Viral abundance and a high proportion of lysogens suggest that viruses are important members of the microbial community in the gulf of trieste

Abstract

Epifluorescence microscopy and transmission electron microscopy were applied to study virioplankton community in the Gulf
of Trieste (northern Adriatic Sea). The total viral abundance was in a range between 2.5 × 109/L and 2.9 × 1010/L and was positively correlated with trophic status of the environment. Viruslike particles were significantly correlated
with bacterial abundance in all samples studied. Correlations with other physicochemical or biological parameters were not
significant. The data suggest that, because of the substantial fraction of tailed viruses present (26%), bacteriophages are
an important component of the virioplankton community in the Gulf of Trieste. The abundance of viruslike particles in the
seawater changed at hour intervals in a range from 1.3 × 109/L to 5.1 × 109/L. A significant fraction (71%) of the bacterial isolates was inducible in vitro by mitomycin C, and a high occurrence (51%) of lysogenic isolates with more than one phage morphotype present in the lysate
was detected. The presence of lysogenic bacteria in the seawater was confirmed in situ with a mitomycin C induction experiment on the natural bacterial population. Results suggest that virioplankton is an abundant
component of the microbial community in the Gulf of Trieste.

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Viral Abundance and a High Proportion of Lysogens Suggest
That Viruses Are Important Members of the Microbial Community
in the Gulf of Trieste
D. Stopar1, A. Cˇerne1, M. Zˇigman1, M. Poljs ˇak-Prijatelj2and V. Turk3
(1) Biotechnical Faculty, Department of Food Technology, University of Ljubljana, Vecna pot 111, SI-1000 Ljubljana, Slovenia
(2) Institute of Microbiology and Immunology, Zaloska 4, SI-1000 Ljubljana, Slovenia
(3) National Institute of Biology, Marine Biological Station, Fornace 41, SI-6330 Piran, Slovenia
Received: 23 May 2002 / Accepted: 26 December 2002 / Online publication: 6 June 2003
Abstract
Epifluorescence microscopy and transmission electron
microscopy were applied to study virioplankton com-
munity in the Gulf of Trieste (northern Adriatic Sea). The
total viral abundance was in a range between 2.5 · 109/L
and 2.9 · 1010/L and was positively correlated with
trophic status of the environment. Viruslike particles
were significantly correlated with bacterial abundance in
all samples studied. Correlations with other physico-
chemical or biological parameters were not significant.
The data suggest that, because of the substantial fraction
of tailed viruses present (26%), bacteriophages are an
important component of the virioplankton community
in the Gulf of Trieste. The abundance of viruslike parti-
cles in the seawater changed at hour intervals in a range
from 1.3 · 109/L to 5.1 · 109/L. A significant fraction
(71%) of the bacterial isolates was inducible in vitro by
mitomycin C, and a high occurrence (51%) of lysogenic
isolates with more than one phage morphotype present in
the lysate was detected. The presence of lysogenic bacteria
in the seawater was confirmed in situ with a mitomycin C
induction experiment on the natural bacterial popula-
tion. Results suggest that virioplankton is an abundant
component of the microbial community in the Gulf of
Trieste.
Introduction
Heterotrophic bacteria assimilate a significant fraction of
the primary production (approximately 50%) in marine
environments [3, 11]. Grazing of the heterotrophic mi-
croorganisms by protists transfer bacterial-derived energy
to a higher trophic level. In contrast, bacterial lysis by
phages may increase the amount of dissolved organic
matter, thus uncoupling the microbial loop from the
higher trophic levels [9]. It is currently estimated that 10–
20% of the bacterial production in marine waters is
converted to dissolved organic matter by phage lysis [35].
Loss of bacterial production through viral lysis is im-
portant for our understanding of the role bacteria play in
eutrophic environment such as the northern Adriatic Sea.
