Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Postfach 100131, 33501 Bielefeld, Germany1
Author for correspondence: Anke Becker. Tel: +49 521 106 5620. Fax: +49 521 106 5626. e-mail: anke.becker{at}genetik.uni-bielefeld.de
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ABSTRACT |
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Keywords: Rhizobium meliloti, exopolysaccharides, type I secretion, Ca2+-binding protein
Abbreviations: ABC, ATP-binding cassette; EPS, exopolysaccharide; EPS I, succinoglycan; EPS II, galactoglucan; HMM, high molecular mass; LMM, low molecular mass; MBP, maltose-binding protein; MCS, multiple cloning site
a Present address: Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
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INTRODUCTION |
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In addition, S. meliloti strains 1021 and 2011 have the ability to produce a second EPS, termed galactoglucan (EPS II), which can replace EPS I in nodule invasion (Glazebrook & Walker, 1989 ; González et al., 1996
; Zhan et al., 1989
). EPS II is composed of alternating glucose and galactose residues which are decorated by acetyl and pyruvyl groups (Glazebrook & Walker, 1989
). Under standard culture conditions in complex media, large amounts of EPS I and almost no EPS II are produced. The production of EPS II was observed in the presence of a mutation in either of the regulatory genes expR (Glazebrook & Walker, 1989
) or mucR (Keller et al., 1995
; Zhan et al., 1989
). In addition, Zhan et al. (1991)
showed that under phosphate-limiting conditions the wild-type strain 1021 produces EPS II. Extra copies of the exp gene cluster responsible for EPS II biosynthesis or extra copies of the transcriptional regulator expG also resulted in the biosynthesis of EPS II (Glazebrook & Walker, 1989
; Rüberg et al., 1999
; Zhan et al., 1989
).
The sequence of the exp gene cluster directing the biosynthesis of EPS II was previously reported and the inferred properties of the encoded gene products implied that these proteins are involved in regulation of exp gene expression, diphospho-nucleotide sugar biosynthesis, polymerization and export (Becker et al., 1997 ). Homologies of the deduced amino acid sequences of ExpD1 and ExpD2 to components of type I secretion systems implied that the N-terminal domain of ExpD1 represents a membrane-spanning domain and the C-terminal portion is the ATP-binding cassette (ABC) domain (Becker et al., 1997
). ExpD2 displays homologies to accessory factors of these secretion systems designated membrane fusion proteins (MFPs). Secretion complexes consisting of an ABC transporter and a MFP in conjunction with an outer-membrane protein are involved in the signal-peptide-independent secretion of proteins which usually contain a C-terminal secretion signal (Dinh et al., 1994
). Most of these secreted proteins contain several aspartate- and glycine-rich nonapeptide tandem repeats involved in the binding of Ca2+ near the C-terminal secretion signal (Baumann, 1994
; Economou et al., 1990
; Ludwig et al., 1988
).
Genes encoding type I secretion systems are usually situated adjacent to the genes encoding the secreted proteins (Fath & Kolter, 1993 ). The protein encoded by expE1, the gene for which is located immediately downstream of the expD1expD2 operon, contains 15 aspartate- and glycine-rich nonapeptide repeats, and the structural characteristics of the C-terminal portion of ExpE1 are similar to those of C-terminal secretion signals (Becker et al., 1997
). These features of ExpE1 suggest that this protein is a candidate to be secreted by a transporter complex involving ExpD1 and ExpD2. In this study we have analysed the role of expD1 and expD2 in secretion of ExpE1 and the involvement of the expD1, expD2 and expE1 genes in EPS II production.
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METHODS |
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Construction of expD1, expD2 and expE1 deletion mutants.
