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Journal of Bacteriology, October 1998, p. 5432-5436, Vol. 180, No. 20
Department of Biology, Massachusetts
Institute of Technology, Cambridge, Massachusetts
02139,1 and
Complex Carbohydrate
Research Center, University of Georgia, Athens, Georgia
30602-47122
Received 22 May 1998/Accepted 3 August 1998
For Sinorhizobium meliloti (also known as
Rhizobium meliloti) AK631 to establish effective symbiosis
with alfalfa, it must be able to synthesize a symbiotically active form
of its K antigen, a capsular polysaccharide containing a Kdo
(3-deoxy-D-manno-octulosonic acid) derivative. Previously
isolated mutants defective in the synthesis of K antigen are resistant
to bacteriophage In order to invade the nodules it
elicits on alfalfa (Medicago sativa), Sinorhizobium
meliloti (also known as Rhizobium meliloti) must
synthesize at least one of three polysaccharides: succinoglycan, exopolysaccharide (EPS) II, or a symbiotically active form of the
strain-specific K antigen (4, 15-17). Strains which produce symbiotically appropriate forms of any one of these three
polysaccharides are capable of forming healthy, nitrogen-fixing
nodules. In contrast, S. meliloti mutants that fail to
synthesize at least one of these polysaccharides in symbiotically
appropriate forms will produce ineffective or Fix The fact that K antigen can replace succinoglycan or EPS II was
discovered through the study of a derivative of S. meliloti Rm41 known as AK631. This strain cannot synthesize succinoglycan or EPS
II due to a mutation in the exoB (galE) gene,
which is required for the synthesis of both of these polysaccharides
(2, 3). However, strain AK631 is still capable of forming
fully effective nodules on alfalfa because AK631 produces a form of K
antigen which can functionally replace succinoglycan or EPS II during
nodule invasion (12, 13, 15-17). So far, two gene regions
that are required for the production of K antigen have been identified.
The genes rkpABCDEFGHIJ are clustered in the rkp-1 region, and rkpZ is located by itself in a
distinct genetic region. However, the fact that none of these genes
were predicted to be glycosyltransferases, as have been found in the
succinoglycan (5, 6, 14) and EPS II (1)
biosynthetic regions, suggested that there must be additional genetic
loci that encode functions required for K-antigen biosynthesis.
The lytic phage Screening for mutations in genes involved in K-antigen synthesis by
selecting for mutants with resistance to phage
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Different Phenotypic Classes of Sinorhizobium
meliloti Mutants Defective in Synthesis of K Antigen
ABSTRACT
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16-3. By screening ca. 100,000 Tn5-mutagenized R. meliloti bacteria for resistance to bacteriophage 16-3, we isolated 119 mutants, 31 of
which could not be complemented by genes previously identified as being
required for K-antigen synthesis. Of these 31 new mutants, 13 were
symbiotically defective and lacked the K antigen. Through genetic and
phenotypic analyses, we have grouped these mutants into four distinct
classes. Although all of these mutants lack the K antigen, many also
have altered lipopolysaccharides (LPS), suggesting that the biochemical
pathways for the synthesis of K antigen and LPS have common enzymatic
steps. In addition, we have found that these and other classes of
K-antigen-defective mutants of S. meliloti AK631 exhibit
unique patterns of sensitivities to phage strains to which the parental
strain was resistant. Our studies have identified new classes of genes
required for both the synthesis of K antigen and the symbiotic
proficiency of S. meliloti AK631. Some of these classes of
genes also play a role in LPS synthesis.
TEXT
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Abstract
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nodules, which contain few bacteria and which are incapable of fixing
nitrogen on alfalfa host plants.
16-3 will infect S. meliloti AK631, which
produces K antigen, but not AK631 mutants which lack K antigen (13). The role that K antigen plays in sensitivity to this
phage is unknown, but in a simple model the K antigen would act as a ligand for a receptor on the phage particle. We therefore reasoned that
resistance to phage 16-3 could be used as a powerful selection factor in isolating additional mutations in genes required for the
synthesis of K antigen. Using this method, we carried out a
large-scale genetic study that resulted in the identification of four
new, phenotypically and genetically distinct classes of K-antigen
mutations which are not complemented by plasmids carrying the
rkp-1 region or the rkpZ+ gene.
