VIROLOGY

114,236-246

New Anabaena
NIEN-TAI

(1981)

and Nostoc Cyanophages

from Sewage

Settling Ponds

HU, TERESA THIEL,2 THOMAS H. GIDDINGS, JR.,

AND C. PETER WOLK4

MSU-DOE Plant Reseawh Laboratory, Michigan State University,

East Lansing, Michigan 4882.4

Received May 13, 1981; accepted June 4, 1981

We have isolated, from sewage settling ponds, 16 cyanophages for heterocyst forming,
filamentous cyanobacteria
of the genera Anabaena and Nostoc. These phages fall into
three groups based on morphology, host range, one-step growth curves, and restriction
digests. On the basis of these criteria they can be distinguished
from cyanophages Al(L), A-4(L), N-l, and AN-10 which we received from other laboratories.
Certain of the
newly described phages are similar in morphology to the short-tailed
LPP cyanophages,
and others to the long-tailed AS cyanophages.

1973), and nine others, A-l(L) through A9(L), were isolated in Leningrad, USSR,
The first cyanophages to be described by screening about 2000 samples from six
and characterized were members of the regions in the USSR (Kozyakov, 1977).
LPP group which attack three genera of Among the cyanobacteria tested, only one,
filamentous cyanobacteria, Lyngbya, Plec- a strain of Anabaena vamabilis, served as
tonema, and Phormidium (Safferman and host for the nine phages of the A(L) series.
Morris, 1963; Safferman et ah, 1969). Cy- Unfortunately, all but two of these phages,
anophages of this group have been isolated A-l(L) and A-4(L), have been lost (S.
many times from different parts of the Kozyakov, personal communication).
world, and are currently the best characBecause we would like to develop a
terized group of cyanophages (Sherman
system for gene transfer by transduction
and Brown, 1978). Several viruses (AS
in heterocyst-forming cyanobacteria, and
group) for the unicellular cyanobacteria
were unsuccessful in our initial efforts
Anacystis and Synechococcus have been
isolated; however, these cyanophages do using N-l, we began a search for more
not appear to be as widely distributed in phages of the proper specificity. Waste
nature as are the LPP phages (Sherman stabilization ponds have been the source
and Connelly, 1976). Viruses which attack of many previously isolated cyanophages
the heterocyst-forming, filamentous cy- (Sherman and Brown, 1978). We screened
anobacteria are the least common: only samples from 60 sewage settling ponds at
30 sites in south-central Michigan for cyone, N-l, has been reported in the United
States (Adolph and Haselkorn, 1971,1972, anophages which would attack any of
three strains of Anabaena, and obtained
16 isolates, all of which proved capable of
1 Present address: Department of Genetics and Cell
infecting several strains of Anabaenu and
Biology, University
of Minnesota, St. Paul, Minn.
Nostoc. These isolates were partially char55108.
acterized, and compared to N-l, A-l(L), A* Present address: Department of Biology, Univer4(L),
and AN-lo. AN-10 is an apparently
sity of Missouri-St.
Louis. St. Louis, MO. 63121.
undescribed
phage which came to us as a
3 Present address: Department
of Molecular, Celcontaminating phage in-the stock of Alular and Developmental
Biology, University
of Col4(L) which we received; we have characorado, Boulder, Colo. 80302.
4 To whom correspondence should be addressed.
terized it together with our isolates from
INTRODUCTION

0042-6822/81/130236-11$02.00/O
Copyright
All rights

0 1981 by Academic Press. Inc.

of reproduction
in any form reserved.

236

NEW Anabaenu AND Nostoc CYANOPHAGES

sewage. The new isolates, including AN10, represent at least four new phages for
Anabaena and Nostoc. By use of restriction
endonucleases, we have shown that certain
genetically closely related cyanophages
exhibit genetic microheterogeneity. Part
of our results have been reported briefly
(Abstracts, Annual meeting of the American Society for Microbiology, 1980, p. 274).

