Alejandro Hernández-Morales

Gene expression of Pht cluster genes and a putative non-ribosomal peptide synthetase required for phaseolotoxin production is regulated by GacS/GacA in Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by watersoaked lesions surrounded by a chlorotic halo resulting from the action of a non-host specific toxin known as phaseolotoxin. This toxin inhibits the enzyme ornithine carbamoyltransferase involved in the arginine biosynthesis pathway. It was previously reported that genes within the Pht cluster were involved in the regulation and synthesis of phaseolotoxin. The GacS/GacA two-component signal transduction system controls important pathogenicity and virulence mechanisms in several Gram-negative bacteria. Tox(-) phenotype gacA(-) and gacS(-) mutants were obtained and gacA(-) transcriptome analysis revealed that this response activator controls expression of genes within the Pht cluster as well as another gene located in a different region in the bacterial chromosome and that has been unambiguously shown to be directly involved in phaseolotoxin biosynthesis. Results presented in this work suggest that phaseolotoxin biosynthesis involve elements within and outside the Pht Cluster, and that the GacS/GacA two-component system exerts control over them.

Pharmacological effects and toxicity of Costus pulverulentus C. Presl (Costaceae)

ETHNOPHARMACOLOGICAL RELEVANCE Costus pulverulentus C. Presl (Costaceae), a species endemic to Mexico, is used for the empirical treatment of cancer, pain, and inflammation. AIM OF THE STUDY The objective of this study was to evaluate the toxicity, as well as the cytotoxic, antinociceptive, anti-inflammatory and sedative effects of an ethanol extract from Costus pulverulentus stem (CPE). MATERIALS AND METHODS The chemical characterization of CPE was performed by Gas chromatography-mass spectrometry (GC-MS). The toxicity of CPE was evaluated using the comet assay (10-1000 µg/ml during 5h) and the acute toxicity test (500-5000 mg/kg p.o. and i.p. during 14 days). The cytotoxic effect of CPE (1-250 µg/ml) on human cancer cells was evaluated using the MTT assay. The antinociceptive effects of CPE (50-200mg/kg p.o.) were evaluated using thermal-induced nociception tests (hot plate and tail flick) and the chemical-induced nociceptive tests (acetic acid and formalin). The sedative activity of CPE (50-200mg/kg p.o.) was evaluated using the ketamine-induced sleeping time test. RESULTS CPE showed the presence of compounds such as campesterol, stigmasterol β-sitosterol, vanillic acid, among others. In the comet assay, CPE at 200 µg/ml or higher concentrations induced DNA damage. In the acute toxicity test, the LD50 estimated for CPE was>5000 mg/kg p.o. or i.p. CEP showed moderate cytotoxic effects on prostate carcinoma cells PC-3 cells (IC50=179 ± 23.2 µg/ml). In the chemical-induced nociception models, CPE (100 and 200mg/kg p.o.) showed antinociceptive effects with similar activity to 100mg/kg naproxen. In the thermal-induced nociception tests, CPE tested at 200mg/kg showed moderate antinociceptive effects by 28% (hot plate test) and by 25% (tail flick test). In the ketamine-induced sleeping time test, CPE showed no sedative effects. CONCLUSIONS C. pulverulents exerts moderate cytotoxic effects in human cancer cells, moderate anti-inflammatory and antinociceptive effects. C. pulverulentus induces antinociceptive effects without inducing sedation.

