The long-chain flavodoxin FldX1 improves the biodegradation of 4-hydroxyphenylacetate and 3-hydroxyphenylacetate and counteracts the oxidative stress associated to aromatic catabolism in Paraburkholderia xenovorans.

BACKGROUND: Bacterial aromatic degradation may cause oxidative stress. The long-chain flavodoxin FldX1 of Paraburkholderia xenovorans LB400 counteracts reactive oxygen species (ROS). The aim of this study was to evaluate the protective role of FldX1 in P. xenovorans LB400 during the degradation of 4-hydroxyphenylacetate (4-HPA) and 3-hydroxyphenylacetate (3-HPA). METHODS: The functionality of FldX1 was evaluated in P. xenovorans p2-fldX1 that overexpresses FldX1. The effects of FldX1 on P. xenovorans were studied measuring growth on hydroxyphenylacetates, degradation of 4-HPA and 3-HPA, and ROS formation. The effects of hydroxyphenylacetates (HPAs) on the proteome (LC-MS/MS) and gene expression (qRT-PCR) were quantified. Bioaugmentation with strain p2-fldX1 of 4-HPA-polluted soil was assessed, measuring aromatic degradation (HPLC), 4-HPA-degrading bacteria, and plasmid stability. RESULTS: The exposure of P. xenovorans to 4-HPA increased the formation of ROS compared to 3-HPA or glucose. P. xenovorans p2-fldX1 showed an increased growth on 4-HPA and 3-HPA compared to the control strain WT-p2. Strain p2-fldX1 degraded faster 4-HPA and 3-HPA than strain WT-p2. Both WT-p2 and p2-fldX1 cells grown on 4-HPA displayed more changes in the proteome than cells grown on 3-HPA in comparison to glucose-grown cells. Several enzymes involved in ROS detoxification, including AhpC2, AhpF, AhpD3, KatA, Bcp, CpoF1, Prx1 and Prx2, were upregulated by hydroxyphenylacetates. Downregulation of organic hydroperoxide resistance (Ohr) and DpsA proteins was observed. A downregulation of the genes encoding scavenging enzymes (katE and sodB), and gstA and trxB was observed in p2-fldX1 cells, suggesting that FldX1 prevents the antioxidant response. More than 20 membrane proteins, including porins and transporters, showed changes in expression during the growth of both strains on hydroxyphenylacetates. An increased 4-HPA degradation by recombinant strain p2-fldX1 in soil microcosms was observed. In soil, the strain overexpressing the flavodoxin FldX1 showed a lower plasmid loss, compared to WT-p2 strain, suggesting that FldX1 contributes to bacterial fitness. Overall, these results suggest that recombinant strain p2-fldX1 is an attractive bacterium for its application in bioremediation processes of aromatic compounds. CONCLUSIONS: The long-chain flavodoxin FldX1 improved the capability of P. xenovorans to degrade 4-HPA in liquid culture and soil microcosms by protecting cells against the degradation-associated oxidative stress.

Diplodia seriata Biocontrol Is Altered via Temperature and the Control of Bacteria.

Grapevine trunk diseases (GTDs) attack the vine's wood, devastating vineyards worldwide. Chile is the world's fourth-largest wine exporter, and Cabernet Sauvignon is one of the most economically important red wine varieties. Botryosphaeria dieback is an important GTD, and Diplodia seriata is one of the main pathogenic species. Biocontrol studies of these pathogens are commonly carried out at different incubation times but at a single temperature. This study aimed to evaluate the biocontrol effect of Chilean PGPB and grapevine endophytic bacteria against D. seriata at different temperatures. We analyzed the biocontrol effect of Pseudomonas sp. GcR15a, Pseudomonas sp. AMCR2b and Rhodococcus sp. PU4, with three D. seriata isolates (PUCV 2120, PUCV 2142 and PUCV 2183) at 8, 22 and 35 °C. Two dual-culture antagonism methods (agar plug diffusion and double plate) were used to evaluate the in vitro effect, and an in vivo test was performed with Cabernet Sauvignon cuttings. In vitro, the greatest inhibitions were obtained using the agar plug diffusion method and at a temperature of 8 °C, where Rhodococcus sp. PU4 obtains a 65% control (average) and Pseudomonas sp. GcR15a a 57% average. At 22 °C, only strains of Pseudomonas sp. show control. At 35 °C, one Pseudomonas strain shows the highest control (38%), on average, similar to tebuconazole (33%), and then Rhodococcus sp. (30%). In vivo, a biocontrol effect is observed against two D. seriata isolates, while the PUCV 2142 proves to be more resistant to control. The biocontrol ability at low temperatures is promising for effective control in the field, where infections occur primarily in winter.

