Assembly of Bacillus subtilis Phage Phi29. 2. Mutants in the Cistrons Coding for the Non-structural Proteins

Abstract

The effect on phage morphogenesis of sus mutations in the cistrons coding for nonstructural proteins has been studied. Mutants in three cistrons analyzed that are involved in phage DNA synthesis, as well as in cistron 16 which codes for a late nonstructural protein, produce prolate capsids which are more rounded at the corners than complete phage heads and have an internal core; they contain the head proteins, the upper collar protein and protein p7, not present in mature phage particles. Mutants in cistron 7 do not produce capsids nor other phage-related structures; this result and the presence of p7 in phage capsids suggest an essential role in capsid assembly for this protein. The protein product of cistron 13 is probably needed for a stable DNA encapsulation since mutants in this cistron produce mainly DNA-free complete phage particles and only about 10 % of uninfective DNA-containing complete phage. Cistron 15 codes for a late, partially dispensable, nonstructural protein which is present in the DNA-free capsids produced after infection with the delayed-lysis mutant sus14(1242), used as the wild-type control, or with mutants in cistrons 9, 11, 12 and 13. Proteins pl5 and p16 are probably involved in the encapsulation of viral DNA in a prohead.It is now well established that DNA encapsulation in several bacteriophages takes place on a preformed head, named prohead, containing some electron-dense internal material which is released when the DNA is encapsulated [I -61.The results presented in the preceding paper suggest that in the Bacillussubtilis phage @29 a structure similar to a prohead is produced after infection with a delayed lysis mutant which behaves like wild-type phage, or with several mutants in cistrons coding for phage structural proteins. In this paper we present evidence on the accumulation of proheadlike structures after restrictive infection with sus mutants in several cistrons involved in phage DNA synthesis and in cistron 16, coding for a late, nonstructural protein (see Fig. I of the preceding paper).Sus mutants in cistron 7, also coding for a late, nonstructural protein, p7, do not produce any kind of phage-related structure suggesting a role for this protein in head morphogenesis. Protein p7 is present in all prohead-like structures and is absent from all DNA-containing heads, suggesting that this protein is released from the phage heads prior to or simultaneously with the encapsulation of DNA. The effect on phage morphogenesis of mutations in cistrons coding for other nonstructural proteins is also de6nzymes Lysozyme or mucopeptide N-acet ylmuramylhydrolase (EC 3 2 1 17), pancreatic ribonudease (EC 3 1 4 23), pancreatic deoxyribonuclease (EC 3 1 4 5) -scribed in this paper. A preliminary account of some of these results has already been given [7].Taking into account the results presented in this and in the preceding paper, a preliminary morphogenetic route for phage @29 assembly is proposed. MATERIALS AND METHODS Bacteria, Phage and MediaThe nonpermissive host, B. subtilis 110NA try--s...

φ29 Family of Phages

“…Nonsuppressor cells infected with this mutant phage produced a reduced phage yield and viral DNA synthesis (50,120,153). In addition, infection with the sus17(112) 29 mutant in solid media gave only very tiny plaques (138).…”

“…Thus, repression of the A2c promoter occurs by overstabilization of the RNA polymerase to this promoter (148). Interestingly, both repression at the A2c promoter and activation of the A3 promoter involve interaction between a region of protein p4 containing Arg 120 and the C-terminal domain of the RNA polymerase ␣ subunit (140-143, 150, 151, 171).…”

“…After packaging, the ATPase protein p16 and the pRNA molecules are also released from the prohead (94) by an unknown mechanism. Next, 6 copies of the lower collar (p11), 3 or 4 copies of the tail knob (p9), and 12 appendages (dimers of protein p12* cleaved from p12 precursor molecules) are assembled sequentially onto a stable, DNA-filled head (45,79,120,168; for a review, see reference 9) (Fig. 1).…”

In vitro assembly of the Bacillus subtilis bacteriophage phi 29.

“…Provisional pathways of 029 morphogenesis have been published (2,8,9). Proheads, the first particle detected, accumulate in the absence of the protein gpI6 (gp, gene product) and are composed of the major head protein (gp8), the head fiber protein (gp8.5), the neck upper collar protein (gplO), and the recyclable "scaffolding-core" protein (gp7); gp7 may catalyze ordered aggregation of the major head protein and be exchanged for the DNA-protein complex during DNA encapsidation.…”

Purification, Properties and Assembly of the Neck‐Appendage Protein of the Bacillus subtilis Phage φ 29

“…All the mutants contained an additional mutation in gene 14, sus14(1242), which produces a delay in the lysis of the infected bacteria [151. Mutant susl4 (1242) was used as wild-type phage [9]. The nomenclature used for the 429 mutants was that adopted by Mellado et al [16].…”