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1 Institute of Cell Biology and Centre for Science at Extreme Conditions, School of Biological Sciences, King's Buildings, University of Edinburgh, Edinburgh EH9 3JR, UK
2 School of Medicine, Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
3 Department of Biological Sciences, Louisiana State University, 202 Life Sciences Building, Baton Rouge, LA 70803, USA
4 College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
Correspondence
Gail P. Ferguson
g.ferguson{at}abdn.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-proteobacterium Sinorhizobium meliloti forms a beneficial symbiosis with leguminous plants such as alfalfa (Medicago sativa) (Niner & Hirsch, 1998
). During this interaction, S. meliloti enters into the legume host via the root hairs and induces the formation of plant-derived structures known as infection threads. The bacteria multiply and traverse down the infection threads, where they are ultimately endocytosed into the plant cell within membrane-bound acidic compartments (Mellor, 1989). Within these compartments, S. meliloti differentiates into a nitrogen-fixing bacteroid, which persists for extensive periods. Although this interaction is beneficial for the legume host and is an important system in its own right, it has also been used to gain insights into the molecular basis of chronic mammalian pathogens such as Brucella species (Ferguson et al., 2004; LeVier et al., 2000). S. meliloti is closely related to Brucella species (Paulsen et al., 2002), and commonalities exist in their infection processes despite the major differences in their eventual outcome on their respective hosts (LeVier et al., 2000).
The BacA protein was found to be essential for the persistence of both S. meliloti and Brucella abortus within their hosts (Glazebrook et al., 1993; LeVier et al., 2000). Based on the distant sequence similarity between BacA and the adrenoleukodystrophy family of eukaryotic proteins, which are thought to be involved in the transport of very-long-chain fatty acids (VLCFA) out of the cytoplasm into peroxisomes, it was subsequently discovered that BacA affects the VLCFA modification of the lipid A in both S. meliloti and B. abortus (Ferguson et al., 2004). Interestingly, in the absence of BacA, 
50 % of the lipid A molecules of S. meliloti and B. abortus lack the lipid A VLCFA modification. In contrast, every lipid A molecule of the parent strain possesses a VLCFA modification. The lipid A is a component of the lipopolysaccharide (LPS), which forms the outermost leaflet of the outer membrane of Gram-negative bacteria. Consequently, these findings led to a model where BacA is involved in the transport of an activated VLCFA out of the cytoplasm that is used to modify the lipid A in the outer membrane (Ferguson et al., 2004). If this model were correct, then the LPS would need to be transported across the inner membrane before it could be modified with the VLCFA. However, the mechanism by which lipid-containing macromolecules such as LPS are transported from their site of synthesis on the inner face of the inner membrane to the outer membrane is poorly understood (Doerrler, 2006; Ruiz et al., 2006).
In Escherichia coli, the inner-membrane MsbA protein is essential (Doerrler et al., 2001). However, since a temperature-sensitive E. coli msbA mutant accumulates LPS in the inner membrane at the non-permissive temperature, this provided evidence that MsbA is involved in the inner-membrane transport of rough LPS, which lacks O-antigen (Doerrler et al., 2001, 2004). The E. coli MsbA protein is also thought to be involved in the transport of phospholipids across the inner membrane (Doerrler et al., 2001). However, there is still some debate about the role of MsbA proteins in phospholipid transport since a Neisseria meningitidis msbA mutant was affected in LPS but not phospholipid transport (Tefsen et al., 2005). Interestingly, the S. meliloti Rm1021 genome (Galibert et al., 2001) encodes multiple proteins (Fig. 1a), which share between 26–34 % identity (47–58 % similarity) and 23–32 % identity (44–54 % similarity) over their entire length with the E. coli and N. meningitidis MsbA proteins, respectively. It was shown previously that the S. meliloti ExsA protein affects the molecular mass distribution of the exopolysaccharide (EPS) succinoglycan and has been proposed to play a role in the transport of succinoglycan across the inner membrane (Becker et al., 1995). Although an S. meliloti exsA mutant forms a symbiosis with legumes, a B. abortus exsA deletion mutant has reduced survival in mice and induced protective immunity against the parent strain (Rosinha et al., 2002). Additionally, the S. meliloti and B. abortus NdvA proteins (known as Cgt in B. abortus) are involved in the inner-membrane transport of a β-1,2-glucan and are essential for the host interaction (Dickstein et al., 1988; Roset et al., 2004; Stanfield et al., 1988). Combined, these findings raised the possibility that the other S. meliloti MsbA-like proteins (Fig. 1A) could also be playing a role in the transport of a polysaccharide or lipid-containing polysaccharide such as LPS and these processes could play an important role in the host interaction.
