California State University, Northridge
Transduction in the Blue-Green
II
A1qa,
SJ'.!::.echo<;:os:..~
e l_~:q<3}!:12.
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Robert Eugene Williams
ScptembAr, 1972
The thesis of Robert Eugene Williams is approved:
I
i
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California State Ur;iversity, Northridge
September, -1972
~
i-~--. ~--' .. --~. -- ---~-·- ~----···-·· --~--~----- -~-- .. ~- ·»-~- ·-----~---~--· -~---·~. --------- ---···"~-·----~~---
ii
--.----
----~--,...,--•. -.q-----~----~- ~~~---- ---·--------~- ---~~ ~-"--... -.~------~-~--·~J
I would like to express my appreciation to Dr. Charles
R. Spotts for
endless time and assistance given me during
the course of this study.
Joyce
t~axv:ell
I would also like to thank Dr.
and Dr. Donald Bianchi for serving on my com-
mittec and for their assistance.
Further acknowledgement
must be given to the California State University, Northridge
Foundation for financial assistance and to Dr. Robert S.
Safferman for samples of his virus SM-1 and his SM-1 antiserum.
I would also like to acknowledge Tim Deakers for
his assistance on the electron microscope and photography
and
~Ja 1t
\~hee 1et~,
Ke 11 ey, Bernadine Pregerson, R. C.
Vo 1ney Hi 11 i ams , Jeff
for collecting water samples.
t~i
Whe£~ l er,
Rodney
11 i ams, and Mike Wi 11 i ams
Last of all, I would like
to express my gratitude to my wife Phyllis for her help in
the preparation of this thesis.
iii
,-~--~--~----~------~~~------·------·-------· --··-··----~~-----..-~--~~·"·-··•···>-~·-~
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TABLE OF CONTENTS
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vi
Abstract .
Introduction
Chapter I.
1
Table 1.
5
I
Table 2.
7
I
Chapter II.
!
I
Materials and Methods
12
. 14
Table 3 . .
Chapter II I.
.
Results. .
27
Table 4.
29
Figure
1
32
Figure
2
I
.
.
.
Figure 3
Table 5.
37
39
....
Figure 4
Figure 5
35
.
42
44
Figure 6
. . 46
Figure 7
48
Figure 8
. 50
Figur·e 9
52
Figure 10.
. 54
Figure 11.
57
Figure 12.
• • • • 59
Figure 13.
62
Figure 14.
64
·jy
Figure 16.
68
Table 6. .
. 71
Chapter IV.
Discussion and Conclusions.
Literature Cited . . . . . . . . . . . • . .
v
72
• 82
r·----------~------~·-·-·-·----~----------------------------~----------·----·-------------·--·--------------·-----------···---·,
'
1.
ABSTRACT
Transduction in the Blue-Green
Alga, ?ynechococcus elongatus
by
Robert Eugene Williams
Master of Science in Biology
September, 1972
Transduction of streptomycin resistance at a frequency
of 4.0 x lo-7 to 6.9 x lo- 7 per cell was observed for the
blue-green alga ?ynechococcus elonaatus.
This report pro-
vides the first direct evidence of gene transfer among the
blue-green algae.
Evidence is presented for the lysogeni-
zation of the unicellular blue-green alga,
~~
elongatus.
The temperate phage has been designated S-lT and found to
have a head capsid tip-to-tip distance of 840 angstroms.
Induction experiments of lysogenic Synechococcus with the
antibiotic, fvlitomycin-C, resulted in the production of virus
as confirmed by electron mic1·oscopy and plaque assay.
The isolation of a new blue-green algal virus S-1
which infects
~- el_~2tus
is described.
~
The new virus
appears to be a hexagon with no obvious tail and having
a head capsid tip-to-tip distance of 710 angstroms based
upon
co~parisons
with latex balls.
Several characteristics
indicate that the virus is d·ist-inct from SM-1 isolated by
I
t. _, __ •'·~- •r•~•--•----~
•p
~-.~~
vi
..
r-----------··-----~---·-----··--·-·~---··--··---------·-··-··-·-·~~--·--····---·-------·--·-·-----·······1
l
l
i
Safferman.
S-1, unlike SM-1, is sensitive to chloroform
and to filtration and is not neutralized by
I
9~-1
antiserum.
'
CHAPTER I
Introduction
Transduction is well known in the bacteria as a means
of gene transfer involving a bacteriophage which acts as a
vector to transmit a segment of host bacterial DNA from an
infected to a non-infected bacterium of the same species.
The blue-green algae and the bacteria are similar in
mor~
pholcgical and physiologicai characteristics and are thought
by many to be closely related (Echlin, 1965; Holm-Hansen,
1968; and Allsopp, 1969).
The process of transduction has
not yet been described among the blue-green algae but one
might expect it to occur because of the similarity of these
two groups and their viruses.
The i so 1ati on of a virus (cyanophage) for a b1ue-gr'een
alga
by Safferman and Morris in 1963, provided the first
indication that blue-green algae were subject to the same
type of virus infections characteristic of bacteria.
There
are many morphological analogies wh·ich may be dravm between
the two types of phage systems.
Adolph and Haselkorn (1971)
found that the cyanophage N-1 resembled the T-even eoliaphages T2 and T4.
The temperate bacteriophages Pl and P2
(Hayes, 1968) and Sp 50 phage of Bacil"lus subtilis (Bradley, 1967) show remarkable similarity to cyanophage AS-1
with the hexagonal capsid and the presence of a tail.
!_.____ , ••
1
In
2
r-·-----·---~-------
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-~----------·---~-------------··-l
a11 cases , cyanophages that have been i so 1a ted conform to
Bradley's (1967) classification of bacteriophages.
I
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Cyanophage LPP-1 and bacteriophage T7 have been comMorphological analysis has shown
pared extensively.
an edge-to-edge capsid distance of approximately 600 angstroms for both T7 (Luftig and Haselkorn, 1968) and LPP-1
(Luftig and Haselkorn, 1967).
Tail measurements showed a
similar type of correlation v!ith a value of 150-200 angstroms by 150 angstroms for both T7 and LPP-1 (Luftig and
Haselkorn, 1967).
Infective activities of cyanophages also seemed to
resemble the activities of bacteriophages in many ways.
The
capacity of blue-green algae to support viral infection is
reduced in the dark which is believed to be attributed to
the inability of the algae to synthesize needed nucleotides
for viral growth in the ·absence of light (Wu, Lewin, and
Werbin, 1967).
This is similar to the phenomenon of 11 abor-
tive infection 11 by the bacteriophage T2 on a
starved-~
coli
cell, where a poor nutritional state of the bacterial host
results in reduced phage growth (Benzer, 1952).
LPP-1 DNA when compared to T7 DNA (Sherman and Haselkorn, l970a) showed a remarkable similarity.
During the
initial stages of infection host DNA was degraded in a
manner·which was similar to that observed for T7 (Sherman
and Haselkorn, l970a).
•. ._
.~
• -. ·-
GC ratios also were found to be simi-~·-~····-·--·-~····-·~--~---·--~--•-•-·~:·~~~
.-•-..·-
~--"--··~
•
-·-·,~·,.__..•~·· --··-·--~·-·~--~~---~~~-~-~~-~~-
'
.......... -.--~A
3
~------------------------~----~----·------------~---·---·-----~--~-----
..-·---..-·---- --·-·-·· ..... i
I
I
for LPP-1 (Luftig and Haselkorn, 1968) varied only slightly
l
from T7 which rtad a value of 1.710 g/ml (Scheldraut et
i
lar with 52 percent for LPP-1 and 48 percent for T7 (Luftig
and Haselkorn, 1968).
DNA bouyant densities of 1.714 g/ml
~.l:_,
1962).
I
The traditional techniques used in the study of bacteriophages have been adapted for cyanophage study; for
I
example absorption experiments and one-step growth experiments (Safferman et
~' 1972).
techniques used by Safferman et_
The 11 enrichment chloroform"
~ (1963
and l964b) and
other researchers are similar to the techniques described
by Adams (1959) for bacteri opha.ges.
It is apparent that cyanophages and bacteriophages
share the same types of properties.
Some characteristics
of the ten known cyanophages are listed in Table 1 and
Table 2.
The existence of virus infection does not necessarily
mean that transduction is possible.
Transduction is gen-
erally found associated with a specialized type of infection where virus is propagated within a host cell without
lytic activity resulting.
Such a
condition~
called lysogeny,
renders the cell immune to superinfection by the phage
residing in that cell and 1s well documented in bacterial
systems·(Barksdale,
1959; Campbell, 1962; and Hayes, 1968).
It is possible through a process of induction to cause lyso-
.
4
Blue-green algae viruses, their hosts and isolators.
l
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----------···--------··-------------------·-·-·-·---.,
CYANOPHAGE
ALGA
\
ISOLATORS
!
l
-~
----i
LPP-10
AS-1
*
i
.,?!
N-1
LPP-1
LPP-2
D-1
Glll or
LPP-lG
C-l
Jl,R-1
SM-·1
LPP2-SPI
Plectonema boryanum (lysogenic)
Anacystis nidulans and Synechococcus cedrorum
Anabaena variablis
Nostoc muscorum
Plectonema luridum var. olicacea~ Phormidium
fareda.rurn, Plectonema boryanum, Plectonerna
ca1othricoides, Plectonema notatum and Lyngbya sp.
Same as LPP-1
Plectonema boryanum, Plectonema calothricoidies
Plectonema notatum, Phormidium faveo1arum
Phormidium luridium var. o1ivaces and Lyngbya sp.
P1ectonema boryanum
Cannon, Shane, and Bush (1971)
Safferman et a1. (1972)
Granha11 and Hofsten (1969)
Adolph and Hase1korn (1971)
Safferman et ~ (1963)
Cylindrospermum sp.
Anabaenopsis raciborskii, Raphidiopsis indica
Synechococcus elongatus, Microcystis aeruginosa
Plectonema boryanum and Phormidium 1uridum
Singh and Singh (1967)
Singh and Singh (1967)
Safferman et al. (1969)
Padan, ShiTO and Oppenheim (1972)
* no symbolhas b-een--des-ignated.
