Endophytic Seed-Associated Bacteria as Plant Growth Promoters of Cuban Rice (Oryza sativa L.).

Cuban rice cultivars INCA LP-5 and INCA LP-7 are widely distributed in Cuba and Caribbean countries. Although there are studies about rhizospheric bacteria associated with these cultivars, there are no reports about their seed-associated bacteria. This study aimed to isolate endophytic bacteria from rice seeds and select those with the greatest plant growth-promoting traits. A total of nineteen bacterial strains from the genera Pantoea, Bacillus, Paenibacillus, and Pseudomonas were isolated from the husk and endosperm of rice seeds. The strains Pantoea sp. S5-1, Pseudomonas sp. S5-38, and Pseudomonas sp. S7-1 were classified as the most promissory to increase rice growth as they demonstrated the presence of multiple plant growth-promoting traits such as the production of auxins, phosphate, and potassium solubilization, the production of siderophores, and the inhibition of the phytopathogen Pyricularia oryzae. The inoculation of strains of Pantoea sp. and Pseudomonas spp. in rice improves the height, root length, fresh weight, and dry weight of the shoot and root after 21 days post-inoculation in hydroponic assays. This study constitutes the first report on Cuban rice cultivars about the presence of endophytes in seeds and their potential to promote seedling growth. Pantoea sp. S5-1, Pseudomonas sp. S5-38, and Pseudomonas sp. S7-1 were selected as the more promising strains for the development of bio-stimulators or bio-inoculants for Cuban rice crops.

Synthetic Biology Toolbox for Antarctic Pseudomonas sp. Strains: Toward a Psychrophilic Nonmodel Chassis for Function-Driven Metagenomics.

One major limitation of function-driven metagenomics is the ability of the host to express the metagenomic DNA correctly. Differences in the transcriptional, translational, and post-translational machinery between the organism to which the DNA belongs and the host strain are all factors that influence the success of a functional screening. For this reason, the use of alternative hosts is an appropriate approach to favor the identification of enzymatic activities in function-driven metagenomics. To be implemented, appropriate tools should be designed to build the metagenomic libraries in those hosts. Moreover, discovery of new chassis and characterization of synthetic biology toolbox in nonmodel bacteria is an active field of research to expand the potential of these organisms in processes of industrial interest. Here, we assessed the suitability of two Antarctic psychrotolerant Pseudomonas strains as putative alternative hosts for function-driven metagenomics using pSEVA modular vectors as scaffold. We determined a set of synthetic biology tools suitable for these hosts and, as a proof of concept, we demonstrated their fitness for heterologous protein expression. These hosts represent a step forward for the prospection and identification of psychrophilic enzymes of biotechnological interest.

Paenibacillus farraposensis sp. nov., isolated from a root nodule of Arachis villosa.

Strain UY79T was isolated from a root nodule of Arachis villosa, collected at the Esteros de Farrapos National Park, Río Negro, Uruguay. Cells were non-motile Gram-variable rods with central to subterminal oval to ellipsoidal endospores that swell the sporangia. Growth was observed in the range of 15-42 °C (optimum, 30 °C), pH 5.0-9.0 (optimum, pH 7.0-8.0) and with up to 3 % (w/v) NaCl (optimum, 1-2 %). Strain UY79T was facultative anaerobic, catalase-positive and oxidase-negative. According to the results of 16S rRNA gene sequence analysis, UY79T belongs to the genus Paenibacillus and is closely related to P. ottowii MS2379T, P. peoriae BD-57T, P. polymyxa ATCC 842T and P. brasilensis PB172T, exhibiting 99.4, 99.0, 99.0 and 98.9% sequence identity, respectively. Average nucleotide identity and digital DNA-DNA hybridization values with the most closely related type strains were 74.3-88.6% and 38.2-48.7 %, respectively. Major fatty acids (>10 %) were anteiso-C15:0, iso-C15:0, and C16 : 0. Menaquinones MK-7 and MK-6 were the only isoprenoid quinones detected. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified glycolipid. Spermidine was the predominant polyamine. The DNA G+C content based on the draft genome sequence was 46.34 mol%. Based on the current polyphasic study, UY79T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus farraposensis sp. nov. is proposed. The type strain is UY79T (=CCM 9147T=CGMCC 1.19038T).