Draft genome sequences of halotolerant Halomonas spp. SpR1 and SpR8, potential plant growth-promoting bacteria associated with Salicornia rhizosphere in a hydrothermal lagoon ecosystem of the Altiplano, Northern Chile.

Halotolerant Halomonas spp. SpR1 and SpR8 are potential plant growth-promoting bacteria (PGPB) isolated from Salicornia rhizosphere in a Chilean Altiplano hydrothermal lagoon. We report draft genomes of Halomonas sp. SpR1 (5.17Mb) and Halomonas sp. SpR8 (4.47 Mb). Both represent potentially novel independent species closely related to Halomonas boliviensis DSM 15516T.

Higher Virulence of Diplodia seriata Isolates on Vines of cv. Cabernet Sauvignon Associated with 10-Year-Old Wood Compared to Young Tissue.

Botryosphaeria dieback (BD) occurs in young and old plants. In the field, the prevalence and severity of the disease increase proportionally with the age of vineyards. Among the pathogens that cause BD, Diplodia seriata is the most prevalent species in Chile and other countries with a Mediterranean climate. To date, no information is available on the susceptibility of adult wood to infection by this pathogen since most of the pathogenicity tests have been carried out on 1- or 2-year-old shoots or detached canes. Therefore, a pathogenicity test was carried out on plants under field conditions, with inoculations in 1-year-old shoots and 2- and 10-year-old wood in grapevine cv. Cabernet Sauvignon. A pathogenicity test was carried out with two isolates of D. seriata. The results for the plants show that D. seriata was significantly more aggressive on the 10-year-old than on the one- or two-year-old tissue, where the lesions were 4.3 and 2.3 cm on average, respectively. These results were compared with the lesions obtained from two-year-old canes after the isolates were activated in grape berries. Also, the Chilean isolates of D. seriata were compared phylogenetically with those from other countries, and no major differences were found between them. Our results are consistent with the damage observed in the field, contributing to the knowledge of the epidemiology of this disease in Mediterranean climates. In the future, the effect observed in cv. Cabernet Sauvignon with D. seriata on virulence at different tissue ages should be tested for other BD-causing agents and wine varieties.

GenoVi, an open-source automated circular genome visualizer for bacteria and archaea.

The increase in microbial sequenced genomes from pure cultures and metagenomic samples reflects the current attainability of whole-genome and shotgun sequencing methods. However, software for genome visualization still lacks automation, integration of different analyses, and customizable options for non-experienced users. In this study, we introduce GenoVi, a Python command-line tool able to create custom circular genome representations for the analysis and visualization of microbial genomes and sequence elements. It is designed to work with complete or draft genomes, featuring customizable options including 25 different built-in color palettes (including 5 color-blind safe palettes), text formatting options, and automatic scaling for complete genomes or sequence elements with more than one replicon/sequence. Using a Genbank format file as the input file or multiple files within a directory, GenoVi (i) visualizes genomic features from the GenBank annotation file, (ii) integrates a Cluster of Orthologs Group (COG) categories analysis using DeepNOG, (iii) automatically scales the visualization of each replicon of complete genomes or multiple sequence elements, (iv) and generates COG histograms, COG frequency heatmaps and output tables including general stats of each replicon or contig processed. GenoVi's potential was assessed by analyzing single and multiple genomes of Bacteria and Archaea. Paraburkholderia genomes were analyzed to obtain a fast classification of replicons in large multipartite genomes. GenoVi works as an easy-to-use command-line tool and provides customizable options to automatically generate genomic maps for scientific publications, educational resources, and outreach activities. GenoVi is freely available and can be downloaded from https://github.com/robotoD/GenoVi.

