Metagenomic Study of Fungal Microbial Communities in Two PDO Somontano Vineyards (Huesca, Spain): Effects of Age, Plant Genotype, and Initial Phytosanitary Status on the Priming and Selection of their Associated Microorganisms.

The study of microbial communities associated with different plants of agronomic interest has allowed, in recent years, to answer a number of questions related to the role and influence of certain microbes in key aspects of their autoecology, such as improving the adaptability of the plant host to different abiotic or biotic stresses. In this study, we present the results of the characterization, through both high-throughput sequencing and classical microbiological methods, of the fungal microbial communities associated with grapevine plants in two vineyards of different ages and plant genotypes located in the same biogeographical unit. The study is configured as an approximation to the empirical demonstration of the concept of "microbial priming" by analyzing the alpha- and beta-diversity present in plants from two plots subjected to the same bioclimatic regime to detect differences in the structure and taxonomic composition of the populations. The results were compared with the inventories of fungal diversity obtained by culture-dependent methods to establish, where appropriate, correlations between both microbial communities. Metagenomic data showed a differential enrichment of the microbial communities in the two vineyards studied, including the populations of plant pathogens. This is tentatively explained due to factors such as the different time of exposure to microbial infection, different plant genotype, and different starting phytosanitary situation. Thus, results suggest that each plant genotype recruits differential fungal communities and presents different profiles of associated potential microbial antagonists or communities of pathogenic species.

A Tar aspartate receptor and Rubisco-like protein substitute biotin in the growth of rhizobial strains.

Biotin is a key cofactor of metabolic carboxylases, although many rhizobial strains are biotin auxotrophs. When some of these strains were serially subcultured in minimal medium, they showed diminished growth and increased excretion of metabolites. The addition of biotin, or genetic complementation with biotin synthesis genes resulted in full growth of Rhizobium etli CFN42 and Rhizobium phaseoli CIAT652 strains. Half of rhizobial genomes did not show genes for biotin biosynthesis, but three-quarters had genes for biotin transport. Some strains had genes for an avidin homologue (rhizavidin), a protein with high affinity for biotin but an unknown role in bacteria. A CFN42-derived rhizavidin mutant showed a sharper growth decrease in subcultures, revealing a role in biotin storage. In the search of biotin-independent growth of subcultures, CFN42 and CIAT652 strains with excess aeration showed optimal growth, as they also did, unexpectedly, with the addition of aspartic acid analogues α- and N-methyl aspartate. Aspartate analogues can be sensed by the chemotaxis aspartate receptor Tar. A tar homologue was identified and its mutants showed no growth recovery with aspartate analogues, indicating requirement of the Tar receptor in such a phenotype. Additionally, tar mutants did not recover full growth with excess aeration. A Rubisco-like protein was found to be necessary for growth as the corresponding mutants showed no recovery either with high aeration or aspartate analogues; also, diminished carboxylation was observed. Taken together, our results indicate a route of biotin-independent growth in rhizobial strains that included oxygen, a Tar receptor and a previously uncharacterized Rubisco-like protein.

Phenotypic plasticity and a new small molecule are involved in a fungal-bacterial interaction.

Nitrogen-fixing bacteria have been extensively studied in the context of interactions with their host plants; however, little is known about the phenotypic plasticity of these microorganisms in nonmutualistic interactions with other eukaryotes. A dual-species coculture model was developed by using the plant symbiotic bacterium Rhizobium etli and the well-studied eukaryote Saccharomyces cerevisiae as a tractable system to explore the molecular mechanisms used by R. etli in nonmutual interactions. Here, we show that the fungus promotes the growth of the bacterium and that together, these organisms form a mixed biofilm whose biomass is ~ 3 times greater and is more structured than that of either single-species biofilm. We found that these biofilm traits are dependent on a symbiotic plasmid encoding elements involved in the phenotypic plasticity of the bacterium, mitochondrial function and in the production of a yeast-secreted sophoroside. Interestingly, the promoters of 3 genes that are key in plant bacteria-interaction (nifH, fixA and nodA) were induced when R. etli coexists with yeast. These results show that investigating interactions between species that do not naturally coexist is a new approach to discover gene functions and specialized metabolites in model organisms.