16-3.
To select
phage-resistant mutants of S. meliloti AK631, bacteria were
mutagenized with Tn5 and plated on LB plates spread with
109 PFU of phage 16-3. Colonies which grew on these
plates were picked and restreaked on plates spread with phage 16-3
to confirm phage resistance. In this manner, 20 independent pools of
bacteria representing approximately 100,000 genomes were screened,
which resulted in 119 phage 16-3-resistant mutants.
16-3 by complementation with either plasmid, and so we
focused on these mutants for further study.
phenotype. The other group
consisted of 18 mutants that formed wild-type, elongated pink nodules
and resulted in healthy green plants. This suggests that these
mutations either do not affect K antigen or else affect K antigen in a
fashion that does not interfere with its symbiotic role. Since it
seemed likely that the mutants in the first group would have mutations
in hitherto-unidentified K-antigen synthesis genes, we focused our
efforts on analyzing the Fix, 16-3-resistant mutants.
Analysis of K antigen and LPS from Fix, phage
16-3-resistant mutants reveals three distinct classes of K antigen
mutations.
We analyzed the K antigen from our novel
Fix, phage 16-3-resistant mutants to determine whether
their inability to form nitrogen-fixing nodules correlated with a
defect in K-antigen production. The cell-associated polysaccharides
were extracted with hot phenol-water, and the water-soluble extracts
were analyzed by polyacrylamide gel electrophoresis (PAGE) and alcian
blue-silver staining (Fig. 1). All 13 of
the Fix mutants we had isolated failed to produce K
antigen, indicating that all of these mutants had defects in previously
unidentified genes involved in K-antigen synthesis.
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mutants into three
phenotypic classes. Mutants in class I lack the K antigen but produce
LPS that appears similar to that produced by the parental strain AK631;
i.e., the rough-LPS (R-LPS) migrates as two diffuse bands (Fig. 1A). It
is important to note that the R-LPS from the parental strain, AK631,
migrates slightly further due to the presence of the K antigens; also,
the rkp-180 mutant appears to have only one band due to less
total carbohydrate being loaded. This is similar to the previously
isolated K-antigen mutations in the rkp-1 region and in the
rkpZ gene. Seven of the 13 mutants, rkp-085,
rkp-143, rkp-162, rkp-163,
rkp-180, rkp-181 (Fig. 1A), and
rkp-063 (Fig. 1B), fall into this class. However, the
rkp-063 mutant has a phenotype which make it distinct from
other members of this class, as will be discussed below. In contrast,
both class II and III mutants produce LPS which is different from that
produced by the parental strain (Fig. 1B). Class II includes the five
mutants rkp-132, rkp-133, rkp-135,
rkp-161, and rkp-172. During the process of
extracting the polysaccharides from these mutants with hot phenol-water, we noticed that the LPS from these mutants had the unusual characteristic of separating predominantly into the phenol phase rather than into the aqueous phase. We confirmed this observation by dialyzing the phenol phase and running the resulting products on
PAGE gels (data not shown). Class III consisted of one
K-antigen-deficient mutant, rkp-205, which was unusual in
that its LPS resembled that found in the exoB+
parent of AK631 rather than that of AK631 itself. Like the
exoB+ strain, the rkp-205 mutant has
only one LPS band which, unlike the LPS of class II mutants, does not
segregate into the phenol phase. The fact that class II and class III
mutants displayed alterations in their LPS as well as being deficient
in K antigen may indicate that the biochemical pathways for K antigen
and LPS have common steps and that these mutations are defects in genes involved in those common steps. The set of 13 Fix,
K-antigen-deficient mutants also shared one other interesting feature.
In every case, they produced a very-high-mobility polysaccharide that migrated in a ladder pattern at the bottom of the lane. The nature
of this material is currently under investigation.
Absence of K antigen alters the susceptibility of S. meliloti AK631 to several phages.
Since AK631 mutants
lacking K antigen are resistant to 16-3, we decided to examine
whether they had altered sensitivity to other S. meliloti
phages (Table 1). With the exception of
16-3 and M12H1, the phages we tested had been isolated based on
their ability to form plaques on another S. meliloti
strain, SU47. We found that none of these phages would form plaques on
AK631, the parental strain in our genetic study. M12H1 is a
derivative of M12 capable of infecting both Rm41, the parental
strain of AK631, and strain SU47.
