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J Bacteriol, January 1998, p. 395-399, Vol. 180, No. 2
Lehrstuhl für Genetik, Fakultät
für Biologie, Universität Bielefeld, 33501 Bielefeld,
Germany
Received 11 August 1997/Accepted 6 November 1997
The production of the acidic exopolysaccharide succinoglycan (EPS
I) by Rhizobium meliloti exoP* mutants expressing an ExoP protein lacking its C-terminal cytoplasmic domain and by mutants characterized by specific amino acid substitutions in the proline-rich motif
(RX4PX2PX4SPKX9IXGXMXGXG)
located from positions 443 to 476 of the ExoP protein was analyzed. The
absence of the C-terminal cytoplasmic ExoP domain (positions 484 to
786) and the substitution of both arginine443 by
isoleucine443 and proline457 by
serine457 within the proline-rich motif resulted in
enhanced production of low-molecular-weight (LMW) EPS I at the expense
of high-molecular-weight (HMW) EPS I. The ratios of HMW to LMW EPS I of
the wild type and mutant strains increased with osmolarity.
The gram-negative soil
bacterium Rhizobium meliloti SU47 (Sinorhizobium
meliloti SU47) and its derivative strains Rm2011 and Rm1021
produce the acidic exopolysaccharide succinoglycan (EPS I), which plays
an important role in the invasion of Medicago sativa root
nodules by R. meliloti (26). EPS I is a polymer of octasaccharide repeating units. Each repeating unit contains seven
glucose molecules and one galactose molecule joined by Twenty-one exo and 2 exs genes involved in the
biosynthesis of EPS I are located in a 27-kb gene cluster on
megaplasmid 2 (7, 26). The combination of genetic and
biochemical approaches allowed the assignment of functions to most of
the exo gene products and resulted in a detailed model for
the biosynthesis of the EPS I repeating unit (26, 31). The
membrane-associated proteins ExoP, ExoQ, and ExoT were determined to be
involved in polymerization and export of EPS I (6, 31).
Recently, González et al. (21) reported evidence that
the ExoQ protein is involved in the biosynthesis of HMW EPS I, whereas
ExoT was suggested to be involved in the synthesis of EPS I
octasaccharide trimers and tetramers. Moreover, ExoP was found to be
essential for the synthesis of HMW and LMW EPS I octasaccharide
multimers (21).
ExoP consists of 786 amino acids and can be divided into an N-terminal
domain (positions 1 to 481), mainly located in the periplasm, and a
C-terminal cytoplasmic domain (positions 482 to 786) (6).
The C-terminal cytoplasmic domain contains a putative ATP binding motif
(6). R. meliloti Rm2011 exoP* mutants
characterized by a deletion of the 3' portion of exoP
encoding the C-terminal cytoplasmic domain produced a reduced amount of
EPS I. In addition, the ratio of HMW to LMW EPS I secreted by these
mutants was decreased (6).
Similarities of the N-terminal domain of ExoP to CLD (Rol and
Wzz) proteins involved in the determination of O-antigen chain length
(33) together with the phenotype of exoP* mutants
suggested that ExoP might be involved in regulating the size
distribution of EPS I (6). Although the amino acid sequences
of the N-terminal ExoP domain and the similar proteins were only weakly
conserved, these proteins displayed structural similarities, since they
were characterized by two putative transmembrane helices and a
conserved proline-rich amino acid motif located on the periplasmic
side, very close to the second membrane helix (2, 6, 28,
32). Bastin et al. (2) proposed that the regulation of
O-antigen chain length by CLD proteins might involve the interaction of these proteins with the O-antigen polymerase. They indicated that the
proline-rich segment of CLD proteins together with conserved glycine
residues of the putative second transmembrane helix may be involved in
a protein-protein interaction of this region with an integral membrane
protein. To date no experimental evidence for the functional
significance of the conserved proline-rich segment has been reported.