Safferman et al. (1969)
Di~aft, Begg, and Stewat't (1970)
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Padan and Shilo (1967)
!
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l
II
I!
!
!
!
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'-------·---·---------
--··--·-·J
(.)1
6
r-~·---·----
. -.._. ____________
----------·-·--·---------~----····--·--------------------··--~-------1
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TABLE 2
Characteristics of known cyanophages
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L.______ .................._. __,_________________ ··~;""-·~·-·--------·--·--·-··--. ---·-----···-~~----.------------·-··- ---···-----··-------J
;"''··~ .... ~-··----·---~-~-~- ... -·~---~~---
'
VIRUS
ISOLATION GEOGRAPHIC
FROM
DISTRIBUTION
*
·----------..
-----·-----------,
---......~~-'----------·
HEAD TYPE TAIL LENGTH GC RATIO
AND SIZE
(ANGSTROMS)
(%)
(ANGSTROMS)
WS
Polyhedral
Fla.
N-·1
L
Wis.
Polyhedral
Anabena
LPP-1
s
Sweden
Mo., Ind.
Ark., Fla.,
N.H., S.D.
Tex., Cal.
Same as
LPP-1
India
India
Ind.
Hexagonal
Polyhedral
i
J.
-/f
I
ws
I
I
2435
53-54
1100
37
100-200
100-200
53-55
920 X 900
550
LPP-2
WS
C-1
AR-1
PW
PW
SM··l
WS
--1
Safferman et
~
!
Adolph and Haselkorn (1971)
Polyhedra 1
not given
Po 1yhedra 1
none
2001
Dl"aft et £.L_ (1970)
600
II
(1972)
Granhall and Hofsten (1969)
Goldstein et al. (1967);
Safferman and Morris (1967);
Schr.eider et al. (1964);
Luftig andHaselkorn (1967)
Safferman, Morris, Sherman and
Hase1korn (1969)
Singh and Singh (1967)
Singh and Singh (1967)
Safferman et ~ (1969)
5
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-
AS-1
I
REFERENCES
!'
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I
i
880
D-1
. Glll or
LPP-lG
LPP-10
LPP2-SPI
SA
Scotland
Isohedra·l
FP
Israel
Po 1yhedra 1
* abbreviations:ws
Fish Pond; L - Lake.
Ind.
Israel
586
600
Waste Stabilization Pond; S - Soil; PH - Polluted
~-------·-·--·---·-·····-----------------------------··--·-
Padan _et
Cannon et a1. (1971)
Padan et al. (1972)
\~ater;
I
~ (1967)
SA - Sewage; FP -
l
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--------------------..--.. ··-··-------------------------·-----·-··------·. ---·----J
.....J
8
,---------·---~-""--------·------··---------·-····--·-------------·--···----------·---~---··--
genic ce 11 s to lose their immunity, whereupon the vegeta-
1
I
tive phage cycle ensues and free phage particles are liberTreatment with UV (Fisher, 1962), thymine starva-
a ted.
tion (Melechon and Skaar, 1962), X-ray, nitrogen mustard,
I
hydrogen peroxide, and organic peroxides (Jacob and Wollman,
1959) all have been used to induce lysogenic cells to produce phages.
Recently lysogeny was demonstrated in the
blue-green alga
(l 972).
by Cannon _et al._ (1971) and Padan et
~
The two cases that have been reported are for the
blue-green algae Plectonema boryanum with the temperate
phages des-ignated LPP-10 (Cannon et
(Padan et al :..' 1972).
~.
1971) and LPP2-SPI
Using an induction procedure similar
Salmone~ll:.?_
to that used by Levine and Borth Hick (1963) for
1
!Yrhimt~ri~~
LT 17 (P22), Cannon et
~
(1971) were able to
demonstrate lysogeny with Mitomycin-C-induction.
al~-
Padan et
{1972) wer'e also able to show lysogeny but failed to
demonstrate induction with UV, X-ray, or Mitomycin-C, the
usual agents used for this purpose.
The presence of a halo
of lys·is surTounding a lysogenic colony when plated on a
lawn of sensitive cells was used initially to indicate the
pr·esence of the lysogenic state (Padan et_
~~-,
1972). Fur-
ther studies resulted in isolation of a thermal sensitive
mutant wh·i ch caul d be induced by changing the temper·ature
from 26°C to 37°C.
The non-inducible state of LPP2-SPI
may be an indication that other methods should be considered
!I -
-~'---'-
. -· -~<P•.
····--·.
,_
--~ ·~
•
~~ ~-..
· - · • • •A- ••-
-
-:*'._·;~~
- . ..- -~~---·--·· ·---- ---~~-- ---------~------·--· --~------ -~.·-·-------··-- .
9
r--w·----···--------·-~~----~--··----·--··-··,.~·---
!
..-··-----·---------,.---···---·····--·-·····-··--·-···-···-·-·--· . ··
I
when screening for lysogenic blue-green algae.
I
then seem advisable to try a wide range of techniques in
1
the hope of finding the correct procedure for induction.
I
It would
Two types of transduction have been recognized in bacterial systems: restricted transduction which involves the
transfer of only one locus adjacent to an integrated prophage; and
un~estricted
or generalized transduction in which
random segments of genetic material are transduced.
Restricted transduction occurss when)during the induetion of a prophage, a segment of bacterial chromosome is
substituted for a portion of viral genome.
When it infects
and lysogenizes a new host this virus brings with it a very
small portion of its prev·ious host with a high frequency of
transfer of only one locus.
It is important to recognize
that restricted transductants are derived only from induetion of lysogenic cells,
~hich
rarely produce infectious
viral progeny unless artificially induced.
All recipients
are found to be lysogenic.
In unrestricted transduction, bacteriophages incorporate random fragments of host DNA in lieu of phage chromosomes by a pr·ocess similar to phenotypic mixing at a low
frequency of transfer for any one locus.
It is not neces-
sary for virus to be integrated into lysogenic state and
the abi 1i ty of bacteria phages to ex hi b·J t this type of transduci ng activity may be independent of
th(~i
r ability to
10
,-------~-----~-----·--·~---·--··-~--~--------~·------~~--1
I
I
I
lysogenize the recipient bacte·t'i a.
Such phages may be i nfec-
tious, transferring their DNA to a new host, but unable to
replicate because of the deficiency in essential virus genes.
Nevertheless, phaqes exhibiting unrestricted transduction
can be induced from non-producing bacteria by procedures
identical to those used in lysogenic induction, and thus is
correlated (at least as far as the ability to be induced)
with an apparent lysogenic state.
In order to demonstrate transduction, it is necessary
to have strains differing in measureable genetic traits so
that gene flow can be demonstrated.
Among the bacteria,
auxotrophic mutants and antibiotic resistance are most often
used for this purpose, and strains carrying these markers
are easily produced and isolated.
It is very difficult to
isolate these types of mutants in the blue--green algae and
it wasn•t until 1964 that Singh and Singh isolated stable
physiological mutants in the blue-green alga Anabaena
cycadeae.
They isolated a non-nitrogen fixing mutant; an
apochlorotic mutant; a mutant deficient in beta-carotenoid;
and a photoheterotrophic nitrogen-fixer.
Since this initial
isolation other mutants have now been isolated, for example
amino acid auxotrophs (Stevens and Van
Baalen~
1970), anti-
biotic resistant mutants (Gupta and Kumar, 1970), a nitrate
reductase deficient mutant (Stevens and Van Baalen, 1970),
and cell division mutants (Kunisawa and Cohen-Bazire, 1970).
I
11
Recently-nineteen mutants requiring
v~rious
growth factors
such as phenylalanine, methionine, biotin, acetate, and
strains deficient in sulphate and nitrate r·eductiun wet'e
isolated by Herdmand and Carr (1972).
It is now accepted that cyanophages and bacteriophages
resemble each other in terms of infection processes and
·resistance to superinfection.
Lysogeny has been demonstrated
in two cases for blue-green algae and is similar to' bacterial lysogeny in
te~ms
of resistcince and infection.
The
demonstration of discernible genetic markers has been shown
to be obtainable for most blue-greens.· If the analogy of
blue-green algae to bacteria systems is carried further, it
seems probable that transduction found in the bacteria
should occur in the blue-green algae.
This study describes the isolation and Gharacteriza-
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tion of a new virulent cyanophage for a unicellular blueThis virus is compared to a previously isolated
green alga.
virus, SM-1 (Safferman et
II.
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same host.
Lysog~~ization
~'
1969), which infects the
is described with a new temperate
phage, and strong evidence for transduction by this
is presented.
v~n..;s
.
CHAPTER II
Materials and Methods
I.
Organisms
Parental Strains
The par·ent strains of blue-gre·en algae and their sources
that were examined in this study are listed in Table 3·.
Several bacteria and one green alga have been included as
test organisms for host range susceptability.
Antibiotic-Resistant Strains
I
Antibiotic-resistant strains were selected by incubating
about 5 x· 1010 wild type L-2-II cells in 100 ml of antibiotic
II
supplemented medium for several days, followed by plating
I
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on supplemented agar using overlays.
I
of colonies, growth curves were initiated to test for resis-
I
tance of strains to increasing concentrations of antibiotics.
I
Following isolation
Resistant strains are listed in Table 3. ·
Cyanophag_es
Cyanophages infecting Synechococcus elonqatus. LB 563
were isolated and designated S-1 and S-lT according to the
nomenclature system of Safferman and Morris (1963).
SM-1
also infecting h elongatus was obtained from the laboratory
of Robert S. Safferman, Environmental Protection Agency,
Cincinnati, Ohio.
._j
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1_.._.__ ~--~-·-~·-~·-~-w.-~--~~----~----·
12
13
..
TABLE 3
Blue-green algae, bacteria, and the green alga isolated
during this study or obtained from other culture collections.
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L.~--··-·"·-..·~··---~-·---~~----· .. ~·--·-····-"·-·"..;.~···-·---"·-----·-·---·------·"------------·----------"-·-···- ..-·----.. ~---·.J
.,-
..
