A glimpse of the endophytic bacterial diversity in roots of blackberry plants (Rubus fruticosus).

The aim of this study was to explore the diversity of culturable bacterial communities residing in blackberry plants (Rubus fruticosus). Bacterial endophytes were isolated from plant roots, and their 16S rDNA sequences were amplified and sequenced. Our results show that the roots of R. fruticosus exhibit low colony forming units of bacterial endophytes per gram of fresh tissue (6 x 102 ± 0.5 x 102). We identified 41 endophytic bacterial species in R. fruticosus by BLAST homology search and a subsequent phylogenetic analysis, belonging to the classes Actinobacteria, Bacilli, Alfaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Predominantly, genera belonging the Proteobacteria (Burkholderia, 29.4%; Herbaspirillum, 10.7%; Pseudomonas, 4.9%; and Dyella, 3.9%), Firmicutes (Bacillus, 42.1%), and Actinobacteria (two isolates showing high identity with the Streptomyces genus, 1.9%) divisions were identified. Fifty percent of the bacterial endophytes produced the phytohormone indole-acetic acid (IAA), eleven of which exhibited higher IAA production (>5.8 mg/mL) compared to the plant growth-promoting strain, Pseudomonas fluorescens UM270. Additionally, the endophytic isolates exhibited protease activity (22%), produced siderophores (26.4%), and demonstrated antagonistic action (>50% inhibition of mycelial growth) against the grey mold phytopathogen Botrytis cinerea (3.9%). These results suggested that field-grown R. fruticosus plants contain bacterial endophytes within their tissues with the potential to promote plant growth and display antagonism towards plant pathogens.

Predicting Functional Interactions Among Genes in Prokaryotes by Genomic Context.

Genomic context methods for finding functions of unannotated genes were implemented very early after the publication of the first few prokaryotic genomes. The ideas behind these methods include gene fusions, conservation of gene adjacency, and the patters of co-occurrence of genes across available genomes. A later addition was the prediction of features related to functional organization, such as operons, stretches of genes co-transcribed into a single messenger RNA. The ideas behind these methods tend to be easy to understand, while the strategies for transforming those basic ideas into predictions can vary in complexity, mostly because genes whose products are known to functionally interact vary in the way they relate to those basic ideas. We present here a view of genomic context methods for predicting functional interactions, with simple examples of their implementation as compared and evaluated using genes whose products are known to functionally interact.

Isolation and characterization of genetic variability in bacteria with ß-hemolytic and antifungal activity isolated from the rhizosphere of Medicago truncatula plants.

In the present study, we analyzed the frequency of hemolytic and antifungal activities in bacterial isolates from the rhizosphere of Medicago truncatula plants. Of the 2000 bacterial colonies, 96 showed ß-hemolytic activities (frequency, 4.8 x 10(-2)). Hemolytic isolates were analyzed for their genetic diversity by using random amplification of polymorphic DNA, yielding 88 haplotypes. The similarity coefficient of Nei and Li showed a polymorphic diversity ranging from 0.3 to 1. Additionally, 8 of the hemolytic isolates showed antifungal activity toward plant pathogens, Diaporthe phaseolorum, Colletotrichum acutatum, Rhizoctonia solani, and Fusarium oxysporum. The 16S ribosomal sequencing analysis showed that antagonistic bacterial isolates corresponded to Bacillus subtilis (UM15, UM33, UM42, UM49, UM52, and UM91), Bacillus pumilus (UM24), and Bacillus licheniformis (UM88). The present results revealed a higher genetic diversity among hemolytic isolates compared to that of isolates with antifungal action.

Microbial electrolysis cell scale-up for combined wastewater treatment and hydrogen production.

This study demonstrates microbial electrolysis cell (MEC) scale-up from a 50mL to a 10L cell. Initially, a 50mL membraneless MEC with a gas diffusion cathode was operated on synthetic wastewater at different organic loads. It was concluded that process scale-up might be best accomplished using a "reactor-in-series" concept. Consequently, 855mL and 10L MECs were built and operated. By optimizing the hydraulic retention time (HRT) of the 855mL MEC and individually controlling the applied voltages of three anodic compartments with a real-time optimization algorithm, a COD removal of 5.7g L(R)(-1)d(-1) and a hydrogen production of 1.0-2.6L L(R)(-1)d(-1) was achieved. Furthermore, a two MECs in series 10L setup was constructed and operated on municipal wastewater. This test showed a COD removal rate of 0.5g L(R)(-1)d(-1), a removal efficiency of 60-76%, and an energy consumption of 0.9Whperg of COD removed.

