GFP-tagged multimetal-tolerant bacteria and their detection in the rhizosphere of white mustard

The introduction of rhizobacteria that tolerate heavy metals is a promising approach to support plants involved in phytoextraction and phytostabilisation. In this study, soil of a metal-mine wasteland was analyzed for the presence of metal-tolerant bacterial isolates, and the tolerance patterns of the isolated strains for a number of heavy metals and antibiotics were compared. Several of the multimetal-tolerant strains were tagged with a broad host range reporter plasmid (i.e. pPROBE-NT) bearing a green fluorescent protein marker gene (gfp). Overall, the metal-tolerant isolates were predominately Gram-negative bacteria. Most of the strains showed a tolerance to five metals (Zn, Cu, Ni, Pb and Cd), but with differing tolerance patterns. From among the successfully tagged isolates, we used the transconjugant Pseudomonas putida G25 (pPROBE-NT) to inoculate white mustard seedlings. Despite a significant decrease in transconjugant abundance in the rhizosphere, the gfp-tagged cells survived on the root surfaces at a level previously reported for root colonisers

Isolation of nucleic acids from the environment - the first step in metagenome analysis

Recently, increasing interest in ecology of microorganisms has been associated with the possibility of direct analysis of microbial community structure due to application of molecular methods based on isolated metagenome DNA. The metagenome approach can provide a cultivation-independent assessment of the largely untapped genetic reservoir of soil or water microbial communities. However, the crucial step in this approach is efficient extraction of high-quality total DNA representing the metagenome of a habitat. The DNA extraction methods for soil habitats are grouped into two major types, i.e. indirect based on the recovery of microbes (e.g. bacterial cells) and their subsequent lysis, and direct lysis of cells in the sample followed by DNA purification. The direct extraction of total DNA from an environmental sample presumably better represents its bacterial or fungal metagenome; hence, this approach has been used more often than the fractionation methods. Although direct extraction of DNA is less labour-intensive and yield more DNA, the recovered DNA fragments are usually smaller than those obtained by the indirect approach are. The fractionation method is advantageous for soil samples containing higher amounts of organic matter or other substances that interfere with DNA isolation. This method is also applied for DNA extraction from water samples. Microbial ecologists currently use different commercially available kits for total DNA isolation from soil or water. However, it would appear that the most efficient method of DNA extraction from environmental samples is still far from being established

Spatial Distribution of Salmonella in Soil near Municipal Waste Landfill Site

Due to the heterogeneous origin of municipal waste, different substrates support the growth of many microorganisms, including those hazardous to humans. In consequence, landfills collecting these refuses are regarded as serious sources of infectious material contaminating the environment. In this study, we aimed to assess how waste may be related to the presence of Salmonella spp. in soil within a landfill and its surroundings. The numbers of these pathogens were estimated in soil samples collected at 17 different stands established in the municipal waste landfill of Barycz (near Kraków, Poland) and the surrounding area. The analysis showed that in all soil samples, Salmonella spp. did not exceed 270 cfu g−1 in dry soil (i.e., the active landfill sector). Salmonella spp. was found in 57% of the tested soil samples in spring, 88% in summer, 45% in autumn, and was not detected in winter. A spatial distribution visualized by graphical maps allowed determination of the influence of the active sector on the surrounding areas. The graphical maps showed the impact of seasons on the spread of Salmonella spp. in the soil near the landfill. Detection and estimation of Salmonella spp. distribution in soil within the landfill area distinctly confirms the hazardous impact of collected wastes on hygienic characteristics of the soil