Rootstock increases the physiological defence of tomato plants against Pseudomonas syringae pv. tomato infection.

Climate change has intensified the infection of tomato plants by pathogens such as Pseudomonas syringae pv. tomato (Pst). Rootstocks may increase plant tolerance to leaf phytopathogens. The aim of this study was to evaluate the effects of the tolerant Poncho Negro (R) tomato rootstock on physiological defence and the role of hydrogen sulfide (H2S) in susceptible Limachino (L) tomato plant responses to Pst attack. Ungrafted (L), self-grafted (L/L), and grafted (L/R) plants were infected with Pst. Rootstock increased the concentration of antioxidant compounds including ascorbate in the scion. Tolerant rootstock induced an increase of H2S in the scion, which correlated with enhanced expression of the SlAPX2 gene. A high accumulation of salicylic acid was observed in Pst-inoculated grafted L/L and L/R plants, but this was higher in L/R plants. The increase of H2S during Pst infection was associated with a reduction of ethylene in L/R plants. Our study indicates that the Poncho Negro rootstock reduced the symptoms of bacterial speck disease in the Limachino tomato plants, conferring tolerance to Pst infection. This study provides new knowledge about the impact of rootstock in the defence of tomato plants against leaf pathogens that could be used in sustainable management of tomato cultivation.

Addition of Trichoderma consortia to Chilean endemic flora compost teas strongly enhances in vitro and in vivo biocontrol of phytopathogenic fungi.

AIM: To evaluate the in vitro and in vivo antifungal capability of diverse compost teas of endemic Chilean flora inoculated with a consortium of fungal strains of Trichoderma spp. (biocontrol agent) against three important phytopathogens: Botrytis cinerea, Fusarium oxysporum, andLasiodiplodia theobromae. METHODS AND RESULTS: Compost teas were obtained from the endemic flora of Chile (Azara celastrina, Citronella mucronate, Cryptocarya alba, Peumus boldus, and Quillaja saponaria). Eleven Trichoderma strains were isolated, and antagonism tests were performed to develop fungal consortiums with biocontrol properties. The biocontrol effect of compost teas inoculated with Trichoderma consortia was also analyzed. The results showed that the teas possess antifungal activity against B. cinerea and F. oxysporum and, to a lower degree, against L. theobromae. In vitro tests showed that Trichoderma consortiums improved the suppressive effect against B. cinerea (94-97%), F. oxysporum (89-92%), and L. theobromae (51-73%). Peumus boldus tea showed the highest suppressive effect against the plant pathogen L. theobromae. In addition, the in vivo assay showed that tomato plants treated only with Trichoderma or compost tea did not show differences in height with regard to control plants. However, when these two treatments were combined, the best performance in plant height and protection against pathogens was observed. CONCLUSIONS: This study indicates that the addition of a consortium of Trichoderma strains with intra- and interspecific incompatibilities significantly improves the inhibitory effect of compost teas in in vitro tests against the plant pathogenic fungi, while in vivo it enhances tomato plant growth and reduces plant disease symptoms.

Cupriavidus metallidurans CH34 Possesses Aromatic Catabolic Versatility and Degrades Benzene in the Presence of Mercury and Cadmium.