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), we constructed and characterized mutants defective in these proteins. In this paper, we report our findings for an S. meliloti Rm1021 msbA2 insertional mutant. We initially focused our efforts on the msbA2 gene since it is borne on the pSymB megaplasmid of S. meliloti Rm1021, which carries a large number of genes whose products are involved in the synthesis of cell-surface carbohydrates (Finan et al., 2001), and is immediately downstream of genes whose products are predicted to be involved in the biosynthesis of a lipid-linked polysaccharide (Fig. 1B). Our characterization determined that the MsbA2 protein is essential for the legume symbiosis, and since the S. meliloti msbA2 insertional mutant has a polysaccharide alteration, we propose that the MsbA2 protein could be involved in the transport of a novel polysaccharide. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. All S. meliloti strains were grown in Luria–Bertani (LB) medium (Sambrook et al., 1982) or on LB agar supplemented with 2.5 mM MgSO4 and 2.5 mM CaCl2 (LB/MC) and 500 µg streptomycin (Sm) ml–1, unless defined otherwise. Where indicated, the LB medium or agar was also supplemented with 200 µg neomycin (Nm) ml–1, 50 µg gentamicin (Gm) ml–1 or hygromycin (Hm) at either 100 (for LB) or 150 µg ml–1 (for LB/MC). The E. coli strains were grown in LB medium or on LB agar supplemented with 50 µg kanamycin (Km) ml–1 or 10 µg Gm ml–1 as defined.
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, pJH104-msbA2in was transferred into S. meliloti Rm1021 by tri-parental mating using E. coli MM294A with the helper plasmid pRK600 and transconjugants were selected on LB/MC Sm Nm agar after incubation at 30 °C for 4 days. To avoid contamination with the E. coli donor strains, a final concentration of 1 mg Sm ml–1 was used in the selection agar. The vector, pJH104, is able to replicate in E. coli but is unable to replicate in S. meliloti, hence the genomic msbA2 gene is disrupted by homologous recombination upon transfer of pJH104-msbA2in into S. meliloti. The disrupted msbA2 gene was transduced into Rm1021 and transductants were selected on LB Nm. All transductants were purified onto LB/MC agar containing the defined antibiotics and the disrupted msbA2 gene was confirmed by PCR using the primers SmmsbA2+155F (5'-CGGAGATGGACAGGCCAAC-3') and pJH104gusR (5'-GAGTTTTTTGATTTCACGGGTT-3'). All transductants gave the expected PCR product of 0.9 kb with this primer pair.
Construction of the bacA-uidA BacA+ fusion strain.
A 0.94 kb fragment of the S. meliloti bacA gene, including upstream region, was amplified from pJG51A (Glazebrook et al., 1993) by PCR using the primers SmBaUS1F-BamHI (5'-TCGAGTTCCTTGCCTGGC-3') and SmBaIN1R-XbaI (5'-TTGATCGCGACGCTGACC-3') and cloned into TOPO using the pCR-2.1 TOPO cloning kit (Invitrogen). After sequencing, the bacA fragment was digested from TOPO using SpeI and XhoI and cloned into pJH104. The resulting pJH104-bacA was then transferred into S. meliloti by tri-parental mating, transconjugants were selected and then the insertion was transduced into Rm1021 exactly as described for the construction of the S. meliloti msbA2 : : pJH104 mutant. The insertion of pJH104-bacA was confirmed by PCR using the primers SmBaUS1F-BamHI and pJH104gusR and all transductants gave the expected PCR product of 1 kb with this primer pair. Additionally, since BacA is essential for the alfalfa symbiosis, the presence of a wild-type copy of the bacA gene in this strain was also confirmed by the ability of the strain to form a successful symbiosis with alfalfa.
Cloning of the S. meliloti msbA2G97A gene.
