1022-7954/00/3612- $25.00 © 2000
Russian Journal of Genetics, Vol. 36, No. 12, 2000, pp. 1454–1457. Translated from Genetika, Vol. 36, No. 12, 2000, pp. 1721–1724.
Original Russian Text Copyright © 2000 by Bidnenko, Akhverdyan, Krylov.
Intracellular development of the
transposable phage (TP) D3112 starts with
DNA replication, which is followed by the synthesis of
the proteins essential for the morphogenesis of the
phage particles. This synthesis depends on the activity
gene, whose product is capable of anti-termi-
nating activity. In this work, we show that TP D3112
contains an additional genome region (marked so far by
mutation, and deﬁned as the
which along with the
gene affects the phage protein
synthesis. The lack of this locus results in the decreased
levels of the late gene transcription.
We used the
PAO1 strain, kindly provided by B. W. Holloway
Bacteriophages of P. aeruginosa.
The phages used
were TP D3112
mutation is located in
gene, which is capable of controlling the repres-
sor and makes the prophage thermoinducible at 42
, which in addition to the
mutation carries the
mutation in the
gene, the pos-
itive regulator of the D3112 late genes transcription; TP
, which in addition to the
tion carries the
mutation ; and E79 virulent
phage, kindly provided by B. W. Holloway (Australia).
The following derivatives of the RP4::D3112 hybrid
plasmids carrying defective prophages (in brackets are
physical coordinates on the plasmid ends in the proph-
age part of the genome) were used: pDT18 (0–1.8);
pDT36 (0–14.5); and pKT31 (0–22.0) .
The methods of working with bacteriophages are
described in .
We used Hottingen agar and broth as com-
plete nutrient media. Minimal media used were M9
agar and broth .
Experiments on preparation of the
were carried out using M9 medium .
are described in  and .
Artiﬁcial lysis of the
cells was car-
ried out by use of lysozyme (ﬁnal concentration of
2 mg/ml) or the E79 virulent phage excess (infection
multiplicity = 100).
Induction of lysogens with thermoinducible proph-
ages was carried out as follows: night culture of
lysogenic bacteria was diluted from 50 to 100 times and
grown up to a concentration of (3–5)
C with aeration and agitation at 100 rpm using the
G76 (New Brunswick, United States) shaker. Next, the
samples were incubated upon active aeration at 42
for 90–120 min.
Isolation of total cellular DNA
is described in .
Transfer of the DNA samples onto nitrocellulose ﬁl-
ters, DNA–DNA hybridization, and preparation of the
P-DNA samples were conducted as in . Obtain-
ing preparations of
H-labeled RNA and RNA–DNA
hybridization were carried out according to  and .
The ts47 mutation does not prevent the lysis of bac-
terial cells after thermoinduction of the PAO1 (D3112
cts15 ts47) lysogen.
isolated by use of the method providing selection of
mutant phages by the early genes, i.e., the mutants
whose lysogenic derivatives were not subjected to lysis
during phage development after thermoinduction were
Analysis of the Phenotypic Effect of the
Transposable Phage D3112
on the Expression of Late Phage Genes
E. M. Bidnenko, V. Z. Akhverdyan, and V. N. Krylov
State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 113545 Russia
Received December 19, 1997; in ﬁnal form, May 4, 2000
—At nonpermissive temperature (42
mutation causes substantial abnormalities in the late
phase of the phage intracellular development. In these conditions DNA of the D3112 phage is detected both in
a free form and integrated into bacterial chromosome. The transcription kinetics in the
mutant at 42
indistinguishable from that typical to other early gene mutants (
): speciﬁcally, the preservation of
the ﬁrst transcription peak along with low activity of late transcription were observed. Similarly to the
mutation-carrying locus is involved in regulating the transcription of the D3112 transposable phage late
genes. It is suggested that the mechanism underlying the action of the
mutation differs from that of the
gene product. One of the possible explanations is based on the fact that the product of the
locus affects the
activity of cellular RNA polymerase via providing more effective recognition of the phage promoters by the
RNA polymerase modiﬁed with the phage protein C.