It was demonstrated in different aqueous environ-
ments that viruses are the most abundant microorgan-
isms, and it was also argued that bacteriophages represent
the major virus class [7, 13, 14, 24, 31, 36]. In various
surface waters up to 109viruses per mL have been
counted [9]. Viral abundance declines from near shore to
the open sea, and with increasing depth [5, 7]. Significant
seasonal fluctuations in virus densities of approximately
two orders of magnitude have been observed [14]. The
experimental evidence for diurnal changes is scant, but
claims that virus abundance can change in the interval of
a couple of hours or in as short a time as 10–20 min have
been made [6]. Several field studies suggested the exist-
ence of a coupling between viruses and bacteria distri-
bution [7]. In earlier studies in the northern Adriatic Sea
it was shown that virus activity might play a significant
role in food web structure under changing trophic con-
ditions [30, 31]. Because virus infection is generally host
specific, viruses can shape the microbial community
structure [29]. In addition, they mediate horizontal gene
transfer by transduction [9, 15, 16].
Bacterial viruses exist in the environment mainly as
free virions or as prophages inside a lysogenic host [2].
Prophages contribute to the total virus production in the
environment when the host cell is induced, but perhaps
even more important, they are capable of altering the
Correspondence to: D. Stopar; E-mail: david.stopar@uni-lj.si
DOI: 10.1007/s00248-002-3009-5dVolume 47, 1–8 (2004)d? Springer-Verlag New York, Inc. 2003
1

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host’s genetic makeup and its physiology, as well as in-
creasing the host’s environmental fitness [23]. Recent
studies indicate that up to 64% of the bacteria isolated
from different marine habitats were lysogenic, and that
the fraction of the lysogenic bacterial isolates varied be-
tween different environments [14]. Examination of the
natural bacterial populations that were inducible with
mitomycin C suggests that the fraction of the lysogenic
bacteria varied from 0.1 to 52% [8, 16, 32].
In this study we follow and correlate virus abundance
with several physical, chemical, and biological parameters
at different locations in the Gulf of Trieste. The total
virus abundance and morphology was determined by
epifluorescence microscopy and transmission electron
microscopy. In addition, we estimate the potential sig-
nificance of lysogeny on virus production both in vitro
and in situ.
Materials and Methods
Sampling Sites.
ological analysis were collected at two stations in the
eastern part of the Gulf of Trieste (northern Adriatic Sea).
One sampling station was located between two subma-
rine outfalls of the Piran wastewater treatment plant
(station PI 16: 45?32.00; 13?32.00) with a water column
depth of 22 m; the second station was situated 1.5 km
away (station F: 45?32.30; 13?33.00) with a depth of 21
m. The Gulf of Trieste is a shallow oligotrophic sea with a
marked temporal cycle. The physiography of the area
include a limited water exchange and low river inflow,
especially during summer. Extreme meteorological con-
ditions may therefore strongly influence the nutrient
balance and the succession of organisms in the food web
[19, 20, 27]. During the years 1998–1999 several ocea-
nographic surveys of the water column at the two marine
stations were undertaken. Viral analyses, presented in this
paper, were performed on 24 May 1999 and 21 July 1999.
On each occasion seawater was sampled by submerged
pump at different depths, according to the vertical pro-
files of temperature, salinity, oxygen, and fluorescence
obtained using a fine-scale multiparameter CTD probe
(Center for Water Research, University of Western Aus-
tralia). Samplings were performed during calm weather
and low tidal currents (the tidal range of sea-surface el-
evation was less than 0.6 m). Basic hydrographic pa-
rameters were determined according to Strickland and
Parson [25]. Nitrite, nitrate, and orthophosphate were
analyzed in filtered seawater samples. Ammonium, total
P, and total N were analyzed in unfiltered seawater
samples using standard colorimetric procedures [12].
Seawater samples for bacterial and bacteriophage strain
isolation were collected separately at different locations
along the coast in the Gulf of Trieste. Seawater samples
for diurnal virus productivity and in situ induction
Seawater samples for chemical and bi-
experiments were collected on-shore in front of the
Marine Biological Station in Piran.
Fecal Coliform Bacteria.
forms was determined using the membrane filtra-
tion method according to the protocols and guidelines
of UNEP/WHO (1994) [28]. Seawater samples were
filtered through 0.45-lm Millipore filters and incu-
bated on m-FC broth base medium at 44.5 ± 0.2?C for
24 h.
The number of fecal coli-
Bacterial Secondary Production.
method (tritiated thymidine incorporation) was used for
bacterial production [10]. Each time, 1.7 mL of unfiltered
seawater was incubated with 250 lCi3H-thymidine L)1,
(sp. act. 80 Ci nmol), Amersham) in 2.0-mL polypro-
pylene microcentrifuge tubes for 1 h at 18?C. All samples
were done in triplicate. Samples with 5% trichloroacetic
acid added prior to the addition of3H-thymidine served
as the blanks. Production based on the amount of
thymidineincorporatedinto
according to Fuhrman and Azam [10].