,~-·-·~~"' ~-~-"
~
STRAINS
-------------·-------REFERENCE--s~~;~;-
!
~
NUMBER
!
l
Blue:green Algae
l
,'t
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Synechococcus elongatus
Synechococcus elongatus Lysogenic (L-2-II)
Synechococcus elongatus Lysogenic Streptomycin Resistant (L-2-II Smr)
Synechococcus elongatus Streptomycin Resistant (smr)
Anacystis nidulans
Gloeocapsa alpicola
Oscillatoria sp.
Plectonema boryanum
LB 563
1550
8 589
18200
IU
CSUN*
CSUN*
CSUN*
IU
IU
CSUN*
ATCC
Bacteria
CSUN
CSUN
CSUN
CSUN
.Sarcina lutea
Eschericha coli Kl2
Arthrobacter atrocyonesis
Micrococcus roseus
Bacillus megaterium
15-2070
i
I
II
II
I
I
CSUN
I
esse
I
!
Green Alga
Chlorella pyrenoidosa
I~
I
Source abbreviatfons:-TO- -rnaTana~lJniverslly Culture Co1lect1on-;-L:SUN -Talifornia-State-Vniversity, Northridge; ATCC - American Type Culture Collection; CBSC - Carolina Biological Supply Company; * - Isolated as a part of this study.
.......
..,-:,
15
II. Cultivation Techniques
!"ledi a
The standard medium used in this study for maintenance
of blue-green alga cultures consisted of a modification of
the medium of
Gerloff~
Fitzgerald, and Skoog (1950) and
contained (per liter): Ca(N0 3) 2 .4H 2o, 0.232 q; K2HP04,
0.01 g; MgS04·?H20, 0.025 g; Na2C03, 0.02 g and ferric
citrate, 0.0035 g.
The medium was supplemented with 2-
amino-2-(hydroxymethyl)-1, 3-propanediol, l .2 g/1, in place
of citric acid and sodium citrate.
trace element solution (Stanier et
the medium prior to sterilization.
One milliliter of a stock
al~,
1971) was added to
This stock solution con-
tained (per liter): H3B0 3 , 2.86 g; MnCl2·4 H20, 1.81 g; ZnS04·
41
?H20, 0.22 g; Na2Mo04·2H 20, 0.39 g; CuS04·5H 20, 0.079 g; and
Co(N0 3) 2 ·6H 20, 0.0494 g. All media were prepared volumet-
j
rically to insure accuracy, adjusted to a pH of 8. 6 with 2N
HCl and sterilized for 15 minutes at a pressure of 15 lbs/in 2.
Agar plates were prepared using 30 ml of standard medium
which was solidified with 1.5 percent bacto-agar.
Overlay
tubes were prepared using 2.5 ml of 0.5 percent agar of standard medium.
!
Bacterial cultures were grown on 2. 75 pet·cent Tryptic
I
Soy Broth without dextrose (Difco) containing 0.2 percent
1
I
yeast
1
I
Streptomyr in s upp 1ementerl p1 ates and 1 i quid media were
I
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e~tract.
'
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.
~
L. ---~"~- ------------- -- ----------------...,-;-------------· _________________ . ________ ------- -----.---------·-------------------·------------ _______,____,
16
r~·--------·
l
prepared using standard medium supplemented with 100 ug/ml
of streptomycin sulfate.
I
Culture Conditions
Liquid cultures were grown in a rotary shaker incubator
(NevJ Brunswick Incubator Shaker-Model G--27) modified to hold
four 15 watt cool white fluorescent (General Electric) lights
and six 14 watt cool white
11
Plant Grow Lights 11 (General Elec-
tric) at a temperature of 30°C and.agitated at 120 rpm.
All
cultures were routinely grown in deLong culture flasks.
All
plating was done by the over·lay method at the temperature
and concentrations previously indicated.
Plates were illuminated with 2-3 cool white fluorescent lights (General Electric) at a temperature of 25-30°C.
All cultures in both liquid and plates received 2000-3000
lux.
Large scale cultivation was carried out in a 14 liter
fermentator (Fermentation Design, Inc.) without aeration
or gassing with carbon dioxide and stirred at a rate of 400
rpm.
Two 40 watt 16 inch circline fluorescent cool white
lamps (General Electric) mounted outside the culture vessel
were used for illumination (Luftig and Haselkorn, 1967).
The apparatus and medium were sterilized at 15 lbs/in 2 for
60 minutes.
I
Initial cell density was approximately 5 x 10 7
cells/ml.
I
L-·-··-~·-·
I
I
:
••
h~•r-- ~ ~--
~ <"r....,..-. ~-~-~"" •~ - · - - · :~ _,.,., ~., ~~·~~·~"
............-......... ·---·· .............. -- ···----.............. --.......................................................-.....................!
17
r~----"~----····----~-------------------------------··-----~--------~-----------------~-l
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·
1
III. Isolation of Cyanophaqes and Lysogenic Strains
1
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Cyanophage Isolation
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Water samples collected as possible sources of virus
II
were placed in screw cap culture tubes and stored in ice
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until they could be placed in refrigeration at 5°C.
Prior
to testing for the presence of virus these samples were filtered twice, first through a filter of 1.2 u pore diameter
then through a 0.45 u pore diameter filter.
Five ml of the
samples of the filtrates were added to 45 ml of suspension
of~ elonga~~
in 125 ml flasks at a final cell density of
1.2 x 107 cells/ml.
Incubation was continued for 14 days;
those cultures showing no lysis were discarded.
The presence of virus was confirmed by assaying for
!
plaques.
This procedure consisted of preparing algal lawns
by centr-ifuging 10 ml of a 5 x 108 to 1 x 109 cells/ml culture at 120 x g for 30 minutes in a Sorvall GLC-1 centrifuge.
The supernatant was decanted and the cells resuspended in
0.4 ml of medium, to which 0.1 ml of a properly diluted lysate
was added.
This mixture was added to an overlay tube and
then plated (Adams, 1959).
days.
Plaques were counted within 3-5
Plaques measured less than 0.5 mm making it extremely
difficult to determine accurately the
Units (PFU).
numbel~
of P1 aque Forming
Estimates of virus titer were therefore gen-
erally·determined by growth curves
using~!..
elonaatus in which
the effect of the virus was measured turbidimetrically.
l
"-·-·-·~~rA<-
F
l
.. ~---~--~-·~,·---~
18
~--·---··-·-···~--·------·-------·-·---·----
!
Prop~_ga t·i on
Cyanophages vJere routinely propagated in 300 ml cultures using 1 liter flasks.
A culture of~ elon~us was
9
started at a density of 2.5 x 10 cells and allo¥/ed to grow
for 2-3 days.
At this t·ime virus was added and incubation
continued until lysis occurred.
Stock suspensions of virus prepared in this manner were
clarified
by
centrifugation :in a Sorvall RC·-2 centrifuge at
5860 x g for 30 minutes, and stored at 5°C without further
treatment.
Clarified lysates prepared in this manner were
tested for virus either by plating or observing lysis upon
addition
to~
elongatus cultures.
For large scale virus production a 14 liter fermenter
was used.
Lysogenic Strains
A lysogenic strain of h elongatus was isolated from a
lysate by selecting organisms insensitive to the original
lytic virus.
Several cultures which showed lysis after treat-
ment with virus samples were allowed to continue to incubate
for an additional 7 days after lysis was evident.
Cells from
cultures which showed evidence of renewed growth during this
period were harvested, washed, and subjected to three successive challenges with the original lytic virus.
Each challenge
of the resistant cells of these cultures was carried out by
I
1.. ...... " ... -----·--··~---·-·· ·-·-· ..... ····-----···,
't
19
r. . . ,. ., . . . . --.. - . . .,. . . .
-~·~·~,.
~-,._.-.__..,.--~~....---_,.-··~---
. ·-,.. . . . . . . . .
-~--~"'""'·~----·a·--·-------~~~-.~~-
....
m-~'-"""'_...,....
______
~-..--.~··
. . . -..
_··~,-,-
~
adding 10 ml of the original clarified lysate to 300 ml
of a suspension of washed cells at a density of 5 x 10 8
l
.....
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cells/ml.
"
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These suspensions were allowed to incubate for 7 days,
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then cultures showing little or no lysis were selected for
the subsequent cha 11 enge round.
After the fi na 1 treatment,
the cells were washed free of virus and carried through
three successive subcultures, starting each time with a small
inoculum of washed cells.
Finally, the cultures were tested
for lysogeny by Mitomycin-C-induction according to the method
of Cannon et
~.:_
(1971).
To l 00 ml of a lysogenic culture,
t~itomyci
n-C was added to give a fi na 1 concentration of 10
~g/ml.
Incubation was allowed to occur for 15 minutes and
then the culture was centrifuged at 3020 x g for 10 minutes
in a Sorval1 RC-2 centrifuge.
in 100 ml of fresh medium.
The pellet was resuspended
Controls consisted of the above
procedure without the addition of MHomycin-C.
The pre-
sence of virus was confirmed by plaque assay and electron
microscopy.
Purification
Viruses were purified by methods s·imilar to those described
by Safferman et_ ~ (1969).
Three hundred ml of freshly pre-
W"ere added aseptically to a growing culture of
~ ~longa~~- at a cell density of 5 x 10 8 cells/ml in a 14 L
pared
~ysate
;
'
;
.. ~ ....... ··-;··-··--··"·· .............. _._ .. ___~-----· ............ "'".-·---·.. '" -- ........ --····--·-· -•"····--.···--·-· . . ····-··---···---.... .J
20
.
r----~·-- -·-·-·-··---~-~----··--·-
!
1
. -------..
fermentation vessel.
·--··-·-----·--------~·-···----·---~-----·-~--
I
When lysis occurred the virus and
remaining cells and debris were concentrated by centrifugation at 25,300 x g using a Sorvall RC-2 centrifuge
equipped with a Szent-Gycirgi and Bl urn continuous flow
system operating at a flow rate of 20-30 ml/min.
