Summary: A modified method for the direct extraction of DNA from alkaline-saline soils with minimum DNA fragmentation and a possible reduction in chimera formation during polymerase chain reaction (PCR) was developed. The commercial extraction kit Power Soil DNA (Mo Bio™ Laboratories, Inc.) was used as a reference technique. The method reported here was based on cell lysis employing ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), and cell disruption with mechanical force with FastPrep-24™ equip followed by one cycle of freezing at -40 °C for 60 min and thawing at 65 °C for 20 min. The extraction method was tested for allophonic soils with large concentrations of organic matter, fulvic and humic acids, electrolytic conductivity (EC) ranging between 2.6 dS m-1 and 39.9 dS m-1, and pH between 8.8 and 10.9. The yield of DNA extracted depended on soil type, i.e., DNA extracted from soil varied between 2.35 (Texcoco-2) to 3.66 (Texcoco-1) μg DNA g-1 soil. The proposed method in this study produced enough DNA with yield and quality for PCR amplification of 16S rDNA when bovine serum albumin (BSA) was added to the reaction buffer. The DNA obtained had sufficient quality and yield for later use for 16S sequencing or possible use in other sequencing technologies, e.g. whole metagenome shotgun sequencing.

Resumen: Se desarrolló un método modificado para la lisis bacteriana y la extracción directa de ADN de suelo alcalino-salino con mínima fragmentación y con esto, evitar posiblemente, la formación de quimeras a partir de pequeños fragmentos de ADN durante la amplificación por PCR. Se utilizó el kit Power Soil DNA (Mo Bio™ Laboratories, Inc.) como técnica de referencia. El método reportado se basó en la lisis celular en presencia de dodecilsulfato de sodio (SDS), ácido etilendiaminotetraacético (EDTA) y lisis celular mecánica empleando un equipo Fast-Prep-24™, seguido de un ciclo de congelación a -40 °C durante 60 min y descongelación a 65 °C durante 20 min. El método de extracción se probó para suelos alofónicos con grandes concentraciones de materia orgánica, ácidos fúlvicos y húmicos, conductividad electrolítica (CE) entre 2.6 dS m-1 y 39.9 dS m-1, y pH entre 8.8 y 10.9. La concentración del ADN extraído de los suelos osciló entre 2.35 (Texcoco-2) y 3.66 (Texcoco-1) µg de ADN g-1 de suelo, y el rendimiento dependió del tipo de suelo empleado. El método produjo ADN lo suficientemente puro para la amplificación del gen 16S rADN por PCR cuando se añadió albúmina de suero bovino (BSA) al buffer de reacción. El ADN obtenido presenta suficiente calidad y rendimiento para su uso posterior para la secuenciación del gen 16S rADN, o para un secuenciamiento directo, i.e. secuenciación del metagenoma.

Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection.

Background: The application of polycyclic aromatic hydrocarbons (PAHs) will affect the bacterial community structure as some groups will be favoured and others not. An alkaline saline soil with electrolytic conductivity (EC) 56 dS m-1 was spiked with anthracene and acetone while their effect on bacterial community structure was investigated. Results: The percentages of Acidobacteria and Actinobacteria decreased over time, while the percentage of Proteobacteria, mostly Xanthomonadales, increased. The percentage of the phylotypes belonging to the Nocardioides, Rhodococcus and Streptomyces, known degraders of PAHs, was larger in the anthracene-amended soil than in the acetone-amended and unamended soil at day 14. The phylotypes belonging to the genera Sphingomonas, also a known degrader of PAHs, however, was lower. Weighted and unweighted PCoA with UniFrac indicated that phylotypes were similar in the different treatments at day 0, but changed at day 1. After 14 days, phylotypes in the unamended and acetone-amended soil were similar, but different from those in the anthracene-spiked soil. Conclusions: It was found that incubating the soil and contaminating it with anthracene changed the bacterial community structure, but spiking the soil with acetone had little or no effect on the bacterial community structure compared to the unamended soil.