A modified TTI
DNA wascalculated
Transmission Electron Microscopy.
matter in freshly collected water samples was, depending
on the sample, concentrated from 50 to 2000 times by a
standard Amicon ultrafiltration with a 30,000 MW cutoff
cartridge filter (Milipore, polyethersulfonate, PM30, dia.
44.5 mm). The retentate was filtered through a mem-
brane filter with pore size 0.2 lm to remove bacteria [26].
Concentrated samples were stored in 2% glutaraldehyde
at 4?C prior to the TEM examination. Carbon-stabilized
Formvar-coated 200-mesh copper grids were placed on a
small drop (20–50 lL) of sample for 3 min. Grids were
then negatively stained with 2% (w/v) phosphotungstic
acid for 3 min and examined on a JEOL 1200EXII-1
TEM. Viral size and morphology were determined using
the measurement function of the TEM, which allows
measurement of a TEM image directly (after calibration)
on the fluorescent screen at a magnification of 30,000 or
50,000. In total more than 400 distinct viruses were
morphologically characterized.
The particulate
Epifluorescence Microscopy.
(BDCs) and viruslike particles (VLPs) were enumerated
by epifluorescence microscopy (Olympus BH2-RFL) in
formaldehyde-preservedwater
staining with SYBR Green-1 nucleic acid fluorescence
stain (Molecular Probes) [22]. No sample used for enu-
meration was preserved in formaldehyde for longer than
6 months. Aliquots of the samples (1–5 mL) were filtered
through Anodisc Al2O3 filters (0.02 lm pore size,
Whatman) with a vacuum pump (?20 kPa). Twenty to
30 fields were viewed, and at least 200 viruses or bacteria
were counted per sample. Blank probes were prepared
Bacterial direct counts
samples(2%), after
2
D. STOPAR ET AL: MICROBIAL COMMUNITY IN THE GULF OF TRIESTE

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regularly (every 5–8 samples). Some concerns that the
SYBR Green-1 method may underestimate viral abun-
dance have been raised in the literature, so the results
presented in this manuscript should be considered as
conservative estimates of VLP [4].
Marine
Bacteria were isolated as CFU from seawater
spread on MWS ZoBell 2216E medium (1.8% agar, 0.1%
yeast extract, and 0.5% peptone, prepared with 75% (v/v)
of 0.2 lm filtered seawater) [37]. Each isolate was puri-
fied by at least three serial transfers of well-separated
colonies. Bacterial isolates were not taxonomically de-
termined.
However, basedon
mentation, and Gram staining, as well as catalase,
oxidase, motility, gelatinase, lipase, lecitinase, optimal
redox potential for growth, and cellulose tests, we in-
ferred that they belong to different taxa (unpublished
data). Twenty-eight fast-growing
with generation time <180 min were used for further
studies.
The seawater for the bacteriophage isolation was
collected and concentrated as described above for the
TEM. A spot test was used to detect the presence of vi-
ruses. Typically 50 lL of the concentrated natural virus
communities was spotted at the center of the agar plate
preseeded with exponentially grown host cells, and in-
cubated at 28?C for at least 48 h. Plates were checked
every 12 h for the presence of a growing clearing zone,
which was indicative of phage growth. Plates that showed
a clearing zone were further tested for plaque formation.
The efficiency of plating was calculated as a fraction of
the total plates which form regular plaques. Clonal iso-
lates of bacteriophages were obtained using standard
plaque purification procedures [24].
