids containing the Gly-Ser utilization region also complemented
GABA utilization. These phenotypes, however, were shown to be
genetically distinct (I. J. Oresnik and M. F. Hynes,
unpublished data).
The megaplasmids of S. meliloti are essentially stable, and
conventional methods for plasmid curing have proven to be unsuccessful for these replicons (2, 3, 24). Conventional mutagenesis and
screening for phenotypes are often unsuccessful due to reiterated DNA
sequences which are found in Rhizobium (20, 31, 34, 35). Methods which have utilized curing or the construction of
defined deletions have been useful in studying cryptic replicons in
Rhizobium (1, 2, 3, 6, 12, 24, 25, 31). In this
work we provide evidence for the curing of pRme2011a. This is, to the
best of our knowledge, the largest replicon cured to date in any
bacterium. By curing this plasmid, we have demonstrated that pRme2011a
does not carry single-copy genes which are essential for cell viability
or for the maintenance of this strain on normal lab media. Thus, by
definition, pRme2011a is a plasmid and not a minichromosome as others
have suggested (29). Extensive probing using markers
localized to the characterized regions of pRme2011a has shown that the
corresponding DNA is absent in strain SmA818. Pulsed-field analyses of
SmA818 have been consistent with the hypothesis that, in this strain,
pRme2011a has been cured. Judging by the size of the pulsed-field
electrophoresis fragments in SmA818, it appears unlikely that genomic
rearrangements larger than 40 kb occurred in either of the remaining
replicons as a consequence of either the deletion or curing events used
to generate this strain.
Surprisingly, although almost one-quarter of the genome of Rm2011 has
been removed, the strain still exhibits growth rates that are identical
to that of the wild type on defined and complex media. This in itself
is remarkable, considering that this replicon is inherently stable and
that large deletions in this plasmid are rarely isolated. This suggests
that there must be reasons other than viability for the maintenance of
this plasmid. Studies addressing these issues as well as more detailed
phenotype determination will presumably be more fruitful following the
complete determination of the genome sequence of Rm1021, which is
currently under way (10).
We thank J. Batut, S. R. Long, M. Barnett, T. Finan, and T. Charles for strains and probes that were used in this study. We are
also very grateful to S. R. Long and M. Barnett for comments on
the manuscript. The advice, support, and encouragement of P. Boistard,
in whose laboratory a small part of the initial work was done, are
gratefully acknowledged.
A high-resolution physical map of pRme2011a (pSymA) has very
recently been published (F. Barloy-Hubler et al. J. Bacteriol. 182:1185-1189, 2000). This paper suggested that genes for the production of the siderophore rhizobactin mapped to the middle of the undeleted region in plasmid pRme2011a14-6, and we have confirmed that strain SmA818 does not produce rhizobactin. It is also
of note that at least four copies of ISRm2011-2 map to the undeleted
region of pRme2011a in strain SmA416.
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