Luc Felicianus Marie Rouws

Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5

BackgroundGluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins.ResultsGluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat.ConclusionThe genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.

Exopolysaccharide Production Is Required for Biofilm Formation and Plant Colonization by the Nitrogen-Fixing Endophyte Gluconacetobacter diazotrophicus

The genome of the endophytic diazotrophic bacterial species Gluconacetobacter diazotrophicus PAL5 (PAL5) revealed the presence of a gum gene cluster. In this study, the gumD gene homologue, which is predicted to be responsible for the first step in exopolysaccharide (EPS) production, was insertionally inactivated and the resultant mutant (MGD) was functionally studied. The mutant MGD presented normal growth and nitrogen (N(2)) fixation levels but did not produce EPS when grown on different carbon sources. MGD presented altered colony morphology on soft agar plates (0.3% agar) and was defective in biofilm formation on glass wool. Most interestingly, MGD was defective in rice root surface attachment and in root surface and endophytic colonization. Genetic complementation reverted all mutant phenotypes. Also, the addition of EPS purified from culture supernatants of the wild-type strain PAL5 to the mutant MGD was effective in partially restoring wild-type biofilm formation and plant colonization. These data provide strong evidence that the PAL5 gumD gene is involved in EPS biosynthesis and that EPS biosynthesis is required for biofilm formation and plant colonization. To our knowledge, this is the first report of a role of EPS in the endophytic colonization of graminaceous plants by a nitrogen-fixing bacterium.

The Bacterial Superoxide Dismutase and Glutathione Reductase Are Crucial for Endophytic Colonization of Rice Roots by Gluconacetobacter diazotrophicus PAL5

Gluconacetobacter diazotrophicus is an aerobic diazotrophic plant-growth-promoting bacterium isolated from different gramineous plants. We showed that reactive oxygen species (ROS) were produced at early stages of rice root colonization, a typical plant defense response against pathogens. The transcription of the pathogen-related-10 gene of the jasmonic acid (JA) pathway but not of the PR-1 gene of the salicylic acid pathway was activated by the endophytic colonization of rice roots by G. diazotrophicus strain PAL5. Quantitative polymerase chain reaction analyses showed that, at early stages of colonization, the bacteria upregulated the transcript levels of ROS-detoxifying genes such as superoxide dismutase (SOD) and glutathione reductase (GR). To proof the role of ROS-scavenging enzymes in the colonization and interaction process, transposon insertion mutants of the SOD and GR genes of strain PAL5 were constructed. The SOD and GR mutants were unable to efficiently colonize the roots, indicated by the decrease of tightly root-associated bacterial cell counts and endophytic colonization and by fluorescence in situ hybridization analysis. Interestingly, the mutants did not induce the PR-10 of the JA-pathway, probably due to the inability of endophytic colonization. Thus, ROS-scavenging enzymes of G. diazotrophicus strain PAL5 play an important role in the endophytic colonization of rice plants.

Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes

Aims:  To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes.

Bradyrhizobium ingae sp. nov., isolated from effective nodules of Inga laurina grown in Cerrado soil

Root-nodule bacteria were isolated from Inga laurina (Sw.) Willd. growing in the Cerrado Amazon region, State of Roraima, Brazil. The 16S rRNA gene sequences of six strains (BR 10250(T), BR 10248, BR 10249, BR 10251, BR 10252 and BR 10253) showed low similarities with currently described species of the genus Bradyrhizobium. Phylogenetic analyses of sequences of five housekeeping genes (dnaK, glnII, gyrB, recA and rpoB) revealed Bradyrhizobium iriomotense EK05(T) to be the closest type strain (97.4% sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [with the major components C16:0 and summed feature 8 (C18:1ω6c/C18:1ω7c)], the slow growth rate and carbon compound utilization patterns supported the assignment of our strains to the genus Bradyrhizobium. Results from DNA-DNA hybridizations and physiological traits differentiated our strains from the closest related species of the genus Bradyrhizobium with validly published names. Sequences of symbiosis-related genes for nodulation (nodC) and nitrogen fixation (nifH) grouped together with those of B. iriomotense EK05(T) and Bradyrhizobium sp. strains BR 6610 (used as a commercial inoculant for Inga marginata in Brazil) and TUXTLAS-10 (previously observed in Central America). Based on these data, the six strains represent a novel species, for which the name Bradyrhizobium ingae sp. nov. is proposed. The type strain is BR 10250(T) ( = HAMBI 3600(T)).

