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Previous Article | Next Article 
Journal of Bacteriology, October 1998, p. 5426-5431, Vol. 180, No. 20
Institute of Genetics, Biological Research
Center, Hungarian Academy of Sciences, H-6701 Szeged,
Hungary1;
Complex Carbohydrate Research
Center, University of Georgia, Athens, Georgia
306022; and
Institut des Sciences
Vegetales, CNRS, F-91198 Gif-sur-Yvette Cedex,
France3
Received 30 April 1998/Accepted 3 August 1998
The production of exopolysaccharide (EPS) was shown to be required
for the infection process by rhizobia that induce the formation of
indeterminate nodules on the roots of leguminous host plants. In
Sinorhizobium meliloti (also known as Rhizobium
meliloti) Rm41, a capsular polysaccharide (KPS) analogous to the
group II K antigens of Escherichia coli can replace EPS
during symbiotic nodule development and serve as an attachment site for
the strain-specific bacteriophage Extracellular carbohydrates produced
by rhizobia play an important role in the development of
nitrogen-fixing symbioses with leguminous plants.
Lipochitooligosaccharide signal molecules (Nod factors) are required
for the formation of a new plant organ, the nodule, where bacteroids,
the endosymbiotic forms of rhizobia, reduce atmospheric nitrogen
(7, 34). Additional surface and extracellular components,
including lipopolysaccharides (LPS), exopolysaccharides (EPS),
capsular polysaccharides (KPS), and cyclic Recently, it has been demonstrated that some rhizobia possess surface
polysaccharides that are analogous to the group II K antigens (KPS) of
Escherichia coli (28, 29), and in S. meliloti this type of surface polysaccharide can also
replace EPS in the infection process (22, 30).
Although the complete chemical structure of the S. meliloti
Rm41 KPS has not yet been determined, available data indicate that it
is composed of disaccharide repeating units of
We have isolated a gene cluster of S. meliloti Rm41
(rkp-1, formerly fix-23) that is required for KPS
production. Mutants affected in this gene cluster are not able to
adsorb the strain-specific bacteriophage The rkp-1 region does not code for proteins participating in
the biosynthesis and polymerization of the KPS precursors. Therefore, to identify novel rkp genes required for these latter
functions, a screening for new mutants was carried out. Since most of
the rkp mutants isolated so far were resistant to phage
Bacterial strains, bacteriophages, plasmids, and growth
conditions.
Strain AK631 carrying the exoB-631 mutation
is a Fix+ Exo
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Novel rkp Gene Clusters of
Sinorhizobium meliloti Involved in Capsular Polysaccharide
Production and Invasion of the Symbiotic Nodule: the rkpK
Gene Encodes a UDP-Glucose Dehydrogenase
Kiss,1
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
16-3. The rkpA to
-J genes in the chromosomal rkp-1 region code for proteins that are involved in the synthesis, modification, and
transfer of an as-yet-unknown lipophilic molecule which might function
as a specific lipid carrier during KPS biosynthesis. Here we report
that with a phage 16-3-resistant population obtained after random
Tn5 mutagenesis, we have identified novel mutants impaired
in KPS production by genetic complementation and biochemical studies.
The mutations represent two novel loci, designated the rkp-2 and rkp-3 regions, which are required for
the synthesis of rhizobial KPS. The rkp-2 region harbors
two open reading frames (ORFs) organized in monocistronic transcription
units. Although both genes are required for normal lipopolysaccharide
production, only the second one, designated rkpK, is
involved in the synthesis of KPS. We have demonstrated that RkpK
possesses UDP-glucose dehydrogenase activity, while the protein product
of ORF1 might function as a UDP-glucuronic acid epimerase.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-glucans, are
required for the infection of the nodule tissue by the microsymbiont.
The symbiotic phenotype of the LPS- and EPS-defective mutants depends
on the type of nodule determined by the host genome (13).