In this study, we analyzed the size distribution of EPS I produced by
R. meliloti exoP* mutants lacking the C-terminal cytoplasmic ExoP domain and mutants characterized by specific amino acid
substitutions in the proline-rich motif of ExoP.
Production of HMW EPS I by R. meliloti exoP* is induced
by increasing the osmotic pressure.
Sodium chloride-induced
variations of HMW and LMW EPS I production by the R. meliloti wild-type strain Rm2011 (14) and mutant Rm
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Specific Amino Acid Substitutions in the
Proline-Rich Motif of the Rhizobium meliloti ExoP Protein
Result in Enhanced Production of Low-Molecular-Weight Succinoglycan at
the Expense of High-Molecular-Weight Succinoglycan
ABSTRACT
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-1,4, -1,3, and -1,6 glycosidic linkages and can be decorated by
acetyl, succinyl, and pyruvyl groups (1, 29). R. meliloti Rm1021 produces a high-molecular-weight (HMW) and a
low-molecular-weight (LMW) form of EPS I (4). Breedveld et
al. (10) reported that an increase of the osmotic pressure
resulted in enhanced production of HMW EPS I at the expense of LMW EPS
I.
P*1 (6) were analyzed (Table
1; Fig. 1).
Mutant RmP*1 contained the exoP* gene encoding a
truncated ExoP protein that comprises amino acids 1 to 484 instead of
the complete ExoP protein (Fig. 2). In GMS medium (34)
supplemented with sodium chloride to an osmolarity of 0.09 to 0.48 osmol/liter no significant difference between the growth rates of these
two strains was observed. Culture supernatants of the exoP*
mutant RmP*1 contained approximately 10 to 30% of the total amount
of EPS I produced by the wild type (Table 1).
TABLE 1.
NaCl-induced osmotic effects on production of HMW EPS I
and LMW EPS I by R. meliloti Rm2011 mutants characterized by
alterations in the ExoP proteina
View larger version (13K):
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FIG. 1.
HMW EPS I production of different R. meliloti
strains. The HMW EPS I production of the R. meliloti Rm2011
wild-type strain (WT), the exoP* mutant Rm P*1
(exoP*), and strain RmexoP carrying the plasmids
pExoP-R443L (R443L) and pExoP-P457S (P457S) in GMS medium supplemented
with sodium chloride to osmolarities of 0.09 to 0.48 osmol/liter is
shown. The percentage of the HMW EPS I fraction in relation to the
total amount of EPS I is given. Values are averages of at least five
independent experiments. Standard deviations were equal to or less than
4%.
P*1 produced
approximately 50% HMW EPS I in relation to the total amount of EPS I.
To verify that EPS I was exclusively secreted by the R. meliloti strains under the growth conditions used, the glucose and galactose contents of the EPS fractions were quantified by enzymatic assays (9, 25) after hydrochloric acid hydrolysis and
neutralization. All EPS fractions tested contained glucose and
galactose in ratios of 6.9:1 to 7.1:1, indicating that these fractions
solely contained EPS I.
The osmotically induced production of HMW EPS I by
exoP* mutants exclusively expressing the N-terminal ExoP
domain indicates that the C-terminal cytoplasmic ExoP domain and
therefore the putative binding and hydrolysis of ATP are not essential
for HMW EPS I production. In addition, the C-terminal domain is not
essential for the osmotically induced changes in the ratio of HMW to
LMW EPS I. If ExoP plays a role in the osmotically induced alteration of this ratio, the presence of the N-terminal ExoP domain is sufficient to promote this change. High salt concentrations may influence the
biosynthesis or the export of HMW or LMW EPS I. This influence may be
exerted on the level of protein expression or stability.
Substitution of specific amino acid residues in the proline-rich
motif of the ExoP protein.
To analyze the relevance of the
proline-rich segment of the R. meliloti Rm2011 ExoP protein,
R. meliloti Rm2011 mutants expressing ExoP proteins
characterized by specific amino acid substitutions in this segment were
constructed. These substitutions were localized in the periplasmic
portion of the ExoP protein (Fig. 2D).
