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Published on: July 11, 2013
Molecular cloning, sequencing, and expression of two late proteins of bacteriophage MB78
V Kolla1, P Datta, M Chakravorty
1Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
Abstract:
Bacteriophage MB78, a virulent phage of Salmonella typhimurium, does not allow other phages, such as P22 and 9NA, to grow in its presence. A detailed physical map of this phage has been constructed in our laboratory. In an ongoing effort to understand the genetics of this interesting phage, various genes were characterized. Here, we report cloning, sequencing, and expression of two late proteins, coded in a SalI-HindIII fragment (SH9), by using the minicell expression system. Further, we performed a kinetic study of phage proteins by infection the host LT2 cells and compared the proteins produced, with proteins obtained by the minicell expression system. Both sets of proteins run exactly parallel and migrated as 14- and 15-kDa proteins on a polyacrylamide gel. The synthesis of these two proteins started 15 min after infection with MB78 and was prominent after 45 min. One of the proteins exhibited 57% homology to the structural protein of mycobacteriophage L5.
Insights
Bacteriophage MB78 inhibits other phages. Researchers cloned and expressed two MB78 late proteins, identifying them as 14- and 15-kDa, with one showing homology to a mycobacteriophage L5 protein.
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- Bacteriophage MB78 is a virulent phage of Salmonella typhimurium.
- MB78 exhibits interference with other phages like P22 and 9NA.
- A physical map of MB78 has been previously established.
Purpose of the Study:
- To characterize genes of bacteriophage MB78.
- To clone, sequence, and express two late proteins encoded by the SH9 fragment.
- To analyze the kinetics and homology of these proteins.
Main Methods:
- Minicell expression system for protein production.
- DNA sequencing of the SalI-HindIII (SH9) fragment.
- Host cell infection (Salmonella typhimurium LT2) for kinetic studies.
- Polyacrylamide gel electrophoresis for protein analysis.
Main Results:
- Two late proteins of 14-kDa and 15-kDa were successfully cloned, sequenced, and expressed.
- Protein synthesis initiated 15 minutes post-infection and was prominent by 45 minutes.
- One identified protein showed 57% homology to a structural protein of mycobacteriophage L5.
Conclusions:
- The SH9 fragment encodes two MB78 late proteins.
- These proteins are synthesized late in the phage infection cycle.
- The homology suggests potential functional or evolutionary links between MB78 and mycobacteriophage L5.
