Download Lysogeny and Transduction in the Marine Environment

Viruses as Regulators in Microbial Systems
Lysogeny and Transduction in the Marine Environment
Paul, J.H., P.K. Cochran, S.C. Jiang
University of South Florida, St. Petersburg, FL 33701
ABSTRACT
Viruses can interact with their hosts in a myriad of ways. Most ecological investigations of
viral/bacterial interactions have focused on the process of bacterial mortality resulting from lytic
infections. We have been interested in the genetic interactions of phages with their bacterial hosts.
Specifically, we have investigated the process of lysogeny, whereby a virus sets up a stable
genetic symbiosis with its host cell. We have found a strong seasonal pattern of occurrence of
lysogeny in the subtropical estuary of Tampa Bay, with the greatest proportion of lysogens in the
spring and summer, with no detectable lysogeny in the winter months. A series of commonly
occurring organic pollutants (polychlorinated biphenyls, polynuclear aromatic hydrocarbons, and
pesticides) were demonstrated to cause prophage induction in natural microbial populations. We
have demonstrated plasmid transfer by transduction using a marine bacterial phage host system in
culture and in ambient microbial populations. Our work demonstrates that viruses can genetically
alter marine microbial populations through the processes of lysogeny and transduction.
Introduction
Over the past decade, viruses have become recognized as the most numerically abundant members
of the planktontic marine microbial flora, at concentrations ranging from 104 to 108/ml [2]. We
now know that approximately 20 to 50% of the bacterial mortality can be caused by viral lysis ,
on the same order of magnitude of that caused by protistan grazing [8]. Models of the Microbial
Loop now show that viruses are an important component of this important oceanic process.
Perhaps less is known concerning the genetic interaction of viruses and their hosts. In this
short communication, we will share some observations on the processes of lysogeny and
transduction in the marine environment, two process which potentially can affect the flow of
genetic information rather than the flow of carbon through marine microbial communities [4, 13,
14].
In lysogeny, a temperate virus sets up a stable symbiosis with its host, resulting in the
integration of the phage genome (termed prophage) into one of the replicons inside the cell. The
prophage genes remain silent until the process of induction, whereby a chemical or physical
factor results in activation of the prophage genes and initiation of the lytic cycle [1]. A survey of
110 marine bacterial isolates indicated that 40% contained inducible prophage [11]. To determine
if lysogeny occurred amongst natural populations of marine bacteria, we have used the mutagen
Mitomycin C to induce natural populations of marine bacteria. Inducible prophage were found
more in eutrophic estuarine environments (80% of the environments tested) compared to
oligotrophic offshore environments (27%), with 1.5 to 38% of the bacterial population being
lysogenized [10].
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Viruses as Regulators in Microbial Systems
Results and Discussion
During 13 month of biweekly sampling at a station in Tampa Bay prophage induction by the addition
of mitomycin C was only detected when water temperatures were greater than 15oC (February to
October; [5]). No induction was detected in November, December, or January (Fig. 1). The percent
of the population lysogenized ranged from 0 to 37%, averaging 6.9%. If 30% of the population was
lysogenized, and 1/3 of these were induced by some event, this would produce ca. 107 phages /ml,
equivalent to 100% of the ambient viral population, indicating that prophage induction could have
a significant effect on ambient viral concentrations and bacterial (and phytoplankton) mortality.
Fig. 1. (Top panel) Viral direct counts (VDC) and bacterial direct counts during a 13 month seasonal study in Tampa
Bay. Notice the low viral counts in December, January, and February. (Bottom panel). Prophage induction by mitomycin
C (squares) and water temperature during the same seasonal study. Notice the absence of inducible prophage in
November, December, January and early February, followed by a “lysogeny bloom” in late February and early March
(p<0.0001). Significant prophage induction (p<0.05) occurred during most samplings from mid-February to October.
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Viruses as Regulators in Microbial Systems
This data clearly shows that in the eutrophic estuary of Tampa Bay, there is some seasonal
control in the expression of induction of natural populations of lysogens. We do not know if this
means that there were no lysogens present in the winter months, or that they were metabolically
inactive and thus not inducible by mitomycin C. We are currently pursuing these hypotheses.
We have also investigated the capability of various environmentally significant pollutants
and physical treatments to elicit prophage induction of natural populations. The purpose of these
studies was to determine the potential for certain pollutants to cause prophage induction. A
second goal was to determine if physical stimuli to which natural populations of lysogens can be
exposed (elevated temperature, pressure, and sunlight) could cause prophage induction, and
thereby increase indigenous phage levels. Such experiments were performed in the Southeastern
Gulf of Mexico, the Dry Tortugas, Key West Harbor, Charlotte Harbor, Tampa Bay, and in the
South Atlantic Bight off Cape Hatteras, between 1994 and 1997 [6, 10]. Natural populations
were either concentrated by Vortex Flow Filtration or used directly. When a chemical agent was
used, the natural population was exposed to the agent for 16 to 24 hr, whereas the physical
treatments were of usually much shorter duration. The results of these studies appear in Table 1.
