High diversity and suggested endemicity of culturable Actinobacteria in an extremely oligotrophic desert oasis

The phylum Actinobacteria constitutes one of the largest and anciently divergent phyla within the Bacteria domain. Actinobacterial diversity has been thoroughly researched in various environments due to its unique biotechnological potential. Such studies have focused mostly on soil communities, but more recently marine and extreme environments have also been explored, finding rare taxa and demonstrating dispersal limitation and biogeographic patterns for Streptomyces. To test the distribution of Actinobacteria populations on a small scale, we chose the extremely oligotrophic and biodiverse Cuatro Cienegas Basin (CCB), an endangered oasis in the Chihuahuan desert to assess the diversity and uniqueness of Actinobacteria in the Churince System with a culture-dependent approach over a period of three years, using nine selective media. The 16S rDNA of putative Actinobacteria were sequenced using both bacteria universal and phylum-specific primer pairs. Phylogenetic reconstructions were performed to analyze OTUs clustering and taxonomic identification of the isolates in an evolutionary context, using validated type species of Streptomyces from previously phylogenies as a reference. Rarefaction analysis for total Actinobacteria and for Streptomyces isolates were performed to estimate species' richness in the intermediate lagoon (IL) in the oligotrophic Churince system. A total of 350 morphologically and nutritionally diverse isolates were successfully cultured and characterized as members of the Phylum Actinobacteria. A total of 105 from the total isolates were successfully subcultured, processed for DNA extraction and 16S-rDNA sequenced. All strains belong to the order Actinomycetales, encompassing 11 genera of Actinobacteria; the genus Streptomyces was found to be the most abundant taxa in all the media tested throughout the 3- year sampling period. Phylogenetic analysis of our isolates and another 667 reference strains of the family Streptomycetaceae shows that our isolation effort produced 38 unique OTUs in six new monophyletic clades. This high biodiversity and uniqueness of Actinobacteria in an extreme oligotrophic environment, which has previously been reported for its diversity and endemicity, is a suggestive sign of microbial biogeography of Actinobacteria and it also represents an invaluable source of biological material for future ecological and bioprospecting studies

Temperature-mediated biosynthesis of the phytotoxin phaseolotoxin by Pseudomonas syringae pv. phaseolicola depends on the autoregulated expression of the phtABC genes

Abstract Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally synthesized between 18˚C and 20˚C, while no detectable amounts are present above 28˚C. The Pht cluster, involved in the biosynthesis of phaseolotoxin, contains 23 genes that are organized in five transcriptional units. The function of most of the genes from the Pht cluster is still unknown and little information about the regulatory circuitry leading to expression of these genes has been reported. The purpose of the present study was to investigate the participation of pht genes in the regulation of the operons coded into the Pht cluster. We conducted Northern blot, uidA fusions and reverse transcription-PCR assays of pht genes in several mutants unable to produce phaseolotoxin. This allowed us to determine that, in P. syringae pv. phaseolicola NPS3121, genes phtABC are essential to prevent their own expression at 28˚C, a temperature at which no detectable amounts of the toxin are present. We obtained evidence that the phtABC genes also participate in the regulation of the phtD, phtM and phtL operons. According to our results, we propose that PhtABC and other Pht product activities could be involved in the synthesis of the sulfodiaminophosphinyl moiety of phaseolotoxin, which indirectly could be involved in the transcriptional regulation of the phtA operon

Transcriptional profile of Pseudomonas syringae pv. phaseolicola NPS3121 in response to tissue extracts from a susceptible Phaseolus vulgaris L. cultivar

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas syringae </it>pv. phaseolicola is a Gram-negative plant-pathogenic bacterium that causes "halo blight" disease of beans (<it>Phaseolus vulgaris </it>L.). This disease affects both foliage and pods, and is a major problem in temperate areas of the world. Although several bacterial genes have been determined as participants in pathogenesis, the overall process still remains poorly understood, mainly because the identity and function of many of the genes are largely unknown. In this work, a genomic library of <it>P. syringae </it>pv. phaseolicola NPS3121 was constructed and PCR amplification of individual fragments was carried out in order to print a DNA microarray. This microarray was used to identify genes that are differentially expressed when bean leaf extracts, pod extracts or apoplastic fluid were added to the growth medium.</p> <p>Results</p> <p>Transcription profiles show that 224 genes were differentially expressed, the majority under the effect of bean leaf extract and apoplastic fluid. Some of the induced genes were previously known to be involved in the first stages of the bacterial-plant interaction and virulence. These include genes encoding type III secretion system proteins and genes involved in cell-wall degradation, phaseolotoxin synthesis and aerobic metabolism. On the other hand, most repressed genes were found to be involved in the uptake and metabolism of iron.</p> <p>Conclusion</p> <p>This study furthers the understanding of the mechanisms involved, responses and the metabolic adaptation that occurs during the interaction of <it>P. syringae </it>pv. phaseolicola with a susceptible host plant.</p

Expression of the gene for resistance to phaseolotoxin (argK) depends on the activity of genes phtABC in Pseudomonas syringae pv. Phaseolicola