The exoP mutant genes were expressed in an R. meliloti
exoP deletion mutant to exclude interference of ExoP proteins
encoded by the endogenous gene and ExoP proteins encoded by the
exoP mutant genes. exoP mutants are usually
characterized by a slow growth rate, which is most likely due to an
accumulation of lipid-linked intermediates of the EPS I biosynthetic
pathway (30). Consistent with this observation, R. meliloti mutants lacking the exoP gene grew slowly and
were not viable in media of low osmolarities (<0.1 osmol/liter) (data
not shown). Therefore, mutant RmexoP, characterized by a deletion
comprising 40 nucleotides of the 3' terminus of the exoN
coding region, the intergenic region between exoN and
exoP, the complete exoP coding region, and 262 nucleotides downstream of the exoP coding region, was
constructed (Fig. 3). The spectinomycin resistance cassette derived from pHP45
(16) was inserted
into the NcoI site located 25 nucleotides downstream of the
deletion site. Mutant RmexoP displayed a growth rate comparable to
the growth rate of the wild type and grew in media of low osmolarities. Mutations in exoN cause a reduction of EPS I production,
since this gene encodes a UDP-glucose pyrophosphorylase that
participates in the synthesis of the nucleotide sugar precursors for
EPS I biosynthesis (5, 20). In exoN mutants the
activity of another pyrophosphorylase that can substitute for ExoN may
account for the synthesis of enough UDP-glucose to produce the reduced
amount of EPS I. Therefore, the additional mutation in the
exoN gene probably reduced the deleterious accumulation of
EPS I biosynthetic intermediates induced by the mutation of
exoP.
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exoP by 
M12-mediated
transduction (17). The resulting mutant, RmPII15, and
mutant RmexoP were used as recipient strains to analyze the effects
of alterations in the proline-rich motif of ExoP on the production of
EPS I.
Seven independent base pair substitutions affecting the amino acid
sequence of the proline-rich segment of ExoP were introduced into
plasmid pSDM-P. This plasmid resulted from the insertion of the
internal 876-bp XhoI-EcoRI fragment of
exoP into the vector pHIP2 (8). A mutagenic
primer (Table 2) carrying the mutation to
be inserted into the exoP gene and a selective primer (Table 2) characterized by a substitution that eliminates the internal EcoRV site of the aacC1 gene of pSDM-P were
simultaneously annealed to one strand of the denaturated plasmid. After
completion of the second strand, using T7 DNA polymerase and T4 DNA
ligase, nonmutated plasmids were excluded from transformation due to
linearization by EcoRV restriction. Plasmids were introduced
into Escherichia coli XLmutS (Stratagene) by transformation,
and nonmutated plasmids were again eliminated from plasmid DNA isolated
from the pool of transformants by EcoRV restriction. After
transformation of E. coli XL1-Blue (12) with this
plasmid pool, single clones were tested for the desired mutation in
exoP by DNA sequencing. Subsequently, the 371-bp
BglII-EcoRI wild-type fragment of the exoP gene of pExoP was replaced by the corresponding
fragment of pSDM-P carrying the base pair substitution. Mutations in
exoP carried by pExoP derivative plasmids were verified by
DNA sequencing. Plasmid pExoP carried the fragment deleted from the
exoP-exoN region of strains RmexoP and RmPII15.
Additionally, it contained the first part of the exoN gene
and a part of the 3' terminus of the exoO coding region
(Fig. 3). Therefore, integration of pExoP containing the wild-type
exoP gene and its derivative plasmids carrying the
exoP mutant genes into the genomes of RmexoP and RmPII15 by homologous recombination occurred upstream of the deletion site, thereby restoring the native structure of the
exoN-exoP region (Fig. 3). The genomic structures of the
exoO-exoN-exoP regions of the mutants were verified by
Southern hybridization. Since ExoP may interact with other proteins
involved in EPS I biosynthesis, the copy number of exoP and
the regulation of exoP gene expression is probably critical
for EPS I production. The effect of specific amino acid substitutions
in ExoP was therefore analyzed using the R. meliloti strains
which carried a single exoP mutant gene at the native site
in the genome.