Bacteriophages andTheirHost Isola-
tion.
colony morphology,pig-
bacterialisolates
Induction
Among isolated bacteria, 28 different fast-growing
bacterial isolates were tested for the presence of an in-
ducible prophage using mitomycin C (Sigma Chemical
Co.). Typically, a colony of a given bacterial isolate was
inoculated in 25 mL of MWS ZoBell 2216E medium and
grown overnight in the dark at 28?C on a shaker (200
rpm). Fresh growth medium was then inoculated with 2%
(v/v) of the bacterial culture that had been grown over-
night. Replicate samples (25 mL) of the mid-log-phase
bacterial culture (OD6600.4–0.6) either were treated by
adding freshly prepared mitomycin C (10 lg/mL deion-
ized water) or were left untreated (control). The final
concentration of mitomycin C in the treated samples was
0.4 lg/mL. Both treated and untreated samples were in-
cubated for 18 h at 28?C in the dark on a shaker (200
rpm). The optical density of the bacterial cultures was
determined at the beginning of the induction and at the
of LysogenicBacteria byMitomycin
C.
end of incubation. Induced samples showed a significant
decrease in optical density at the end of incubation, which
was indicative of bacterial lysis. After the incubation both
treated and untreated samples were centrifuged (12,000
rpm, 5 min), and supernatants were fixed with glutaral-
dehyde (final concentration 2%) and stored at 4?C prior
to TEM examination. In the untreated samples there was
on average less than one VLP per view field. Only bacterial
lysates that had a significantly increased number of VLPs
after mitomycin C treatment, as detected by TEM, were
treated as lysogenic. Polylysogeny of bacterial isolates was
determined based on the presence of more than one
morphologically different VLP in the lysate. We treated as
morphologically different those VLPs that differed more
than 30 nm in virus capside diameter (i.e., <30 nm, 30–60
nm, 60–90 nm, >90 nm) or were members of a different
symmetry group.
Induction of Natural Lysogenic Bacterial Popula-
To determine induction of the natural bacterial
population in the coastal waters under aerobic condi-
tions, replicate coastal water samples either were treated
by mitomycin C (final concentration 0.4 lg/mL) or were
left untreated (control). Samples were incubated for 24 h
in the dark, at room temperature, with vigorous shaking
(200 rpm). After incubation, samples were fixed with
formaldehyde (final concentration 2%) and stored at
4?C, prior to BDC and VLP enumeration by SYBR Green
I epifluorescence microscopy.
tion.
Results and Discussion
VLP in Relation to Several Physical Chemical and Bio-
logical Parameters.
In this study we follow and correlate
virus abundance with several physical, chemical, biolog-
ical, and nutrient parameters. In addition, we comment
on the potential significance of lysogeny on virus pro-
duction both in vitro and in situ in the Gulf of Trieste.
Virus densities during the entire period of the study
varied between 2.5 · 109/L and 2.9 · 1010/L in the Gulf of
Trieste. These values are of the same order of magnitude
but are somewhat smaller than those previously reported
for the northern Adriatic Sea (1.2 · 109to 8.7 · 1010)
Table 1. Virus morphology in the Gulf of Trieste as determined
by transmission electron microscopy
Virus
morphology
Virus
family
% of virus in
the class
Head size
(nm)
Tail
size (nm)
TailedMyoviridae
Siphoviridae
Podoviridae
n.d.
12100 ± 25
50 ± 20
55 ± 20
50 ± 25
80 ± 70
120 ± 90
10 ± 5.0
3
11
74Nontailed
aSeawater was concentrated 2000 times prior to the observation. To obtain
statistics 400 VLP were characterized. n.d., could not be uniquely deter-
mined.
D. STOPAR ET AL: MICROBIAL COMMUNITY IN THE GULF OF TRIESTE
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[30]. The difference could be attributed to the differ-
ent method of enumeration used by the two groups
(i.e., SYBR Green 1 vs TEM), as well as different trophic
status of the sampling locations. In all samples the
VLPs exceeded the BDCs. The ratio VLP/BDC varied
between 5 and 22. This ratio is comparable with most
virioplankton data in aquatic ecosystems (i.e., see Table 1
in [36]).
To study the effect of sewage inflow environment on
VLP abundance, we compared the eutrophic PI-16 sta-
tion located at the submarine sewage outfall with a
control F station. The spreading of the sewage in the
water column was detected by observing vertical salinity
and nutrient concentrations. Sewage dispersal was con-
firmed by the presence of fecal coliforms and higher
ammonium concentrations in the sewage plume few
Figure 1. Vertical concentration profiles
of ammonium (h) and fecal coliforms
(d) for the experimental station PI-16
(panels A and B), and experimental
station F (panels C and D). Samples were
taken on 24 May 1999 and 21 July 1999.