237

Gromov via P. Desjardins (also designated

PCC 7118; ATCC 27892, supposedly derived
from PCC 7118, differs morphologically
and physiologically from the Myers strain
which we received from Gromov and from
PCC 7118 which we received from R.
Rippka of the Pasteur Institute); Nostoc
linckia, from the late R. N. Singh, Department of Botany, Banaras Hindu University, Varanasi, India; A. variabilis Indiana University Strain 1444 (formerly
MATERIALS
AND METHODS
misidentified as Anabaena jtos-aquae;
ATCC 29413); A. variabilis FD, a strain
Media. Cyanobacteria were grown in the derived from 1444 and resistant to 40C
liquid medium (AA) of Allen and Arnon (Currier and Wolk, 1978); Nostoc spp.
(1955) diluted eightfold (AA/8), or in AA/
ATCC 29105, ATCC 29106, and ATCC
8 supplemented with 2.5 mM KN03 and 2.5 29131, and Anabaena spp. ATCC 27898 and
mM NaN03 (AA/8 + NOs). Phage plaque ATCC 29151, all from ATCC; and A. varassays were done and plate lysates made iabilis strains M-2 and M-3, and Nostoc
on AA medium solidified with 1% agar muscorum strain M-131 from T. Ichimura,
(Difco Laboratories, Detroit, Mich.) puri- Institute of Applied Microbiology, Univerfied by the method of Braun and Wood sity of Tokyo (M-2 and M-3, which came
(1962). Cyanophages were maintained in to the University of Tokyo from Allen and
cyanophage (CP) buffer containing 5 mM from Myers, respectively, may both have
MgClz, 5 mM CaClz, 10 mM NaCl, and 10 been derived from the Myers strain of A.
mM N-tris(hydroxymethyl)methyl-2-amivariabilis). Strains of A. variabilis (Myers)
noethanesulfonic acid (TES), pH 7.0. The resistant to cyanophages AN-20 and ANstandard saline citrate phosphate buffer 22 and of Anabaena sp. PCC 7120 resistant
(SSCP) used in hybridization experiments
to cyanophage AN-13 were isolated by
contained 120 mM NaCl, 15 mM sodium picking and streaking, to remove free
citrate, 13 mM KHzPOI, and 1 mM EDTA, phage, those colonies which grew within
pH 7.2 (Benton and Davis, 1977).
plaques on agar plates.
Phage strains. R. Haselkorn, University
None of the phages grew in the following
of Chicago, sent us N-l. A-l(L) and A-4(L) strains of cyanobacteria: Cylindrospercame from B. V. Gromov, Leningrad State mum lichenifmze ATCC 29412, derived
University, via P. Desjardins, University
from UTEX B 1828; C. Zicheniforme UTEX
of California, Riverside. The A-4(L) stock 2014; Plectonema boryanum UTEX 594;
which we received from Desjardins con- Anabaena doliolum, from R. N. Singh via
tained phages of large and small plaque R. D. Simon, University of Rochester; Nossize. Each purified phage has retained its tot ATCC 27895, Nostoc ATCC 27897, Anphenotype through repeated transfers. The abaena ATCC 27899, Nostoc ATCC 29168,
phage which forms large plaques corre- Anabaena ATCC 29208, and Nostoc ATCC
sponds in its properties to A-4(L) de- 29411; Nostoc PCC 7807, from R. Rippka;
scribed by Gromov and co-workers (Khu- Anabaena cylindrica strain M-l, from
dyakov and Gromov, 1973; Kozyakov, Fogg via Myers and then T. Ichimura;
1977). We designate the phage which forms Nostoc strains 2 and 3 from T. C. Shen,
small plaques AN-10 (see Introduction).
University of Malaya, Kuala Lumpur; AnCyanobacterial host strains. Those strains abaena strains 39, 40, 286, 287, 311, 312,
tested which were hosts for one or 314, and 317 and Nostoc strains 154 and
more of the phages described here were: 247 from G. S. Venkataraman, Indian AgAnabaena sp. PCC 7120 (ATCC 27893), ob- ricultural Research Institute, New Delhi;
tained from R. Haselkorn; Anabaena var- Anabaena strain L-31 from J. Thomas,
iabilis (Myers strain) received from B. V. Bhabha Atomic Research Center, Bombay,

238

HU ET AL.

