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Appl Environ Microbiol, June 1998, p. 2308-2312, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Seasonal Abundance of Lysogenic Bacteria in a
Subtropical Estuary
Pamela K.
Cochran and
John H.
Paul*
Department of Marine Science, University of
South Florida, St. Petersburg, Florida 33701
Received 9 June 1997/Accepted 13 March 1998
 |
ABSTRACT |
Seasonal changes in the abundance of inducible lysogenic bacteria
in a eutrophic estuarine environment were investigated over a 13-month
period. Biweekly water samples were collected from Tampa Bay, Fla., and
examined for prophage induction by mitomycin C treatment. At the
conclusion of the study, we determined that 52.2% of the samples
displayed prophage induction, as indicated by significant increases in
viral direct counts compared with uninduced controls. Samples that
displayed prophage induction occurred during the warmer months
(February through October), when surface water temperatures were above
19°C, and no induction was observed in November, December, or
January. This study presents clear evidence that there is seasonal
variation in the number of inducible lysogenic bacteria in an estuarine
environment.
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TEXT |
Lysogeny is the process by which a
DNA-containing phage maintains a stable symbiosis with its host and is
the alternative to lytic replication (1). Lysogeny can be
achieved by integration of the viral genome into the host chromosome
and transmission of the integrated DNA (termed a prophage) to progeny
cells during division. Alternatively, lysogeny can occur by plasmid
formation, and the viral DNA is then perpetuated as an extrachromosomal
element. A cell carrying a prophage is termed a lysogen (1).
The lysogenic state may continue for many generations until the
prophage is spontaneously activated or until induction by mutagenic
agents, such as UV radiation or mitomycin C, occurs (1). The
inducing agents then trigger the host's DNA repair mechanisms and
cause initiation of viral replication and host lysis (1).
Wilcox and Fuhrman (13) and Weinbauer and Suttle
(12) reported that the majority of viruses found in the
marine environment are lytic and that lysogenic bacteriophages are
quantitatively insignificant in coastal waters. Jiang and Paul
(7), however, showed that 43% of the bacteria isolated from
various marine environments were lysogenic, as determined by prophage
induction with mitomycin C. An examination of natural bacterial
populations that were inducible by mitomycin C indicated that the
proportion of bacteria that were lysogenic ranged from 1.5 to 38%
(8). We extended these studies to determine the relative
proportion of lysogenic bacteria as a function of season in the
estuarine environment of Tampa Bay, Fla., with the intention of
relating abundance to various physical, chemical, and biological
parameters. We show here that there is a strong seasonal variation in
the number of inducible lysogens in this subtropical estuarine
environment.
Sampling sites.
Water samples were collected biweekly from
Tampa Bay, Fla., at the St. Petersburg city pier for 13 months, from
October 1995 to October 1996. The site selected for sampling was on the
western shore of the bay and about midway down the bay and was
influenced by urban runoff from the city of St. Petersburg.
Induction of lysogenic bacteria by mitomycin C.
Replicate
seawater samples (25 ml) either were treated by adding mitomycin C (1 µg ml
1) or were left untreated (controls). Both types
of samples were incubated for 24 h at room temperature in the dark
and then fixed with 2% glutaraldehyde and stored at 4°C before
bacterial direct counts and viral direct counts were determined. The
fixed samples used for viral enumeration were concentrated by
ultracentrifugation onto 200-mesh Formvar-coated copper grids at
160,070 × g for 60 min at 4°C (2, 3). The
grids were then negatively stained with 2% uranyl sulfate
(Polysciences, Inc., Warrington, Pa.), rinsed with distilled water, and
air dried. Viruslike particles were counted at a magnification of
×50,000 with a Hitachi model 7100 transmission electron microscope.
The samples used for bacterial enumeration were stained with
2,4-diamidino-2-phenylindole (DAPI) (Sigma Chemical Co., St. Louis,
Mo.) overnight, and 1 ml of each sample was filtered onto an Irgalan
black-stained, 25-mm-diameter, 0.2-µm-pore-size Nuclepore filter and
counted by epifluorescence microscopy as described elsewhere
(9).
