Glycobiology Advance Access originally published online on September 6, 2006
Glycobiology 2006 16(12):1181-1193; doi:10.1093/glycob/cwl042
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Overproduction and increased molecular weight account for the symbiotic activity of the rkpZ-modified K polysaccharide from Sinorhizobium meliloti Rm1021
2 Department of Genetics, Biology VI, Bielefeld University, Postfach 100131, 33501 Bielefeld, Germany; and
3 Spectrométrie de Masse, Fluorescence et Chimie Bioanalytique Laboratoire des IMRCP 118, Route de Narbonne, F-31062 Toulouse-Cedex, France
1 To whom correspondence should be addressed; e-mail: larissa{at}genetik.uni-bielefeled.de
Received on April 12, 2006; revised on August 28, 2006; accepted on August 31, 2006
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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K polysaccharides (KPSs) of Sinorhizobium meliloti strains are strain-specific surface polysaccharides analogous to the group II K antigens of Escherichia coli. The KR5 antigen of strain AK631 is a highly polymerized disaccharide of pseudaminic and glucuronic acids. During invasion of host plants, this K antigen is able to replace the structurally different exopolysaccharide succinoglycan (EPS I) and promotes the formation of a nitrogen-fixing (Fix+) symbiosis. The KPS of strain Rm1021 is a homopolymer of 3-deoxy-D-manno-2 octulosonic acid (Kdo). The Kdo polysaccharide is covalently linked to the lipid anchor, has a low molecular weight (LMW), and is symbiotically inactive. On introduction of the Rm41-specific rkpZ gene into strain Rm1021, a modified KPS is expressed that is able to substitute EPS I during symbiosis with the host plant. To better understand the nature of modification conferred by rkpZ, we performed a structural analysis of the KPS using nuclear magnetic resonance (NMR), electrospray ionization–mass spectrometry (ESI–MS), and gas chromatography (GC–MS). The modified KPS retained primary polyKdo structure, but its degree of polymerization (DP) and level of production were increased significantly. In contrast to the wild-type polyKdo, only a part of polyKdo was lipidated. Shorter polysaccharide chains were lipid-free, whereas longer polysaccharide chains were lipidated. Sinorhizobium meliloti Rm1021 was found to carry two paralogs of rkpZ. Both genes are involved in polyKdo production, but they only show partial functional activity as compared with the rkpZ of Rm41.
Key words: electrospray ionization–mass spectrometry / host invasion / lipid anchor / rkpZ / surface polysaccharide
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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Sinorhizobium meliloti is a Gram-negative soil bacterium that elicits nitrogen-fixing nodules on the roots of alfalfa and related leguminous plants. Sinorhizobium meliloti has the capacity to produce various cell-surface polysaccharides, including lipopolysaccharide (LPS), capsular polysaccharide (K antigen and K polysaccharide [KPS]), two exopolysaccharides (EPS I and EPS II), and cyclic glucans. In contrast to other cell-envelope components, the structure of S. meliloti K antigen is strain specific (Reuhs et al., 1998
). Up to now, the genetics and structure of KPS biosynthesis were only investigated in S. meliloti strain Rm41 and its derivative AK631 (Kiss et al., 2001). Little is known about KPS of S. meliloti strain Rm1021. However, this strain deserves special attention, because its whole genome sequence is available (Galibert et al., 2001), and it is a symbiotic partner of the model legume plant Medicago truncatula.
K antigens of S. meliloti and S. fredii typically comprise highly polymerized disaccharide repeating units of a hexose and 3-deoxy-D-manno-2 octulosonic acid (Kdo) or another 1-carboxy-2-keto-3-deoxy sugar (Reuhs et al., 1993, 1998). These surface polysaccharides are structurally analogous to group II K antigens of Escherichia coli (Reuhs et al., 1993). Sinorhizobium meliloti strain Rm41 produces KR5 antigen consisting of disaccharide repeating units composed of glucuronic and pseudaminic acids, which are decorated by butyryl and acetyl residues (Reuhs et al., 1993, 1998). K antigen of Rm41 can be easily visualized in sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) or deoxycholate (DOC)–PAGE using Alcian blue silver staining. The KR5 antigen is symbiotically active and is able to replace EPS I during infection of alfalfa nodules (Williams et al., 1990; Reuhs et al., 1995). Thus, an exoB mutant of Rm41 (strain AK631), which does not produce EPS I but KPS, induces pink nitrogen-fixing nodules on the roots of its host plants.
