195
J. gen. Virol. 0979), 45, I95-2oo
Printed in Great Britain
Pilus-specific, Lipid-containing Bacteriophages PR4 and PR772:
Comparison of Physical Characteristics of Genomes
By W. F. C O E T Z E E AND P. J. B E K K E R
M R C Unit.for Microbial Genetics, University of Pretoria, PO Box 2034, Pretoria,
Republic of South Africa
(Accepted 3 May 1979)
SUMMARY
The genomes of pilus-specific, lipid-containing phages PR4 and PR772 were
studied electron microscopically. An identical mol. wt. of IO-9× IO~ was obtained.
The genomes are unique (non-permuted) and have cohesive ends. From the
similarities in size and denaturation maps of the genomes and failure to demonstrate non-homology in heteroduplexes, reported morphological ambiguities
were clarified. The known serological difference between the phages could not be
related to non-homology of their genomes. It is concluded that phages PR4 and
PR772 are the same phage.
INTRODUCTION
Pilus-specific phages PR3, PR4 (Stanisich, 1974; Bradley & Rutherford, 1975) and PR772
(Coetzee et al. I979) have recently been studied extensively. Phages PR3 and PR 4 have been
compared with respect to their morphology, buoyant density, sensitivity to chloroform,
inactivation by antiserum, adsorption to host cells, host range, growth on infected bacteria,
efficiency of plating and plaque morphology. The only difference observed was in their
growth characteristics, reflected in plaque morphology. Hence they can be viewed as plaquetype mutants of the same virus (Bradley & Rutherford, I975). Attachment of PR 4 to pili
specified by plasmids of the P-I, N and W incompatibility groups has been demonstrated
(Bradley & Rutherford, I975; Bradley, I976; Bradley & Cohen, I977).
Following the isolation of phage PR772 on Proteus mirabilis strain PMsoo6 nal-r(R772),
this plasmic-specific phage was compared with phage PR4. The phages proved to be
identical in the above respects, except for differences in morphology and inactivation by
antiserum (Coetzee et al. I979). Although they are hexagonal and exhibit a thick inner
layer, presumably lipid, the diam. of PR4 has been reported as 65 nm v. 53±3 nm for that
of PR772. Phage PR4 reportedly has a tail of variable length whereas PR772 has no tail.
Antisera produced with phage PR772 in three rabbits showed inactivation constants (K)
of 6"76, 4"86 and 8.32 per min and when used against PR4, K-values of 0.68, 0.20 and 0"34
per min, respectively, were obtained. The difference in K-value is consistently one order in
magnitude (Coetzee et al. I979).
In order to elucidate the above ambiguities, the genomes of phages PR4 and PR772 were
studied electron microscopically. We report here on mol. wt., homoduplex, heteroduplex
and denaturation mapping analyses.
METHODS
General. Escherichia coli J62-I (R772) was used as host for phages PR4 and PR772, all
from the laboratory stocks of this Unit. The methods described by Adams (I956) and
Coetzee (I974) were used.
oo22-I317/79/oooo-3686 $o2.oo~)
I979 SGM
Downloaded
from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 00:51:00
I96
W. F. C O E T Z E E A N D P. J. B E K K E R
Purification of phage and nucleic acid extraction. These were done as described by Coetzee
et al. 0979) and Olsen et al. (I974).
DNA microscopy. The methods of Coetzee & Pretorius (t979), involving benzylalkyldimethylammonium chloride (BAC) were used. Molecules were photographed at an instrumental magnification of 68oo, by means of a Philips EM 300 electron microscope.
Data processing. Photographic negatives were projected in a rear-view fashion and were
traced by means of an Electronic Graphics Calculator (Numonics Corporation, Lansdale,
Pennsylvania) interfaced with a Hewlett Packard 9825A calculator. Computer simulations
of molecules were plotted and integrated to yield denaturation maps.
Mol. wt. determinations. These were done electron microscopically relative to the length
04-8/zm) of the Proteus mirabilis phage 5006 M genome of mol. wt. 30"7 IO6 (Pretorius
& Coetzee, 1979).
RESULTS
Electron microscopic appearance of genomes
Linear and circular forms were observed. In both cases multiple lengths of an apparent
monomer were encountered. Incubation at 55 °C for Io min in the presence of 5 ~ formaldehyde converted all circles and concatenates to the linear monomeric length. This
suggests that both genomes are unique and have cohesive ends. Examples of such genomes
are known, e.g. A (Davidson & Szybalski, I97I) and the lambdoid phages 2I, 80, 82, 424
and 434 (Yamagishi et al. 1965; Baldwin et al. 1966; Kaiser & Wu, 1968).