The S. meliloti msbA2 gene was amplified by PCR using the primers SmmsbA-90F-XbaI (5'-CCGTCTAGATCGCGCGGCTTGCCATG-3') and SmmsbA2+1832R-XbaI (5'-CCGTCTAGAATGGACAAGCGGCTGGGTCTT-3') and digested with XbaI. The digested msbA2 gene was ligated into the XbaI site of pJN105 (Newman & Fuqua, 1999), under control of the pBAD promoter, and transformants were selected on LB Gm supplemented with 0.1 % (w/v) glucose to repress transcription. After screening 60 transformants by digestion, we identified one clone containing an msbA2 gene insert. Sequencing of the cloned msbA2 gene revealed a point mutation (G97A) resulting in an asparagine residue instead of a serine at amino acid position 33 of the MsbA2 protein.
Polysaccharide isolation and analysis.
Cultures of the defined strains were grown to late-exponential phase in LB/MC with the appropriate antibiotics and diluted into 5 l LB/MC to an OD600 0.1. The cultures were then grown for 48 h to late exponential phase (OD600 2), and the cells were harvested by centrifugation at 6000 r.p.m. for 10 min (Sorvall RC-3B). The crude polysaccharides were extracted from the cell pellets using a hot water/phenol procedure (Reuhs et al., 1994) and were then treated with DNase (20 µg ml–1), RNase (100 µg ml–1) and proteinase K (100 µg ml–1). After lyophilizing, the polysaccharide preparations were resuspended in distilled water to a concentration of 10 mg ml–1. After centrifugation (4500 g for 15 min), the supernatant was ultracentrifuged (100 000 g, 8 °C) for 3.5 h. The purified polysaccharide pellet was then washed twice with distilled water, freeze-dried and then 3 µg aliquots analysed by deoxycholate (DOC)-PAGE/silver staining as described previously using either periodate or alcian blue (0.005 % w/v) (Reuhs et al., 1994).
Fluorescence microscopy and histochemical staining for polyphenolics.
Polyphenolics were identified using previously published methods with modifications (Vasse et al., 1993). In brief, for both methods, root nodules were sliced longitudinally and then fixed immediately in 2.5 % (w/v) glutaraldehyde and 10 mM PIPES (pH 7.2) for 1 h. For the fluorescent microscopy, the nodule slices were then cleared by soaking in 50 % (v/v) bleach for 3 min, washed three times with sterile distilled water and then visualized for fluorescent polyphenolics by microscopy (10x magnification, Zeiss Axioskop) using UV excitation. For the histochemical staining, after fixing, the nodule slices were immersed in 0.04 % (w/v) potassium permanganate for 1 h, rinsed with 10 mM PIPES (pH 7.2), and then stained with 0.01 % (w/v) methylene blue for 2 min. The nodule slices were then immersed in 50 % (v/v) bleach for 3 min and visualized using bright-field optics (10x magnification, Zeiss Axioskop). Images were then processed by Axio Vision software.
β-Glucuronidase (GUS) assay.
Cultures of the defined strains were grown to late-exponential phase in LB/MC media with the appropriate antibiotics. An aliquot of culture (100 µl) was centrifuged, washed and resuspended in 0.75 ml lysis buffer [50 mM sodium phosphate buffer pH 7.0, 2.6 % (v/v) β-mercaptoethanol, 0.1 % (v/v) Triton X-100 and 1 mg lysozyme ml–1]. One hundred microlitres of chloroform was added and the mixture was incubated at 37 °C for 10 min prior to the addition of 100 µl 10 mM p-nitrophenyl-β-D-glucuronide. The samples were incubated at 37 °C until the appearance of a yellow colour and then the reaction was stopped by the addition of 400 µl 2 M 2-amino-2-methylpropandiol. The cell debris was removed by centrifugation (microfuge, 13 000 r.p.m., 5 min) and then the GUS activity of the supernatant was determined by measuring the A415. Units of GUS activity were then calculated using the Miller equation [A415/time (min)xvolume (ml)xOD600)]x1000 (Miller, 1972).
S. meliloti-alfalfa interaction experiments.
To determine the ability of S. meliloti to form a successful symbiosis with alfalfa, 3-day-old seedlings were inoculated with 1 ml S. meliloti culture, resuspended to an OD600 0.05 in sterile water, on Jensen's agar as described previously (Leigh et al., 1985). The plates were incubated at 25 °C and then plant growth and nodule morphology were determined at different stages throughout a 4 week period. To confirm that the mutants were not reverting during the symbiosis, individual nodules were removed, surface sterilized with 50 % (v/v) bleach and crushed in 200 µl LB/MC supplemented with 5 % (w/v) glucose. The bacteria were then serially diluted and 10 µl aliquots from each dilution were spotted in triplicate onto LB/MC Sm agar (to determine total number of bacteria extracted) and LB/MC agar containing the appropriate antibiotic to select for the mutant strain. In all cases, we observed equivalent numbers of colonies on both selective and non-selective agar, indicating that the mutants were not reverting.