Chlorophyll a determination.
Water samples (50 ml)
for a chlorophyll a analysis were filtered onto Whatman type
GF/F filters and stored frozen until analysis. The filters were
extracted in the dark overnight in methanol, and the chlorophyll
a content was fluorometrically determined by the method of
Holm-Hansen and Riemann (6).
Statistical analysis.
Multisample regression and comparison by
analysis of variance and two-sample comparison with a t test
were performed by using Statgraphics software (Manugistics Inc.,
Rockville, Md.). Statistical comparisons of viral direct count and
bacterial direct count data were performed by using the averages of the
values from four grids per replicate slide per sample.
Figure 1A shows the seasonal variations
in salinity and temperature in Tampa Bay. The water temperature dropped
7°C between the first two sampling dates and continued to drop to an
unseasonably low 13°C during February. In general, the water
temperatures were below 20°C for the winter season and began to
increase in April. Salinity followed rainfall, which was quite erratic
during the winter and spring of 1996 (data not shown). The peaks in
salinity during the first half of the study were associated with months in which there was very little rainfall. The summer rains, which normally decrease salinity during the summer, were not as great as in
previous years (the 1995 rainfall for June through October was 46.37 in., and the 1996 rainfall for June through October was 27.33 in.
[National Climatic Data Center, Asheville, N.C.]).
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FIG. 1.
(A) Seasonal variation in salinity and temperature. Both
parameters were measured at the time of sample collection. (B) Total
viral direct counts (VDC) and total bacterial direct counts (BDC)
measured during the 13-month seasonal study. Viral direct counts were
determined by negative staining and transmission electron microscopy.
Bacterial direct counts were determined by DAPI staining and
epifluorescence microscopy. DC, direct counts. (C) Chlorophyll
a (Chla) concentrations and VBR (V/B Ratio) in Tampa Bay
during the 13-month seasonal study.
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|
A seasonal pattern in viral and bacterial abundance was observed (Fig.
1B), and the virus concentrations were lowest in the
winter and highest
in late fall and summer. There was a decrease
in viral abundance (from
1.59 × 10
7 to 0.52 × 10
7 viruses
ml
1) between December and January, which corresponded
with a decrease
in water temperature. This trend continued until the
first February
sample, which contained the lowest concentration of
viruses (0.186
× 10
7 viruses ml
1) and
had the lowest recorded water temperature (13°C). An increase
in
bacterial direct counts coincided with a decrease in viral
direct
counts during the winter, with the bacterial numbers increasing
from
2.4 × 10
6 to 6.79 × 10
6 cells
ml
1 and the viral numbers decreasing from 1.88 × 10
7 to 0.52 × 10
7 viruses
ml
1. The increase in bacterial abundance was preceded by
increases
in the water temperature from 13 to 27°C by the end of May.
The
average bacterial direct count in this study was 4.42 × 10
6 ± 1.5 × 10
6 cells ml
1.
The chlorophyll
a values were lowest during the winter
months (3.85 ± 1.67 µg liter
1) and highest during
the summer months (9.27 ± 4.25 µg liter
1) (Fig.
1C). A chlorophyll
a peak (6 ± 0.54 µg
liter
1) was observed with the first February sample,
which coincided
with the coldest water temperature recorded (13°C)
and the lowest
viral direct count (0.186 × 10
7
viruses ml
1).
The virus-to-bacterium ratio (VBR) (
5,
14) is used to
characterize the relationship between bacterial and viral communities.
In the seasonal study, the VBR ranged from 0.305 to 9.29, with
the
highest values obtained at the two October sampling times
(Fig.
1C).
The seasonal pattern of the VBR most closely resembled
the seasonal
distribution of the viral direct counts (Fig.
1B),
with the highest VBR
coinciding with the highest viral counts
and the lowest VBR coinciding
with the lowest temperature recorded
(13°C) and the lowest viral
direct counts (0.186 × 10
7 viruses
ml
1).
