Rhizobium sp. Strain NGR234 Possesses a Remarkable Number of Secretion Systems

Abstract

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogenfixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.Diverse soil bacteria interact with plants in ways that range from symbiotic to pathogenic. Symbiotic Eubacteria (both alpha-and betaproteobacteria, collectively called rhizobia) form nitrogen-fixing associations of tremendous environmental importance (41, 66). Although some rhizobia are able to reduce atmospheric nitrogen to ammonia under saprophytic, free-living conditions, the reduced oxygen tensions found within the intracellular environment of specialized organs called nodules, maximizes this process (16). As legume roots penetrate the soil, they come in contact with rhizobia. Symbiotic interactions are initiated by the exchange of diverse molecules between the partners. Among them, plants liberate flavonoids into the rhizosphere that upregulate rhizobial genes. As a result, lipo-chito-oligo-saccharidic Nod factors are produced that trigger the nodulation pathway in susceptible legumes. Then, in many hosts, rhizobia enter the roots through root hairs, make their way to the cortex, multiply and fill the intracellular spaces of mature nodules. Centripetal progression of rhizobia into the plant and their maturation into nitrogen-fixing symbiosomes depends on the continued exchange of diverse signals.Many, but not all of these signals have been identified; one sure way to take stock of what is necessary for effective symbiosis is to sequence the partners. We began this work by assembling overlapping sets of cosmids (contigs) of the microsymbiont Rhizobium sp. strain NGR234 (hereafter NGR234) (63), which enabled us to elucidate the nucleotide sequence of the symbiotic (pNGR243a) plasmid (29). Similar techniques permitted the assembly of sections of th...

Functional Analysis of the Type 3 Effector Nodulation Outer Protein L (NopL) from Rhizobium sp. NGR234

“…strain NGR234 (20) possesses a functional type 3 secretion system (21), which can affect nodulation of host plants either positively or negatively. Up to now, four nodulation outer proteins (Nops) of NGR234 have been identified as type 3 effectors, namely NopL (21-24), NopP (25,26), NopT (27,28), and NopM (28).…”

Functional analysis of the nifQdctA1y4vGHIJ operon of Sinorhizobium fredii strain NGR234 using a transposon with a NifA-dependent read-out promoter

“…The gene rpsL codes for the 30S ribosomal subunit protein S12 and is the first gene of the well conserved 'str' chromosomal operon, which includes the essential housekeeping genes rpsG, fusA1 and tufA. The major sigma subunit SigA (RpoD) of NGR234 is encoded by the chromosomal gene NGR_c22670 (Schmeisser et al, 2009). PnodA comprises the NodD1-dependent and nod-box NB8 regulatory element that controls the flavonoid-inducible and symbiotically essential nodABCIJnolOnoeI operon of pSfrNGR234a (Kobayashi et al, 2004;Perret et al, 1999).…”

“…Mutant strains deleted in nifA were constructed as follows. A fragment of 3083 bp which spans nifA and covers positions 154 653 to 157 731 of pSfrNGR234a (GenBank NC_000914.2) (Freiberg et al, 1997;Schmeisser et al, 2009) was amplified by PCR using primers NifA-1 and NifA-2 with pXB296 cosmid DNA (Perret et al, 1991) as template. The resulting PCR product was digested by SpeI and cloned into a modified pBluescript KS + (Stratagene) in which the EcoRI and BamHI sites were removed.…”

“…More recently, in vitro assays showed that NifQ donates molybdenum to a putative NifEN/NifH complex during synthesis of the nitrogenase FeMo-cofactor (Hernandez et al, 2008). Of the two copies of dctA found in NGR234, dctA1 is encoded by pSfrNGR234a (van Slooten et al, 1992), while dctA2 is carried by the 2 430 033 bp megaplasmid pSfrNGR234b, and together with dctB and dctD forms the conserved Dct locus of NGR234 (Schmeisser et al, 2009). Initially, dctA1 was reported to be non-essential for the growth of NGR234 in free-living conditions but required for symbiotic nitrogen fixation with M. atropurpureum, L. leucocephala and V. unguiculata (van Slooten et al, 1992).…”

“…Bioinformatic analysis using the conserved binding sites for NifA and s 54 identified 16 potential NifA-activated promoters in the 536 165 bp plasmid pSfrNGR234a (previously pNGR234a), the symbiotic plasmid of Sinorhizobium fredii strain NGR234 (Freiberg et al, 1997). In addition to pSfrNGR234a, the genome of NGR234 comprises a chromosome of 3 925 702 bp that is predicted to encode 3633 ORFs, and a megaplasmid pSfrNGR234b (pNGR234b) of 2 430 033 bp with 2342 ORFs (Schmeisser et al, 2009). Transcriptional analyses confirmed that the 16 predicted NifA-s 54 -dependent promoters of pSfrNGR234a direct the expression of more than 50 genes, many of which are clustered within a 54 kb region centred around the nifHDK1nifENXy4vQ operon (Perret et al, 1999.…”