India; Nostoc spongiaefomze, isolated from

sewage by T. Thiel; N. muscorum UTEX
387; and 26 additional heterocyst-forming
cyanobacteria isolated from sewage.
Isolation of cyanophages. Samples from
sewage settling ponds were passed through
0.4-pm pore-size Nuclepore filters. Portions of 3 to 5 ml were supplemented with
AA medium to a final concentration
of
AA/g, and were inoculated with liquid cultures of three cyanobacterial
strains, A.
variabilis
(IUCC 1444, ATCC 29413), Anabaena sp. (PCC 7120), and (supplementing with nitrate to 5 ml@ the Myers strain
of A. variabilis.
After about 2 weeks of
incubation, samples of these cultures were
spotted on lawns of the same organisms.
Zones of lysis which appeared on the lawns
were picked, resuspended in AA/g, and
sterilized by filtration
through Millipore
filters (type HA, 0.45~pm pore size) or by
chloroform. We serially diluted these suspensions and assayed for discrete plaques
indicative of phage. Based on this criterion, we isolated 16 cyanophages from the
sewage pond samples. We named the
phages according to the convention established by Safferman and Morris (1963),
using the initials of the generic names of
the hosts, Anabaena and Nostoc (AN), followed by arbitrary
numbers to indicate
different isolates (although not necessarily different phages). The phages, deposited with the American Type Culture Collection, have been given the following
designations: AN-10 is ATCC 27893-B14;
AN-11, ATCC 27893-B13; AN-12, ATCC
27893-B12; AN-13, ATCC 27893-Bll; AN14, ATCC 27893-BlO; AN-15, ATCC 29106Bl; AN-16, ATCC 27893-B9; AN-17, ATCC
27893-B8; AN-18, ATCC 27893-B7; AN-19,
ATCC 27893-B6; AN-20, ATCC 27892-B2;
AN-21, ATCC 27893-B5; AN-22, ATCC
27892-Bl; AN-23, ATCC 27893-B4; AN-24,
ATCC 27893-B3; AN-25, ATCC 27893-B2;
AN-26, ATCC 27893-Bl; A-l(L),
ATCC
27893-B16; A-4(L), ATCC 27892-B3; and N1, ATCC 27893-B15.
Preparation
of phage stocks. Twice in
succession, single, well-separated
plaques
of each phage were picked, diluted, and
replated. Plate lysates were made by mixing approximately
500-1000 plaque-form-

ing units (PFU) with lo* cyanobacterial

cells in a 0.5% soft agar overlay. Plates,
incubated at 30 in the light, showed confluent lysis in about 3 days. Phages were
eluted by overlaying the agar with 5 ml
of CP buffer, and then removing the buffer
overlay after overnight incubation at 4.
This extraction
was repeated, and the
combined extracts were st.ored over a few
drops of chloroform
at 4. Phage titers
varied from 2 X 10 to 4 X 10 PFU/ml.
Host range experiments. The host range
of each phage was determined
by spot
tests on the cyanobacterial
strains indicated above. Phage stocks at a concentration of 2 X lo3 or 10 phage/ml were placed
in the wells of a Steers replicator device
(Melrose Machine Shop, Woodlyn, Pa.) and
spotted (10 ~1 per spot) onto lawns of cyanobacteria containing lo7 to lo8 cells in
a 0.5% agar overlay. After lawns had
grown sufficiently
to detect lysis (3 to 7
days) a spot was recorded as either lysed
or not. Those hosts which were lysed by
both concentrations of phage were considered to susceptible to the phage, those
which were lysed only by a high concentration of phage were scored as partially
susceptible, whereas the absence of lysis
indicated resistance of that strain to the
phage being tested. We observed lysis
from without (killing of the host at a high
multiplicity
of infection,
but without
growth of the phage in that host) of some
of the cyanobacterial
hosts. This was distinguished from susceptibility
or partial
susceptibility,
as defined above, by the fact
that zones of lysis increased in size with
time for susceptible hosts, but remained
a constant size when cells were lysed from
without.
One step growth curves. We grew A. variabilis (Myers strain) to l-2 x 10 cells/ml
and concentrated the cultures to 5 X lo7
cells/ml in CP buffer. Phage were added
in a small volume of CP buffer to give a
final multiplicity
of infection (MOI) of 10m2
PFU/cell, and the mixture was incubated
at 30 in the dark for 1 hr. Infected cells
were washed twice in CP buffer to remove
unadsorbed phage, and the washes were
assayed for phage to determine the efficiency of adsorption. The efficiency varied