Heavy metal co-contamination in crude oil-polluted environments may inhibit microbial bioremediation of hydrocarbons. The model heavy metal-resistant bacterium Cupriavidus metallidurans CH34 possesses cadmium and mercury resistance, as well as genes related to the catabolism of hazardous BTEX aromatic hydrocarbons. The aims of this study were to analyze the aromatic catabolic potential of C. metallidurans CH34 and to determine the functionality of the predicted benzene catabolic pathway and the influence of cadmium and mercury on benzene degradation. Three chromosome-encoded bacterial multicomponent monooxygenases (BMMs) are involved in benzene catabolic pathways. Growth assessment, intermediates identification, and gene expression analysis indicate the functionality of the benzene catabolic pathway. Strain CH34 degraded benzene via phenol and 2-hydroxymuconic semialdehyde. Transcriptional analyses revealed a transition from the expression of catechol 2,3-dioxygenase (tomB) in the early exponential phase to catechol 1,2-dioxygenase (catA1 and catA2) in the late exponential phase. The minimum inhibitory concentration to Hg (II) and Cd (II) was significantly lower in the presence of benzene, demonstrating the effect of co-contamination on bacterial growth. Notably, this study showed that C. metallidurans CH34 degraded benzene in the presence of Hg (II) or Cd (II).

The OxyR and SoxR transcriptional regulators are involved in a broad oxidative stress response in Paraburkholderia xenovorans LB400.

BACKGROUND: Aerobic metabolism generates reactive oxygen species that may cause critical harm to the cell. The aim of this study is the characterization of the stress responses in the model aromatic-degrading bacterium Paraburkholderia xenovorans LB400 to the oxidizing agents paraquat and H2O2. METHODS: Antioxidant genes were identified by bioinformatic methods in the genome of P. xenovorans LB400, and the phylogeny of its OxyR and SoxR transcriptional regulators were studied. Functionality of the transcriptional regulators from strain LB400 was assessed by complementation with LB400 SoxR of null mutant P. aeruginosa ΔsoxR, and the construction of P. xenovorans pIZoxyR that overexpresses OxyR. The effects of oxidizing agents on P. xenovorans were studied measuring bacterial susceptibility, survival and ROS formation after exposure to paraquat and H2O2. The effects of these oxidants on gene expression (qRT-PCR) and the proteome (LC-MS/MS) were quantified. RESULTS: P. xenovorans LB400 possesses a wide repertoire of genes for the antioxidant defense including the oxyR, ahpC, ahpF, kat, trxB, dpsA and gorA genes, whose orthologous genes are regulated by the transcriptional regulator OxyR in E. coli. The LB400 genome also harbors the soxR, fumC, acnA, sodB, fpr and fldX genes, whose orthologous genes are regulated by the transcriptional regulator SoxR in E. coli. The functionality of the LB400 soxR gene was confirmed by complementation of null mutant P. aeruginosa ΔsoxR. Growth, susceptibility, and ROS formation assays revealed that LB400 cells were more susceptible to paraquat than H2O2. Transcriptional analyses indicated the upregulation of the oxyR, ahpC1, katE and ohrB genes in LB400 cells after exposure to H2O2, whereas the oxyR, fumC, ahpC1, sodB1 and ohrB genes were induced in presence of paraquat. Proteome analysis revealed that paraquat induced the oxidative stress response proteins AhpCF and DpsA, the universal stress protein UspA and the RNA chaperone CspA. Both oxidizing agents induced the Ohr protein, which is involved in organic peroxide resistance. Notably, the overexpression of the LB400 oxyR gene in P. xenovorans significantly decreased the ROS formation and the susceptibility to paraquat, suggesting a broad OxyR-regulated antioxidant response. CONCLUSIONS: This study showed that P. xenovorans LB400 possess a broad range oxidative stress response, which explain the high resistance of this strain to the oxidizing compounds paraquat and H2O2.

The OxyR and SoxR transcriptional regulators are involved in a broad oxidative stress response in Paraburkholderia xenovorans LB400