Light and transmission electron microscopy (TEM).
The light microscopy and TEM of the nodules were performed as described previously (Krusell et al., 2005). The nodules were halved and fixed in 2.5 % (w/v) glutaraldehyde in 0.1 M sodium cacodylate (pH 7.0) overnight at 4 °C. The fixed nodules were either dehydrated in an ethanol series and embedded in LR White acrylic resin (Agar Scientific) for light microscopy or post-fixed in 1 % osmium tetroxide (w/v), dehydrated in an ethanol series, and then embedded in Durcupan epoxy resin (Sigma-Aldrich) for conventional TEM. Semi-thin sections (1 mm) and ultrathin sections (70 nm) were taken from the resin-embedded samples (LR White and Durcupan) using a Reichert Ultracut E ultramicrotome. The semi-thin sections were collected on glass slides and stained with 1 % toluidine blue in borax (w/v) and viewed and photographed using a Zeiss Axioskop optical microscope fitted with an AxioCam digital camera (Carl Zeiss Imaging). The ultrathin sections for conventional TEM were collected on pioloform-coated copper grids and stained with uranyl acetate (10 min) and lead citrate (5 min) before being viewed with a JEOL 1200 EX transmission electron microscope.
Membrane localization of phosphate-containing lipids.
S. meliloti strains were grown at 30 °C to an OD600 of 0.5–0.6 in LB/MC containing appropriate antibiotics then labelled for 20 min with 32Pi [2 µCi ml–1 (74 kBq ml–1)] before the cells were cooled on ice and recovered by centrifugation at 4 °C for 10 min at 4000 g. Spheroplasts were prepared by treatment with EDTA/lysozyme and lysed by sonication. Washed membranes were isolated by centrifugation at 100 000 g and separated into inner- and outer-membrane fractions using a 30–60 % sucrose gradient (Doerrler et al., 2004; Osborn & Munson, 1974). Portions of gradient fractions were subjected to mild acid hydrolysis in 0.4 ml 12.5 mM sodium acetate, pH 4.5, 1 % SDS to release the lipid A from the LPS core sugars, then extracted by the addition of 1 ml chloroform/methanol (1 : 1) to yield a two-phase solution. This treatment has no effect on phospholipids and allows the LPS and phospholipids to be analysed simultaneously (Zhou et al., 1998). The aqueous upper phase was discarded and the lower phase was washed with fresh upper phase and dried. Alternatively, LPS was recovered free from phospholipids due to its insolubility (5 min, 20 000 g) in a single-phase Bligh–Dyer solution, chloroform/methanol/H2O (1 : 2 : 0.8) (Bligh & Dyer, 1959; Nishijima & Raetz, 1979) prior to mild acid treatment as described above. Lipid species were dissolved in chloroform/methanol (4 : 1), spotted onto silica gel 60 TLC plates (Merck) and resolved using the solvent chloroform/pyridine/88 % formic acid/H2O (50 : 50 : 16 : 5). Plates were analysed using a Phosphorimager equipped with IQMac software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) using an internal fragment of the msbA2 gene cloned into the suicide vector, pJH104 (Davis & Walker, 2007). The msbA2 gene is carried on one of the two megaplasmids, pSymB, in the S. meliloti genome (Galibert et al., 2001), immediately downstream of two potential glycosyltransferases genes and an acyltransferase gene (Fig. 1B). Disruption of genes with pJH104 also creates a transcriptional fusion to the uidA gene, which encodes GUS. Once constructed, the msbA2 : : pJH104 disruption was then transduced into Rm1021 to create Rm1021 msbA2 : : pJH104t. To investigate whether the MsbA2 protein was playing a role in the S. meliloti–legume symbiosis, alfalfa seedlings were inoculated with cultures of either the parent strain, Rm1021, or the mutant strain on Jensen's agar, which lacks nitrogen and carbon sources. Plant growth and nodule development were monitored after 4 weeks (Fig. 2). In contrast to inoculation with the parent strain, which resulted in healthy, dark green alfalfa plants with a mean height of 14 cm (n=19), seedlings inoculated with the S. meliloti msbA2 : : pJH104t mutant were stunted [mean height 2 cm (n=21)] and the leaves were paler green/yellowish, indicative of an unsuccessful symbiosis (Fig. 2A and B, respectively). Additionally, compared to the pink, elongated, nitrogen-fixing root nodules induced by the parent strain, the root nodules induced by the Rm1021 msbA2 : : pJH104t mutant were shorter and white, with brown tinges, indicative of a potential plant defence response (Fig. 2C and D, respectively). The same defective plant phenotypes were also observed for additional Rm1021 msbA2 : : pJH104 transductants and the original Rm1021 strain where the msbA2 : : pJH104 insertion was created (data not shown). Therefore, combined, these findings provide evidence that the MsbA2 protein is essential for S. meliloti to form a successful symbiosis with alfalfa.