NEW Anabaem AND Nosh

among phage strains from 80 to 99%, but

was usually greater that 90%. We diluted
the infected cells 10m4and 10e5into flasks
of AA/8 + NOs, which were then shaken
in the light at 30. Samples removed
hourly were assayed for phage on a lawn
containing lo* cells of the same host per
plate in a 0.5% agar overlay. In some experiments, filaments of A. variabilis were
fragmented to an average filament length
of 1.5 cells by bath cavitation of culture
suspensions (Wolk and Wojciuch, 1973).
The fragments were washed, concentrated
to 5 X lo7 cells/ml in CP buffer, and used
as the host in one-step growth experiments done as described above.
Electron rnicroscolry. Phage stocks were
concentrated to 5 X lOlO- X 1Ol PFU/ml
by differential centrifugation. A droplet
of concentrated phage was applied to a
grid coated with Formvar and carbon.
After 1 min, most of the fluid was drawn
off with filter paper. A drop of stain, either
0.5% uranyl acetate (pH 4.5) or 1% ammonium molybdate (pH 7.0), was applied
before the grid dried completely. Excess
stain was removed quickly with filter paper. Both stains contained 25 pg bacitracm/ml, added as a wetting agent. Samples
were examined and photographed at a
magnification of 80,000 X in a Phillips EM
300 electron microscope which had been
calibrated with a diffraction grating. Dimensions were measured directly from
negatives using a 7 X viewing lens equipped
with a micrometer grating.
PurQication of cyanophages and isolation of phage DNA. Cyanophages were purified by sedimentation at high speed
(15,000 rpm for 4 hr in the type 19 rotor
of a Beckman L2-65B, or at 20,000 rpm for
2 hr in the SS-34 rotor of a Sorvall RC2B) followed by equilibrium ultracentrifugation in CsCl [final concentration of 44%
(w/w); Beckman L2-65B, SW65,40,000 rpm
for 24 hr]. Phage DNA was isolated from
the purified phage by extraction with redistilled phenol (saturated with 1 hf Tris,
pH 8.0), followed by precipitation with
ethanol.
32P labeling of cyanophage DNAs. Cyanophage DNAs were labeled with [a-

CYANOPHAGES

239

32P]deoxycytidine 5-triphosphate (490 Ci/

mmol from New England Nuclear, or approx 2000-3000 Ci/mmol from Amersham)
by nick translation (Rigby et al., 1977) using a nick translation kit purchased from
New England Nuclear or Bethesda Research Laboratories, Inc. The reaction was
stopped, and the DNA was extracted with
phenol (redistilled and saturated with 10
m&f Tris, pH 7.8, containing 1 mM EDTA)
and then precipitated with ethanol. Specific activities of the DNA at the time
when hybridization was performed, determined from trichloroacetic acid-precipitable counts, ranged from 3.0 X lo5 to
1.2 X lo7 cpm/pg. Each hybridization was
performed with three preparations of nicktranslated DNA from AN-24 and one preparation from AN-13. Hybridization with
DNA from plaques transferred to nitrocellulose membranes was done according
to Benton and Davis (1977).
endonuclease digestions.
Restriction
EcoRI and Hind111 were purchased from
Miles Laboratories, Inc., and from Bethesda Research Laboratories, Inc., and
were used according to the suggestions of
the suppliers. Reaction mixtures containing 0.5-2 gg of DNA, enzyme in excess, and
reaction buffer in a final volume of 25 ~1
were incubated at 37 for periods of time
sufficient to allow digestions to go to completion. The reactions were then stopped
by addition of 5 ~1 of 5% SDS, 25% glycerol, and 0.025% bromophenol blue followed by heating at 65 for 10 min. Restriction fragments of known size (Thomas
and Davis, 1975; Murray and Murray,
1975) were derived from the digestion of
X DNA by EcoRI, HindIII, or both.
Gel electrophoresis. Agarose or gradient
polyacrylamide gels (Jeppesen, 1974) 14
cm wide x 12 cm long X 3 mm thick were
run in a Model 220 vertical slab gel apparatus (Bio-Rad Laboratories) with Trisborate buffer (90 mM Tris, 90 mM H3B03,
2.5 mM Na2EDTA, pH 8.3). After completion of electrophoresis, the gels were
stained with ethidium bromide, visualized
with a uv transilluminator,
and photographed with a Polaroid MP3 Land Camera using a red filter.

