Length quantization of DNA partially expelled from heads of a bacteriophage T3 mutant

Abstract

DNA packaging of phages phi29, T3 and T7 sometimes produces incompletely packaged DNA with quantized lengths, based on gel electrophoretic band formation. We discover here a packaging ATPase-free, in vitro model for packaged DNA length quantization. We use directed evolution to isolate a five-site T3 point mutant that hyper-produces tail-free capsids with mature DNA (heads). Three tail gene mutations, but no head gene mutations, are present. A variable-length DNA segment leaks from some mutant heads, based on DNase I-protection assay and electron microscopy. The protected DNA segment has quantized lengths, based on restriction endonuclease analysis: six sharp bands of DNA missing 3.7–12.3% of the last end packaged. Native gel electrophoresis confirms quantized DNA expulsion and, after removal of external DNA, provides evidence that capsid radius is the quantization-ruler. Capsid-based DNA length quantization possibly evolved via selection for stalling that provides time for feedback control during DNA packaging and injection.

Testing a proposed paradigm shift in analysis of phage DNA packaging

“…The sucrose shrank E. coli 16 (The normal internal osmotic pressure of E. coli B is over 3x lower 17 ) and increased intracellular concentration of osmolytes, RNAs and proteins 18 . This T3 adaptation produced three mutations, based on outsourced whole genome Illumina sequencing, 19 with sequence changes determined via breseq software 20 : (1) gene 2.5 single-stranded DNA binding protein, A16T (GCT: ACT); (2) gene 12 tail protein, T514A (ACT: GCT) and (3) gene 16 internal core stack/cylinder protein, P613L (CCA: CTA). The mutant, called T3 Su-1 , had latent period of 3.5–4.0 hr in 0.94 M sucrose-supplemented medium and 1.1–1.3 hr in medium without sucrose.…”

“…We then purified (1) T3 Su-1 capsid I (procapsid; Fig. 1a), (2) DNA-detached capsid II, and (3) T3 Su-1 phage, by centrifugation in cesium chloride density gradients., 19,21 Most wild type capsid II has previously been found to have detached from DNA, post lysis. DNA-detached capsid II was produced with kinetics of a phage precursor 22 …”

“…Per ml of lysate, 1.0 × 10 10 phage particles were produced, 1% infective, in contrast to 30–40% infective wild type phage. T3 Su-1 phage particles were quantified by optical density at 260 nm 23 after purification in cesium chloride density gradients 19,21 . The T3 Su-1 capsid I had the radius of wild type capsid I ± 4%, as determined by native gel electrophoretic sieving (technique of reference 19; data not shown).…”

Three-step channel conformational changes common to DNA packaging motors of bacterial viruses T3, T4, SPP1, and Phi29

“…The finding of the common discrete 3-step conformational change in T3, T4, SPP1 and phi29 implies a universal property of all portals. It is possible that the three gating steps may also correspond to the quantized steps of partial genome ejection observed in T3 (Serwer et al, 2014), and the partial packaging intermediates observed in phi29 (Bjornsti et al, 1983). …”

31 Genetic and electrostatic maps of bacteriophage lambda genome

“…The mature capsid of a tail-free phage T3 head (30 nm in radius) contracts when it partially expels DNA in quantized lengths (Serwer et al, 2014). In the current study, we use (1) genetics via directed evolution and (2) hydration-based buoyant density centrifugation to isolate a previously obscure T3 intermediate that has incompletely packaged DNA and a capsid with a Nycodenz-and negative stain (PTA)-impermeable shell that varies in appearance.…”