Early studies performed on an exoB mutant of S. meliloti Rm1021 using size exclusion chromatography led to the conclusion that this strain produces high-molecular-weight (HMW) Kdo-rich KPS (Reuhs et al., 1995). Recent studies of S. meliloti Rm1021 surface polysaccharides using mass spectroscopy identified only low-molecular-mass KPS (Fraysse et al., 2005). It is a homopolymer of Kdo covalently linked to the lipid anchor. The degree of polymerization (DP) ranges from 10 to 20. KPS of Rm1021 is symbiotically inactive, because exopolysaccharide-deficient (exo–) mutants of Rm1021, in contrast to those of Rm41, are unable to form a nitrogen-fixing symbiosis with the host plant alfalfa (Niehaus and Becker, 1998). The symbiotic deficiency of exoB mutants of Rm1021 could be partially suppressed by plasmid pMW23 carrying the rkpZ gene (former lpsZ) from strain Rm41 (Williams et al., 1990; Reuhs et al., 1995). This gene is absent in the genome of Rm1021 (Williams et al., 1990). Inactivation of the rkpZ gene in AK631 resulted in a Fix– phenotype, altered phage sensitivity, as well as altered size distribution and reduced affinity of KPS to nitrocellulose (Williams et al., 1990; Reuhs et al., 1995). RkpZ was annotated as a chain-length determination protein. However, considering the alterations in phage sensitivity and the fact that HMW KPS exhibits a reduced affinity to nitrocellulose, the role of RkpZ in KPS expression may be more complex than modifying the size range. In case of the Rm1021 exoB mutant, the expression of RkpZ from Rm41 was associated with partial restoration of the symbiotic phenotype, changes in phage sensitivity, and size modification of the KPS. Moreover, the modified KPS showed reactivity against a polyclonal antiserum recognizing KR5 antigen of Rm41 (Reuhs et al., 1995). Thus, it seemed likely that rkpZ-mediated modification involved not only the size but the structure of the KPS as well.
To resolve this issue, we analyzed the structure of the modified KPS and studied the symbiotic suppression in more detail. We demonstrate here that no modifications of the primary structure were observed in the KPS of Rm1021 on introduction of a plasmid carrying the heterologous rkpZ gene. However, quite a number of changes in the mode of expression of polyKdo were detected. These included an increased rate of polymerization, increased level of production, and reduced lipidation. In addition, we report here that two rkpZ paralogs present in S. meliloti Rm1021 are involved in KPS production but cannot promote such high level of polymerization and surface expression as rkpZ from Rm41.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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Expression of pMW23 in S. meliloti SU47 background leads to overproduction and an increased DP of polyKdo
Plasmid pMW23 is a medium-copy-number plasmid that carries a 4-kb genomic fragment from Rm41 including rkpZ, rkpS, and a part of rkpT (Figure 6). In contrast to rkpZ, rkpS and rkpT are not required for the expression of KR5 antigen (Kiss et al., 2001
). Previously, the ability of pMW23 to suppress the symbiotic deficiency of an EPS– mutant was studied in an exoB derivative of strain Rm1021 (Williams et al., 1990; Reuhs et al., 1995). The Rm1021 strain is closely related to strain Rm2011, and both of them represent spontaneous streptomycin-resistant mutants of the native S. meliloti strain SU47. In many respects they are identical. However, recently, certain differences were found in the ability to interact with host plants (Wais et al., 2002) and in gene expression and protein profiles (Djordjevic et al., 2003; Krol and Becker, 2004). Therefore, we analyzed the effect of pMW23 on K antigen expression not only in Rm1021 but also in Rm2011. As exopolysaccharide-deficient recipients, exoY instead of exoB mutants were used, because the latter is known to have pleiotropic phenotypes (Niehaus and Becker, 1998). This is connected with the involvement of exoB in the biosynthesis of a sugar nucleotide precursor required for biosynthesis of EPS I, EPS II, and LPS (Niehaus and Becker, 1998). An exoY mutation exclusively blocks the expression of EPS I, because exoY encodes the first glycosyltransferase of the EPS I biosynthesis pathway (Niehaus and Becker, 1998). To check if the pleiotropic nature of an exoB mutation had an influence on pMW23-conditioned phenotypes, we transferred pMW23 also to an Rm2011 exoB mutant.
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SDS–PAGE analysis of ethylenediaminetetraacetic acid–triethylamine (EDTA–TEA) cell extracts obtained from transconjugants carrying pMW23 revealed the presence of a high amount of KPS (Figure 1). It appeared as a homogenous smear extending from the band of R-LPS to the upper edge of the gel, indicating that the size of this KPS varied from low-molecular-weight (LMW) form to HMW form. The most intensive Alcian blue staining was observed around the band of S-LPS, in the middle-molecular-weight (MMW) range (Figure 1). As expected, the exoB mutant had an altered LPS pattern. However, its KPS looked the same as that of the exoY mutant and of the wild-type strain. No differences conditioned by the Rm1021 or Rm2011 genetic background were found.
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To characterize polysaccharide(s) produced by the transconjugants, crude polysaccharide extracts from strains Rm2011exoY/pMW23, Rm1021exoY/pMW23, and 2011 exoB/pMW23 were analyzed using 1H NMR and 1H-13C correlation spectroscopy. Each spectra showed the presence of Kdo. However, the obtained data were not sufficient for the unambiguous determination of the linkage. Therefore, the HMW fraction of the crude extract was purified from L839, employing S300 size exclusion chromatography. This fraction was subjected to dialysis and extraction steps (to eliminate DOC present in the eluent). The purity of this fraction was checked using 1H NMR. As the spectrum exhibited mostly sugar-like signals, 1H–13C heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments were done. HSQC allowed the assessment of the 1H and 13C chemical shifts of each carbohydrate position. All measured values are very close to those published by Fraysse and others (2005)
(Table I). The 
> 0.5 ppm between the two H3 bands attests the ß pyranoside conformation of the Kdo subunit (Kohlbrenner and Fesik, 1985). The 13C value of C7 appeared quite downfield (70.92 ppm) but not enough to demonstrate that position 7 is involved in the osidic junction. Therefore, a HMBC experiment was performed. Determination of J2 and mostly J3 correlations between 1H and 13C allowed the sequencing of the sugar subunit and demonstrated clearly that H7 is a neighbor to C8 and C6 but also to C2 at 100.3 ppm (Figure 2). This correlation unambiguously supports a 2–7 linkage of the Kdo subunits within the HMW KPS. As C2 correlates only with H7 and H3, it can be concluded that the osidic junction is exclusively ß2–7.