Pellets
containing virus, cells, and debris were collected, resuspended in 40 ml of standard medium and refrigerated at 5°C
for 12 hours.
To remove virus from cells and heavier debris,
three cycles of differential centrifugation were carried out
by centrifuging at 25,300 x g for 45 minutes, each time resuspending the final pellet containing virus in 40 ml of standard medium.
At the completion of the last cycle, the top
fluffier layer containing the virus was separated from the
bottom firm layer consisting mainly of cells and cell debris
by gently swirling and decanting.
Each layer was then cen-
trifuged at 3090 x g for 15 minutes to remove cell debris.
The pellets were discarded and the supernatant was centrifuged at 25,300 x g for 45 minutes to sediment the virus.
The final pellet containing virus was resuspended in 2.0 ml
of standard medium and stored at 5°C.
Density gradient centrifugation was carried out in linear sucrose gradients prepared by mixing 12.5 ml each of
0.2 M and 1.0 M sucrose solutions in a gradient maker.
. ·-1
One
ml samples of virus were layered on top of the gradients
which were then centrifuged in a Spinco Model L centrifuge
21
·------~~--«------.1
.--~--------·-----
!
at 24,000 rpm for 15 minutes using a SW 25.1 Rotor.
II
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Grad-
ients were fractionated by removing successive 1.0 ml samp1es from the bottom of the tubes by means of a cannula
attached to a tuberculin syringe.
collected by this method
f~·om
A total of 25 samples were
each gradient.
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The absorbance
of each sample \vas then determined at 260 m1-1 and 280 mJ..l on
the Beckman DU Spectophotometer.
Those samples with a UV-
absorption characteristic of a nucleoprotein were stored
at
soc
for 24 hours (Safferman et
~.
1969).
Sucrose was removed by dialysis aga·inst 500 volumes of
standard medium for 3 hours.
After this per·iod the medium
was discarded and replaced with an equal volume of fresh
medium for an additional 14 hours.
The purified virus was
then collected and stored in a sterile container at S°C.
Lytic activity was assayed by addition of 0.1 ml of a virus
to a cultw~e containing 5 x 108 cells/ml of~ elongatus.
IV.
Cyanophage Characterization
Electron Microscopy
Combined fractions taken from major peaks of purified
virus were prepared for electron microscopy by allowing one
drop to stand for 3-4 minutes on 200 mesh grids coated with
one percent formvar.
Alternatively some samples were allowed
to evaporate to dryness.
Material
from cultures beginning to undergo lysis was
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,-~----prepared for examination by centrifuging at 2140 x g for
20 minutes in a Sorvall GCL-1 centrifuge.
The supernatant
was decanted and the pellet resuspended in the original
volume of distilled water.
One drop of this suspension was
placed on a grid and lysis allowed to continue uninterrupted
for one hour.
The grid was then touched to filter paper to
draw off excess liquid and allowed to dry.
All grids were fixed with one percent osmium vapor for
one minute, then stained by placing one drop of one percent
uranyl acetate (UA) on the grid for 3-4 minutes.
The grids
were then touched to filter paper to remove excess stain
and allowed to dry thoroughly before examination on a Carl
Zeiss Electron Microscope Model 9A.
The negative stain
phosphotungstic acid (PTA) at a concentration of four percent was also used in a manner similar to that of UA.
Virus measurements Were based on comparisons with latex
particles with a diameter of 0.109
~ ~
0.0027 u (Pelco)
Electron Microscopy Supplies) prepared in the same manner
as virus preparations.
Host Range Sensitivity
Cyanophages v1ere tested for their abi 1ity to infect
various hosts by adding suspensions of virus to liquid cultures and observing for lysis.
Five ml samples were added
to 45 ml of an actively growing culture and the effect on
23
r·--~,-.,--~-~-------·-~~-----·----~---·~-----·------·-·---------·-----l
cell
gt~owth
determined turbidimetrically.
Control cultures
I
prepared with five ml of water in place of virus were treated
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in a similar manner.
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Serological Procedure
Sf4-l anti serum was obtai ned from the laboratory of
Robert S. Safferman (Evironmental Protection Agency, Cincinnati~
Ohio).
Neutralization tests were carried out in a manner simi-
lar to that used by Safferman et
~h
(1969).
Antiserum was
diluted l/10, 1/100, and l/1000 with a standard suspension
of virus and allowed to react for 60 minutes at 25°C.
One
ml samples each of the treated virus suspensions were added
to Nephelo culture flasks containing 14 ml of a suspension
of~..:..
elongatus at a cell dens·ity of approximately 1.5 x
108 cells/ml.
Controls received no antiserum.
The effect
of treated virus on cell growth was followed turbidimetrically
for six days.
·pH Sensitivity
pH sensitivity of cyanophages was examined by methods
similar to those employed by Safferman and Morris (1964a).
Five ml of virus was added to 50 ml of medium which was then
adjusted to the desired pH using either 40 percent NaOH or
2N HCl
~
to 8.6.
After 60 minutes of exposure, the pH was readjusted
Virus suspensions were then brought up to 100 ml
24
r'-----~-----------~--------------------~----·------o·----------~--,.----'-·---~1
I
with standard medium and the virus activity assayed by inoculating 20 ml of virus suspension with 5.0 ml of a culture
of ~ el ongatus containing l x 107 cell s/ml.
Effect of virus on cell growth was followed turbidimetrically for three days.
Thermal Sensitivity
A modi fi cation of the method of Safferman and
~,1orri s
{l964a) for testing thermal stability was developed using
20 ml of medium containing 5.0 ml of stock virus.
Virus
samples were subjected to various temperatures in a waterbath for one hour.
They were then allowed to equilibrate
to room temperature and inoculated with 0.5 ml of h elongatus
to give a density of approximately 2 x 107.
Effect of virus
on cell growth was followed turbidimetrically for three
days.
Sensitivity to Filtration
The effect of filtration on virus samples was determined
by fi 1 teri ng 10 ml of virus through a 0. 45
ter.
1-1
mi 11 i pore fi 1-
Five ml samples of virus were added to Nephelo culture
flasks containing 20 ml of standard medium with a cell density of approximately 4 x 108 cells/ml.
on ce 11 growth was fo 11 owed
The effect of virus
tu1~bi d imetri ca lly
for 4-5 days.
25
r--..--.-.. -..
.
--------·~- ·-------------------~----------------~--------,
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Chlorofor·m Sensitivity
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Chloroform sensitivity was determined by adding sam-
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ples of virus to 1.0 ml spectroquality chloroform.
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This
suspension was shaken vigorously on a vortex mixer for 35 minutes and the virus was allowed to remain in contact
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with the chloroform for 24 hours.
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was decanted into sterile petri dishes and allowed to
After 24 hours, the virus
aerate for 1 l/2 hours to remove chloroform.
I
Five rnl of
this sample was then added to a Nephelo culture flask con-
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taining 20 ml of a culture of a cell density of approximately 4 x 108 cells/ml. Effect of virus on cell growth was
!
followed turbidimetdcal1y
V.
evm~y
24 hours for five days.
Transduction Assay
Transduction of antibiotic resistance was carried out
by adding five ml of a freshly prepared and clarified Mitomycin-C-induced lysate of resistant lysogenic cells to 20
ml of a culture of wild type cells at a final density of
4 x 10 8 cells/ml. These cultures were then incubated under
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growth conditions for a period sufficient to allow 1-2 generations of growth, the samples were then plated on several replicate plates of selective media.
The mutant fre-
quency v.'as determined in order to distinguish betv.,reen transductants and mutations.
Such controls were treated in a
similar manner as transducing cultures except sterile medium
'····
""'
.......................................
''"'
"'"
-
I
___
··.·;~·"'''""" -·- .. ---·--· -·-.. --- .. ----·-· ·---- ..................... ,,..,,,.. ···---.... ..........-. ·-------,-· _______ .
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..)
26
r-~-------~---
-------------~--~-------~·]
was added in place of clarified transducing lysate.
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Clarified transducing lysates were assayed for the
pt'esence of antibiotic-resistant cells by direct plating
of the lysate onto selective media.
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II
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L__ -·····--- ............ ·--·--·-··· ...... -------···•;. -----~---··· .............._....................~- ---·--------·~---------·--·-----------··· ................ ---···--·--······ ............... l
CHAPTER III
Results
Cyanophage Isolation
Table 4 lists 58 water samples collected as possible
sources of virus.
These samples were obtained from a large
geographic area which included California, Oregon, Nevada,
Hawaii, and Louisiana.
samples produced lysis
It was found that eight of these
of~
elongatus.
An attempt was made to collect water samples from a
variety of ecological niches.
This survey involved 2xami-
nation of rivers, streams, reservoirs, ponds, and sewage
effluents.
Analysis showed that of the eight samples indi-
cating lytic activity five of these could be attributed to
running water; three samples from stationary water were
found to produce lytic activity.
Tapia Park sample II was chosen for further study when
lytic activity was demonstrated to be viral in nature by
formation of plaques and by visualization of virus particles
by electron microscopy.
Electron micrographs of a lysate
produced by this sample (Figure 1) confirms the presence
of cyanophage.
The virus from this sample was designated
S-1 according to the nomenclature system established by Safferman and Morris (1963).
27
28
r·-
~···~-
............
~·--·------·
. ··· ................._______ -·---
--~
.......... . . -...............
~
~.-·----------------~-·-
..----·------·· ------·-........................................- ..1
:
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TABLE 4
Origin of water samples tested for lytic activity vs. water
characteristics. The table indicates where 58 water samples
wer·e collected from and whether the water was stationary or
running. Each sample was then tested for the presence of
virus, lr.Jith those samples showing lytic activity indicated
by +.
