Ecological Research Series
EFFECTS OF CYANOPHA6E SAM-1
UPON MICROCYSTIS AERUGINOSA
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
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EPA-600/3-77-079
July 1977
EFFECTS OF CYANOPHAGE SAM-1 UPON MICROCYSTIS AERUGINOSA
by
D. L. Parker, G. P. Jansen, and L. Corbett
University of Wisconsin - Oshkosh
Oshkosh, Wisconsin 54901
Project P5J1190-J
Project Officer
William E. Miller
Special Studies Branch
Corvallis Environmental Laboratory
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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DISCLAIMER
This report has been reviewed by the Special Studies Branch, U.S.
Environmental Protection Agency, and approved for publication. Approval does
not signify that the contents necessarily reflect the views and policies of
the U.S. Environmental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
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FOREWORD
Effective regulatory and enforcement actions by the Environmental Protection
Agency would be virtually impossible without sound scientific data on pollu-
tants and their impact on environmental stability and human health. Respon-
sibility for building this data base has been assigned to EPA's Office of
Research and Development and its 15 major field installations, one of which
is the Corvallis Environmental Research Laboratory (CERL).
The primary mission of the Con/all is Laboratory is research on the effects of
environmental pollutants on terrestrial, freshwater, and marine ecosystems;
the behavior, effects and control of pollutants in lake systems; and the
development of predictive models on the movement of pollutants in the bio-
sphere.
This report centers around the isolation, culture and use of viruses as
regulators of blue-green algal blooms in natural waters.
A. F. Bartsch
Director, CERL
m
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ABSTRACT
Cyanophage SAM-1, which infects Synechococcus cedrorum, Anacystis
nidulans and certain strains of Microcystis aeruginosa has been isolated from
sewage. The host range of cyanophage SAM-1 differs from those of other
reported cyanophages. Phage SAM-1 stocks are rapidly inactivated at temper-
atures above 45°C or at pH values below 4 or above 10. The simultaneous
application of chloroform and of agitation reduces viral infectivity to 20%
of the original infectivity. The SAM-1 multiplication cycle has a latent
period of 10 hours, a rise period of an additional 6 hours, and eclipse
period of 6 hours, and an average burst size of 90 plaque-forming units per
infected cell. Electron micrographs show SAM-1 virions consisting of poly-
hedral head and a contractile tail with a distinctive terminal structure. The
properties of cyanophage SAM-1 are compared with those of other cyanophages.
Four distinct morphological types of M_. aeruginosa have been isolated
from the Winnebago Pool, Wisconsin. These types "breed true" upon cultiva-
tion and can be enumerated in water samples, by counting of characteristic
colonies growing on Jansen's agar. The virus-sensitivities, frequencies on
different dates, frequencies at different stations and other properties of
these ri. aeruginosa types are discussed.
The potential role which cyanophage SAM-1 may play in the limitation or
in the production of Microcystis blooms is discussed in terms of virus-
* sensitivities and their relationship to possible lysogeny.
This report was sumbitted in fulfillment of purchase order #P5J1190J by
Dr. Parker under the sponsorship of the U.S. Environmental Protection Agency.
This report was completed as of July 15, 1976.
IV
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CONTENTS
FOREWORD
ABSTRACT
LIST OF TABLES
LIST OF FIGURES
1. INTRODUCTION
2. MATERIALS AND METHODS.
Maintenance of Algal Cultures
Measurement of Light Intensity
Standard Growth Conditions
Standard Plaque Assay
Spot Test for virus-sensitivity of Blue Green Algae
Isolation and Propagation of Cyanophage SAM-1
Unialgal cultures of M_. aeruginosa (morphological types)
3. RESULTS 19
Host range of cyanophage SAM-1
Plaque morphologies and rates of plaque appearance
Stability of cyanophage SAM-1
SAM-1 Multiplication Cycle: One-step growth experiment
SAM-1 Multiplication Cycle: Premature lysis experiment
Electronmicrographs of cyanophage SAM-1
Properties of ML aeruginosa cultures from the Winnebago pool
Virus-sensitivities of M_. aeruginosa morphological types
Frequency of M_. aeruginosa morphological types at different
stations and times
Unexplained lytic phenomena
4. DISCUSSION 45
Properties of cyanophage SAM-1
Topics for further experimentation
5. REFERENCES 49
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LIST OF TABLES
Table Page
1 Properties of certain cyanophages 2
2 Comparison of cyanophages of Chroococales 3
3 Algal cultures 9
4 Jansen's medium 12
5 Host ranges of several cyanophages 20
6 SAM-1 plaques on susceptible hosts 21
7 Stability of cyanophage SAM-1 in water, MgCl,, and NaCl 22
8 Comparison of M_. aeruginosa morphological types 35
VI
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LIST OF FIGURES
Figure Page
1 The Winnebago Pool 7
2 Locations of stations 8
3 Stability of phage SAM-1 at different temperatures 23
4 Stability of phage SAM-1 at different pH values 25
5 Sensitivity of phage SAM-1 to chloroform and to agitation 26
6 SAM-1 multiplication cycle 27
7 Electronmicrograph of SAM-1 virion 29
8 Electronmicrograph of SAM-1 virion 30
9 Electronimicrograph of SAM-1 virion with contracted tail 30
10 Colony of M_. aeruginosa Cl, on Jansen's agar 31
11 Colony of M_. aeruginosa C2, on Jansen's agar 31
12 Colony of M_. aeruginosa C3, on Jansen's agar 32
13 Colony of M_. aeruginosa C4, on Jansen's agar 32
14 Culture of M^. aeruginosa Cl, in liquid medium 33
15 Culture of M. aeruginosa C2, in liquid medium 33
16 Culture of M_. aeruginosa C3, in liquid medium 34
17 Culture of M^. aeruginosa C4, in liquid medium 34
18 Frequency of M_. aeruginosa cells at different stations 37
19 Frequency of M_. aeruginosa types, station 1 38
20 Frequency of M_. aeruginosa types, station 2 39
21 Frequency of M. aeruginosa types, station 3 40
vii
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LIST OF FIGURES (Con't.)
Figure Page
22 Dissolved oxygen at each station 41
23 Water temperature at each station 42
24 Secchi disc readings at each station 43
25 Unidentified lytic agent affecting M_. aeruginosa Jl 44
26 Unidentified lytic agent affecting Synechococcus sp. 44
vi n
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SECTION 1
INTRODUCTION
The susceptibility of the Cyanophyta, or blue-green algae, to viral
infection was first established by Safferman and Morris (1963), when they
reported the isolation of cyanophage LPP-1. Since that time, the study of
cyanophages has expanded sufficiently to be the topic of several recent
review articles (Brown, 1972; Safferman, 1973; Padan and Shilo, 1973).
Tables 1 and 2 summarize the properties of certain cyanophages which
have been extensively characterized. These viruses can be grouped on the
basis of host range, serologic cross reactions and structure of the virion,
also called the extracellular virus particle. The virions of all cyanophages
thus far examined contain linear, double-stranded DNA and show a "head-plus-
tail" structure (Brown, 1972: Padan and Shilo, 1973). Three general types of
virion tails have been observed: (a) the short, non-contractile, but perhaps
extendable, tails or collars of viruses LPP-1, LPP-2 and SM-1, (b) the longer
contractile tails of viruses N-l and AS-1, and (c) the long, non-contractile
tail of virus S-l (Brown, 1972; Safferman, 1973; Padan and Shilo, 1973).
The several cyanophage groups each have rather narrow host ranges (Table
1). Taking into account the existence of resistant variant cells and of host
range mutant viruses, this specificity of the cyanophages could aid in iden-
tification and in typing of the Cyanophyta. Thus, the cyanophage might be a
useful tool in reorganization of cyanophyte systematics, in the same way that
Staphylococcus phages have been used for typing strains of S^. aureus and
other related species (Williams and Rippon, 1953).
Ecological studies indicate that cyanophages have a wide geographic
distribution (Safferman and Morris, 1964b; Padan and Shilo, 1969; Shane,
1971; Shilo, 1971, 1972; Singh, 1973). Of twelve waste stabilization ponds
sampled, Safferman and Morris (1967) detected LPP viruses in eleven ponds.
Similar viruses have been isolated from widely distributed sites in the
United States (Jackson, 1967; Safferman, 1968; Jackson and Sladecek, 1970;
Shane, 1971; Cannon, et^ al_., 1974), from fish ponds in Israel (Padan and
Shilo, 1969), from stabilization ponds and streams in Scotland (Daft et al..
1970) and from water bodies in India (Singh and Singh, 1967). Viruses of the
SM-1, MA-1 group have been isolated both in the United States (Safferman ejt
al., 1969) and in Russia (Goryushin and Chaplinskaya, 1966, 1968). Cyano-
phage AS-1 has been recovered at least twice, once from Florida by Safferman
e_t al_., (1972) and again from Missouri by Sherman (1975). Cyanophages are
characteristically more prevalent in polluted or eutrophic waters than in
less polluted or hypotrophic waters (Safferman and Morris, 1964b, 1967; Singh
and Singh, 1967; Padan and Shilo, 1969; Singh, 1971; Safferman et. al.., 1972;
Shane e_t aj_., 1972; Shilo, 1972; Singh, 1973, 1974; Cannon et al_., 1974).
1
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TABLE 1 PROPERTIES OF CERTAIN CYANOPHAGES
Virus
LPP-1
LPP-2
N-l
MA-1
SM-1
AS-1
Reference
Safferman & Morris
(1963)
Safferman et al . ,
(1969)
Adolph & Haselkorn
(1971)
Chaplinskaya &
Goryushin (1966)
Safferman et al . ,
(1969T
Safferman et al . ,
(1972)
*
Source
waste stabilization
pond, Indiana
waste stabilization
ponds, U.S.
lake, Wisconsin
reservoir, U.S.S.R.
waste stabilization
pond, Indiana
waste stabilization
pond, Florida
Known Hosts
Lyngbya, Plectonema
Phormidium
Lyngbya, Plectonema
Phormidium
Nostoc muscorum
Microcystis aeruginosa
Microcystis pulverea
Microcystis muscicola
Synechococcus elongatus
Microcystis aeruginosa
Anacystis nidulans
Synechococcus cedrorum
Anacystis sp.
Known Serologic
Cross reactions
LPP-1G (GUI)
LPP-1D (D-l)
several LPP-1
isolates
LPP-2-SPI
several LPP-2
isolates
itself only
?
itself only
AS-1M
S-l Adolph & Haselkorn
(1973)
Synechococcus sp.
itself only
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TABLE 2 COMPARISON OF CYANOPHAGES OF CHROOCOCCALES
CO
S-l
VIRION STRUCTURE
Head
Edge-edge diameter 50 nm
Tail non-contractile
Length, width 140 nm
Tail length/head diameter 2.8
Tail length/tail width
STABILITY OF
VIRION
In disti 1 led water
Mg required
pH range of
greatest stability
Temperature range of
greatest stability
Inactivation
SM-1 AS-1
icosahedron polyhedron
(icosahedron?)
