Js. Oh et al., Isolation of chloroform-resistant mutants of filamentous phage: Localization in models of phage structure, J MOL BIOL, 287(3), 1999, pp. 449-457
Interaction of fd or M13 filamentous phage with a chloroform/water interfac
e 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 treat
ment of wild-type phages at pH 8.2 and 4 degrees 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 e
xhibited variable sensitivity to chloroform, but all contracted much more s
lowly than wild-type phage during treatment at 4 degrees C. They all contra
cted like wild-type phage at 37 degrees 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 S50
T locus is on the inside. The M28L and I37T loci are buried in a mostly hyd
rophobic region in the middle. Although these four mutants are spread out r
adially, 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 solven
t-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 corre
sponding steps in phage penetration, and, in the reverse order, for phage a
ssembly. (C) 1999 Academic Press.