29
r~·-----------·--·---~~-----------~----~------------------~---~--·-·
I
SAMPLE LOCATION
t
WATER
CHARACTERISTICS
LYTIC
ACTIVITY
CALi FORNI A
Santa Clara River I
Santa Clara River II
Santa Clara River II I
Piru Creek
Santa Paula Creek I
Santa Paula Creek II
Santa Paula Creek III
Lion Canyon Creek I
Lion Canyon Creek II
San Antonio Creek
Tapia Park I
Tap·ia Park II
Tapia Park II I
Tapia Park IV
Lake Sherwood I
Lake Sherwood II
Arroyo Simi I
Arroyo Simi II
Arroyo Simi II I
Reseda Park I
Reseda Park (L.A. River) II
Reseda Park (L.A. River) III
Osborne Bridge I
Osborne Birdge II
Hansen Lake~ East Shore I
Hansen Lake Stream II
Gold Creek Bridge I
Gold Creek Bridge II
Gold Creek
Dry Canyon Creek
Malibu Canyon Creek
Vason Park I
Vason Park II
Lake Enchanto I
Lake Enchanto II
Matilija Creek
Matilija Dam
Water Fall (Hwy. 33)
Sespe Creek Bridge
Reyes Creek
Guyana River
\~a 1kel~ River I
Wa 1ker River II
Running
Stationary
Stationary
Running
Slow Running
Slow Running
Stationary
Running
Running
Running
Stationary
Effluent
Stationary
Stationary
Lake
Lake
Effluent
Stationary
Stationary
Lake
Running
Running
Running
Running
Lake
Running
Stationary
Stationary
Running
Stationary
Running
Running
Running
Stationary
Stationary
Running
Stationary
Running
Running
Running
Running
Running
Stationary
+
+
+
+
+
+
-------,
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----
~-----·-~·--·-·
-·-------~----------·----------------.
i
TABLE 4 (Continued)
1
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SAMPLE LOCATION
I
WATER
LYTIC
CHARACTERISTICS ACTIVITY
OREGON
Jordan Valley
Wolf Creek I
Wolf Creek II
North Power Road
Anthony Lake
Hanies (stream) I
Hanies (stream) II
Bi 11 's SvJamp
Bi 11' s Ditch
Running
Running
Running
Running
Lake
Running
Running
Sta'ti onary
Running
NEVADA
Eagle Lake
Upper Truckee River
Lake
Ru:ming
LOUISIANA
Caddo Lake
Shrivaport
Lake
Running
HAWAII
Maui Kai
Honokavai
Stationary
Stationary
+
+
31
FIGURE 1
Eiectron microscopy of S. e1ongatus showing presence of virus
S-1, prepared by the enrichment-iysis-electt'on microscopy
technique. This photograph confirms the presence of cyanophage, S-1. Magnification 18)000 X.
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..
~-·••··~-~~-·--_,~
--~-fi
_
.
.
__,,~~-~---...·~-- --~~-•·..,.r··--~ -~~·-·---·-~-~.,~------
-..-----..
--~~-·~·-·~~ --~-··-----~;~--.--~-·- -'-'~r.
~· -...- ~ _,_
•w- --.• ,..• _.,_,,- ••
~---~ ... ~-·...-..,"·~···-·
l
...... -~-"
33
r·-------------------------- ---------------------
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----------------------------------------------------------------------------------------------~
J:ysogen·i.£_Strai ns
A lysogenic strain
of~ !'lOQ_ga~_l:l:'i. was
isolated from
a lysate resulting from treatment of Tapia Park Water Sample
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II by selecting surviving organisms from the lysate insensitive to cyanophage S-1 by the procedure described in Materials and Methods.
The strain isolated by this procedure
(L-2-II) was resistant to lysis by the original virus and
was demonstrated to be lysogenic by Mitomycin-C-induction.
Figure 2 shows the results of such an experiment where
strain L-2-II was induced.
Samples taken routinely through-
out the induction procedure were examined by electron microscopy for virus production.
Virus was present in suffi-
ci ent quantities to observe after about three days and was
designated S-lT (Figure 3).
Host Ra.n_ge Sensitiv-ity of S-1
Organisms tested for susceptabil ity to S-1 and the
results are listed in Table 5.
The parent strain of S.
!'long_~tus_ LB 563 and the smr mutant were the only strains
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found to be subject to the lytic infection of S- i.
Serological
Relationship~
S-1 virus was treated with SM-1 antiserum to detect
any antigenic rel ati onshi p between these two vi ruses.
The
treatments were carried out with diffel'ent concentrations
of antiserum ranging from 1/10 dilution to 1/1000 dilution.
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-·--------·--·-----·----·--------.. -·-··-·-···----·--·-·-·-·---------·-······---.-----·-······l
i
FIGURE 2
Mi tomyci n-C-i nducti on of the lysogenic strain i,_ e 1ongatus
L-2-ll. Mitomycin-C was added to one culture of S. elongatus L-2-II to give a final concentration of 10 ug/~
Cultures induced in this manner (o-o) were followed tur·bidimetrically for 5 days showing the lysis of L-2-II and
release of S-lT. A second untreated culture vias used as
a control (~).
35
1.20
1.00
~
E
t:
0
N
<0
Cl!
u
0.80
t:
"'
..Q
S0
Vl
.0
'"
~
_;:;>
0.60
·~
Vl
<=
Cl!
D
t:
··-.,_,
0
"'
0.40
~
:::1
"0
Cl..
0.20
1
2
3
4
Number of Days After Treatment
5
36
FIGURE 3
Mitomycin-C-induced lysates of L elonoatus L-2-II. The
sample vtas prepared for electron microscopy by centrifugation of the lysate at 1240 x g for 30 minutes and resuspension of the pellet in the original volume of water. One
drop of this suspension was applied to 200 mesh grids coated
with formvar and allowed to stand for 1 hour. The preparations wer·e then fixed for 1 minute in 1% osmi urn vapor fo 1lowed by staining with 1% uranyl acetate for 3-4 minutes.
Magnification 70,000 X.
38
... --·-···-···········---·-·-··- -----·-····----------·· -·· ·----------·· -------- -··--- ·-··- ···---------·····-···- ..1
i
TABLE 5
Host range spectrum of S-1 cyanophage.
39
,--------··------_-----·-·-----·------
H·~-----------,
i
1
ORGANISM
Anacystis nidulans 1550
Synechococcus elongatus LB 563
Synechococcus elongatus smr
Synechococcus elongatus L-2-II
Synechococcus elongatus L-2-II smr
Gloeocapsa alpicola B 589
Oscillatoria sp.
Plectonema boryanum 18200
Sarcina lutea
E. coli
Arthobacter sp.
Micrococcus roseus
Baci 11 us mega teri urn
Chlorella pyrenoidosa 15-2070
SUSCEPTIBLE TO
S-1 LYSIS
+
+
!
I.... - _ _____
..
-
·········- ..... .. ··-·.
----,.,-·-·--····- . ···- ·-·· -----------·--···-··-- .......... -- - ····------ ------·- ·-· -- ---·-.- ........ __ i,
40
Effects of the antiserum on S-1 and SM-1 are shown in Figures
4 and 5 respectively.
A comparison readily indicates that
S-1 is not neutralized and as a result lytic activity is
still evident.
SM-1, however, was neutralized completely
losing its ability to cause lysis .
.Morphology of S-1 and S-lT
Electron micrographs of negatively stained (UA) cyanophages, S-1 and S-lT, reveal a hexagonal capsid with no
visible tail (Figure 6).
They apparently belong to the
same morphological class as SM-1.
Most of the heads appear
positively stained, whereas the outer protein coat appears
in negative contrast as shown in Figures 6 and 7. Cyanophages in association with host cell appear to represent
a homogenous preparation (Figure 7).
attached
to~
Figure 8 shows S-1
elongatus using the enrichment-lysis-elec-
tron microscopy techniques with phages appearing to repre-
' sent consistent and uniform particles. S-lT (Figure 9)
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obtained from the lysogenic strain L-2-II also shows simi1a r consi s tancy.
ferman et
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~
St·1-1, the cyanophage i soia ted by Saf-
(1969) was prepared by the same methods and
is illustrated in Figure 10 for comparison.
Cyanophage measurements were based on over 100 independent virus.measurements in several fields, with the mean
I
and standard deviations determined for S-1, S-lT, and SM-1.
L----~----·-··--·--
_j
41
........ ·-·· ······-···-----···-· ··--···-··--·-··-.... -··--·--······--·--···-·--------· ··-· ... ---·--····· ·-·· "1
I
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FIGURE 4
Sensitivity of S-1 to Sl~-1 antiserum. Antiserum 11as
di'luted l/10 (.Ir-A); 1/100 (<'>---<'>.); l/1000 (o-o); with
a standard suspension of S-1 virus and allowed to react
for 60 minutes at 25°C. The remaining activity was
determined by assaying the ability to lyse a culture of
~- elO!J.9.9tus.
The points represent turbidimetric measurements taken every 24 hours for five days. The following
controls wel'e used: S-1 virus untreated Iii-Ill and a culture
of h _g]_onqatus with no virus added o-o .
. . ........ ------- -- ... ·····-----------·-. _____ ,.,,_ _________________ .... ----- .. -------
'
-----~-
--·
-----
..
---------
----------~~-------------
!
-.-------····---------------·-'
42
-~--------.~----~------·-----------------·-----------·-
1.40
1.20
~
E
s::
0
N
0.0
1.00
•
OJ
u
s::
,_"'
.0
0
Vl
.0
"'
~
0.80
.iS
·~
Vl
s::
([)
Cl
c
0
...,
·~
"'
"'"-
~
0
0..
"~>~-----~~~
-----a
1
3
4
Number of Days After Treatment
I
[____
2
5
I
I'
·--- --- ... -
!
···-·-·····--------------- ·------.. ------------~-- ------- ------------------------- -------------- ----------·--·-- ______________ .J
43
FIGURE 5
Sensitivity of SM- 1 to SM-1 anti serum. Anti serum was
diluted l/10 (.&.----A); l/100 (Lx-il.); l/1000 (o-o) with
a standard suspension of SM-1 virus and allowed to react
for 60 minutes at 25°C. The remaining activity was determined by assaying the ability to lyse a culture of S.
e1ongat.us. The points represent turbi di metric readTngs
taken every 24 hours for five days. The following controls
were used: SM-1 virus untreated~ and a culture of S.
el ongatu2_ with no virus added o-o .