BACKGROUND: Aerobic metabolism generates reactive oxygen species that may cause critical harm to the cell. The aim of this study is the characterization of the stress responses in the model aromatic degrading bacterium Paraburkholderia xenovorans LB400 to the oxidizing agents paraquat and H 2 O2. METHODS: Antioxidant genes were identified by bioinformatic methods in the genome of P. xenovorans LB400, and the phylogeny of its OxyR and SoxR transcriptional regulators were studied. Functionality of the transcriptional regulators from strain LB400 was assessed by complementation with LB400 SoxR of null mutant P. aeruginosa ΔsoxR, and the construction of P. xenovorans pIZ oxyR that overexpresses OxyR. The effects of oxidizing agents on P. xenovorans were studied measuring bacterial susceptibility, survival and ROS formation after exposure to paraquat and H 2 O2. The effects of these oxidants on gene expression (qRT PCR) and the proteome (LC-MS/MS) were quantified. RESULTS: P. xenovorans LB400 possesses a wide repertoire of genes for the antioxidant defense including the oxyR , ahpC , ahpF , kat , trxB , dpsA and gorA genes, whose orthologous genes are regulated by the transcriptional regulator OxyR in E. coli . The LB400 genome also harbors the soxR, fumC , acnA , sodB , fpr and fldX genes, whose orthologous genes are regulated by the transcriptional regulator SoxR in E. coli . The functionality of the LB400 soxR gene was confirmed by complementation of null mutant P. aeruginosa Δ soxR . Growth, susceptibility, and ROS formation assays revealed that LB400 cells were more susceptible to paraquat than H2O2. Transcriptional analyses indicated the upregulation of the oxyR , ahpC1 , katE and ohrB genes in LB400 cells after exposure to H2O2, whereas the oxyR , fumC , ahpC1 , sodB1 and ohrB genes were induced in presence of paraquat. Proteome analysis revealed that paraquat induced the oxidative stress response proteins AhpCF and DpsA, the universal stress protein UspA and the RNA chaperone CspA. Both oxidizing agents induced the Ohr protein, which is involved in organic peroxide resistance. Notably, the overexpression of the LB400 oxyR gene in P. xenovorans significantly decreased the ROS formation and the susceptibility to paraquat, suggesting a broad OxyR regulated antioxidant response. CONCLUSIONS: This study showed that P. xenovorans LB400 possess a broad range oxidative stress response, which explain the high resistance of this strain to the oxidizing compounds paraquat and H2O2.

First Report of bacterial speck caused by Pseudomonas syringae pv. tomato Race 1 affecting tomato in different Regions of Chile.

In Chile, tomato is one of the most widely cultivated vegetables, with around 5,000 ha for fresh market and 8,000 ha for processing industry. During recent years, symptoms of bacterial speck caused by Pseudomonas syringae pv. tomato, have been observed more frequently in tomato plants in different regions of Chile. This pathogen was first identified in Chile in 1987 (Latorre & Lolas, 1988) and the presence of an apparent new variant was reported in 2004 (Besoain et al. 2004). To characterize the pathogen that was affecting this crop, samples of diseased tomato plants were taken in three regions of Chile. The samples were collected in 2016 in Northern Chile in Lluta Valley from the Arica y Parinacota Region, and in Central Chile, in 2014 in Limache from Valparaíso Region and in 2015 in Pichidegua from O´Higgins Region. Affected tomato plants exhibited dark brown to black lesions surrounded by yellow halos in the leaves, and dark brown to black lesions in the stems, pedicels, and peduncles. Plants tissues were macerated, and the suspension was spread on King's B medium, resulting in fluorescent colonies visualized under 366 nm UV light. LOPAT tests results of three selected isolates from different Regions, were: levan production (+), oxidase reaction (-), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+) (Lelliot et al. 1966). Molecular identification was carried out by amplification and sequence analysis of housekeeping genes cts, encoding citrate synthase, gyrB, encoding DNA gyrase B, and rpoD, encoding sigma factor 70 (Hwang et al. 2005; Sarkar & Guttmann 2004) (GenBank Accessions No. OK001658-OK001666). BLAST analysis of cts and rpoD genes of the three isolates resulted in a match with a 100% identity (919 bp and 491 bp respectively) with Pseudomonas syringae pv. tomato strain B13-200 (GenBank: CP019871.1). BLAST analysis of gyrB gene of two isolates resulted in a match with a 100% identity (684 bp) and one isolate with 99.85% (683 bp) with Pseudomonas syringae pv. tomato strain B13-200. To identify the race 1, each strain was inoculated in five tomato plants cv. San Pedro, susceptible to both races of P. syringae pv. tomato, and cv. Rio Grande, resistant to race 0. The tomato plants were slightly wounded with a metal sponge and then sprayed with the bacterial suspension (108 CFU mL-1) of each isolate, including the reference strain DC3000 (race 0). Negative controls were sprayed with water. The plants inoculated with Chilean strains in both cv. San Pedro and cv. Rio Grande, showed symptoms of bacterial speck after 7 days. Plants inoculated with DC3000 strain showed symptoms only in cv. San Pedro, whereas control plants remained asymptomatic. Strains were re-isolated from symptomatic plants and identified by gene sequence analyses as Pseudomonas syryngae pv. tomato. This is the first report of Pseudomonas syryngae pv. tomato race 1 in Chile. Race 1 was previously reported in Canada (Lawton and MacNeill. 1986), in Italy (Buonaurio et al. 1996), in California (Arredondo and Davis 2000), in Portugal (Cruz et al. 2010), and in other states in the USA and countries in South America, Europe, Africa, and Australia, becoming the most commonly isolated race today (Cai et al 2011). These results will be the base for future studies of epidemiology, characterization, and virulence in order to explain the outbreak of this disease and the severity of symptoms observed.