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) (Galibert et al., 2001), it was unlikely that the insertion of pJH104 into the msbA2 gene was exerting a polar effect on these genes. Additionally, another study had independently identified that disruption of the Rm1021 msbA2 gene with a transposon insertion also prevented a successful legume symbiosis (J. Griffitts & S. Long, unpublished). Unfortunately, we were unsuccessful in our attempts to clone a wild-type copy of the S. meliloti msbA2 gene into either TOPO (Invitrogen) or the broad-host-range vectors pMS03 (Keating et al., 2002) and pJN105 (Newman & Fuqua, 1999) in E. coli. However, we were able to clone a mutated form of the msbA2 gene (pmsbA2G97A) into pJN105 (Newman & Fuqua, 1999), under control of an arabinose-inducible promoter in the presence of 0.1 % (w/v) glucose. The cloned S. meliloti msbA2 gene contained a point mutation (G97A), which produced a mutated form of the MsbA2 protein with a substitution of an asparagine, instead of a serine residue, at amino acid position 33. We discovered that, although 7/15 plants inoculated with the S. meliloti msbA2 : : pJH104t mutant containing pmsbA2G97A in the presence of 0.1 % (w/v) arabinose were light green/yellow with only brown nodules (7.0±0.7) and white nodules (2±3), 8/15 plants were dark green and had on average 2.6±1.0 pink nodules per plant root (Fig. 2E and F, respectively). Since pJN105 had never been used previously to complement in planta defects of S. meliloti mutants, the variability in these results could be due to differences in the diffusion of arabinose into the root nodule to induce transcription of the msbA2 gene. However, we determined that increasing the percentage of arabinose in the Jensen's agar had a detrimental effect on plant growth regardless of the strain used for inoculation (data not shown). In contrast, no pink nodules were observed on plants inoculated with the S. meliloti msbA2 : : pJH104t mutant containing the control plasmid, pJN105, in the presence of 0.1 % (w/v) arabinose (n=9, data not shown). Consequently, despite our inability to clone the wild-type msbA2 gene, these data provide further support that the MsbA2 protein is important for the legume symbiosis and suggest that the serine at residue 33 is not critical for MsbA2 function.
The S. meliloti msbA2 insertional mutant is defective in host cell entry
To investigate more precisely the host defect of the Rm1021 msbA2 : : pJH104t mutant, the root nodules induced by either the parent or the mutant strain were analysed by light microscopy and TEM, 2 and 4 weeks after inoculation (Figs 3 and 4, respectively). Unlike the nodules induced by the parent strain after 2 weeks, which were surrounded by an endodermis and were packed throughout the different developmental zones with S. meliloti bacteroids (Fig. 3A and B, respectively), the nodules induced by the Rm1021 msbA2 : : pJH104t mutant after 2 weeks had a significantly thickened endodermis and were devoid of bacteroids (Fig. 3C and D, respectively). The differences between the nodules induced by either the parent strain or the Rm1021 msbA2 : : pJH104t mutant strain were even more pronounced 4 weeks post-infection (Fig. 4A, B, C). Interestingly, we determined that, in the absence of MsbA2, S. meliloti appeared to be randomly colonizing the root hair cells (Fig. 4D). Additionally, within the Rm1021 msbA2 : : pJH104t mutant-induced nodules, we observed abnormal infection threads, which appeared to be dramatically thickened, were packed full of bacterial cells and had penetrated through the root hair (Fig. 4E and F). Randomly colonized root hair cells and swollen, abnormal infection threads were never visualized in the nodules induced by the parent strain (data not shown). Therefore, our observations show that the Rm1021 msbA2 : : pJH104t mutant is defective in host cell entry and that the interaction with the host is aborted whilst the bacterial cells are within the infection thread.
