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Transfection in Micromonospora spp
J L Caso1, C Hardisson, J E Suárez
1Departamento de Biología Funcional, Universidad de Oviedo, Spain.
Abstract:
The introduction of bacteriophage DNA into Micromonospora protoplasts, resulting in the production of infective viral progeny, is reported. Transfection was affected by several factors. We observed that it reached a maximum when protoplasts from young mycelium (15 h old) were used. Maximum transfection took place when polyethylene glycol (PEG) was added to the mixtures at a final concentration of 20% (vol/vol) and did not occur at PEG concentrations under 10% or over 35%. The addition of positively charged liposomes to the mixtures was essential, since no transfectants were detected in the absence of liposomes at any PEG concentration. When DNA was present in nonlimiting amounts, a maximum efficiency of around 10(-3) to 10(-4) PFU per protoplast was obtained. The efficiency per DNA molecule showed a constant value of around 10(-4) to 10(-5) PFU, but the data suggest that transfection could be achieved by a single DNA molecule. The method proved to be equally efficient for the DNAs of at least five Micromonospora bacteriophages. On the contrary, we failed to transfect five of seven Micromonospora strains. These data suggest that only a minor subpopulation of protoplasts is competent and that the main factors influencing the transfection of Micromonospora protoplasts are neither the characteristics nor the origin of the DNA but the properties and status of the protoplasts.
Insights
Bacteriophage DNA transfection into Micromonospora protoplasts was optimized using specific polyethylene glycol (PEG) concentrations and liposomes. Protoplast properties, not DNA, are key for successful viral progeny production.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Micromonospora are important antibiotic-producing bacteria.
- Bacteriophages are viruses that infect bacteria and can be used as tools in molecular biology.
- Efficiently introducing foreign DNA into Micromonospora protoplasts is crucial for genetic manipulation and bacteriophage research.
Purpose of the Study:
- To report the successful introduction of bacteriophage DNA into Micromonospora protoplasts.
- To identify factors influencing the efficiency of this transfection process.
- To understand the limitations and potential of this method for genetic studies.
Main Methods:
- Protoplast isolation from Micromonospora mycelium.
- Treatment with bacteriophage DNA in the presence of polyethylene glycol (PEG) and liposomes.
- Optimization of PEG concentration and protoplast age.
- Assessment of infective viral progeny production (PFU).
Main Results:
- Transfection efficiency was maximized with 15-hour-old mycelium and 20% PEG.
- Positively charged liposomes were essential for successful transfection.
- Maximum efficiency reached 10(-3) to 10(-4) PFU per protoplast.
- The method worked for multiple bacteriophage DNAs but only with a subset of Micromonospora strains.
Conclusions:
- Micromonospora protoplast competence, rather than DNA characteristics, is the primary determinant of transfection success.
- A small subpopulation of protoplasts appears to be competent for DNA uptake.
- This method provides a foundation for further genetic studies of Micromonospora and their bacteriophages.