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M7, M9, M10, M11, M12, M14, and M19. Strains with these mutations also show a slight sensitivity to M6, but efficient plaque formation did
not occur. These data indicate that the production of K antigen by
strain AK631 protects it against infection by many S. meliloti phages to which it would otherwise be susceptible. K
antigen does confer sensitivity to 16-3, however, indicating that
while K antigen offers AK631 protection against some phages, this
polysaccharide renders the strain susceptible to others.
New K-antigen mutants have distinct phage sensitivity profiles
which support the mutant classifications determined by PAGE and suggest
an additional mutant class.
Like the mutants containing defects in
the rkp-1 region analyzed above, the 13 novel
Fix K-antigen mutants we isolated are more sensitive to
many of the phages tested (Table 1). Like mutants rkpA674,
rkpI634, and rkpJ671, most of our mutants are
very sensitive to M7, M11, M12, and M19. These phages will
form plaques on all of our mutants. However, the phage sensitivity
profiles of our mutants are different from those of rkp-1
mutants in certain respects. For example, in every case, our mutants
were more resistant to M7, M10, and M14 than the
rkp-1 mutants we tested. The patterns of phage sensitivity shown by our mutants also differ between classes (Table 1).
M7 and resistance to both M9 and
M10. Also, this mutant resembles the class III mutants in being
resistant to M14. Based on these results and on the results of the
following sections, we have placed mutant rkp-063 into a
distinct class termed class IV.
Different classes of K-antigen-deficient mutants show distinct phenotypes when streaked on Congo red plates. Congo red dye has been found to bind many types of polysaccharides (19). It has been reported that when streaked on M9-mannitol plates containing 0.05% Congo red, AK631 will form salmon-colored colonies (18). In contrast, an AK631 rkpZ mutant forms dark-red colonies under these conditions (18). We therefore investigated whether this assay could be used to distinguish AK631 strains which had deficiencies in producing K antigen from those that did not. As expected, when we streaked an rkpZ mutant on Congo red plates, it formed dark-red colonies as did the rkpA674, rkpI634, and rkpJ671 mutants. Surprisingly, when we streaked our newly isolated mutants on Congo red plates, they had two distinct phenotypes (Table 2). Mutants from classes III and IV formed dark-red colonies similar to the ones formed by the rkpZ and rkpJ671 mutants. Mutants from classes I and II, however, formed salmon-colored colonies which were almost identical in color to those of AK631, the parental strain. The fact that mutant rkp-063 stains red whereas the class I mutants do not support our decision to place this mutant in a separate class.
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Genetic analysis of K-antigen mutations.
While performing
the experiments described above, we became aware that Kereszt
et al. (7, 8) had also isolated a series of
16-3-resistant mutants, which were Fix on
alfalfa and failed to produce K antigen, that fell into two complementation groups, which they called rkp-2 and
rkp-3 (7, 8). Mutations in the
rkp-2 region, were complemented by the cosmid pAT330;
mutations in the rkp-3 region, was complemented by the
overlapping cosmids pAT399 and pAT401 (7, 8). Kereszt et al. generously provided us with these three cosmids prior to publication (7, 8). We were thereby able to perform
complementation tests with the mutants that we had isolated by
determining whether these cosmids would restore 16-3 sensitivity to
these mutants.
16-3 by complementation with pAT399 and
pAT401 and were thus in the rkp-3 region, while mutants we determined as belonging to class II were complemented by pAT330 or in
the rkp-2 region. These data indicate that the classes that we had assigned on the basis of phenotypes represent distinct genetic
classes of mutants as well.
Our class III and IV mutants were not complemented by the cosmids
pAT330, pAT399, and pAT401. To analyze these mutants further, we first
screened an Rm41 library for cosmids that complemented the sensitivity
of mutants rkp-063 and rkp-205 to M12.