67 nm 90 nm
short, if present contractile sheath
rigid core
base plate
243.5 nm, 22.5 nm
2.7
10.8
stable not stable
no no
pH 5-11 pH 4-10
4-40C
55 C 60 C
SAM-1
polyhedron
(icosahedron?)
contractile sheath
rigid core
terminal structure
-
2.5
9
stable
no
pH 4-10
4-45C
55 C
REFERENCES Adolph and
Saf ferman et al . Saf ferman et al .
Haselkorn (1973)
(1969)"
(1972)"
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Several cyanophages have been isolated from water samples taken during algal
blooms or fish kills (Goryushin and Chaplinskaya, 1966, 1968; Singh, 1967;
Padan and Shilo, 1969, 1973).
The widespread occurrence of cyanophages suggests that they may signif-
icantly influence the cyclic succession of cyanophyte populations in nature.
Viruses might affect cyanophyte populations in several ways. First, free
virus particles (virions) persisting in a body of water might infect and lyse
susceptible host cells, thus limiting the host density (Safferman and Morris,
1964b, 1967; Goryushin and Chaplinskaya, 1966, 1968; Padan and Shilo, 1969;
Jackson and Sladecek, 1970). Second, several cyanophages can enter "lyso-
geny," in which a non-infectious viral entity persists inside infected cells
without producing any major effect on cell growth, division or survival
(Singh ejt al_., 1969, 1972; Cannon et al_., 1971, 1972; Padan ejt al_. , 1972;
Cannon, 1975; Rimon et. al_., 1975). Certain environmental conditions can
simultaneously "induce" virus multiplication and subsequent cell lysis within
most cells of a lysogenic population, with the result that a dense and appar-
ently healthy culture can suddenly lyse (Cannon, 1975; Rimon et_ al_., 1975).
Such induction of virus multiplication in lysogenic cultures could play a
role in the rapid decline of algal blooms, or it could be involved in the
production of certain apparent blooms, characterized by moribund cells float-
ing on the water surface. In the latter case, lysis of a dense but dispersed
population could cause the cells to float upward and become concentrated at
the surface. Evidence concerning the potential roles of virions and of
lysogenic cells in cyanophyte population dynamics is considered in the follow-
ing two paragraphs.
That an equilibrium lytic infection involving persisting virions may
limit cyanophyte populations has been proposed by Safferman and Morris (1967)
and by Padan and Shilo (1969), both of whom observed that susceptible algal
species were never dominant in ponds containing significant concentrations of
virions infecting those species. Safferman and Morris (1964) demonstrated
that cyanophage LPP-1 could limit Plectonema populations under conditions
simulating natural algal blooms. A similar phenomenon was reported by Jackson
and Sladecek (1970), who suggested that naturally-occurring virions in 5000-
gallon (18,900 L) sewage treatment tanks were responsible for their repeated
inability to produce Plectonema blooms in these tanks. Goryushin and Chaplin-
skaya (1966, 1968) observed that certain areas of the Kremenchug reservoir
had clear areas even during the height of a Microcystis bloom and fhat these
areas contained a virus infecting Microcystis. Furthermore, Russian investi-
gators have reported success in using this virus to clear Microcystis blooms
in a large reservoir (Padan and Shilo, 1973).
Lysogeny by cyanophages LPP-1 D, LPP-2SPI, AS-1 and N-l has been reported
(Singh et. al_., 1969, 1972; Cannon et al_., 1971, 1972; Padan et. al_., 1972;
Cannon, 1975; Rimon ejt al_., 1975). The probability that infected cells will
enter the lysogenic cycle rather than the lytic viral multiplication cycle is
frequently increased by environmental stress factors such as antibiotics,
HgCl2, CuSOit, and nitrogen starvation (Cannon ejt al_., 1972; Cannon, 1975).
Lysogenic cells carrying a temperature sensitive mutant of LPP-2SPI can be
induced to produce virus by increasing the growth temperature (Rimon et al.,
1975). Lysogenic cells carrying LPP-1D, AS-1 or N-l can be induced to
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release virus by the addition of mitomycin C (Cannon ejt al_., 1971, 1972;
Cannon, 1975). Lysogeny may play a significant role in the persistence of
cyanophages in certain habitats. It might explain the rapid decline or rapid
formation of certain algal blooms. Lysogeny might also influence determina-
tions of viral host ranges, because lysogenic cells can not ordinarily be
lysed by a virus which they are carrying, and thus would incorrectly be
considered not to be a host for that virus.
In the present communication, a new cyanophage infecting Synechococcus
cedrorum, Anacystis nidulans and Microcystis aeruginosa is described. In
accordance with the established system of nomenclature (Safferman and Morris,
1963), this virus has been designated cyanophage SAM-1. Because of the
unusual observation that only certain M_. aeruginosa isolates are sensitive to
SAM-1 virus, several additional M_. aeruginosa strains have been isolated,
classified into four morphological types, and tested for virus-sensitivity.
The potential role which cyanophage SAM-1 may play in the limitation or in
the production of Microcystis blooms is discussed in terms of these virus-
sensitivities and in relation to possible lysogeny.
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SECTION 2
MATERIALS AND METHODS
Lake Winnebago, which borders the eastern side of Oshkosh, Wisconsin,
U.S.A., was the major source of the Cyanophyta. Lake Winnebago is the
largest body of water within the boundaries of the state and is one of the
largest freshwater lakes in the United States. Birge and Juday (1914) termed
Lake Winnebago a glacial dam lake having an area of 208.3 mi2 (557.7 km2),
and a maximum depth of 6.4 m. The lake has a continuous mixing pattern
thoughout the ice-free months of the year. Lake Winnebago is part of the
"Winnebago Pool," a complex, shallow reservoir having an area of 265 mi2 (708
km2) and comprising Lake Butte des Morts, Winneconne and Poygan (Figure 1).
The pool is in the watershed of the Fox and Wolf Rivers, which join in Lake
Butte des Morts and which exit Lake Winnebago through the lower Fox River to
Green Bay and Lake Michigan (Sloey, 1970).
The Fox-Wolf watershed comprises some 6,000 mi2 (15,530 km2) and is the
largest basin to Lake Michigan (Sloey, 1970). The Winnebago Pool retains
some 25 x 109 ft3 (70 x 106 m3) of water (Olcott, 1966) and is presently the
source of domestic water for the cities of Oshkosh, Neenah-Menasha and
Appleton, which have a combined population of 150,000 (Sloey, 1970).
Three stations were sampled for both viral and planktonic algal content
during this study (Figure 2). Stations one and two are located in the Fox
River; upstream, station 1, and downstream, station 2, from the city of
Oshkosh's sewage treatment plant waste discharge. Station three is located
in Lake Winnebago near Oshkosh1s water departments pretreatment basin.
The Fox-Wolf River System is the greatest contributor of nutrients to
Lake Winnebago (EPA, 1974). In addition, there are numerous smaller tribu-
taries which further contribute to the input of nutrients. These sjnaller
tributaries flow through various types of ecological habitats bordering the
lake, notably urban, agricultural and marsh areas. The Winnebago-Pool is
hypereutrophic (Sloey, 1970); and supports growth of numerous cyanophytes,
each of which exhibits a particular seasonal fluctuation (Sloey and Blum,
1972).
MAINTENANCE OF ALGAL CULTURES
Table 3 lists each algal culture, its source and its reference number in
the University of Wisconsin-Oshkosh culture collection. Isolation of M_.
aeruginosa morphological types Cl, C2, C3 and C4 is considered under tFe
appropriate heading below.
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Fox River (Lower)
Lake Butte des
Morts
Fox River (Upper)
Figure 1. The Itfinne&ago Pool, east-central Wisconsin (Sloey, 1970).
Scale: 1:250,000 ;
Fond
du Lac
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co
OSHKOSH
WI^NEBAGO COUNTY
WISCONSIN
Figure 2. Locations of stations 1, 2, and 3. Symbols: 0 Station 1;f2J Station 2;/3\ Station 3.
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TABLE 3 ALGAL CULTURES
UNIALGAL CULTURES (NOT AXENIC)
AXENIC CULTURES DERIVED FROM THAT CULTURE
Name as arrived at UW-0#
UW-0 Unialgal
Source
Axenic culture
Derived by
uw-o#
Axenic
Synechococcus elongatus S2
IU #563
Synechococcus cedrorum SI
IU #1191
Synechococcus cedrorum J3
J3
Anacystis nidulans Acl
IU #625
Anacystis sp. (Probably J4
nidulansj J4
Gloecapsa calcarea Gl
Til den 1898
Merismopedia elegans Mel
Osci1latoria subbrevis Os 1
Schmidle 1901b
Anabaena flos-aquae
"[tyngb.T Cte Brebisson
R. Safferman
E.P.A.
Cincinnati 7/15/74
R. Safferman
E.P.A.
Cincinnati
7/15/74
Lake Winnebago
G. P. Jansen
4/15/73
R. Safferman
E.P.A.
Cincinnati
7/15/74
Lake Winnebago
G. P. Jansen
3/20/73
Lake Winnebago
G. P. Jansen
8/15/74
Lake Winnebago
G. P. Jansen
8/15/74
Lake Winnebago
G. P. Jansen
8/15/74
Lake Winnebaqo
G. P. Jansen
6/10/73
L. Corbett
G. P. Jansen
J. McBain
L. Corbett
G. P. Jansen
L. Corbett
L. Corbett
G. P. Jansen
J. McBain
G. P. Jansen
J. HeBain
S2C2
S1G1
SI MB!
S1C1
J3G1
J3C1
AclCl
J4G1
OslMBl
AblMBl
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(TABLE 3 CON'T)
UNIALGAL CULTURES (NOT
Name as arrived at
UW-0
Nostoc muscorum
C. A. Agardh 1812
Phormidium sp.
Euglena gracilus*
Klebs 1883
Microcystis aeruginosa
Kuetzing
Microcystis aeruginosa
Kuetzing
Microcystis aeruginosa
Kuetzing
Microcystis aeruginosa
Kuetzing
Microcystis aeruginosa
Kuetzing
Microcystis aeruginosa
Kuetzing; UC#7005
Microcystis aeruginosa
Kuetz. f. minor Elenkin
Synechococcus cedrorum
Saug.
Synechococcus dtedrorum
Saug. ; IU #1191
Synechococcus elongatus
Naegeli; IU #563
AXENIC)
uw-o#
Unialgal
Nl
PI
El
Cl
C2
C3
C4
Jl
M2
M4
J3G1
S1G1
S2G2
AXENIC CULTURES
Source
Lake Winnebago
G. P. Jansen
7/4/73
Lake Winnebago
G. P. Jansen
10/10/73
Lake Winnegago
G. P. Jansen
8/5/74
L. Corbett
8/27/75
L. Corbett
8/27/75
L. Corbett
L. Corbett
8/27/75
G. P. Jansen
10/10/72
P. J. Reilly
U. of Nebraska
7/23/73
E.P.A.