4
D. STOPAR ET AL: MICROBIAL COMMUNITY IN THE GULF OF TRIESTE

Page 5

meters above the bottom, as shown in Fig. 1. At the
control station F there was an increase in ammonium
concentration toward the bottom, but no increase in the
fecal coliforms in July sampling. There was, however, an
increase in both the ammonium concentration and the
total fecal coliforms toward the bottom in the May
sampling. The concentrations of the other nutrients were
comparable between the two stations [21]. VLP abun-
dance at the two experimental stations was correlated
with different physicochemical and microbiological
Figure 2. Vertical abundance
profiles of viruslike particles (VLP)
(h), bacteria direct counts (BDC)
(d), and bacterial productivity (BP)
(horizontal bars) for experimental
stations PI-16 (panels A and B) and
experimental station F (panels C and
D). Samples were taken on 24 May
1999 and 21 July 1999. VLP and
BDC abundances were determined
with epifluorescence of SYBR Green
1 stained samples.
D. STOPAR ET AL: MICROBIAL COMMUNITY IN THE GULF OF TRIESTE
5

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parameters. There was consistently a significant positive
correlation between VLP abundance and BDC at the
PI-16 station (r was 0.9 and 0.93 for the May and July
samplings, respectively). Similarly, VLP abundance cor-
related with BDC at the F station (r was 0.9 and 0.85 for
the May and July samplings, respectively). Bacterial
productivity did not correlate significantly with VLP (r <
0.7). The correlations between VLP abundance and
temperature, O2, PO4
N, or fecal coliforms were weak and not significant (r <
0.7).
VLP abundance, BDC, and bacterial productivity at
the PI-16 and F stations along the depth profiles for the
May and July sampling are given in Fig. 2. The VLP
abundance was, assuming the independent nature of
samples across the depth at the two stations and using
paired t-test, significantly higher in the May than in the
July sampling (p = 0.05). In addition, on average a three-
fold higher VLP abundance along the depth profile was
detected at the PI-16 station, which is enriched with
ammonium, comparedto
(p = 0.05). It is interesting to note that at the depths
where both fecal coliforms and ammonium concentra-
tions were increased, there was no corresponding increase
in VLP abundance. This suggests that increased VLP
abundance at the PI-16 station as compared to the F
station is likely to be the consequence of the increased
bacterial abundance and productivity and not due to the
3), total P, NO2
), NO3
), NH4
+, total
thecontrolF station
influx of VLP from the sewage, although the effect is
small.
The stability of virus population at short time in-
tervals in seawater samples was followed in May 2000 for
70 h (see Fig. 3). The temperature of the seawater was
19?C and the salinity was 36 psu. VLP abundance varied
from 1.3 · 109/L to 5.1 · 109/L during the observed
period. The VPL abundance fluctuations of adjacent time
points were on average less than two standard deviations
apart, which suggests that virus population is rather
stable on a short time scale. In the same period BDC
varied from 1.36 · 108/L to 4.69 · 108/L. The BDC
abundance peak was followed by a peak in VLP abun-
dance. Such a phase shift may be incidental and due to
small-scale physical changes in the environment, which
we could not discriminate.
Morphology of the virioplankton community in the
Gulf of Trieste was studied by TEM. All the viruses were
arbitrarily divided into four classes based on virus head
size. Approximately 13% of all detected viruses belonged
to the <30 nm class; 50% of all viruses were in the 30–60
nm class, 25% in the 60–90 nm class, and 12% in the >90
nm class. As shown in Table 1, 26% of all viruses had tails
belonging to bacteriophage families: Podoviridae, Myov-
iridae, and Syphoviridae. The fraction of estimated bac-
teriophages is considered to be conservative, because
sample manipulation may have caused some virus dam-
age that resulted in loss of the tail. It is also likely that
because of the staining procedure used in TEM prepa-
rations some short tails were not observed. We have
confirmed the presence of bacteriophages by isolation of
phage particles from the seawater; however, the efficiency
of plating was less than 30%. The majority of viruses were
not tailed and had icosahedral morphology, but could
not be classified based solely on morphological charac-
teristics.
Figure 3. Temporal variation of viruslike particles (VLP) (j), and
bacteria direct counts (BDC) (D) in seawater beginning at 12:00
noon local time on 10 May 2000. Light and dark periods are in-
dicated at the bottom of the figure. Estimates of experimental error
are given as error bars, which represent standard deviation of the
measurement.