The 7633 bp KpnIEcoRI fragment of the exp gene cluster of S. meliloti 2011 (Becker et al., 1997 ) containing a deletion that ranged from nt 36 to nt 1756 of the 1770 nt expD1 coding region (expD1
361756) was cloned into the multiple cloning site (MCS) of suicide vector pK18mobG (Katzen et al., 1999
). Start and stop codons of the remaining expD1 coding region were in-frame due to the presence of some nucleotides from the pK18mobG MCS. The same procedure was followed for the deletion of large parts of the expD2 and expE1 genes. The 4182 bp BglIIEcoRI fragment and the 6292 bp BalIHindIII fragment of the exp gene cluster containing respectively, a deletion ranging from nt 37 to nt 1009 of the 1422 nt expD2 coding region (expD2
371009) and a deletion ranging from nt 2 to nt 616 of the 660 nt expE1 coding region (expE1
2616) were inserted into the MCS of pK19mobG or pK18mobG. The plasmids obtained were designated pLM82-4, pLM87-1 and pLM811-3, characterized by deletions in expD1, expD2 and expE1, respectively. These plasmids were mobilized from strain E. coli S17-1 (Simon et al., 1983
) to S. meliloti 2011. Homogenotization of these mutations was carried out as described by Katzen et al. (1999)
.
Plasmids pLM91-1 and pLM93-2 were obtained by insertion of a 2268 bp PvuIIEcoRI fragment containing expE1 under the control of its own promoter into the broad-host-range vector pSUP104 (Priefer et al., 1985 ) and suicide vector pK18mob (Schäfer et al., 1994
), respectively. These plasmids were transferred to RmLM9835 and RmLM9836. Plasmid pLM811-1 was obtained by cloning a 3927 bp BalI fragment containing expD1 and expD2 under the control of the promoter of the expD operon into vector pK18mobG (Katzen et al., 1999
). This plasmid was mobilized to SmLM9828 and SmLM9832.
To obtain plasmid pExpE7lacZ a promoterless lacZGm interposon (Becker et al., 1995 ) was inserted in sense orientation into the EcoRV site of expE7 cloned into pK18mob. This plasmid was homogenotized to Rm101 (Becker et al., 1997
) and SmLM9909. These strains were grown until OD600 0·50·6 in LB medium and assayed for ß-galactosidase activity as described by Miller (1972)
.
All plasmid integrations into the genome and homogenotizations were verified by Southern hybridizations.
Purification of recombinant ExpE1.
The 980 bp BalI fragment from pAB56-9 (Table 1) containing expE1 was inserted into the BamHI site of pWH844 (Schirmer et al., 1997
) blunted by S1 nuclease. Correct fusion between the expE1 structural gene and the (His)6-encoding region of pWH844 was verified by DNA sequencing. The resulting plasmid was designated pLM75-2 and transferred to E. coli SURE (Stratagene).
Purification of ExpE1 was performed using the QIAexpressionist protein purification system (Qiagen) with some modifications. After lysis, the cell-free extract was loaded onto a nickel-nitrilotriacetic acid (Ni-NTA) agarose column (Superflow) equilibrated in lysis buffer. The column was washed with 6 vols buffer A (50 mM Tris/HCl, 300 mM NaCl, 10%, v/v, glycerin, pH 7·5) and 6 vols buffer B (100 mM Tris/HCl, 500 mM NaCl, pH 8·9). Bound proteins were eluted with buffer B by using a step gradient containing 00·5 M imidazole. Proteins from collected fractions were visualized by vertical SDS-PAGE with 15% acrylamide (Laemmli, 1970 ) stained with Coomassie brilliant blue R250. The most purified fractions of (His)6ExpE1 were pooled, concentrated in an Ultrafree Concentrator (molecular mass cutoff, 10 kDa; Millipore) and then separated on a Sephacryl S200 HR column (16 mm diameter, 600 mm length) according to the manufacturers recommendations (Pharmacia) with buffer B as the mobile phase. The eluted (His)6ExpE1 was associated with a major protein peak as confirmed by SDS-PAGE. The purification of (His)6ExpE1 to electrophoretic homogeneity was achieved by preparative gel electrophoresis (model 491 Prep Cell; Bio-Rad) according to the manufacturers instructions. N-terminal protein sequencing was performed by the Max-Plank-Institut for Molecular Physiology.
Affinity purification of anti-ExpE1 antibodies.