For Sinorhizobium meliloti (also known as Rhizobium
meliloti), which induces indeterminate nodules on the roots of its
symbiotic partner, alfalfa (Medicago sativa), EPS has been
reported to be involved in the infection process while the importance
of the LPS has not yet been demonstrated. The production of EPS I
containing octasaccharide repeating units of seven glucoses and a
galactose, which are substituted with pyruvyl, acetyl, and succinyl
groups, was shown to be necessary for the development of the infection
thread and the invasion process. It has also been shown that S. meliloti has a latent capability to produce a second type of
exopolysaccharide, EPS II, containing a glucose-galactose disaccharide
subunit carrying pyruvyl and acetyl modifications. A second-site
mutation allows several EPS I-nonproducing bacteria to synthesize EPS
II and helps to overcome their invasion defect in some host plants,
suggesting that EPS II can replace EPS I in the infection process
(17).
-keto-3,5,7,9-tetradeoxy-5,7-diaminonulonosic acid, which
is a Kdo (3-deoxy-D-manno-2-octulosonic acid)-like
molecule, and of 4-deoxy-4-amino-hexuronic acid (27).
The isolated KPS from S. meliloti shows a banding
pattern on polyacrylamide gels that corresponds to the different
degrees of polymerization that are controlled by the rkpZ
gene product (30).
16-3 and, in an
Exo
background, cannot invade the nodule. The
rkp-1 region harbors 10 open reading frames (ORFs) that are
designated the rkpA to -J genes and whose
predicted protein products exhibit a high degree of similarity to the
different components and domains of fatty acid synthases as well as to
enzymes taking part in the modification and transfer of lipid molecules
to capsular polysaccharides (15, 22). These gene products
might be involved in the synthesis of a putative lipid anchor of the
polysaccharide or, more likely, in the production of a specific lipid
carrier required for the biosynthesis of the KPS.
16-3, new mutations affecting KPS synthesis could be collected by
isolating additional phage-resistant derivatives. Here we report the
identification of two novel gene clusters, designated the
rkp-2 and rkp-3 regions, that are involved in the
production of the capsular polysaccharide. Moreover, genetical and
biochemical analysis of the rkp-2 region is presented.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
derivative of S. meliloti Rm41 (24). Strains AT211, AT212, AT231, AT232, AT233, AT234, AT578, PP2936, and PP2937 are
KPS and/or LPS derivatives of AK631 that
carry Tn5 and plasmid insertions in the rkp-2 or
rkp-3 region, as indicated below (see Fig. 2 and 3).
E. coli JM109 was used in the cloning procedures
(38) which involved the following plasmids: pUC19
(38), pK18/19 (23), and pOK12 (37).
Isolation of the S. meliloti mutants defective
in KPS production and identification of the rkp-2 and
rkp-3 regions.
Random transposon Tn5
mutagenesis of S. meliloti AK631 was carried out by
using the mobilizable suicide plasmid pSUP1011 (35). The
kanamycin- and streptomycin-resistant transconjugants were selected on
minimal medium, and aliquots (1 × 108 to 5 × 108 cells) from 10 mutagenized populations (2,000 to 3,000 transconjugants/population) were infected by bacteriophage 16-3 (at
a multiplicity of infection 10) and plated. Phage-resistant colonies
were tested in phage adsorption experiments as described previously
(26). For the isolation of the complementing cosmid clones,
the genomic libraries were introduced into the rkp mutants
and plant assays were used to identify the clones carrying the
wild-type genes as described earlier (24).
Directed Tn5 and plasmid integration mutagenesis. Tn5 mutagenesis of the cosmid clone pAT330 was carried out as described earlier (26). The isolated Tn5 insertions were introduced into the AK631 genome by homologous recombination by the method of Ruvkun and Ausubel (31). Positions of the insertions (Tn5-202, Tn5-207, and Tn5-238) are shown below (see Fig. 3A).