Mol. wt. of genomes
The lengths of PR4 molecules were measured relative to the length of the phage 5006 M
genome on the same photographic plates. The average value for the length ratio PR 4: 5006 M
was found to be 0.35-+-0.02 (s.d.). Assuming the mol. wt. of phage 5006 M to be 30"7 × Io 6
(Pretorius & Coetzee, I979) the mol. wt. of phage PR4 is Io'9× IOe. Similar measurements on PR772 genomes yielded values of o - 3 5 i o . o i (s.d.) and 1o"9 × Io e, respectively.
There is no significant difference in moi. wt. between the phage genomes.
Partial denaturation
Partially denatured molecules of PR4 and PR772 DNA were monomeric. Computer
simulations of such molecules resulted in Fig. I(a), z(a), respectively. Sixty molecules of
each phage facilitated construction of denaturation maps (Fig I b, zb).
These results confirm the uniqueness of both phage genomes. The similarity of the denaturation maps (Fig. I b, 2b) is evident. They do not reveal any significant difference in
broad characteristics between the genomes of phages PR4 and PR772, as minor discrepancies may be attributed to a difference in the average extent of denaturation (22 ~o for
PR4 and 3I ~o for PR772 ).
Duplex analyses
Homoduplex preparations of PR4 and PR772 DNA contained high yields of double
stranded molecules. These were indistinguishable from the untreated preparations and this
supports the above suggestions as to the uniqueness of the genomes.
Heteroduplexes of PR 4 and PR772 D N A molecules likewise yielded double-stranded
molecules. These were scrutinized for regions of non-homology but none was observed.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 00:51:00
Comparison of PR4 and PR772 genomes
I97
n
M
,,j
n
I
.
[
~
.
.
.
M
wmr'1
i_
rl
M
i'm
.
,,._....:,
.
r'9
a-m
Pm~m
:. .
MI
n
m-m
,
q
n
n
__
.
i-I
M
ri
~m
N_
[
|
[
M
fq
m,q
.
.
M
rl
.
~ql
n
n
IMI
M
M
M
M
|
.
lll,m rl _
I ~
r'm'm
i,--q ~
.
m'n'm
19
Ig'9
Ilm
re"
M
I"ml ~m _
.
M
~m,~
M
.
I~
R
.l---m
I ¢ , - i i=,R
m=~.
M
.~i
n
M
I
.
l~m
=
l'm
I,M.
•
M
M
l
.
I ~
r"l~
-m
,-,
n
~
rl
M
~
|il
•
ill.
"
n r
~-'1'
m~l
~
n
rm
n
•
r-v
•
Ill
rl
r-i
•
l
.
I
~
M
= i,m,,!
9
n
nJ
rl
r'!
1"9
~1
n
,,
M
M
l
I
rl
M I
•'
I
n
M
i-1
rlM
mlmM
j
r'll
~I
~
'-A
nM
M
n
Ill
rl
m
- ~
I
I'!
H
m'z
.
h
1
M
f--ll
rl
.rm
i~
m.m-m
$-m
f"l'm
IM
•
M
"
~
rl
.
m-.~ m=m
e'~
~e--,-m M
~-'I
.M
rll
rl
I m,l.~
h
.
l
.
If~
M
,-q
n
l~.m
°d
M
M
,----,-
n l
M
n
r ~
"
..q
n
n
r-'-i
M
l .
.
I~
i
.
.
.
~
~
mr"l
19
M
IPm
|'q-
l
l
--
l
.
~
~
II"
.
rl'l'l
Im=~m
.
M
~
I
r=-I
l
M
I
I'll
n
M
I
',--I
M
M
M
M
M
M
M
~'~1
~
M
=
n
•
n
n
I
M
M
n
n
(a)
(b)
50
40
"5
o
E 30
j
C~
Z 20;
lOi
I
0-1
i
|
l
0"2
0"3
0"4
Fractional
|
0'5
genome
|
0-6
|
0.7
|
0"8
|
0"9
1"0
length
Fig. z. (a) Computer simulations of 60 partially denatured PR4 D N A molecules, (b) denaturation
map deduced from the data in (a).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 00:51:00
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 00:51:00
•(~) u! ~l~poql tuotj po3npop dCeu
u o ! l ~ m l e u o p (q) 'soln3OlOm V N G "~LL~Id po~nl~uap ,~ll~p~d o9 jo suop~lntu!s Jomdt.uo~) (v) "~ '$!A
O.l
6.0
I
~-0
L.0
I
t.=,
t=J
!
!
ql~uaI amou~ l~UO.tl3~l
9.0
S.0
17-0
I
I
E-0
I
E-0
I
[.0
I
~
- 01
-o~
oz
o
(q) -
.•
t.__.i
ii
I
I
i
~
~
I
I /
I..,
i
i
I
I I
l
U
! I..I
L=_J
i
I I
l.=,
. -. u.