240

HU ET AL.
TABLE 1
HOST RANGES

OF CYANOPHAGES

Myers

No&c

ATCC
29105

(also
A?Zabaena

muacc-

PCC 7120

PCc7120
AN-13

rum
M-131)

Myers
AN&+
or AN22!

N-l

+c

A-l(L)**

NO&C
Cyanophage
(and source
host)

A?l&l.W2a

ATdlWTUl

AWbW?Ul

NO&C

NO8tOC

linekin

ATCC
29151

ATCC
29131

AK%
29196

M-3

ATCC
29413

ATCC
27898

k
*

M-2

No&c

_t

+
+

+
+

+
*

+
k

AN-W

AN-15

AN-ll,AN-12.
AN-13,AN-14,
AN-WAN-l?,
AN-l&AN-19,
AN-21,AN-23,
AN-24,AN-25,
AN-B

zt

AN-206
AN@

+
*

+
2

+
+

+
+

A-4(L)b

-These cyanophages were grmvn on Anabaena FCC 7120.

bThese cyanophages were grown on the Myers strain of A. variabilis.
c +. Lysis of cyanobacteria by both high and low concentrations of cyanophage; -, no lysis of cyanobacteria by either high or low concentrntions
of cyanophage; +, lysis of cyanobackria only by high concentrations of cyanophage.

RESULTS

Host range. The 16 new phage isolates

and the four we received from other laboratories were tested for lysis of 64 cyanobacteria. The phage susceptibility of
the 14 hosts which were used to place the
phages into groups is given in Table 1. All
of the cyanobacterial strains which were
lysed by any of the phages are members
of the genera Anabaena and Nostoc. Lysis
of these hosts was not caused by lysis from
without based upon the criterion described
in Materials and Methods.
The newly described phages (including
AN-lo) may be classified into four groups
on the basis of morphology, host range,
relative plating efficiency on two or more
susceptible hosts, and electrophoretic patterns of fragments derived from digestion
of their DNAs by restriction endonucleases. Thirteen of the phages fall into one
group; all grow well on most of the sus-

ceptible hosts, giving large, clear plaques.

Only AN-15 infects No&c sp. ATCC 29106,
producing small clear plaques. AN-20 and
AN-22, although closely related (see below), differ in plaque morphology: young
plaques of AN-20 are clear, whereas those
of AN-22 are turbid. Older, larger plaques
of both phages are clear. In addition, when
AN-22 is grown on the Myers strain of A.
variabilis, it plates on Anabaena sp. PCC
7120 with about 10e2of the efficiency with
which it plates on the host in which it was
grown, whereas similarly grown AN-20
plates with only slightly lower efficiency
on PCC 7120 than on the Myers strain of
A. variabilis. AN-10 produces very small,
clear plaques and is distinguishable from
the other AN phages both by plaque morphology and host range. On the basis of
host range alone it is not possible to distinguish it from the previously described
phages A-l(L) and N-l. Our findings confirm a previous report (Rippka et &, 1979)

NEW Armbama

AND No&c

Tlrn. (ham,

FIG. 1. One-step growth curves of cyanophages in

intact filaments of A. variabi~is (Myers strain) at an
MO1of 10d2 PFU/cell.