The pure ExpE1 containing the (His)6 tag was used for immunization of rabbits (Eurogentec). The serum obtained was subjected to immunoaffinity purification using the 85 C-terminal amino acids from ExpE1 fused in-frame to the maltose-binding protein (MBP). To obtain this fusion protein, the last 258 nt of expE1 were amplified by PCR (upper primer 5'-GGCAGCGATATCTTCGTT-3'; lower primer 5'-CTATCAGTGGACAGTGAAGTAGTT-3') and cloned into the StuI site of vector pMAL-p (New England Biolabs) resulting in pLM76-4. Purification of MBPExpE1C-term was performed according to a standard protocol (New England Biolabs) with some minor modifications. The osmotic-shock fluid containing the fusion protein was loaded onto an amylose resin column and the buffers used in the washing steps did not include Tween 20. The eluted protein was applied to a SDS-PAGE and electroblotted onto a nitrocellulose membrane. After protein staining with Ponceau S, the band corresponding to the MBPExpE1C-term fusion was removed and used as an immobilized antigen. Antibody affinity purification was performed as described by Harlow & Lane (1988) .
45Ca2+ binding.
To test the calcium-binding activity of ExpE1, the corresponding gene was amplified by PCR (upper primer 5'-ATGGCCACTTTGGAAGG-3'; lower primer 5'-CTATCAGTGGACAGTGAAGTAGTT-3') and cloned in-frame with the MBP gene from pMAL-p, producing pLM76-2. This fusion protein (MBPExpE1) was purified by affinity chromatography as described above. MBP was cleaved from ExpE1 by protease factor Xa (2 d at 4 °C) resulting in the removal of vector-derived residues attached to ExpE1. The protein mixture containing ExpE1 was subjected to SDS-PAGE, transferred onto a nitrocellulose membrane and treated as described by Maruyama et al. (1984) to identify Ca2+-binding proteins.
Immunodetection of proteins.
Proteins separated by SDS-PAGE were electroblotted onto nitrocellulose membranes by the procedure of Towbin et al. (1979) . Immunodetection was performed according to standard protocols (Harlow & Lane, 1988
).
Analysis of ExpE1 in cell extract and culture supernatant.
S. meliloti strains in the late-exponential growth phase (OD600 1·52·0) were grown in MOPS-buffered medium supplemented with 0·1 mM phosphate. Cells were centrifuged for 5 min at 18000 g at 4 °C and the pellets were solubilized in SDS sample buffer to yield a preparation of total cellular proteins. Culture supernatants were separated from the cells by centrifugation (20000 g, 4 °C, 30 min) and a protease-inhibitor cocktail (EDTA free; Boehringer) was added. A volume of 8 ml of each supernatant was concentrated by filtration (Ultrafree Concentrator) and mixed with SDS sample buffer. Preparation of outer-membrane proteins from S. meliloti was performed according to a protocol described by Poxton et al. (1985) . Protein concentrations were determined by using the Bio-Rad Protein Assay with BSA as a standard.
Analysis of extracellular carbohydrates in culture supernatants.
S. meliloti strains were grown at 30 °C for 10 d in MOPS-buffered medium supplemented with 0·1 mM phosphate. Cells were removed by centrifugation (20000 g, 1 h) and the culture supernatants were desalted by dialysis (molecular mass cutoff 1 kDa) against water for 4 d, followed by EPS concentration by lyophilization. High- (HMM) and low-molecular-mass (LMM) fractions were separated by gel-filtration chromatography (twice on GFC 4000-8 and once on GFC 300-8, column size 300x7·7 mm; Machery-Nagel; flow rate, 0·8 ml min-1; 200 mM sodium chloride, 200 mM sodium phosphate buffer, pH 7·0). Fractions were detected using a differential refraction index detector. Total carbohydrates were quantified by the HCl/L-cysteine method (Chaplin & Kennedy, 1986 ). Glucose and galactose content of the HMM and LMM fractions was quantified by enzymic assays after hydrolysis with hydrochloric acid and subsequent neutralization (Beutler, 1984
; Kunst et al., 1984
).