.
i
i
|
|
I
I
i
11~,
~
I.~.,
I
.-
~
i
I ~
i
I
~
n
.
I
I
L~I
,
t.....,
.
t
i.J
,
,
,
!
,
,'-' ,.
,i
I
I
!f
I
l...I
~
,
t._..J
I--,
!
I
I.--I
~
|
I
~
I
~.l
i
L.--J
I--I
I
¢------=~
i
!
|
I
,
l i l
~
I
I
I
!
I
|
I
ai
L-I
I.~
I
I
I
it
U
. . . .
l..l
I
t
I
I
U
L__I
I--I
t
I
,
i
,
I
~
' - ~
.
U
,
I--I
L_=,
U l
l.J
I
e.=..,
. ,...,
.
I--I
~
I..I
~
I
~
i
~
h.,
.~
L.I
I--I
t..I ~
I L...=I
t.i
~,,%
~
~
,
m
Lil
~
l
l I
I
I
I
I
I
!
I
tl
I ~
l...--I
!
,5-',
U
,~
i
U
,'-:
l
i
I.J
i
II
L3
,~
,
e..,
tl
I.
IL--.J ,-J
,,.,
|
'
,
~"~'i_s
,==,
II
n
•
u
u
~
•
I
l'J
I.J
u
I
. ",~
I-II
l
i
,
II
~
i t
,
I
b
L...J
LI
I.l
n
, ~ ' .'-l
~
n
I
|
t~--~
II----ll
, . = . . 1l - - ' l ~ J
S
t...l
i J
I.
!
,
i
i i
~
~
,i
U
sl
i
LJ
|
.
i
I=I
,
#
i
U
I-,
1--J
i
~
i
I
I
i
!
i
,
~
,
I
I
,
~ , . J
b
i
I
,..,
~
~'
•
:.
L....
0
•
I
I-.J
u3)I~38 "f "d CINV H3Z±303 ":1 "A~
~6I
Comparison o f P R 4 and PR772 genomes
I99
DISCUSSION
The above results indicate that the genomes of phages PR4 and PR772 are unique, that
they have cohesive ends and identical mol. wt. Within the limits of electron microscopic
detectability, non-homology between the phage genomes could not be demonstrated.
It has been shown (Vollenweider et al. I975) that when the benzylalkyldimethylammonium
chloride monolayer technique of the present experiments is used, denaturation can be
detected during unstacking of base pairs, prior to physical strand separation. This emphasizes the high resolution that can be expected in partial denaturation and heteroduplex
experiments. Non-homology can be resolved to about 2o0 base pairs (unpublished observations). Consequently, if the reported morphological and serological differences between
phages PR4 and PR772 are related to differences in their respective genomes, such differences have to be small. We suggest that as for morphology, this is not likely.
Firstly, the discrepancy in head size, 65 nm for PR 4 v. 53 nm for PR772, manifests itself
in the outer layer, which consists of protein (Bradley & Rutherford, I975; Coetzee et aL
I979). In view of the similarity in genome size and phage buoyant density (Coetzee et al.
I979) it follows that the difference in diam. necessitates an almost twofold difference in
phage mass. This has to be accounted for by proteins in the capsid. However, the geometric
and physico-chemical limitations on the number and character of the constituents of
icosahedrally symmetrical phage heads would necessitate the presence of proteins in the
large capsid that differ from those in the small capsid (Caspar & Klug, t962). Conceivably
the genetic sequence for such proteins would exceed 2o0 base pairs, which would have been
detectable in our heteroduplex experiments. We conclude that the reported size difference
between these phages should be viewed with caution.
Secondly, it is well known that a number of phage gene products are required for tail
synthesis and assembly. These genes may occupy a considerable portion of the phage
genome, e.g. phages//(Szybalski & Herzkowitz, I97I), T2, T4 and T6 (Kim & Davidson,
7974). The present findings on size and sequence homology of the genomes of phages PR 4
and PR772 lead to the conclusion that no difference exists between these phages as for the
existence of tails. However, in view of the relatively small size of their genomes, we favour
the notion of Coetzee et al. 0979) that tail-like structures observed on some PR772 particles
are in fact broken-off pili to which the phages had attached. This would account for the
variable lengths of tail-like structures on PR4, as observed by Bradley & Rutherford (I975).
Antigenic determinants are thought to be small and consist of 5 to 2o amino acid residues
(Davis et al. 1973). Consequently, if an altered protein is to account for the observed difference in serum neutralization rate constant between phages PR4 and PR772, it would
imply a nucleotide sequence difference < 60 nucleotide pairs - clearly beyond our electron
microscopic detectability.