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pMW23-modified KPS comprises a mixture of lipidated and non-lipidated polyKdo
As recently reported, S. meliloti strain Rm1021 produces lipidated polyKdo (Fraysse et al., 2005
). In this study, we characterized KPS of Rm2011 using electrospray ionization–mass spectrometry (ESI–MS). Figure 3A shows MaxEnt3 deconvolution of the multicharged spectrum obtained from Rm2011 crude extract. The KPS was resolved as a series of ion peaks with a mass difference of 220 amu. This distribution corresponds to KPS molecules containing a lipid anchor and an increasing number of Kdo residues, up to 22. The lipid anchor was identified as a negatively single-charged molecular ion at mass/charge (m/z) of 622. It was shown to be a phospholipid (Fraysse et al., 2005). It should be noted that in the LMW range, where oligoKdo comprises less than seven sugar residues, lipidated oligoKdo was accompanied by small amounts of lipid-free oligoKdo (Fraysse et al., 2005). The same was observed in this study for strain Rm2011 (data not shown). Possibly, these short Kdo chains resulted from partial sample degradation during ESI–MS analysis.
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ESI–MS analysis of the crude extract from strain Rm2011exoY/pMW23 also revealed a series of ion peaks with a mass difference of 220 amu (Figure 3B and C). However, these peaks corresponded exclusively to lipid-free polyKdo. ESI–MS was able to detect single-, double-, and triple-charged molecular ions (insert in Figure 3B). As in case of Rm1021 and Rm2011, free oligoKdo with a DP < 7 may result from source degradation. However, molecules with a higher DP most likely represent native size variation of free polyKdo. This may be inferred from the observation that the corresponding molecular ions have gaps in the regularity of the distribution and do not show a typical degradation profile. Similar spectra were obtained for three other strains carrying plasmid pMW23. These results are summarized in Table II.
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To preserve the integrity of larger KPS molecules, we decreased cone and Rf voltages in ESI–MS. Using an MS resolution of 20,000, molecules bearing up to 12 charges in the range from 500 to 2000 m/z were detected. As in the case of smaller molecules (Figure 3D), no 622 m/z shift was observed on the deconvoluted molecular masses (data not shown). To test whether lipidated KPS escaped detection by MS, we subjected the crude polysaccharide extract to chromatography using polymyxin B. It is known that this cationic peptide has affinity not only to lipid A but also to anionic phospholipids (Teuber and Bader, 1976
). As expected, LMW polyKdo expressed by Rm1021 and Rm2011 was almost entirely retained on the polymyxin B phase. When the modified KPS was tested, a significant proportion of KPS, especially in the MMW and HMW size range, was affinity-bound to polymyxin B (data not shown). This finding provided evidence for the presence of a lipid anchor in the modified KPS.
In the search for the lipid anchor, we performed mild acid hydrolysis of polysaccharide extracts from Rm2011/pMW23 with 1% acetic acid. This was done to cleave the bond between the saccharide subunit and the lipid anchor. In addition, acid hydrolysis released lipid A from LPS that was also present in our crude extracts. The negative-ion mass spectrum of the lipid fraction displayed quasi exclusively ion peaks at m/z 622.54 and 594.4 (Figure 3D). Both of them corresponded to the lipid anchor of KPS (Fraysse et al., 2005). Ion peaks representing the lipid A moiety of LPS were small compared with the peak at m/z 622.54, indicating that the latter lipid was present at a higher amount. An increased production of the lipid anchor was found in all other S. meliloti strains harboring plasmid pMW23 (Table II). Although the signal of the lipid 622 was very strong, its total amount constituted only 1–5% of the crude extract. This estimation was made as described in Materials and methods.
To identify which saccharidic moiety was attached to the lipid anchor, we separated the polysaccharide crude extract by size exclusion chromatography on a Sephacryl S300 column (Figure 4A). This allowed the purification of HMW, MMW and LMW forms of KPS. Following acid hydrolysis and lipids extraction with chloroform, lipids were subjected to ESI–MS analysis. Peaks at m/z 579 and 622 corresponding to the lipid anchor were detected in all three fractions (Figure 4B–D). In the case of HMW polysaccharide, except for residual DOC, only lipid anchor was detected (Figure 4B). In the case of MMW and LMW polysaccharide, in addition to the lipid anchor, many other lipids were detected, including fatty acids derived from lipid A and phospholipids (Figure 4C and D). Gas chromatography (GC–MS) analysis of the saccharidic part of the purified polysaccharide samples proved that they contained only Kdo (Figure 4E). To summarize, the modified KPS comprised a mixture of lipidated and non-lipidated polyKdo. The latter was much more abundant in the LMW fraction. Thus, the failure to detect lipidated molecular ions in ESI–MS may be related to their signal suppression by well-responding lipid-free molecular species prevailing in the LMW range.
