I53
J. gen. ViroL (I974), 22, I53-I57
Printed in Great Britain
Bacteriophages and Cryptic Lysogeny in Achromobacter
(Accepted 14 September I973)
SUMMARY
The characteristics of 4 closely related bacteriophages of halotolerant, collagenolytic strains of Achromobacter isolated from cured hides are described. All
phages cause lysogenic conversion. Achromobacter sp. 2 is shown to be a cryptic
lysogen for phage ~3 and has a low rate of spontaneous liberation which can be
increased by treatment with mutagens. An explanation for the cryptic lysogeny is
proposed.
Bacteriophages of Achromobacter have not been previously described. We report the
isolation and characterization of four Achromobacter phages. Achromobacter sp. 2 is shown
to be stably lysogenic for a prophage which does not confer immunity to superinfection.
This strain, therefore, belongs to the class of cryptic lysogens which are stable in the absence
of functional repressor. Evidence is presented that the mechanism of cryptic lysogeny does
not involve a prophage deletion but is similar to that described by Krizsanovich (I973) in
Proteus mirabilis.
Methods and media were as described by Thomson & Woods (I973). The halotolerant,
collagenolytic hide bacteria included Achromobacter spp. I to 8 (Thomson, Woods &
Welton, 1972), A. iophagus (Welton & Woods, 1973) and a further I I Achromobacter strains.
Bacteriophages for Achromobacter are rare and only four were isolated. Phage ~I was isolated from the supernatant fluids of thirty hide soak solutions (Thomson et al. 1972). Phage
c~3a was isolated from one spontaneous plaque on 250 lawns of Achromobacter sp. 2. For
u.v. induction of the 20 Achromobacter strains (Krizsanovich, 1973) a Hanovia lamp with an
output of 7"2 ergs mm 2 sec-1 was used. Phage c~2was induced from Achromobacter sp. 9 and
~3b from Achromobacter sp. 2. Each phage plated only on Achromobacter spp. 2, 3 and 4
giving turbid plaques with distinct cloudy centres.
Electron micrographs of high titres of phage (Brenner & Home, 1959) showed that all had
hexagonal heads with no visible neck (Fig. 0. The tail lengths varied slightly: ~I, 3009 A;
e~2, 3867 A; ~3a, 3450 A and o~3b 3178 A. The proximal half of the tail showed cross striations and short tail fibres originated near the tip. Phage nucleic acid was shown to be 2-DNA
by the acridine orange staining method of Bradley (I966) and the Burton modification
(Burton, I956) of the Dische diphenylamine method. Base compositions of the DNA samples (Table I) were calculated from their thermal denaturation temperatures (Marmur &
Doty, 1962). For antiphage serum preparation concentrated phage was further purified by
zone electrophoresis (Poison & Russell, I967). The neutralization constants of the antiphage
serum obtained from injected rabbits (Eisenstark, I967) showed that the phages were closely
related serologically (Table I). Single-step growth curves (Eisenstark, i967) revealed
characteristic long latent periods (Table I). The phages plated with very low efficiencies on
partially resistant, non-lysogenic bacteria isolated from turbid plaques. Samples of high
titre phage (c. lo 11 p.f.u./ml) were unstable when stored at 4 °C in the presence of chloroform, showing a loss of infectivity of two logs after one week. Under these conditions
stability was maximal between pH 7 and pH 8. The kinetics of phage inactivation at 60 °C
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 20:26:25
I54
Short communications
Fig. i. Electron micrographs of the c~phages. (a) c~r, (b) ~2, (C) c~3a, (d) c~3b.
in phage buffer pH 7"8 (Eisenstark, I967) were biphasic (Fig. 2), suggesting the presence of
two components of different heat sensitivity (Wilkowske, Nelson & Parmelee, I954). The
phages were relatively resistant to u.v. inactivation (Table I). Further study of the response
to u.v. by ~3a (Krizsanovich, i973) showed that free phage was more resistant to irradiation
than vegetative phage (Fig. 3). The kinetics of inactivation in both cases were one-hit and
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 20:26:25
Short communications
I55
Table I. Phage characteristics
Phages
k valu~min -~*
G+C (%)
Latent period
(min)
Averageburst
size
U.v.
irradiationt
~I
~2
~3a
~3b
247
230
300
30o
42'56
40'97
45'73
45"58
85
Ioo
too
9o
380
300
230
300
950
~I95
II52
I317
* Neutralization constants with cc3aantiserum.
t u.v. dose (ergs mm-2) required for 75 % inactivation.
0
I
1
I
1
1
-1"75!
,-5
,'5
-~ -1"5
!
3
~JJ
~h
G
-4
-5
0
I
5
I
10
I
20
Time (rain)
Fig. 2.
- 1.25
-1
30
500 1000 t 500 2000
U.\. dose (ergs mm 2)
Fig. 3.
Fig. 2. Thermal inactivation of the ~ phages at 60 °C. 0 - - 0 , ~xI; A - - A , ~2; E - - I I , c~3a;
0 - - 0 , a3b.
Fig. 3. U.v. inactivation of free phage. =3a (O--O) and vegetative phage c~3a(O--Q).
biphasic. This is similar to phage P22 (Garen & Zinder, I955) but different from Proteus
mirabilis phages (Krizsanovich, I973).
