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Extending filamentous phage host range by the grafting of a heterologous receptor binding domain
R Marzari1, D Sblattero, M Righi
1International School for Advanced Studies (SISSA), Trieste, Italy.
Abstract:
fd and IKe are two similar filamentous phage which infect their hosts by means of pili found on the host membrane: fd infects bacteria bearing F pili, whereas IKe infects bacteria bearing N or I pili. Infection is mediated by the gene 3 protein (g3p), which of the nine proteins found in both phage is the most diverse. Previous attempts to incorporate g3p from one phage into the other by complementation have been unsuccessful [Bross et al. (1988) J. Gen. Microbiol. 134, 461-471]. Here we have grafted different parts of IKe g3p to the end of fd g3p and so augmented the host range of fd phage. We show that phage bearing such chimeric g3p are able to infect bacteria bearing both N and F pili providing they contain at least the receptor domain of IKe g3p, the infection of N bearing bacteria occurring at a level 70,000 times greater than background. This level of infection can be increased tenfold by including the glycine-rich domain as well. Addition of the penetration domain does not improve the level of infection above that of the receptor domain alone, indicating that the fd penetration domain is functional in the infection of bacteria bearing either N or F pili. Similarly derived fd phagemid also show increased infection of bacteria bearing N pili, albeit at much lower levels, suggesting that efficient infection requires more than one functional g3p on the surface of the phage.
Insights
Researchers engineered filamentous phage fd by grafting parts of phage IKe's gene 3 protein (g3p). This successfully expanded the host range of fd phage to infect bacteria with N pili, demonstrating the importance of specific g3p domains.
Area of Science:
- Microbiology
- Virology
- Molecular Biology
Background:
- Filamentous phages fd and IKe infect bacteria via host pili.
- Phage infection relies on the diverse gene 3 protein (g3p).
- Previous attempts to combine g3p functions between fd and IKe failed.
Purpose of the Study:
- To augment the host range of fd phage by modifying its gene 3 protein (g3p).
- To investigate the role of different domains of the IKe g3p in host range expansion.
Main Methods:
- Grafting different domains of IKe g3p onto the fd g3p.
- Testing the infectivity of engineered fd phage on bacteria with F and N pili.
- Analyzing the contribution of IKe g3p receptor and glycine-rich domains.
Main Results:
- Chimeric fd phage with IKe g3p receptor domain infected N-pili bacteria 70,000 times greater than background.
- Including the glycine-rich domain increased infection levels tenfold.
- The fd penetration domain was functional for both N and F pili infection.
Conclusions:
- The receptor domain of IKe g3p is crucial for expanding fd phage host range to N-pili bacteria.
- Efficient phage infection may require multiple functional g3p molecules on the phage surface.
- Engineering phage g3p offers a viable strategy for altering phage host specificity.