Qualitative changes in proteins contained in outer membrane vesicles produced by Rhizobium etli grown in the presence of the nod gene inducer naringenin.

In this work, we compared the proteomic profiles of outer membrane vesicles (OMVs) isolated from Rhizobium etli CE3 grown in minimal medium (MM) with and without exogenous naringenin. One-hundred and seven proteins were present only in OMVs from naringenin-containing cultures (N-OMVs), 57 proteins were unique to OMVs from control cultures lacking naringenin (C-OMVs) and 303 proteins were present in OMVs from both culture conditions (S-OMVs). Although we found no absolute predominance of specific types of proteins in the N-, C- or S-OMV classes, there were categories of proteins that were significantly less or more common in the different OMV categories. Proteins for energy production, translation and membrane and cell wall biogenesis were overrepresented in C-OMVs relative to N-OMVs. Proteins for carbohydrate metabolism and transport and those classified as either general function prediction only, function unknown, or without functional prediction were more common in N-OMVs than C-OMVs. This indicates that naringenin increased the proportion of these proteins in the OMVs, although NodD binding sites were only slightly more common in the promoters of genes for proteins found in the N-OMVs. In addition, OMVs from naringenin-containing cultures contained nodulation factor.

New Insights into the Mechanisms of Action of Topical Administration of GLP-1 in an Experimental Model of Diabetic Retinopathy.

The main goals of this work were to assess whether the topical administration of glucagon-like peptide-1 (GLP-1) could revert the impairment of the neurovascular unit induced by long-term diabetes (24 weeks) in diabetic mice and to look into the underlying mechanisms. For that reason, db/db mice were treated with eye drops of GLP-1 or vehicle for 3 weeks. Moreover, db/+ mice were used as control. Studies performed in vivo included electroretinogramand the assessment of vascular leakage by using Evans Blue. NF-κB, GFAP and Ki67 proteins were analyzed by immunofluorescence (IF). Additionally, caspase 9, AMPK, IKBα, NF-κB, AKT, GSK3, ß-catenin, Bcl-xl, and VEGF were analyzed by WB. Finally, VEGF, IL-1ß, IL-6, TNF-α, IL-18, and NLRP3 were studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. We found that topical administration of GLP-1 reverted reactive gliosis and albumin extravasation, and protected against apoptosis and retinal dysfunction. Regarding the involved mechanisms, GLP-1 exerted an anti-inflammatory action by decreasing NF-κB, inflammosome, and pro-inflammatory factors. In addition, it also decreased VEGF expression. Furthermore, GLP-1 promoted cell survival by increasing the anti-apoptotic protein Bcl-xl and the signaling pathway Akt/GSK3b/ß-catenin. Finally, Ki67 results revealed that GLP-1 treatment could induce neurogenesis. In conclusion, the topical administration of GLP-1 reverts the impairment of the neurovascular unit by modulating essential pathways involved in the development of diabetic retinopathy (DR). These beneficial effects on the neurovascular unit could pave the way for clinical trials addressed to confirm the effectiveness of GLP-1 in early stages of DR.

Proteins in the periplasmic space and outer membrane vesicles of Rhizobium etli CE3 grown in minimal medium are largely distinct and change with growth phase.