Cowpea Nodules Harbor Non-rhizobial Bacterial Communities that Are Shaped by Soil Type Rather than Plant Genotype

Many studies have been pointing to a high diversity of bacteria associated to legume root nodules. Even though most of these bacteria do not form nodules with legumes themselves, it was shown that they might enter infection threads when co-inoculated with rhizobial strains. The aim of this work was to describe the diversity of bacterial communities associated with cowpea (Vigna unguiculata L. Walp) root nodules using 16S rRNA gene amplicon sequencing, regarding the factors plant genotype and soil type. As expected, Bradyrhizobium was the most abundant genus of the detected genera. Furthermore, we found a high bacterial diversity associated to cowpea nodules; OTUs related to the genera Enterobacter, Chryseobacterium, Sphingobacterium, and unclassified Enterobacteriacea were the most abundant. The presence of these groups was significantly influenced by the soil type and, to a lesser extent, plant genotype. Interestingly, OTUs assigned to Chryseobacterium were highly abundant, particularly in samples obtained from an Ultisol soil. We confirmed their presence in root nodules and assessed their diversity using a target isolation approach. Though their functional role still needs to be addressed, we postulate that Chryseobacterium strains might help cowpea plant to cope with salt stress in semi-arid regions.

Bradyrhizobium stylosanthis sp. nov., comprising nitrogen-fixing symbionts isolated from nodules of the tropical forage legume Stylosanthes spp.

The introduction of legumes and nitrogen-fixing bacteria in tropical areas under pasture is a key factor for improvement of soil fertility. However, there are still very few studies concerning the symbionts of tropical forage legumes. We performed a polyphasic study with three strains representing the genus Bradyrhizobium (BR 446T, BR 510 and BR 511) isolated from the tropical perennial forage legume of the genus Stylosanthes. On the basis of 16S rRNA gene sequences, the three strains showed highest similarity with B. huanghuaihaiense, and in the analysis of the intergenic transcribed spacer (ITS) they showed less than 93.4 % similarity to all described species of the genus Bradyrhizobium. Multilocus sequence analysis (MLSA) with three, four or five (dnaK, glnII, gyrB, recA and rpoB) housekeeping genes confirmed that the BR strains belong to a distinct clade, with <96.5 % nucleotide identity with other members of the genus Bradyrhizobium. Average nucleotide identity (ANI) of genome sequences between strain BR 446T and B.huanghuaihaiense was below the threshold for species circumscription (90.7 %). DNA-DNA hybridization resulted in ΔTm values over 6.7 °C with the most closely related species. Similarities among the BR strains and differences from other species were confirmed by rep-PCR analysis. Interestingly, the BR strains were grouped in the analysis of nifH and nodC genes, but showed higher similarity with B. iriomotense and B. manausense than with B.huanghuaihaiense, indicating a different evolutionary history for nitrogen-fixation genes. Morpho-physiological, genotypic and genomic data supported that these BR strains represent a novel species for which the name Bradyrhizobium stylosanthis sp. nov. is suggested. The type strain is BR 446T (=CNPSo 2823T=HAMBI 3668T=H-8T), isolated from Stylosanthes guianensis.

Endophytic Bradyrhizobium spp. isolates from sugarcane obtained through different culture strategies.

