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Linking the lytic and lysogenic bacteriophage

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physiology and their variability in coastal
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ARTICLE in ENVIRONMENTAL MICROBIOLOGY · MARCH 2013

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Environmental Microbiology (2013) 15(9), 2463–2475 doi:10.1111/1462-2920.12120

Linking the lytic and lysogenic bacteriophage cycles to

environmental conditions, host physiology and their
variability in coastal lagoons

C. F. Maurice,† C. Bouvier, R. de Wit and T. Bouvier* Introduction

Université de Montpellier 2, Laboratoire Ecologie des
Viruses are the most abundant biological entities in
Systèmes Marins Côtiers ECOSYM UMR5119
aquatic systems, outnumbering prokaryotic cells by at
CNRS-Ifremer-IRD, case 093. Place Eugène Bataillon,
least one order of magnitude (Bergh et al., 1989;
34095 Montpellier cedex 5, France.
reviewed by Weinbauer, 2004). This high abundance is
sustained by two principal replication cycles, the lytic and
Summary the lysogenic cycles. The lytic cycle is characterized by a
rapid production of new viral particles in the system upon
Changes in environmental conditions and pro-
the host’s lysis, whereas the lysogenic cycle allows the
karyote physiology can strongly affect the dynamics
integration of the viral genome as a prophage within the
of both the lysogenic and lytic bacteriophage re-
bacterium (Ackermann and Dubow, 1987; reviewed by
plication cycles in aquatic systems. However, it
Weinbauer, 2004). This prophage will remain in the lys-
remains unclear whether it is the nature, amplitude
ogenic bacteria until an inducing event takes place, trig-
or frequency of these changes that alter the phage
gering the onset of viral production. Through lysogeny, the
replication cycles. We performed an annual survey
host gains immunity to superinfection by closely related
of three Mediterranean lagoons with contrasting
phages and acquisition of antibiotic resistance and/or
levels of chlorophyll a concentration and salinity to
toxin encoding sequences through phage conversion.
explore how these cues and their variability influ-
Such acquired traits increase prokaryotic fitness under
ence either replication cycle. The lytic cycle was
specific environmental conditions (reviewed by Wommack
always detected and showed seasonal patterns,
and Colwell, 2000; Chibani-Chennoufi et al., 2004;
whereas the lysogenic cycle was often undetected
Weinbauer, 2004). However, lysogenic cells become sus-
and highly variable. The lytic cycle was influenced
ceptible to a variety of inducing agents, and it is often
by environmental and prokaryotic physiological
suggested that there could be a metabolic cost associated
cues, increasing with concentrations of dissolved
with the replication of the integrated viral genome
organic carbon, chlorophyll a, and the proportion of
(reviewed by Paul, 2008).
respiring cells, and decreasing with the proportion
Studies with cultured isolates and natural bacterio-
of damaged cells. In contrast, lysogeny was not
plankton communities indicate that lytic infection is gen-
explained by the magnitude of any environmental or
erally found in productive environments, characterized by
physiological parameter, but increased with the
high host abundance and activity, allowing for the fast
amplitude of change in prokaryote physiology. Our
production of numerous new viral particles. In contrast,
study suggests that both cycles are regulated by
lysogeny is proposed to enable the survival of the host
distinct factors: the lytic cycle is dependent on
and prophage under unfavourable conditions, such as low
environmental parameters and host physiology,
prokaryote abundance and primary production (reviewed
while lysogeny is dependent on the variability of
by Wommack and Colwell, 2000; Chibani-Chennoufi
prokaryote physiology. This could lead to the con-
et al., 2004). Studies that address simultaneously both
trasting patterns observed between both cycles in
cycles and their prevalence in aquatic systems are less
aquatic systems.
numerous, and have resulted in contrasting ecological
scenarios. Temporal and spatial studies have demon-
strated an inverse correlation between the lytic and
Received 7 June, 2012; accepted 5 March, 2013. *For correspond- lysogenic cycles, suggesting that environmental charac-
ence. E-mail tbouvier@univ-montp2.fr; Tel. (+33) 4 67 14 40 32; Fax teristics allow the predominance of one cycle (Weinbauer
(+33) 4 67 14 37 19. †Present address: FAS Center for Systems
Biology, Harvard University, 52 Oxford Street, Cambridge, MA 02138, et al., 2003; Colombet et al., 2006; Pradeep Ram and
USA. Sime-Ngando, 2010); others have reported a positive
© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology
2464 C. F. Maurice, C. Bouvier, R. Wit and T. Bouvier

