Brief Article
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World J Virol. Nov 12, 2013; 2(4): 152-159
Published online Nov 12, 2013. doi: 10.5501/wjv.v2.i4.152
Genetic analysis of structural proteins in the adsorption apparatus of bacteriophage epsilon 15
Jared A Guichard, Paula C Middleton, Michael R McConnell
Jared A Guichard, Paula C Middleton, Michael R McConnell, Department of Biology, Point Loma Nazarene University, San Diego, CA 92106, United States
Author contributions: McConnell MR designed and performed the genetic mapping and DNA sequencing experiments and also directed undergraduate researchers; Guichard JA and Middleton PC, in performing compositional analyses of particles produced by nonsense mutant viruses and the wt parent strain under non-permissive growth conditions; McConnell MR interpreted the resulting data and wrote the manuscript.
Supported by The NIH-AREA Grant, No. 1R15GM52696-01; the NSF-RUI Grant, No. DMB-8608480; the Howard Hughes Medical Research Institute (two grants to the PLNU Biology Department); Research Associates of PLNU (a 300 member alumni support group); and the PLNU Administration
Correspondence to: Michael R McConnell, PhD, Department of Biology, Point Loma Nazarene University, 3900 Lomaland Drive, San Diego, CA 92106, United States. mmcconne@pointloma.edu
Telephone: +1-619-8492304 Fax: +1-619-8492598
Received: June 30, 2013
Revised: September 20, 2013
Accepted: October 15, 2013
Published online: November 12, 2013
Processing time: 133 Days and 15.9 Hours
Abstract

AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15 (E15) using genetic and biochemical methodology

METHODS: Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under non-permissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins. These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of 35S-methionine and co-purified along with E15wt phage on CsCl block gradients.

RESULTS: Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive CsCl buoyant density centrifugation.

CONCLUSION: The portal ring (gp4) of E15 is bound to tail spikes (gp20) and the tail tube (gp15 and gp17); gp17’s attachment requires both gp15 and gp16.

Keywords: Epsilon15; Virion structure; Salmonella phages

Core tip: Epsilon 15 (E15) is a temperate, serotype-converting bacteriophage that specifically infects group E1 Salmonellae bacteria. This paper presents genetic and biochemical evidence regarding the identities and positional relationships of the proteins that comprise the tail tube structure of E15. As such, it makes a small contribution towards what may someday be a fuller understanding, not only of how E15 stabilizes its packaged DNA, but also, how it triggers release of its DNA when the phage encounters a susceptible Salmonella host cell.