II
.... -. -~----~~---·----------------------····-.>~--·-·'- . --------------------------------- --·'·--·- .... --···--- ----- ··---- ··-·····----------------·- ____ j
I
44
-------------l
r·----------------------------------------
I
1
1.40
I!
1.20
~
E
<=
0
N
1.00
.
\D
"'u<=
,_"'
L:l
0
0.80
Vl
L:l
"'>,
~
-1-'
·~
Vl
s::
"'
0
0.60
<=
0
·~
+'
"'
"'
~
0.
0
0-
0.40
----111
1
I
I
I
2
3
4
5
Number of Days After Treatment
I- - - - -- - - -- ------ - -·- -- --- ·.·---··· ··--···-··· ---···-·····----------·-··············- ----------·····----------- -·-··· -- ·--~-------------------1
45
.. --··-- ········-----------~--- --------------------~----- -------·-·····-------------
---- ······--------------·-. --------------- --·-·
·;
I
··FIGURE 6
Electron micrographs of negatively stained cyanophages
representative of S-1, S-lT, and SM-1.
A.
B.
C.
D.
S-1 cyanophage at a magnification of 154,000 X.
S··l cyanophage at a magnification of 84,000 X.
S-lT cyanophage at a magnification of 154,000 X.
St~-1 cyanophage at a magnification of 154,000 X.
The line represents a scale of 1000 angstroms.
I
II
I'
----- ....... ······-· ,_, .. ,. ··-·· . ···- ................ --------- ..,-:- ---------- ····---------·-·· -------· ----- ·----- -- ---~------ ----------.-------------- _________ .. ---------·-..l
A
8
c
D
47 .
~--··-·-··-------·--·-··--··---·-·--·-----------····-
··---·-····-·--·-------·--------·-····-·-··--·---··· ··-· ···---··1
I
I
FIGURE 7
Parti c1 es of purified cyanophage S-1. E1 ectron micrograph
shows S-1 cyanophages stained with 1% UA. ~1agnification
84,000 X.
'·-------~----------~--- -------------·-~---·-
i
. ------ ---- ---- ···<;,·;----..---·-·--·-- --------- _____ ,. ____________ ------------------------.-- ·-·-- -- . -------·· ----··· ···------ -----·····'
49
•<- - -
--~-- ~-~---·-··
------------·· ----·-·········· --------····-·-
••• . - - . - - - - - - - - - - - - - - - - - - . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - · - " " ' - - - - - - -
~
'
FIGURE 8
Particles of S-1 cyanophage attached to _h elongatus cell.
A.
S-1 is attached to a cell of S. elongatus.
tion "13,500 X.
B.
h elongatus cell shown at a higher magnification with
virus attached.
Magnifica-
Magnification 45,000 X.
I
I
..... -- ----·--------· --··-·- ------- --+·" -- ·-------- -- -------·
'
i
..... ------------ ----- . -··-····----------------------------------------------·---.!•
51
FIGURE 9
Particles of S-lT cyanophage attached
ce 11 .
to~
elongatus L-2-II
A.
The temperate phage, S-lT, obtained from a culture of
L-2-11 which had been induced with t~itomycin-C. Magnification 13,500 X.
B.
The same cell is shown at a higher magnification.
Magnification 45,000 X.
. .... ..
------~-
......... -... ---------------------1!-J.------·····----------~----- ------
B
------------------------------------------
53
. - .. -------- -----------------··--·· ----------------------------------- ---- ----- ---- ·------- -----------------------------------1
FIGURE 10
Particles of SM-1 cyanophage attached
nification 56,000 X.
to~
elongatus.
Mag-
.. -- ------ ------ ----·-7·---------- ----- ---- .. ----- --- --------------- . ------ ... - -- ---- - ---- . -·· -- -- ----- - --- .-- ... ____!
55
r ::::::::~,~::~:::~:::~::~:·:,~:::.:~·::~:,:·:;~ ~~ .
stroms
t
31 angstroms.
A schematic diagram of S-1 is
silown in Figur·e 11, indicating morphological structure and
dimensions.
SM-1 (Figure 60) was analyzed in a manner simi-
lar to S-1: its hexagonal capsid was found to be 895 angstroms + 31 angstroms by 764 angstroms! 35 angstroms, a
value corresponding closely to the measurement of 880
angstroms made by Safferman
~ ~
(1969).
For compara-
tive purposes a schematic diagram of SM-1 is shown in
Figure 11.
The temperate phage S-lT (Figure 6C) was found
to have a head of 840 angstroms
angstroms
t
49 angstroms.
t 37 angstroms by 710
Figure 11 shows a schematic
representation of the morphology of S-lT.
Sensitivity of S-1 and SM-1 to Environmental Conditions
The effects of pH, temperature, filtration, and ch 1Ol"Oform treatment on viruses were assayed by determining the
effect of these agents on the abi 1i ty of the virus to lyse
a suspension of _0_,_
.§_l_C2.!_1_gat~.
The effect of pH on virus activity is shown in Figtwe
12.
S-1
\~as
found to be stab 1e between pH 5 and pH 11 .
Treatment of the virus at pH 2.9 greatly decreased activity
and a similar decrease in activity 1-1as observed at pH 12.5.
S-1 was found to be stable to treatment at temperatures
56
. . . ··-· -------------·-·· ----·--·-··-·· --··· --·- --····-···-·-···-······--··---- --·-···--· ----···-·--······-- --· -··-·--·-··--·-··- . . -··-· -· ····-· ---·r
I
I
FIGURE 11
Schematic diagrams of S-1, S-lT, and SM-1.
A.
The structu1·e of S-1 cyanophage isolated from Tapia
Park, California.
B.
The temperate virus S-lT is shown.
C.
The structure of cyanophage SM-1 is shown which was
obtained from Robert S. Safferman, Environmental Protection Agency.
57
r·-..----------------------------·-------------------------------..--------------1
I
I
.
,------""\
'
I
I
I
\
\
'
'' \
I
}
f-
(
~
I
I
\
Vl
'\
.
\
I
'---- -- _,'
I
I
0
~
,...__
l
'
"'
~
I
<><(
I
r---- - ,
0178
I
\
\
I
'\ \
I
I
I
I
\
~
(
I
:E
\
''
I
VJ
'' \
I
I
\.._
_____
u
I
_/
I
)
""
\0
"""
,...__
1
'g' S68
I
,-------..
\
'
I'
I
\
<><(
I
I
~
>
I
I
(
'
Vl
'\
L()
L()
LO
I
I
I
'\
'--------'
""
f----
\t
1
ZLL
I
L. ------- . . . . . . . . -------..................... ----.. . ,._,.______________ . . . . . . . . . . . . . . . . . . -.. . . . . . . . . . . _________________ . . . . . . . . . . . . -------.. . . . __
_i
58
FIGURE 12
Sensitivity of S-1 to pH. Virus suspensions were treated
for 60 minutes at the pH indicated, then their remaining
activity determined by assaying the ability to lyse a culture of~ elongatus. The points represent final turbidimetric readings determined after 72 hours.
59
r-··----·--·-·----------·-·--·------..------·------------···-·-------------1
I
-
I
I
I
N
:r:
0.
N
0
<:1'
.•
0
(mu
i
L.
LO
M.
M
0
LO
N
0
N
0
0
0
0
ozg
'a:Jueq.wsqe) r.:nsuaa
LO
0
0
0
uo~:pqndod
0
60
~-------------------------------------------------------------
of 30°C to 70° C but was substantially reduced in activity
,1
by treatment above this.
Figure 13 shows the results of
temperature treatment.
Figures 14 and 15 show the effect of filtering virus
through a millipore filter membrane with a pore size of
0.45 u.
It can be observed that S-1 lost considerable acti-
vity when filtered (Figure 14). The loss was more scdous
when increasingly larger quantities of virus were filtered.
SM-1, in comparison, showed no such reduction in activity
when filtered in a similar manner (Figure 15).
The effect of ch 1oroform treatment on S·-1 and SM-1 is
shown in Figure 16.
It is evident from this figure that
S-1 is sensitive to chloroform with the loss of activity.
SM-1, when treated in a similar manner, shows no such effect.
Transduction of Streptomycin Resistance
Transducing activity was sought by treating wild type
cultures
of~
elongatus with Mitomycin-C-induced lysates.
Streptomycin resistant (Smr) mutants of L-2-I I when
tested for resistance were found to be completely resistant
to 200
~g/ml
even after being maintained through a series
of 20 liquid subculturings in the absence of the drug.
Wild
type S. elongatus strains were tested for sensitivity to
streptomycin and were found to be completely inhibited at
a concentration of 0.02 ug/ml.
I
~--····-
..
. . ······----···-------·
·-···----.--·-··-,,~-,--·
'"' '"" '--------' -- -----------..,. --- -------------·----------- ----- ---·------____!
61
FIGURE 13
Sensitivity of S-1 to temperature. Virus suspensions were
treated for 60 minutes at the temperature indicated. Their
remain·ing act·ivity was then determined by assaying the ability
to lyse a culture of h e·longatus. The points represent
final turbidimetric readings determined after 72 hours.
I
J
62
-------------- ---------------------------···-·--------------·-···--------··1
I
0.40
~
E
t:
0
N
1.0
.
0.30
C)
l)
t:
..."'
.n
0
Vl
.n
"'
~
.,_,>,
·~
Vl
<::
OJ
0.20
C>
<:
0
.,_,
·~
"'
~
:::!
0.
0
0..
