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Journal of Bacteriology, October 2006, p. 6943-6952, Vol. 188, No. 19
0021-9193/06/$08.00+0     doi:10.1128/JB.00651-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: Subcellular Localization and Possible Involvement in Exopolysaccharide Export

Malgorzata Marczak,1 Andrzej Mazur,1 Jaroslaw E. Król,1 Wieslaw I. Gruszecki,2 and Anna Skorupska1*

Department of General Microbiology, Institute of Microbiology and Biotechnology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland,1 Department of Biophysics, Institute of Physics, Maria Curie-Sklodowska University, Maria Curie-Sklodowska Sq. 1, 20-031 Lublin, Poland2

Received 8 May 2006/ Accepted 15 July 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Surface expression of exopolysaccharides (EPS) in gram-negative bacteria depends on the activity of proteins found in the cytoplasmic membrane, the periplasmic space, and the outer membrane. pssTNOP genes identified in Rhizobium leguminosarum bv. trifolii strain TA1 encode proteins that might be components of the EPS polymerization and secretion system. In this study, we have characterized PssN protein. Employing pssN-phoA and pssN-lacZ gene fusions and in vivo acylation with [3H]palmitate, we demonstrated that PssN is a 43-kDa lipoprotein directed to the periplasm by an N-terminal signal sequence. Membrane detergent fractionation followed by sucrose gradient centrifugation showed that PssN is an outer membrane-associated protein. Indirect immunofluorescence with anti-PssN and fluorescein isothiocyanate-conjugated antibodies and protease digestion of spheroplasts and intact cells of TA1 provided evidence that PssN is oriented towards the periplasmic space. Chemical cross-linking of TA1 and E. coli cells overproducing PssN-His6 protein showed that PssN might exist as a homo-oligomer of at least two monomers. Investigation of the secondary structure of purified PssN-His6 protein by Fourier transform infrared spectroscopy revealed the predominant presence of ß-structure; however, {alpha}-helices were also detected. Influence of an increased amount of PssN protein on the TA1 phenotype was assessed and correlated with a moderate enhancement of EPS production.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rhizobia secrete polysaccharides with little or no cell association, termed exopolysaccharides (EPS). They have been shown to be critical factors in symbiotic interactions of rhizobia with leguminous plants that form the indeterminate type of nodules (2). Accumulating data suggest that exopolysaccharides may be involved in several steps of nodule development, such as invasion, bacterial release from infection threads, bacteroid development, suppression of plant defense response, and protection against plant antimicrobial compounds (15, 67).

Surface polysaccharides of Escherichia coli have provided models for synthesis and translocation of extracellular polysaccharides in other bacteria (72, 73). There are two pathways used for assembly of capsules in gram-negative bacteria (73). The first involves polymerization of sugar precursors at the inner leaflet of the inner membrane and the subsequent transport of the nascent polymer through the inner membrane by an ABC-2 transporter (5). In the case of group 1 and 4 capsules, undecaprenol diphosphate-linked oligosaccharide repeat units are formed at the cytoplasmic face of the inner membrane, flipped across the inner membrane, and polymerized in a process involving Wzx and Wzy proteins (54, 72). The length of the polymer chain is controlled by Wzc protein, a member of the cytoplasmic membrane periplasmic auxiliary (MPA1) family of proteins (52). Wzc is a tyrosine autokinase, which is dephosphorylated by its cognate phosphatase Wzb; Wzc cycling between phosphorylated and dephosphorylated forms is thought to be crucial for its function (74). Wza protein, a member of the outer membrane auxiliary (OMA) family of proteins, was shown to be a component of the capsule translocation machinery forming ring-like channel structures (12, 49). In the polymerization and translocation of succinoglycan (EPS I) produced by Sinorhizobium meliloti, ExoT, ExoQ, and ExoP proteins are involved (21). ExoT shows similarity to Wzx proteins and is involved in synthesis/secretion of the low-molecular-weight EPS I, while ExoQ (Wzy-like protein) is indispensable for production of high-molecular-weight succinoglycan (21). ExoP is an autophosphorylating protein tyrosine kinase and was proposed to have a dual role in EPS biosynthesis: enzymatic, by catalyzing the formation of dimers of EPS I octasaccharide units; and structural, by forming a complex with ExoQ and ExoT proteins (21, 50, 59).

pssTNOP and pssL genes identified in Rhizobium leguminosarum bv. trifolii strain TA1 encode proteins that might constitute similar polymerization/translocation machinery (44). PssT, an inner membrane protein with homology to Wzy, most probably controls EPS polymerization (42). PssP protein (MPA1 family) comprises 2 transmembrane (TM) segments and a large periplasmic loop, which shows high propensity for folding into coiled-coil structures possibly engaged in interactions with the outer membrane component of the EPS transporter. PssP is required for exopolysaccharide biosynthesis and determination of the degree of EPS polymerization (43). PssL is a Wzx-like protein that most probably acts in flipping the lipid-linked oligosaccharides to the outer leaflet of the cytoplasmic membrane (44).