Corvallis
12/5/73
G. P. Jansen
E.P.A.
Cincinnati
7/15/74
E.P.A.
Cincinnati
7/15/74
DERIVED FROM THAT CULTURE
Axenic culture
Derived by
G. P. Jansen
J. McBain
~
~
L. Corbett
L. Corbett
L. Corbett
L. Corbett
G. P. Jansen
Stanier et al. ,
1971
-
G. P. Jansen
Starr, 1964
Starr, 1976
UW-0#
Axenic
N1G1
NIMBI
"
"
-
-
-
-
-
~
-
J3G1
S1G1
S2G2
*Green alga
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Table 4 gives the composition of Jansen's medium. Jansen's liquid
medium is prepared by mixing 2 parts of sterile solution A, 5 parts of
sterile solution B and 3 parts of sterile glass-distilled water. Jansen's
agar is prepared by mixing 2 parts of sterile solution A, 5 parts of sterile
solution B and 3 parts of sterile, melted 3.33% w/v Bacto agar (Difco Co).
Solutions A and B are autoclaved separately and stored in opaque containers.
Jansen's cycloheximide agar is Jansen's agar containing 30 yg of cyclo-
heximide (Sigma Co.) per ml. This concentration of cycloheximide inhibits
the growth of eukaryotes but not of prokaryotes.
Tris hydroxymethylamino ethane (Tris) was dissolved to 0.01 M in glass
distilled water, adjusted to pH 8.2 and autoclaved.
Measurement of light intensity
A Panlux light meter (Gossen-Ascor Co.) was used. The light intensity
for liquid cultures was determined at the culture's minicus, at the outside
surface of the glass container. The light intensity for petri plate cultures
was determined at the agar surface on which the colonies were growing, by
placing the light meter inside the plastic bag and either 1 mm below the agar
surface (for inverted plates) or at the level of the agar surface (for non-
inverted plates).
Standard growth conditions
All cultures were propagated at 26-27°C and were illuminated from above
by Westinghouse CW 40 fluorescent lamps. Petri plates were incubated in
plastic bags, which were folded once at their openings and which contained
moist paper towels below the petri plates. Unless otherwise specified,
cultures were not agitated. Erlenmeyer flasks, vials and test tubes contain-
ing liquid cultures were filled to 1/5 of their maximum capacity and were
loosely capped. Vials and test tubes are incubated on their sides, at 20°
from the horizontal. Sterile media, sterile glassware and aseptic technique
were used throughout.
Standard plaque assay
Ten-fold serial dilutions of the virus suspension were prepared in
Jansen's liquid medium. One tenth ml of each viral dilution was added to 4
ml of a 3-day-old culture containing 1 x 108 cells/ml of the host organism.
After adsorption for 1 hour at 26°C, the virus-host mixture was combined with
5 ml of melted (48°) Jansen's agar, then poured into a 15 x 100 mm petri
plate already containing 25 ml of solidified Jansen's agar. The plates were
incubated top-side-up overnight at 120 ft-c and at standard growth conditions,
then inverted and incubated as before. The number of plaque forming units/ml
(pfu/ml) was calcualted from the number of plaques and the dilution factor,
with S_. cedrorum J3G1 as the host. The pfu/ml for phages AS-1 and SM-1 were
similarly calculated, except that the hosts were S_. cedrorum S1G1 and S_.
elongatus S2C2 respectively.
11
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TABLE 4 JANSEN'S MEDIUM
SOLUTION A
NaN03
Na2HP04 . 12H20
K2HP04 . 3 H20
Na9SiO~ . 5 H90
L- O <_
MgS04 . 7 H20
MgCl2 . 6 H20
Tricine buffer (Sigma Chem. Co.)
2x distilled water
1 N NaOH
SOLUTION B
Fed, . 6 H?0
•J L-
CaCl2 . 2 H20
Na,., ethyl enedi ami netetraacetate
H3B03
MnS04. 4 H20
ZnS04 . 7 H20
(NHJ, Mo7 0,. . 4H90
40 / <14- L.
CuS04 . 5 H20
Co(N03)2 . 6 H20
A12 K(S04)4 . 24 H20
Ni S04(NH4)2 . 6 H20
KI
2 x distilled water
JANSEN'S LIQUID MEDIUM
Solution A
Solution B
2 x distil led water
JANSEN'S AGAR MEDIUM
Solution A
Solution B
3.33% w/v Bacto agar (Difco Co.)
670 mg
17 mg
50 mg
75 mg
100 mg
50 mg
250 mg
200 ml
to pH 8.3
5.00 mg
42.00 mg
4.00 mg
3.20 mg
2.40 mg
0.30 mg
0.08 mg
0.13 mg
0.15 mg
0.47 mg
0.20 mg
0.09 mg
500 ml
•
200 ml
500 ml
300 ml
200 ml
500 ml
300 ml
12
-------
Spot Test for Virus-Sensitivity of Blue-green Algae
Four ml of a 3-day-old culture of the appropriate alga was mixed with 5
ml of melted (48°C) Jansen's agar, then poured into a 15 x 100 mm petri plate
already containing 25 ml of solidified Jansen's agar. Alternatively, fila-
mentous algae were sometime filtered onto type HA Millipore filters which
were placed algae-side-up on the solidfied Jansen's agar. One drop of virus
suspension, diluted to give 105 pfu/ml in plaque assays with S^ cedrorum
J3G1, was applied to a localized spot on the plate or filter. The plate was
incubated as for the standard plaque assay. The host was considered virus-
sensitive if lysis was observed in the spot within 14 days.
Isolation and Propagation of Cyanophage SAM-1
The virus was isolated from an untreated sewage sample entering the
Oshkosh, Wisconsin, sewage treatment plant. This sewage sample would be
expected to contain municipal and industrial wastes, run-off water, and a
considerable fraction of chlorinated water from Lake Winnebago, since the
Oshkosh municipal water comes from this lake.
The virus was isolated and cloned following a procedure modified from
that of Saffermen (1968). One hundred ml of the sewage sample, which had
been filtered through a 0.45 ym pore-size (type HA) Millipore filter, was
added to 900 ml of a 3-day-old, exponentially-growing culture of Synechococcus
cedrorum J3G1 in Jansen's liquid medium. The culture was incubated at 150
rev/min, at 150 ft-c and at standard growth conditions. Lysis, characterized
by bleaching and decreased turbidity of the culture, was apparent by the
ninth day of incubation, at which time the culture, was centrifuged at 6000
rev/min (5860 x g average) for 15 minutes, to separate cells and debris from
the potentially virus-containing supernate. The supernate was filtered
through a 0.45 y Millipore filter, and stored at 4°C. The pellet was resus-
pending in 100 ml of 0.01 M Tris buffer, pH 8.2, containing 17% v/v of chloro-
form. The resuspended pellet was shaken at 200 rev/min for 10 minutes, then
allowed to settle for 20 minutes at 4°C. The original supernate and the
supernate from the resuspended pellet were combined, centrifuged at 5860 x g
for 15 minutes, and then filtered through 0.22 ym pore-size (type GS) Milli-
pore filter.
The filtrate was assayed for virus by the standard plaque assay with S_.
cedrorum J3G1 as the host organism. A viral clone was obtained by removing
the agar from the center of a well-isolated plaque and suspending this virus-
containing agar in 2 ml of Jansen's liquid medium. After the suspension had
been stored overnight at 4°C, it was centrifuged at 5860 x g for 15 minutes
and the supernate was assayed for virus by the standard plaque assay. This
cloning procedure from randomly-selected, isolated plaques was repeated four
times in series.
Propagation of Cyanophage SAM-1: liquid medium method--
Cultures of S^. cedrorum J3G1 containing between 5 x 107 and 1 x 108
cells/ml were infected with a final concentration of 1 x 106 pfu of virus/ml,
then incubated at standard conditions and 60 rev/min in the dark. After 1
13
-------
hour in the dark, the culture was incubated at 100 rev/min and 150 ft-c,
under standard growth conditions, for 3 to 5 days, until lysis occurred.
Chloroform was added to 17% v/v. After sedimentation of the lysate at 5860 x
g for 15 minutes, the supernate was filtered through a .45 vm Mi 11ipore
membrane and stored at 4°C.
Propagation of Cyanophage SAM-1: plate method—
One tenth ml of a virus suspension containing 105 pfu/ml was added to 4
ml of a 3-day-old culture of S_. cedrorum J3G1, then incubated in the dark at
26°C and 60 rev/min. After 1 hour, the virus-host mixture'was added to 4 ml
of melted (48°C) Jansen's agar, poured, and incubated as for the standard
plaque assay. After confluent lysis of the host lawn developed, 10 ml of
sterile 0.01M Tris buffer, pH 8.2, was dispensed onto the agar surface. The
plates were incubated for 60 minutes at 26°C. The buffer and the top agar
layer were carefully scraped into a 1000 ml beaker. After the contents of
the beaker had been allowed to settle for 75 mintues at 4°C, the buffer was
carefully decanted and centrifuged at 5860 x g for 15 min at 4°C. The
supernate was filtered through a .45 ym Millipore membrane and stored at 4°C.
Propagation of Cyanophages AS-1 and SM-1--
The procedures were identical to those used for cyanophage SAM-1, except
that the hosts for phages AS-1 and SM-1 were S^. cedrorum S1G1 and S_. elongatus
S2C2, respectively.
Stability of Cyanophage SAM-1 at Different pH Values--
Samples of Jansen's liquid medium were adjusted to different pH values
by the addition of 5M NaOH or 5M HC1. Ten ml of the medium at a particular
pH was added to a tube containing 0.1 ml of a virus suspension originally at
pH 8. The final virus concentration was 2 x 106 pfu/ml. Each mixture was
incubated at 26°C for 1 hour, then brought to pH 8 by a 1/10 dilution into pH
8 Jansen's liquid medium. Serial 10-fold dilutions in pH 8 Jansen's liquid
medium were immediately assayed for virus by the standard plaque assay.
Multiplication Cycle of Cyanophage SAM-1: one-step growth and premature
lysis experiments--
*•
A three-day-old culture of S_. cedrorum J3G1 in Jansen's liquid medium
was diluted into an equal volume of Jansen's liquid medium supplemented with
0.1 M NaCl. This diluted culture, containing 2 x 108 cells/ml, was incubated
at 150 rev/min, 150 ft-c and standard growth conditions. After 30 minutes of
incubation, phage SAM-1 was added to a final concentration of 4 x 105 pfu/ml.