In order to determine whether Achromobacter sp. 2 was irtducible and thereby explain the
origin of ~3a and c~3b, forty samples of this strain were tested for induction by u.v. irradiation. Mitomycin C (MC, Calbiochem., San Diego, California. B grade) and N-Methyl-Nnitroso-N'-nitroguanidine (nitrosoguanidine, N G ; Koch-Light Laboratories, England)
induction was performed on IO ml log phase cells incubated for 3 h with 5 #g/ml MC or
3 #g/ml N G and on 400 ml log phase cultures containing 2"5/zg MC/ml or 3/~g NG/ml
incubated for 5 h and 9 h, respectively. Supernatant fluids of the 4oo ml cultures were purified and concentrated by cycles of differential centrifuging and tested for phage. Controls
were carried out without the addition of MC or NG. Temperature-sensitive inducible prophages were investigated by incubating lawns of Achromobacter sp. 2 at 25 °C and 42 °C.
Induction by ageing was tested by incubating cultures for 3 weeks at 3o °C and testing
supernatant fluids for phage. Only treatment of 400 mt cultures with MC and N G resulted
in the induction of phage. The high titres obtained (4 x ~o1 to 5"3 x lO5 p.f.u./ml) were due to
the phage being able to re-infect its parent strain. Controls of untreated 400 ml cultures were
shown to be free of phage when assayed and examined electron-microscopically. Phages
c~3a and ~3b both originated from Achromobacter sp. 2 by rare events, but as phage can be
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 20:26:25
156
Short communications
induced by MC and NG, the possibility of phage contamination is eliminated. The induction
of Achromobacter sp. 2 yielded phages which were not distinguished as either ~3a or ~3b and
are, therefore, referred to as ~3. They are very similar and it is concluded that they are variants
of the same cryptic prophage of A chromobacter sp. 2. This strain, with its very low frequency
of spontaneous and u.v. inducibility and sensitivity to superinfection, may be termed a
cryptic lysogen (Fischer-Fantuzzi & Calef, 1964).
Cryptic lysogens have been isolated from a number of bacterial species (Krizsanovich,
I973). Most are due to extensive deletions including the repressor gene or the site of action
of the immunity repressor. They are not inducible but can be recovered by superinfection
with related phage. Cryptic lysogeny in Aehromobacter sp. 2, as in Proteus mirabilis (Krizsanovich, 1973) must have a different cause for, although the cryptic prophage does not
synthesize repressor, vegetative ~3 can be recovered and lysogenize the parent strain. The
repressor gene must, therefore, be present but only expressed by the vegetative phage. The
MC and N G inducibility of the cryptic lysogen argues against an extensive prophage deletion. A simple explanation is that of Krizsanovich (I973), whereby a defective enzyme
necessary for prophage excision and/or vegetative phage development is produced by the
cryptic prophage and can be reverted or suppressed by mutagens. Lack of functional repressor in the cryptic lysogen could be due to the phage integration site lying within the repressor
gene and causing its disruption.
From the colonies in the centre of turbid plaques, stable lysogens were isolated which were
u.v.-inducible and immune to superinfection by all four phages. The phages caused lysogenic
conversion preventing lysogens from re-adsorbing phage. These strains may thus be referred
to as double lysogens. The reconstitution of the repressor gene in double lysogens may be
caused by superinfecting phage lysogenizing in tandem with the cryptic prophage.
This work was supported by the Shell (S.A., Ltd.) Research Fellowship of J.A.T. and a
research grant from the South African Council for Scientific and Industrial Research. The
authors wish to thank Mr R. Cross for the electron micrographs of c~I, c~2 and ~3b and
D r P. Appelbaum and Mr N. Hugo of Pretoria University for that of c~3a.
Department of Botany & Microbiology
Rhodes University, Grahamstown
South Africa
JENNIFER A. THOMSON
D. R. WOODS
REFERENCES
BRADLEY, D. E. (1966). The fluorescent staining of bacteriophage nucleic acids. Journal of General Microbiology 44, 383-391 •
BRENNER, S. & HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of
viruses. Biochimica et biophysica acta 34, lO3-110.
~URTON, K. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochemical Journal 62, 315-323.
EISENSTARK, A. (1967). Bacteriophage techniques. In Methods in Virology, vol. I, pp. 449-524. Edited by
K. Maramorosch and H. Koprowski. New York and London: Academic Press.
FISCHER-FANTUZZI, L. & CALEF, E. 0964). A type of ,~ prophage unable to confer immunity. Virology, 23,
209-216.
GAREN, A. & ZINDER, N. D. (I955). Radiological evidence for partial genetic homology between bacteriophage
and host bacteria. Virology x, 347-376.
KRlZSANOVlCH, K. (1973). Cryptic lysogeny in Proteus mirabilis. Journal of General Virology I9, 311-32o.
MARMUR,J. & DOTY,P. (1962). Determination of the base composition of deoxyribonucleic acid from its
thermal denaturation temperature. Journalof Molecular Biology 5, lO9-I 18.
POLSON,A. & RUSSELL,B. (I967). Electrophoresis of viruses. In Methods in Virology, vol. 2, pp. 391-426.
Edited by K. Maramotsch and H. Koprowski. New York and London: Academic Press.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 20:26:25
Short communications
I57
THO~aSON, J. A. & WOODS, D. R. (I973). Properties of sphaeroplasts o f a halotolerant A chromobacter strain a n d
their infection with bacteriophage deoxyribonucleic acid. Journal of General Microbiology 74, 71-76.
THOMSON, J. a., WOODS, D. R. & WELTON, R. L. 0972). CoIlagenolytic activity of aerobic halophiles f r o m hides.
Journal of General Microbiology 7 o, 315 319.
WELTON, R.L. & WOODS, D.R. (~973)- Halotolerant collagenolytic activity o f Aehromobaeter iophagus.
Journal of General Microbiology 75, I 9 I - t 9 6 .
WILKOWSKE, H. H., NELSON, F. E. & PARMEr,~E, C. E. (I954). H e a t inactivation of bacteriophage strains active
against lactic streptococci. Applied Mierobiology ~, 250-253.
(Received I I July x 9 7 3 )
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 20:26:25