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A high yielding mutant of mycobacteriophage L1 and its application as a diagnostic tool
S Chatterjee1, M Mitra, S K Das Gupta
1Department of Microbiology, Bose Institute (Centenary Building), P1/12 C.I.T. Scheme VIIM, 700054, Calcutta, India.
Abstract:
L1 is a lysogenic phage of mycobacteria, which along with L5 and D29 constitute a closely linked family of homoimmune mycobacteriophages. These phages can be potentially used for genetic engineering of mycobacteria and diagnosis of mycobacterial infection. The effectiveness of such phage based systems depends on the efficiency with which they infect and grow within target cells. While working with phage L1c1ts which is a temperature sensitive mutant of phage L1, we observed that high yielding phage stocks were generated by repeated passage through the host, Mycobacterium smegmatis. A plaque purified mutant L1-P2, obtained from one such high yielding stock, when analyzed further was found to infect host cells with increased efficiency. The DNA obtained from L1-P2 was examined by restriction digestion, and it was observed that spontaneous loss of DNA fragment from the right arm, which encodes early regulatory factors, had occurred. It has been further demonstrated that the high yielding property of the mutant phage could be utilized to increase the sensitivity of mycobacteriophage-based detection systems.
Insights
A mutant mycobacteriophage, L1-P2, shows increased infection efficiency in Mycobacterium smegmatis. This high-yielding phage property can enhance mycobacteriophage-based detection systems for improved sensitivity.
Area of Science:
- Microbiology
- Virology
- Molecular Biology
Background:
- Mycobacteriophages, including L1, L5, and D29, are closely related and can be used for genetic engineering and diagnosing mycobacterial infections.
- The efficacy of phage-based systems relies on efficient infection and replication within host cells.
Purpose of the Study:
- To investigate the increased phage yield observed during serial passage of a temperature-sensitive mutant of L1 phage (L1c1ts) in Mycobacterium smegmatis.
- To characterize a high-yielding mutant (L1-P2) derived from this process and assess its potential for improving phage-based detection systems.
Main Methods:
- Serial passage of phage L1c1ts in Mycobacterium smegmatis to generate high-yielding stocks.
- Isolation and plaque purification of a high-yielding mutant (L1-P2).
- Analysis of L1-P2 DNA using restriction digestion to identify genetic modifications.
Main Results:
- Repeated passage of L1c1ts in M. smegmatis resulted in high-yielding phage stocks.
- The plaque-purified mutant L1-P2 exhibited enhanced infection efficiency in host cells.
- Restriction digestion revealed a spontaneous deletion of a DNA fragment from the right arm of L1-P2, which encodes early regulatory factors.
Conclusions:
- The L1-P2 mutant demonstrates improved phage yield and infection efficiency due to a specific DNA deletion.
- The enhanced high-yielding property of L1-P2 can be leveraged to increase the sensitivity of mycobacteriophage-based diagnostic and detection systems.