Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: subcellular localization and possible involvement in exopolysaccharide export

Abstract

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 beta-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.

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.

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.

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 β-sheets, 1,660 cm⁻¹ for α-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.

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 β-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.

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.

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.

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) (▪) (A); the activity of an inner membrane marker enzyme, NADH oxidase (expressed in μmol min⁻¹ ml⁻¹) (▴) (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).

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.

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.

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.

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.