The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571

<p>Abstract</p> <p>Background</p> <p>Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. <it>Azorhizobium caulinodans </it>ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with <it>Sesbania rostrata</it>. The host is a fast-growing, submergence-tolerant tropical legume on which <it>A. caulinodans </it>can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem.</p> <p>Results</p> <p>The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for <it>A. caulinodans</it>. Phylogenetic analyses show that the diazotroph <it>Xanthobacter autotrophicus </it>is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor.</p> <p>Conclusion</p> <p>The genome analysis reveals that <it>A. caulinodans </it>is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make <it>A. caulinodans </it>an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.</p

The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus

Bacterial infection of interior tissues of legume root nodules is controlled at the epidermal cell layer and is closely coordinated with progressing organ development. Using spontaneous nodulating Lotus japonicus plant mutants to uncouple nodule organogenesis from infection, we have determined the role of 16 genes in these two developmental processes. We show that host-encoded mechanisms control three alternative entry processes operating in the epidermis, the root cortex and at the single cell level. Single cell infection did not involve the formation of trans-cellular infection threads and was independent of host Nod-factor receptors and bacterial Nod-factor signals. In contrast, Nod-factor perception was required for epidermal root hair infection threads, whereas primary signal transduction genes preceding the secondary Ca2+ oscillations have an indirect role. We provide support for the origin of rhizobial infection through direct intercellular epidermal invasion and subsequent evolution of crack entry and root hair invasions observed in most extant legumes

Contribution of NFP LysM Domains to the Recognition of Nod Factors during the Medicago truncatula/Sinorhizobium meliloti Symbiosis

The root nodule nitrogen fixing symbiosis between legume plants and soil bacteria called rhizobia is of great agronomical and ecological interest since it provides the plant with fixed atmospheric nitrogen. The establishment of this symbiosis is mediated by the recognition by the host plant of lipo-chitooligosaccharides called Nod Factors (NFs), produced by the rhizobia. This recognition is highly specific, as precise NF structures are required depending on the host plant. Here, we study the importance of different LysM domains of a LysM-Receptor Like Kinase (LysM-RLK) from Medicago truncatula called Nod factor perception (NFP) in the recognition of different substitutions of NFs produced by its symbiont Sinorhizobium meliloti. These substitutions are a sulphate group at the reducing end, which is essential for host specificity, and a specific acyl chain at the non-reducing end, that is critical for the infection process. The NFP extracellular domain (ECD) contains 3 LysM domains that are predicted to bind NFs. By swapping the whole ECD or individual LysM domains of NFP for those of its orthologous gene from pea, SYM10 (a legume plant that interacts with another strain of rhizobium producing NFs with different substitutions), we showed that NFP is not directly responsible for specific recognition of the sulphate substitution of S. meliloti NFs, but probably interacts with the acyl substitution. Moreover, we have demonstrated the importance of the NFP LysM2 domain for rhizobial infection and we have pinpointed the importance of a single leucine residue of LysM2 in that step of the symbiosis. Together, our data put into new perspective the recognition of NFs in the different steps of symbiosis in M. truncatula, emphasising the probable existence of a missing component for early NF recognition and reinforcing the important role of NFP for NF recognition during rhizobial infection

Structural characterization of extracellular polysaccharides of Azorhizobium caulinodans and importance for nodule initiation on Sesbania rostrata

During lateral root base nodulation, the microsymbiont Azorhizobium caulinodans enters its host plant, Sesbania rostrata, via the formation of outer cortical infection pockets, a process that is characterized by a massive production of H2O2. Infection threads guide bacteria from infection pockets towards nodule primordia. Previously, two mutants were constructed that produce lipopolysaccharides (LPSs) similar to one another but different from the wild-type LPS, and that are affected in extracellular polysaccharide (EPS) production. Mutant ORS571-X15 was blocked at the infection pocket stage and unable to produce EPS. The other mutant, ORS571-oac2, was impaired in the release from infection threads and was surrounded by a thin layer of EPS in comparison to the wild-type strain that produced massive amounts of EPS. Structural characterization revealed that EPS purified from cultured and nodule bacteria was a linear homopolysaccharide of alpha-1,3-linked 4,6-O-(1-carboxyethylidene)-D-galactosyl residues. In situ H2O2 localization demonstrated that increased EPS production during early stages of invasion prevented the incorporation of H2O2 inside the bacteria, suggesting a role for EPS in protecting the microsymbiont against H2O2. In addition, ex planta assays confirmed a positive correlation between increased EPS production and enhanced protection against H2O2

Biosynthesis of Azorhizobium caulinodans Nod factors. Study of the activity of the NodABSC proteins by expression of the genes in Escherichia coli

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Exo-Oligosaccharides of Rhizobium sp. Strain NGR234 Are Required for Symbiosis with Various Legumes

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with β-1,3, β-1,4, β-1,6, α-1,3, and α-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGRΩexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGRΩexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)(5)(GlcA)(2)PvGal. When used as inoculants, both the exo-deficient mutants and NGRΩexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, ∼50 μg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-β-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes

SrSymRK, a plant receptor essential for symbiosome formation

The symbiosis between legumes and rhizobia is essential for the nitrogen input into the life cycle on our planet. New root organs, the nodules, are established, which house N(2)-fixing bacteria internalized into the host cell cytoplasm as horizontally acquired organelles, the symbiosomes. The interaction is initiated by bacterial invasion via epidermal root hair curling and cell division in the cortex, both triggered by bacterial nodulation factors. Of the several genes involved in nodule initiation that have been identified, one encodes the leucine-rich repeat-type receptor kinase SymRK. In SymRK mutants of Lotus japonicus or its orthologs in Medicago sp. and Pisum sativum, nodule initiation is arrested at the level of the root hair interaction. Because of the epidermal block, the role of SymRK at later stages of nodule development remained enigmatic. To analyze the role of SymRK downstream of the epidermis, the water-tolerant legume Sesbania rostrata was used that has developed a nodulation strategy to circumvent root hair responses for bacterial invasion. Evidence is provided that SymRK plays an essential role during endosymbiotic uptake in plant cells

Role of BacA in Lipopolysaccharide Synthesis, Peptide Transport, and Nodulation by Rhizobium sp. Strain NGR234▿

BacA of Sinorhizobium meliloti plays an essential role in the establishment of nitrogen-fixing symbioses with Medicago plants, where it is involved in peptide import and in the addition of very-long-chain fatty acids (VLCFA) to lipid A of lipopolysaccharide (LPS). We investigated the role of BacA in Rhizobium species strain NGR234 by mutating the bacA gene. In the NGR234 bacA mutant, peptide import was impaired, but no effect on VLCFA addition was observed. More importantly, the symbiotic ability of the mutant was comparable to that of the wild type for a variety of legume species. Concurrently, an acpXL mutant of NGR234 was created and assayed. In rhizobia, AcpXL is a dedicated acyl carrier protein necessary for the addition of VLCFA to lipid A. LPS extracted from the NGR234 mutant lacked VLCFA, and this mutant was severely impaired in the ability to form functional nodules with the majority of legumes tested. Our work demonstrates the importance of VLCFA in the NGR234-legume symbiosis and also shows that the necessity of BacA for bacteroid differentiation is restricted to specific legume-Rhizobium interactions