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pMW23-mediated suppression of the Fix– phenotype of S. meliloti exopolysaccharide-deficient derivatives of SU47
Sinorhizobium meliloti exopolysaccharide-deficient mutants are unable to infect host plants and elicit the formation of empty non-fixing pseudonodues (Niehaus and Becker, 1998
). Owing to the presence of the rkpZ gene, plasmid pMW23 suppressed the Fix– phenotype of S. meliloti strain Rm6903 (Rm1021 exoB::631) (Williams et al., 1990). To ensure that our strains were also able to form a nitrogen-fixing symbiosis with alfalfa, we performed plant tests with Medicago sativa cv. Europe. Ten days after inoculation, alfalfa plants inoculated with strains Rm1021 or Rm2011 displayed pink nitrogen-fixing nodules, whereas plants inoculated with mutants carrying pMW23 displayed only white non-fixing nodules. Twenty days after inoculation, a few of the nodules on the latter plants turned pink (Table III). The number of pink nodules steadily increased, which resulted in improved growth of shoots. Owing to the increased nitrogen supply, the plants became green (Table III). The effect of plasmid pMW23 on the symbiotic performance was stronger in derivatives of Rm2011 than in derivatives of Rm1021 (Table III). Probably, this is connected with the above-mentioned intrinsic differences between Rm1021 and Rm2011 (Wais et al., 2002; Djordjevic et al., 2003; Krol and Becker, 2004).
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In contrast to strains Rm1021 and Rm2011, exo mutants expressing pMW23 elicited three types of nodules: (1) white non-fixing (Figure 5A), (2) pink nitrogen-fixing (Figure 5B), and (3) "double-decker" nodules consisting of a non-fixing base and a pink lobe protruding from it (Figure 5C and D). White nodules were usually the most abundant, comprising 88–94% of the total number of nodules. "Double-decker" pseudonodules were first described by Niehaus and others (1993)
. They resulted from aberrant late infection of nodule tissue by the exopolysaccharide-deficient mutant Rm0540 (the same one which was used in our study). We also observed such nodules on roots of plants inoculated with exoY or exoB mutants (Figure 5F). It should be noted that, similar to non-fixing nodules (Figure 5A and E), double-decker nodules elicited either by pMW23-harboring strains or by exo mutants had brown spots on their non-fixing lobes (Figure 5C, D, and F). These brown spots are the manifestations of plant defense reactions leading to the accumulation of phenolic compounds in the cortical cells of nodules (Niehaus et al., 1993).
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As a control, we constructed a rkpZ mutant of AK631. The introduction of pMW23 into this strain resulted in the complete restoration of the symbiotic phenotype. Taking into account that AK631 does not produce exopolysaccharides but only KPS, it is remarkable how effective the symbiosis between AK631 and alfalfa was. Thus, there was not only a quantitative (Williams et al., 1990
) but also a qualitative difference in symbiotic properties of Rm41 and SU47 derivatives. The delayed timing of nodulation, the low ratio of pink nodules, the presence of double-decker nodules, and the manifestation of a plant defense response suggest that expression of pMW23 in exo– mutants of SU47 derivatives just increases the odds of abnormal invasion.
Paralogs of rkpZ are present in the S. meliloti Rm1021 genome
Previously, it was reported that strain Rm1021 does not carry a rkpZ gene (Williams et al., 1990). However, the S. meliloti Rm1021 genome contains three paralogs of this gene (S. meliloti GenDB genome project is available at https://www.cebitec.uni-bielefeld.de/groups/nwt/sinogate). All three genes (rkpZ1, rkpZ2, and Smb21014) are located in the pSymB gene region that contains many other genes probably involved in KPS production (Becker et al., 2005). One of these genes, smb21014, is truncated. Its open reading frame encodes a protein of only 126 amino acid residues, whereas RkpZ consists of 440 amino acid residues. Two other paralogs, rkpZ1 and rkpZ2, may encode functional proteins (432 and 343 amino acids residues, respectively) because they share significant similarities with RkpZ over three domains (PD347417, PD489235, and PD025031), characteristic for KpsC/LipA family of proteins (ProDom database; Servant et al., 2002).
In contrast to the rkpZ gene of Rm41, which does not cluster with other rkp genes, the rkpZ1 coding sequence of Rm1021 overlaps with a 5' end of coding sequence of rkpT1 by four nucleotides (Figure 6). There is also a short overlap between the rkpT1 and rkpS coding sequences, indicating that rkpZ1, rkpT1, and rkpS may be co-transcribed (Figure 6). The proteins deduced from rkpT1 and rkpS were predicted to encode components of the KPS export machinery. To study the role of rkpZ1 in KPS production in strain Rm1021, we inactivated rkpZ1 by site-directed integration of plasmid pK19mob2BMH. Upon integration, the coding sequence of rkpZ1 was interrupted, and the downstream genes were placed under the control of the lac promoter reading out of the plasmid. This was done to avoid a polar effect of the mutation on the expression of rkpS and rkpT1. The resulting mutant lost the ability to produce KPS. To confirm that this KPS deficiency was caused by the inactivation of rkpZ1, we performed complementation analysis. A genomic fragment containing rkpZ1 was cloned into the replicative vector pPHU231 and introduced into the rkpZ1 mutant (Figure 6A). The resulting strain expressed the same KPS as the wild type (Figure 6B). These results demonstrate that rkpZ1 is functionally active and that it is required for the expression of KPS in Rm1021.
