Diversity and antimicrobial potential of the culturable rhizobacteria from medicinal plant Baccharis trimera Less D.C.

Plant microbiota is usually enriched with bacteria producers of secondary metabolites and represents a valuable source of novel species and compounds. Here, we analyzed the diversity of culturable root-associated bacteria of the medicinal native plant Baccharis trimera (Carqueja) and screened promising isolates for their antimicrobial properties. The rhizobacteria were isolated from the endosphere and rhizosphere of B. trimera from Ponta Grossa and Ortigueira localities and identified by sequencing and restriction analysis of the 16S rDNA. The most promising isolates were screened for antifungal activities and the production of siderophores and biosurfactants. B. trimera presented a diverse community of rhizobacteria, constituted of 26 families and 41 genera, with a predominance of Streptomyces and Bacillus genera, followed by Paenibacillus, Staphylococcus, Methylobacterium, Rhizobium, Tardiphaga, Paraburkholderia, Burkholderia, and Pseudomonas. The more abundant genera were represented by different species, showing a high diversity of the microbiota associated to B. trimera. Some of these isolates potentially represent novel species and deserve further examination. The communities were influenced by both the edaphic properties of the sampling locations and the plant niches. Approximately one-third of the rhizobacteria exhibited antifungal activity against Sclerotinia sclerotiorum and Colletotrichum gloeosporioides, and a high proportion of isolates produced siderophores (25%) and biosurfactants (42%). The most promising isolates were members of the Streptomyces genus. The survey of B. trimera returned a diverse community of culturable rhizobacteria and identified potential candidates for the development of plant growth-promoting and protection products, reinforcing the need for more comprehensive investigations of the microbiota of Brazilian native plants and habitats.

Revealing potential functions of hypothetical proteins induced by genistein in the symbiosis island of Bradyrhizobium japonicum commercial strain SEMIA 5079 (= CPAC 15).

BACKGROUND: Bradyrhizobium japonicum strain SEMIA 5079 (= CPAC 15) is a nitrogen-fixing symbiont of soybean broadly used in commercial inoculants in Brazil. Its genome has about 50% of hypothetical (HP) protein-coding genes, many in the symbiosis island, raising questions about their putative role on the biological nitrogen fixation (BNF) process. This study aimed to infer functional roles to 15 HP genes localized in the symbiosis island of SEMIA 5079, and to analyze their expression in the presence of a nod-gene inducer. RESULTS: A workflow of bioinformatics tools/databases was established and allowed the functional annotation of the HP genes. Most were enzymes, including transferases in the biosynthetic pathways of cobalamin, amino acids and secondary metabolites that may help in saprophytic ability and stress tolerance, and hydrolases, that may be important for competitiveness, plant infection, and stress tolerance. Putative roles for other enzymes and transporters identified are discussed. Some HP proteins were specific to the genus Bradyrhizobium, others to specific host legumes, and the analysis of orthologues helped to predict roles in BNF. CONCLUSIONS: All 15 HP genes were induced by genistein and high induction was confirmed in five of them, suggesting major roles in the BNF process.

Indole-3-acetic acid production via the indole-3-pyruvate pathway by plant growth promoter Rhizobium tropici CIAT 899 is strongly inhibited by ammonium.

Like many rhizobia, Rhizobium tropici produces indole-3-acetic acid (IAA), an important signal molecule required for root hair infection in rhizobia-legume symbioses. However, the IAA biosynthesis pathway and its regulation by R. tropici are still poorly understood. In this study, IAA synthesis and the effects of mineral N in IAA production by R. tropici CIAT 899 were verified by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). Furthermore, expression of genes related to IAA biosynthesis and metabolism were evaluated by RT-qPCR. Results indicated that IAA production by CIAT 899 was 12 times lower in the presence of [Formula: see text] . Moreover, it was found that indole-3-pyruvate (IPyA) is the major IAA biosynthesis intermediate. Genes y4wE, lao and iorA were identified by analysis of R. tropici genome in silico and were upregulated by tryptophan, indicating a possible role of these genes in IAA biosynthesis by CIAT 899. In conclusion, we show that IPyA is the major pathway for IAA biosynthesis in CIAT 899 and that its production is strongly inhibited by [Formula: see text] . Although present results arose from in vitro experiments, they provide new insight into the role of nitrogen in early events related to legume nodulation.

Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis.

Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and provides evidence for the involvement of an L-amino acid oxidase gene cluster in the biosynthesis of IAA. Furthermore, we showed that the mutant strains with reduction in IAA biosynthesis ability, in consequence of the lower transcription levels of genes of the lao cluster, had remarkable effects on development of rice roots.

Tn5 insertion in the tonB gene promoter affects iron-related phenotypes and increases extracellular siderophore levels in Gluconacetobacter diazotrophicus.

TonB-dependent receptors in concert with the TonB-ExbB-ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5(T). Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP(tonB)) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.

Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean.

BACKGROUND: The soybean-Bradyrhizobium symbiosis can be highly efficient in fixing nitrogen, but few genomic sequences of elite inoculant strains are available. Here we contribute with information on the genomes of two commercial strains that are broadly applied to soybean crops in the tropics. B. japonicum CPAC 15 (=SEMIA 5079) is outstanding in its saprophytic capacity and competitiveness, whereas B. diazoefficiens CPAC 7 (=SEMIA 5080) is known for its high efficiency in fixing nitrogen. Both are well adapted to tropical soils. The genomes of CPAC 15 and CPAC 7 were compared to each other and also to those of B. japonicum USDA 6T and B. diazoefficiens USDA 110T. RESULTS: Differences in genome size were found between species, with B. japonicum having larger genomes than B. diazoefficiens. Although most of the four genomes were syntenic, genome rearrangements within and between species were observed, including events in the symbiosis island. In addition to the symbiotic region, several genomic islands were identified. Altogether, these features must confer high genomic plasticity that might explain adaptation and differences in symbiotic performance. It was not possible to attribute known functions to half of the predicted genes. About 10% of the genomes was composed of exclusive genes of each strain, but up to 98% of them were of unknown function or coded for mobile genetic elements. In CPAC 15, more genes were associated with secondary metabolites, nutrient transport, iron-acquisition and IAA metabolism, potentially correlated with higher saprophytic capacity and competitiveness than seen with CPAC 7. In CPAC 7, more genes were related to the metabolism of amino acids and hydrogen uptake, potentially correlated with higher efficiency of nitrogen fixation than seen with CPAC 15. CONCLUSIONS: Several differences and similarities detected between the two elite soybean-inoculant strains and between the two species of Bradyrhizobium provide new insights into adaptation to tropical soils, efficiency of N2 fixation, nodulation and competitiveness.

Fast induction of biosynthetic polysaccharide genes lpxA, lpxE, and rkpI of Rhizobium sp. strain PRF 81 by common bean seed exudates is indicative of a key role in symbiosis.

Rhizobial surface polysaccharides (SPS) are, together with nodulation (Nod) factors, recognized as key molecules for establishment of rhizobia-legume symbiosis. In Rhizobium tropici, an important nitrogen-fixing symbiont of common bean (Phaseolus vulgaris L.), molecular structures and symbiotic roles of the SPS are poorly understood. In this study, Rhizobium sp. strain PRF 81 genes, belonging to the R. tropici group, were investigated: lpxA and lpxE, involved in biosynthesis and modification of the lipid-A anchor of lipopolysaccharide (LPS), and rkpI, involved in synthesis of a lipid carrier required for production of capsular polysaccharides (KPS). Reverse transcription quantitative PCR (RT-qPCR) analysis revealed, for the first time, that inducers released from common bean seeds strongly stimulated expression of all three SPS genes. When PRF 81 cells were grown for 48 h in the presence of seed exudates, twofold increases (p < 0.05) in the transcription levels of lpxE, lpxA, and rkpI genes were observed. However, higher increases (p < 0.05) in transcription rates, about 50-fold for lpxE and about 30-fold for lpxA and rkpI, were observed after only 5 min of incubation with common bean seed exudates. Evolutionary analyses revealed that lpxA and lpxE of PRF81 and of the type strain of R. tropici CIAT899(T)clustered with orthologous Rhizobium radiobacter and were more related to R. etli and Rhizobium leguminosarum, while rkpI was closer to the Sinorhizobium sp. group. Upregulation of lpxE, lpxA, and rkpI genes suggests that seed exudates can modulate production of SPS of Rhizobium sp. PRF81, leading to cell wall changes necessary for symbiosis establishment.