Figure 4. In situ induction of the natural bacterioplankton com-
munities by mitomycin C. VLP (black bar) and BDC (white bar) at
the beginning of the experiment (to) and after 24 h for control
(mitC )) and for the mitomycin C treated sample (mitC +). VLP
and BDC abundance were determined with epifluorescence of
SYBR Green 1 stained samples.
6
D. STOPAR ET AL: MICROBIAL COMMUNITY IN THE GULF OF TRIESTE

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Lysogeny and Polylysogeny in the Gulf of Trieste.
indicated above, there is a significant fraction (at least
26%) of the bacteriophage particles in the total virio-
plankton community in the Gulf of Trieste. There are two
main bacteriophage reproduction strategies: lytic and
lysogenic. In the literature there is considerable disa-
greement about the prevalence and importance of ly-
sogeny in marine environments [15–17, 32, 36]. The
presence of temperate viruses in the Gulf of Trieste was
tested on 28 different fast-growing bacterial isolates by
induction with mitomycin C. The TEM observations of
lysates indicated that the most abundant class of tem-
perate phage head size was 30–60 nm. Approximately
two-thirds of all the temperate bacteriophages were
tailed. In total 71% of the bacterial isolates were induced
upon treatment with mitomycin C. This is considered to
be a conservative estimate, since mitomycin C does not
induce all known temperate phages [15]. With the test we
have also missed those strains that produces viruses at
low rates. The fraction of the inducible bacteria is nev-
ertheless higher than that observed by Jiang and Paul
(1998) [17], who reported from 25% to 64% inducible
bacterial isolates from different oligotrophic and eu-
trophic environments.
The relatively high fraction of lysogenic bacterial
isolates determined in vitro in the Gulf of Trieste may be
biased by the selection procedure during bacteria isola-
tion that favored fast-growing aerobic heterotrophic
bacteria, which may be more susceptible to the action of
the mitomycin C. It is also possible that the relatively low
host density in the Gulf of Trieste (?105BDC mL)1) is
inadequate for a successful virulent phage population
growth. For instance, if the total number of bacteria is
105/mL and there are 10 different host bacteria species,
which is probably an underestimate, the average host
density for a phage would be 104/mL. Such a host density
is considered to be at the lower limit for a successful lytic
virus population growth [1, 33]. In the natural marine
environment we expect the number of different bacterial
hosts to be higher, further decreasing the available host
population for viral attack. One should be careful, how-
ever, not to extrapolate this to biofilms or interfaces,
where local concentrations of host populations can be
significantly higher and supportive of virulent phage
growth. Nevertheless, survival of temperate phages is
generally less dependent on host cell density [18]. Lower
host density may actually increase the propensity for
lysogeny [34].
Of the bacterial lysates obtained by treatment with
mitomycin C, 51% had more than one morphologically
different phage. Phages differed in capsid diameter,
symmetry group, and the presence and size of the tail.
Some phages contained broken tails and empty phage
headlike structures. It is also possible that some of the
particles were bacteriocins. No systematic survey of the
As
infectivity of the viruses present in the lysate was per-
formed, because of the lack of nonimmune bacterial
hosts. Thus it is possible that some of the morphotypes
detected were defective virus particles and not different
morphotypes. In spite of that, different symmetry groups
of viruses present in the lysate would argue that mor-
phologically different phages were induced.
In order to confirm the presence of inducible bacteria
in the natural environment in situ induction experiments
with the natural bacterial community were performed
under aerobic conditions. After 24 h of incubation VLP
abundancewassignificantlyhigherinsamplestreatedwith
mitomycin C compared to the control samples. The dif-
ference was attributed to the phage production as a result
of prophage induction. The increase in VLP and decrease
in BDC abundance is shown in Fig. 4. Relative to the
control sample, a 4.3-fold increase in VLP abundance and
2.3-fold decrease in BDC abundance was observed after 24
h of incubation. These results suggest that lysogenic bac-
teria are present in the Gulf of Trieste, and consequently,
temperate phages may be an important component of the
virioplankton community. In the event of prophage in-
duction in the presence of an inducing agent (i.e., UV),
significant bacterial lysis may occur with a subsequent
increase in the amount of dissolved organic matter that
enters the microbial loop and modulates its dynamics.