A rkpZ2 mutant was also constructed by plasmid integration. This gene seems to be transcribed independently of a downstream gene, which is oriented in the opposite direction. Therefore, plasmid integration into the rkpZ2 coding sequence should knock out only this gene. Similar to the rkpZ1 mutant, the rkpZ2 mutant lost the ability to express KPS (data not shown). Thus, despite structural similarity, RkpZ1 and RkpZ2 proteins cannot replace each other, and both are required for surface expression of polyKdo.
Functional relationships of RkpZ paralogs
Using rkpZ or rkpZ1 mutants and complementing plasmids with respective genes, we determined whether these genes can replace each other. When pMW23 carrying rkpZ from Rm41 was expressed in the rkpZ mutant of AK631, the production of KR5 antigen was restored completely (Figure 6B). However, when this plasmid was expressed in the rkpZ1 mutant of Rm1021, the detected KPS showed a different pattern from that of Rm1021. Instead of LMW KPS, a polysaccharide with a broader size range distribution and increased molecular weight was observed (Figure 6B). The size range was similar to that of KPS produced by strain Rm1021/pMW23, but the level of production was significantly reduced. Thus, the rkpZ gene from Rm41 was able to restore partly KPS production in the rkpZ1 mutant of Rm1021 and caused a higher rate of its polymerization.
When the plasmid pPHU115 carrying rkpZ1 from Rm1021 was transferred to the rkpZ mutant of AK631, no restoration of KPS production was observed (Figure 6B), implying that the Rm1021 rkpZ1 gene was unable to replace rkpZ in the Rm41 genetic background.
To analyze the roles of rkpZ1 and rkpZ in the lipidation of Rm1021 KPS, lipid fractions of acid-hydrolyzed crude extracts obtained from the rkpZ1 mutant of Rm1021 and pMW23-complemented derivatives were analyzed using ESI–MS (Table II). No lipid 622 was detected in the crude extract from rkpZ1 mutant, and only a residual amount of this lipid was detected in the extract from the exoY rkpZ1 double mutant. All strains carrying pMW23, regardless of the presence or absence of the rkpZ1 mutation, produced significant amounts of lipid 622 (Table II).
Complementation studies were also performed with the rkpZ2 mutant. When the mutant acquired the pPHU115 plasmid carrying rkpZ1, there was no complementation. When the mutant acquired pMW23 plasmid carrying rkpZ, similar to the rkpZ1/pMW23 transconjugant, it expressed a modified KPS.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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This study showed that the introduction of plasmid pMW23 into S. meliloti SU47 derivatives resulted in the modified expression of the recently identified LMW KPS (Fraysse et al., 2005
). Nuclear magnetic resonance (NMR), ESI–MS, and GC–MS analyses proved that LMW KPS and pMW23-modified KPS consist of ß-(2
7)-linked Kdo. Whereas wild-type KPS was expressed at a relatively low level and its molecular weight varied from 3 to 6 kDa, the modified KPSs was expressed at a very high level and had a broader size-range variation, starting from 3 to 30 kDa. Additionally, in contrast to the wild-type polysaccharide, which was homogenously lipidated, the modified one comprised a mixture of lipidated and non-lipidated polymers. The lipid anchor was detected in all fractions of the modified KPS. However, LMW fraction (3–6 kDa) comprised mainly lipid-free polyKdo, whereas MMW and LMW fractions comprised mainly lipidated polyKdo. According to the model of KPS expression suggested by Reuhs and others (1995) for S. meliloti Rm41, (1) the initial polymerization process in the cytoplasm leads to the production of a LMW polysaccharide primer of 4–7 kDa. (2) Upon attachment of the lipid encoded by the chromosomal rkp-1 gene cluster, further polymerization and export of the polysaccharide to the cell surface are enabled. Probably, abundantly produced LMW polyKdo represents such a primer which is then lipidated, elongated, and translocated to the cell surface.
RkpZ and its paralogs belong to the same family of proteins as KpsC of E. coli and LipA of Neisseria meningitidis. Earlier, KpsC and LipA were annotated as proteins involved in the lipidation of capsular polysaccharides, which is an important prerequisite for their export to the cell surface (Roberts, 1996). However, recently, it was found that LipA is not required for attachment of the lipid to KPS but is essential for the translocation of lipidated KPS to the cell surface (Tzeng et al., 2005). Inactivation of the lipA gene did not affect biosynthesis, polymerization, and intracellular accumulation of lipid-free HMW KPS in N. meningitidis. In contrast, a kpsC mutation had a severe effect on the biosynthesis of K5 polysaccharide in E. coli (Bronner et al., 1993; Rigg et al., 1998). In this organism, KPS biosynthesis and export proteins assemble into a multiprotein complex (Bronner et al., 1993; Rigg et al., 1998). It is likely that in the absence of KpsC, the function of the complex was disturbed.