Incluye material complementarioThe bacterium Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally produced between 18 degrees C and 20 degrees C, while no detectable amounts are present above 28 degrees C. Phaseolotoxin is an effective inhibitor of ornithine carbamoyltransferase (OCTase) activity from plant, mammalian and bacterial sources and causes a phenotypic requirement for arginine. To protect the cell from its own toxin, P. syringae pv. phaseolicola synthesizes a phaseolotoxin-resistant OCTase (ROCT). The ROCT is the product of the argK gene and is synthesized only under conditions leading to phaseolotoxin synthesis. The argK gene is included in a chromosomal fragment named Pht cluster, which contains genes involved in the synthesis of phaseolotoxin. The aim of the present work was to investigate the possible involvement of other genes included in the Pht cluster in the regulation of gene argK. We conducted transcriptional analyses of argK in several mutants unable to produce phaseolotoxin, transcriptional fusions and electrophoretic mobility shift assays, which allowed us to determine that genes phtABC, located within the Pht cluster, participate in the transcriptional repression of gene argK at temperatures not permissive for phaseolotoxin biosynthesis. This repression is mediated by a protein present in both toxigenic and nontoxigenic strains of P. syringae and in E. coli, and requires the coordinated participation of phtA, phtB and phtC products in order to carry out an efficient argK repression.This work was funded by grants from CONACYT (Consejo Nacional de Ciencia y Tecnología; http://www.conacyt.mx), research grant SEP-2006-C01-49958/24089 to AAM and SA (Postdoctoral scholarship), and from the Spanish Plan Nacional I+D+I grant AGL2008-55311-CO2-01 (Ministerio de Ciencia e Innovación; http://micinn.es/), co financed by FEDER, to JM

Producción de conidios en sustratos sólidos y mutación del hongo entomopatógeno Hirsutella citriformis mediante metanosulfonato de etilo

El Huanglonging (HLB) está ocasionando grandes pérdidas en cítricos causada por la bacteria Candidatus liberibacter y transmitida por el insecto Diaphorina citri. Una de las alternativas es el control del vector usando hongos entomopatógenos como Hirsutella citriformis, el cual presenta algunas desventajas como su lento crecimiento y baja conidiación. En este trabajo se determinaron como objetivos, obtener alta producción de conidios en varios sustratos vegetales, así como conocer la concentración y tiempos de exposición de un agente mutagénico como el metanosulfonato de etilo (MSE) sobre la cepa silvestre necesarios para generar cepas mutantes al azar que mostraran características mejoradas. H. citriformis fue cultivada en dos sustratos vegetales arroz y avena y sometida a varias concentraciones de MSE y diferentes tiempos. Se obtuvo un total de 104 colonias mutantes las cuales se caracterizaron y seleccionaron en base a su velocidad de crecimiento radial, producción de conidios y germinación respecto a la cepa silvestre. Cuatro cepas mutantes fueron seleccionadas para futuros estudios.Huanglonging disease (HLB) is causing large losses in citrus which is caused by the bacterium Candidatus liberibacter and transmitted by the insect Diaphorina citri. One alternative is the control of vector using entomopathogenic fungi as Hirsutella citriformis, however this fungus has some drawbacks as slow growth and low conidiation. Therefore, in this work objectives such as obtaining higher production of conidia in various plant substrates as well as to determine the concentration and exposure times of a mutagenic agent such as ethyl methane sulfonate (EMS) on the wild strain to generate mutant strains at random that showed improved features were performed. A total of 104 mutant colonies were obtained which were characterized and selected based on their rate of radial growth, conidia production and germination compared to the wild strain, this analysis allowed us to select four mutant strains for future studies including bioassays against the insect vector

Analysis of Customer Behaviour in the Cash & Carry Wholesale

Import 04/11/2015Tato bakalářská práce se zabývá analýzou nákupního chování mezi dvěma segmenty - vietnamskými a českými zákazníky, v ostravské prodejně Makro. Hlavním cílem práce je najít a porovnat rozdílné chování mezi těmito dvěma segmenty, ale i jejich podobnosti při nákupu v prodejně. Dílčím cílem je zjistit, proč zákazníci nenakupují v ostravské prodejně Makro častěji.This bachelor work focuses on the analysis of shopping behavior between the two segments - the Vietnamese and Czech customers in Ostrava Makro. The main objective is to find and compare the different behavior between these two segments, but also their similarities when customers buying in a store. Partial aim is to find out why customers do not buy in Ostrava Makro more often.116 - Katedra marketingu a obchoduvelmi dobř