The infected culture was incubated at 25°C and 80 rev/min in the dark, during
a 1 hour "adsorption period." After the adsorption period, 1 ml samples of
the infected culture were diluted 10"2 and 10"4 in Jansen's liquid medium, to
prevent futher virus adsorption to host cells. The two diluted cultures,
each consisting of 100 ml in a 500 ml Erlenmeyer flask, were incubated at 150
rev/min, 150 ft-c. and standard growth conditions. At regular intervals,
samples were withdrawn from each culture, diluted if necessary, and assayed
for plaque-forming units. Other samples from each culture were prematurely
14
-------
lysed by the addition of chloroform, as follows: every 30 minutes, a 1 ml
sample was removed from each culture and transferred to a 15 ml centrifuge
tube containing 0.1 ml of chloroform. This chloroform-containing suspension
was vigorously agitated on a Vortex-Genie mixer (Scientific Industries, Inc.)
at maximum speed for 15 seconds. The suspensions were centrifuged at 5000
rpm for 5 minutes, diluted when necessary, and assayed for plaque forming
units.
Sensitivity of Cyanophage SAM-1 to Chloroform and to Agitation—•
A virus stock was diluted to contain 105 pfu/ml. Sixty ml amounts of
diluted virus stock were transferred to each of nine 250 ml Erlenmeyer flasks
at 4°C. Ten ml of chloroform was added to each of 3 flasks, which were then
allowed to stand undisturbed for 30 min at 4°C. Ten ml of 0.01 M Tris buffer,
pH 8.2, was added to each of three flasks, which were then agitated at 150
rev/min for 60 sec and subsequently allowed to stand for 30 min at 4°C. Ten
ml of chloroform was added to each of three flasks, which were then agitated
at 150 rev/min for 60 sec and subsequently allowed to stand for 30 min at
4°C. The infective virus in each flask was assayed using the standard plaque
assay.
Partial Purification of Cyanophage SAM-1 Stocks--
Step-type sucrose gradients were prepared by carefully layering 2.0 ml
of each appropriate sucrose solution (10%, 20%, 25%, 30%, 35% and 40% surcrose
in 0.01 M Tris buffer, pH 8.2) into 1.6 cm x 10.2 cm cellulose nitrate
centrifuge tubes (Beckman Instruments, Palo Alto, California) while the tubes
were tilted 60° from the vertical. The gradients were stored undisturbed for
24 hr at 4°C, allowing diffusion to form a quasi-continuous sucrose gradient.
After 24 hr, a 1.8 ml sample of a previously-cloned cyanophage stock was
layered onto the top of the gradient. The tubes were placed in the SW27.1
rotor of a Beckman Model L5-50 ultra-centrifuge and centrifuged at 24,000 rpm
(120,000 x g) for 20 min.
One ml fractions from the gradients were collected, scanned for absorp-
tion of ultraviolet light (at 260 and 280 nm) and analyzed for infective
virus by the standard plaque assay. Fractions containing virus were pooled
and dialyzed overnight against 0.01 M Tris buffer, pH 8.2. This dialyzed
preparation was centrifuged a second time and dialyzed again by the same
procedure. Any insoluble material was removed by centrifugation for 15 min
at 1000 x g.
Electron Microscopy--
Virus suspensions which had been partially purified by surcrose-gradient
ultracentrifugation and dialysis overnight against 0.01 M Tris buffer, pH
8.2, were used. A small droplet of virus suspension was deposited on the
center of a carbon- and Colloidon-coated, 200 mesh copper grid. After 90
sec, and without removing any of the droplet, the grid was inverted onto a
solution (at pH 4.5) of 2% w/v uranyl acetate (Mallinckrodt Chem., St. Louis,
Mo.), and 0.15% w/v polymixin B sulfate (Sigma Chemicals, St. Louis, Mo.).
After 5 min, all excess liquid was carefully removed by touching a piece of
15
-------
filter paper to the edge of the grid. The specimen was allowed to air dry
for 5 min before observation in a R.C.A. model EMU-36 transmission electron
microscope.
Determination of M_. aeruginosa Frequency at Different Stations and on Differ-
ent Dates--
Two water samples, one from 12.7 cm below the water surface and another
from 12.7 cm above the bottom, were periodically removed with a Wildo Corp.
horizontal sampler, at the three stations indicated on Figure 2. These
stations were: (a) station 1, at the end of Knasta's boat dock, 3.0 m from
the south bank of the Fox River and 213 m upstream from the junction of the
Fox River and Campbell Creek, which is Oshkosh's sewage treatment plant
outlet; (b) station 2, 0.6 m from the south bank of the Fox River and 0.9 m
downstream from the junction with Campbell Creek, and (c) station 3, 1.5 m
from the shore of Miller's Bay in Lake Winnebago, 21 m south of the southeast
corner of the Oshkosh Water Department pretreatment basin.
The M_. aeruginosa cells in each sample were counted by microscopic
observation in a Sedgwick-Rafter cell. If necessary, aggregates were dis-
persed so that individual cells could be counted. Estimates of cell concen-
trations obtained by this procedure are termed microscopic counts.
The M^. aeruginosa cells (or aggregates) capable of forming colonies on
Jansen's agar were counted as follows. Appropriate volumes of each sample,
chosen to give 100-200 cyanophytes/plate, were spread on the surface of
Jansen's cycloheximide agar in 15 x 100 mm petri plates. Samples containing
less than 200 cyanophytes/ml were filtered onto 0.45 ym Millipore filters
which were placed algae-side-up on the Jansen's cycloheximide agar. The
plates were incubated at 150 ft-c and standard growth conditions. After 1 to
2 weeks, the plates were inspected at 3x or 6x magnification under a dissect-
ing microscope and colonies typical of M_. aeruginosa were counted. Counting
was performed while the colonies were still small and before filamintous
cyanophytes spread over the plates. Although F4. aeruginosa colonies grown on
Jansen's agar can be readily identified by an experienced observer, the
identification's were confirmed by microscopic observation, under lOOOx
magnification, of cells aseptically removed from representative colonies.
Each M_. aeruginosa cell (or aggregate) which yielded one colony on Jansen's
agar was considered to be one colony-forming unit. Estimates of M_. aeruginosa
concentrations obtained by this procedure are termed viable counts.
Unialgal cultures of M. aeruginosa morphological types
Several distinct M_. aeruginosa colony types were observed on Jansen's-
agar-containing petri plates used in obtaining M_. aeruginosa viable counts.
Cells from representative colonies of each colony type were aseptically
removed with a flamed inoculating loop and transferred into 10 ml screw-cap
vials, each containing 2 ml of Jansen's liquid medium. The vials were
incubated for 2 weeks, at 150 ft-c and at standard growth conditions. As
soon as these cultures showed a green layer of growth at the surface of the
medium, cells from the surface growth were cloned by streaking on Jansen's
a.g = r. using a flamed inoculating loop and bacteriological streaking procedures
16
-------
(Rouf, 1970). The plates were inverted and incubated under standard condi-
tions and at 150 ft-c, until colonies formed. Cells from well-isolated M_.
aeruginosa colonies were streaked onto another plate containing Jansen's agar
medium, and this cloning procedure was continued until the culture was
unialgal. In cases in which repeated streaking failed to yield unialgal
cultures, a 15 ml conical centrifuge tube containing 8 ml of a culture in
Jansen's liquid medium was agitated for 2 minutes at the maximum speed of a
Vortex-Genie mixer, and then centrifuged at 5000 x g for 5 minutes.' Most M_.
aeruginosa cells moved to the top of the tube, whereas other cyanophytes
usually moved to the pellet. Cells were removed from the top of the centri-
fuge tube, resuspended in 8 ml of Jansen's liquid medium and centrifuged once
more. Cells from the top of the second centrifuge tube were both streaked
onto Jansen's agar and inoculated into a 10 ml screw-cap vial containing 2 ml
of Jansen's liquid medium. Various combinations of centrifugation, streaking
on agar and cultivation in liquid media (required periodically because M_.
aeruginosa does not grow indefinitely on agar media) produced unialgal
cultures.
Cultures were considered unialgal if only M_. aeruginosa could be observed
after very careful microscopic observation of liquid cultures, including
observation of capsular material. Senescent liquid cultures were carefully
monitored for contaminating algae and for green growth below the surface of
the medium, usually indicative of contaminating algae. The cultures were
streaked on Jansen's agar, to observe for colonies not typical of M_. aeru-
ginosa.
Photography of M_. aeruginosa Morphological Types--
A Pentax Co. "Spot-Matic" camera mounted on a Zeiss model RA-38 micro-
scope and containing Ektachrome High Speed (EH135) film was used. Specimens
were illuminated from below by the microscope's light system. Colonies on
agar were also illuminated from the side, by two 500 W Sylvania Photo-Ect
lamps, at 30 cm from opposite sides of the specimen.
The photographed colonies on Jansen's agar had been cultured for 10
days, at 80 ft-c and at standard growth conditions. The petri plate lid was
removed; a characteristic colony was centered under the microscope's low
power (10x) objective and photographed. The colonies were magnified 10 times
by the microscope and approximately 6-8 times by enlargement of the prints.
The cells photographed in Jansen's liquid medium had been cultured for
10 days at 150 ft-c, in 10 ml vials and at standard growth conditions. Two
drops of each culture were placed on a microscope slide, covered with a
coverslip, and photographed under the oil-immersion objective lens. Cells on
the photographic prints were magnified 100 times by the microscope and
approximately 6-8 times by enlargement of the prints.
Measurements of Dissolved Oxygen, Water Temperature and Water Clarity--
The dissolved oxygen in each water sample was determined as described by
the American Public Health Association (1971). Water temperature was measured
with a Yellow Springs Inst. Co. Tele-thermometer, by lowering the instrument's
17
-------
probe to the position of each water sample. Water trubidity was measured by
raising a Secchi disc (Sloey, 1970) through the water and recording the
distance at which the disc first became visible.
-------
SECTION 3
RESULTS
The SAM-1 virus was isolated from an unchlorinated sewage sample. It
was initially detected through the lysis of a laboratory culture of Syne-
chococcus cedrorum J3, on which host it was also cloned four times in series,
each time by preparing a viral stock from a well-isolated plaque.
HOST RANGE OF CYANOPHAGE SAM-1
To determine the host range of cyanophage SAM-1, plaque assays and "spot
tests" involving both diluted and undiluted SAM-1 stocks were performed with
a variety of algal hosts from the UW-0 algal culture collection (Table 3).
The results are summarized in Table 5. Cyanophage SAM-1 produced plaques
only with Synechococcus cedrorum (strains SIGI and J3GI), Anacystis m'dulans
(strains AclCl and J4GI) and Microcystis aeruginosa (strain Jl only). It did
not produce observable plaques with any of the other organisms tested, even
when undiluted virus suspensions were used. Although phage SAM-1 was capable
of lysing strain Jl of M_. aeruginosa, it did not lyse M_. aeruginosa strains
M2 and M4.
For purposes of comparison, plaque assays and spot tests on the same
hosts were also performed with cyanophages AS-1 and SM-1 cyanophages which
also infect members of Chroococcales. These data are shown in Table 5, which
also list previously-published host range data for another cyanophage of
Chroococcales, cyanophage S-l. It can be seen that the host range of cyano-
phage SAM-1 is related to, but distinct from, the host ranges of cyanophages
AS-1, SM-1 and S-l.