In conclusion, we have detected high virus abundance
in the Gulf of Trieste. Depending on the environment and
season, there was between 108and 1010viruses per liter in
the Gulf of Trieste. VLP abundance consistently correlated
with BDC in all samples studied, whereas other physical,
chemical and biological parameters only weaklycorrelated
or did not correlate with VLP abundance. We therefore
conclude that the virus community in the Gulf of Trieste
is rather stable. Bacteriophages represent a significant
fraction of the virus community. Furthermore, lysogeny is
widespread among bacterial isolates. It appears that the
majority of lysogenic bacterial isolates in this environment
are polylysogenic. A relatively high occurrence of lysogeny
among bacterial isolates and in the natural bacterial
community in the Gulf of Trieste suggests that temperate
phages contribute to the total virus production and may
in addition be instrumental in the distribution of geno-
typic as well as phenotypic traits among the bacterial
community.
Acknowledgments
This research was supported by the Slovenian Ministry of
Science and Technology (No. L1-1608-0490).
References
1. Abedon, ST, Herschler, TD, Stopar, D (2001) Bacteriophage latent-
period evolution as a response to resource availability. Appl En-
viron Microbiol 67: 4233–4241
D. STOPAR ET AL: MICROBIAL COMMUNITY IN THE GULF OF TRIESTE
7

Page 8

2. Ackermann, HW, DuBow, MS (1987) Viruses of Prokaryotes. CRC
Press, Boca Raton, FL
3. Azam, F, Fenchel, T, Field, J, Gray, JG, Meyer-Reil, LA, Thingstad,
T (1983) The ecological role of water-column microbes in the sea.
Mar Ecol Prog Ser 10: 257–263
4. Bettarel, Y, Sime-Ngando, T, Amblard, C, Laveran, H (2000) A
comparison of methods for counting viruses in aquatic systems.
Appl Environ Microbiol 66: 2283–2289
5. Boehme, J, Frischer, ME, Jiang, SC, Kellogg, CA, Pichard, S, Rose,
JB, Steinway, C, Paul, JH (1993) Viruses, bacterioplankton and
phytoplankton in the southeastern Gulf of Mexico: distribution
and contribution to oceanic DNA pools. Mar Ecol Prog Ser 97: 1–
10
6. Bratbak, G, Heldal, M, Thingstad, TF, Tuomi, P (1996) Dynamics
of virus abundance in coastal seawater. FEMS Microbiol Ecol 19:
263–269
7. Cochlan, WP, Wikner, J, Steward, GF, Smith, DC, Azam, F (1993)
Spatial distribution of viruses, bacteria and chlorophyll a in neritic,
oceanic and estuarine environments. Mar Ecol Prog Ser 92: 77–87
8. Cochran, PK, Paul, JH (1998) Seasonal abundance of lysogenic
bacteria in a subtrophical estuary. Appl Environ Microbiol 64:
2308–2312
9. Fuhrman, JA (1999) Marine viruses and their biogeochemical and
ecological effects. Nature 399: 541–548
10. Fuhrman, JA, Azam, F (1982) Thymidine incorporation as a
measure of heterotrophic bacterioplankton production in ma-
rine surface waters: evaluation and field results. Mar Biol 66: 109–
120
11. Fuhrman, JA, Suttle, CA (1993) Viruses in marine planktonic
systems. Oceanography 6: 51–63
12. Grasshoff, K, Ehrhardt, M, Kremling, K (1983) Methods of Sea-
water Analyses. Verlag Chemie, Weinheim
13. Hara, S, Koike, I, Terauchi, K, Kamiya, H, Tanoue, E (1996)
Abundance of viruses in deep oceanic waters. Mar Ecol Prog Ser
145: 269–277
14. Jiang, SC, Paul, JH (1994) Seasonal and diel abundance of viruses
and occurrence of lysogeny/bacteriocinogeny in the marine envi-
ronment. Mar Ecol Prog Ser 104: 163–172
15. Jiang, SC, Paul, JH (1996) Occurrence of lysogenic bacteria in
marine microbial communities as determined by prophage in-
duction. Mar Ecol Prog Ser 142: 27–38
16. Jiang, SC, Paul, JH (1998) Gene transfer by transduction in the