For an induction to have occurred, a significant increase in viral direct counts (by TEM)
compared to an untreated control incubated in the same was found.
Table 1. Induction of Indigenous Marine Lysogens by Chemical and Physical Agents
Inducing Agent
No. of Expts.
Performed
No. of Positive
Inductions
Ave. % Viral
Increase
% Positive
inductions
(efficiency)
Mitomycin C1
33
16
348.8
48.5
UV radiation2
12
6
218.4
50
Sunlight3
9
0
N/A
0
Temperature4
6
2
256.2
33.3
Pressure5
3
0
N/A
0
PAH chemicals6
11
8
206.3
73
PCB Mixture7
4
3
552
75
Arochlor12488
4
3
570
75
Pesticide
Mixture9
6
3
570
50
Bunker C fuel
oil10
16
1
343
6.3
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Viruses as Regulators in Microbial Systems
Inducing Agent
No. of Expts.
Performed
No. of Positive
Inductions
Ave. % Viral
Increase
% Positive
inductions
(efficiency)
Trichloroethylene11
6
1
205.9
17
Mitomycin C was added at a concentration of 1 µg/ml in most instances.
UV radiation was administered for 30 s (254 nm; 14.76 mJ cm-2)
3
Sunlight exposure was for 15 min between 1200 and 1600 hr in summer in Florida
4
Temperature treatments that resulted in positive induction were for 30 min@ 30oC or 42oC.
5
Pressure treatment was by lowering the sample to 827 or 2000 m in a sealed microcentrifuge tube.
6
PAH chemicals are the polynuclear aromatic hydrocarbons naphthalene, phenanthrene, and pyrene, with effective
concentrations equal to their solubility in seawater, or 22.5, 0.72, and 0.08 µg/ml
7,8
PCB (polychlorinated biphenyl) mixture and Arochlor 1248 were added at a concentration of 1 µg/ml
9
Pesticide mixture included lindane, DDD, DDT, BHC, dieldrin, endosulfan I, endrin, hepochlor, and methoxychlor at a
combined concentration ranging from 0.01 to 1 µg/ml depending on the experiment.
10
Bunker C fuel oil was added at a range of concentrations (0.01 to 10 µg/ml) depending on the experiment
11
Trichloroethylene was added as a solution at 4%, v/v.
1
2
The highly mutagenic polynuclear aromatic hydrocarbons and polychlorinated biphynyls
(PCB’s) were the most efficient in causing prophage induction of natural populations, while
sunlight, pressure, and bunker C fuel oil were least effective. Prophage induction is believed to be
caused by stimulation of excision-repair mechanisms which result in excision of the prophage
genome from the host chromosome [1]. Although UVC radiation was relatively efficient in
prophage induction, there apparently was not high enough energy of UV in sunlight to cause
prophage induction. The pesticides were equally as efficient in causing prophage induction as
mitomycin C, which has been our standard for detection of lysogeny in the marine environment.
The fact that a temperature increase from 27o to 30oC resulted in prophage induction indicates
that relatively mild treatments can cause phage release. Temperature increases to 40oC are known
to cause induction of λ prophage [3]. The fact that elevated temperature and pollutants such as
PAH’s, PCB’s, and pesticides cause prophage induction in natural populations suggests that such
processes could in part be causing the elevated phage abundances seen in eutrophic estuaries,
particularly in the summer months.
To investigate the potential for gene transfer by transduction in the marine environment,
we examined the potential for broad host range, non-conjugative plasmids to be transferred using
temperate phage-host systems. The first system employed use the φHSIC/HSIC phage host
system (PHS) while the second employed φD1B/D1B PHS. Both of these were isolated from
Mamala Bay, Oahu [9]. The plasmid used in transduction studies was pQSR50 [7], a 14.4 kb
derivative of R1162 and Tn5 that encodes for kanamycin and streptomycin resistance. The
plasmid was put into the HSIC and D1B hosts by triparental mating to yield the plasmidcontaining donor strains HOPE-1 and HOPE-2, respectively. For a transduction assay with these
strains, a phage lysate was made from each of the donors (HOPE-1 and HOPE-2), the lysate
filtered, then exposed to UV to reduce the infectivity to approximately 1%. The lysates were
DNAse I treated to ensure that no extracellular plasmid DNA was present that might cause
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Viruses as Regulators in Microbial Systems
transformation. The results of the study appear in Table 2.