correlation, suggesting that continuous induction events
aliment the pool of visibly infected cells (Bettarel et al.,
2008; Maurice et al., 2010); and finally others have
reported no correlation between these cycles (Boras
et al., 2009). These results highlight our limited under-
standing of the natural processes regulating phage repli-
cation cycles.
Changes in environmental conditions can alter viral
dynamics and replication cycles (reviewed by Wommack
and Colwell, 2000; Weinbauer, 2004), but it is unclear
whether this is due to the nature of the environmental
perturbation, its amplitude and/or frequency, or through its
impact on the prokaryote host. In addition, a given vari-
able can affect the lytic and lysogenic cycles in dissimilar
ways. For example, high growth rates generally associ-
ated with lytic replication can also correspond to an induc-
tion signal for the prophage, in order to maximize viral
production (Gottesman and Oppenheim, 1994; Paul,
2008). Similarly, low levels of prokaryote activity usually
associated with lysogeny can also lead to the induction of
the prophage before the host’s lysis and loss of the viral
genome (Clarke, 1998; Maurice et al., 2010). These dis-
crepancies emphasize that the variables regulating the
lysogenic and the lytic replication cycles in aquatic
systems remain poorly understood.
Observing the dynamics of phage replication cycles in
natural systems with strong environmental variability
would provide an initial framework to characterize the
relationship between environmental variability and phage
replication cycles. These systems harbour prokaryote
communities that are very diverse in their physiology,
activity and community structure (as observed in estuar-
ies or coastal waters; del Giorgio and Bouvier, 2002;
Riemann and Middelboe, 2002; Hewson and Fuhrman,
2004). Constant fluctuations in the host physiology and
activity could constitute unfavourable conditions for the
phage communities and promote the establishment of
lysogeny (Paul, 2008). Alternatively, these environmental
fluctuations could promote inducing events and high rates
of successful viral and host encounters, thereby favouring
lytic infections. However, to our knowledge, there is no in
situ evidence linking environmental variability and phage
replication cycles so far.
Mediterranean lagoons are shallow water bodies
subject to major periodic nutrient inputs from human
activities [agriculture, extensive urbanization (Castel
et al., 1996; Ifremer, 2008)], as well as irregular and
strong periods of rainfall in autumn and spring, followed
by long periods of dryness in summer. These systems
thus go through great temporal and spatial environmental
changes, differing in their amplitude and nature, and
thereby constitute model ecosystems to study the impor-
tance of environmental variability in phage replication
cycles. Over the course of 1 year, we sampled three
Mediterranean coastal lagoons characterized by contrast-
ing annual variability in: salinity; dissolved organic carbon
(DOC); total nitrogen (TN) and phosphorus (TP); and
chlorophyll a (Chl a) concentrations. These variables are
all recognized for their impact on prokaryote physiology
(del Giorgio and Bouvier, 2002; Hewson and Fuhrman,
2004) and viral replication cycles (Cochran and Paul,
1998; Weinbauer, 2004; Bettarel et al., 2009). We aimed
to determine: (i) the type of pattern (negative/positive
correlation or no pattern) occurring between the lytic and
lysogenic cycles in coastal lagoons, and (ii) how environ-
mental variables, the host’s physiology, and their variabil-
ity over short (seasonal) and long (annual) timescales
might explain the patterns of occurrence and proportions
of both cycles.
Results
Environmental variables and annual variability
Three coastal Mediterranean lagoons, Leucate, Bages
and Gruissan, were sampled every 3 weeks, and the
means and ranges of values of all environmental
variables measured are reported in Table 1. Mean
annual DOC concentrations were significantly different
among lagoons (ANOVA, F = 5.060, P = 0.011; Table 1),
with higher concentrations in Bages (Tukey Kramer,

Table 1. Annual means and ranges of environmental and chemical parameters in the three lagoons.