The results in Table 5 apply to unialgal, but not necessarily axenic
cultures. The names of the nonaxenic host cultures are followed by an
asterik.
PLAQUE MORPHOLOGIES AND RATES OF PLAQUE APPEARANCE ON DIFFERENT HOSTS
Cyanophage SAM-1 formed plaques with both axenic and nonaxenic cultures
of S^. cedrorum and A_. nidulans (Table 6). Axenic cultures of M. aeruginosa
Jl have not been isolated. Plaques consistently became visible earlier on
axenic cultures than on nonaxenic cultures of the same strain. Plaques on
nonaxenic cultures exhibited bacterial microcolonies inside the otherwise
clear plaques. With axenic cultures, no microcolonies were seen inside the
clear plaques.
Table 6 lists the plaque morphologies produced by cyanophage SAM-1 on
different susceptible hosts. For most hosts, clear plaques with sharply
19
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TABLE 5. HOST RANGES OF SEVERAL CYANOPHAGES
Host Test Organisms
Virus
Cyanopnyta S-l*
Synechococcus strain NRC-1 +
SM-10 AS-10 SAM-1#
U.R.a
Synechococcus elongatus ILJ 563 +
Microcystis aeruginosa.NRC-1 +
Anacystis sp. IU 1549 g -<£ -i -i U.R.
Anacysits nidulans IU 625, 1550 - - + +
Synechococcus cedrorum IU 1191 + +
Synechococcus sp. J-3 U.R. + +
Microcystis aeruginosa J-1 U.R. +
Microcystis aeruginosa 1036 -
Microcystis aeruginosa 7005 -
Microcystis aeruginosa EPA U.R.
Anabaena flos-aquae U.R. -
Nostoc muscorum U.R. -
Phormidium ambiguum U.R. -
S-l* SM-10 AS-1@ SAM-1#
Bacteria
Escherichia sp. U.R. -
Bacillus sp. U.R. -
Aerobacter sp. U.R. -
Aeromonas sp. U.R. -
Acaligenes sp. U.R.
Proteus sp. U.R. -
Pseudomonas sp. U.R. -
Serratia sp. U.R. -
Streptococcus sp. U.R. -
Contaminants #1-#9 from U.R. -
Microcystis aeruginosa Jl
*Host range data from Saffermen and Haselkorn, 1973.
(^Viruses kindly provided by R. S. Safferman, E.P.A., Cincinnati, Ohio.
#Isolated from sewage treatment plant, Oshkosh, Wisconsin.
gHost range data from Safferman et_. al_., 1972.
Unknown reaction (Results not available)
+Plaque formation
-No plaque formation
20
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TABLE 6 PLAQUES ON SUSCEPTIBLE HOST STRAINS
Susceptible Host
Synechococcus cedrorum
IU 1191 (SI)
Synechococcus cedrorum
IU 1191 (S1G1)
Synechococcus cedrorum J3
Synechococcus cedrorum J3G1
Anacystis nidulans
IU 625, 1550 (Ac!)
Anacystis nidulans
IU 625, 1550 (AclCl)
Anacystis sp.-J4
Anacystis sp. J4G1
*Microcystis aeruginosa Jl
Culture
Condition
Ua
AXC
U
AX
U
AX
U
AX
U
Rate of Plaque
Formation
3-4 days
2-3 days
3 days
2-3 days
4-5 days
3-4 days
4-5 days
4 days
10-12 days
Plaque Diameter
Size
1-2 mm
2 mm
3-4 mm
4-5 mm
2-3 mm
2-3 mm
2-3 mm
2-3 mm
1-2 mm
Plaque
Morphology
C/Mb
Translucent
C/M
Translucent
C/M
Translucent
C/M
Translucent
Turbid
*Axenic cultures of M_. aeruginosa J-1 have not been isolated.
Ua = Unialgal culture.
C/M = Clear having microcolonies of bacteria inside plaque.
AXC = Axenic culture.
-------
defined edges were observed. The one exception was M. aeruginosa J1. with
which the plaques were turbid. Unlysed cells of M_. aeruginosa, as well as
bacterial cells, could be seen inside these turbid plaques.
The rate of plaque formation varied among host genera (Table 6). Cul-
tures of S_. cedrorum and of A_. nidulans formed plaques in a maximum of five
days under" the standard plaque assay conditions. For M_. aeruginosa Jl, the
illumination during incubation had to be decreased to 80 ft-c. At higher
illumination, the cells would yellow and die before plaques developed. At
decreased illumination, turbid plaques on M. aeruginosa Jl began to develop
after 12 to 15 days.
STABILITY OF CYANOPHAGE SAM-1
Clarified stocks of cyanophage SAM-1 in Jansen's liquid medium were
stored at 4°C and assayed periodically by the standard plaque method. Only
a slight loss in viral titer was detected after storage for 15 months.
Suspensions of phage SAM-1 showed no significant decrease in pfu/ml when
stored for 24 hours at 4°C in either distilled water or 0.01 M Tris buffer,
pH 8.2 (Table 7). The concentration of plaque forming units was neither
increased nor decreased when phage SAM-1 suspensions'were stored and absorbed
to host cells in Jansen's medium supplemented with 0.1 M NaCl plus 0.001 M
MgCl2 (Table 7).
TABLE 7 STABILITY OF SAM-1 CYANOPHAGE IN VARIOUS SOLUTIONS
Experiment Original Infectivity (pfu/ml) after exposure to:
Number Infectivity
(pfu/ml)
0.1 M NaCl + Distilled water 0.01 M Tris
0.001 M MgCl2
1*
2**
4.0 x 107
7.4 x 109
2.0 x 107
4.0 x 109
3.4 x 107
7.3 x 109
4.2 x 107
8.3 x 109
* Purified SAM-1 cyanophage was diluted 100-fold with the appropriate
solution; the mixtures were incubated for 24 hours at 4°C and assayed for
infectivity.
** Purified SAM-1 cyanophage was dialyzed against three 1000-fold volumes of
the appropriate solution, during 48 hours, and then assayed for infectiv-
ity.
Portions of a SAM-1 stock in Jansen's medium were maintained at different
temperatures for 1 hour. No change in infectivity, measured by the standard
plaque assay, was observed for samples held at temperatures equal to or below
45°C (Figure 3). Samples held at 50°C, 55°C and 60°C for 1 hour retained,
22
-------
1000
100 --
10--
-i—l-
20
25
30
35
40
45
50
55
60
TEMPERATURE °C
Figure 3: Stability of Cyanophage SAM-1 at different temperatures.
Samples of a viral stock were maintained at appropriate
temperatures for 1 hr, then assayed by the standard plate
assay.
23
-------
respectively, 80%, 12% and 0.0002% of the original infectivity.
Portions of a viral stock were adjusted to different pH values and stored
for 1 hour at 25°C. Infectivity, measured as pfu/ml, was unaltered in all
samples maintained at or between pH 4 and pH 10 (Figure 4). Samples main-
tained at pH 11 retained 25% of the original infectivity. Samples maintained
at pH 3 or at pH 12 retained less than 0.001% of the original infectivity.
Figure 5 suggests certain synergistic effects of agitation and of
chloroform on the stability of cyanophage SAM-1. When a viral stock was
agitated at 150 rev/min for 60 seconds and then allowed to stand undisturbed
for 30 minutes, the viral infectivity decreased to 70% of the original value.
When chloroform was added to 17% v/v, no change in infectivity was observed
after 30 minutes. When these same conditions of agitation and of chloroform-
ing were applied simultaneously, the infectivity decreased to 20% of the
original value.
SAM-1 MULTIPLICATION CYCLE: ONE-STEP GROWTH EXPERIMENT
The SAM-1 viral multiplication cycle was studied with a one-step growth
experiment very similar to design to the original experiment of Ellis and
Dulbruck (1939). SAM-1 virus was absorbed to the host, S_. cedrorum strain
J3G1, for 1 hour in the dark in an absorption flask containing Jansen's
liquid medium supplemented with 0.1 M NaCl. The culture was thereafter
cultured under standard conditions and assayed for pfu/ml at one hour inter-
vals for 24 hours (Figure 6). In this study, 90% of the virus adsorbed during
the 1 hour dark adsorption period; the end of the adsorption period is con-
sidered to be the time when illumination was resumed. The first virus to be
released from the infected cells appeared 10 hours after illumination was
resumed, as is indicated by the increase in pfu/ml at that time. Thus, the
latent period lasted 10 to 11 hours under these conditions. The rise period,
or time of cell lysis, continued to the sixteenth hour after resumed illumina-
tion. An average of 90-100 plaque forming units of virus was released per
infected cell.
SAM-1 MULTIPLICATION CYCLE: PREMATURE LYSIS EXPERIMENT
At 30-minute intervals after infection, certain samples of the same
infected culture used for the one-step growth experiment were lysed pnemature-
ly by the addition of chloroform and assayed for virus by the standard plaque
assay (Figure 6). In this premature-lysis experiment, no detectable virus
was observed for the first 6 hr after resumed illumination, suggesting that
the eclipse period of the SAM-1 multiplication cycle is 6 to 7 hr long. That
intracellular virus accumulated within the host cells between 6 to 14 hr after
resumed illumination is indicated by the presence of more plaque forming units
in chloroform-treated, prematurely-lysed samples than in unchloroformed
samples taken at the same times after illumination.
24
-------
107 n-
106 «
105 -
10*
10
P'FU.
ML
10'
4—4
10 11
pH Values
Figure 4.
Stability of Cyanophage SAM-1 at different pH values.
Samples of a viral stock were adjusted to the desired
pH and stored at 25 C for 1 hr, then assayed by the
standard plate assay.
25
-------
o
ui
u.
100
90
80
70
60
50
« 40 -
30
20 --
10 -•
NO AGITATION + CHLOROFORM
AGITATION ONLY
AGITATION + CHLOROFORM
Figure 5.
TIME IN MINUTES
Sensitivity of Cyanophage SAM-1 to chloroform and to
agitation. Samples of a viral stock were maintained
under appropriate conditions for 30 min, then assayed
by the standard plate assay.
30
26
-------
108 J.
PFUs
ML
10' --
106 --
10* --
10"
PREMATURE LYSIS CURVE
O ONE-STEP CURVE
' i ' I i | ' i i 1 » I >
8 10 12 14 16 18 20
HOURS AFTER ADSORPTION
Figure 6. SAM-1 multiplication cycle, one-step growth curve and
premature lysis curve, with Synechococcus cedrorum J3G1
as the host.
27
-------
ELECTRONMICROGRAPHS OF CYANOPHAGE SAM-1
Transmission electron micrographs of both negatively- and positively-
stained viral preparations reveal phage-like particles (virions) with a
polyhedral head and slightly flexible tail (Figures 7-9). The head of phage
SAM-1 exhibits the familiar hexagonal outline of the other cyanophage heads.
