NopB, a type III secreted protein of Rhizobium sp. strain NGR234, is associated with pilus-like surface appendages

Abstract

Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGROmegarhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.

FIG. 1.

Identification of NopB as a TTSS-dependent secreted protein (A) Extracellular proteins of apigenin-induced cultures of Rhizobium sp. strain NGR234 and NGRΩrhcN (a TTSS mutant) were electrophoretically separated by one-dimensional SDS-15% PAGE. Molecular masses of the marker proteins are indicated on the right. The identified Nops are named on the left. A 16-kDa protein labeled as Nop16 was unique to induced wild-type NGR234 and absent in NGRΩrhcN. (B) 2D gel electrophoresis of total extracellular proteins from apigenin-induced cultures of NGR234 (left panel) and NGRΩrhcN (right panel). Each sample contained 75 μg of protein and was IEF for a total of 100,000 Vh. The IPG strips were then placed on top of SDS-12% PAGE gels and after separation were stained with silver nitrate. Black circles indicate the (approximately 16-kDa) protein spots that were subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry.

FIG. 2.

Construction and analysis of a nopB mutant. (A) Genetic organization of part of the TTSS locus of NGR234 showing the location of nopB. A 4-kb XhoI-BamHI fragment of pXB110 was cloned in pJQ200SK. A nonpolar mutant was constructed by inserting uidA (Gus gene), digested by SmaI, into the SmaI site of nopB (pMSG2). The black arrow shows the direction of the transcription of the Gus gene. pMSG4 is pLAFR6 containing a HindIII-XbaI fragment of pXB110 that was used to complement the nopB mutant. (B) Silver-stained SDS-15% PAGE of proteins isolated from the supernatants of apigenin-induced cultures of NGR234, NGRnopB::uidA, and NGRnopB::uidA (pMSG4). Bacterial cells were grown in RMS for 40 h. Molecular mass markers (Mwt) in kilodaltons are shown at the left. (C) In parallel, the samples were transferred to PVDF membranes, and immunological identification of the Nops was performed by probing the membranes with antibodies raised against NopX, NopL, and NopA.

FIG. 3.

Amino acid alignment of NopB homologues. NopB of NGR234, NopB of USDA257, blr1812 of B. japonicum USDA110, and m1r8743 of M. loti MAFF303099 were aligned by using ClustalV. Amino acids common to all the NopB homologues are marked in red. The consensus sequence is shown in bold below the alignment, with the three most conserved domains highlighted in yellow. The positions of the peptides used for antibody production are shown in green above the alignment.

FIG. 4.

Detection of NopB in NGR234 and derivatives. Extracellular proteins of induced NGR234, NGRΩrhcN, NGRΩnopL, NGRΔnopP, NGRnopB::uidA, and NGRnopB::uidA (pMSG4) were resolved by SDS-15% PAGE and electrophoretically blotted to PVDF membranes. Immunological detection of NopB was carried out using anti-NopB antibodies at a 1:1,000 dilution.

FIG. 5.

Analysis of isolated surface appendages of NGR234. Isolation of the proteins associated with the NGR234 surface appendages was performed using two different media: RMS (left panel) and YEM (right panel). The proteins were separated by SDS-15% PAGE, and stained with silver nitrate. In parallel, the separated proteins were transferred to PVDF membranes and probed with antibodies against NopX, NopL, NopB, NopA, and flagellin. Lanes 1, isolated surface filaments from apigenin-induced cultures of NGR234; lanes 2, NGRnopB::uidA.

FIG. 6.

Electron micrographs of the NGR234 surface appendages. Transmission electron micrographs showing flagella, pili, and other filaments of negatively stained cells of noninduced cultures of NGR234 (A) and apigenin-induced cultures of NGR234 (B), NGRΩrhcN (C), and NGRnopB::uidA (D). The flagella are marked with arrowheads, while the pilus-like structures are marked with white arrows. The bacterial cells were grown in YEM on carbon-Formvar-coated gold grids at 28°C and stained with 1% phosphotungstate. Scale bar, 1 μm.

FIG. 7.

Immunogold localization of NopB. TTSS pili were marked using NopB antibodies and secondary antibodies tagged with 10-nm gold particles. The NGR234 cells were grown in YEM containing apigenin on carbon-coated electron microscope gold grids for 40 h at 28°C. After this, they were incubated with preimmune serum (A and B) or anti-NopB antibodies (C). The grids were then incubated with goat anti-rabbit antibodies labeled with 10-nm gold particles (black particles) and then stained with 1% phosphotungstate. The flagella are marked with arrowheads, while the pilus-like structure is marked with a white arrow. Scale bar, 0.25 μm.