Phage I3vir is a sheath-tailed phage (Prozesky, de Klerk & Coetzee, I965, P1. I, fig. 5) which arises from single clones of Proteus mirabilis strain 13 (Coetzee ...
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J. gen. Virol. 0 9 7 o ) , 9 , 2 4 7 - 2 4 9 Printed in Great Britain
Properties of Proteus mirabilis phage 13vir (Accepted 7 September 197o) Phage I3vir is a sheath-tailed phage (Prozesky, de Klerk & Coetzee, I965, P1. I, fig. 5) which arises from single clones of Proteus mirabilis strain 13 (Coetzee & Sacks, I96o). It forms large clear plaques on strain 13. The phage may carry a constitutive mutation in an operator gene which controls development of the prophage in strain 13. It is not inducible with ultraviolet light or mitomycin C but is often present in overnight broth cultures of strain 13 . The phage is particularly troublesome in that strain 13 is the host of transducing phage 34 (Coetzee & Sacks, 196o ) and lysates of the latter are usually contaminated with phage I 3vir. Table
1. Fate of ~5 I3vir. 13 DNA labelled with a2p after infection of Proteus mirabilis hosts Host strains
Experimental conditions Phage e.o.p. M.o.i.
Adsorption i5 min. (~) Cells lysed with sodium lauryl sulphate. Treated with cold 2 ~o perchloric acid: Adsorption mixture o min. (counts/min.) Insoluble 32p after 20 min. (counts/min.) Soluble 8zp after 2o min. (counts/min.) Perehloric acid soluble asp (9/o)
I3
5006
I "0 2'0
IO -9 I "2
99"9
99'9
I23087 77052 46035 8'4
I55646 27328 I28318 48'6
Overnight broth cultures were centrifuged and starved by suspending in a equal volume of o.ol M-MgSO4 for 2 hr. Two ml. of the suspension were then infected at a final m.o.i, of about 2 with purified 3~P-labelled phage and total radioactivity measured. After adsorption for 15 min. incubation mixtures were diluted threefold in prewarmed broth and incubation with aeration continued for a further 2o min. Samples were then lysed with sodium lauryl sulphate for 2 min. at 7o° followed by treatment with cold perchloric acid. When high titre ~ 13vir. 13 (prepared by infecting a broth culture of strain 13) is spotted on a lawn of a locally isolated P. mirabilis strain 5oo6 the bacteria are killed but serial dilutions of the phage only sporadically form plaques. This latter phenomenon was investigated by methods previously used to study a system of abortive infection (Coetzee & Smit, 1969). More than 99 ~ of ~5 I3vir. 13 adsorbed to strain 5oo6 within IO min, at 37 °. Within 15 min. of adsorption to strain 5oo6 about 48 ~ of ~ 13vir. 13 D N A was extractable in acid-soluble form (Table I). The 8 ~ breakdown of D N A in the productive host 13 is slightly higher than that encountered for ~ 34. I3 in the same host (Coetzee & Smit, i969). These results indicate that ~ I3vir. 13 D N A penetrates strain 5oo6 but is rapidly degraded. Transmission experiments (Table 2) showed that only 3"8 x lO .5 of cells of strain 5oo6 infected with ~ I3vir. 13 yielded phage. This phage formed plaques on strain 13 but not on strain 5oo6; thus, phage 13 is restricted in strain 5oo6 and the infecting particles which escape restriction do not receive the specificity of this strain. Only 2.6 x io -~ of infected cells yielded phage which plated on strain 5oo6. This phage is thought to result from a mutation in phage I3vir which is concerned with the processes of restriction and modification of strain 5oo6 and is named I7-2
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I3vir.5oo6. It plates with equal efficiency on both strains and is neutralized at the same rate as ~ I3vir. 13 by an antiserum against the latter. Phage I3vir. 13 can transduce various auxotrophs of strain 5oo6 to prototrophy at rates of about 4 × IO-S/P .f.u. adsorbed. The optimum m.o.i, of phage is about 5. Many transductant colonies are nibbled due to liberation of phage 13 vir. 5oo6. Nibbling can be overcome by addition of phage I3vir antiserum to transduction mixtures at the end of the adsorption period. It is possible to transduce an arginine-requiring auxotroph of strain 5oo6 to streptomycin resistance and prototrophy in successive experiments with a ~b ~3vir. I3str-r lysate. T a b l e 2.
M.o.i.
Adsorbed phage (~)
0.8
996
Single cycle growth ~ I3vir. I3 in strain Free phage after antiserum