Sequence similarity searches identified the PssN protein, the focus of this study, as a putative lipoprotein (41), a member of the OMA family of proteins (52) that might act in translocation of polysaccharides through the outer membrane. Except for the in silico predictions, the precise role of PssN remains unknown. The main goal of the present study was to establish the subcellular localization of PssN in TA1 and to examine its structural/functional features indicated by computer analyses.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains, plasmids, media, and growth conditions. The bacterial strains, plasmids, and primers used in this study are listed in Table 1. Escherichia coli strains were cultured in Luria-Bertani (LB) medium at 37°C (61) and Rhizobium strains were grown in TY medium at 28°C (3), unless otherwise stated. Antibiotics were used at the following final concentrations: ampicillin, 100 µg ml–1; kanamycin, 40 µg ml–1 [except for 25 µg ml–1 used for propagation of E. coli M15(pREP4)]; and gentamicin, 10 µg ml–1 for Rhizobium and 5 µg ml–1 for E. coli.


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  		TABLE 1. Strains, plasmids, and primers used in this study

 
DNA and bioinformatics methods. For plasmid and total DNA isolation, restriction enzyme digestion, agarose electrophoresis, and cloning, standard techniques were employed (61). Sequencing was performed using BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystem) and ABI PRISM 310 sequencer. Sequence data were analyzed with DNASTAR-Lasergene analysis software. Database searches were performed using the FASTA program (53) available at the European Bioinformatics Institute (Hinxton, United Kingdom). Signal sequence predictions were done with the DOLOP (39) and Lipo P 1.0 (30) programs. Secondary structure and topology predictions were done with SSCP (13) and Pred-TMßß (1). For prediction of subcellular localization, the PSORTb v.2.0 (17), CELLO v.2.5 (75), and PSLpred (4) programs were used.

Overproduction and purification of PssN-His6 protein. For purification of PssN with a C-terminal His6 tag, a DNA fragment matching the whole pssN gene was amplified by PCR using the PssNFwSph and PssNRvC primers (Table 1) and pUC98 plasmid as a template. The amplification product was cloned into the SphI and BglII sites of pQE70 vector (Qiaexpress; QIAGEN), resulting in pQC32 plasmid. The fusion junctions were verified by DNA sequencing. E. coli strain M15 was used for propagation of the pQC32 plasmid. Overproduction of PssN-His6 protein was induced with 0.5 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) for 4 h. Cell lysates were subjected to chromatography on Ni-nitrilotriacetic acid (NTA) agarose (QIAGEN) according to the manufacturer's instructions. Fractions containing PssN-His6 protein were pooled and concentrated by ultrafiltration. The procedure allowed isolation of approximately 0.2 mg of PssN-His6 protein from 500 ml of E. coli M15 culture.

FTIR. Samples for Fourier-transform infrared spectroscopy (FTIR) contained 50 µg ml–1 PssN-His6 protein in H2O. Infrared absorption spectra were recorded with an FTIR spectrometer from Bruker Optik, Germany; model Vector 33, supplied with an attenuated total reflection accessory. Before and during the measurements, the instrument was purged with argon. The protein was deposited on a ZnSe crystal support in the form of a partially dried layer. Typically 30 interferograms were collected, Fourier transformed, and averaged. Absorption spectra in the region between 4,000 and 600 cm–1, at a resolution of 1 data point at every 0.6 cm–1, were obtained using a clean crystal as the background. Spectral analysis was performed with Grams32AI software (version 7.2) from ThermoGalactic. All measurements were done at 20°C. Determination of the secondary structure of the protein was based on data from the literature (68).

Anti-PssN antibody preparation. For preparation of polyclonal anti-PssN antibodies, PssN carrying N-terminal His6 tag was purified. A DNA fragment covering the pssN gene lacking the first 102 nucleotides (the predicted signal sequence) was amplified by PCR using Pfu polymerase, PssNFw and PssNRv primers (Table 1), and pUC98 plasmid as a template. The PCR product was cloned into pQE32 vector (Qiaexpress; QIAGEN), resulting in pQN42. The fusion junction was verified by DNA sequencing. His6-PssN fusion protein was purified under denaturing conditions from E. coli JM101(pQN42) according to the supplier's instructions. Polyclonal anti-PssN serum was prepared essentially as described by Janczarek and Skorupska (26).

Immunoblotting. Proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were electroblotted onto polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore). The detection procedure was performed as described by de Maagd et al. (10). Polyclonal goat anti-rabbit and anti-mouse antibodies conjugated with alkaline phosphatase, mouse anti-His5 antibodies, and goat anti-alkaline phosphatase antibodies were purchased from Sigma, QIAGEN, and Polysciences, respectively. Bands were visualized with nitroblue tetrazolium chloride-5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Roche).