that N-l lyses A. variabilis PCC 7118,and

Anabaena spp. ATCC 27893 (PCC 7120),
ATCC 27898 (PCC 6411), and ATCC 29151
(PCC 7119).
One-step growth experiments. Representative one-step growth curves for the new
cyanophages, as well as for A-l(L) and A4(L), are given in Fig. 1. The phages fall
into two groups with respect to time of
lysis: phages with a large-plaque morphology, such as A-4(L), AN-20, AN-22,
and those representatives of the group
which includes AN-24 and which have
been tested (only data for AN-24 are given
here), lyse within a few hours of infection.
Those phages which produce small plaques,
namely AN-15, AN-lo, and A-l(L), lyse
several hours later. The latent period of
A-4(L) at 25 was reported to be 2.5 hr
(Khudyakov and Gromov, 1973). That we
observed a much shorter latent period may
be explained by the facts that our experiments were done at 30 and that infection
began during the hour-long adsorption
period.
In a filamentous host, burst sizes can be
difficult to determine from one-step growth
curves done at low MOI. Even when very
few host cells are present per milliliter,

241

CYANOPHAGES

phages released from one cell in a filament

can infect adjacent cells in the same filament. Because the effective cell concentration is high, lysis may be prolonged and
ever-increasing yields of phage with time
may be observed. In some experiments,
particularly those using the rapidly lysing
phages AN-20 and AN-22, synchrony of
the initial lytic events gave evidence of
multiple cycles of infection. A one-step
growth curve of AN-20 using fragments
of host cell filaments containing primarily
one or two cells had a form typical of
growth curves for phages in unicellular
hosts (Fig. 2).
Phuge size and morphology. The newly
isolated cyanophages could be divided into
two morphological groups, those with long,
contractile tails (Figs. 3A-C), and those
with very short tails (Fig. 3D). The longtailed phages, AN-10 and AN-15, were
morphologically similar and resembled
the previously described cyanophages N1 (Adolph and Haselkorn, 1973) and Al(L) (Kozyakov et al., 1972). The capsids
had a hexagonal projection and were connected to the tail by a narrow neck (Fig.

11 (how.)

FIG. 2. One-step growth curves of cyanophage AN20 in normal and in fragmented filaments of A. wwiabilis (Myers strain) at an MO1 of 10e2 PFU/cell.

242

HU ET AL.

3A). A base plate with some indication of

attached pins was generally observed. A
phage with a contracted sheath and a protruding core is shown in Fig. 3B. Staining
with ammonium
molybdate
apparently
stimulated
the contraction
of the phage
tails; many such images were seen with
this strain, whereas most of the particles
in the preparations
stained with uranyl
acetate remained uncontracted and intact.
Some differences in overall length and
in the side-to-side diameter of the capsids
of the phages were evident (Table 2). Al(L) (Fig. 3C) was significantly
longer than
the other phages; our measurements show
it to be larger than reported previously
(Kozyakov, 1977). The diameter of the
capsid of A-4(L), 62 nm, was larger than
that of the other short-tailed
phages. The
tails of the short-tailed phages were rarely
observed and their structure was difficult
to resolve. They appear to be tubes with
a length of about 20 nm (Fig. 3D). The tail
morphology
presumably
changes during
injection of the DNA. It is conceivable that
these short tails emerge from the capsids
during injection and are therefore visible
TABLE

PHYSICAL CHARACTERISTICS OF CYANOPHAGES

Cyano@we
N-l
A-l (L)
AN-10
AN-15
AN-24
AN-20
AN-22
A-4 (L)d

Total particle
length
(nm)
165
134
168
162

+
f
f
+

4
6
5
3

(6)*
(12)
(6)
(9)

Side-toside capsid
diameter
(nm)
62
66
60
63
50
51
54
62

f 2
+ 4
f 4
+ 4
f 1
2~ 2
? 3
k 2

(6)
(12)
(6)
(9)
(10)
(6)
(12)
(6)

EcoRI
fragments
visualized
9
10
9
9
2
Not cleaved
Not cleaved
ND

Measurements
according to Adolph and Haselkorn (1973) are 61.4 nm edge-to-edge capsid diameter
and 100 nm length of tail.
b Mean f SD (number of measurements).
Measurements according to Kozyakov (1977) are
60 nm vertex-to-vertex
capsid diameter and 82.5 nm
length of tail.
d According to Khudyakov and Gromov (1973), the
capsid diameter is 56 nm.
Not determined.

