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

BMC Genomics BioMed Central Research article Open Access Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae Martin Krehenbrink1,2 and J Allan Downie*1 Address: 1Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK and 2Current address: Unité de Génétique Moléculaire, Institut Pasteur 25, rue du Dr. Roux, 75724 Paris Cedex 15, France Email: Martin Krehenbrink - ; J Allan Downie* - * Corresponding author Published: 29 January 2008 BMC Genomics 2008, 9:55 doi:10.1186/1471-2164-9-55 Received: 6 September 2007 Accepted: 29 January 2008 This article is available from: http://www.biomedcentral.com/1471-2164/9/55 © 2008 Krehenbrink and Downie; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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. Background Rhizobium leguminosarum bv. viciae is a Gram-negative soil bacterium which forms a mutualistic symbiosis with legumes, resulting in nitrogen-fixing root nodules. This symbiotic relationship is initiated by an exchange of signals between the two partners. While the general features of this signal exchange are common to all rhizobia-plant symbioses, differences in the signalling molecules allow only certain bacterium-plant combinations to lead to a successful symbiosis. Plant-made flavonoids released into Page 1 of 19 (page number not for citation purposes) BMC Genomics 2008, 9:55 the rhizosphere [1,2] induce rhizobia to make specific signalling molecules known as Nod factors, which are four or five β-1,4 linked N-acetyl-glucosamine residues with a fatty acid residue replacing the N-acetyl group at the nonreducing end. This basic structure can carry various substituents (such as acetyl, methyl, carbamoyl, sulfuryl and various glycosyl groups) depending on the rhizobial strain, and many strains synthesise a limited variety of Nod factors [3,4]. In addition to Nod factors, secreted proteins can contribute to nodulation ability. NodO is a protein secreted via a Type I secretion system (PrsDE) by some rhizobia and heterologous expression of nodO could extend the host range of some rhizobial strains [5,6]. Type I secretion systems transport proteins from the cytoplasm across both membranes to the extracellular space and are usually composed of three gene products: an ATPase of the ATPbinding cassette (ABC) protein family; a membrane fusion protein which spans the periplasm and links the inner and outer membranes [7]; and an outer membrane protein [8]. Their substrate proteins usually carry tandem nonapeptide glycine-rich repeats known as RTX (repeat in toxin) motifs which form a β-roll structure stabilised by coordinated Ca2+ ions [9,10]. In R. leguminosarum and some other rhizobia, both glycanases involved in bacterial exopolysaccharide (EPS) processing and rhizobial adhesion proteins are secreted via a Type I secretion system (PrsDE) in addition to NodO [6,11-16]. Secreted cell-wall degrading enzymes may help to erode plant cell walls enhancing infection and in Rhizobium leguminosarum biovars trifolii and viciae, cell-associated pectinolytic and cellulolytic enzymes have been found [12,13,17]. Sinorhizobium meliloti has been reported to induce the production of polygalacturonase in alfalfa roots [18]. Other rhizobia make use of Type III and Type IV protein secretion systems to inject effector proteins directly into the host plant cells where they modify plant signalling pathways [19-22]. Type III secretion systems translocate proteins across both membranes using ATP. They are assembled from over 20 proteins, many of which closely resemble proteins involved in flagellar biogenesis [23,24]. In some cases, the flagellar apparatus has been shown to export proteins not related to the flagellum [25]. The proteins are normally delivered into the eukaryotic cytoplasm, where they then alter the host metabolism to suit the bacterial needs [26]. In Rhizobium sp. NGR234 several proteins including NopX, NopA, NopB, NopP and NopL, were shown to be secreted via a Type III system upon flavonoid induction [21,27-29]. The Vir system of Agrobacterium tumefaciens is a wellknown Type IV secretion system involved in the transfer of DNA and protein to the plant cell. The Type IV secretory http://www.biomedcentral.com/1471-2164/9/55 machinery is encoded by the virB1–11 genes [30]. A related system in Mesorhizobium loti delivers effector proteins into plants during nodule initiation [20]. The twin arginine translocase pathway (TAT) pathway exports folded proteins [31,32] often with posttranslational modifications [31,33-35] and does not recognise unfolded proteins [36]. TAT substrates carry an N-terminal tripartite signal sequence (...truncated)