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Journal of Bacteriology, June 2000, p. 3582-3586, Vol. 182, No. 12
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Megaplasmid pRme2011a of Sinorhizobium
meliloti Is Not Required for Viability
Ivan J.
Oresnik,
Shu-Lin
Liu,
Christopher K.
Yost, and
Michael F.
Hynes*
Department of Biological Sciences, University
of Calgary, Calgary, Alberta T2N 1N4, Canada
Received 8 November 1999/Accepted 21 March 2000
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ABSTRACT |
We report the curing of the 1,360-kb megaplasmid pRme2011a from
Sinorhizobium meliloti strain Rm2011. With a positive
selection strategy that utilized Tn5B12-S containing the
sacB gene, we were able to cure this replicon by successive
rounds of selecting for deletion formation in vivo. Subsequent Southern
blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were
consistent with the hypothesis that the resultant strain was indeed
missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number
of substrates as a sole source of carbon on defined media.
| |
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
Tn
5B12-S. Single colonies were conjugally mated with
Agrobacterium tumefaciens UBAPF2 to identify those
colonies which
carried a Tn
5B12-S insertion on
pRme2011a
14-6. 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 Tn
5B12-S insertion on pRme2011a
14-6 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 pRm2011a
14-6
(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 pRme2011
14-6 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 pRme2011
14-6 had indeed been cured from
SmA818, plasmid DNA from SmA146 carrying pRme2011
14-6
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).
| |
ACKNOWLEDGMENTS |
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.
This work was supported by an NSERC grant to M.F.H.
| |
ADDENDUM IN PROOF |
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.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada. Phone: (403) 220-8473. Fax: (403) 289-9311. E-mail:
hynes{at}ucalgary.ca.
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Journal of Bacteriology, June 2000, p. 3582-3586, Vol. 182, No. 12
0021-9193/00/$04.00+0
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