Paenibacillus sp. Strain UY79, Isolated from a Root Nodule of Arachis villosa, Displays a Broad Spectrum of Antifungal Activity.

A nodule-inhabiting Paenibacillus sp. strain (UY79) isolated from wild peanut (Arachis villosa) was screened for its antagonistic activity against diverse fungi and oomycetes (Botrytis cinerea, Fusarium verticillioides, Fusarium oxysporum, Fusarium graminearum, Fusarium semitectum, Macrophomina phaseolina, Phomopsis longicolla, Pythium ultimum, Phytophthora sojae, Rhizoctonia solani, Sclerotium rolfsii, and Trichoderma atroviride). The results obtained show that Paenibacillus sp. UY79 was able to antagonize these fungi/oomycetes and that agar-diffusible compounds and volatile compounds (different from HCN) participate in the antagonism exerted. Acetoin, 2,3-butanediol, and 2-methyl-1-butanol were identified among the volatile compounds produced by strain UY79 with possible antagonistic activity against fungi/oomycetes. Paenibacillus sp. strain UY79 did not affect symbiotic association or growth promotion of alfalfa plants when coinoculated with rhizobia. By whole-genome sequence analysis, we determined that strain UY79 is a new species of Paenibacillus within the Paenibacillus polymyxa complex. Diverse genes putatively involved in biocontrol activity were identified in the UY79 genome. Furthermore, according to genome mining and antibiosis assays, strain UY79 would have the capability to modulate the growth of bacteria commonly found in soil/plant communities. IMPORTANCE Phytopathogenic fungi and oomycetes are responsible for causing devastating losses in agricultural crops. Therefore, there is enormous interest in the development of effective and complementary strategies that allow the control of the phytopathogens, reducing the input of agrochemicals in croplands. The discovery of new strains with expanded antifungal activities and with a broad spectrum of action is challenging and of great future impact. Diverse strains belonging to the P. polymyxa complex have been reported to be effective biocontrol agents. Results presented here show that the novel discovered strain of Paenibacillus sp. presents diverse traits involved in antagonistic activity against a broad spectrum of pathogens and is a potential and valuable strain to be further assessed for the development of biofungicides.

Draft Genome Sequence of Paraburkholderia sp. UYCP14C, a Rhizobium Strain Isolated from Root Nodules of Calliandra parvifolia.

Here, we present the draft genome sequence of strain UYCP14C, a rhizobium isolated from Calliandra parvifolia nodules. The assembled genome size was around 9.8 million bp, containing 9,031 predicted protein-coding sequences, including several symbiotic and nitrogen fixation genes. UYCP14C appears to be a novel species of the plant growth-promoting Paraburkholderia genus.

Highly conserved nucleotide motifs present in the 5'UTR of the heme-receptor gene shmR are required for HmuP-dependent expression of shmR in Ensifer meliloti.

Heme may represent a major iron-source for bacteria. In the symbiotic nitrogen-fixing bacterium Ensifer meliloti 1021, iron acquisition from heme depends on the outer-membrane heme-receptor ShmR. Expression of shmR gene is repressed by iron in a RirA dependent manner while under iron-limitation its expression requires the small protein HmuP. In this work, we identified highly conserved nucleotide motifs present upstream the shmR gene. These motifs are widely distributed among Alpha and Beta Proteobacteria, and correlate with the presence of HmuP coding sequences in bacterial genomes. According to data presented in this work, we named these new motifs as HmuP-responsive elements (HPREs). In the analyzed genomes, the HPREs were always present upstream of genes encoding putative heme-receptors. Moreover, in those Alpha and Beta Proteobacteria where transcriptional start sites for shmR homologs are known, HPREs were located in the 5'UTR region. In this work we show that in E. meliloti 1021, HPREs are involved in HmuP-dependent shmR expression. Moreover, we show that changes in sequence composition of the HPREs correlate with changes in a predicted RNA secondary structure element and affect shmR gene expression.