Figure
2 and Table
1 show the results of induction by
mitomycin C of indigenous lysogenic bacteria from the Tampa Bay
sampling
site. Based on our previous criterion for prophage induction
in
environmental samples, whereby an increase in phage counts must
be
accompanied by a decrease in bacterial counts (
8), 39% of
the samples exhibited prophage induction. However, we feel that
small
changes in bacterial direct counts caused by prophage induction
might
not be detected by the methods currently used for bacterial
enumeration. Therefore, we assumed that positive induction occurred
when there was a significant increase in viral direct counts compared
with the control. On the basis of this criterion, 52.2% of the
samples
resulted in prophage induction. If all decreases in bacterial
concentration in response to mitomycin C were considered
prophage-induced
lysis, then 0 to 41% of the bacterial population
contained inducible
prophage (Table
1).
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FIG. 2.
(A) Percent change in viral abundance during the
seasonal study. The percent change was calculated by dividing the
mitomycin C-treated sample value by the control sample value and
subtracting 100 to determine the percent increase or decrease in
relation to the control sample. (B) Percent difference in bacterial
direct counts from the control sample value. The percent decrease was
calculated by subtracting the ratio of mitomycin C-treated bacterial
direct counts to control bacterial direct counts from 100. Statistically significant increases or decreases (95% confidence
interval) are indicated by asterisks.
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TABLE 1.
Induction of indigenous lysogenic bacteria present in
seasonal samples from the St. Petersburg city pier, Tampa
Bay, Fla.a
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All of the samples that displayed positive induction were obtained
during the summer and fall of the seasonal study, which
suggests that
temperature or some other biological factor controlled
by temperature
may control prophage induction. The estimated burst
sizes (based on
changes in viral direct counts divided by changes
in bacterial direct
counts) for these samples ranged from 2.1
to 15.5. A second approach
used to determine the percentage of
lysogenic bacteria in the marine
environment is to assume an average
burst size (ca. 30) and calculate
the number of lysogens by dividing
the increased virus numbers by this
burst size (
8). The percentage
of lysogenic bacteria
calculated from the average burst size ranged
from 2.1 to 37.3%. The
results of both methods used to determine
the percentage of lysogenic
bacteria are presented in Table
1.
The multiple regression analysis of the seasonal study data showed that
the percent increase in virus abundance was positively
correlated with
salinity (
r = 0.52), with positive induction occurring
at salinities above 23 ppt and only one sample induced below this
level
of salinity. Salinity was also positively correlated with
temperature
(
r = 0.65). These results imply that the variation
in
viral abundance could be explained by the variation in salinity
and
temperature and are consistent with previous reports for this
marine
environment (
7). The induced viral direct counts were
positively correlated with the control viral counts (
r = 0.73)
and the total viral direct counts (
r = 0.57),
which implies that
a large viral population favors lysogeny. This
relationship has
also been observed in offshore studies on the
occurrence of lysogeny
(
8).
In order to determine the significance of lysogeny in the marine
environment, a seasonal study was performed to measure the
abundance of
lysogens in a eutrophic estuary, Tampa Bay, Fla.
Previous studies on
lysogeny in the marine environment brought
into question the
significance of this process for bacterial mortality
or phage
production (
8,
12). Weinbauer and Suttle (
12)
reported that 0.07 to 4.4% of the total bacterial community was
lysogenic, whereas Jiang and Paul (
8) showed that up to 38%
of the bacterial population was lysogenized in various estuarine
environments. In this study, we determined that 0 to 37.3% (average,
6.9%) of the indigenous bacteria present in the Tampa Bay estuarine
environment were lysogenic, as calculated by using an average
burst
size of 30 (
8). The percentage ranged from 0 to 41%
(average,
11.7%) when bacterial mortality was used to determine the
lysogenic
fraction (this method assumed that the increase in viral
numbers
and the decrease in bacterial numbers in the induced samples
were
caused solely by lysogenic induction).
Seasonal studies on viral abundance in various lake and marine
environments have indicated that the greatest viral concentrations
occur during the spring and summer, suggesting that there is a
possible
correlation with water temperature (
2,
4,
7,
11). This
phenomenon was also observed in our seasonal study.