~
"'
<:
·~
LL
0.10
-
30
40
50
60
70
80
90
100
110
Temperature oc
I
,
l.. ·········----------·-···· ·- ... ··- .•;;· .... --- ---- -------------·-· ----------------- ............ ·····-·-···-----···-··---···-·-----·----· ..... I
63
,--------------------- ------------------·---------·-----------··---------···-·····-····-···----- ..... --- ···- .. -.. -- - ·-- ---1
I
I
I
FIGURE 14
The sensitivity of S-1 to filtration. The effect of filtration on S-1 was determined by f-i 1teri ng virus through
a 0. 45 JJ mi 11 i pore filter membrane and determining the
remaining activity by assaying the ability to lyse a culture of S. elongatus. The points reoresent turbidimetric
readings taken every 24 hours over a six day period. The
fo 11 owing amounts of virus were tested in this manner:
5. 0 ml of filtered S- 1 , b-i:J. ; 0. 5 ml of filtered S- 1 ,
o--o . The fo 11 owing contro 1s ~;ere used which were not fi 1tered: 5.0 ml of untreated S-1, ~ ; 0.5 ml of untreated
S-1 , 111-111 ; no virus in a culture of i:_ e 1ongatus, o-o .
l_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - · ;-· · - - - - -· · · ·- . . .- - - · · · ·-- - - -~- - - - - - - - · · · - -- - - -
!
I
64
------·-··-·· ············-···-·---·-·-·----------·
--···-···-----·---·--·-·----------~
1. 00
'
0.90
0.80
~0.70
I~
~~
~
[j,,_ o.6o I
lo
IV>
,.a
~ ..:::.o. 5o
1
>,
!::=
IV>
'"'
' OJ
lo
1.~ 0.40
IE
1::>
1~0.30
0.20 -
0.10
1
2
3
4
Number of Days After Treatment
5
6
65
,--------------------------- ·------------------- --------------------- -----· --· -·- ---------··------------ ------·--··--- ----·- ---1
I
I
I
FIGURE 15
The sensitivity of SM-1 to filtration. The effect of filtration on SM-1 was determined by filtering virus through
a 0. 45 1-1 mi 11 i pore filter membrane and determining the
remaining activity by assaying the ability to lyse a culture of S. elongatus. The points represent turbidimetric
readings taken every 24 hours over a six day period. The
following amounts of virus were tested in this manner: 5. 0
m1 of fi 1 tered SM- 1 , f:r--1:::,. ; 0. 5 m·l of fi 1tered St1-l D-D .
The following controls were used which were not filtered:
5.0 ml of untreated St·1-1, k--A ; 0.5 ml of untr·eated SM-1,
111-111 ; and no virus in a culture of i,_ elongatus, o-o .
I
L_ _____ .. ----·-·------ -------- -------·····- -- ---------------- --- -.. ----------
··-------~---·-- . ·-·- --· .. -.. -·-·--- .. -·· .-- . -··· -- ....... - -·· j
I
66
r--·----------------------- ------- ------------------------- -----------------------------..
'T
I
0.90
0.80
~
E
0.70
<:
0
I~
I~
0.60
<=
n:l
J:l
S0
Vl
J:l
n:l
I;
I·~
i
I
Vl
<=
QJ
10
"'0
•c·
+-'
n:l
~
::l
0.
0
0-
0.20
0.10
1
I
I
I
L_____ -
2
3
4
5
6
Number of Days After Treatment
;
....• ~-- ------·-···---.- ....... ··············-·· -------------------------------------------·· •• --
'
......•••.!
67
i
.... --- ---------··· -----
---~·- -~---···
------- ----------------------------- - ---- ·- ····--------- ---- .... ------- -- -- . . . . . ........... ._ ... ---- ---- --· ------------ -1
'
FIGURE 16
Sensitivity of S-1 and SM-1 to chloroform. Virus suspensions of S-l and SM-1 were treated v1i th ch 1oroform for 24
hours. The remaining acti v·i ty was determined by assaying
the ability to lyse a culture of~- ~ongatus. The points
represent turbidimetric readings taken every 24 hours for
6 days. S-1 treated with chloroform~; S-1 control
o-o; SM-1 treated with chloroform A-A ; SM-1 control
br--1:.; and no virus in a culture of S. eloQ_gatus o--o .
........ -·····-············· -·-'··; . -......... ··-··· ............... -----····· .. -·- . .. ......... ... ... . .... ----· .····· ....... -·····-.J
68
~------·--·----------------------------·----··---------·--·-·-------··-·-··--·-·1
1.00 -
E
0.80
"'
0
0.0
"'
.
QJ
u
"'"',._
.0
0
0.60
Ill
.0
"'
·~
~
·~
Ill
"'
"'
+'
"'
:::>
QJ
0
0.40
0
·~
~
A
0.
0
0...
0.20
1
2
3
4
5
6
Number of Days After Treatment
!
I
........
···--·--·- •···•······ . ·····-·
--,~·-·------"-·-
·····--
. - -·- · · · - · -· . . - - -· · - · · - .- --· ·- · · - ·- · - - · ----·------·---·--·- .-
69
r··- ----------------------------------------------------------------------------------------,
I
The treated wild type cells were allowed to incubate
I
I
under growth conditions for a 1-2 generation growth period
I
to permit expression of transduced marker before plating
l
directly onto the medium containing streptomycin.
The
Il
results of two such experiments are summarized in Table 6,
i
I
clearly showing the transduction of smr resistance.
In
every case the number of resistant cells in vi rl!s tr·eated
cultures are approximately an order of magnitude greater
than that from untreated cultures.
-~
----··-- .. -·····----.-
··---~---··-
-·· -.. -----•.-;·; .
·--··--··--·
.•. ··-·-·.
!I
I
!
70
TABLE 6
Transduction of antibiotic resistance in the blue-green alga
h elongatus. Five milliliters of freshly prepared and clarified Mitumycin-C-induced lysates of smr strains of L-2-II
were added to 20 ml of a culture of w·i l d type ce 11 s to give
a final density of 4 x 108 cells/ml. These cultures were
incubated under growth conditions for a period sufficient to
allow 1-2 generations of growth, then samples were plated on
several plates of selective medium. Controls consisted of
a portion of the original culture which received sterile
medium in place of the transducing lysate. The transducing
lysates themselves were assayed for the presence of antibiotic-resistant cells by direct plating of the lysates onto
selective media. A total of 2.5 ml of each lysate was
plated d·irectly: no colonies appeared on any of the plates.
l. ..
·--·····--····--·--······ ---~--~·~-~------------------------
·····--··-·---·..--- ______,. __ ----- ---. ·---- ---·-···-··--- -·-------···-
.
...... ··--·· .,_,
71
CONTROL
(NO VIRUS)
I
rI
Total No. Cells
I
Plated
VIRUS
TREATED
Expt. 2
17. 5 x 1o8
34 x 1o8
Expt. 1
63
589
Expt. 2
164
1078
Cells in Population Expt_._1
Expt. 2
(x 1o8 )
3.6
69
4.8
40
I
Exo!:__j_
8.5
X
34
X
I
Total No. Resistant
I
I
Colonies
I
I
I
I
I
Fraction Resistant
108
10 8
CHAPTER IV
Discussion and Conclusions
The evidence presented in this paper clearly demonstrates the isolation of. the cyanophage S-1, lysogeny of
~
elongatus with the temperate phage designated S-lT,
and transduction of a genetic marker.
During the course of this study a variety of water
samples were surveyed in an attempt to isolate a cyanophage
which could ultimately be used in a transduction study.
Although lysogeny had already been demonstrated in the filamentous blue-green alga Plectonema boryanum (Cannon et
~.
1971 and Padan et al., 1972), I required a unicellular bluegreen alga in order to avoid .Procedural difficulties which
would be
encountere~
genetic marker.
in recording the transduction of a
The· choice
of~
the demonstration by Safferman et
elongatus was based upon
~
(1969) that this blue-
green alga was· subject to lytic infection and it was suspected that it might be susceptible to more than one type
of virus.
This suspicion was supported by the
fi~dings
that Plectonema boryanum is infected by two different phages
0
LPP-l (Safferman and Morris, 1963) and LPP-2 (Safferman et
al., 1967).
A variety of ecological niches were surveyed to include
as many sources of virus as possible.
Eight samples out of
II
1"·-·-·
···--···--------- - - - - - - - - - - - · - · - · - - - - - - - - - - - - - - - · - - - - - - - - - - · - - - - · - - - - - - - - - · - - - · - - - - - -
72
I
I
I
, ..... --. ---- ...... ------- .. ---·· · · · -------·--·---- -····· -----------·--------------------------------------------1
'
I
58 (shown in Table 4)
i
activity.
\~ere
found to have some form of lytic
In addition to viral infection, the activity
could have been the result of chemical contamination or bac-
I
I
terial pathogens (Stewart and Brown, 1969 and Draft and
II
Stewart, 1971).
I
I
I
i
I
For this reason it was felt advisable to
confirm the suspected virus by electron microscope.
The
number of samp 1es was too sma 11 to draw a significant correlation between the presence of lytic activity and the ecologica·l niche from which it came.
Lytic activity was found
in both stationary and flowing waters.
Because of the vari-
ety of niches in which lytic activity was found, S- l and SM-1
type viruses are apparently wide spread.
There is probably a
greater likelihood that they wi 11 be i so"l a ted from sewage
treatment plants like the majority of the cyanophages previously isolated (Table 2).
The Tapia Park sample which was
chosen for further study was collected from the effluent of a
new sewage treatment plant for Los Angeles, California.
It was
observed that the discharge fed into a stream north of the plant
which contained a rich variety of alga life.
The assay used by most researchers in the study of
these viruses has involved the "enrichment-chloroform technique" used by Safferman and Morris (1963).
I found that S-1,
when treated with chloroform for 24 hours (Figul'e 16) lost all
of its ·activity, thus indicating that the so 1e use of this
method would exclude those viruses which are sensitive to
l. .. ... _, ____ ·····--·
»- •
-
- - - - - - - · - - - - - - "'"
·-
-
··;-~ ~--·
I
...... -- .... --· ......................... ----·-····-·-·-······--·-··- ··- ___I
74
1····----·------------··----------·-···-·-·-··----·--··-··--·---·------·----------l
I
ch 1oro form.
Even a ten minute exposure to ch 1oro form
results in a loss of activity.