marine environment. Appl Environ Microbiol 64: 2780–2787
17. Jiang, SC, Paul, JH (1998) Significance of lysogeny in the marine
environment: studies with isolates and a model of lysogenic phage
production. Microb Ecol 35: 235–243
18. Lenski, RE (1988) Dynamics of interactions between bacteria and
virulent bacteriophage. Adv Microb Ecol 10: 1–44
19. Malej, A, Mozetic ˇ, P, Malac ˇic ˇ, V, Turk, V (1997) Response of
summer phytoplankton to episodic meteorological events (Gulf of
Trieste, Adriatic Sea). Mar Ecol 18: 273–288
20. Mozetic ˇ, P, Fonda-Umani, S, Cataletto, B, Malej, A (1998) Sea-
sonal and interannual plankton variability in the Gulf of Trieste
(northeren Adriatic). J Mar Sci 55: 711–722
21. Mozetic ˇ, P, Malacic, V, Turk, V (1999) Ecological characteristics
of seawater influenced by sewage outfall. Ann Ser Hist Nat 9: 177–
190
22. Noble, RT, Fuhrman, JA (1998) Use of SYBR Green I for rapid
epifluorescence counts of marine viruses and bacteria. Aquat
Microb Ecol 14: 113–118
23. Ohnishi, M, Kurokawa, K, Hayashi, T (2001) Diversification of
Escherichia coli genomes: are bacteriophages the major contribu-
tors? Trends Microbiol 9: 481–485
24. Sambrook, J, Fritsch, EF, Maniatis, T (1989) Molecular Cloning: A
Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY
25. Steward, GF, Smith, DC, Azam, F (1996) Abundance and pro-
duction of bacteria and viruses in the Bering and Chukchi Sea. Mar
Ecol Prog Ser 131: 287–300
26. Strickland, JHD, Parson, TR (1972) A Practical Handbook of
Seawater Analysis. Fish Res Bd Canada Bull
27. Suttle, CA, Chan, AM, Cottrell, MT (1991) Use of ultrafiltration to
isolate viruses from seawater which are pathogens of marine
phytoplankton. Appl Environ Microbiol 57: 721–726
28. Turk, V, Mozetic ˇ, P, Malej, A (2001) Seasonal variability in phy-
toplankton and bacterioplankton distribution in the semi-enclosed
temperate Gulf (Gulf of Trieste, Adriatic Sea). Ann Ser Hist Nat 11:
53–64
29. UNEP/WHO (1994) Determination of faecal coliforms in sea
water by the membrane filtration (MF) culture method. UNEP,
Copenhagen
30. Van Hannen, EJ, Zwart, G, Van Agterveld, MP, Gons, HJ, Ebert, J,
Laanbroek, HJ (1999) Changes in bacterial and eukaryotic com-
munity structure after mass lysis of filamentous cyanobacteria
associated with viruses. Appl Environ Microbiol 65: 795–801
31. Weinbauer, MG, Fuks, D, Peduzzi, P (1993) Distribution of viruses
and dissolved DNA along a coastal trophic gradient in the
northern Adriatic Sea. Appl Environ Microbiol 59: 4074–4081
32. Weinbauer, MG, Fuks, D, Puskaric, S, Peduzzi, P (1995) Diel,
seasonal, and depth-related variability of viruses and dissolved
DNA in the northern Adriatic Sea. Microb Ecol 30: 25–41
33. Weinbauer, MG, Suttle, CA (1996) Potential significance of ly-
sogeny to bacteriophage production and bacterial mortality in
coastal waters of the Gulf of Mexico. Aquat Microb Ecol 62: 4374–
4380
34. Wiggins, BA, Alexander, M (1985) Minimum bacterial density
for bacteriophage replication: Implications for significance of
bacteriophages in natural ecosystems. Appl Environ Microbiol 49:
19–23
35. Wilcox, RM, Fuhrman, JA (1994) Bacterial viruses in coastal sea-
water: lytic rather than lysogenic production. Mar Ecol Prog Ser
114: 35–45
36. Wilhelm, S, Suttle, C (1999) Viruses and nutrient cycles in the sea.
Bioscience 49: 781–788
37. Wommack, EK, Colwell, RR (2000) Virioplankton: viruses in
aquatic ecosystems. Microbiol Mol Biol Rev 64: 69–114
38. ZoBell, CE (1946) Marine Microbiology. Chronica Botanica,
Waltham, MA
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