Construction of pssN-phoA/lacZ gene fusions and reporter enzyme assays. For each fusion, a forward primer annealing upstream of the pssN promoter sequence (NphoAFw or NlacZFw) and one of the reverse primers (NphoA53, NlacZ53, NphoA149, or NlacZ149) annealing within the pssN coding region were used to amplify DNA fragments that were further cloned into BamHI and HindIII sites of the reporter plasmids pUCphoA and pNM480. The fusion plasmids given in Table 1 were named after the position of the last PssN residue in the in-frame fusion. Fusion junctions in all of the plasmids were verified by DNA sequencing. Measurements of reporter enzyme activities were performed in E. coli strain ET8000. The alkaline phosphatase activity assay was conducted according to a method of Manoil (40), and that of ß-galactosidase was performed essentially as described by Miller (47), with the activity expressed in Miller units.

In vivo acylation with [3H]palmitate. E. coli M15(pQC32) was grown in LB medium to an optical density at 600 nm (OD600) of 0.5. The cells were then washed with an equal volume of M1 minimal medium (61), resuspended in the same volume of M1, supplemented with 1% (wt/vol) Casamino Acids, and incubated with vigorous shaking at 37°C for 0.5 h. Following that, the overproduction of PssN-His6 protein was induced with 0.5 mM IPTG (final concentration). Simultaneously, [3H]palmitic acid (specific activity, 45 Ci mmol–1; MP Biomedicals, Inc.) was added to the culture (20 µCi ml–1 final concentration). After 4 h, the cells were harvested and resuspended in 2x SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membrane, followed by immunodetection with anti-His5 antibodies and exposition onto X-ray film for 3 weeks at –70°C.

Isolation of TA1 total membrane fraction and sucrose density gradient centrifugation. Cell membrane isolation was performed essentially as described by Que-Gewirth et al. (58). TA1 cells from a 500-ml culture in TY medium were resuspended in lysis buffer (50 mM HEPES, 0.5 mM EDTA, pH 7.5) and lysed by three passages through a French press (SLM-Amico Instruments, Thermo Spectronic, Rochester, N.Y.) at 18,000 lb/in2. The membranes were recovered by ultracentrifugation at 100,000 x g for 1 h. The soluble protein fraction (the supernatant) was retained. The membranes (the pellet) were homogenized in 1 ml of lysis buffer, layered on top of a seven-step sucrose gradient described by Guy-Caffey et al. (22), and centrifuged for 20 h at 114,000 x g. Efficacy of the membrane isolation procedure was confirmed by measuring the activity of NADH oxidase (51) and the content of lipopolysaccharide (LPS) by immunoblotting with anti-LPS serum specific for strain TA1 (27). The turbidity (OD600) of each fraction was measured to confirm the presence of membrane fragments.

Indirect immunofluorescence. Binding of anti-PssN polyclonal antibodies to intact TA1 cells and spheroplasts was investigated by indirect immunofluorescence assay according to the method described by Hu et al. (24). The TA1 strain was grown in TY medium to an OD600 of 0.6 to 0.8 and washed twice with phosphate-buffered saline (PBS) buffer. An aliquot of washed cells was converted into spheroplasts by incubation in a mixture of 50 mM Tris-Cl, 20% sucrose, 5 mM EDTA, and 2 mg ml–1 lysozyme (1 h on ice). Intact cells and the spheroplasts were resuspended in PBS with 1% (wt/vol) bovine serum albumin or PBS with 1% bovine serum albumin and 20% (wt/vol) sucrose, respectively, and incubated at room temperature for 1 h (blocking step). The cells and spheroplasts were subsequently incubated with polyclonal anti-PssN serum (1:1,000), followed by incubation with anti-rabbit immunoglobulin G (IgG) antibodies conjugated with fluorescein isothiocyanate (FITC) (1:150 dilution). Each incubation was followed by three extensive washes with PBS (or PBS-20% sucrose in the case of spheroplasts). Ten microliters of cell suspension was transferred onto a microscopic slide covered with poly-L-lysine and air dried. Five microliters of 50% glycerol was then applied onto the slide, covered with coverslip, and examined under a confocal microscope (Zeiss LSM 5 Pa Axiovert 200 M) at a x1,000 magnification. Control samples were incubated only with the secondary, FITC-conjugated, antibodies. Five representative fields were examined for each specimen.

Construction of plasmid vector bearing pssN under the control of the psyn promoter. A DNA fragment containing the pssN open reading frame (starting with ATG initiating codon and ending with an in-frame stop codon) was PCR amplified using Pfu polymerase, PssNFwSph and PssNRvSt primers, and pUC98 plasmid as a template. The product was cloned into SmaI and BamHI sites of pK19mobGII vector, resulting in pKN12. Then, the SalI-XbaI fragment from pBKD11 plasmid, carrying the psyn promoter-operator cartridge and lacIq gene, was cloned into pKN12, resulting in pKN12-PO, which was then digested with SalI-SacI. The fragment harboring the pssN gene under the control of the psyn promoter was cloned into pBBRMCS-5, giving plasmid pWLN1. The plasmid was transferred to the TA1 wild-type strain by electroporation (18).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of pssN gene and purification of His6-tagged PssN protein. PssN was overproduced in E. coli as an N-terminally His6-tagged protein, purified under denaturing conditions (data not shown) and used for immunization of rabbits. For the analysis of PssN secondary structure, its C-terminally His6-tagged derivative was overproduced and purified under native conditions. The presence of a His-tagged epitope in eluted proteins was confirmed by Western blotting with anti-His5 antibodies (data not shown). The latter fusion protein had a molecular mass of approximately 43.5 kDa (Fig. 1), which corresponded to a prediction allowing for both the cleavage of the putative lipoprotein signal sequence and the extension of the C terminus with the His tag.