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Size distribution of succinoglycan produced by strains characterized by specific mutations in the proline-rich motif of the ExoP protein. Since the osmotic pressure of the culture medium influences the ratio of HMW to LMW EPS I produced by R. meliloti (10), the size distributions of EPS I produced by the wild-type Rm2011, the expA1 mutant RmAR1015, and the strains characterized by specific mutations in the proline-rich motif of ExoP in media of various osmolarities were compared. After cultivation of the merodiploid R. meliloti strains the presence of the plasmid carrying the various exoP genes was verified by comparison of the total cell titer to the titer of neomycin-resistant cells. In all cases the total cell number corresponded to the number of neomycin-resistant cells. This indicates that during cultivation the plasmid was not lost in a significant number of cells by homologous recombination.
As a control, the EPS I production of the wild-type Rm2011 and the expA1 mutant RmAR1015 was compared to that of the exoP deletion mutant RmexoP and the exoP
deletion-expA1 mutant RmPII15, both carrying plasmid
pExoP with the wild-type exoP gene. No significant differences between the growth rates, the total amount of EPS I in the
culture supernatant, and the ratio of HMW to LMW EPS I secreted to the
medium were observed for the wild-type Rm2011 and RmexoP carrying
pExoP (Table 1) or for RmAR1015 and RmPII15 containing pExoP (data
not shown). In addition, all EPS fractions obtained from culture
supernatants of these strains contained glucose and galactose in the
ratios of 6.9:1 to 7.1:1, indicating that the sole exopolysaccharide in
these fractions was EPS I. These results show that the mutants
RmexoP and RmPII15 in combination with derivative plasmids of
pExoP containing exoP mutant genes can be used to analyze
the effects of the different mutations in exoP on the
production of EPS I.
Compared to strains RmexoP and RmPII15, containing an
integrated wild-type copy of exoP, these strains displayed
no significant differences in growth rate and the total amount of EPS I
isolated from the culture supernatants when carrying the
exoP mutant genes. The ratios of glucose to galactose of
6.9:1 to 7.1:1 determined for the HMW and LMW EPS I fractions of these
strains indicated that EPS I was the sole exopolysaccharide in these
fractions.
Virtually the same ratio of HMW to LMW EPS I was determined for
RmexoP carrying pExoP plasmids with exoP mutant genes as for RmPII15 carrying the identical plasmids. With respect to the
ratio of HMW to LMW EPS I, these strains could be grouped into two
classes. Substitution of arginine443 by
leucine443 (R443L) and proline457 by
serine457 (P457S) resulted in enhanced production of LMW
EPS I at the expense of HMW EPS I (Table 1, Fig. 1). On the other hand,
strains carrying the mutation R443L or P457S produced more HMW EPS I
than did mutant RmP*1 (Fig. 1) and as much EPS I as the wild type
(Table 1). The phenotype of these mutants therefore supports the
hypothesis that the reduced EPS I production by exoP*
mutants results from alterations in the stability or the amount of
either exoP* transcripts or the N-terminal ExoP domain.
These alterations might be explained either by the absence of the part
of the exoP gene encoding the C-terminal domain or by the
absence of this domain.
In contrast to the substitutions R443L and P457S, substitutions of
proline451 by serine451 (P451S),
proline454 by serine454 (P454S),
serine456 by asparagine456 (S456N),
lysine458 by methionine458 (K458M), and
lysine459 by isoleucine459 (K459I) resulted in
a ratio of HMW EPS I to LMW EPS I similar to that determined for the
wild type (data not shown).
