L. Arlene Porteous

Recovery of bulk DNA from soil by a rapid, small-scale extraction method

We describe an extraction method that yields up to 25 µg of DNA from 1 gram of soil. Samples are processed within 48 h. The purified DNA is 20–25 kb in size and can be digested by restriction enzymes. The soil is vortex-mixed with 6 mL mixing buffer in an Oakridge tube. Polyvinyl polypyrrolidone is added to adsorb the humic compounds that interfere with restriction enzyme activity. The entire soil homogenate is treated with lysozyme and Novozym, followed by sodium dodecyl sulfate to lyse the cells. The crude DNA extract is separated from the soil debris by low-speed centrifugation. Finally, the DNA is purified by CsCl gradient centrifugation and extracted with Geneclean.

A simple mini-method to extract DNA directly from soil for use with polymerase chain reaction amplification

A rapid, simple method is used that yields amplifiable fungal and bacterial DNAs directly from soil. DNA is separated from soil contaminants by electrophoresis in low-melting-temperature agarose and used directly in polymerase chain reaction amplification. Fifty 20-mg samples can be processed in one day. Fragments of 16S and 18S ribosomal RNAs are amplified by polymerase chain reaction with DNA extracted from the soil. Universal primers are used that are capable of amplifying ribosomal DNA from a wide variety of bacteria and fungi. Eubacterial and fungal primers are used that are capable of distinguishing between eubacterial and fungal DNAs. Restriction enzyme digests are performed on amplified DNA fragments from five soil samples.

Multiple enzyme restriction fragment length polymorphism analysis for high resolution distinction of Pseudomonas (sensu stricto) 16S rRNA genes.

Members of the genus Pseudomonas (sensu stricto) are important phytopathogens and agents of human infections, while other strains and species have beneficial bioremediation and biocontrol activities. Traditionally, these important species have been difficult to differentiate phenotypically; thus, rRNA lineage analyses have often been invoked. In this report, a newly developed approach is described to rapidly detect and distinguish fluorescent Pseudomonas isolates: PCR amplification of a Pseudomonas-specific 990-bp ribosomal RNA gene (rDNA) fragment [Appl. Environ. Microbiol. 64 (1998) 2545.] coupled with multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of AluI, HinfI, RsaI, and Tru9I incubated at 37 degrees C. The method distinguished 116 published sequences and 47 reference strains of authentic Pseudomonas representing 28 nomenspecies. A total of 55% (64/116) of the sequences analyzed by MERFLP were grouped into distinct phylogenetic clusters including Pseudomonas putida, P. syringae, P. aeruginosa, P. stutzeri, and P. fluorescens. The utility of the MERFLPs was confirmed when 100% (33/33) of the above named control reference strains were correctly placed into their phylogenetic clusters. The environmental relevance of the MERFLP method was confirmed when 67% of 28 forest and agricultural soil-derived presumptive Pseudomonas environmental clones and isolates were placed into the five major pseudomonad clusters, one clone fell into the P. agarici cluster, and five clones clustered near related pseudomonads. These data demonstrated that the PCR-MERFLP protocol provides an efficient and powerful tool for distinguishing isolates and rDNA gene libraries of environmental Pseudomonas species.

Evaluation of a method to measure conjugal transfer of recombinant DNA in soil slurries

Release of recombinant microbes into the environment necessitates an evaluation of their ability to transfer genetic material. The present report evaluates a method to detect conjugal DNA plasmid transfer in soil slurries under various environmental conditions. DonorPseudomonas cepacia containing pR388::Tn1721 andP. cepacia recipient cultures were coincubated in soil slurries containing autoclaved or natural soil and treated with one or more of 14 experimental conditions. Conjugal mating frequency (transconjugants per initial donor) ranged from 4.8×10−1 to 1.9×10−7. Highest numbers of transconjugants, 1.5×107 colony forming units/ml soil slurry, were observed following incubation at 35°C with an enriched nutrient supplement added to the soil. Low numbers of transconjugants, 103 colony forming units/ml soil slurry, were observed when mating pairs were subjected to low nutrient or pH stress even though initial donor and recipient populations were maintained at high levels. This test system provides a simple way to estimate effects of changing environmental factors on plasmid transfer rates and on the survival of recombinant microorganisms. By use of soil collected from sites proposed to receive genetically engineered microorganisms, preliminary risk assessments can be obtained regarding the potential for gene transfer and microorganism survival with this soil slurry test system.

Bracken (Pteridium aquilinum L.) frond biomass and rhizosphere microbial community characteristics are correlated to edaphic factors

Bracken is a broadly distributed weedy fern common in disturbed habitats. Frond and rhizosphere soil samples were obtained from bracken growing in three clearcut locations in the Willamette National Forest in western Oregon. The highest frond biomass was correlated with soil having the highest total %N, lowest Fe content and oldest geological age. Based on analysis of variance of principal component scores for patterns of utilization of substrates on Biolog GN plates, metabolic profiles of rhizosphere microbial communities of bracken differed significantly between locations. Utilization of carbohydrates and phosphorylated compounds was positively correlated with organic matter (OM) and total N and negatively correlated with extractable Fe and Mn content of soil. Carboxylic acid utilization was positively correlated with pH and OM and negatively correlated with extractable Mn and P content of soils. Pseudomonas rDNA fingerprints of bracken rhizosphere samples suggested that the diversity of pseudomonads at the location with the most acidic (pH 5.5) soil (Burnside Road) differed from those at less acidic (pH 6.2 and 6.1) locations (Falls Creek and Toad Road). Mycorrhizal infection of bracken was lowest at Falls Creek, the location with the highest %N soil content. Our results suggest that bracken frond biomass and rhizosphere microbial community characteristics are correlated with local edaphic factors such as soil chemistry and geological age.