Table 2. Transduction of plasmid pQSR50 with isolates as recipients.
Donor
Transducing
Phages
Recipient
MOI1
Frequency of Transduction
No. of
No. of
Transduct/
Transduct/
2
PFU
CFU3
HOPE-1
UV-treated TφHSIC
HSIC (10 ml)
0.01
5.13x10-9
4.02x10-10
HSIC (100 ml)
0.01
1.33x10-7
6.8x10-10
0.05
1.33x10-8
6.8x10-10
0.5
<2.6x10-11
—
HOPE-2
Untreated TφHSIC
HSIC (10 ml)
0.01-10
ND4
UV-treated and
untreated
φD1B
D1B (10 ml)
0.01-5
ND
1
MOI is multiplicity of infection
PFU is plaque forming units (number of phages)
3
CFU is colony forming units (number of hosts)
4
ND is not detected
2
Putative transductants were found only when HOPE-1 was used as the donor strain and
when the T-φHSIC lysate had been UV-treated. No transfer was found when untreated T-φHSIC
was the donor or when the φD1B/HOPE-2 PHS system was used. Additionally, no transfer was
found when the MOI was greater than 0.05. This may have been because of the lytic effects at
such high MOI’s. To verify that the transductants contained the pQSR50 plasmid, colony lifts and
plasmid minipreps of the putative transductants were probed with the nptII probe generated from
pQSR50 [7]. Colony lifts and dot-blots of plasmid DNA from HOPE-1 donor strain and putative
transductants hybridized strongly with the nptII probe while those from the recipients did not
(data not shown). Additionally, the transductants expressed the kanamycin/streptomycin
resistance while the recipients did not. We also had made a gene probe to the φHSIC genome by
randomly cloning a 100 bp AccI restriction fragment into a Riboprobe vector and labeled it by in
vitro transcription. All putative transductants hybridized to the probe while the HOPE-1 donor
and HSIC recipient did not. This indicated that the transductants had either integrated the phage
genome (were lysogenized) or that the phage genome was also maintained as a plasmid. All
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Viruses as Regulators in Microbial Systems
transductants were tested for sensitivity to the φHSIC virus and all were immune, suggesting that
the transductants indeed were lysogenized.
Transduction assays were then performed with the indigenous bacterial community as
recipient. This was accomplished by concentrating the marine bacterial population from 20 to 100
L of seawater to 50 ml. One ml of transducing lysate was added to 10 ml of the concentrated
microbial population for 10 min, then the mixture filtered and plated on selective media (seawater
agar plus kanamycin and streptomycin). Putative transductant colonies were hybridized to the
nptII probe and those that hybridized subjected to miniprep and restriction analysis. Unlike the
transduction experiments with isolates only (Table 2), there often were indigenous bacteria
present resistant to kanamycin and streptomycin, and some of these bacteria hybridized with the
nptII probe. Therefore, only experiments where there were no indigenous bacteria hybridizing to
the nptII probe were examined further for potential transduction. The results of such experiments
appear in Table 3.
Table 3. Transduction of indigenous marine bacteria.
Recipient
Sampling site
Donor
Transducing
phages
Transduction
frequency
(trans./PFU)
Transduction
detection limit
(trans./PFU)
Mouth of Tampa
Bay (27o 35'N,
82o43'W)
HOPE-1
T-φHSIC
ND1
1.74x10-10
HOPE-1
UV-treated
T−φHSIC
ND
1.74x10-10
HOPE-2
T-φD1B
1.57x10-8
1.74x10-10
HOPE-2
UV-treated, TφD1B
3.7x10-8
3.7x10-8
Gulf of Mexico,
24o55'N,85o32'W,
1500 m depth
1
ND is not detected
Only two environments yielded putative transductants, and of these, only the T-φD1B
phage and HOPE-2 donor system resulted in a positive response. The transductants were verified
by PCR amplification of portions of the plasmid followed by restriction digestion and
hybridization to the nptII probe [12].
To summarize, we have found a clear seasonal pattern in the distribution of lysogens in
Tampa Bay, with none detected in the winter, and an abundance throughout the rest of the year.
A series of pollutants and physical agents were found to result in prophage induction of natural
populations of marine lysogens, and such induction may be the reason that such large viral
populations are present in eutrophic estuaries. Transduction of plasmid DNA occurred with
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Viruses as Regulators in Microbial Systems
coincident lysogenization using a temperate marine phage host system, and transfer to indigenous
flora by transduction has been documented.
Acknowledgements
This research was supported by grants from the National Science Foundation to JHP and a Knight
Fellowship to SCJ.
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