Two of seven amino acid substitutions in the proline-rich motif of ExoP
affected EPS I production, indicating that most of the conserved
residues of this motif can be exchanged without influencing the
function of ExoP. This is in accordance with the observation that in
the periplasmic portion of ExoP and the homologous proteins identified,
only one residue of the proline-rich motif is invariant. Interestingly,
the exchange of this invariant residue, proline457, with
serine457 (P457S), which is located within the highly
conserved central SPK motif (Fig. 2), resulted in enhanced production
of LMW EPS I at the expense of HMW EPS I. Essentially the same effect
resulted from the exchange of arginine443 with leucine443 (R443L). The positively charged
arginine443 is highly conserved in the proteins involved in
lipopolysaccharide, enterobacterial common antigen, and
exopolysaccharide biosynthesis but not in proteins involved in the
production of capsular polysaccharides.
The cytoplasmic localization of the C-terminal ExoP domain is
not affected by the mutations R443L and P457S.
By analyzing
translational fusions to reporter genes, we demonstrated that the
C-terminal domain of ExoP is located in the cytoplasm (6).
To test whether the mutations R443L and P457S, which caused enhanced
production of LMW EPS I, affected the cytoplasmic localization of the
C-terminal ExoP domain, a lacZ reporter gene was inserted
into plasmids pExoP, pExoP-R443L, and pExoP-P457S, resulting in
translational fusions to the exoP genes. The fusion site of
the ExoP proteins and the LacZ monitor protein was located at amino
acid position 773 of the ExoP proteins in their cytoplasmic domains.
R. meliloti RmexoP carrying plasmid pExoP, pExoP-R443L, or pExoP-P457S with the exoP-lacZ translational fusions
displayed -galactosidase activities of 80 to 86 U. These activities
correspond to the activity previously determined for a strain carrying
the LacZ monitor protein fused to amino acid 773 of the wild-type ExoP
protein (6). Since the LacZ monitor protein displays
-galactosidase activity only when fused to cytoplasmic portions of
membrane proteins (22), these results indicate that the
amino acid substitutions R443L and P457S did not affect the cytoplasmic
localization of the C-terminal ExoP domain.
-galactosidase activities mediated by ExoP-R443L-LacZ,
ExoP-P457S-LacZ, and wild-type ExoP-LacZ fusion proteins argue against
an alteration of the amount or the stability of the ExoP protein and in
favor of structural alterations of the protein, although it cannot be
excluded that the lacZ fusion stabilized either the
exoP mRNA or the ExoP-LacZ fusion protein. The cytoplasmic
localization of the C-terminal domain of the ExoP-R443L and ExoP-P457S
mutant proteins indicate that a structural alteration of the ExoP
protein would not involve the destruction of its second transmembrane
helix.
Conclusions. We were able to show that specific mutations within a proline-rich segment of ExoP which is conserved in a number of proteins involved in polysaccharide biosynthesis can drastically alter the ratio of HMW to LMW EPS I. This supports our previous proposition that ExoP influences the ratio of HMW to LMW EPS I in R. meliloti. This ratio may be altered by influencing the biosynthesis or the export of HMW or LMW EPS I. This may include the release of EPS I polymerization products from the polymerization apparatus.
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ACKNOWLEDGMENTS |
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This work was supported by grant Pu 28/19-1 from Deutsche Forschungsgemeinschaft.
We thank J. E. González for sharing unpublished results and H. Küster for critical reading of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany. Phone: 49 521-106-5607. Fax: +49 521-106-5626. E-mail: hippo{at}genetik.uni-bielefeld.de.
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REFERENCES |
|---|
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Top
Abstract
Text
References
|
|---|
| 1. | Aman, P., M. McNeil, L. Franzen, A. G. Darvill, and P. Albersheim. 1981. Structural elucidation using HPLC-MS and GLC-MS of the acidic polysaccharide secreted by Rhizobium meliloti strain 1021. Carbohydr. Res. 95:263-282. |
| 2. | Bastin, D. A., G. Stevenson, P. K. Brown, A. Haase, and P. R. Reeves. 1993. Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosome and fatty acid synthetase, with a novel mechanism for determining chain length. Mol. Microbiol. 7:725-734[Medline]. |
| 3. |
Batchelor, R. A.,
P. Alifano,
E. Biffali,
S. I. Hull, and R. A. Hull.
1992.