Identification and expression analysis of multiple FRO gene copies in Medicago truncatula.

Iron (Fe) is an essential element for plant growth. Commonly, this element is found in an oxidized form in soil, which is poorly available for plants. Therefore, plants have evolved ferric-chelate reductase enzymes (FRO) to reduce iron into a more soluble ferrous form. Fe scarcity in plants induce the FRO enzyme activity. Although the legume Medicago truncatula has been employed as a model for FRO activity studies, only one copy of the M. truncatula MtFRO1 gene has been characterized so far. In this study, we identified multiple gene copies of the MtFRO gene in the genome of M. truncatula by an in silico search, using BLAST analysis in the database of the M. truncatula Genome Sequencing Project and the National Center for Biotechnology Information, and also determined whether they are functional. We identified five genes apart from MtFRO1, which had been already characterized. All of the MtFRO genes exhibited high identity with homologous FRO genes from Lycopersicon esculentum, Citrus junos and Arabidopsis thaliana. The gene copies also presented characteristic conserved FAD and NADPH motifs, transmembrane regions and oxidoreductase signature motifs. We also detected expression in five of the putative MtFRO sequences by semiquantitative RT-PCR analysis, performed with mRNA from root and shoot tissues. Iron scarcity might be a condition for an elevated expression of the MtFRO genes observed in different M. truncatula tissues.

Isolation and molecular characterization of a novel strain of Bacillus with antifungal activity from the sorghum rhizosphere.

We looked for bacterial strains with antifungal activity in the sorghum rhizosphere. A prescreening procedure to search for hemolytic activity among the isolated strains allowed us to detect good fungitoxic activity in a bacterial isolate that we named UM96. This bacterial isolate showed strong growth inhibition in bioassays against the pathogens Diaporthe phaseolorum, Colletotrichum acutatum, Rhizoctonia solani, and Fusarium oxysporum. The supernatant of isolate UM96 also showed strong hemolytic activity, which was not observed in the protease-treated supernatant. However, the supernatant that was treated with protease had similar antagonistic effects to those exhibited by the supernatant that was not treated with this enzyme. These results suggest that a bacteriocin-like compound is responsible for the hemolytic activity; whereas, as far as antifungal effect is concerned, an antibiotic of nonribosomal origin, such as a lipopeptide, might be acting. Further molecular characterization by partial 16S rDNA sequencing placed isolate UM96 in a cluster with Bacillus amyloliquefaciens; however, the highest identity match found in databases of Bacillus species was 91% identity. This suggests that Bacillus sp UM96 might be a novel species.

Phylogenetic analysis reveals gene conversions in multigene families of rhizobia.

Gene families are an important and intrinsic trait of rhizobial species. These gene copies can participate in non-reciprocal recombination events, also called gene conversions. Gene conversion has diverse roles, but it is usually implicated in the evolution of multigene families. Here, we searched for gene conversions in multigene families of six representative rhizobial genomes. We identified 11 gene families with different numbers of copies, genome location and function in CFN42 and CIAT652 strains of Rhizobium etli, Rhizobium sp NGR234, Mesorhizobium loti MAFF303099, Sinorhizobium meliloti 1021, and Bradyrhizobium japonicum USDA110. Gene conversions were detected by phylogenetic inference in the nifD and nifK gene families in R. etli. Sequence analysis confirmed multiple gene conversions in these two gene families. We suggest that gene conversion events have an important role in homogenizing multigene families in rhizobia.

Diversity of bacterial endophytes in roots of Mexican husk tomato plants (Physalis ixocarpa) and their detection in the rhizosphere.

Endophytic bacterial diversity was estimated in Mexican husk tomato plant roots by amplified rDNA restriction analysis and sequence homology comparison of the 16S rDNA genes. Sixteen operational taxonomic units from the 16S rDNA root library were identified based on sequence analysis, including the classes Gammaproteobacteria, Betaproteobacteria, Actinobacteria, and Bacilli. The predominant genera were Stenotrophomonas (21.9%), Microbacterium (17.1%), Burkholderia (14.3%), Bacillus (14.3%), and Pseudomonas (10.5%). In a 16S rDNA gene library of the same plant species' rhizosphere, only common soil bacteria, including Stenotrophomonas, Burkholderia, Bacillus, and Pseudomonas, were detected. We suggest that the endophytic bacterial diversity within the roots of Mexican husk tomato plants is a subset of the rhizosphere bacterial population, dominated by a few genera.