only in the postinjection

the phages.

morphology

of

In situ hybridization. Cyanophages AN10 through AN-26, N-l, and A-l(L), fixed
and denatured
on nitrocellulose
membranes, were incubated with 32P-labeled
DNA from AN-13 and AN-24. According
to the results of in situ hybridization,
these two cyanophages share DNA sequence homology with each other and with
all of the other cyanophages which have
the same host range, but not with AN-20,
AN-22, N-l, A-l(L),
AN-IO, and AN-15
(data not shown).
Treatment with restriction

endonucle-

uses. The DNAs purified from cyanophages

AN-lo, AN-13, AN-15, AN-M, AN-20, AN22, AN-24, N-l, and A-l(L) were treated
with restriction endonucleases EcoRI and
HindIII,
and the products of digestion analyzed by gel electrophoresis. The EcoRIcleavage patterns of the DNAs from AN13, AN-18, and AN-24 appeared indistinguishable (Fig. 4); their Hind111 patterns,
however, differed slightly (Fig. 5). Band
b from AN-13 (Fig. 5, lane A) appeared
slightly brighter, relative to band a, than
did the corresponding
band from AN-18
(lane B) and AN-24 (lane C). In addition,
band c from AN-24 appeared broader than
from AN-13 or AN-18, suggesting that a
restriction
fragment of slightly different
size is present in AN-24. Further digestion
of HindIII-treated
AN-24 DNA with EcoRI
resulted in the loss of band 1 and the appearance of two new bands (data not
shown). The DNAs from AN-20 and AN22 were cleaved into fragments of similar
size by HindIII,
but were not cleaved by
EcoRI. Only the products of cleavage of
AN-22 by Hind111 are shown, in Fig. 5. The
DNAs from N-l, A-l(L), AN-lo, and AN15, treated with EcoRI or HindIII,
generated patterns (Figs. 6 and 7) quite different from those of the other cyanophages tested.
DISCUSSION

Sewage settling ponds were a good source

of cyanophages for strains of Anabaena
and Nostoc. Samples from 60 ponds at 30
sites gave 16 phages, which came from 15

FIG. 3. (A) Phage AN-15 stained with uranyl acetate. The hexagonal outline of the capsid, the
tail and the base plate are resolved. 360,000X. (B) Phage AN-15 stained with ammonium molybdate.
The tail has contracted leaving a shortened sheath and a long, protruding core. 360,000X. (C) Phage
A-l(L) stained with ammonium molybdate. 360,000X. (D) Phage AN-22 stained with uranyl acetate.
A short tail is visible (arrow). 360,000X.
243

244

HU ET AL.

FIG. 4. DNAs from cyanophages (A) AN-13, (B)

AN-18, and (C) AN-24 separated on a 0.7% agarose
gel with (+) or without (-) treatment for 2.5 hr with
restriction
endonuclease EcoRI (40 unitsing DNA).
Electrophoresis
was performed at a constant current
of 40 mA for 2 hr. The positions of molecular weight
standards (units in megadaltons),
derived from X
DNA by digestion with EcoRI, are indicated.

ponds at 12 sites. Thirteen of the phages

are indistinguishable
on the basis of host
range, and their presumptive
close relationship was confirmed by in situ hybridization and, in certain cases, by electrophoretic separation of products of digestion
of the phage DNAs by restriction
endonucleases. It therefore appears that in the
geographical area in which we sampled,
this is the predominant
group of phages
which can lyse Anabaena sp. PCC 7120 or
the Myers strain of A. variabilis. We simultaneously
enriched for and then purified Nz-fixing cyanobacteria,
from the
same ponds, capable of growing on AA
+ 1% agar. Such cyanobacteria
were recovered at a frequency of about 10 to 100
colony-forming
units per milliliter.
However, none that we tested were susceptible
to any of the cyanophages, nor have we
found any evidence that the cyanobacteria
are lysogens. The identity of the natural
hosts for the phages is therefore unknown.
All of the phages infect strains that
have been designated Anabaena and Nostot (Rippka et al., 1979). Sherman and
Brown (1978) discuss the problems of classifying phages according to the hosts identified by current cyanobacterial taxonomy.
They point out that both for LPP phages