Herbaspirillum seropedicae Differentially Expressed Genes in Response to Iron Availability.

Herbaspirillum seropedicae Z67 is a nitrogen-fixing endophyte that colonizes many important crops. Like in almost all organisms, vital cellular processes of this endophyte are iron dependent. In order to efficiently acquire iron to fulfill its requirements, this bacterium produces the siderophores serobactins. However, the presence in its genome of many others iron acquisition genes suggests that serobactins are not the only strategy used by H. seropedicae to overcome metal deficiency. The aim of this work was to identify genes and proteins differentially expressed by cells growing in low iron conditions in order to describe H. seropedicae response to iron limitation stress. For this purpose, and by using a transcriptomic approach, we searched and identified a set of genes up-regulated when iron was scarce. One of them, Hsero_2337, codes for a TonB-dependent transporter/transducer present in the serobactins biosynthesis genomic locus, with an unknown function. Another TonB-dependent receptor, the one encoded by Hsero_1277, and an inner membrane ferrous iron permease, coded by Hsero_2720, were also detected. By using a proteomic approach focused in membrane proteins, we identified the specific receptor for iron-serobactin internalization SbtR and two non-characterized TonB-dependent receptors (coded by genes Hsero_1277 and Hsero_3255). We constructed mutants on some of the identified genes and characterized them by in vitro growth, biofilm formation, and interaction with rice plants. Characterization of mutants in gene Hsero_2337 showed that the TonB-dependent receptor coded by this gene has a regulatory role in the biosynthesis of serobactins, probably by interacting with the alternative sigma factor PfrI, coded by gene Hsero_2338. Plant colonization of the mutant strains was not affected, since the mutant strain normally colonize the root and aerial part of rice plants. These results suggest that the strategies used by H. seropedicae to acquire iron inside plants are far more diverse than the ones characterized in this work. In vivo expression studies or colonization competition experiments between the different mutant strains could help us in future works to determine the relative importance of the different iron acquisition systems in the interaction of H. seropedicae with rice plants.

The Irr and RirA Proteins Participate in a Complex Regulatory Circuit and Act in Concert To Modulate Bacterioferritin Expression in Ensifer meliloti 1021.

In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the smc03787 open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787 We demonstrate that bfr gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA is inversely correlated to that of smc03787 Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr expression and that additional unknown factors are involved in iron-dependent regulation.IMPORTANCEE. meliloti belongs to the Alphaproteobacteria, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied Gammaproteobacteria In this work we analyzed the effect of rirA and irr mutations on bfr gene expression. We demonstrate the effect of an irr mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance bfr gene expression.

Draft Genome Sequence of Cupriavidus UYMMa02A, a Novel Beta-Rhizobium Species.

We present the draft genome of Cupriavidus UYMMa02A, a rhizobium strain isolated from root nodules of Mimosa magentea The assembly has approximately 8.1 million bp with an average G+C of 64.1%. Symbiotic and metal-resistance genes were identified. The study of this genome will contribute to the understanding of rhizobial evolution.

Essential Genes for In Vitro Growth of the Endophyte Herbaspirillum seropedicae SmR1 as Revealed by Transposon Insertion Site Sequencing.

The interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil. Herbaspirillum seropedicae strain SmR1, belonging to the betaproteobacteria, is an endophyte that colonizes crops, including rice, maize, sugarcane, and sorghum. Different approaches have revealed genes and pathways regulated during the interactions of H. seropedicae with its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools. Here we successfully employed a combination of in vivo high-density mariner Tn mutagenesis and targeted Tn insertion site sequencing (Tn-seq) in H. seropedicae SmR1. The analysis of multiple gene-saturating Tn libraries revealed that 395 genes are essential for the growth of H. seropedicae SmR1 in tryptone-yeast extract medium. A comparative analysis with the Database of Essential Genes (DEG) showed that 25 genes are uniquely essential in H. seropedicae SmR1. The Tn mutagenesis protocol developed and the gene-saturating Tn libraries generated will facilitate elucidation of the genetic mechanisms of the H. seropedicae endophytic lifestyle. IMPORTANCE: A focal point in the study of endophytes is the development of effective biofertilizers that could help to reduce the input of agrochemicals in croplands. Besides the ability to promote plant growth, a good biofertilizer should be successful in colonizing its host and competing against the native microbiota. By using a systematic Tn-based gene-inactivation strategy and massively parallel sequencing of Tn insertion sites (Tn-seq), it is possible to study the fitness of thousands of Tn mutants in a single experiment. We have applied the combination of these techniques to the plant-growth-promoting endophyte Herbaspirillum seropedicae SmR1. The Tn mutant libraries generated will enable studies into the genetic mechanisms of H. seropedicae-plant interactions. The approach that we have taken is applicable to other plant-interacting bacteria.

Improved glycerol to ethanol conversion by E. coli using a metagenomic fragment isolated from an anaerobic reactor.

Crude glycerol obtained as a by-product of biodiesel production is a reliable feedstock with the potential to be converted into reduced chemicals with high yields. It has been previously shown that ethanol is the primary product of glycerol fermentation by Escherichia coli. However, few efforts were made to enhance this conversion by means of the expression of heterologous genes with the potential to improve glycerol transport or metabolism. In this study, a fosmid-based metagenomic library constructed from an anaerobic reactor purge sludge was screened for genetic elements that promote the use and fermentation of crude glycerol by E. coli. One clone was selected based on its improved growth rate on this feedstock. The corresponding fosmid, named G1, was fully sequenced (41 kbp long) and the gene responsible for the observed phenotype was pinpointed by in vitro insertion mutagenesis. Ethanol production from both pure and crude glycerol was evaluated using the parental G1 clone harboring the ethanologenic plasmid pLOI297 or the industrial strain LY180 complemented with G1. In mineral salts media containing 50 % (v/v) pure glycerol, ethanol concentrations increased two-fold on average when G1 was present in the cells reaching up to 20 g/L after 24 h fermentation. Similar fermentation experiments were done using crude instead of pure glycerol. With an initial OD620 of 8.0, final ethanol concentrations after 24 h were much higher reaching 67 and 75 g/L with LY180 cells carrying the control fosmid or the G1 fosmid, respectively. This translates into a specific ethanol production rate of 0.39 g h(-1) OD(-1) L(-1).

Novel Cupriavidus Strains Isolated from Root Nodules of Native Uruguayan Mimosa Species.

UNLABELLED: The large legume genus Mimosa is known to be associated with both alphaproteobacterial and betaproteobacterial symbionts, depending on environment and plant taxonomy, e.g., Brazilian species are preferentially nodulated by Burkholderia, whereas those in Mexico are associated with alphaproteobacterial symbionts. Little is known, however, about the symbiotic preferences of Mimosa spp. at the southern subtropical limits of the genus. In the present study, rhizobia were isolated from field-collected nodules from Mimosa species that are native to a region in southern Uruguay. Phylogenetic analyses of sequences of the 16S rRNA, recA, and gyrB core genome and the nifH and nodA symbiosis-essential loci confirmed that all the isolates belonged to the genus Cupriavidus However, none were in the well-described symbiotic species C. taiwanensis, but instead they were closely related to other species, such as C. necator, and to species not previously known to be symbiotic (or diazotrophic), such as C. basilensis and C. pinatubonensis Selection of these novel Cupriavidus symbionts by Uruguayan Mimosa spp. is most likely due to their geographical separation from their Brazilian cousins and to the characteristics of the soils in which they were found. IMPORTANCE: With the aim of exploring the diversity of rhizobia associated with native Mimosa species, symbionts were isolated from root nodules on five Mimosa species that are native to a region in southern Uruguay, Sierra del Abra de Zabaleta. In contrast to data obtained in the major centers of diversification of the genus Mimosa, Brazil and Mexico, where it is mainly associated with Burkholderia and Rhizobium/Ensifer, respectively, the present study has shown that all the isolated symbiotic bacteria belonged to the genus Cupriavidus Interestingly, none of nodules contained bacteria belonging to the well-described symbiotic species C. taiwanensis, but instead they were related to other Cupriavidus species such as C. necator and C. pinatubonensis These data suggest the existence of a higher diversity within beta-rhizobial Cupriavidus than was previously suspected, and that Mimosa spp. from Sierra del Abra de Zabaleta, may be natural reservoirs for novel rhizobia.

