Laura S. England

Bacterial survival in soil: Effect of clays and protozoa

Abstract There are numerous interacting biological, physical and chemical factors that affect survival of both indigenous and introduced bacteria in soil. Three important factors are clay type, clay content and predation by soil protozoans. These factors are important when considering release of non-engineered and genetically engineered microorganisms (GEMs) into soil. In this review we examine the influence of clays and predatory activity of protozoans on bacterial survival. These aspects are related because protozoa may ingest clay particles as well as bacteria. In addition, clays may protect bacteria from predation by protozoans by increasing the number of protective microhabitats available to bacteria in soil.

Persistence of Pseudomonas aureofaciens strains and DNA in soil

Abstract Persistence of a 630-bp fragment of a Tn7- lac fusion element inserted chromosomally in Pseudomonas aureofaciens Ps3732RNL11 was assessed in non-autoclaved and autoclaved sandy loam soil. PCR-amplified 630-bp DNA fragments, from DNA extracts of inoculated, non-autoclaved soil, were observed at 2 and 4 wk after inoculation. DNA extracts of inoculated, autoclaved soil subjected to PCR yielded the expected 630-bp DNA bands at 2, 4, 8, 12, 18 and 24 wk after inoculation. Our results showed that extracellular DNA can persist in soil for extended periods. Survival of P. aureofaciens Ps3732RNL11 and Ps3732RN (parental wildtype) in soil was assessed over 44 wk. After 30 wk in non-autoclaved soil, Ps3732RNL11 and Ps3732RN cells initially inoculated into soil at log 4.3 and log 4.1 cells g −1 dry soil, respectively, were not detectable by viable plating. Ps3732RNL11 and Ps3732RN cells inoculated at log 8.1 and log 7.9 cells g −1 dry soil, respectively, were detectable by plating after 44 wk.

Real-time polymerase Chain reaction quantification of the transgenes for Roundup Ready corn and roundup ready soybean in soil samples

A method for quantification of recombinant DNA for Roundup Ready (RR) corn and RR soybean in soil samples is described. Soil DNA from experimental field samples was extracted using a soil DNA extraction kit with a modified protocol. For the detection and quantification of recombinant DNA of RR corn and RR soybean, a molecular beacon and two pairs of specific primers were designed to differentially target recombinant DNA in these two genetically modified crops. Soil DNA extracts were spiked with RR corn or RR soybean DNA, and recombinant DNA was quantified using real-time PCR with a molecular beacon. As few as one copy of RR corn genome or one copy of RR soybean genome was detected in the soil DNA extract.

Recombinant and wild‐type Pseudomonas aureofaciens strains introduced into soil microcosms: effect on decomposition of cellulose and straw

The effect of a genetically engineered Pseudomonas aureofaciens (Ps3732RNL11) strain (GEM) and the parental wild‐type (Ps3732RN) on decomposition of cellulose paper, straw and calico cloth was assessed after 18 weeks incubation in laboratory soil microcosms. Effect(s) of inoculum density (103, 105, and 108 cells/ g dry soil) and single versus multiple bacterial inoculations were also investigated. Cellulose paper was completely decomposed after 18 weeks in all treatments. There were no significant differences (95% level), between treatments, in percentage decomposition of either straw or calico cloth. Recovery of the GEM at 18 weeks, using viable plating, was limited to treatments originally receiving 108 cells/g dry soil. Log 1.8 CFU/g dry soil were recovered from the single dose treatment while log 4.2 CFU/g dry soil were recovered from the multiple dose treatment Biolog metabolic tests were used to determine if the GEM or parental wild‐type had any effect on overall carbon utilization in soil. Results suggested they did not. Detection of the recombinant lacZY gene sequence in soil using PCR suggested the possibility of viable but nonculturable cells and/or persistence of chromosomal DNA.

Microbial diversity in soil: effect of releasing genetically engineered micro-organisms

This review examines the potential for change in microbial diversity, with the emphasis on bacteria, in soil resulting from the introduction of genetically engineered microorganisms (GEMs). With the advent of GEMs came the impetus for new technologies to recover these micro‐organisms from soil and to assess their effects on microbial diversity. This review also presents general aspects of and genetic approaches to accessing bacterial diversity in the environment.

Recombinant and wild‐type Pseudomonas aureofaciens strains in soil: survival, respiratory activity and effects on nodulation of whitebean Phaseolus vulgaris L. by Rhizobiutn species

Survival and respiratory activity of a genetically engineered Pseudomonas aureofaciens Ps3732RNL11 were compared to the parental wild‐type P. aureofaciens Ps3732RN in loam and sandy loam soils over 17‐ and 28‐day periods. Survival and respiratory activity of P. aureofaciens Ps3732RNL11 was not statistically significantly different from that of P. aureofaciens Ps3732RN. Soil texture had an effect on respiratory activity; carbon dioxide evolution was significantly higher in the sandy loam soil. This effect was observed on days 2, 10 and 18 but not on day 24. The presence of P. aureofaciens Ps3732RNL11 and Ps3732RN did not significantly affect growth of whitebean (Phaseolus vulgaris L.) in vermiculite, loam, or sandy loam soils. There was no significant difference (95% level) in numbers of nodules produced in the presence of P. aureofaciens Ps3732RNL11 and Ps3732RN as a result of the symbiotic relationship between Rhizobium phaseoli and the whitebean roots in vermiculite. Enumeration of nodules on whitebean roots in loam and sandy loam soils was not conducted due to difficulties in removing intact roots from the soils.

The Microbial DNA Cycle in Soil

Upon microbial cell death and lysis in soil, the free or naked DNA is exposed to the dynamic environment of the soil. The DNA can be enzymatically degraded by nucleases (DNases), bind to soil components, genetically transform competent bacterial cells and be a nutrient for other microorganisms. In this article we discuss the dual role of DNA as genetic material and as a nutrient source in the soil environment.

Persistence of naturally occurring and genetically modified Choristoneura fumiferana nucleopolyhedroviruses in outdoor aquatic microcosms.

BACKGROUND To assess the persistence of genetically modified and naturally occurring baculoviruses in an aquatic environment, replicate (three) outdoor, aquatic microcosms were spiked with spruce budworm viruses [Ireland strain of Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) and the recombinant CfMNPVegt(-)/lacZ(+)] at a rate of 1.86 x 10(10) occlusion bodies (OBs) m(-2) of surface area. The presence of virus in water samples collected at various times after inoculation was determined by PCR amplification of baculoviral DNA extracted from OBs. RESULTS Although UV radiation rapidly degrades baculoviruses under natural conditions, both viruses persisted above the level of detection (>100 OBs 450 microL(-1) of natural pond water) for at least 1 year post-inoculation, with little difference between the viruses in their patterns of persistence. CONCLUSION The present microcosm study suggests that occlusion bodies of baculoviruses can persist in the flocculent layer of natural ponds. On disturbance, OBs could re-enter the main water column and thus be available for transport to new locations. Implications for environmental risk assessment are discussed.

Bacterial starvation-survival and a method to determine carbon utilisation by soil micro-organisms (famine or feast).

Bacteria are ubiquitous in the natural environment, even when nutrients are limiting and they are in a physiological state of starvation-survival. How bacterial cells survive for extended periods of time when the nutrients and energy needed for growth and cell division are insufficient is examined. A method for determining carbon utilisation patterns in soil extracts is included. This determines the diverse range of carbon substrates metabolised by soil microorganisms when they are extracted from soil samples and incubated under conditions suitable for rapid metabolism. This practical also highlights the diversity of carbon substrate utilisation in environmental soil samples.