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RESULTS |
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As described for (His)6ExpE1, ExpE1 expressed in E. coli or S. meliloti also migrated abnormally in SDS-PAGE. ExpE1 has a predicted Mr 22117, but it migrated at approximately 30 kDa (Fig. 1b, lanes 1 and 2).
ExpE1 binds Ca2+
Since ExpE1 contains 15 aspartate- and glycine-rich nonapeptide repeats, the ability of ExpE1 to bind Ca2+ was assayed. The (His)6ExpE1 protein could not be used for this assay due to the capacity of the histidine tag to bind divalent cations, therefore the expE1 gene was expressed as a fusion with MBP in E. coli SURE. The E. coli cell extract was subjected to affinity chromatography and we observed, after SDS-PAGE, that beside the fusion protein MBPExpE1 at 64 kDa, another intense band running at approximately 42 kDa was present (Fig. 2a, lane 1) that possibly represented the MBP alone. Such an observation was also reported by Maina et al. (1988)
and can be explained by premature termination or cleavage of the fusion protein. Incubation of this MBPExpE1 eluted fraction with the protease factor Xa resulted in the release of ExpE1. This cleavage gave a 42 kDa band corresponding to the released MBP and a 30 kDa band corresponding to ExpE1 (Fig. 2a
, lane 3). Due to premature termination or cleavage of the fusion protein and some degradation of ExpE1, the concentration of MBP was higher than that of ExpE1 (Fig. 2a
, lane 3). Since the cleavage with factor Xa was not complete, a 64 kDa band corresponding to the non-cleaved MBP-ExpE1 was still present (Fig. 2a
, lane 3). The eluted proteins together with protein cell extract from E. coli expressing the expE1 gene were tested for binding of 45Ca2+. A band migrating at approximately 64 kDa that corresponded to the fusion protein MBPExpE1 (Fig. 2b
, lanes 1 and 3) and a band migrating at 30 kDa representing ExpE1 (Fig. 2b
, lane 3) was detected due to binding of 45Ca2+. In this assay, ExpE1 was also detected in the protein cell extract from E. coli expressing the native ExpE1 protein (Fig. 2b
, lane 4) but no signal was obtained from E. coli cell extract lacking the expE1 gene (data not shown). The observation that in Fig. 2b
, lane 1, we obtain a radioactive signal only for the MBPExpE1 fusion but no signal with the cell extract in which MBP was expressed alone (Fig. 2b
, lane 2), indicates that the binding of Ca2+ is due to ExpE1 and not to other E. coli proteins that may co-migrate with ExpE1.
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Under standard conditions, Rm2011 produces EPS I and only traces of EPS II, if any. However, in a mucR or expR background and under phosphate-limiting conditions, synthesis of EPS II is stimulated (Glazebrook & Walker, 1989 ; Keller et al., 1995
; Zhan et al., 1991
). To overexpress the exp genes leading to the biosynthesis of EPS , the mucR101spc mutation (Becker et al., 1997
) was transferred to each of the deletion mutants. To block biosynthesis of EPS I, a lacZaacC1 insertion in the exoY gene (Keller et al., 1995
) encoding the galactose-1-phosphate transferase that catalyses the first step in the synthesis of the EPS I subunit (Reuber & Walker, 1993
), was additionally transferred to the deletion mutants. The strains obtained were designated SmLM9815 (Rm2011
expD1exoY), SmLM9828 (Rm2011
expD1exoYmucR), SmLM9831 (Rm2011
expD2exoY), SmLM9832 (Rm2011
expD2exoYmucR), SmLM9835 (Rm2011
expE1exoY) and SmLM9836 (Rm2011
expE1exoYmucR) (Table 1
). Blocking EPS I and EPS II production was necessary to enable the high-efficiency removal of cells from culture supernatants and to facilitate the concentration of proteins. Therefore, strains RmAR1157 (Rm2011 expA1exoY) and SmLM8311 (Rm2011 expA3exoYmucR) were used to determine if ExpE1 is secreted. Detection of ExpE1 in the supernatant of S. meliloti strains was additionally complicated by proteolysis. Several bands migrating at sizes lower than 30 kDa reacted with the anti-ExpE1C-term antibody (data not shown). This effect was enormously reduced by adding a protease-inhibitor cocktail to the culture supernatant immediately after removal of the cells.