Based on various electron micrographs and preliminary measurements with
calibrated polystyrene latex spheres, the head of SAM-1 virus appears to be
approximately 85 nm in edge-to-edge diameter.
A long, narrow tail extends from one of the vertices of the SAM-1 head
(Figures 7 and 8). The tail structure is marked by numerous stripes roughly
perpendicular to the axis of the tail. In most micrographs, the tail can be
observed to consist of at least three parts: an outer sheath, an inner core
and a terminal structure characterized by concentric rings. Pins and fine
"whiskers" may extend from the distal end of the tail. The tail sheath is
frequently observed either 1/2 or 3/4 contracted (Figure 9), even though the
virion is not absorbed to host cells. In such cases, the tail core can
usually be observed protruding from the distal end of the tail sheath.
Although the size of the SAM-1 virion has not yet been precisely mea-
sured, ratios of the lengths and widths of various virion components can be
readily determined from Figures 7 to 9 and from similar electron micrographs.
Table 2 presents these ratios and compares them with comparable ratios for
cyanophages AS-1, SM-1 and S-l.
PROPERTIES OF M. aeruginosa CULTURES FROM THE WINNEBAGO POOL
Because of the paradoxical observation (Table 5) that cyanophage SAM-1
could lyse M^. aeruginosa strain Jl, which had been isolated from the Winnebago
pool, but could not lyse M_. aeruginosa strains M2 and M4, which had been
isolated from other sources, it was decided to: (a) isolate further M_.
aeruginosa cultures from the Winnebago pool and (b) determine the effect of
cyanophage SAM-1 on these cultures. Several unialgal cultures of M_. aeru-
ginosa were isolated from the Fox River and from Lake Winnebago. Microscopic
examination of these water samples suggested that more than one M_. aeruginosa
morphological type was present. Furthermore, four distinct types of N[.
aeruginosa colonies could be detected when organisms from the water samples
were cultured on Jansen's agar (Table 8, Figures 10-13). Colonies of ea£h
type could readily be identified and counted, both: (a) on petri plates*
containing Jansen's agar medium supplemented with cycloheximide and inoculated
with a fresh water sample and (b) on plates containing Jansen's agar medium
and inoculated with unialgal laboratory cultures. Cells from each colony
type on agar showed a characteristic cell morphology and a characteristic
cell aggregation pattern when cultured in Jansen's liquid medium (Table 8,
Figures 14-17). Thus, at least some of the different morphological types
originally observed in the water samples appeared to retain detectable morpho-
logical differences when cultured. The existence of easily recognizable
colony types on the agar medium was of particular significance, because
colonies of different morphological types could be counted on plates inocu-
lated with field samples, thus allowing quantisation of the number of colony-
forming units of different M_. aeruginosa morphological types present in a
particular water sample.
28
-------
Figure 7. Virion of cyanophage SAM-1
29
-------
Figure 8. Virion of cyanophage SAM-1
4
Figure 9. Virion of cyanophage SAM-1, with a
partially-contracted tail sheath.
30
-------
Figure 10.
r
A 10-day colony of M_. aeruginosa strain
Cl (morphological type 1) growing on
Jansen's agar medium. Magnified approxi'
mately 80 times, including enlargement
of print.
Figure 11. A 10-day colony of M_. aeruginosa strain
C2 (morphological type 2) growing on
Jansen's agar medium. Magnified
approximately 80 times, including
enlargement of print.
31
-------
Figure 12.
•^WjUK^
A 10-day colony of M_. aeruginosa strain
C3 (morphological type 3) growing on
Jansen's agar medium. Magnified approxi-
mately 70 times, including enlargement
of print.
Figure 13. A 10-day colony of M_. aeruginosa strain
C4 (morphological type 4) growing on
Jansen's agar medium. Magnified approxi-
mately 80 times, including enlargement of
print.
32
-------
Figure 14. A 10-day culture of M_. aeruginosa strain
Cl (morphological type 1), growing in
Jansen's liquid medium. Magnified
approximately 800 times, including
enlargement of print.
Figure 15. A 10-day culture of M. aeruginosa strain
C2 (morphological type 2), growing in
Jansen's liquid medium. Magnified
approximately 800 times, including
enlargement of print.
33
-------
Figure 16,
A 10-day culture of M. aerug.inosa strain
C3 (morphological type 3), growing in
Jansen's liquid medium. Magnified
approximately 600 times, including
enlargement of print.
Figure 17. A 10-day culture of M_. aeruginosa strain
C4 (morphological type 4), growing in
Jansen's liquid medium. Magnified
approximately 800 times, including
enlargement of print.
34
-------
TABLE 8. COMPARISON OF M. AERUGINOSA MORPHOLOGICAL TYPES
CO
01
COLONY MORPHOLOGY
ON JANSEN'S AGAR MEDIUM
(14 day old cultures)
Morpho- Culture Colony
logical diameter
type
1 Cl 1 mm
2 C2 2 mm
Mean
cell
d i am .
4 ym
4 ym
CELL MORPHOLOGY
IN JANSEN'S LIQUID MEDIUM
(7 day old cultures)
Colony Mean cell
description diameter
dark green smooth 5 ym
not spreading
olive green granular 4 ym
Other Properties
less capsule
pseudovacuoles
much capsule
C3
C4
Jl
M2
M4
3 mm
4 mm
2 mm
spreading glossy
mucoid
6 ym dark green granular
very glossy
6 ym light olive
green granular
very spreading
glossy
5 ym olive green
raised
glossy
prominent
pseudovacuoles
7-8 ym less capsule
prominent
pseudovacuoles
5 ym most capsule
5 ym much capsule
prominent
pseudovacuoles
5 ym moderate capsule
pseudovacuoles
4 ym moderate capusle
Dseudovacuoles
-------
Table 8 summarizes the colony and cell .morphologies of the M^. aeruginosa
unialgal cultures Cl, C2, C3, and C4, each of which represents one of the four
observed M_. aeruginosa morphological types. For comparison, Table 8 also
gives the morphologies of the three M_. aeruginosa strains Jl, M2 and M4,
strains already present in the UW-0 culture collection. Figures 10 through
13 show photomicrographs of typical colonies formed on Jansen's agar by the
proposed morphological types. Figures 14 through 17 present photomicrographs
of 10-day-old cultures of the different morphological types grown in Jansen's
liquid medium.
VIRUS-SENSITIVITIES OF M. aeruginosa MORPHOLOGICAL TYPES
Spot tests with cyanophages AS-1 and SM-1 were performed to determine
the virus-sensitivities of M_. aeruginosa cultures Cl, C2, C3 and C4. None of
these cultures showed detectable lysis when exposed to either virus.
FREQUENCY OF M. aerugionsa MORPHOLOGICAL TYPES AT DIFFERENT STATIONS AND TIMES
At four-week intervals during August through November, 1975, near-surface
and near-bottom water samples were removed from two stations on the Fox River
and from one station in Lake Winnebago, at the locations indicated in Figure
2. Individual M_. aeruginosa cells in these samples were counted microscopi-
cally in a Sedgwick-Rafter cell, allowing calculation of the number of J4.
aeruginosa cells per ml of each sample (Figure 18). Although different
morphological types were observed in the water samples, the morphological
types were not counted separately in this preliminary study.
Appropriate volumes of each water sample were also inoculated onto
Jansen's cycloheximide agar medium. The number of colonies corresponding to
each M_. aeruginosa morphological type were counted, allowing calculation of
the number of colony-forming units (viable units) of each morphological type
present in each water sample. Figures 19 through 21 give these data for
stations 1 through 3 respectively.
Figure 22 gives the dissolved oxygen in each water sample. Figure 23
shows the near-surface and near-bottom water temperatures at each station and
at each sampling time. Figure 24 presents water turbidity, measured as Secchi
disc depth, for each station and each sampling time.
»
UNEXPLAINED LYTIC PHENOMENA
During the isolation of various unialgal cultures and during the mainten-
ance of stock cultures, unexplained lysis was frequently observed. In many
cases, the lytic agent formed plaques on the otherwise confluent growth of
the algal host in Jansen's agar medium. Figure 25 shows the plaques produced
by one such unidentified lytic agent infecting M. aeruginosa Jl. Figure 26
shows areas of lysis produced by another unidentified lytic agent, occurring
on the confluent growth part of a petri plate streaked with Synechococcus sp.
Although these lytic agents have as yet been inadequately characterized, they
are briefly mentioned here to emphasize the frequency with which lytic phenom-
ena were observed at certain stations and to suggest possibilities for further
experimentation.
36
-------
CELLS
PER
ML
1050 •
900 -
750 •
600 .
450
300 •
150 -
0
750 -
600 -
450 -
300 -
150 -
0
0
Figure 1!
(A) STATION 1
— -X
(B) STATION 2
—„ -y
(C) STATION 3
10
20
DAYS
Frequency of M_. aerugi'nosa cells, from direct microscopic
counts. Day 0 was August 20, 1975. (A) Station 1;
(B) Station 2; (C) Station 3. Symbols: o, near-surface
sample; x, near-bottom sample. (Pilot study)
37
-------
220
200
180 '
160
COLONY-
FORMING
UNITS
140
120
100
80
60
20
20
30
50
60
70
DAIS
Figure 19. Frequency of M_. aeruginosa colony-forming units, for
near-surface samples removed at different times from
Station 1. Day 0 was August 20, 1975.
Symbols: o, morphological type 1;Q, morphological
type 2;A, morphological type 3;<0>« morphological
type 4. (Pilot study)
38
-------
50 -
COLONT-
30 •
FORMING
UNITS
PER
ML 20
10 -
10
20
30
DAYS
50
Figure 20. Frequency of M_. aeruginosa colony-forming units, for
near-surface samples removed at different times from
Station 2. Day 0 was August 20, 1975.
Symbols: o, morphological type 1;Q. morphological
type 2; A, morphological type 3;, morphological
type 4. (Pilot study)
39
-------
350 •
300 '
250
COLQNI-
FQRMING
UNITS 200
ML
150
100
50
10
20
30
-40
50
60
70
DAYS
Figure 21
Frequency of M_. aeruginosa colony-forming units, for
near-surface samples removed at different times from
Station 3. Day 0 was August 20, 1975.
Symbols: o, morphological type 1; D, morphological
type 2; A, morphological type 3;<£>, morphological
type 4. (Pilot study)
40
-------
DISSOLVED
OXYGEN
CngA)
11
10
9 •
8 .
7 .
6
11
10
9
8
7
6
11
10
9
8
(A)
(B)
•«
10
20
30
50
60
DAYS
70
Figure 22. Dissolved oxygen in near surface and near-bottom water
samples. (A) Station 1; (B) Station 2; (C) Station 3.
Symbols: o, near-surface samples; x, near-bottom
samples. (Pilot study) Day 0 was August 20, 1975.