Analyses of Virulence Genes of Clavibacter michiganensis subsp. michiganensis Strains Reveal Heterogeneity and Deletions That Correlate with Pathogenicity.

Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial canker of tomato. Differences in virulence between Cmm strains have been reported. The aim of this study was the characterization of nine Cmm strains isolated in Chile to reveal the causes of their differences in virulence. The virulence assays in tomato seedlings revealed different levels of severity associated with the strains, with two highly virulent strains and one causing only mild symptoms. The two most virulent showed increased cellulase activity, and no cellulase activity was observed in the strain causing mild symptoms. In three strains, including the two most virulent strains, PCR amplification of the 10 virulence genes analyzed was observed. In the strain causing mild symptoms, no amplification was observed for five genes, including celA. Sequence and cluster analyses of six virulence genes grouped the strains, as has been previously reported, except for gene pelA1. Gene sequence analysis from the genomes of five Chilean strains revealed the presence of deletions in the virulence genes, celB, xysA, pat-1, and phpA. The results of this study allow us to establish correlations between the differences observed in disease severity and the presence/absence of genes and deletions not previously reported.

Genome-Wide Metabolic Reconstruction of the Synthesis of Polyhydroxyalkanoates from Sugars and Fatty Acids by Burkholderia Sensu Lato Species.

Burkholderia sensu lato (s.l.) species have a versatile metabolism. The aims of this review are the genomic reconstruction of the metabolic pathways involved in the synthesis of polyhydroxyalkanoates (PHAs) by Burkholderia s.l. genera, and the characterization of the PHA synthases and the pha genes organization. The reports of the PHA synthesis from different substrates by Burkholderia s.l. strains were reviewed. Genome-guided metabolic reconstruction involving the conversion of sugars and fatty acids into PHAs by 37 Burkholderia s.l. species was performed. Sugars are metabolized via the Entner-Doudoroff (ED), pentose-phosphate (PP), and lower Embden-Meyerhoff-Parnas (EMP) pathways, which produce reducing power through NAD(P)H synthesis and PHA precursors. Fatty acid substrates are metabolized via ß-oxidation and de novo synthesis of fatty acids into PHAs. The analysis of 194 Burkholderia s.l. genomes revealed that all strains have the phaC, phaA, and phaB genes for PHA synthesis, wherein the phaC gene is generally present in ≥2 copies. PHA synthases were classified into four phylogenetic groups belonging to class I II and III PHA synthases and one outlier group. The reconstruction of PHAs synthesis revealed a high level of gene redundancy probably reflecting complex regulatory layers that provide fine tuning according to diverse substrates and physiological conditions.

Diffusible Compounds Produced by Hanseniaspora osmophila and Gluconobacter cerinus Help to Control the Causal Agents of Gray Rot and Summer Bunch Rot of Table Grapes.