The RkpZ protein was not required for the expression of the capsule in the S. meliloti Rm41 exoB background (Reuhs et al., 1995). It was suggested to modify the polymerization process by promoting the export of smaller polysaccharides (Reuhs et al., 1995). When studies with Rm41 KPS were performed, no lipid anchor was detected (Reuhs et al., 1993). However, our results demonstrate that it is very difficult to reveal the anchor, especially in highly polymerized KPS, because it represents only 1–5% of total polysaccharide. It would be of interest to analyze KR5 antigen using our approaches and to determine whether LMW and HMW KPSs differ in the level of lipidation.
Complementation and mutation studies with RkpZ and its paralogs showed that both RkpZ1 and RkpZ2 are required for KPS expression in strain 1021; however, they seem to be partially defective compared with RkpZ. Whereas RkpZ promotes a high rate of the polysaccharide polymerization and expression, RkpZ1 and RkpZ2 promote only partial polymerization and poor expression. Probably, being expressed together, these intracellular proteins complement each other and constitute a functional unit that stabilizes the nascent lipo-polyKdo chain and directs it to the export machinery. When the RkpZ protein is present in addition to RkpZ1 and RkpZ2, the process of chain elongation and translocation of the KPS to the cell surface proceed much more effectively. RkpZ enables surface expression of Kdo chains with an increased DP even in the absence of either RkpZ1 or RkpZ2. However, the production of the polysaccharide is reduced, implying that RkpZ from Rm41 does not fit perfectly to the protein complex involved in polyKdo biosynthesis and export.
Considering the role of RkpZ in the modification of KPS expression in Rm1021 and related strains, we may conclude the following. Although the primary structure of the modified KPS remained without changes, the cell surface might have acquired new properties because of the increased length and density of anchored polysaccharides and because of the production of lipid-free LMW polyKdo. Probably, this is the reason for the altered phage sensitivity and altered symbiotic phenotype.
Our analysis of symbiotic phenotypes of pMW23-suppressed exo mutants (Rm2011 exoY, Rm2011 exoB, and Rm1021 exoY) showed that although they acquired the ability to form few nitrogen-fixing nodules, they still elicited non-fixing nodules with evident signs of plant defense response. Evidently, RkpZ-modified polyKdo cannot functionally replace EPS I but helped rhizobia to utilize aberrant ways of infection. PolyKdo is unable to mediate a signaling function as proposed for EPS I during symbiosis (Battisti et al., 1992) but may restore certain cell-surface properties, such as net charge and hydrophobicity, which enable host invasion and colonization.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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Bacterial strains and growth conditions
Strains and plasmids used in this work are summarized in Table IV. Sinorhizobium meliloti strains were incubated at 30°C either on LB or Vincent medium (Sambrook et al., 1989
; Somasegaran and Hobben, 1994). Escherichia coli strains were incubated at 37°C on LB medium. Antibiotics were added at the following concentrations (µg/mL): neomycin (Nm) 120, streptomycin (Sm) 600, and tetracycline (Tc) 8 for S. meliloti; ampicillin (Ap) 100, kanamycin (Km) 50, and Tc 5 for E. coli.
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Construction of recombinant S. meliloti strains by conjugation and transduction
Plasmids were transferred to S. meliloti strains by conjugation using the mobilizing E. coli strain S17-1 as a donor. General transduction was performed using phage
M12 as described previously (Finan et al., 1984).
Cloning and DNA manipulations
All DNA manipulations were carried out using standard techniques (Sambrook et al., 1989) or according to manufacturers’ instructions.
Construction of rkpZ, rkpZ1, and rkpZ2 mutants
An intragenic fragment of the rkpZ gene from AK631 was amplified using Taq polymerase and the primer pair rkpZ-431up (5'-GCTAAAGCTGTTGGGTCTGC-3') and rkpZ-431dn (5'-CGACCGCTAGAGATAGTGGC-3'). The polymerase chain reaction (PCR) product was first inserted into vector pGEM-T-Easy, then recovered using the EcoRI endonuclease, and ligated into the mobilizable suicide vector pK19mob2. The resulting plasmid pK19m-rkpZ was transferred by conjugation into S. meliloti strain AK631. Transconjugants were selected on LB plates containing Nm and Sm. To verify that the genomic rkpZ sequence was disrupted by the plasmid, we analyzed the transconjugants by PCR using the above-mentioned primers in combination with primers directed to the beginning and to the end of the rkpZ coding sequence: rkpZ-1323up (5'-CTGAAATAAGAGCGGCCAAG-3') plus rkp3–431dn and rkpZ-431up plus rkpZ1323-dn (5'-ATCGACATTAATCGCTTCGG-3'). This resulted in the amplification of recombinant DNA fragments flanking the integrated vector.