The virus numbers
were greatest during the summer months (average,
1.32 × 10
7 viruses ml
1) and smallest during the
winter (0.57 × 10
7 viruses ml
1). With
the onset of warmer water temperatures during the spring
and summer,
positive induction was observed. When the water temperature
was 19°C
or greater, the percent increase in virus numbers was
significantly
greater than the increase in the control, which
allowed detection of
prophage induction. Decreases in bacterial
numbers compared with the
control also corresponded with these
episodes of induction. With the
onset of winter and cooler water
temperatures (the water temperature
decreased from 25 to 13°C),
no induction was observed. Similar
observations were made for
Lake Superior (
10), suggesting
that bacterial activity controlled
by temperature may be a significant
factor in prophage induction.
Once initial warming occurred in February
(the water temperature
increased from 13 to 19°C), a chlorophyll
a peak was observed,
and then 2 weeks later (when the next
sample was obtained), a
"lysogeny bloom" was observed and there was
the greatest measured
percent increase in induced virus numbers
compared with the control
(a 379% increase). However, a significant
increase in inducible
prophage was observed only in the viral counts. A
concomitant
decrease in bacterial numbers was not observed. If the
initial
bacterial counts and not the control counts at the end of the
experiment were used, a significant decrease (
P < 0.05) in the
bacterial counts was observed.
Our data indicate that there is a strong interaction between the
presence of inducible lysogens and the total viral direct
counts. In
fact, the greatest percent increase in inducible prophage
(second
February 1996 sample) corresponded with the initiation
of the seasonal
increase in viral direct counts. This could be
interpreted as a
seasonal regulatory switch, when lysogens go
from a dormant state to
inducibility. In cultures, induction does
not occur readily in starved,
slowly growing, or nonexponentially
growing cells (
1). The
dormant state, which we hypothesize
is related to low activity, is
caused by cooler water temperatures
or lack of dissolved organic carbon
or both. In fact, the increase
in viral direct counts observed
seasonally may be in part a result
of prophage induction and the attack
and lysis of sensitive hosts
by induced prophages. For example, if on
average 30% of the population
is lysogenized and a portion of the
lysogenized cells (say one-third)
are induced by some environmental
event, then this could result
in 1.2 × 10
7 viruses
produced per ml, which is close to the total viral counts
measured. If
only 8% of the cells are lysogenized and one-half
of these cells are
induced, there would be 4.8 × 10
6 viruses produced
per ml, still a significant proportion of the
viral population.
We do not know what environmental conditions might lead to large-scale
induction of natural populations. Ackermann and DuBow
(
1)
list a variety of treatments shown to cause prophage induction
in
bacterial cultures, including a range of DNA-damaging agents,
radiation, heat, and pressure, as well as other phages and plasmids.
We
have demonstrated that induction of natural populations by
aromatic
hydrocarbon pollutants and elevated temperatures occurs
(
8).
It is possible that naturally occurring processes and
anthropogenic
activities cause large-scale prophage induction
and that these events
can contribute significantly to the total
viral direct counts and the
seasonal variability in such counts.
In fact, the widespread
observation of extremely high viral direct
counts in estuarine samples
may be the result of induction of
prophages by anthropogenic or natural
causes. Estuaries are often
stressed by pollutants, and a range of
environmental pollutants
have been shown to result in prophage
induction (
8). Naphthalene,
phenanthrene, and pyrene all
cause prophage induction in natural
populations of marine bacteria. It
is interesting that the greatest
induction (percent increase in
prophage) occurred during the second
seasonal induction in February. In
the summer, a smaller increase
in phage was observed, suggesting that a
proportion of the indigenous
lysogens might be induced by anthropogenic
and naturally occurring
agents found in the estuary studied.
| |
ACKNOWLEDGMENTS |
This research was supported in part by grants OCE 9502775, OCE
9214614, and BIR-9512122 from the National Science Foundation.
We are grateful to Sunny Jiang for valuable suggestions and comments
during the course of this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Marine Science, University of South Florida, 140 7th Ave. South, St. Petersburg, FL 33701. Phone: (813) 553-1130. Fax: (813) 553-1189. E-mail: jpaul{at}seas.marine.usf.edu.
| |
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Appl Environ Microbiol, June 1998, p. 2308-2312, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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