Figure 1
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  		FIG. 1. Overproduction of PssN in E. coli as a C-terminally His6-tagged protein. Proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue. Lanes: 1, proteins from uninduced E. coli M15(pQC32) cells; 2, proteins after induction with IPTG; 3, PssN-His6 protein eluted from Ni-NTA agarose. Molecular mass standards (kDa) are shown to the left.

 
Occurrence and secondary structure of PssN protein. Database searches revealed the presence of PssN protein homolog-encoding genes in the genomes of many gram-negative bacteria, including Rhizobium (Table 2). The majority of these proteins have been classified as OMA, and the respective genes are found in clusters coding for proteins engaged in transport of exopolysaccharides and capsular polysaccharides (52). We evaluated the occurrence of PssN-like proteins cross-reacting with anti-PssN antibodies in different rhizobial species. In all of the examined species, specific bands that corresponded in size to PssN protein (i.e., ~43 kDa) were observed (Fig. 2).


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  		TABLE 2. Homologues of PssN protein identified in gram-negative bacteria by FASTA searches

 

Figure 2
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  		FIG. 2. Occurrence of PssN-like proteins in different rhizobial species analyzed by Western immunoblotting with anti-PssN serum. Lanes: 1, R. leguminosarum bv. trifolii TA1; 2, R. leguminosarum bv. viciae 3841; 3, Rhizobium etli USDA9032; 4, Agrobacterium tumefaciens GMI9023; 5, S. meliloti SU47; 6, M. loti HAMBI1129; 7, Bradyrhizobium japonicum USDA110.

 
The OMA proteins are proposed to be ß-barrel proteins that transport polysaccharides across the outer membrane (OM) (52). PssN secondary structure analysis revealed the presence of 7.4 to 15.8% {alpha}-helices, 34.4 to 36.9% ß-sheets, and 47.3 to 58.2% turns. In a predicted topological model of PssN, the protein comprises two transmembrane (TM) ß-strands at amino acid positions 37 to 45(47) and 57(61)-69(71), with an external loop between them and the C-terminal part of the polypeptide exposed to the periplasm. We investigated the secondary structure of PssN-His6 protein by FTIR (7). Analysis of the spectra yielded the following approximate values: 15% {alpha}-helices (maximum at 1,660 cm–1), 43% ß-sheets (maximum at 1,681 cm–1 and 1,649 cm–1), 40% turns (maximum at 1,669 cm–1), and 2% aggregated structures (maximum at 1,634 cm–1) (Fig. 3). Although the presence of His6 tag in a recombinant protein may result in minor deviations in the recorded secondary structure content from the wild-type protein (49), the results were in good agreement with the secondary structure predicted in silico and indicated that PssN may not be a typical ß-barrel-shaped protein.


Figure 3
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  		FIG. 3. Secondary structure of recombinant PssN-His6 protein. The graph represents the FTIR spectrum of PssN-His6 in the amide I region. The original spectrum (thick line) is presented along with the Gaussian deconvolution components (thin lines). Numbers over each component refer to the position of the maximum (cm–1) corresponding to particular secondary structures detected: i.e., 1,669 cm–1 for turns, 1,681 cm–1 and 1,649 cm–1 for ß-sheets, 1,660 cm–1 for {alpha}-helices, and 1,634 cm–1 for aggregates. A contribution of individual secondary structure to the spectrum, representing a fraction of each form, was calculated as a surface beneath a spectral component.

 
PssN is a lipoprotein. In silico analysis of PssN predicted the presence of an N-terminal signal peptide that conforms to the rules established for prokaryotic lipoprotein signal peptides (30, 70). Similarly to many members of the OMA family, PssN has a carboxy-terminal signal peptidase II cleavage site (lipobox "LASC"). We verified PssN trafficking to the periplasm due to its N-terminal signal by employing a reporter gene fusion approach. Four fusion plasmids with 5' segments of pssN gene and promoterless and signal sequenceless phoA or promoterless lacZ genes were constructed (Fig. 4A). Reporter enzyme assays in E. coli ET8000 producing hybrid proteins revealed a high level of alkaline phosphatase activity but no detectable ß-galactosidase activity (Fig. 4B). In Western analysis with anti-alkaline phosphatase antibodies, two hybrid proteins of predicted molecular weights were detected (Fig. 4C). These results evidenced that the putative signal sequence detected in the N-terminal part of PssN indeed promoted translocation of the protein to the periplasm.