Brazilian sugarcane has been shown to obtain part of its nitrogen via biological nitrogen fixation (BNF). Recent reports, based on the culture independent sequencing of bacterial nifH complementary DNA (cDNA) from sugarcane tissues, have suggested that members of the Bradyrhizobium genus could play a role in sugarcane-associated BNF. Here we report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies: direct isolation on culture media and capture of Bradyrhizobium spp. using the promiscuous legume Vigna unguiculata as trap-plant. Both strategies permitted the isolation of genetically diverse Bradyrhizobium spp. isolates, as concluded from enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S ribosomal RNA, nifH and nodC sequence analyses. Several isolates presented nifH phylotypes highly similar to nifH cDNA phylotypes detected in field-grown sugarcane by a culture-independent approach. Four isolates obtained by direct plate cultivation were unable to nodulate V. unguiculata and, based on PCR analysis, lacked a nodC gene homologue. Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a nodule environment for BNF. Therefore, this study brings further evidence that Bradyrhizobium spp. may play a role in sugarcane-associated BNF under field conditions.

Rhizobium altiplani sp. nov., isolated from effective nodules on Mimosa pudica growing in untypically alkaline soil in central Brazil

Root nodule bacteria were isolated from nodules on Mimosa pudica L. growing in neutral-alkaline soils from the Distrito Federal in central Brazil. The 16S rRNA gene sequence analysis of 10 strains placed them into the genus Rhizobium with the closest neighbouring species (each with 99 % similarity) being Rhizobium grahamii, Rhizobium cauense, Rhizobium mesoamericanum and Rhizobium tibeticum. This high similarity, however, was not confirmed by multi-locus sequence analysis (MLSA) using three housekeeping genes (recA, glnII and rpoB), which revealed R. mesoamericanum CCGE 501T to be the closest type strain (92 % sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [with majority being C19 : 0 cyclo ω8c and summed feature 8 (C18 : 1ω7c/C18 : 1ω6c)], DNA G+C content (57.6 mol%), and carbon compound utilization patterns supported the placement of the novel strains in the genus Rhizobium. Results of average nucleotide identity (ANI) differentiated the novel strains from the closest species of the genus Rhizobium, R. mesoamericanum, R. grahamii and R. tibeticum with 89.0, 88.1 and 87.8 % similarity, respectively. The symbiotic genes essential for nodulation (nodC) and nitrogen fixation (nifH) were most similar (99-100 %) to those of R. mesoamericanum, another Mimosa-nodulating species. Based on the current data, these 10 strains represent a novel species of the genus Rhizobium for which the name Rhizobium altiplani sp. nov. is proposed. The type strain is BR 10423T (=HAMBI 3664T).

Gluconacetobacter diazotrophicus exopolysaccharide protects bacterial cells against oxidative stress in vitro and during rice plant colonization

Background and aimsThe endophytic bacterium Gluconacetobacter diazotrophicus produces exopolysaccharides (EPS) that are required for biofilm formation and colonization of rice seedlings. Here we investigated whether EPS produced by the G. diazotrophicus strain Pal5 protects the bacteria against free radicals.MethodsEPS-mediated protection of Pal5 cells against oxidative damage was evaluated by cell counting and fluorescence microscopy. Rice seedling inoculation studies were performed to investigate the antioxidant activity of EPS during plant colonization. The expression of three bacterial antioxidant genes during plant colonization was also monitored.ResultsFree radical activities were reduced in vitro by the addition of Pal5 EPS. An EPS-defective Pal5 mutant was hypersensitive to H2O2 and addition of purified EPS reversed this phenotype. Addition of EPS at the inoculation time increased colonization efficiency by the mutant strain and a similar effect was observed after addition of the antioxidant ascorbic acid. qPCR profiles of sodA, gor, and katE gene expression in the mutant confirmed the role of EPS during the initial plant colonization.ConclusionsOur results indicated that EPS produced by G. diazotrophicus protects the bacterial cells against oxidative stress in vitro and during colonization of rice plants.