Rhizobium etli CE3 grown in succinate-ammonium minimal medium (MM) excreted outer membrane vesicles (OMVs) with diameters of 40 to 100 nm. Proteins from the OMVs and the periplasmic space were isolated from 6 and 24 h cultures and identified by proteome analysis. A total of 770 proteins were identified: 73.8 and 21.3 % of these occurred only in the periplasm and OMVs, respectively, and only 4.9 % were found in both locations. The majority of proteins found in either location were present only at 6 or 24 h: in the periplasm and OMVs, only 24 and 9 % of proteins, respectively, were present at both sampling times, indicating a time-dependent differential sorting of proteins into the two compartments. The OMVs contained proteins with physiologically varied roles, including Rhizobium adhering proteins (Rap), polysaccharidases, polysaccharide export proteins, auto-aggregation and adherence proteins, glycosyl transferases, peptidoglycan binding and cross-linking enzymes, potential cell wall-modifying enzymes, porins, multidrug efflux RND family proteins, ABC transporter proteins and heat shock proteins. As expected, proteins with known periplasmic localizations (phosphatases, phosphodiesterases, pyrophosphatases) were found only in the periplasm, along with numerous proteins involved in amino acid and carbohydrate metabolism and transport. Nearly one-quarter of the proteins present in the OMVs were also found in our previous analysis of the R. etli total exproteome of MM-grown cells, indicating that these nanoparticles are an important mechanism for protein excretion in this species.

Overproduction of Sinorhizobium meliloti ArgC (N-acetyl-gamma-glutamyl phosphate reductase) promotes growth delay and inefficient nodules.

argC encodes N-acetyl-gamma-glutamyl phosphate reductase, the enzyme that catalyzes the high-energy-consuming third step in the arginine synthesis pathway. A comparative analysis revealed two translation start sites in argC from Sinorhizobium meliloti. To determine whether both protein versions are synthesized in the organism and their functional role, we obtained genetic constructs with one (1S) or two (2S) start sites, with promoters of low (pspeB) or high (plac) transcriptional rate. The constructs were transferred to the S. meliloti 1021 derivative argC mutant strain. Both protein versions were found in the free-living proteomes, but only ArgC 1S showed post-translational modification. Expression levels from argC 1S were five times higher than those of 2S, when transcribed by plac, and in concordance, its protein activity was 3-fold greater. The overexpression of both versions under plac delayed cellular growth. Inoculation of Medicago sativa plants with the S. meliloti strain harboring the argC 1S under plac induced nodulation but not nitrogen fixation. However, the strain with the argC 2S under the same promoter had a positive phenotype. Overproduction of ArgC protein for the synthesis of arginine induced physiological and symbiotic effects.

Genomic studies of nitrogen-fixing rhizobial strains from Phaseolus vulgaris seeds and nodules.

BACKGROUND: Rhizobia are soil bacteria that establish symbiotic relationships with legumes and fix nitrogen in root nodules. We recently reported that several nitrogen-fixing rhizobial strains, belonging to Rhizobium phaseoli, R. trifolii, R. grahamii and Sinorhizobium americanum, were able to colonize Phaseolus vulgaris (common bean) seeds. To gain further insight into the traits that support this ability, we analyzed the genomic sequences and proteomes of R. phaseoli (CCGM1) and S. americanum (CCGM7) strains from seeds and compared them with those of the closely related strains CIAT652 and CFNEI73, respectively, isolated only from nodules. RESULTS: In a fine structural study of the S. americanum genomes, the chromosomes, megaplasmids and symbiotic plasmids were highly conserved and syntenic, with the exception of the smaller plasmid, which appeared unrelated. The symbiotic tract of CCGM7 appeared more disperse, possibly due to the action of transposases. The chromosomes of seed strains had less transposases and strain-specific genes. The seed strains CCGM1 and CCGM7 shared about half of their genomes with their closest strains (3353 and 3472 orthologs respectively), but a large fraction of the rest also had homology with other rhizobia. They contained 315 and 204 strain-specific genes, respectively, particularly abundant in the functions of transcription, motility, energy generation and cofactor biosynthesis. The proteomes of seed and nodule strains were obtained and showed a particular profile for each of the strains. About 82 % of the proteins in the comparisons appeared similar. Forty of the most abundant proteins in each strain were identified; these proteins in seed strains were involved in stress responses and coenzyme and cofactor biosynthesis and in the nodule strains mainly in central processes. Only 3 % of the abundant proteins had hypothetical functions. CONCLUSIONS: Functions that were enriched in the genomes and proteomes of seed strains possibly participate in the successful occupancy of the new niche. The genome of the strains had features possibly related to their presence in the seeds. This study helps to understand traits of rhizobia involved in seed adaptation.