An intragenic fragment of the rkpZ1 gene from S. meliloti Rm1021 was amplified using Taq polymerase and the primer pair SMb20834l (5'-GGTTCCACGTAAGCTT CCGCGAACTCAGAGCAAGCGG-3') and SMb20834r (5'-GCGATTACCCTGTACACCGATAGGCGCCGGCA AAAAG-3'). The underlined nucleotides represent HindIII and BsrGI restriction sites, respectively, added for convenience of cloning. Gene-specific sequences in both oligonucleotide primers start 2 bp downstream of the restriction sites. This pair of primers belongs to the set of primers used for the construction of an S. meliloti Rm1021 DNA microarray (Rüberg et al., 2003). The amplified DNA fragment was ligated into mobilizable suicide vector pK19mob2BMH using HindIII and Bsr GI restriction sites. The resulting plasmid was conjugated into Rm1021. Transconjugants were selected on LB medium containing Nm and Sm. To verify that the genomic rkpZ1 sequence was disrupted by the integrated vector, we analyzed the transconjugants by PCR using two pairs of primers combining gene-specific primers and primers directed to the vector: univM13 (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') plus SMb20834l and revM13 (5'-AGCGGATAACAATTTCACACAGGA-3') plus SMb20834r. This resulted in the amplification of the target genomic sequences plus adjacent stretches of nucleotides from the integrated vector.
To knock out the rkpZ2 gene, we amplified its internal fragment using Taq polymerase and the primer pair SMb20823l (5'-GGTTCCACGTAAGCTTCCGAAAGC GCGTGCTGGTCAT-3') and SMb20823r (5'-GCGA-TTACCCTGTACACCGTCGTCACGTTGATGCCCC-3'). HindIII and BsrGI restriction sites are underlined. The amplified DNA fragment was ligated into a mobilizable suicide vector pK19mob2
HMB using HindIII and BsrGI restriction sites. Further steps of mutant construction and verification were the same as for the rkpZ1 mutant.
Cloning of the rkpZ1 gene in a replicative vector
To clone the rkpZ1 gene, we excised a 2.0-kb KpnI–PstI DNA fragment from the shotgun sequencing clone smbp5d05z05e01448 and ligated into the broad host range vector pPHU231. The resulting plasmid pPHU115 contained the rkpZ1 coding sequence plus 433 bp upstream of the start codon and 300 bp downstream of the stop codon. The rkpZ1 gene was placed behind the lac promoter reading out of the vector. Plasmid pPHU115 was used for complementation analysis of rkpZ1 and rkpZ mutants. Conjugational transfer of pPHU115 into S. meliloti strains was performed as described above. Transconjugants were selected on LB plates containing Tc and Sm.
Extraction and visualization of KPS and LPS in SDS–PAGE
KPS and LPS from S. meliloti strains were isolated using the TEA–EDTA extraction procedure (Valverde et al., 1997) omitting the polymyxin B purification step. KPS/LPS samples were fractionated by SDS–PAGE. After electrophoresis in 16.5% acrylamide gels, KPS and LPS were visualized by Alcian blue silver staining. The gel was prestained with 0.005% Alcian blue in a 40% ethanol and 5% acetic acid solution for 0.5 h and then stained in a fresh Alcian Blue solution overnight. Consecutive silver nitrate staining was performed, as described by Hitchcock and Brown (1983), omitting the oxidation step (Kiss et al., 1997).
Large-scale purification of LPS and KPS
Overnight cultures of S. meliloti strains were diluted 1:100 into liquid Vincent medium. The cultures (1 L each) were incubated with shaking (160 rpm) for 24 h at 37°C until a cell density of (A600) 1.0 was reached. Growth was stopped by the addition of 10–6 M sodium azide. Cells were collected by centrifugation for 30 min at 10,000 g at 4°C. The bacterial pellets were subjected to a hot-phenol water extraction as described by Carlson and others (1978). The water phase (
400 mL) was reduced by rotational evaporation to 100 mL and dialyzed three times at 1:100 in water adjusted to pH 7.8 with NH4OH (to minimize Na+ and Ca2+ associations and to optimize solubilization) using a membrane with 12,000 Da cutoff. After complete elimination of phenol traces, RNAse, DNAse, and proteinase K digestion was performed. Then, samples were dialyzed again under the same conditions and freeze-dried.
NMR spectroscopy
NMR spectra were acquired on a Bruker Avance 500-MHz spectrometer outfitted with cryosonde TCI. All recordings were made at 298K. The HMW KPS, obtained by S300 exclusion chromatography and dialysis, was dissolved in D2O 100% (11.2 mg/mL) after three exchanges with D2O. The assignment of the 1H and 13C chemical shifts was achieved by 1H 13C Heteronuclear Single Quantum Correlation (hsqcetgpsi sequence) and compared with published data (Kohlbrenner and Fesik, 1985; Fraysse et al., 2005). To determine the linkage, we performed a HMBC experiment (hmbcgplpndqf sequence). This permitted to determine junction points between a proton and carbons that are separated by two or three bonds (J2, J3).
Preparation of lipid samples for ESI–MS analysis
The absence of phospholipids in the crude extract was checked by ESI–MS measurements (see ESI–QTOF MS analysis). When phospholipids were still present, we performed an additional dialysis. To release lipid anchor moieties from lipidated polysaccharides (lipidated polyKdo and LPS), we treated the crude extract sample (1 mg/mL) with 1% acetic acid for 1 h at 100°C. Then, the sample was centrifuged, and the precipitate was washed three times with water and twice with methanol. When lipid concentration was too low, it was extracted two times using chloroform in a 1:3 (v/v) ratio with vortexing.