Figure 4
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  		FIG. 4. Overview of pssN gene fusion experiment. (A) Physical and genetic map of the R. leguminosarum bv. trifolii TA1 region encompassing the pssT and pssN genes. E, EcoRI; P, PstI; H, HindIII, K, KpnI. PCR products covering the pssN gene fragments shown underneath were cloned into reporter plasmids, pUCphoA and pNM480. (B) Alkaline phosphatase and ß-galactosidase activities for the PssN-PhoA and PssN-LacZ hybrid proteins assayed in E. coli strain ET8000; values given are the averages from at least three independent experiments; For background, the PhoA and LacZ activities in ET8000(pUCphoA) and ET8000(pNM480), respectively, are given. (C) Western blotting analysis with anti-alkaline phosphatase antibodies of E. coli ET8000 expressing PssN(53)-PhoA and PssN(149)-PhoA hybrid proteins. Positions of molecular mass standards (kDa) are shown at the left.

 
To obtain biochemical evidence for lipid modification of PssN protein, we cultured the wild-type TA1 strain in the presence of [3H]palmitate; however, the efforts to label PssN in TA1 cells did not result in detectable labeling. Thus, E. coli M15(pQC32) was labeled and the crude cell lysate was subjected to SDS-PAGE, immunoblotting, and autoradiography. The cells carrying pQE70 vector were labeled and analyzed as well, to distinguish PssN-His6 from other labeled proteins. The autoradiograms revealed the presence of a labeled band with a size of ~43.5 kDa (Fig. 5A and B, lane 2). The band corresponded to the recombinant PssN-His6 and was missing from the control strain carrying a vector without the pssN insert (Fig. 5A and B, lane 1), thus confirming that PssN is lipid modified in E. coli.


Figure 5
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  		FIG. 5. In vivo acylation of PssN protein in E. coli cells. Total proteins isolated from E. coli M15 labeled with [3H]palmitate were probed with anti-His5 antibodies (A) and exposed to X-ray film (B). Lanes in both panels A and B are as follows: lane 1, E. coli M15(pQE70), lane 2, E. coli M15(pQC32). Positions of molecular mass (kDa) standards (M) are shown to the left.

 
PssN is outer membrane associated. Fatty acid acylation at the amino terminus serves to tether most lipoproteins to the membrane. Western blot analysis of fractionated TA1 cells revealed PssN to be confined mainly to the membrane fraction (TM), although some protein was also detected in the soluble fraction (S) (Fig. 6A). Whether the lipoprotein will be directed to the outer membrane or retained in the inner membrane depends on the identity of the amino acid following the invariant cysteine. Typically, lipoproteins lacking an Asp residue at position +2 of the mature protein are localized to the outer membrane (64). A +2 Thr residue in PssN supported its outer membrane association predicted in silico by the PSORTb, CELLO, and PSLpred programs. To determine the location of PssN in the particular membrane, detergent extraction of total membranes was performed (14, 24). After sarcosyl and Triton X-100 treatment, PssN was found to predominate in detergent-insoluble fractions (OM) (Fig. 6B and C). Second, total membranes were resolved into inner and outer membrane fractions by sucrose density gradient centrifugation. NADH oxidase activity was concentrated in the upper, inner membrane fractions, with little activity in the lower fractions (Fig. 7A), while LPS was concentrated in the lower, outer membrane fractions (Fig. 7B), confirming the efficiency of fractionation. Western blotting with anti-PssN antibodies revealed that PssN protein cofractionated mainly with the outer membrane (Fig. 7C).


Figure 6
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  		FIG. 6. Subcellular localization of the PssN protein in R. leguminosarum bv. trifolii TA1. Western blotting analysis of TA1 cell fractions: (A) S, fraction containing soluble cytoplasmic and periplasmic proteins; TM, total membrane fraction containing inner and outer membrane proteins. (B) IM, sarcosyl-soluble proteins; OM, sarcosyl-insoluble proteins. (C) IM, Triton X-100-soluble proteins; OM, Triton X-100-insoluble proteins. The samples have been standardized so that they represent equivalent numbers of cells.

 

Figure 7
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  		FIG. 7. Subcellular localization of the PssN protein in R. leguminosarum bv. trifolii TA1. Total membranes of TA1 were separated into inner and outer membranes by isopycnic sucrose density gradient centrifugation. Fractions of 0.6 ml were collected from the top of the gradient and assayed for the presence of membrane fragments (OD600) ({blacksquare}) (A); the activity of an inner membrane marker enzyme, NADH oxidase (expressed in µmol min–1 ml–1) ({blacktriangleup}) (A); the presence of LPS by Western blotting with anti-LPS polyclonal serum specific for TA1 (B); and the presence of PssN protein by Western blotting with anti-PssN antibodies (C).

 
PssN forms oligomers. PssN protein's propensity to form oligomeric structures was investigated by means of in vivo chemical cross-linking with formaldehyde (55, 65). Formaldehyde treatment of TA1 cells generated species migrating as ~85-kDa, ~130-kDa, and ~170-kDa bands in addition to the 43-kDa-band of PssN protein. These forms corresponded to two, three, and four times the molecular mass of PssN (Fig. 8). After formaldehyde cross-linking of E. coli M15(pQC32) cells and PssN-His6 purification, two bands corresponding to monomeric and tetrameric form of the protein were detected on SDS-PAGE gels and immunoblots of protein fractions eluted from the Ni-NTA agarose resin (Fig. 8).