and for some AS phages the hosts are

misclassified, and that those strains attacked by one phage are probably closely
related. It is possible that the strains of
Anabaena and Nostoc which are hosts for
our phages are more closely related than
their generic classifications would suggest,
and should be considered congeneric.
The phages are of two morphological
types: those with long, contractile
tails
which resemble in general morphology,
but are smaller than, the long-tailed AS
phage ASl-M
(Sherman and Connelly,
1976); and those with short tails which are
virtually identical to the LPP phages (Safferman et ah, 1969). The long-tailed phages
have relatively long latent periods of 7 to
11 hr and produce small plaques. The
smaller, short-tailed
phages lyse more
quickly and produce large plaques.
Digestion of phage DNAs with restriction endonucleases having specific cleavage sites is a simple and sensitive approach to screening for differences in their
base sequences. Differences (see b and c,
Fig. 5) between the patterns of fragments
resulting from digestion of the DNAs from
AN-13, AN-18, and AN-24 by HindIII,
although slight, were reproducible, suggesting that these phages are not identical.

!!!!
14.26
2.46

1.42
: I::;
as6

FIG. 5. DNA fragments from HindIII digestion (10

units of HindIII/rg
DNA, 18 hr) of DNAs from (A)
AN-13, (B) AN-18, (C) AN-24, (D) AN-22, and (E) X
digested also with EcoRI are shown following separation on a 2.5-7.5% gradient polyacrylamide
gel.
Electrophoresis
was run at a constant voltage of 90
V for 5 hr.

NEW Andaena

AND No&x

245

CYANOPHAGES

FIG. 6. Fragments
from (A) A-l(L), (B) N-l, (C) AN-lo, and-from
a separately run gel-(D)
AN-15 DNAs, treated (+) or not (-) with EcoRI (40 units/pg DNA, 2.5 hr), and then subjected to
electrophoresis
on 0.7% agarose gels at a constant current of 40 mA for 2 hr. The positions of
molecular weight standards (units in megadaltons), derived from X DNA by digestion with EcoRI,
are indicated.

The small nucleotide-sequence difference

exposed by restriction endonuclease digestion could have gone undetected by other,
less sensitive analytical techniques. The
Hind111 digestion patterns of the DNAs
from AN-20 and AN-22, phages which

-Md
2.46
I 42
I 36
1.12
0.96

show slight differences in plaque morphology and in plating efficiency on Anabaena sp. PCC 7120 (ATCC 27893), appear
similar. Their genetic difference thus appears to be very small, possibly only in the
nucleotide sequence of a single gene. The
DNA from neither of these phages was
susceptible to EcoRI. Because the patterns
of fragments generated from the DNAs of
AN-lo, N-l, A-l(L), and AN-15 by treatment with EcoRI and Hind111 are quite
disparate, it seems likely that these cyanophages are not closely related despite
their numerous similarities.

:::03
0.33

7. Fragments from (A) AN-15, (B) N-l, (C)

AN-lo, and (D) A-l(L), digested by Hind111 (10 units/
pg DNA, 18 hr), and then subjected to gel electrophoresis on a 2.5-7.5% gradient of polyacrylamide
at a constant voltage of 90 V for 5 hr. Lane E shows
the products of digestion of X DNA by EcoRI plus
HindIII.
The positions of corresponding
molecular
weight standards for lane A are indicated.
FIG.

ACKNOWLEDGMENTS
We thank Julie Peter and Jennifer Kraus for assistance in gathering samples and for other technical
assistance, and H. J. Kung, J. Nilson, and A. Ilan for
helpful discussions. This work was supported by the
U. S. Department
of Energy under Contract DEAC02-76ERO-1338, by National Science Foundation
Grant PCM 77-00808, and by the Science and Education Administration
of the U. S. Department
of
Agriculture
under Agreement
5901-0410-8-0166-0,
ORD 21919, from the Competitive
Research Grants
Office.
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