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). The production of these compounds in plant tissues can be detected either by their autofluorescence or by histochemical staining (Vasse et al., 1993). To investigate whether the Rm1021 msbA2 : : pJH104t mutant induces a plant defence response in alfalfa, the nodules induced by the mutant strain were analysed by fluorescence microscopy and by bright-field microscopy after histochemical staining (Fig. 5A and B, respectively). As controls, nodules from alfalfa seedlings inoculated with the parent strain were also analysed under the same conditions (Fig. 5C and D, respectively). The nodules induced by the Rm1021 msbA2 : : pJH104t mutant had a dramatically increased autofluorescence and stained blue with potassium permanganate/methylene blue, indicating the accumulation of polyphenolics, whereas the nodules induced by the parent strain had less autofluorescence and were not stained. Combined with the microscopy analysis showing that the nodule endodermal layer is dramatically thickened in response to the Rm1021 msbA2 : : pJH104t mutant strain (Fig. 4C), these results provide strong evidence that the Rm1021 msbA2 : : pJH104t mutant induces a heightened defence response in alfalfa.
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, 2004; Tefsen et al., 2005), suggesting that MsbA2 could also be involved in the transport of a lipid-linked polysaccharide such as LPS. Additionally, the msbA2 gene is located adjacent to two potential glycosyltransferease genes and an acyltransferase gene in the Rm1021 genome (Fig. 1B), providing further support that MsbA2 could be involved in the transport of a lipid-linked polysaccharide. Since previous studies have determined that the lipid A molecules of S. meliloti are modified with phosphate groups (Gudlavalleti & Forsberg, 2003; Sharypova et al., 2003), we labelled the lipid A and phospholipids of the S. meliloti parent and msbA2 : : pJH104t mutant by growth in the presence of 32Pi. However, analysis of the 32Pi-labelled lipids from either the inner- or outer-membrane fractions by TLC (data not shown), showed that disruption of the msbA2 gene was not affecting the membrane localization of the either the lipid A or phospholipids. Additionally, using classical physiology tests, such as an increased sensitivity to detergents such as SDS and DOC, which have been successfully used to identify S. meliloti mutants with LPS alterations (Ferguson et al., 2002, 2004), we observed no difference in the sensitivity of the parent strain and Rm1021 msbA2 : : pJH104t mutant toward these agents on LB agar (data not shown). Additionally, small-scale analysis of the LPS by SDS-lysis followed by SDS-PAGE/periodate/silver staining revealed no LPS alterations between the parent strain and Rm1021 msbA2 : : pJH104t mutant (data not shown), suggesting that if there were LPS alterations in this mutant strain then these could not be detected using this methodology. Therefore, these data suggest that the MsbA2 protein is either not expressed in free-living S. meliloti or is not essential for the transport of phosphate-containing lipids such as LPS across the inner membrane.
Since disruption of the msbA2 gene with pJH104 created a transcriptional fusion to the uidA gene, encoding GUS, we investigated whether the msbA2 gene was being transcribed in free-living S. meliloti by assaying for GUS activity (Fig. 6A). As controls we also determined the GUS activity of the free-living parent strain and an S. meliloti Rm1021 strain containing a pJH104 insertion in the bacA gene [Rm1021 bacA : : pJH104t (BacA+)] (Fig. 6A). Transcription of the bacA gene was used as a positive control since we have shown previously that deletion of the bacA gene in S. meliloti results in an LPS alteration in free-living S. meliloti (Ferguson et al., 2002). Our GUS assay results provided evidence that the msbA2 gene is being transcribed in free-living S. meliloti, although to a lower level than the bacA gene (Fig. 6A). Therefore, these findings suggest that MsbA2 is not essential for the transport of either LPS or phospholipids in S. meliloti.