41
-------
DEGREES
CELSIUS
19 •
18 -
17
16
15 •
13 •
12 •
11 •
10
10
20
30 40
DAYS
50
60
70
Figure 23. Water temperature at each station. Day 0 was
August 20, 1975. Symbols: o, Station 1;
D, Station 2; A, Station 3. (Pilot study)
42
-------
180 -
160 •
140 -
SECCHI
120 •
DEPTH
100 -
(cm)
80 -
60 -
fr
-* _-..-, «.«43
20 -
DAYS
6'0
Figure 24.
Secchi disc readings at each station. Day 0 was August 20, 1975,
Symbols: o, Station 1; D, Station 2; A, Station 3. (Pilot study)
43
-------
Figure 25. Plaques produced by an unidentified
lytic agent infecting NL aeruginosa Jl
Figure 26. Plaques produced by an unidentified
lytic agent infecting Synechococcus sp.
44
-------
SECTION 4
DISCUSSION
PROPERTIES OF CYANOPHAGE SAM-1
The isolation and characterization of a lytic agent infecting certain
blue-green algae is herein described. That this lytic agent is a virus can
be concluded from: (a) its morphology as observed in electron micrographs,
(b) the occurrence of an eclipse period during its multiplication cycle, (c)
its filterability through 0.45 ym pore-size filters and, with lower effici-
ency, through 0.22 ym pore-size filters, and (d) its sedimentation properties
during ultracentrifugation. Cyanophages are designated by the initials of the
generic names of their hosts, to which arabic numerals are added to indicate
different isolates or different subgroups (Safferman et^ al_., 1963). Accord-
ingly, the newly isolated virus has been designated cyanophage SAM-1, since
it infects Synechococcus cedrorum, Anacystis nidulans and one strain of
Microcystic aeruginosa.
Cyanophage SAM-1 differs from other reported cyanophages in virion
morphology (Table 2) and in host range (Table 5). It most resembles cyano-
phage AS-1, but can be distinguished from this virus by differences in host
range, heat-sensitivity, virion size and virion tail structure.
Some confusion exists in the taxonomy of certain SAM-1 susceptible
organisms. Thus, Anacystis has been classified along with Synechococcus into
typological group IA by Stanier ejt a_l_. (1971), who suggested that Anacystis
be reclassified as Synechococcus. Krauss (1958) has also commented that
Anacystis nidulans is probably more accurately a Synechococcus. The related-
ness of these two organisms is also suggested by the susceptibility of both
organisms to cyanophages SAM-1, AS-1 and S-l, since most bacteriophages show
a host range limited to closely related organisms (Luria and Darnell, 1967).
In contrast to cyanophages AS-1 and S-l, cyanophages SAM-1 and SM-1
infect the additional host Microcystis aeruginosa, which belongs to typologi-
cal group III C, not to group IA, of Stanier ejt al_., (1971). M_. aeruginosa
differs markedly from SL cedrorum and A. nidulans, in both cell morphology
and growth properties. Cells of M_. aerugniosa are large, 3-5 ym in diameter,
pseudovacuolated, coccoid and form relatively large, mucoid colonies on
Jansen's agar medium (Table 8). Cells of the latter two hosts are smaller,
lack pseudovacuoles, are bacillary and form pinpoint colonies on Jansen's
agar medium. M. aeruginosa cultures have remained morphologically distinct
from S^. cedrorum or A. nidulans cultures under a variety of growth conditions
and during long-term laboratory cultivation.
45
-------
The ability of cyanophages SAM-1 and SM-1 to infect not only Microcystis
but also Synechococcus is of considerable interest. Future experiments may
indicate that Microcystis is more related to Synechococcus than would be
expected from cell morphology, plane of cell division, and colony formation.
Alternatively, it may be found that the two genera are relatively unrelated,
but that they nevertheless share certain viral receptors or other properties
which allow infection by the same virus.
Because of the dissimilarity between M_. aeruginosa and the other hosts
of cyanophage SAM-1, it is prudent to critically evaluate the evidence that
the virus actually infects M_. aeruginosa. Cyanophage SAM-1 can be propagated
by serial transfers in M_. aeruginosa cultures, indicating that viral multipli-
cation, not just cell lysis, can occur. Although rigorous verification
awaits axenic cultures of M_. aeruginosa, no evidence suggests that cyanophage
SAM-1 infects contaminating bacteria rather than the algae or that the cul-
tures carried contaminating type IA algae. The M_. aeruginosa Jl culture used
for the phage-sensitivity tests had been freed of all contaminating bacteria
except one organism, an Arthrobacter species, pure cultures of which are
insensitive to phage SAM-1. Furthermore, none of the bacteria isolated from
earlier, more heavily contaminated, cultures of strain Jl could be infected
with cyanophage SAM-1 (Table 5). Extreme care was taken to ensure that M_.
aeruginosa Jl cultures did not contain other algae, including type IA organ-
isms, which have been reported to sometimes occur as endophytes in the Micro-
cystis envelope (Prescott, 1962). Healthy and senescent cultures were exten-
sively examined microscopically in a search for contaminating type IA cyano-
phytes, either free-floating or in the mucilage surrounding M_. aeruginosa
cells. Cultures were streaked on agar media, to detect colonies not typical
of M_. aeruginosa. Liquid cultures were screened for growth below the surface
of the medium, since M_. aeruginosa grows at the surface whereas type IA
organisms characteristically grow throughout the medium or settle to the
bottom. The lawns around SAM-1 plaques were observed for contaminating type
IA organisms. On the basis of these and other tests, the culture was uni-
algal. The sensitivity of the tests was such that they would have detected
one type IA contaminant per at least 103 M_. aeruginosa cells, a density of
potential contaminants too low to account for the virus sensitivity. Never-
theless, it would perhaps be wise to further screen the M_. aeruginosa Jl
culture for type IA contaminants, using even more sensitive procedures, to
absolutely eliminate this possibility.
In contrast to the clear plaques produced by phage SAM-1 infecting S_.
cedrorum or A. nidulans, turbid plaques were formed by SAM-1 virus infecting
M_. aeruginosa. Turbid plaques could arise from several conditions, some of
which are:(a) lysongeny, in which cells carry the virus in a latent, non-
infectious form and are not killed by the virus, (b) poor absorption of virus
to host cells, (c) failure of virus to kill host cells, (d) inability of
virus to replicate in slowly-dividing sensescent cells, and (e) lysis inhibi-
tion of the type observed with T-even bacteriophages (Luria and Darnell,
1967). This topic requires further investigation, especially because of its
pertinence to the potential role played by cyanophage SAM-1 in the ecology of
1M. aeruginosa populations.
46
-------
Although cyanophage SAM-1 did infect M_. aeruginosa strain Jl, it did not
detectably infect six other M_. aeruginosa strains (Table 8). Since four of
the virus-insensitive strains, as well as the one virus-sensitive strain,
were all isolated from the Winnebago pool, it is unlikely that any geograph-
ical factors determines why some strains are virus-sensitive. Some possible
explanations of this varying virus-sensitivity are as follows: (a) the
turbid plaques produced by phage SAM-1 on M_. aeruginosa Jl suggest lysogeny.
Lysogenic (virus-carrying) cells are usually "immune" (not lysed) by a virus
which they are already carrying. If this were the case with M_. aeruginosa
and phage SAM-1, then most M_. aeruginosa cultures would be carriers of and
therefore not lysed by cyanophage SAM-1; strain Jl would be non-lysogenic and
therefore virus-sensitive, (b) M_. aeruginosa cultures tend to lose most of
their mucilage upon prolonged laboratory cultivation. Mucilage could inter-
fere with absorption of virus to host cells. Strain Jl had been cultured for
several years when its sensitivity to phage SAM-1 was first examined; strains
Cl, C2, C3, and C4 had been cultured for only.a few weeks and appeared to
possess more mucilage. In contradiction of this idea, strains M2 and M4 were
both insensitive to phage SAM-1 and yet were established laboratory strains
almost devoid of mucilage when cultivated in Jansen's medium, (c) the presence
of a given virus in a particular water system may exert a strong selective
pressure favoring virus-resistant algal mutants. Cyanophage SAM-1 and M_.
aeruginosa strains Jl, Cl, C2, C3 and C4 were all isolated from the Winnebago
pool. Strain Jl was isolated in October, 1972, approximately 7 months before
cyanophage SAM-1 was; strains Cl through C4 were isolated in August and
September of 1975, 2-1/2 years after phage SAM-1. Thus, strain Jl could
represent a virus-insensitive strain present before phage SAM-1 became widely
distributed in the Winnebago pool; strains Cl through C4 could represent
virus-resistant varities which became dominant as the virus exerted selective
pressure. All three of the above hypothesis (lysogeny, mucilage or mutations)
can be readily tested by standard virological experiments designed to deter-
mine why some M_. aeruginosa strains are virus-sensitive and others are not.
The outcome of any cyanophyte-cyanophage interaction depends not only on
inherent properties of both phage and host, but also on fluctuations in
external conditions and on the appearance of viral or algal mutants. Never-
theless, the cyanophage may show promise as a biological control agent.
Before the potential of cyanophage SAM-1 for biological control can be
evaluated, it will be necessary to understand, among other things: (a) the
factors which make some M_. aeruginosa strains, but not other strains, sensi-
tive to phage SAM-1, (b) the reasons why phage SAM-1 produces turbid plaques,
an indication of incomplete cell lysis, with M_. aeruginosa, (c) the conditions
which favor cell-to-cell transmission of the virus in nature and (d) what
effects phage SAM-1 has on endogenous strains of M_. aeruginosa under the
environmental conditions of a particular water system.
The enumeration of M_. aeruginosa morphological types by the counting of
characteristic colonies grown on Jansen's cycloheximide agar offers a new
method for quantitating M_. aeruginosa types in water samples. Its main
advantage is that it detects morphological types which "breed true" upon
cultivation; it is not affected by different cell metabolic states or growth
conditions in various water samples. This method quantitates "viable" M_.
aeruginosa cells or aggregates (with viability being defined as the ability
47
-------
to grow under specific culturing conditions) and thus is a potential tool for
identifying viable cells among a mixture of viable and non-viable organisms.
Thus, the ratio of "total" M_. aeruginosa cells (from microscopic counts) to
"viable" M_. aeruginosa units (from colony counts on agar media) increased
with time between August and November, 1975, as the M_. aeruginosa populations
declined and approached sensescence (Figures 18-21).
Of the four M_. aeruginosa morphological types observed (Table 8), two
probably correspond to f. major (Wittr.) Elenkin and two to f. minor Elenkin
.(Elenkin, 1924). Other important properties of the different types, including
bloom formation and toxin production, remain to be elucidated.