For plasmid integration mutagenesis, DNA fragments were cloned into vector pK18 and derivatives obtained after single homologous recombination with pAT330 were isolated in a Rec+ E. coli strain. The resultant constructions were introduced into the S. meliloti genome in the same way as the Tn5 insertions were, by selecting for the antibiotic (kanamycin) resistance of pK18.DNA manipulation. Standard procedures, including DNA isolation, restriction enzyme digestion, radioactive labelling of DNA, agarose gel electrophoresis, DNA ligation, and transformation of E. coli, were performed by using conventional methods (32).
For sequencing, appropriate restriction fragments were subcloned and their nucleotide sequences were determined by the dideoxy nucleotide chain termination method (33) by using [35S]dATP and the T7 sequencing kit from Pharmacia. Additional sequence information was obtained from the Tn5-flanking regions of different insertions by using a Tn5-specific oligonucleotide primer (14). Computer analysis of the sequence data was carried out by using the PC/Gene microcomputer software (designed by Amos Bairoch, IntelliGenetics) and the Genetics Computer Group (GCG) software package of the University of Wisconsin (8). Amino acid similarity searches were performed by using the Blast programs (1) at the National Center for Biotechnology Information, National Library of Medicine, Bethesda, Md., and the FASTA program (21).Plant tests. Assays for the symbiotic properties of the mutant S. meliloti strains were carried out with alfalfa (Medicago sativa L. Nagyszénási) plantlets as described earlier (24).
Preparation and analysis of polysaccharides. Extraction of the surface polysaccharides by a modified hot phenol-water method and DOC-polyacrylamide gel electrophoresis (PAGE) analysis of the samples were carried out as described elsewhere (15, 28).
Cell fractionation. S. meliloti cells were harvested from a 500-ml overnight culture and washed with 100 mM Tris-HCl buffer (pH 8.5) containing 1 mM MgCl2. The pellet was then suspended in 10 ml of buffer containing 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 20 µg of DNase I and RNase A per ml. Bacteria were disrupted by passing them three times through a French press (12,000 lb/in2). The suspension was centrifuged at 15,000 × g for 30 min to remove unbroken cells, and the supernatant was centrifuged at 140,000 × g for 2 h. Protein concentrations of the supernatants (soluble fraction) were estimated by using the Bio-Rad (Richmond, Calif.) assay with bovine serum albumin as a standard.
UDP-glucose dehydrogenase assay. Enzyme activity was assayed with a Shimadzu spectrophotometer in the double-beam mode at 25°C to measure the rate of change of optical density at 340 nm caused by the NAD reduction accompanying the oxidation of UDP-glucose to UDP-glucuronic acid. The assay mixture contained 100 mM Tris-HCl buffer (pH 8.5), 1 mM MgCl2, 5 mM dithiothreitol, 2 mM NAD, 1 mM UDP-glucose, and 200 to 400 mg of the soluble protein fractions in a 1-ml volume. One unit of UDP-glucose dehydrogenase is defined as the amount of enzyme that catalyzes the reduction of 1 micromole of NAD per minute (18).
Nucleotide sequence accession number. A DNA sequence of 3,673 bp has been registered in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under accession no. AJ222661. Coordinates in this publication are identical with those of the database record.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Identification of novel regions required for KPS production.
To isolate genetic determinants involved in the biosynthesis of the
precursors as well as in the polymerization of the rhizobial KPS,
random Tn5 mutagenesis was carried out in the S. meliloti Rm41 derivative strain AK631. For selection of potential
mutants, we used phage 16-3, which had previously been shown
to be unable to adhere to the surfaces of the KPS-defective
strains harboring a mutation in the rkp-1 region
(26). In phage inactivation experiments, 20 isolates
resistant to phage 16-3 were assayed for their capacity to adsorb to
the phage particles. Ten mutants were not able to bind the phages.
These strains were further screened in genetic complementation
experiments for mutations affecting as-yet-unidentified rkp
regions or genes. The phage adsorption capability of six mutants (AT211, AT212, AT231, AT232, AT233, and AT234) was not restored by the
cosmid clone pPP428 carrying the rkp-1 region; therefore, these mutants were retained for further biochemical and genetic studies.