Nucleotide sequences of the genes regulating O-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: protein homology and functional complementation.
J. Bacteriol.
174:5228-5236 |
| 4. |
Battisti, L.,
J. C. Lara, and J. A. Leigh.
1992.
Specific oligosaccharide form of the Rhizobium meliloti exopolysaccharide promotes nodule invasion in alfalfa.
Proc. Natl. Acad. Sci. USA
89:5625-5629 |
| 5. | Becker, A., A. Kleickmann, W. Arnold, M. Keller, and A. Pühler. 1993. Identification and analysis of the Rhizobium meliloti exoAMONP genes involved in exopolysaccharide biosynthesis and mapping of promoters located on the exoHKLAMONP fragment. Mol. Gen. Genet. 241:367-379[Medline]. |
| 6. | Becker, A., K. Niehaus, and A. Pühler. 1995. Low molecular weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal and a missing C-terminal domain. Mol. Microbiol. 16:191-203[Medline]. |
| 7. | Becker, A., H. Küster, K. Niehaus, and A. Pühler. 1995. Extension of the Rhizobium meliloti succinoglycan biosynthesis gene cluster: identification of the exsA gene encoding an ABC transporter protein, and the exsB gene which probably codes for a regulator of succinoglycan biosynthesis. Mol. Gen. Genet. 249:487-497[Medline]. |
| 8. |
Becker, A.,
S. Rüberg,
H. Küster,
A. A. Roxlau,
M. Keller,
T. Ivashina,
H.-P. Cheng,
G. C. Walker, and A. Pühler.
1997.
The 32-kb exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products.
J. Bacteriol.
179:1375-1384 |
| 9. | Beutler, H. O. 1984. Lactose and D-galactose: UV-method, p. 104-112. In H. U. Bergmeyer (ed.), Methods of enzymatic analysis, 3rd ed., vol. 6. Verlag Chemie, Weinheim, Germany. |
| 10. | Breedveld, M. W., L. P. T. M. Zevenhuizen, and A. J. B. Zehnder. 1990. Osmotically induced oligo- and polysaccharide synthesis by Rhizobium meliloti SU47. J. Gen. Microbiol. 136:2511-2519. |
| 11. | Bugert, P., and K. Geider. 1995. Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora. Mol. Microbiol. 15:917-933[Medline]. |
| 12. | Bullock, W. C., J. M. Fernandez, and J. M. Short. 1987. XL1-Blue: a high efficient plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. BioTechniques 5:376-379. |
| 13. | Capage, M. A., D. H. Doherty, M. R. Betlach, and R. van der Slice. October 1987. Recombinant-DNA mediated production of xanthan gum. Patent International publication no. WO87/05938. |
| 14. | Casse, F., C. Boucher, J. S. Julliot, M. Michel, and J. Dénarié. 1979. Identification and characterization of large plasmids in Rhizobium meliloti using agarose gel electrophoresis. J. Bacteriol. 113:229-242. |
| 15. | Chaplin, M. F., and S. F. Kennedy. 1986. . Carbohydrate analysis. A practical approach. IRL Press, Washington, D.C. |
| 16. | Fellay, R., J. Frey, and H. Krisch. 1987. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria. Gene 52:147-154[Medline]. |
| 17. |
Finan, T. M.,
E. Hartwieg,
K. LeMieux,
K. Bergman,
G. C. Walker, and E. R. Signer.
1984.
General transduction in Rhizobium meliloti.