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). The polysaccharides isolated into the aqueous and the phenol phases were then resolved by DOC-PAGE and treated with either periodate or alcian blue followed by silver staining. Using the periodate method, we determined that in the absence of MsbA2, a new high-molecular-mass polysaccharide band appeared in the phenol-phase-extracted material and there was also a reduction in the intensity of a lower molecular mass band (Fig. 6B). Differences were also observed in the phenol-phase-extracted material from the parent and msbA2 : : pJH104t mutant strain using the alcian blue method (Fig. 6C). In contrast, no reproducible differences were observed in the aqueous-phase-extracted material from the parent and S. meliloti msbA2 : : pJH104 mutant strains using either staining method (data not shown). However, these findings provide evidence that disruption of the msbA2 gene in S. meliloti affects the polysaccharide content. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), a sole disruption of the msbA2 gene was sufficient to prevent S. meliloti Rm1021 from forming a successful symbiotic relationship with its plant host. We determined that in the absence of MsbA2, the S. meliloti–legume infection was aborted at the level of the infection threads and the bacterial cells were unable to be endocytosed into the host cells but instead remained within abnormally swollen infection threads. It appears that in the absence of MsbA2, S. meliloti induces a response in the plant host more characteristic of a pathogen, causing browning of the plant tissue, a substantial thickening of the plant endodermis, preventing bacterial entry into the plant cell, and a heightened production of polyphenolic defence compounds. Since similar heightened plant defence responses, accompanied by aborted infection threads, have also been observed during the infection of legumes with S. meliloti and Rhizobium leguminosarum mutants, which are known to have alterations in their polysaccharides (Niehaus et al., 1993, 1998; Perotto et al., 1994), our findings suggest that the polysaccharide alteration observed by PAGE analysis in the S. meliloti msbA2 mutant could be responsible for at least some of the symbiotic defects of this mutant.
Since the E. coli MsbA protein affects the transport of phosphate-containing lipids such as LPS across the inner membrane, we initially investigated whether the S. meliloti msbA2 mutant was also affected in the transport of phosphate-containing lipids. However, although the lipid A component of S. meliloti LPS is bisphosphorylated (Gudlavalleti & Forsberg, 2003; Sharypova et al., 2003), we did not observe any differences in the membrane localization of 32Pi-containing lipids in the S. meliloti msbA2 insertional mutant relative to the parent strain (data not shown), suggesting that MsbA2 is not essential for the transport of either phosphorylated LPS or phospholipids. Consistent with this, we did not observe any altered sensitivities of the S. meliloti msbA2 mutant relative to the parent strain towards cell envelope disrupting agents usually associated with LPS mutants.
Although we determined that the MsbA2 protein is not essential for the transport of LPS and phospholipids in free-living S. meliloti, we cannot rule out at this stage that one or more of the other potential MsbA-like proteins in S. meliloti (Fig. 1A) could be masking a role for MsbA2 in the transport of LPS and/or phospholipids. With the exception of ExsA and NdvA, the functions of the other potential MsbA-like proteins in S. meliloti have not been extensively investigated. Our phylogenetic analysis showed that the MsbA1 and Y02836 proteins show the highest degree of similarity to the E. coli and N. meningitidis MsbA proteins (Fig. 1A). However, our preliminary analyses suggest that MsbA1 is not essential for the legume symbiosis and neither an S. meliloti msbA1 nor an msbA1/msbA2 double mutant was affected in the transport of phosphate-containing lipids (V. L. Marlow, A. Haag, S. Beck, W. T. Doerrler & G. P. Ferguson, unpublished data). However, the MsbA1 and Y02836 proteins are 91 % similar (84 % identical) to each other, suggesting that Y02836 may be able to compensate for loss of MsbA1. Consequently, future studies will focus on the creation and characterization of S. meliloti mutants with deletions in multiple msbA-like genes, so that their functions can be thoroughly elucidated. Additionally, since the MsbA1 and MsbA2 proteins are only 44 % similar (26 % identical), this suggests that the msbA2 gene should be reannotated in future studies once the precise function of the MsbA2 protein is determined.