RmAR1157 grown under phosphate-limiting conditions was assayed for the presence of ExpE1 in cells and supernatant using the anti-ExpE1C-term antibody. The result indicated that ExpE1 is present both in the cell extract (Fig. 3a, lane 1) and the supernatant (Fig. 3b
, lane 1). This implies that ExpE1 is secreted into the growth medium.
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Since ExpE1 is predicted to be secreted via a signal peptide-independent pathway, it was tested whether this protein is secreted via a transport system involving ExpD1 and ExpD2 that display homologies to components of type I protein-secretion complexes (Becker et al., 1997 ). The double mutants SmLM9815 (Rm2011
expD1exoY) and SmLM9831 (Rm2011
expD2exoY) grown under phosphate-limiting conditions were tested for the presence of ExpE1 in cells and supernatants. Immunodetection showed the absence of ExpE1 in the growth medium (Fig. 3b
, lanes 2 and 3) of both mutants, indicating that ExpE1 is secreted by a transporter system involving ExpD1 and ExpD2. The protein was found in the cell extract of SmLM9831 (Rm2011
expD2exoY) (Fig. 3a
, lane 3), but curiously only a very weak signal at the detection limit was observed in the cell extract of SmLM9815 (Rm2011
expD1exoY) (Fig. 3a
, lane 2). The ß-galactosidase activity of a translational expE1lacZ fusion expressed in the wild-type background was comparable to the values obtained in the expD1 or expD2 mutant. This implies that the deletions in expD1 and expD2 did not influence the transcription or translation of expE1. ExpE1 was not detected in the cells or the supernatant of SmLM9835 (Rm2011
expE1exoY) (Fig. 3a
and 3b
, lanes 4).
To restore the ability of SmLM9835 to express ExpE1, pLM91-1 and pLM93-2 carrying the expE1 gene under the control of its own promoter were transferred to SmLM9835. Strain SmLM9901 contained the replicating plasmid pLM91-1 whereas strain SmLM9903 carried plasmid pLM93-2 integrated into the genome. The ExpE1 protein was detected in cell extracts and culture supernatants of both strains (Fig. 3a, b
, lanes 5 and 6). The introduction of the multicopy plasmid pLM91-1 in SmLM9815 (SmLM9906) resulted in the detection of ExpE1 in the cell extract, but not in the supernatant (0. 3a
, b
, lanes 7). Since a very small amount of ExpE1 protein was detected in the cell fraction of SmLM9815, accumulation of ExpE1 in this mutant might only occur if it is overexpressed. Secretion of ExpE1 was also assayed in the deletion mutants carrying additionally an exoY mutation and a mucR mutation that stimulated the expression of the exp genes. The results of ExpE1 detection in these triple mutants were consistent with those described above, with the exception of strain SmLM9828 (Rm2011
expD1exoYmucR), in which ExpE1 was detected in the cells (data not shown).
ExpD1, ExpD2 and ExpE1 are required for the synthesis or secretion of EPS II
To study the involvement of the secreted protein ExpE1 and its transport system in EPS II secretion, S. meliloti strains were used that carried mutations in exoY and mucR and were grown in low-phosphate medium. Under these conditions, exp genes are maximally expressed (Rüberg et al., 1999 ). Using gel-filtration chromatography, HMM and LMM fractions present in the culture supernatants of strains carrying deletions in expE1, expD1 and expD2 were separated. No HMM fraction was detected in the culture supernatants of expE1, expD1 and expD2 deletion mutants indicating the absence of extracellular HMM-EPS II (Table 2
).
