Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae

Abstract

Background: Proteins secreted by bacteria play an important role in infection of eukaryotic hosts. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. Proteins secreted during the infection process by some rhizobial strains can influence infection and modify the plant defence signalling pathways. The aim of this study was to systematically analyse protein secretion in the recently sequenced strain Rhizobium leguminosarum bv. viciae 3841.

Results: Similarity searches using defined protein secretion systems from other Gram-negative bacteria as query sequences revealed that R. l. bv. viciae 3841 has ten putative protein secretion systems. These are the general export pathway (GEP), a twin-arginine translocase (TAT) secretion system, four separate Type I systems, one putative Type IV system and three Type V autotransporters. Mutations in genes encoding each of these (except the GEP) were generated, but only mutations affecting the PrsDE (Type I) and TAT systems were observed to affect the growth phenotype and the profile of proteins in the culture supernatant. Bioinformatic analysis and mass fingerprinting of tryptic fragments of culture supernatant proteins identified 14 putative Type I substrates, 12 of which are secreted via the PrsDE, secretion system. The TAT mutant was defective for the symbiosis, forming nodules incapable of nitrogen fixation.

Conclusion: None of the R. l. bv. viciae 3841 protein secretion systems putatively involved in the secretion of proteins to the extracellular space (Type I, Type IV, Type V) is required for establishing the symbiosis with legumes. The PrsDE (Type I) system was shown to be the major route of protein secretion in non-symbiotic cells and to secrete proteins of widely varied size and predicted function. This is in contrast to many Type I systems from other bacteria, which typically secrete specific substrates encoded by genes often localised in close proximity to the genes encoding the secretion system itself.

Figure 1

Unrooted dendrogram of R. l. bv viciae 3841 proteins with similarity to PrsD. The bar represents 0.1 amino acid changes; predicted functions for proteins are given.

Figure 2

Domains found in proteins prediced to be secreted via a Type I system. Coloured elements represent repeated sequences. Red denotes clusters of repeated RTX nonapeptides; the numbers of repeats are indicated. A: Zn, Zn-metalloprotease domain. B: Blue boxes represent single repeated domains; gly, glycosyl hydrolase domain; dark grey, TPT repeat; hatched area marks sequence similarity. C: Blue boxes represent single cadherin domains. D:. Blue, repeated sequence 1; magenta, repeated sequence 2. E: NodO protein shown for comparison. Bar represents 100 amino acid residues.

Figure 3

The R. l. bv. viciae 3841 tatC mutant exhibits anomalous infection of pea nodules. Nodules formed on pea plant roots (cv. Frisson) three weeks after inoculation with wild type R. l. bv. viciae 3841 (A, C) and the tatC mutant A1052 (B, D). A and B are images of fresh nodules, C and D were nodules preserved in 70% ethanol before staining with SYTO 13 dye. Structures of high DNA content (bacteria and plant cell nuclei) are stained yellow. The nodule tip is at the bottom.

Figure 4

The tatC mutant of R. l. bv. viciae 3841 is altered in the culture supernatant protein profile, but is very similar to the wild type in its periplasmic protein profile. R. l. bv. viciae 3841 and the tatC mutant (A1052) were grown for three days in 100 ml TY (periplasm) or for four days in 100 ml Y-mannitol medium (culture supernatant). Periplasmic proteins were prepared by cold hypo-osmotic shock, ~30 μg were loaded per lane. Culture supernatant proteins were precipitated with TCA and protein representing 20 ml of culture were loaded in each lane.

Figure 5

Culture supernatant proteins from extracellular protein secretion system mutants. Cells were grown in Y-mannitol medium for 4 days. Proteins were precipitated with trichloroacetic acid. A, R. l. bv. viciae 3841 wild type; B, toaD mutant; C, tobD mutant; D, prsD mutant; E, virB6 mutant; F, autA mutant; G, autB mutant; H, autC mutant. Protein bands missing in the lane representing the prsD mutant are marked (*).

Figure 6

Protein bands submitted to MALDI-ToF MS analysis. Supernatant proteins were separated by SDS-PAGE and stained with colloidal Coomassie G. The indicated protein bands present in the wild type were excised and analysed by MALDI-ToF MS fingerprinting. Protein band missing in the culture supernatant of the prsD mutant (A898) are indicated by red arrows.