Isolation of chloroform-resistant mutants of filamentous phage: localization in models of phage structure

Abstract

Interaction of fd or M13 filamentous phage with a chloroform/water interface induces morphological change, contracting the filaments sequentially into shortened rods (I-forms), and then into spheroidal particles (S-forms). To further investigate this phage contraction, 34 and 26 chloroform-resistant isolates of fd and M13, respectively, were selected after chloroform treatment of wild-type phages at pH 8.2 and 4°C. DNA sequencing of gene VIII of the 34 fd isolates revealed five different mutants: these were D5H, M28L, V31L, I37T, and S50T. All 26 M13 isolates were I37T. These mutants exhibited variable sensitivity to chloroform, but all contracted much more slowly than wild-type phage during treatment at 4°C. They all contracted like wild-type phage at 37°C. Site-directed mutagenesis showed that the indicated single mutations carried the chloroform resistance. In structural models of the phage, the D5H locus is on the outside and the S50T locus is on the inside. The M28L and I37T loci are buried in a mostly hydrophobic region in the middle. Although these four mutants are spread out radially, they are localized in the axial direction into a thin disk in the model. The last mutant locus, V31L, is out of this disk, but this locus is proximal to the M28L and I37T loci and also in contact with the surface via a deep hydrophobic hole or depression. These five mutants, their locations, and their variable affects on contraction suggest that chloroform-induced contraction involves a specific mechanism rather than a generalized solvent-induced denaturation and that the critical structural changes occur in a localized level in the phage. These results add weight to suggestions that the sequential contraction of filaments → I-forms → S-forms mimic corresponding steps in phage penetration, and, in the reverse order, for phage assembly.