Transcriptional profile of P. syringae pv. phaseolicola NPS3121 at low temperature: Physiology of phytopathogenic bacteria

BackgroundLow temperatures play key roles in the development of most plant diseases, mainly because of their influence on the expression of various virulence factors in phytopathogenic bacteria. Thus far, studies regarding this environmental parameter have focused on specific themes and little is known about phytopathogenic bacteria physiology under these conditions. To obtain a global view regarding phytopathogenic bacteria strategies in response to physiologically relevant temperature changes, we used DNA microarray technology to compare the gene expression profile of the model bacterial pathogen P. syringae pv. phaseolicola NPS3121 grown at 18°C and 28°C.ResultsA total of 236 differentially regulated genes were identified, of which 133 were up-regulated and 103 were down-regulated at 18°C compared to 28°C. The majority of these genes are involved in pathogenicity and virulence processes. In general, the results of this study suggest that the expression profile obtained may be related to the fact that low temperatures induce oxidative stress in bacterial cells, which in turn influences the expression of iron metabolism genes. The expression also appears to be correlated with the profile expression obtained in genes related to motility, biofilm production, and the type III secretion system.ConclusionsFrom the data obtained in this study, we can begin to understand the strategies used by this phytopathogen during low temperature growth, which can occur in host interactions and disease development.

The PhtL protein of Pseudomonas syringae pv. phaseolicola NPS3121 affects the expression of both phaseolotoxin cluster (Pht) and Non-Pht encoded genes.

Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight disease in bean, produces a toxin known as phaseolotoxin, whose synthesis involves the products of some of the genes found within the Pht region. This region, considered a pathogenicity island, comprises 23 genes arranged in five transcriptional units: two single-gene units (argK, phtL) and three arranged as operons (phtA, phtD, phtM), most with unknown function. In P. syringae pv. phaseolicola, maximal expression of most of the genes encoded in the Pht region and the synthesis of phaseolotoxin require the product of the phtL gene, of unknown function but that has been proposed to have a regulatory role. In order to evaluate the role of phtL gene in P. syringae pv. phaseolicola, we performed a comparative transcriptional analysis with the wild type and a phtL(-) mutant strains using microarrays. The microarray data analysis showed that PhtL regulates the expression not only of genes within the Pht region, but also alters the expression of genomic genes outside it, indicating that this gene has been integrated into the regulatory machinery of the bacterium. The expression changes of many of those genes were confirmed by RT-PCR. This study also demonstrated the importance of the PhtL protein in the process of iron response, and suggests that the effect of PhtL on the expression of pathogenicity related, respiration and oxidative stress genes, observed in this study, appears to be indirect through its influence on the Fur protein expression.

The antinociceptive effects of a standardized ethanol extract of the Bidens odorata Cav (Asteraceae) leaves are mediated by ATP-sensitive K+ channels

ETHNOPHARMACOLOGICAL RELEVANCE Bidens odorata Cav (Asteraceae) is used for the empirical treatment of inflammation and pain. AIM OF THE STUDY This work evaluated the in vitro and in vivo toxicity, antioxidant activity, as well as the anti-inflammatory and antinociceptive effects of an ethanol extract from Bidens odorata leaves (BOE). MATERIALS AND METHODS The in vitro toxicity of BOE (10-1000µg/ml) was evaluated with the comet assay in PBMC. The in vivo acute toxicity of BOE (500-5000mg/kg) and the effect of BOE (10-1000µg/ml) on the level of ROS in PBMC were determined. The in vivo anti-inflammatory activity of BOE was assessed using the TPA-induced ear edema in mice. The antinociceptive activities of BOE (50-200mg/kg p.o.) were assessed using the acetic acid and formalin tests. The antinociceptive mechanism of BOE was determined using naloxone and glibenclamide. RESULTS BOE lacked DNA damage, and showed low in vivo toxicity (LD50 > 5000mg/kg p.o.). BOE inhibited ROS production (IC50 = 252.13 ± 20.54µg/ml), and decreased inflammation by 36.1 ± 3.66%. In both antinociceptive test, BOE (200mg/kg) exerted activity with similar activity than the reference drugs. CONCLUSION B. odorata exerts low in vitro and in vivo toxicity, antioxidant effects, moderate in vivo anti-inflammatory activity, and antinociceptive effects mediated by ATP-sensitive K+ channels.