In S. meliloti Rm1021, succinoglycan is the symbiotically active form of EPS and is essential for infection thread formation (Niehaus et al., 1998; Pellock et al., 2000). Previous studies have shown that mutants affected in succinoglycan production often affect calcofluor fluorescence (Long et al., 1988). However, we also did not observe any differences between the calcofluor fluorescence of the S. meliloti parent and msbA2 insertional mutant strain, suggesting that succinoglycan could still be transported in the absence of MsbA2 (data not shown). Additionally, a previous study showed that the S. meliloti exsA gene forms part of the succinoglycan biosynthetic gene cluster and ExsA has been proposed to be involved in the transport of succinoglycan (Becker et al., 1995). Consequently, further biochemical studies will be needed to determine the precise polysaccharide(s) affected in the S. meliloti msbA2 mutant. However, since the compositional and structural analyses of S. meliloti polysaccharides to date have focused on the aqueous-phase-extracted material (Ferguson et al., 2002, 2004, 2005; Gudlavalleti & Forsberg, 2003; Sharypova et al., 2003), extensive future studies will be necessary to enable the characterization of the phenol-phase polysaccharides from both the parent and the msbA2 mutant strains. Since the msbA2 gene appears to be closely associated with two glycosyltransferase genes and an acyltransferase gene (Fig. 1B), it is interesting to speculate that these genes form an operon and that the MsbA2 protein is involved in the transport of a novel as-yet-unidentified polysaccharide. Therefore, the polysaccharide alteration we observe in the absence of MsbA2 could be due to the intracellular accumulation of this species or to compensatory changes in the S. meliloti polysaccharide content resulting from the absence of this polysaccharide species. Interestingly, non-polar single deletion mutants in the upstream genes do not appear to affect the legume symbiosis (J. Griffitts & S. Long, unpublished), suggesting that either the novel polysaccharide is not essential for the legume symbiosis or another glycosyltransferase or acyltransferase encoded in the S. meliloti genome (Galibert et al., 2001) can substitute for their function. Additionally, since the importance of the msbA2 gene in the S. meliloti Rm1021–legume symbiosis was not identified in a previous study (Charles & Finan, 1991), which analysed large deletion mutants of pSymB (120–600 kb deletions), this suggests that the host defect observed in our msbA2 insertional mutant could be due to the cytoplasmic accumulation of the novel polysaccharide, encoded by the upstream genes, which somehow interferes with the legume interaction. Thus, future studies investigating defined combinations of mutants in this region will be necessary to rule out this possibility. Additionally, since the Rm1021 strain background used in this study contains an insertion sequence in the expR gene, which is required for the production of symbiotically active EPS II (Pellock et al., 2001), further studies will be necessary to determine whether disruption of the msbA2 gene will result in the same symbiotic phenotypes in an S. meliloti Rm8530 strain, which has an intact expR gene.
If the altered polysaccharide observed in the S. meliloti msbA2 mutant is responsible for the symbiotic defect, the question remains as to how this polysaccharide alteration could affect the host interaction. It has been suggested previously that S. meliloti polysaccharides are essential to suppress the plant defence response and enable proper infection thread development (Niehaus et al., 1993, 1998; Perotto et al., 1994). Consequently, since we observed that the S. meliloti msbA2 mutant produced an elevated plant defence, the altered polysaccharides in this mutant may be less effective at suppressing the defence response and the plant would perceive this mutant as a pathogen rather than a symbiont. Interestingly, wild-type S. meliloti induce a heightened plant defence response and abnormal infection threads when inoculated onto a nip (numerous infections and polyphenolics) mutant of the legume Medicago truncatula (Veereshlingam et al., 2004), suggesting that there could be some interplay between the nip locus in the host plant and bacterial-produced polysaccharides in suppressing the plant defence response. Polysaccharides are also thought to play a critical role in the interaction of Brucella species with their host. For example, cyclic glucans are proposed to be necessary for the intracellular trafficking of Brucella species within their hosts (Arellano-Reynoso et al., 2005). Additionally, a B. abortus bacA mutant is defective in chronic infection (LeVier et al., 2000), has an altered LPS (Ferguson et al., 2004) and induces a greater amount of pro-inflammatory cytokines than the parent strain (Parent et al., 2007), suggesting that suppression or avoidance of host immune responses by wild-type LPS is necessary for B. abortus to form a chronic infection.
In summary, these studies have shown that the MsbA2 protein is essential for S. meliloti to form a symbiosis with legumes and suggest that it may play a role in the transport of a lipid-linked polysaccharide.
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ACKNOWLEDGEMENTS |
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Edited by: M. F. Hynes
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 9 November 2007;
revised 9 January 2008;
accepted 13 January 2008.
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