). Moreover, light microscopic
analysis of the ineffective symbiotic nodules revealed that they were
incapable of infecting the nodules (Inf) (data not
shown).
|
16-3 and were
Fix, indicating that the mutant phenotypes are due to
Tn5 insertions (Fig. 2).
|
Genetic analysis of the rkp-2 region.
To delimit
the genes in the rkp-2 region, directed
lambda::Tn5 mutagenesis was carried out by using
the complementing cosmid clone pAT330. Transposon insertions were
introduced into the genome of AK631 by homologous recombination, and
the recombinants were tested for phage 16-3 sensitivity and
KPS and LPS production, as well as for symbiotic properties. Only
one additional transposon insertion (Tn5-207), located in
the vicinity of the original mutation (Tn5-AT231), resulted
in a phage-resistant phenotype (Fig. 3A). However, during the homogenization, we realized that in some cases (including Tn5-AT231) it was difficult to obtain bona fide
homogenotes and, moreover, that these strains formed small colonies.
One possible explanation is that the mutations are in genes that affect
the viability (fitness) of the cells.
|
16-3; in the
phage-resistant strains, no capsular polysaccharide was detectable,
while the banding patterns of KPS isolated from the phage-sensitive
mutants were indistinguishable from that of AK631.
In plant nodulation assay, all of the KPS-defective mutants exhibited
the Fix phenotype on alfalfa, while strains altered only
in LPS production were capable of establishing nitrogen-fixing
symbiosis (Fig. 3A).
The rkp-2 region encodes proteins similar to enzymes involved in the metabolism of nucleotide diphospho-sugars. To identify the functions of the genes and gene products involved in the formation of both LPS and KPS, appropriate overlapping restriction fragments of the SphI-BamHI fragment (Fig. 3A), as well as the flanking regions of Tn5 from the insertion mutants, were subcloned and the entire region was sequenced. Computer-aided analysis of the sequence data revealed the presence of two ORFs transcribed in the same direction (Fig. 3A). Codon preference analysis confirmed that the codon usage in these ORFs is in accordance with most of the S. meliloti genes sequenced to date. Weak similarities to the E. coli promoter consensus were found upstream of both ORFs. ORF1 was followed by short inverted repeats preceding several thymidine residues, while an 11-bp-long inverted repeat was found after the translational stop codon of ORF2. These sequences might serve as transcription termination signals.
The first ORF is located between nucleotide positions 307 and 1332 and starts with an ATG initiation codon. A potential ribosomal binding site is situated 5 bp upstream of the initiation codon. The putative translational product of ORF1 contains 341 amino acid residues and has a calculated molecular mass of 38,082 Da. Comparison of the amino acid sequence with protein (SwissProt, PIR) and pretranslated nucleic acid (GenBank) databases revealed significant similarities to different epimerases involved in the epimerization of nucleotide diphospho-sugars. The most definite homology was found with the CapI protein (51% identity, 67% similarity), which is involved in the capsular polysaccharide production of Staphylococcus aureus and which participates in the biosynthetic pathway leading to the synthesis of N-acetyl-aminogalacturonic acid (19). Similar scores resulted from comparisons with a putative epimerase encoded in the chain length determinant (cld) region of E. coli (2) and the UDP-galacturonic acid epimerase protein encoded by the cap1J gene of Streptococcus pneumoniae (20). A limited, 22.9% amino acid identity was also found with the S. meliloti ExoB protein which had previously been shown to posses UDP-glucose epimerase activity (6). The second ORF is 1,314 bp in length and starts at nucleotide position 1479 with an ATG initiation codon that is preceded by a putative ribosomal binding site sequence. The predicted protein consists of 437 amino acid residues and has a calculated molecular size of 47,229 Da. This protein exhibits a high degree of similarity to different nucleotide diphospho-sugar dehydrogenases. The highest amino acid sequence identities (more than 50%) were detected with UDP-glucose dehydrogenases identified in other organisms, like a Synechocystis sp. (12). The sequence analysis of the DNA regions flanking the Tn5 insertions (Fig. 3) revealed that only mutations in ORF2 resulted in KPS, 16-3 phage-resistant, and Fix
phenotypes; therefore, the corresponding gene was designated rkpK; mutations in ORF1 affected only the production of LPS,
and this ORF is referred to hereafter as a rhizobial LPS gene,
lpsL.