In view of the large body of evidence regarding the similarity of phages PR 3, PR4 and
PR772, we conclude that they are in fact the same phage. The only (serological) difference
between phages PR4 and PR772 may be attributed to a minor difference in the amino acid
sequence of otherwise identical capsid proteins.
The authors are grateful to Professor J. N. Coetzee for valuable suggestions during the
preparation of the manuscript.
REFERENCES
ADAMS, M. H. (1956). M e t h o d s of s t u d y o f bacterial viruses. Methods in Medical Research z, 1-73.
BALDWIN, g. L., BARRAND, g., FRITSCH, A., GOLDTHWAIT, D. A. & JACOB, F. 0966). Cohesive sites on the D N A s
f r o m several temperate coliphages. Journal of Molecular Biology 17, 343-357.
BRAOLEV, D. E. (I976). A d s o r p t i o n o f the R-specific bacteriophage P R 4 to pill determined by a d r u g resistance
plasmid of the W compatibility group. Journal of General Microbiolog) 95, 181-I 8 5.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 00:51:00
200
W. F. C O E T Z E E A N D P. J. B E K K E R
BRADLEY, D. E. & COHEN, D. R. (1977). Adsorption of lipid containing bacteriophages PR4 and PRDI to pili
determined by a P-I incompatibility group plasmid. Journal of General Microbiology 98, 619-623.
BRADLEY, D. E. & RUTHERFORD, E. L. (1975). Basic characterization of lipid-containing bacteriophage specific
for plasmids of the P, N and W compatibility groups. Canadian Journal of Microbiology 2x, 152-163.
CASPAR, D. L. O. & KLUG, A. (1962). Physical principles in the construction of regular viruses. Cold Spring
Harbor Symposia on Quantitative Biology 27, 1-24.
COETZEE, J. N. (1974). High frequency transduction of Kanamycin resistance in Proteus mirabilis. Journal
of General Microbiology 84, 285-296.
COETZEE, J. N., LECATSAS, G., COETZEE, W. F. & HEDGES, R. W. (I979). Properties of R plasmid R772 and the
corresponding pilus-specific phage PR772. Journal of General Microbiology xxo, 263-273.
COETZEE, W. F. & PRETOR1US,G. H. J. 0979). Factors which influence the electron microscopic appearance of
DNA when benzyla[kyldimethyt-ammonium chloride is used. Journal of Ultrastructure Research 67,
33-39.
DAVIDSON,N. & SZYBALSKI,W. (197 I). Physical and chemical characteristics of lambda DNA. In The Bacteriophage Lambda, pp. 45-82. Edited by A. D. Hershey. New York: Cold Spring Harbor Laboratory.
DAVIS, B. D., DULBECCO, R., EISEN, H. N., GINSBERG, H. S., WOOD, W. B. & McCARTY, M. (~973)- In Microbiology,
PP- 354-445- Hagerstown: Harper and Row.
KAISER,A. O. & WU, R. (I968). Structure and function of DNA cohesive ends. Cold Spring Harbor Symposium
on Quantitative Biology 33, 729-734.
KIM, J. & DAVIDSON,N. 0974)- Electron microscope heteroduplex study of sequence relations of Tz, T4 and
T6 bacteriophage DNAs. Virology 59, 93-11 I.
OLSEN, R. n., S~AK,J. S. & GRAY, R. H. (I974). Characteristics of PRDI, a plasmid-dependent broad host range
DNA bacteriophage. Journal of Virology x4, 689-699.
PRETORIUS, G. H. W. & COETZEE, W. F. (I979). Proteus mirabilis phage 5oo6M: a physical characterization.
Journal of General Virology 45 (in the press).
STANISlCH, V. A. 0974)- The properties and host range of male-specific bacteriophages of Pseudomonas
aeruginosa. Journal of General Microbiology 84, 332-342.
SZYBALSKI,W. & HERZKOWlTZ, I. (197I). Lambda genetic elements. In The Bacteriophage Lambda, pp. 778779. Edited by A. D. Hershey. New York: Cold Spring Harbor Laboratory.
VOLLENWEIDER,H. J., SOGO,J. M. & KOLLER,T. (1975). A routine method for protein-free spreading of doubleand single-stranded nucleic acid molecules. Proceedings of the National Academy of Sciences of the
United States of America 72, 83-87.
YAMAGISHI, H., NAKAMURA,K. & OZEKI, H. (i965). Cohesion occurring between D N A molecules with temperate phage ~8o and 2t or ~58L Biochemical and Biophysical Research Communications ~o, 727-732.
(Received IO April 1979)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 00:51:00