Testing a proposed paradigm shift in analysis of phage DNA packaging

ABSTRACT

We argue that a paradigm shift is needed in the analysis of phage DNA packaging. We then test a prediction of the following paradigm shift-engendering hypothesis. The motor of phage DNA packaging has two cycles: (1) the well-known packaging ATPase-driven (type 1) cycle and (2) a proposed back-up, shell expansion/contraction-driven (type 2) cycle that reverses type 1 cycle stalls by expelling accidentally packaged non-DNA molecules. We test the prediction that increasing the cellular concentration of all macromolecules will cause packaging-active capsids to divert to states of hyper-expansion and contraction. We use a directed evolution-derived, 3-site phage T3 mutant, adapted to propagation in concentrated bacterial cytoplasm. We find this prediction correct while discovering novel T3 capsids previously obscure.

KEYWORDS:

• bacteriophage assembly
• capsid dynamics
• electron microscopy
• permeability-based fractionation
• ultracentrifugation

Abbreviations

C-CII	=	conventionally appearing capsid II
EM	=	electron microscopy
NHD	=	Nycodenz high density
NLD	=	Nycodenz low density

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Wen Jiang, Purdue University, for comments on a preliminary draft of this manuscript. We thank Dr. James J. Bull for providing access to the facility used for sequencing DNA, at The University of Texas at Austin. We thank Drs. Jeffrey Barrick and Borries Demeler for assistance with downloading and running breseq software.

Funding

This work was supported by the Welch Foundation (AQ-764). The Welch Foundation did not participate in the research design, writing and submission of this manuscript.