Effect of co-inoculation with mycorrhiza and rhizobia on the nodule trehalose content of different bean genotypes.

Studies on Rhizobium-legume symbiosis show that trehalose content in nodules under drought stress correlates positively with an increase in plant tolerance to this stress. Fewer reports describe trehalose accumulation in mycorrhiza where, in contrast with rhizobia, there is no flux of carbohydrates from the microsymbiont to the plant. However, the trehalose dynamics in the Mycorrhiza-Rhizobium-Legume tripartite symbiosis is unknown. The present study explores the role of this tripartite symbiosis in the trehalose content of nodules grown under contrasting moisture conditions. Three wild genotypes (P. filiformis, P. acutifolis and P. vulgaris) and two commercial genotypes of Phaseolus vulgaris (Pinto villa and Flor de Mayo) were used. Co-inoculation treatments were conducted with Glomus intraradices and a mixture of seven native rhizobial strains, and trehalose content was determined by GC/MS. The results showed a negative effect of mycorrhizal inoculation on nodule development, as mycorrhized plants showed fewer nodules and lower nodule dry weight compared to plants inoculated only with Rhizobium. Mycorrhizal colonization was also higher in plants inoculated only with Glomus as compared to plants co-inoculated with both microsymbionts. In regard to trehalose, co-inoculation negatively affects its accumulation in the nodules of each genotype tested. However, the correlation analysis showed a significantly positive correlation between mycorrhizal colonization and nodule trehalose content.

Myosin I phosphorylation is increased by chemotactic stimulation.

Directed cell migration occurs in response to extracellular cues. Following stimulation of a cell with chemoattractant, a significant rearrangement of the actin cytoskeleton is mediated by intracellular signaling pathways and results in polarization of the cell and movement via pseudopod extension. Amoeboid myosin Is play a critical role in regulating pseudopod formation in Dictyostelium, and their activity is activated by heavy chain phosphorylation. The effect of chemotactic stimulation on the in vivo phosphorylation level of a Dictyostelium myosin I, myoB, was tested. The myoB heavy chain is phosphorylated in vivo on serine 322 (the myosin TEDS rule phosphorylation site) in chemotactically competent cells. The level of myoB phosphorylation increases following stimulation of starving cells with the chemoattractant cAMP. A 3-fold peak increase in the level of phosphorylation is observed at 60 s following stimulation, a time at which the Dictyostelium cell actively extends pseudopodia. These findings suggest that chemotactic stimulation results in increased myoB activity via heavy chain phosphorylation and contributes to the global extension of pseudopodia that occurs prior to polarization and directed motility.

Arachidonate and related unsaturated fatty acids selectively inactivate the guanine nucleotide-binding regulatory protein, Gz.

Gz is a member of the family of trimeric guanine nucleotide-binding regulatory proteins (G proteins), which plays a crucial role in signaling across cell membranes. The expression of Gz is predominately confined to neuronal cells and platelets, suggesting an involvement in a neuroendocrine process. Although the signaling pathway in which Gz participates is not yet known, it has been linked to inhibition of adenylyl cyclase. We have found that arachidonate and related unsaturated fatty acids suppress guanine nucleotide binding to the alpha subunit of Gz. This inhibition of nucleotide binding by cis-unsaturated fatty acids is specific for Gz alpha; other G protein alpha subunits are relatively insensitive to these lipids. The IC50 for inhibition by the lipids closely corresponds to their critical micellar concentrations, suggesting that the interaction of the lipid micelle with Gzalpha is the primary event leading to inhibition. The presence of the acidic group of the fatty acid is critical for inhibition, as no effect is observed with the corresponding fatty alcohol. While arachidonic acid produces near-complete inhibition of both GDP and guanosine 5-(3-O-thio)triphosphate binding by Gzalpha, release of GDP from the protein was unaffected. Furthermore, the rate of inactivation of Gzalpha by arachidonate is essentially identical to the rate of GDP release from the protein, indicating that GDP release is required for inactivation. These observations indicate that the mechanism of inactivation of Gzalpha by unsaturated fatty acids is through an interaction of an acidic lipid micelle with the nucleotide-free form of the protein. Although the physiologic significance of this finding is unclear, similar effects of unsaturated fatty acids on other proteins involved in cell signaling indicate potential roles for these lipids in signal modulation. Additionally, the ability of arachidonate to inactivate this adenylyl cyclase-inhibitory G protein provides a molecular mechanism for previous findings that treatment of platelets with arachidonate results in elevated cAMP levels.