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The total sugar content of the EPS fractions was determined (Table 2). A high sugar content in the culture supernatant correlated with the detection of a HMW fraction with the exception of strain SmLM9832. In the culture supernatant of this strain a LMW fraction was exclusively detected. In this LMW fraction glucose but no galactose was detected, implying that strain SmLM9832 secreted no LMW EPS II and might have secreted a high amount of cyclic glucans. In the culture supernatants of strains SmLM9828 and SmLM9836 a LMW fraction containing a low amount of total sugars was exclusively detected (Table 2
). To confirm that the HMW fraction produced by the strains secreting ExpE1 contained EPS II, the glucose and galactose content was quantified. The HMW fractions from RmAR9007 TD101, SmLM9836::pLM93-2, SmLM9828:: pLM811-1 and SmLM9832::pLM811-1 contained glucose and galactose in the ratio of approximately 1:1 (Table 2
), indicating that these fractions contained EPS II. Glucose, but no galactose was detected in the LMW fractions of strains SmLM9828 and SmLM9836, indicating the possible presence of cyclic glucans but no LMW EPS II as was already suggested for the LMW fraction of mutant SmLM9832 (Table 2
).
Since ExpE1 might function extracellularly in EPS II biosynthesis or secretion, we tested if ExpE1 could be supplied in trans to stimulate HMW-EPS II production by mutant SmLM9836. This strain, which is unable to produce EPS II due to the deletion of expE1 was co-cultivated with SmLM8311, which secretes ExpE1 but does not produce EPS II due to a mutation in expA3 encoding a glycosyltransferase. After 10 d growth in low-phosphate medium, no HMW fraction was detected in the culture supernatant (Table 2). This was also confirmed by the low sugar content in the culture supernatant.
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DISCUSSION |
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An amphipathic -helix close to the C terminus and 15 Ca2+-binding domains were predicted for ExpE1 (Becker et al., 1997
). The repeat sequence GGXGXDXUX (where U is any hydrophobic amino acid) was found in most of the members of a family of bacterial proteins that are secreted by type I secretion systems (Binet et al., 1997
). The three-dimensional structure of several of these secreted proteins demonstrated that the repeated motif binds Ca2+ in a parallel ß-roll structure (Baumann et al., 1993
; Baumann, 1994
). Baumann et al. (1993)
suggested that the Ca2+-binding regions may have a role in folding of the molecule after transmembrane translocation. In the absence of Ca2+ this structure is unstable and could facilitate membrane translocation of the polypeptide in an unfolded form. The presence of Ca2+ in the extracellular medium could induce the polypeptide to fold in the right tertiary structure. Another possible role for the calcium domains was proposed for haemolysin A and the adenylate cyclase toxin from Bordetella pertussis (Bakás et al., 1998
; Rose et al., 1995
). In both cases, it was suggested that binding of Ca2+ may induce a conformational change that would result in surfacing hydrophobic portions of the protein. The increased hydrophobic surface probably favours cell-membrane binding that appears to be essential for the lytic process.
The Mr estimated by SDS-PAGE for ExpE1 or (His)6ExpE1 showed an abnormal value. In both cases, this value was 810 kDa higher than the Mr predicted by its amino acid sequence. The discrepancy in the predicted and observed Mr in SDS-PAGE was also reported for NodO from R. leguminosarum, a protein that shows some sequence similarity with ExpE1 (Economou et al., 1990 ) and other acidic proteins (Kaufman et al., 1984
). Size fractionation of the (His)6ExpE1 by gel filtration indicated that this protein purifies as a dimer. The same observation was also made for NodO (Economou et al., 1990
).
expD1 and expD2 were required for the secretion of ExpE1. Since ExpD1 and ExpD2 are homologous to ABC transporters and membrane-fusion proteins, respectively, of type I secretion systems, we propose that ExpE1 is secreted by a type I secretion system involving ExpD1 and ExpD2. In a genetic background allowing strong expression of expE1, ExpE1 accumulated in the cell if expD1 or expD2 were deleted. Under conditions allowing a medium level expression of expE1 this was also the case in an expD2 mutant but not in an expD1 mutant. Since expression of an expE1lacZ translational fusion was not affected in the expD1 deletion mutant, the low amount of ExpE1 detected might have resulted from degradation. Hence, the absence of ExpD1 may destabilize or its presence may stabilize ExpE1 in the cell. It is possible that ExpE1 was also degraded in an expD1 mutant when it was overexpressed, but due to the high amount of ExpE1 produced, this degradation might not have occurred at a relevant level.