Figure 8
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  		FIG. 8. Analysis of PssN oligomerization. For in vivo cross-linking of PssN, R. leguminosarum bv. trifolii TA1 and E. coli M15(pQC32) cells were treated with 1% formaldehyde and proteins were analyzed by Western blotting. (Left panel) TA1 whole-cell lysates. (Right panel) Purified PssN-His6 protein. The numbers 37 and 100 indicate the temperature (°C) of incubation in sample buffer prior to electrophoresis. The latter treatment causes the release of cross-links in the case of formaldehyde-treated samples. Positions of monomeric, dimeric, trimeric, and tetrameric forms of PssN are indicated as N1, N2, N3, and N4, respectively. Positions of molecular mass standards (in kDa) are indicated to the left.

 
Proteins involved in EPS synthesis and transport are proposed to form heterocomplexes. We tested whether PssP protein involved in EPS polymerization was contributing to the oligomeric structures observed in a case of TA1 cross-linking. Di-, tri-, and tetrameric forms of PssN were found in R. leguminosarum bv. trifolii strain P22 (Exo– pssP mutant) cells treated with formaldehyde (data not shown). These forms did not differ in their electrophoretic mobility from those observed in TA1. This suggested that PssP protein did not contribute to the observed complexes; however, it did not exclude the interaction with other protein(s) to form a heterocomplex.

PssN lipoprotein is not surface exposed. A lipoprotein may be either periplasmic or exposed to the extracellular environment. To probe the PssN protein's topology, its sensitivity to externally added proteases was assessed (37). Trypsin treatment of total membrane extract resulted in the disappearance from Western blots of the band corresponding to the full-length PssN accompanied by the appearance of a band of ~35 kDa (Fig. 9A). In the case of proteinase K, complete degradation of PssN protein was observed (Fig. 9B). Treatment of intact TA1 cells with trypsin did not result in detectable loss of the PssN protein band intensity, while proteinase K treatment resulted in a minor decrease (Fig. 9A and B). Trypsin treatment of TA1 spheroplasts resulted in the disappearance of the 43-kDa band and appearance of the ~35-kDa-band seen during membrane digestion, while proteinase K treatment resulted in an almost complete degradation of the protein (Fig. 9A and B). These results suggested that PssN is not a surface-exposed lipoprotein, and it is likely to be associated with the inner leaflet of the outer membrane, with part of the protein exposed to the periplasm. Indirect immunofluorescence with FITC-conjugated antibodies further supported the proposed topology. Incubation of intact cells with anti-PssN and anti-rabbit antibodies conjugated with FITC did not result in detectable fluorescence, even when the residual EPS was removed with 0.5 M NaCl (Fig. 10A) or the Exo– strain 133 was tested (data not shown). When the same procedure was applied to TA1 spheroplasts, intense fluorescence was observed (Fig. 10B). Neither intact cells nor spheroplasts were labeled with FITC conjugate when anti-PssN serum was omitted during the first incubation (data not shown).


Figure 9
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  		FIG. 9. Analysis of PssN susceptibility to proteases. Intact cell, spheroplast, and total membrane samples were treated with trypsin at 50 µg ml–1 (A) and proteinase K at 50 µg ml–1 (B) at room temperature and analyzed by Western immunoblotting with anti-PssN antibodies. Numbers under each panel indicate the time of incubation with enzymes (in hours). Stars indicate positions of ~35-kDa bands that appeared after incubation with trypsin.

 

Figure 10
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  		FIG. 10. Confocal laser microscopy of R. leguminosarum bv. trifolii TA1 intact cells and spheroplasts incubated with anti-PssN antibodies and anti-rabbit FITC-conjugated antibodies. Matching phase-contrast and immunofluorescent images (left and right panels, respectively) of intact cells (A) and spheroplasts (B) show labeling of spheroplasts and no labeling of intact cells.

 
Analysis of PssN protein function in TA1. To test the involvement of PssN in EPS transport, we aimed to create a pssN mutant of the TA1 wild-type strain both by integration of a plasmid carrying a fragment of the pssN gene and by allelic replacement with a construct containing an antibiotic cassette instead of the pssN gene. However, we were not able to select desirable transconjugants, which indirectly indicated an essential role of PssN protein for the viability of TA1. Thus, an attempt was made to construct a conditionally lethal mutant with the pssN gene expressed from an IPTG-inducible psyn promoter. The pssN gene was cloned downstream of the lacIq-psyn cartridge, resulting in plasmid pWLN1, which was introduced into TA1 cells. The level of PssN protein assessed by Western immunoblotting appeared to be higher in TA1(pWLN1) than in TA1 even in the absence of the inducer (Fig. 11) and increased by approximately two times after induction with IPTG for 24 h (Fig. 11).