ESI–QTOF MS analysis
Electrospray ionization/quadrupole-time-of-flight analysis mass spectrometry (ESI–QTOF MS) was performed in the negative and positive ion mode using a Q-Tof Ultima Instrument (Waters, Manchester, UK). To analyze crude polysaccharide extracts, we dissolved samples at 10 ng/µL concentration in a 50:50:0.001 (v/v/v) mixture of 2-propanol, water, and TEA and sprayed at a flow rate of 10 µL/min. Capillary entrance voltage was set to 3.0 kV and dry gas temperature to 120°C; cone: 60 V, Rf lens: 40 V, MS profile (m/z 150 [time 20%], m/z 900 [time 60%], ramping 20%). To analyze lipids, we dissolved samples at 50 ng/µL concentration in a CHCl3/MeOH/H2O (60:30:5) made to 5 mM in TEA. MS conditions were similar to the previous ones, but cone voltage was 100 V and Rf lens 80 V. The spectra, which showed several charge states for each component, were charge deconvoluted using the MaxEnt 3 software (MassLynx, Waters). Mass numbers given refer to the monoisotopic molecular masses.
Evaluation of the mass ratio of the lipid anchor to the entire polysaccharide
Before the MS measurements, the weight ratio of the liophylized lipid fraction to the liophylized crude extract was determined. Using ESI+ and ESI–, the "purity" of the 622 and 624 ions, respectively, was checked, and the contribution of the lipid anchor to the lipid fraction was calculated. The mass ratio of the lipid 622 to the entire polysaccharide molecule was calculated based on the assumption that the molecular mass of the latter is about 40 kDa (based on results of DOC–PAGE analysis).
GC–MS analysis of glycosidic constituents
The determination of acidic sugars was performed by methanolysis (1 M HCl in MeOH, 80°C, overnight) followed by derivatization with 10% heptafluorobutyric anhydride (HFBA) in anhydrous acetonitrile at 60°C for 2 h (Zanetta et al., 1999). Derivatives were identified and quantified by comparison with authentic standards in GC and GC–MS.
The GC chromatograms were performed on a column/FID CP9000 (Varian, Palo Alto, CA) gas chromatograph, using an OV-1 capillary column (50 m x 0.25 µm x 0.1 µm). The temperature program was 70°C (initial temperature) up to 300°C (final temperature) at 3°C min–1. The vector gas was helium at 130 kPa.
GC–MS analysis was recorded on a 5973N instrument (Hewlett Packard, Houston, TX) in the positive EI mode (ionization energy: 70 eV), split injector (ratio 1:100) at temperature 250°C, gas: He, 1 Bar. Temperature program is as follows: 90°C for 2 min, up to 300°C (3°C/min), 300°C during 10 min. Capillary column: HP5-MS (30 m Hewlett Packard 0.25 µm x 0.1 µm;). Transfer line 280°C.
Characterization of symbiotic phenotypes
The ability of pMW23 to suppress the Fix– phenotype of S. meliloti exo mutants was examined by the inoculation of alfalfa (M. sativa cv. Europe) seedlings with transconjugants carrying pMW23. Plant growth conditions were as previously reported (Sharypova et al., 2003). To confirm that the passage through the host plant does not affect the properties of S. meliloti strains, we re-isolated bacteria from root nodules (as described by Sharypova et al., 2003) and checked their phenotypes on selective plates and in SDS–PAGE. The analysis was performed for Rm2011, Rm2011exoY (Rm0540), Rm0540/pMW23, and Rm2011exoB/pMW23 (at least three nodules for each strain). To exclude the influence of the plasmid on symbiotic and KPS phenotypes, exo mutants carrying an empty pRK404 vector were constructed and shown to retain the symbiotic properties of the recipient strains.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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We thank Peter Putnoky and Antonio Lagares for providing the plasmid pMW23, Natalija Pobigaylo for providing strain 2011mTn5_STM.4.04.E11, Eva Schulte-Berndt for assistance with construction of rkpZ1 and rkpZ2 mutants, and Dieter Kapp for help with photographing of nodules. This work was supported by grant 031U213D from Bundesministerium für Bildung und Forschung, Germany, and grant number JC4128 from "Ministère de l’éducation nationale, de l’enseignement supérieur et de la recherché", France.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsConflict of interest statementReferences |
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None declared.
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Abbreviations |
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DOC, deoxycholic acid; DOC–PAGE, deoxycholate–polyacrylamide gel electrophoresis; DP, degree of polymerization; EPS I, exopolysaccharide I; ESI–QTOF MS, electrospray ionization–quadrupole-time-of-flight mass spectrometry; GC–MS, gas chromatography–mass spectrometry; HMBC, heteronuclear multiple-bond correlation; HMW, high molecular weight; HSQC, heteronuclear single-quantum correlation; Kdo, 3-deoxy-D-manno-2 octulosonic acid; KPS, K polysaccharide or capsular polysaccharide; LMW, low molecular weight; LPS, lipopolysaccharide; m/z, mass/charge; MMW, middle molecular weight; NMR, nuclear magnetic resonance; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis; TEA–EDTA, triethylamine–ethylenediaminetetraacetic acid
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