I
I
II
i
I
I
I
Cons ·j der-i nq the extremely
low titer of virus found in nature it is conceivable that
if the "chloroform-enrichment technique" had been used in the
initial isolation, S-1 would probably have been excluded.
It
is advisable for future studies to exclude chloroform from
initial isolation techniques.
The enri chment-lys i s-e 1ectron-mi croscopy method provided a rapid indication of the presence of virus and simultaneously gave information about relative morphology, sizP.,
and the degree of homology with other viruses.
This pro-
cedure (Figures 1, 3, 8, 9, and 10) demonstrated the ease
with which one can make rapid surveys of many possible
sources of virus.
Filtration of samples of virus to purify and clarify
lysates is a common method adopted by · researchers when studying
phages (Adams, 1959).
The original isolation of LPP-1 (Saf-
ferman and Morris, 1963) and the isolation of St1-l by Safferman et
~
membrane.
(1969) were both carried out by using a millipore
The same procedure when attempted in this study
for S-1 drastically reduced virus activity (Figure 14).
Reduced activity also resulted when S-1 was filtered through an
ultrafine sintered-glass filter.
manner·sho~led
SM-1 treated in a similar
an insignificant reduction of activity.
For-
tunately during the initial isolation procedure sufficient
! ....
75
r-···-·-····-······-····-····-·-----------·-··--·-·-·--··---------·-··------------·-----,
v·irus passed through the membrane unhanned.
Such an
I
initial isolation procedure could have eliminated
'
other viruses sensitive to filtration from being recovered.
i
I
f
The sensitivity to filtration may be the result of
attraction of S-1 to the millipore membrane or the sheer
force of filtration may be enough to
vity.
c~use
a loss of acti-
Similar responses are observed for other viruses
such as the enteroviruses studied by Konowalchuk and Speirs
(1971)
0
Extreme difficulty was found in determining the presence or absence of plaques 11hich were used to estimate
the activity of the virus which is the normal method used
for bacteriophages.
I reported that S-1 had plaques
measuring less than 0.5 mm making it impossible to accurately
determine the activity of the virus by this method.
Attempts
to increase the plaque size by varying both the media and
the concentration of a1ga 1 1awns fai 1ed.
Turbidimetric
readings from growth curves were used as an alternative
·method to give some quantitative measurements of the effect
of S-1.
It was also possible to get semiquantitative
estimates of virus titer from growth rate experiments,
based upon the time required for lysis to occur.
The
enri chment-iysi s-·e 1ectron microscopy technique showed
its value by allowing for the proof of virus without
the necessity for p1aque counts .
... .
~--·--------------
--.---
-- ...
76
r·-----------------------------------------------------------------------------------------------1
I
.
Safferman (personal communication) has
tndtc~.ted
that all virus isolated for this host so far are SM··l
type.
He therefore were ·interested in determining the
characteristics of S-1 to see if it was of this type.
The morphology, serology, and the sensitivity to environmental conditions were tested to see whether these two
viruses differed in any major aspect.
Morphological analysis using UA showed that S-1 was
significantly sma 11 er than the reported measurements for
SM-1 (Safferman
~
.'D_,_, 1969).
He therefore prepared and
II
measured SM-1 under the same conditions as a means of con-
I
firming the accuracy of our measurements.
I
The values we
obtained for SM-1 did not differ significantly from those
l
l
reported by Safferman et _tD_,_ (1969).
Both cyanophages conform to Bradley's (1967) classification system because of their hexagonal capsid without
a tail.
Recently, evidence has been presented to indicate
the presence of a stub for SM-1 (personal communication,
Robert S. Safferman).
It is of interest that S-1 was destroyed with the
negative stain PTA and this appears to be a property found
in other cyanophages (Luftig and Haselkorn, 1967).
SM-1,
in contrast, was not affected by as much as 4 percent PTA
as shovin by Sa fferman _et _tD_,_ (1969).
pH sensitivity of S-1 was similar to that found for
--,·.-~
I
.. -------------- ----------- ------------ ---- ------- .----- -----------------------------------_1
77
SM-1 (Safferman --et al., 1969) and LPP-1 (Safferman and
Morris, 1964).
A pH range of 5-11 was found to yield
uninhibited lysis, with a pH of 8.6 yielding optimum
results.
Most phages appear to have similar pH ranges
and it has been suggested that this is probably due to the
fact that the host tends to grow in an alkaline environment
(Safferman, 1968).
Thermal sensitivity was found to be different for
SM-1 and S-1.
It was observed that S-1 was relatively
s_table up to 70°C.
Above 70°C quantitative data indi-
cates denaturation.
Safferman et
~
(1969) found that
0.001 percent of the infectivity remained at 55°C which
is in marked contrast to S-1.
The existence of a thermo-
phile in the Synechococcus genus suggest that S-1 or some
other virus may be able to infect those cells capable of
such extreme temperature.
The availability of antiserum for
SM~l
made serological
testing possible and proved to be a determining factor as
.
to whether S-1 and ·sM-1 were related.
I
SM-1 was found to
be completely neutral"ized in the pr·esence of the antiserum
(Figure 15).
S-1 when treated in a similar manner (Figure
14) showed no neutralization, indicating that there was
no antigenetic relationship between SM-1 and S-1, thus
confirming the distinctiveness of the two phage systems.
I conclude from the data on antiserum effect, thermal
i
l _________________________________________________________
78
r···--···-·· ······-·--···- ·-··-··----···--·-·----·--·-------·---·-----·---·-------·------------·--------·-·----·----1
I
I
I
sensitivity, pH sensitivity, morphology, filtration and
1
chloroform sensitivity that S-1 is a new virus, unrelated
I
to
St~-1 .
The lysogenic strain
of~
elongatus_ was isolated
through a series of challenges with the strain L-2-II
selected on the basis of its resistance to superinfection
from S-1.
To prove that
~
elongatus L-2-II represented
a new lysogenic strain attempts were made to induce virus
production.
Difficulties were encountered when induction was tried
with peroxide, UV irradiation, and temperature shift experiments.
It was only after trying Mi tomyci n-C in a manner
similar to Cannon et
( Fi 9ure 2).
~
(1971) that induction was achieved
Electron micrographs taken after induction
showed the temperate phage, S-lT.
The observation of virus
from an induced lysogenic strain of alga was possible only
after a several-day lag period.
S-lT was found to be similar to S-1 and SM-1 in relation to its over all morphology.
The only apparent dif-
ference v1as in the size of the phage.
This apparently
repre-
sents a different cyanophage although extensive characterization has not yet been carried out.
Transduction of streptomycin resistance was initially
studied· by a series of preliminary experiments.
Once lyso-
geny was confirmed and the processes of induction standar-
79
~--------·-·····--··-------- .. - · - - · - - - - - - · · - - - - - - · - · · - - · - · · •..-.c............. - ••
---------------------.--l
I
dized it was possible to make accurate determinations as
to whether Sn{ resistance vws transduced.
Preliminary
experiments clearly indicated that there was from 4-20
times as many swr co 1oni es showing up when the culture
was treated with a smr lysate as when untreated.
I thus assumed that transduction was possible and proceeded to standardize the methodology.
It became evident
that a lag period was required in order to
esta~lish
plete expression of streptomycin resistance.
type
~-'-
~!hen
com-
wild
e 1ongat~ ce 11 s were added to s- 1T with the smr
marker and then challenged immediately, the number of transductants was drastically reduced.
The need for complete
expression before challenging has also been described in the
bacteria (Hayes, 1968).
Blue-green algae in genera 1 are
known to have very long generation times, depending upon the
culture conditions.
I determined that to ensure the great-
est degree of transduction as possible a period of 1-2 generations was necessary to show complete resistance.
I
Two experiments have been summarized in Table 6 showing
i
the transduction of Smr resistance to wild type i:_ elonqatus
at a frequency of 4.0 x 16- 7 to 6.9 x 10- 7 per cell. The
I
number of antibiotic-resistant colonies indicated in the
table probably does not represent the actual number of trans-
I
ductants, since the cultures had been allowed to grow during
virus treatment . There is also the possibility that some
·;~
I
... ..... .... .. . . ........ ·- '• ...... -- ...... - --- ......_... ,..... -----··· ·····-·····-···-··---·--- ................. __ _!
BO
r---------------------------·--------------------------------------·--------------·--·----------1
I
I
I
I
I
I
I
I
I
r
.
of the res 'istant co ·r oni.es represent carryover of parent ce 11 s
in the virus preparation.
virus sterility control
However, the sensitivity of the
1~as
such that only a small number
of cells were likely to have been included in the transducing lysate; even with as many as 2 generations of
growth before p1ati ng, the carryover would be ins i gni ficant with respect to the actual numbers of resistant cells
found.
It is not possible to determine what type of transduction is involved because only one genetic marker is
available.
An attempt to isolate a penicillin resistant
mutant indica ted that this mutant was unstable.
It is
likely that generalized transduction was the mechanism
because of the restrictiveness and the high frequency of
transfer observed with specialized transduction.
The best
test would be a comparison of the effect of transduction
between two independent markers from the same lysate.
In
such a case restricted transduction would give rise to only
one marker, while unrestricted transduction would see both
markers transduced.
I have proven that the isolation of S-1 from Tapia
Park, California is a distinct and new cyanophage for h
elongatus.
The lysogenization of strain h elongatus L-2-II
is confirmed through e1ectron microscopy and
induction.
\ ____ ··-··-· ·---~---·- ------
~1i tomyci n-C-
The transfer of smr resistance by phage S-lT
.. --. - ----- ..... - ····--.-··-···-- ---·------ "" -----
I
--------·-. --------·········--- -~-------- -- •.. _. .....•• --------------------·· ·----------· ...... ____ j
81
r· -- -------------------------------------------------------------------------------------------1
appears to parallel the action of transduction as found in
the bacteria.
The evidence for transduction of streptomycin
resistance presented in this thesis represents the first
significant demonstration of gene transfer in the blue-green
algae.
r····---------------·--·-··-···-----·-···------·-··-····---······------······--·--- --·----------------------------····-----·-·········
I
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