Operon analysis by plasmid integration mutagenesis.
Sequence
analysis revealed that the two ORFs that are transcribed in the same
direction are separated by 150 bp between the translation stop codon of
lpsL and the ATG initiation codon of rkpK. To
determine whether the transcription of the two ORFs is coupled or
whether rkpK represents a monocistronic transcription unit,
plasmid integration mutagenesis was carried out. To perform this
mutagenesis, the 665-bp HincII fragment, carrying the 3' part of lpsL, the intergenic region with the putative
promoter, as well as the 5' part of rkpK, was cloned into
the pK18 vector in both orientations (AT593 and AT594). These
constructs were then introduced into the AK631 genome as
described in Materials and Methods. The resultant mutants (PP2936 and
PP2937) were sensitive to phage 16-3 and had a Fix+
phenotype, suggesting that KPS production was not affected by these
mutations. Since it is unlikely that in both strains a vector-specific promoter would drive the transcription of the rkpK gene, we
concluded that there is a promoter located in the intergenic region.
These results are in agreement with previous findings where mutations in lpsL induced by either Tn5 insertion or
plasmid integration had no polar effect on rkpK, i.e., the
KPS production in the lpsL mutants was not affected.
RkpK is a UDP-glucose dehydrogenase.
Soluble protein
preparations from the wild-type and different mutant bacteria were
assayed for UDP-glucose dehydrogenase activity as described in
Materials and Methods. The specific enzyme activities measured were
1.82 and 0.97 mU mg1 min1 in the
control strain AK631 and in a plasmid integration mutant of ORF1
(AT578), respectively. In strains carrying the
Tn5-AT231 or Tn5-207 insertion in the
rkpK gene, no UDP-glucose dehydrogenase activity could be
detected. When the complemeting cosmid clone, pAT330, was introduced
into the rkpK mutants, the enzyme activity was restored and
it was about ninefold higher than that of the wild-type strain.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
In this paper, we report on the isolation of novel genomic regions
required for the capsular polysaccharide production of S. meliloti Rm41. This surface component had previously been shown to
be involved in the attachment of the strain-specific bacteriophage 16-3 to the bacterial surface and to be able to replace EPS during symbiotic nodule development (22, 26). In contrast to EPS II, KPS production of S. meliloti is independent of the
presence of a second mutation, like mucR or expR,
as in the case of EPS II (10, 39), and KPS can replace EPS
in the invasion process not only on alfalfa and Melilotus
albus but also on all hosts tested to date (M. sativa
subsp. valria, Medicago media, Medicago truncatula, Melilotus officinalis, and
Trigonella coerulea) (26).
The genetic determinants required for the synthesis of extracellular polysaccharides in S. meliloti have already been identified (4, 11), but much less data are available on those required for KPS production. The rkpA to -J genes in the chromosomal rkp-1 (formerly fix-23) region code for proteins that may participate in the synthesis, modification, and transfer of an as-yet-unknown lipophilic molecule which might serve as a specific lipid carrier during KPS biosynthesis (15, 22), while the rkpZ gene harbored by a megaplasmid affects the degree of KPS polymerization (30). No genes that are involved in the synthesis and polymerization of the precursors, as well as in the export of the mature polymer, have been described so far.
Our earlier observation (26) that mutations in the
rkp-1 region abolished phage 16-3 adsorption offered a
simple way to identify other genetic determinants of KPS synthesis. We
screened for mutants defective in KPS production in a phage-resistant
population obtained after random transposon Tn5 mutagenesis
of S. meliloti AK631. Several novel mutants isolated in
this way were shown to be defective in both KPS production and nodule
invasion. The mutations affected two novel rkp gene clusters
that were designated the rkp-2 and rkp-3 regions.