HmuS and HmuQ of Ensifer/Sinorhizobium meliloti degrade heme in vitro and participate in heme metabolism in vivo.

Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a Kd value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-ß and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.

Serobactins-mediated iron acquisition systems optimize competitive fitness of Herbaspirillum seropedicae inside rice plants.

Herbaspirillum seropedicae Z67 is a diazotrophic endophyte able to colonize the interior of many economically relevant crops such as rice, wheat, corn and sorghum. Under iron-deficient conditions, this organism secretes serobactins, a suite of lipopetide siderophores. The role of siderophores in the interaction between endophytes and their plant hosts are not well understood. In this work, we aimed to determine the importance of serobactins-mediated iron acquisition systems in the interaction of H. seropedicae with rice plants. First we provide evidence, by using a combination of genome analysis, proteomic and genetic studies, that the Hsero_2345 gene encodes a TonB-dependent receptor involved in iron-serobactin complex internalization when iron bioavailability is low. Our results show that survival of the Hsero_2345 mutant inside rice plants was not significantly different from that of the wild-type strain. However, when plants were co-inoculated at equal ratios with the wild-type strain and with a double mutant defective in serobactins synthesis and internalization, recovery of mutant was significantly impaired after 8 days post-inoculation. These results demonstrate that serobactins-mediated iron acquisition contributes to competitive fitness of H. seropedicae inside host plants.

High-quality permanent draft genome sequence of the Parapiptadenia rigida-nodulating Cupriavidus sp. strain UYPR2.512.

Cupriavidus sp. strain UYPR2.512 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Parapiptadenia rigida grown in soils from a native forest of Uruguay. Here we describe the features of Cupriavidus sp. strain UYPR2.512, together with sequence and annotation. The 7,858,949 bp high-quality permanent draft genome is arranged in 365 scaffolds of 369 contigs, contains 7,411 protein-coding genes and 76 RNA-only encoding genes, and is part of the GEBA-RNB project proposal.

High-quality permanent draft genome sequence of the Parapiptadenia rigida-nodulating Burkholderia sp. strain UYPR1.413.

Burkholderia sp. strain UYPR1.413 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Parapiptadenia rigida collected at the Angico plantation, Mandiyu, Uruguay, in December 2006. A survey of symbionts of P. rigida in Uruguay demonstrated that this species is nodulated predominantly by Burkholderia microsymbionts. Moreover, Burkholderia sp. strain UYPR1.413 is a highly efficient nitrogen fixing symbiont with this host. Currently, the only other sequenced isolate to fix with this host is Cupriavidus sp. UYPR2.512. Therefore, Burkholderia sp. strain UYPR1.413 was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the GEBA-RNB project. Here we describe the features of Burkholderia sp. strain UYPR1.413, together with sequence and annotation. The 10,373,764 bp high-quality permanent draft genome is arranged in 336 scaffolds of 342 contigs, contains 9759 protein-coding genes and 77 RNA-only encoding genes.

Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation.

With the aim of improving current ethanologenic Escherichia coli strains, we screened a metagenomic library from bovine ruminal fluid for cellulolytic enzymes. We isolated one fosmid, termed Csd4, which was able to confer to E. coli the ability to grow on complex cellulosic material as the sole carbon source such as avicel, carboxymethyl cellulose, filter paper, pretreated sugarcane bagasse, and xylan. Glucanolytic activity obtained from E. coli transformed with Csd4 was maximal at 24 h of incubation and was inhibited when glucose or xylose were present in the media. The 34,406-bp DNA fragment of Csd4 was completely sequenced, and a putative endoglucanase, a xylosidase/arabinosidase, and a laccase gene were identified. Comparison analysis revealed that Csd4 derived from an organism closely related to Prevotella ruminicola, but no homologies were found with any of the genomes already sequenced. Csd4 was introduced into the ethanologenic E. coli MS04 strain and ethanol production from CMC, avicel, sugarcane bagasse, or filter paper was observed. Exogenously expressed ß-glucosidase had a positie effect on cell growth in agreement with the fact that no putative ß-glucosidase was found in Csd4. Ethanol production from sugarcane bagasse was improved threefold by Csd4 after saccharification by commercial Trichoderma reesei cellulases underlining the ability of Csd4 to act as a saccharification enhancer to reduce the enzymatic load and time required for cellulose deconstruction.

Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes.

A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40 °C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.

Identification and structural characterization of serobactins, a suite of lipopeptide siderophores produced by the grass endophyte Herbaspirillum seropedicae.

Herbaspirillum seropedicae Z67 is a diazotrophic endophyte able to colonize the interior of many economically relevant crops such as rice, wheat, corn and sorghum. Structures of siderophores produced by bacterial endophytes have not yet been elucidated. The aim of this work was to identify and characterize the siderophores produced by this bacterium. In a screening for mutants unable to produce siderophores we found a mutant that had a transposon insertion in a non-ribosomal peptide synthase (NRPS) gene coding for a putative siderophore biosynthetic enzyme. The chemical structure of the siderophore was predicted using computational genomic tools. The predicted structure was confirmed by chemical analysis. We found that siderophores produced by H. seropedicae Z67 are a suite of amphiphilic lipopeptides, named serobactin A, B and C, which vary by the length of the fatty acid chain. We also demonstrated the biological activity of serobactins as nutritional iron sources for H. seropedicae. These are the first structurally described siderophores produced by endophytic bacteria.

New betaproteobacterial Rhizobium strains able to efficiently nodulate Parapiptadenia rigida (Benth.) Brenan.

Among the leguminous trees native to Uruguay, Parapiptadenia rigida (Angico), a Mimosoideae legume, is one of the most promising species for agroforestry. Like many other legumes, it is able to establish symbiotic associations with rhizobia and belongs to the group known as nitrogen-fixing trees, which are major components of agroforestry systems. Information about rhizobial symbionts for this genus is scarce, and thus, the aim of this work was to identify and characterize rhizobia associated with P. rigida. A collection of Angico-nodulating isolates was obtained, and 47 isolates were selected for genetic studies. According to enterobacterial repetitive intergenic consensus PCR patterns and restriction fragment length polymorphism analysis of their nifH and 16S rRNA genes, the isolates could be grouped into seven genotypes, including the genera Burkholderia, Cupriavidus, and Rhizobium, among which the Burkholderia genotypes were the predominant group. Phylogenetic studies of nifH, nodA, and nodC sequences from the Burkholderia and the Cupriavidus isolates indicated a close relationship of these genes with those from betaproteobacterial rhizobia (beta-rhizobia) rather than from alphaproteobacterial rhizobia (alpha-rhizobia). In addition, nodulation assays with representative isolates showed that while the Cupriavidus isolates were able to effectively nodulate Mimosa pudica, the Burkholderia isolates produced white and ineffective nodules on this host.