The expE1, expD1 and expD2 genes were shown to be required for the synthesis or secretion of EPS II. Since secretion of ExpE1 required expD1 and expD2, ExpD1 and ExpD2 may be indirectly required for EPS II production. A direct involvement of ExpD1 and ExpD2 in EPS II production independent of their function in secretion of ExpE1 might be the secretion of EPS II or its precursors. An involvement of type I secretion systems in the secretion of polysaccharides was described for S. meliloti and Agrobacterium tumefaciens cyclic (1,2)-ß-glucans and E. coli K1 capsular polysaccharide (Bliss & Silver, 1996 ; Breedveld & Miller, 1994
). The NdvA and ChvA proteins from S. meliloti and A. tumefaciens share homologies with bacterial ABC transporter proteins, and mutants in these genes did not secrete cyclic (1,2)-ß-glucans to the periplasmic compartment and to the extracellular medium. In E. coli K1, sialic acid is synthesized and polymerized in the cytoplasm, and linking of the synthetic and transport pathways is possibly performed by the proteins KpsC and KpsS that may escort the polySia chains to the transport machinery. This machinery is composed of a type I secretion system comprising KpsM, KpsT and KpsE, that together with the periplasmic protein KpsD and an unknown outer-membrane protein, are responsible for the translocation of the capsular polysaccharide across the inner and outer membranes. Since ExpD1 and ExpD2 resemble components of type I protein-secretion systems and we have demonstrated that these two proteins are required for secretion of ExpE1, it appears rather unlikely that the ExpD1/ExpD2 type I transporter system is additionally involved in the secretion of a polysaccharide or polysaccharide precursors.
Supplying ExpE1 in trans in a co-cultivation experiment did not restore the ability of a S. meliloti expE1 mutant to produce extracellular EPS II. The ExpE1 protein might not be stable enough in the culture medium to correct the deficiency of an expE1 mutant in EPS II synthesis or to fulfil its function ExpE1 has to be synthesized and secreted by the cell that synthesizes EPS II. ExpE1 is similar to NodO from R. leguminosarum which was found to insert into liposomes and when added to planar lipid bilayers, formed cation-selective channels that allowed the movement of monovalent cations across the membrane (Sutton et al., 1994 ). Sutton et al. (1994)
suggested that the role of NodO in nodulation might be the formation of pores in the plant plasma membrane. ExpE1 and NodO mainly consist of the nonapeptide repeats and the C-terminal 85 aa of ExpE1 display no similarities to NodO. Therefore, functions of these two proteins cannot necessarily be suggested based on these sequence similarities.
Since ExpE1 was not detected in the outer-membrane fraction, it seems to be rather unlikely that ExpE1 forms pores in this membrane that are involved in the secretion of EPS II. On the other hand, for technical reasons ExpE1 was recovered from cells and supernatants of S. meliloti strains that were unable to produce EPS I and EPS II. Therefore, we cannot exclude the possibility that the localization of ExpE1 in the wild-type depends on the presence of EPS I or EPS II. Weisgerber & Troy (1990) reported that immunoprecipitated polySia obtained from labelled E. coli K1 membranes was found to be associated with a protein of 20 kDa which has an unknown function. Hence, the hypothesis that ExpE1 mediates the secretion of EPS II by binding to the nascent EPS II chain might be conceivable. Although it was shown that ExpE1 is required for EPS II synthesis or secretion, the role of this protein and its transport system in EPS II production remains unknown, and further studies will be required to understand their function.
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ACKNOWLEDGEMENTS |
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Received 12 May 2000;
accepted 19 May 2000.
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