Apaf-1 inhibitors protect from unwanted cell death in in vivo models of kidney ischemia and chemotherapy induced ototoxicity.

BACKGROUND: Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need. METHODOLOGY AND PRINCIPAL FINDINGS: The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia. CONCLUSIONS: Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases.

Nitrogen-fixing rhizobial strains isolated from common bean seeds: phylogeny, physiology, and genome analysis.

Rhizobial bacteria are commonly found in soil but also establish symbiotic relationships with legumes, inhabiting the root nodules, where they fix nitrogen. Endophytic rhizobia have also been reported in the roots and stems of legumes and other plants. We isolated several rhizobial strains from the nodules of noninoculated bean plants and looked for their provenance in the interiors of the seeds. Nine isolates were obtained, covering most known bean symbiont species, which belong to the Rhizobium and Sinorhizobium groups. The strains showed several large plasmids, except for a Sinorhizobium americanum isolate. Two strains, one Rhizobium phaseoli and one S. americanum strain, were thoroughly characterized. Optimal symbiotic performance was observed for both of these strains. The S. americanum strain showed biotin prototrophy when subcultured, as well as high pyruvate dehydrogenase (PDH) activity, both of which are key factors in maintaining optimal growth. The R. phaseoli strain was a biotin auxotroph, did not grow when subcultured, accumulated a large amount of poly-ß-hydroxybutyrate, and exhibited low PDH activity. The physiology and genomes of these strains showed features that may have resulted from their lifestyle inside the seeds: stress sensitivity, a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, a homocitrate synthase (usually present only in free-living diazotrophs), a hydrogenase uptake cluster, and the presence of prophages. We propose that colonization by rhizobia and their presence in Phaseolus seeds may be part of a persistence mechanism that helps to retain and disperse rhizobial strains.

argC Orthologs from Rhizobiales show diverse profiles of transcriptional efficiency and functionality in Sinorhizobium meliloti.

Several factors can influence ortholog replacement between closely related species. We evaluated the transcriptional expression and metabolic performance of ortholog substitution complementing a Sinorhizobium meliloti argC mutant with argC from Rhizobiales (Agrobacterium tumefaciens, Rhizobium etli, and Mesorhizobium loti). The argC gene is necessary for the synthesis of arginine, an amino acid that is central to protein and cellular metabolism. Strains were obtained carrying plasmids with argC orthologs expressed under the speB and argC (S. meliloti) and lac (Escherichia coli) promoters. Complementation analysis was assessed by growth, transcriptional activity, enzymatic activity, mRNA levels, specific detection of ArgC proteomic protein, and translational efficiency. The argC orthologs performed differently in each complementation, reflecting the diverse factors influencing gene expression and the ability of the ortholog product to function in a foreign metabolic background. Optimal complementation was directly related to sequence similarity with S. meliloti, and was inversely related to species signature, with M. loti argC showing the poorest performance, followed by R. etli and A. tumefaciens. Different copy numbers of genes and amounts of mRNA and protein were produced, even with genes transcribed from the same promoter, indicating that coding sequences play a role in the transcription and translation processes. These results provide relevant information for further genomic analyses and suggest that orthologous gene substitutions between closely related species are not completely functionally equivalent.

Synthesis and evaluation of diverse analogs of amygdalin as potential peptidomimetics of peptide T.

Peptide T (ASTTTNYT) is a promising molecule to prevent the neuropsychometric symptoms of patients suffering AIDS and for the treatment of psoriasis. In order to fully prove its therapeutic benefits, efforts were put forward to design peptidomimetics of the peptide. In this direction, in a recent computational study the natural product amygdalin was identified as a prospective peptidomimetic of the peptide and later proved to exhibit a similar chemotactic profile to the peptide. However, the cyanide moiety of amygdalin provides to the molecule a toxic profile. The present study reports the synthesis of a set of amygdalin analogs lacking the cyanide group with improved chemotactic profiles.