Gray and summer bunch rot are important diseases of table grapes due to the high economic and environmental cost of their control with synthetic fungicides. The ability to produce antifungal compounds against the causal agents Botrytis, Aspergillus, Penicillium, and Rhizopus of two microorganisms isolated from table grapes and identified as Hanseniaspora osmophila and Gluconobacter cerinus was evaluated. In dual cultures, both biocontrol agents (together and separately) inhibited in vitro mycelial growth of these pathogens. To identify the compounds responsible for the inhibitory effect, extractions were carried out with organic solvents from biocontrol agents separately. Through dual cultures with pathogens and pure extracts, only the hexane extract from H. osmophila showed an inhibitory effect against Botrytis cinerea. To further identify these compounds, the direct bioautography technique was used. This technique made it possible to determine the band displaying antifungal activity at Rf = 0.05-0.2. The compounds present in this band were identified by GC-MS and compared to the NIST library. The most abundant compounds, not previously reported, corresponded to alkanes, ketones, alcohols, and terpenoids. H. osmophila and G. cerinus have the potential to control the causal agents of gray and summer bunch rot of table grapes.

Development and thermochemical characterization of an antioxidant material based on polyhydroxybutyrate electrospun microfibers.

The use of antioxidants such as curcumin (Cur) or quercetin (Que) in biomedical and biotechnological applications has been studied owing to their capability to prevent oxidative stress and inhibit free radicals. Using polyhydroxybutyrate (PHB) electrospun fibers is presented as a proper option to encapsulate curcumin and quercetin due to its biocompatibility and biodegradability characteristics. Electrospun fibers were obtained dissolving commercial PHB in chloroform:N,N-dimethylformamide (DMF) (4:1) at 7% m/V, and adding two different concentrations of antioxidant (Cur, and Que) 1%m/m, and 7% m/m. These polymeric solutions were electrospun at different conditions and the obtained fibers were characterized by scanning electron microscopy (SEM), thermogravimetric (TGA) analysis, and Fourier transform infrared spectroscopy (FT-IR). The curcumin and quercetin releases into phosphate buffer saline (PBS) at pH 7.4 were obtained in vitro and measured by spectrophotometry. Antioxidant activities were measured by spectrophotometry in a microplate reader using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Fibers obtained with different formulations presented a chemical composition in accordance with PHB according to FTIR spectra, the diameters fluctuate between 0.761 ± 0.123 and 1.803 ± 0.557 µm, with qualities over 0.95 according to their morphology, and the melting temperature resulted near 178 °C according to the bibliography. The crystallinity of fibers decreases while curcumin or quercetin concentration increases for the studied interval, indeed, quercetin showed a higher impact on the relative crystallinity of fibers. Antioxidant activity of active compounds is maintained after encapsulation in PHB electrospun fibers, and quercetin resulted in near four times antioxidant activity compared to curcumin according to DPPH analysis.

Ecophysiological Features Shape the Distribution of Prophages and CRISPR in Sulfate Reducing Prokaryotes.

Sulfate reducing prokaryotes (SRP) are a phylogenetically and physiologically diverse group of microorganisms that use sulfate as an electron acceptor. SRP have long been recognized as key players of the carbon and sulfur cycles, and more recently, they have been identified to play a relevant role as part of syntrophic and symbiotic relations and the human microbiome. Despite their environmental relevance, there is a poor understanding about the prevalence of prophages and CRISPR arrays and how their distribution and dynamic affect the ecological role of SRP. We addressed this question by analyzing the results of a comprehensive survey of prophages and CRISPR in a total of 91 genomes of SRP with several genotypic, phenotypic, and physiological traits, including genome size, cell volume, minimum doubling time, cell wall, and habitat, among others. Our analysis discovered 81 prophages in 51 strains, representing the 56% of the total evaluated strains. Prophages are non-uniformly distributed across the SRP phylogeny, where prophage-rich lineages belonged to Desulfovibrionaceae and Peptococcaceae. Furthermore, our study found 160 CRISPR arrays in 71 SRP, which is more abundant and widely spread than previously expected. Although there is no correlation between presence and abundance of prophages and CRISPR arrays at the strain level, our analysis showed that there is a directly proportional relation between cellular volumes and number of prophages per cell. This result suggests that there is an additional selective pressure for strains with smaller cells to get rid of foreign DNA, such as prophages, but not CRISPR, due to less availability of cellular resources. Analysis of the prophage genes encoding viral structural proteins reported that 44% of SRP prophages are classified as Myoviridae, and comparative analysis showed high level of homology, but not synteny, among prophages belonging to the Family Desulfovibrionaceae. We further recovered viral-like particles and structures that resemble outer membrane vesicles from D. vulgaris str. Hildenborough. The results of this study improved the current understanding of dynamic interactions between prophages and CRISPR with their hosts in both cultured and hitherto-uncultured SRP strains, and how their distribution affects the microbial community dynamics in several sulfidogenic natural and engineered environments.