Temperature, the GacS/GacA system, and host metabolites regulate the type VI secretion system of Pseudomonas syringae pv. phaseolicola

The recently discovered type VI secretion system (T6SS) is implicated in the pathogenic and/or virulence processes of diverse bacteria. The expression pattern of the T6SS differs among different organisms and also depends on several environmental factors. We initiated a study of the conditions that influence T6SS gene expression in Pseudomonas syringae pv. phaseolicola NPS3121. Our results indicate that low temperatures and plant extracts impact the expression of T6SS genes and that the process is subject to regulation by the GacS/GacA two-component system.

Oxidative stress regulates the expression of the Pht cluster genes involved in phaseolotoxin synthesis in Pseudomonas syringae pv. phaseolicola NPS3121

This study evaluated the role of oxidative stress on the expression of Pht cluster genes involved in phaseolotoxin synthesis in Pseudomonas syringae pv. phaseolicola. Results demonstrate that the expression of Pht cluster genes is regulated by oxidative stress in a manner dependent of the ROS present in the cell. The presence of H2O2 and Paraquat, influences on the expression of the Pht cluster genes in function of the compound and of the concentration evaluated, demonstrating that expression of Pht genes is part of the oxidative stress response in P. syringae pv. phaseolicola NPS3121.

ORIGINALAislados de Bacillus provenientes de la rizósfera de cactus incrementan la germinación y la floración en Mammillaria spp. (Cactaceae)Bacillus isolates from rhizosphere of cacti improve germination and bloom in Mammillaria spp. (Cactaceae)

Cacti are the most representative vegetation of arid zones in Mexico where rainfall is scarce, evapotranspiration is high and soil fertility is low. Plants have developed physiological strategies such as the association with microorganisms in the rhizosphere zone to increase nutrient uptake. In the present work, four bacterial isolates from the rhizosphere of Mammillaria magnimamma and Coryphantha radians were obtained and named as QAP3, QAP19, QAP22 and QAP24, and were genetically identified as belonging to the genus Bacillus, exhibiting in vitro biochemical properties such as phosphate solubilization, indoleacetic acid production and ACC deaminase activity related to plant growth promotion, which was tested by inoculating M. magnimamma seeds. It was found that all isolates increased germination from 17 to 34.3% with respect to the uninoculated control seeds, being QAP24 the one having the greatest effect, accomplishing the germination of viable seeds (84.7%) three days before the control seeds. Subsequently, the inoculation of Mammillari zeilmanniana plants with this isolate showed a positive effect on bloom, registering during two months from a one year period, an increase of up to 31.0% in the number of flowering plants compared to control plants. The characterized Bacillus spp. isolates have potential to be used in conservation programs of plant species from arid zones.

Aislados de Bacillus provenientes de la rizósfera de cactus incrementan la germinación y la floración en Mammillaria spp. (Cactaceae)

Cacti are the most representative vegetation of arid zones in Mexico where rainfall is scarce, evapotranspiration is high and soil fertility is low. Plants have developed physiological strategies such as the association with microorganisms in the rhizosphere zone to increase nutrient uptake. In the present work, four bacterial isolates from the rhizosphere of Mammillaria magnimamma and Coryphantha radians were obtained and named as QAP3, QAP19, QAP22 and QAP24, and were genetically identified as belonging to the genus Bacillus, exhibiting in vitro biochemical properties such as phosphate solubilization, indoleacetic acid production and ACC deaminase activity related to plant growth promotion, which was tested by inoculating M. magnimamma seeds. It was found that all isolates increased germination from 17 to 34.3% with respect to the uninoculated control seeds, being QAP24 the one having the greatest effect, accomplishing the germination of viable seeds (84.7%) three days before the control seeds. Subsequently, the inoculation of Mammillari zeilmanniana plants with this isolate showed a positive effect on bloom, registering during two months from a one year period, an increase of up to 31.0% in the number of flowering plants compared to control plants. The characterized Bacillus spp. isolates have potential to be used in conservation programs of plant species from arid zones.