J. Bacteriol.
159:120-124 |
| 18. | Frosch, M., U. Edwards, K. Bousset, B. Kraube, and C. Weisgerber. 1991. Evidence for a common molecular origin of the capsule gene loci in gram-negative bacteria expressing group II capsular polysaccharides. Mol. Microbiol. 5:1251-1263[Medline]. |
| 19. | Glazebrook, J., and G. C. Walker. 1989. A novel exopolysaccharide can function in place of the calcofluor-binding exopolysaccharide in nodulation of alfalfa by Rhizobium meliloti. Cell 56:661-672[Medline]. |
| 20. |
Glucksmann, M. A.,
T. L. Reuber, and G. C. Walker.
1993.
Genes needed for the modification, polymerization, export, and processing of succinoglycan by Rhizobium meliloti: a model for succinoglycan biosynthesis.
J. Bacteriol.
175:7045-7055 |
| 21. | González, J. E., G. M. York, and G. C. Walker. 1996. Rhizobium meliloti exopolysaccharides: synthesis and symbiotic function. Gene 179:141-146[Medline]. |
| 22. | Hennessey, E. S., and J. K. Broome-Smith. 1993. Gene-fusion techniques for determining membrane-protein topology. Curr. Opin. Struct. Biol. 3:524-531. |
| 23. | Huang, J., and M. Schell. 1995. Molecular characterization of the eps gene cluster of Pseudomonas solanacearum and its transcriptional regulation at a single promoter. Mol. Microbiol. 16:977-989[Medline]. |
| 24. | Kroll, J. S., B. Loynds, L. M. Brophy, and E. R. Moxon. 1990. The bex locus in encapsulated Haemophilus influenzae: a chromosomal region involved in capsule polysaccharide export. Mol. Microbiol. 4:1853-1862[Medline]. |
| 25. | Kunst, A., B. Draeger, and J. Ziegenhorn. 1984. UV-methods with hexokinase and glucose-6-phosphate dehydrogenase, p. 163-172. In H. U. Bergmeyer (ed.), Methods of enzymatic analysis, 3rd ed., vol. 6. Verlag Chemie, Weinheim, Germany. |
| 26. | Leigh, J. A., and G. C. Walker. 1994. Exopolysaccharides of Rhizobium: synthesis, regulation and symbiotic function. Trends Genet. 10:63-67[Medline]. |
| 27. |
Meier-Dieter, U.,
K. Barr,
R. Starman,
L. Hatch, and P. D. Rick.
1992.
Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of entobacterial common antigen.
J. Biol. Chem.
267:746-753 |
| 28. |
Morona, R.,
L. Van Den Bosch, and P. A. Manning.
1995.
Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri.
J. Bacteriol.
177:1059-1068 |
| 29. |
Reinhold, B. B.,
S. Y. Chan,
T. L. Reuber,
A. Marra,
G. C. Walker, and V. N. Reinhold.
1994.
Detailed structural characterization of succinoglycan, the major exopolysaccharide of Rhizobium meliloti Rm1021.
J. Bacteriol.
176:1997-2002 |
| 30. |
Reuber, T. L.,
S. Long, and G. C. Walker.
1991.
Regulation of Rhizobium meliloti exo genes in free-living cells and in planta by using TnphoA fusions.
J. Bacteriol.
173:426-434 |
| 31. | Reuber, T. L., and G. C. Walker. 1993. Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Cell 74:269-280[Medline]. |
| 32. | Stevenson, G., A. Kessler, and P. R. Reeves. 1995. A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants. FEMS Microbiol. Lett. 125:25-30. |
| 33. | Whitfield, C., P. A. Amor, and R. Köplin. 1997. Modulation of the surface architecture of Gram-negative bacteria by the action of surface polymer:lipid A-core ligase and by determinants of polymer chain length. Mol. Microbiol. 23:629-638[Medline]. |
| 34. | Zevenhuizen, L. P. T. M. 1986. Selective synthesis of polysaccharides by Rhizobium trifolii strain TA-1. FEMS Microbiol. Lett. 35:43-47. |
J Bacteriol, January 1998, p. 395-399, Vol. 180, No. 2
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