However, we were unable to isolate any complementing cosmids to the
mutations in these strains by using this strategy. We also attempted to complement the 16-3 resistance phenotype directly, by replica plating colonies containing cosmids from the library onto plates spread with 16-3 and looking for clones which were sensitive to this phage. We screened several thousand clones by this strategy but
were unable to isolate any cosmids which complemented mutant rkp-063 or rkp-205. In addition, we provided
Putnoky and his colleagues with our class III and class IV mutants, and
they also failed to isolate complementing cosmids (11).
Because our Fix, 16-3-resistant mutants had been
isolated from a population of R. meliloti cells mutagenized
with the transposon Tn5, we expected that the majority of
the mutants that we had isolated would carry recessive loss-of-function
alleles caused by the insertion of Tn5 and thus provide us
with a selectable cloning marker. However, when we transduced the
Tn5 insertions in mutants rkp-063 and
rkp-205 into AK631, in each case we found that the mutation
causing the 16-3-resistant phenotype was unlinked to the
Tn5 insertion.
Four classes of novel K-antigen mutants.
After screening
approximately 100,000 Tn5-mutagenized derivatives of
S. meliloti AK631 for mutants which are resistant to 16-3, we successfully isolated 13 novel K-antigen-deficient mutants. Genetic and phenotypic analyses placed these mutants into four distinct
classes (Table 2). Two of these classes, class III and class IV, each
contain only one member and cannot be complemented by a cosmid carrying
any known genes required for K-antigen synthesis, including those
described in the accompanying report (7). On PAGE gels,
rkp-205, the class III mutant, has its own distinct phenotype. Like many of the other K-antigen mutants, rkp-205
produces no K antigen. However, the LPS from this mutant migrates
differently from other K-antigen mutants that exhibit LPS defects and,
in fact, resembles that observed for strain Rm41, the
exoB+ parent of AK631. In contrast, our
class IV mutant rkp-063 looks similar to the class I mutants
in that it produces no K antigen and has no observable differences in
LPS from that of the parental strain AK631. In addition, the class III
and class IV mutants have phage resistance profiles that are distinct
from the rest of our mutant classes, as well as from each other.
M10 and M14 and are resistant to M9. We have determined that these mutations lie in the
region defined by Kereszt et al. as rkp-3 (7, 8),
based on the finding that these mutants are complemented by the
overlapping cosmids pAT399 and pAT401.
The effects of class II mutations are less specific than those of the
class I mutations. Not only do they abolish K-antigen synthesis, but
they also have striking effects on LPS synthesis. In addition to
producing no K antigen, the majority of the LPS from these mutants
segregates into the phenol phase during the extraction process,
indicating that it is altered from the LPS of the parental strain. In
this respect, these mutations are different from those in any of the
previously characterized K-antigen synthesis genes. This class of
mutants also has its own profile of phage resistances and
sensitivities. Through complementation tests, we have determined that
these mutations are located in the rkp-2 region. As
described in the accompanying paper, Kereszet et al. (7, 8)
sequenced the rkp-2 region and found it to contain two open
reading frames organized in monocistronic transcription units. Although
they found both of these genes to be required for normal LPS
production, only the second one, designated rkpK, is
involved in K-antigen synthesis. They have found that RkpK possesses
UDP-glucose dehydrogenase activity. Presumably this enzyme is needed
for the synthesis of both K antigen and LPS.
It is not yet clear why we were unable to obtain complementing cosmids
to the class III and IV mutations. One explanation could be that the
phenotypes of these mutants are not the result of simple recessive null
mutations but are instead caused by dominant mutations, complex
rearrangements, or multiple mutations. Alternatively, these genes may
not be present in our cosmid library because they are toxic when
present in multiple copies. The fact that these mutations were
not caused by Tn5 insertions may reflect difficulty in
obtaining null mutations in these genes, perhaps because they are
essential. This possibility is supported by the facts that these
mutations fell into the smallest groups in our original screen and that
class III and class IV mutants were not obtained by Kereszt et al.
(7, 8).
We thank Peter Putnoky and his colleagues for their willingness
to share data and plasmids prior to publication and Juan
González, Hai-Ping Cheng, and Greg York for their useful
discussions.
This work was supported by Public Health Service grant GM31030 from the
National Institutes of Health.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139. Phone: (617) 253-6716. Fax: (617) 253-2643. E-mail: gwalker{at}mit.edu.
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Journal of Bacteriology, October 1998, p. 5432-5436, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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