Figure 11
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  		FIG. 11. Overproduction of PssN protein in R. leguminosarum bv. trifolii TA1(pWLN1). The number of copies of PssN protein in TA1(pWLN1) was assessed by Western immunoblotting and compared to the wild-type strain; designations –IPTG and +IPTG indicate either no induction or 24 h of induction with 1 mM IPTG, respectively. The samples have been standardized so that they represent equivalent numbers of cells.

 
As the attempts to delete the chromosomal copy of pssN gene in the TA1(pWLN1) strain were unsuccessful, we examined the affect of additional PssN copies on TA1 symbiotic phenotype and EPS production. The TA1 and TA1(pWLN1) strains were each used to inoculate Trifolium pratense seedlings (66). Pink elongated nodules were observed on plants, and no differences in nodule number and nodulation kinetics were detected between the two strains. All plants appeared green, indicating efficient nitrogen fixation, and the green wet masses of plants inoculated with TA1 and TA1(pWLN1) did not differ. The amount of extracellular EPS in the TA1(pWLN1) strain was measured (43) and compared to the amount produced by the wild-type strain. The culture supernatant of TA1(pWLN1) grown in the presence of IPTG contained 37.1 ± 6.4 µg of total carbohydrate per mg of cell pellet (36.0 ± 4.5 µg mg–1 in noninduced culture), whereas the culture supernatant of the wild-type TA1 strain contained 29.3 ± 3.2 µg mg–1 of total carbohydrate after 5 days of cultivation in 79CA medium with 0.5% glycerol as the carbon source. This finding indicated that, in comparison to the wild-type strain, the production of EPS by TA1(pWLN1) was increased to ca. 123% and 126% in the absence and presence of the inducer, respectively.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have identified the pssN gene located within the pss region of R. leguminosarum bv. trifolii TA1 (41). Here, the PssN protein was described as a member of the OMA family of proteins (52) involved in surface expression of polysaccharides in gram-negative bacteria and a good candidate for the protein forming a channel structure that is engaged in translocation of exopolysaccharide across the outer membrane. Most of the OMA proteins are predicted to be lipoproteins. Previously, the lipoprotein character of WzaK30 of E. coli was evidenced (12). Lipoproteins are synthesized in the cytoplasm and exported through the inner membrane by the general secretory pathway (56). Their processing takes place on the periplasmic side of the inner membrane and involves transfer of a diacylglyceride to the cysteine, cleavage of the signal peptide, and the final acylation of the {alpha}-amino group of the N-terminal cysteine (62). Analysis of the constructed PssN-PhoA/LacZ fusions served to demonstrate that PssN protein contains such an export signal recognized by a secretory pathway. Both PssN-PhoA hybrid proteins, regardless of the PssN polypeptide length, were transported to the periplasm. Employing in vivo acylation with [3H]palmitate, we have shown that PssN is acylated in E. coli cells. Exploiting the fact that the lipoprotein-processing enzymes are widely conserved in gram-negative bacteria, we used E. coli overproducing PssN-His6 protein as an R. leguminosarum bv. trifolii TA1 surrogate for labeling experiments. Although the obtained result does not prove that the PssN native form is lipidated identically in TA1 cells, it provides unambiguous evidence that the protein possesses signals involved in lipoprotein processing. The function of the lipid moiety in OMA proteins has not been investigated in detail. However, it was shown for Wza that the acylation, although not required for targeting to the OM, is still indispensable for formation of stable multimeric channels (49).

PssN, although predominantly found in the membrane fraction, was also detected in the soluble protein fraction. However, it was shown that lipoproteins that are tethered to the membrane only by the lipid moiety could be removed from the membrane during cell fractionation (57). Fractionation of membranes with detergents and their separation in the sucrose gradient revealed that PssN predominated in detergent-insoluble and LPS-enriched fractions, confirming its outer membrane association predicted in silico. Other proteins of this family were found localized in the outer membrane, e.g., CtrA, EpsA, Wza (12, 16, 25), and, recently, KpsD protein, which was previously believed to be a periplasmic protein (45). However, in the latter case, the protein is included in the OMA family but shares only local homology with their members (52). PssN protein also repeatedly appeared in the inner membrane fractions. This may result from interaction(s) with other, inner membrane protein(s), association with the peptidoglycan (38), or association with the outer membrane only through the lipid moiety (9).