SUGGESTED TOPICS FOR FURTHER EXPERIMENTATION
Several topics for worthwhile future experimentation are:
(1) the possibility that cyanophage SAM-1 may lysogenize (enter a
latent state within) M_. aeruginosa, with possible effects on the
disappearance of formation of algal blooms,
(2) the factors making certain, but not all, M_. aeruginosa strains
sensitive to cyanophage SAM-1,
(3) refinement of the plate counting method for enumeration and identi-
fication of different M_. aeruginosa morphological types,
(4) counting of different M_. aeruginosa morphological types, for water
samples removed at different dates and from different stations in
the Winnebago pool (Figures 18 through 24 present data from a pilot
study),
(5) investigation of other properties, such as bloom formation and
toxin production, of the different M_. aeruginosa morphological
types, and
(6) further characterization of cyanophage SAM-1, including characteri-
zation of its nuclei acid and of virion proteins, identification of
host cell receptor sites, investigation of antigenic cross-reactions
with other cyanophages, and examinations of the effects of environ-
mental conditions on the host-virus interaction.
48
-------
SECTION 5
REFERENCES
Adolph, K. W. and R. Haselkorn. 1971. Isolation and characterization of a
virus infecting the blue-green algal Nostoc muscorum. Virology. 46:200-
208. ~
Adolph, K. W. and R. Haselkorn. 1973. Isolation and characterization of a
virus infecting a blue-green alga of the genus Synechococcus. Virology.
54^:230-236.
American Public Health Association. 1971. Standard Methods for the Examina-
tion of Water and Waste Water (13th ed.), Washington, D. C. 874 pp.
Birge, E. A. and C. Juday. 1914. The inland lakes of Wisconsin. Wisconsin
Geology and National History Survey, Madison, Wisconsin. 137 pp.
Brown, R. M., Jr. 1972. Algal viruses, Advances Virus Research. l_7:243-277.
Cannon, R. and M. Shane. 1972. The effect of antibiotic stress on protein
synthesis in the establishment of lysogeny of Plectonema boryanum.
Virology. 4^:130-133.
Cannon, R., M. Shane and V. Bush. 1971. Lysogeny of a blue-green alga
Plectonema boryanum. Virology. 45:149-153.
Cannon, R., M. Shane and E. DeMichele. 1974. Ecology of blue-green algal
viruses. Jour. Environ. Eng. Div., ASCE. 100:1205-1211.
Daft, M. J., J. Begg, and W. D. P. Stewart. 1970. A virus of blue-green
algae from freshwater habitats of Scotland. New Phytol. 69^:1029-1038.
Elenkin, A. A. 1924. De spec, duabus gen. Microcystis Kuetz. notula. Not.
Syst. Inst. Crypt. Horti bot. Petropol. 3_:12-15.
Ellis, E. L. and M. DelbrUeck. 1939. The growth of bacteriophage. J. Gen.
Physiol. 22^:365.
E.P.A. 1974. National Eutrophication Survey working paper series Report on
Lake Winnebago; Winnebago, Fond Du Lac, and Calumet Counties, Wisconsin
EPA region V, working paper #57, PNERL Corvallis, Oregon.
Goryushin, V. A. and S. M. Chaplinskaya, 1966. Existence of viruses of blue-
green algae. Mikrobiol. Zh (Kiev) 28^:94-97. (In Russian with English
summary).
49
-------
Goryushin, V. A. and S. M. Chaplinskaya. 1968. Finding of the viruses of
blue-green algae. "Blooming of Water", Scientific Thought Publishing
House, Kiev. pp. 171-174.
Jackson, D. F. 1967. Interaction between algal populations and viruses in
model pools—a possible control of algal blooms. ASCE Ann. Nat. Water
Resources Eng., preliminary draft. 17 pp.
Jackson, D. F. and V. Sladecek. 1970. Algal viruses-eutrophication control
potential. YaleSci. Mag. 44:16-22.
Krauss, R. W. 1958. Physiology of the fresh-water algae. Ann. Rev. Plant
Physiol. i:207-244.
Luria, S. E. and J. E. Darnell, Jr. 1967. General virology, John Wiley and
Sons, Inc., New York.
Olcott, P. G. 1966. Geology and water resources of Winnebago County, Wiscon-
sin. U.S. Geol. Surv. Water Supply Paper No. 1814, U.S. Government
Printing Office, Washington, D. C.
Padan, E. and M. Shilo. 1969. Distribution of cyanophages in natural
habitats. 17th Congress, Intern. Soc. Applied and Theoretical Limnology.
Verh. Internat. Verin. Limnol. 17:747-751.
Padan, E. and M. Shilo. 1973. Cyanophages—viruses attacking blue-green
algae. Bacteriol. Reviews 3^:343-370.
Padan, E., M. Shilo and N. Kislev. 1967. Isolation of "cyanophages" from
freshwater ponds and their interaction with Plectonema boryanum.
Virology 3^:234-246.
Padan, E., M. Shilo and A. B. Oppenheim. 1972. Lysogeny of the blue-green
alga Plectonema boryanum by LPP-2 SPI cyanophage. Virology 47:525-526.
Prescott, G. W. 1970. Algae of the Western Great Lakes. Wm. C. Brown, Pub.,
Dubuque, IA. 978 pp.
Rimon, A. and A. B. Oppenheim. 1975. Heat induction of the blue-gfeen alga
Plectonema boryanum lysogenic for the cyanophage SPI cts 1.
Virology 64_:454-463.
Rouf, M. A. 1970. Laboratories exercises in general microbiology. Castle
Pierce Printing Company, Oshkosh, Wisconsin. 128 pp.
Safferman, R. S. 1968. Virus diseases in blue-green algae. Ir± Daniel F.
Jackson (ed.), p. 429-439. Algae, man and the environment. Syracuse
University Press, New York.
Safferman, R. S. 1973. Phycoviruses. Ir± The Biology of Blue-green Algae.
N. G. Carr and B. A. Whitton, eds., Blackwell Sci. Pub., Oxford.
50
-------
Safferman, R. S., T. 0. Diener, P. R. Desjardins and M. E. Morris. 1972.
Isolation and characterization of AS-1, a phycovirus infecting the blue-
green algae Anacystis nidulans and Synechococcus cedrorum. Virology
47^:105-113.
Safferman, R. S. and M. E. Morris. 1963. Algal virus: isolation. Science
140:679-680.
Safferman, R. S. and M. E. Morris. 1964. Control of algae with viruses. J.
Amer. Water Works Assoc. 56_:1217-1224.
Safferman, R. S. and M. E. Morris. 1967. Observations on the occurrence,
distribution and seasonal incidence of blue-green algal viruses. Appl.
Microbiol. 1_5:1219-1222.
Safferman, R. S., M. E. Morris, L. A. Sherman and R. Haselkorn. 1969.
Serological and electron microscopic characterization of a new group of
blue-green algal viruses (LPP-2). Virology 29^775-780.
Safferman, R. S., I. R. Schneider, R. L. Steere, M. E. Morris and T. Diener.
1969. Phycovirus SM-1: a virus infecting unicellular blue-green algae.
Virology 3_7_: 386-395.
Shane, M. S. 1971. Distribution of blue-green algal viruses in various types
of natural waters. Water Research 5_:711-716.
Sherman, L. A. 1975. Replication and control of AS-1. Abstr. Conf. on Blue-
green Algae and their Viruses, Newark, Delaware.
Shilo, M. 1971. Biological agents which cause lysis of blue-green algae.
Mitt. Internat. Verin. Limnol. 19:206-213.
Shilo, M. 1972. The ecology of cyanophages. Bamidgeh 24:76-82.
Singh. P. K. 1973. Occurrence and distribution of cyanophages in ponds,
sewage and rice fields. Arch, fur Mikrobiol. 89:169-172.
Singh, R. N. and P. K. Singh. 1967. Isolation of cyanophages from India.
Nature 216:1020-1021.
Singh, R. N. and P. K. Singh. 1972. Transduction and lysogeny in blue-green
algae. I_n_ Taxonomy and Biology of Blue-green Algae, T. V. Desikachary,
Ed., Univ. of Madras Press, pp. 258-261.
Singh, R. N., P. K. Singh and P. K. Varanasi. 1969. Lysogeny and induction
of lysis in blue-green algae and their viruses. Proc. 56th Int. Sci.
Congress, p. 272.
Sloey, W. E. 1970. The limnology of hypereutrophic Lake Butte des Morts.
Wisconsin Proc. 13th Conf. Great Lakes Research, 951-968.
51
-------
Sloey, W. E. and J. Blum. 1972. The algae of the Winnebago Pool and some
tributary waters. Trans. Wisconsin Acad. Sci. Arts Letters. 60:125-
145.
Stanier, R. Y., R. Kunisawa, M. Mandel and G. Cohen-Bazire. 1971. Purifica
tion and properties of unicellular blue-green algae [order Chroococ-
cales). Bacteriol. Rev. 35:171-205.
Starr, R. C. 1964. The culture collection of algae at Indiana University.
Amer. J. Bot. 51_:1013-1044.
Williams, R. E. 0. and J. E. Rippon. 1953. Bacteriophage typing of strains
of Staphylococcus aureus from various sources. Lancet 264:510-514.
52
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
. REPORT NO.
EPA-60Q/3-77-079
2.
3. RECIPIENT'S ACCESSION NO.
. TITLE AND SUBTITLE
Effects of Cyanophage SAM-1 upon Microcystis aeruglnosa
5. REPORT DATE
July 1977
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
D. L. Parker, G. P. Jansen and L. Corbett
8. PERFORMING ORGANIZATION REPORT NO.
. PERFORMING ORGANIZATION NAME AND ADDRESS
University of Wisconsin - Oshkosh
Oshkosh, Wisconsin 54901
10 PROGRAM ELEMENT NO.
1BA031
11. CONTRACT/GRANT NO.
P5J1190-J
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency -- Corvallis, OR
Corvallis Environmental Research Laboratory
200 S. W. 35th St.
Corvallis, Oregon 97330
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The isolation, propagation and application of Cyanophage SAM-1 to control blue-green
algal blooms is discussed.
Cyanophage SAM-1, which infects Synechococcus cedrorum, Anacystis nidulans and certain
strains of Microcystis aeruginosa has been isolated from sewage. The host range of
cyanophage SAM-1 differs from those of other reported cyanophages. Phage SAM-1 stocks
are rapidly inactivated at temperatures above 45°C or at pH values below 4 or above 10.
The simultaneous application of chloroform and of agitation reduces viral infectivity
to 20% of the original infectivity. The SAM-1 multiplication cycle has a latent period
of 10 hours, a rise period of an additional 6 hours, and eclipse period of 6 hours, and
ah average burst size of 90 plaque-forming units per infected cell. Electron micro-
graphs show SAM-1 virions consisting of polyhedral head and a contractile tail with a
distinctive terminal structure. The properties of cyanophage SAM-1 are compared with
those of other cyanophages.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
cyanophage
viruses
blue-green algae
eutrophication
isogeny
021
04A
18. DISTRIBUTION STATEMENT
RELEASE UNLIMITED
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
61
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
53
it U.S. GOVERNMENT PRINTING OFFICE: 1977-798-356/182 REGION 10
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