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Survival of a plasmid-bearing strain of Bacillus subtilis introduced into compost
W Amner1, A J McCarthy, C Edwards
1Department of Genetics and Microbiology, University of Liverpool, UK.
Abstract:
Survival of Bacillus subtilis strain 168 containing plasmid pAB224, which carries a gene for tetracycline resistance, was studied in mushroom compost under mesophilic and thermophilic conditions. Stable populations of B. subtilis were maintained as spores in both sterile and fresh mushroom compost incubated at 37 degrees C. At 65 degrees C, the introduced B. subtilis populations declined during incubation but spores were still detectable after 28 d. Survival at the higher temperature was greater in fresh than in sterile compost. There was no apparent loss of plasmid pAB224 or plasmid-determined phenotype from the introduced B. subtilis population at either incubation temperature. The frequency of tetracycline resistance in the indigenous Bacillus population was very low (10(-5), but some tetracycline-resistant isolates contained plasmid DNA. Four plasmid DNA profiles were found associated with five Bacillus phenotypes, and some evidence for homology with pAB224 was found. However, pAB224 was found to be a suitable marker for release studies because it was easily recovered, readily distinguished from indigenous plasmids on agarose gels, and was maintained in compost-grown B. subtilis 168 in the absence of any selective pressure.
Insights
Bacillus subtilis strain 168 with plasmid pAB224 showed stable survival in mushroom compost at 37°C. At 65°C, survival decreased but spores persisted, with plasmid pAB224 proving a reliable marker for release studies.
Area of Science:
- Microbiology
- Environmental Science
- Biotechnology
Background:
- Assessing the survival and stability of genetically modified microorganisms in environmental matrices is crucial for risk assessment.
- Mushroom compost presents a unique environment with varying temperatures and microbial communities.
- Understanding plasmid stability in introduced bacteria is essential for contained field release studies.
Purpose of the Study:
- To evaluate the survival of Bacillus subtilis 168 containing the tetracycline resistance plasmid pAB224 in mushroom compost under different temperature conditions.
- To determine the stability of plasmid pAB224 and its associated phenotype in B. subtilis within the compost environment.
- To assess the suitability of pAB224 as a marker for tracking introduced bacteria in environmental studies.
Main Methods:
- Incubation of sterile and fresh mushroom compost inoculated with B. subtilis 168 (pAB224) at 37°C (mesophilic) and 65°C (thermophilic).
- Enumeration of viable B. subtilis populations and detection of spores over time.
- Analysis of plasmid DNA profiles in surviving B. subtilis and indigenous Bacillus populations using agarose gel electrophoresis.
- Investigation of potential homology between pAB224 and indigenous plasmids.
Main Results:
- Stable populations of B. subtilis 168 (pAB224) were maintained as spores at 37°C in both sterile and fresh compost.
- At 65°C, B. subtilis populations declined, but spores remained detectable for 28 days, with higher survival in fresh compost.
- Plasmid pAB224 and its tetracycline resistance phenotype remained stable in the introduced B. subtilis population at both temperatures.
- The frequency of tetracycline resistance in indigenous Bacillus was low, though some isolates contained plasmids, with limited homology to pAB224.
Conclusions:
- Bacillus subtilis 168 harboring plasmid pAB224 demonstrates significant survival as spores in mushroom compost across mesophilic and thermophilic conditions.
- Plasmid pAB224 is stably maintained in B. subtilis within the compost environment, even without selective pressure, making it a suitable marker.
- The distinct nature of pAB224 allows for easy differentiation from indigenous plasmids, supporting its use in environmental release studies of genetically modified bacteria.