OMA proteins were proposed to be ß-barrel channel proteins (52, 73). FTIR analysis of PssN-His6 revealed that the protein is folded mainly into ß-sheets (43%), although {alpha}-helical structures were also detected (15%). Similar results were obtained for WzaK30 protein; circular dichroism analysis revealed that it does not comprise solely ß-structure (49). Thus, the secondary structure of both proteins indicates a structure distinct from OM porins (32). Still, high ß-sheet content suggested that PssN could be an integral OM protein. The protein was found to be susceptible to both trypsin and proteinase K treatment in spheroplasts but appeared to be insensitive to trypsin treatment in intact cells (even after pretreatment with EDTA; data not shown). Proteinase K treatment of intact cells resulted in a small decrease in PssN protein band intensity in the Western blot, but it might be simply due to an increased OM permeability caused by the digestion of other membrane proteins. Indirect immunofluorescence of intact cells and spheroplasts confirmed that PssN is not likely to be exposed to the external medium, as labeling of the intact cells was not observed, while spheroplasts fluoresced intensely. The simplest interpretation of these experiments could be that PssN is tethered to the outer membrane by a lipid moiety and the entire polypeptide, or at least its part, is exposed to the periplasm. In silico analysis indicated that PssN might have two transmembrane ß-strands and that both ends of the polypeptide might be directed towards the periplasm. Thus, it cannot be ruled out that the results of protease digestion and immunofluorescence reflect the lack of a trypsin digestion site in the surface-exposed loop or loops of PssN and/or absence of antibodies against the surface epitopes in the anti-PssN serum. If so, the topology of PssN would be reminiscent of that described for CtrA of Neisseria meningitidis, with the N-terminal part of the protein embedded in the membrane and the C-terminal part forming a periplasmic domain (16). {alpha}-Helices detected in PssN could be involved in association with murein and/or other protein(s).

It has been reported that Wza protein forms oligomeric structures resembling secretins (49). Cross-linking of intact TA1 cells with formaldehyde resulted in detection of oligomers with molecular weights two, three, or four times the molecular weight of PssN protein, with a putative trimeric form as the predominant one. However, cross-linking of E. coli M15(pQC32) cells allowed us to purify PssN-His6 protein only in a tetrameric form. Chemical cross-linking of intact E. coli cells suggested formation of a transmembrane complex minimally containing outer membrane protein Wza and the inner membrane tyrosine autokinase Wzc (49). Some TA1 proteins, e.g., PssP, are promising candidates for PssN-interacting partners that could contribute to the observed oligomeric forms. However, the same pattern of bands corresponding to various oligomers of PssN was detected in R. leguminosarum bv. trifolii strain P22 ({Delta}pssP) (data not shown). This suggested that either all of the observed forms were homo-oligomers and the oligomerization was independent of the presence of PssP or the observed oligomers represented complexes of PssN and other unidentified protein(s).

The numerous unsuccessful attempts to mutate the pssN gene in strain TA1 made it impossible to unambiguously verify its involvement in EPS extrusion from the cell. Mutations have been made in several OMA proteins: e.g., AmsH of Erwinia amylovora (6), VexA of Salmonella enterica serovar Typhi (23), and Wza of E. coli (11, 12). On the other hand, failures in obtaining mutants with mutations in genes encoding OMA proteins have been described for cpxD of Actinobacillus pleuropneumoniae and bexD of Haemophilus influenzae (35, 71). Contrary to the latter, viable nonencapsulated mutants of both strains were obtained for genes encoding MPA proteins, i.e., cpxC and bexC. An analogous result was previously obtained for strain TA1. Deletion of the pssP gene encoding an MPA protein resulted in an Exo– mutant (43). Based on this, it was concluded that mutation in pssN might be lethal for TA1.

Investigation of the phenotype of TA1(pWLN1) revealed that additional copies of PssN protein did not alter symbiotic properties of strain TA1. However, when the amount of EPS secreted to the culture medium was analyzed, it appeared that the supernatant of the TA1(pWLN1) culture contained more EPS than that of TA1. Taking into account that the growth kinetics of both strains did not differ (suggesting that PssN overproduction did not exert a toxic effect on TA1 cells; data not shown), it is reasonable to suspect PssN involvement in EPS synthesis and/or transport. Considering PssN localization and topology, one might expect that the number of existing channel structures per cell positively correlates with the amount of exopolysaccharide molecules that can be threaded through the outer membrane. However, it cannot be excluded that the increased EPS secretion results from a stress response caused by overexpression of an outer membrane protein (46).

The presented studies concerning PssN localization, topology, and characterization as OMA lipoprotein, together with our previous results on localization and function of PssT, PssL, and PssP proteins, provide a basis for further experiments that can enlighten how the EPS secretion apparatus actually works.


   ACKNOWLEDGMENTS
 
We thank Igor Konieczny from the Division of Molecular Biology (Inter-Collegiate Faculty of Biotechnology, Gdansk, Poland) for help with gradient experiments, Jacek Kuzmak from the National Veterinary Research Institute (Pulawy, Poland) for anti-PssN antibody production, and Ryszard Russa from the General Microbiology Department (UMCS, Lublin, Poland) for helpful and encouraging discussions.

This work was supported by grant no. 2 P04A 034 26 from the Polish Committee for Scientific Research.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of General Microbiology, Institute of Microbiology and Biotechnology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland. Phone: 48 81 537 59 72. Fax: 48 81 537 59 59. E-mail: genet{at}biotop.umcs.lublin.pl. Back


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 Discussion
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Journal of Bacteriology, October 2006, p. 6943-6952, Vol. 188, No. 19
0021-9193/06/$08.00+0     doi:10.1128/JB.00651-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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