Genetic analysis of the rkp-2 region revealed the presence
of two ORFs which are transcribed in the same direction. The phenotypes of the different Tn5 and plasmid integration mutants
indicated that transcription of these ORFs is independent, i.e., they
form monocistronic transcription units. Interestingly, the analysis of
the mutants of this region showed that while both genes are required
for normal LPS production, only the second ORF (rkpK) is
involved in the synthesis of KPS. The predicted protein products of the two ORFs exhibited a high degree of similarity to enzymes involved in the metabolism of nucleotide diphospho-sugars.
The product of ORF1 (lpsL) showed a high degree of homology
to epimerases encoded by the capI and cap1J genes
of Staphylococcus aureus and Streptococcus
pneumoniae, respectively. These epimerases are involved in the
formation of UDP-galacturonic acid (derivatives) (19, 20).
RkpK had a high degree of identity with different UDP-glucose dehydrogenases. By using a biochemical assay, we demonstrated that RkpK
functions as a UDP-glucose dehydrogenase. Our results suggest
that these two enzymatic activities are probably required for the
formation of UDP-galacturonic acid from UDP-glucose, which is the
source of galacturonic acid during LPS synthesis. The disaccharide repeating unit of the KPS in S. meliloti Rm41 is
composed of 3,5,7,9-tetradeoxy-5,7-diamino-2-nonulonosic acid and of an
as-yet-undetermined 4-deoxy-4-amino-hexuronic acid which might be
4-deoxy-4-amino-glucuronic acid (27). Based on the
biochemical data obtained here as well as on previous reports, we
concluded that the biosynthesis of the latter component involves enzymatic steps catalyzed by the RkpK protein as well as by the protein
products of different exo genes (Fig.
4). As the first step,
glucose-6-phosphate is converted to glucose-1-phosphate by the
phosphoglucomutase enzyme encoded by the exoC gene. The mutation in this gene affects the synthesis of most of the rhizobial polysaccharides (EPS I, EPS II, LPS, and cyclic -glucan) including KPS, since, in contrast to other exo mutants of
S. meliloti Rm41, the exoC mutant is
Fix, indicating the lack of KPS (5). The
reversible conversion of glucose-1-phosphate to UDP-glucose is
catalyzed by the UDP-glucose pyrophosphorylase encoded by the
exoN gene. Since the exoN mutation does not
inhibit EPS production completely, a second enzymatic activity that can
replace ExoN must be present in S. meliloti (3). Thus, the amount of KPS produced by an exoN
mutant is expected to be somewhat smaller than that of the wild type.
UDP-glucose is oxidized by the RkpK protein to form UDP-glucuronic
acid, which is in turn converted to the aminohexuronic acid by enzymes
that might be encoded in the rkp-3 region.
|
In contrast to the results with the rkp-2 region, the random mutagenesis experiment resulted in more mutations in the rkp-3 region and the mutations are situated within a relatively large DNA region covered by two cosmid clones. These results suggest that the rkp-3 region may carry the majority of the genes necessary for the biosynthesis of the disaccharide subunits of KPS. Our preliminary Tn5 mutagenesis and sequencing results support this idea. The K antigens of rhizobia determined so far show strain-specific variation, although they possess a common structural motif (9), suggesting that several rkp genes in the newly identified regions are probably strain specific and uncommon even in the different S. meliloti isolates. The structure of KPS and the presence of different rkp genes in the microsymbiont may be important determinants of the successful invasion of a given host plant.
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ACKNOWLEDGMENTS |
|---|
We are grateful to E. Sárai and Z. Liptai for skillful technical assistance.
This work was supported by grant OTKA T 016674 and by the U.S.-Hungarian Science and Technology Joint Fund no. 513.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary. Phone: 36 62 432 232. Fax: 36 62 433 503. E-mail: putnoky{at}everx.szbk.u-szeged.hu.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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