Anticancer potential of Thevetia peruviana fruit methanolic extract

Abstract Background: Thevetia peruviana (Pers.) K. Schum or Cascabela peruviana (L.) Lippold (commonly known as ayoyote, codo de fraile, lucky nut, or yellow oleander), native to Mexico and Central America, is a medicinal plant used traditionally to cure diseases like ulcers, scabies, hemorrhoids and dissolve tumors. The purpose of this study was to evaluate the cytotoxic, antiproliferative and apoptotic activity of methanolic extract of T. peruviana fruits on human cancer cell lines. Methods: The cytotoxic activity of T. peruviana methanolic extract was carried out on human breast, colorectal, prostate and lung cancer cell lines and non-tumorigenic control cells (fibroblast and Vero), using the MTT assay. For proliferation and motility, clonogenic and wound-healing assays were performed. Morphological alterations were monitored by trypan blue exclusion, as well as DNA fragmentation and AO/EB double staining was performed to evaluate apoptosis. The extract was separated using flash chromatography, and the resulting fractions were evaluated on colorectal cancer cells for their cytotoxic activity. The active fractions were further analyzed through mass spectrometry. Results: The T. peruviana methanolic extract exhibited cytotoxic activity on four human cancer cell lines: prostate, breast, colorectal and lung, with values of IC50 1.91 ± 0.76, 5.78 ± 2.12, 6.30 ± 4.45 and 12.04 ± 3.43 μg/mL, respectively. The extract caused a significant reduction of cell motility and colony formation on all evaluated cancer cell lines. In addition, morphological examination displayed cell size reduction, membrane blebbing and detachment of cells, compared to non-treated cancer cell lines. The T. peruviana extract induced apoptotic cell death, which was confirmed by DNA fragmentation and AO/EB double staining. Fractions 4 and 5 showed the most effective cytotoxic activity and their MS analysis revealed the presence of the secondary metabolites: thevetiaflavone and cardiac glycosides. Conclusion: T. peruviana extract has potential as natural anti-cancer product with critical effects in the proliferation, motility, and adhesion of human breast and colorectal cancer cells, and apoptosis induction in human prostate and lung cancer cell lines, with minimal effects on non-tumorigenic cell lines. Keywords: Cytotoxic activity, Anti-proliferative activity, Motility, Apoptosis, Human cancer cells, Flavonoid, Cardiac glycoside

Systematic bioprospection for cellulolytic actinomycetes in the Chihuahuan Desert: isolation and enzymatic profiling

The quest for microbial cellulases has intensified as a response to global challenges in biofuel production. The efficient deconstruction of lignocellulosic biomass holds promise for generating valuable products in various industries such as food, textile, and detergents. This article presents a systematic bioprospection aimed at isolating actinomycetes with exceptional cellulose deconstruction capabilities. Our methodology explored the biodiverse oligotrophic region of Cuatro Cienegas, Coahuila, within the Chihuahuan Desert. Among the evaluated actinomycetes collection, 78% exhibited cellulolytic activity. Through a meticulous screening process based on enzymatic index evaluation, we identified a highly cellulolytic Streptomyces strain for further investigation. Submerged fermentation of this strain revealed an endoglucanase enzymatic activity of 149 U/mg. Genomic analysis of strain Streptomyces sp. STCH565-A revealed unique configurations of carbohydrate-active enzyme (CAZyme) genes, underscoring its potential for lignocellulosic bioconversion applications. These findings not only highlight the significance of the Chihuahuan Desert as a rich source of cellulolytic microorganisms but also offer insights into the systematic exploration and selection of high-performing cellulolytic microorganisms for application in diverse environmental contexts. In conclusion, our bioprospecting study lays a foundation for harnessing the cellulolytic potential of actinomycetes from the Chihuahuan Desert, with implications for advancing cellulose deconstruction processes in various industries. The findings can serve as a blueprint for future bioprospecting efforts in different regions, facilitating the targeted discovery of microorganisms with exceptional cellulosic deconstruction capabilities

In vitro anticancer activity of methanolic extract of Granulocystopsis sp., a microalgae from an oligotrophic oasis in the Chihuahuan desert

With the purpose of discovering new anticancer molecules that might have fewer side effects or reduce resistance to current antitumor drugs, a bioprospecting study of the microalgae of the Cuatro Cienegas Basin (CCB), an oasis in the Chihuahuan desert in Mexico was conducted. A microalgae was identified as Granulocystopsis sp. through sequencing the rbcL gene and reconstruction of a phylogenetic tree, and its anticancer activities were assessed using various in vitro assays and different cell lines of human cancers, including lung, skin melanoma, colorectal, breast and prostatic cancers, as well as a normal cell line. The values of IC50 of the microalgae methanolic extract using the MTT assay were lower than 20 μg/ml, except that in the lung cancer line and the normal cell line. In vitro, the microalgae extract caused the loss of membrane integrity, monitored by the trypan blue exclusion test and exhibited marked inhibition of adhesion and cell proliferation in cancer cell lines, through the evaluation of the clonogenic assay. Also, typical nuclear changes of apoptotic processes were observed under the microscope, using the dual acridine orange/ethidium bromide fluorescent staining. Finally, the microalgae extract increased the activity of caspases 3 and 7 in skin melanoma, colon, breast and prostate cancer cells, in the same way as the apoptotic inductor and powerful antitumoral drug, doxorubicin. This study shows the anticancer activity from Granulocystopsis sp., a microalgae isolated from the CCB