The LPS O-antigen in photosynthetic Bradyrhizobium strains is dispensable for the establishment of a successful symbiosis with Aeschynomene legumes

The photosynthetic bradyrhizobia are able to use a Nod-factor independent process to induce nitrogen-fixing nodules on some semi-aquatic Aeschynomene species. These bacteria display a unique LPS O-antigen composed of a new sugar, the bradyrhizose that is regarded as a key symbiotic factor due to its non-immunogenic character. In this study, to check this hypothesis, we isolated mutants affected in the O-antigen synthesis by screening a transposon mutant library of the ORS285 strain for clones altered in colony morphology. Over the 10,000 mutants screened, five were selected and found to be mutated in two genes, rfaL, encoding for a putative O-antigen ligase and gdh encoding for a putative dTDP-glucose 4,6-dehydratase. Biochemical analysis confirmed that the LPS of these mutants completely lack the O-antigen region. However, no effect of the mutations could be detected on the symbiotic properties of the mutants indicating that the O-antigen region of photosynthetic Bradyrhizobium strains is not required for the establishment of symbiosis with Aeschynomene

A phylogenetic framework of the legume genus Aeschynomene for comparative genetic analysis of the Nod-dependent and Nod-independent symbioses

Background : Among semi-aquatic species of the legume genus Aeschynomene, some have the property of being nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the synthesis of Nod factors. Knowledge of the specificities underlying this Nod-independent symbiosis has been gained from the model legume Aeschynomene evenia but our understanding remains limited due to the lack of comparative genetics with related taxa using a Nod factor-dependent process. To fill this gap, we combined different approaches to perform a thorough comparative analysis in the genus Aeschynomene. Results: This study significantly broadened previous taxon sampling, including in allied genera, in order to construct a comprehensive phylogeny. In the phylogenetic tree, five main lineages were delineated, including a novel lineage, the Nod-independent clade and another one containing a polytomy that comprised several Aeschynomene groups and all the allied genera. This phylogeny was matched with data on chromosome number, genome size and low-copy nuclear gene sequences to reveal the diploid species and a polytomy containing mostly polyploid taxa. For these taxa, a single allopolyploid origin was inferred and the putative parental lineages were identified. Finally, nodulation tests with different Bradyrhizobium strains revealed new nodulation behaviours and the diploid species outside of the Nod-independent clade were compared for their experimental tractability and genetic diversity. Conclusions: The extended knowledge of the genetics and biology of the different lineages sheds new light of the evolutionary history of the genus Aeschynomene and they provide a solid framework to exploit efficiently the diversity encountered in Aeschynomene legumes. Notably, our backbone tree contains all the species that are diploid and it clarifies the genetic relationships between the Nod-independent clade and the Nod-dependent lineages. This study enabled the identification of A. americana and A. patula as the most suitable species to undertake a comparative genetic study of the Nod-independent and Nod-dependent symbioses

Characterization of the common nodulation genes of the photosynthetic Bradyrhizobium sp. ORS285 reveals the presence of a new insertion sequence upstream of nodA

We isolated and characterized nodA genes from photosynthetic and non-photosynthetic rhizobia nodulating the legume genus #Aeschynomene, and found that the nodA sequence from photosynthetic stem-nodulating bacteria was phylogenetically distant from the other already described nodA genes. Characterization of the photosynthetic strain ORS285 common nod gene cluster (nodABC) showed, upstream of nodA, the presence of a new insertion sequence element belonging to the IS3 family and specific to a group of photosynthetic strains from #Aeschynomene. (Résumé d'auteur

A glutamate synthase mutant of Bradyrhizobium sp. strain ORS285 is unable to induce nodules on Nod factor-independent Aeschynomene species

International audienceAbstract The Bradyrhizobium sp. strain ORS285 is able to establish a nitrogen-fixing symbiosis with both Nod factor (NF) dependent and NF-independent Aeschynomene species. Here, we have studied the growth characteristics and symbiotic interaction of a glutamate synthase (GOGAT; gltD::Tn5 ) mutant of Bradyrhizobium ORS285. We show that the ORS285 gltD::Tn5 mutant is unable to use ammonium, nitrate and many amino acids as nitrogen source for growth and is unable to fix nitrogen under free-living conditions. Moreover, on several nitrogen sources, the growth rate of the gltB::Tn5 mutant was faster and/or the production of the carotenoid spirilloxanthin was much higher as compared to the wild-type strain. The absence of GOGAT activity has a drastic impact on the symbiotic interaction with NF-independent Aeschynomene species. With these species, inoculation with the ORS285 gltD::Tn5 mutant does not result in the formation of nodules. In contrast, the ORS285 gltD::Tn5 mutant is capable to induce nodules on NF-dependent Aeschynomene species, but these nodules were ineffective for nitrogen fixation. Interestingly, in NF-dependent and NF-independent Aeschynomene species inoculation with the ORS285 gltD::Tn5 mutant results in browning of the plant tissue at the site of the infection suggesting that the mutant bacteria induce plant defence responses

A quorum-quenching approach to identify quorum-sensing-regulated functions in Azospirilum lipoferum

International audienceA quorum-quenching approach was exploited in order to identify functions regulated by quorum-sensing (QS) in the plant growth-promoting bacterium Azospirillum lipoferum. The AttM lactonase from Agrobacterium tumefaciens was shown to enzymatically inactivate N-acyl homoserine lactones (AHLs) produced by two A. lipoferum strains. The targeted analysis of several phenotypes revealed that in strain B518, a rice endophyte, AHL inactivation abolished pectinase activity, increased siderophore synthesis and reduced indoleacetic acid production (in stationary phase) but no effect was observed on cellulase activity or on swimming and swarming motilities. None of the tested phenotypes appeared to be under QS regulation in strain TVV3 isolated from the rice rhizosphere. Moreover, AHL inactivation had no deleterious effect on the phytostimulatory effect of the two strains in vitro. A global proteomic approach revealed little modification of protein patterns when comparing attM-expressing TVV3 and the wild-type strain, but numerous proteins appeared to be regulated by the AHL-mediated QS system in strain B518. Several proteins identified by MSeMS analysis were revealed to be implicated in transport (such as OmaA) and chemotaxis (ChvE). Altogether, the results indicate that in A. lipoferum, QS regulation is strain-specific and is dedicated to regulating functions linked to rhizosphere competence and adaptation to plant roots