Microbial Diversity of Psychrotolerant Bacteria Isolated from Wild Flora of Andes Mountains and Patagonia of Chile towards the Selection of Plant Growth-Promoting Bacterial Consortia to Alleviate Cold Stress in Plants.

Cold stress decreases the growth and productivity of agricultural crops. Psychrotolerant plant growth-promoting bacteria (PGPB) may protect and promote plant growth at low temperatures. The aims of this study were to isolate and characterize psychrotolerant PGPB from wild flora of Andes Mountains and Patagonia of Chile and to formulate PGPB consortia. Psychrotolerant strains were isolated from 11 wild plants (rhizosphere and phyllosphere) during winter of 2015. For the first time, bacteria associated with Calycera, Orites, and Chusquea plant genera were reported. More than 50% of the 130 isolates showed ≥33% bacterial cell survival at temperatures below zero. Seventy strains of Pseudomonas, Curtobacterium, Janthinobacterium, Stenotrophomonas, Serratia, Brevundimonas, Xanthomonas, Frondihabitans, Arthrobacter, Pseudarthrobacter, Paenarthrobacter, Brachybacterium, Clavibacter, Sporosarcina, Bacillus, Solibacillus, Flavobacterium, and Pedobacter genera were identified by 16S rRNA gene sequence analyses. Ten strains were selected based on psychrotolerance, auxin production, phosphate solubilization, presence of nifH (nitrogenase reductase) and acdS (1-aminocyclopropane-1-carboxylate (ACC) deaminase) genes, and anti-phytopathogenic activities. Two of the three bacterial consortia formulated promoted tomato plant growth under normal and cold stress conditions. The bacterial consortium composed of Pseudomonas sp. TmR5a & Curtobacterium sp. BmP22c that possesses ACC deaminase and ice recrystallization inhibition activities is a promising candidate for future cold stress studies.

Chemical Composition, Antioxidant and Anticancer Activities of Leptocarpha rivularis DC Flower Extracts.

An evaluation of antioxidant and anticancer activity was screened in Leptocarpha rivularis DC flower extracts using four solvents (n-hexane (Hex), dichloromethane (DCM), ethyl acetate (AcOEt), and ethanol (EtOH)). Extracts were compared for total extract flavonoids and phenol contents, antioxidant activity (2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), ferric reducing antioxidant potential (FRAP), total reactive antioxidant properties (TRAP) and oxygen radical absorbance capacity (ORAC)) across a determined value of reduced/oxidized glutathione (GSH/GSSG), and cell viability (the sulforhodamine B (SRB) assay). The most active extracts were analyzed by chromatographic analysis (GC/MS) and tested for apoptotic pathways. Extracts from Hex, DCM and AcOEt reduced cell viability, caused changes in cell morphology, affected mitochondrial membrane permeability, and induced caspase activation in tumor cell lines HT-29, PC-3, and MCF-7. These effects were generally less pronounced in the HEK-293 cell line (nontumor cells), indicating clear selectivity towards tumor cell lines. We attribute likely extract activity to the presence of sesquiterpene lactones, in combination with other components like steroids and flavonoids.