Bacteriophage infection The poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsAGFP. ManY, an E. coli protein that is required for phage lambda DNA injection, was found to localize the bacterial poles as well. Furthermore, labelling of lambda DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to lambda's developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation. A phage display system to study protein-protein interactions Analyzing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2lambda) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro . Bait and prey are displayed as fusions to the surface of phage lambda that are marked with different selectable drug resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)(4) and (Gly-Arg)(4) peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library. The unique usefulness to 2lambda is also described. Bacterial detection by the use of QD-nanocomplexes With current concerns of antibiotic-resistant bacteria and biodefense, it has become important to rapidly identify infectious bacteria. Traditional technologies involving isolation and amplification of the pathogenic bacteria are time-consuming. We report a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides specific detection of as few as 10 bacterial cells per milliliter in experimental samples, with an approximately 100-fold amplification of the signal over background in 1 h. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium, or are highly infectious, e.g., Bacillus anthracis. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are discussed.
| Bair, Catherine L; Oppenheim, Amos; Trostel, Andrei et al. (2008) A phage display system designed to detect and study protein-protein interactions. Mol Microbiol 67:719-28 |
| Edgar, Rotem; McKinstry, Michael; Hwang, Jeeseong et al. (2006) High-sensitivity bacterial detection using biotin-tagged phage and quantum-dot nanocomplexes. Proc Natl Acad Sci U S A 103:4841-5 |
| Rao, Srinivas; Hu, Stella; McHugh, Louise et al. (2005) Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide. Proc Natl Acad Sci U S A 102:11993-8 |
| Scholl, Dean; Adhya, Sankar; Merril, Carl (2005) Escherichia coli K1's capsule is a barrier to bacteriophage T7. Appl Environ Microbiol 71:4872-4 |
| Vitiello, Christal L; Merril, Carl R; Adhya, Sankar (2005) An amino acid substitution in a capsid protein enhances phage survival in mouse circulatory system more than a 1000-fold. Virus Res 114:101-3 |
| Adhya, Sankar; Black, Lindsay; Friedman, David et al. (2005) 2004 ASM Conference on the New Phage Biology: the 'Phage Summit'. Mol Microbiol 55:1300-14 |
| Scholl, D; Kieleczawa, J; Kemp, P et al. (2004) Genomic analysis of bacteriophages SP6 and K1-5, an estranged subgroup of the T7 supergroup. J Mol Biol 335:1151-71 |
| Merril, Carl R; Scholl, Dean; Adhya, Sankar L (2003) The prospect for bacteriophage therapy in Western medicine. Nat Rev Drug Discov 2:489-97 |
| Gupta, Amita; Onda, Masanori; Pastan, Ira et al. (2003) High-density functional display of proteins on bacteriophage lambda. J Mol Biol 334:241-54 |