Proteomic Analysis of Soybean [Glycine max (L.) Merrill] Roots Inoculated with Bradyrhizobium japonicum Strain CPAC 15.

This research intended to analyze the expression pattern of proteins in roots of the Brazilian soybean cultivar Conquista when inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. At ten days after bacterial inoculation, whole-cell proteins were extracted from roots and separated by 2-D gel electrophoresis. Comparative analysis revealed significant changes in the intensity of 37 spots due to the inoculation (17 up-regulated and 20 down-regulated proteins), identified by MALDI-TOF/TOF-TOF. Identified proteins were associated with COG functional categories of information storage and processing, cellular processes and signaling, metabolism, and also in the "poorly characterized" and "not in COG" categories. Among the up-regulated proteins, we identified sucrose synthase (nodulin-100), ß-tubulin, rubisco activase, glutathione-S-transferase, a putative heat-shock 70-kDa protein, pyridine nucleotide-disulphideoxidoreductase and a putative transposase. Proteomic analysis allowed for the identification of some putative symbiotic functions and confirmed the main biological processes triggered in the nitrogen-fixing symbiosis with soybean.

Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.).

BACKGROUND: Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. RESULTS: Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. CONCLUSIONS: Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean and other legume hosts.

A simple, economical and reproducible protein extraction protocol for proteomics studies of soybean roots.

Sample preparation is a critical step in two-dimensional gel electrophoresis (2-DE) of plant tissues. Here we describe a phenol/SDS procedure that, although greatly simplified, produced well-resolved and reproducible 2-DE profiles of protein extracts from soybean [Glycine max (L.) Merril] roots. Extractions were made in three replicates using both the original and simplified procedure. To evaluate the quality of the extracted proteins, ten spots were randomly selected and identified by mass spectrometry (MS). The 2-DE gels were equally well resolved, with no streaks or smears, and no significant differences were observed in protein yield, reproducibility, resolution or number of spots. Mass spectra of the ten selected spots were compared with database entries and allowed high-quality identification of proteins. The simplified protocol described here presents considerable savings of time and reagents without compromising the quality of 2-DE protein profiles and compatibility with MS analysis, and may facilitate the progress of proteomics studies of legume-rhizobia interactions.

The nodC, nodG, and glgX genes of Rhizobium tropici strain PRF 81.

Rhizobium tropici is a diazotrophic microsymbiont of common bean (Phaseolus vulgaris L.) that encompasses important but still poorly studied tropical strains, and a recent significant contribution to the knowledge of the species was the publication of a genomic draft of strain PRF 81, which revealed several novel genes [Pinto et al. Funct Int Gen 9:263-270, 2009]. In this study, we investigated the transcription of nodC, nodG, and glgX genes, located in the nod operon of PRF 81 strain, by reverse-transcription quantitative PCR. All three genes showed low levels of transcription when the cells were grown until exponential growth phase in the presence of common-bean-seed exudates or of the root nod-gene inducer naringenin. However, when cells at the exponential phase of growth were incubated with seed exudates, transcription occurred after only 5 min, and nodC, nodG, and glgX were transcribed 121.97-, 14.86-, and 50.29-fold more than the control, respectively, followed by a rapid decrease in gene transcription. Much lower levels of transcription were observed in the presence of naringenin; furthermore, maximum transcription required 8 h of incubation for all three genes. In light of these results, the mechanisms of induction of the nodulation genes by flavonoids are discussed.

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

BACKGROUND: Gluconacetobacter 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. RESULTS: Gluconacetobacter 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. CONCLUSION: The 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.