Oxygen DOC
Temperature concentration Chl a concentration
Lagoon (°C) (% saturation) Salinity TP (mM) TN (mM) (mg Chl a l-1) (mg C l-1)

Leucate 15.5 84.2 35.1 0.5 23.2 1 258.4

(4.5–26.8) (23.7–168.0) (28.6–38.1) (0.3–0.9) (17.5–29.0) (0.12–2.21) (121.8–531.7)
Bages 15 88.1 34.7 2.4 46.3 4.2 442.2
(4.7–25.3) (25.3–192.5) (24.5–39.4) (0.5–6.4) (25.9–86.0) (0.4–19.6) (194.8–930.6)
Gruissan 16.2 98.9 32.1 2.1 38.6 4.3 309.4
(5.7–27.7) (27.6–190.6) (20.0–42.7) (0.3–13.1) (14.8–102.6) (0.4–17.3) (123.7–601.7)
ANOVA (F, P value) 0.140, 0.870 0.643, 0.531 1.577, 0.219 2.736, 0.077 3.070, 0.057 3.884, 0.028 5.06, 0.011

Chl a: chlorophyll a concentration; DOC: dissolved organic carbon; TP: total phosphate concentration; TN: total nitrogen concentration. ANOVAs
(F and P values) for the effect of lagoon on the environmental and chemical parameters during the annual survey are also reported.

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475
 Bacteriophage replication strategies in lagoons 2465

45 P = 0.009). As expected, the range and variability of salin-

A Leucate
Bages ity and Chl a values were distinct across lagoons (Fig. 1A
Gruissan
40 and B). Leucate remained in an oligotrophic state,
whereas Bages and Gruissan fluctuated from oligotrophic
to eutrophic conditions, as reflected by the Chl a concen-
35
trations. Chl a concentrations were significantly different
Salinity

among lagoons (ANOVA, F = 3.884, P = 0.028; Table 1)

30 with higher concentrations in Bages than in Leucate
(Tukey Kramer, P = 0.027). All three lagoons were mix-
25 opolyhaline according to the Venice system (Anonymous,
1959), and showed seasonal salinity fluctuations. Salinity
fluctuated most in Gruissan (from 20 to 43). The mean
20
values of the other environmental variables were not sig-
nificantly different among the three lagoons (Table 1). The
15 index of annual environmental variability (see Experimen-
20 tal procedures) increased from 2.5 in Leucate to 3.5 in
B Bages, and reached a maximum of 4.6 in Gruissan
(Fig. 1C). This index was significantly different among
lagoons (Bartlett test, P = 0.014). Collectively, these
Chl a (μg Chl a l )

15 results indicate a ranking of the lagoons based on their

-1

environmental variability from low (Leucate), to interme-

diate (Bages), to high variability (Gruissan).
10
Viral and prokaryote community dynamics
In addition to the environmental parameters, we also
5
determined the abundance of the prokaryote and viral
communities using flow cytometry and epifluorescence
microscopy respectively. Their dynamics in the three
0 lagoons are shown in Fig. 2. Prokaryote and viral abun-
Feb Apr Jun Aug Oct Dec Feb dances were significantly different among the three
Time (m) lagoons (ANOVA, F = 12.37, P < 0.0001 for the prokaryo-
tes, and F = 16.82, P < 0.0001 for the viruses). The mean
5
C annual prokaryote and viral abundances in Bages were
6.7 ¥ 106 prokaryotes ml-1 and 2.7 ¥ 108 viral particles
Calculated index of annual

Salinity
environmental variability

Oxygen
4 TP
ml-1, which were significantly higher than in the other two
TN lagoons (Tukey Kramer, P = 0.022 for the prokaryotes,
Chl a
DOC and P = 0.021 for the viruses). The prokaryote abun-
3 dances in Gruissan (4.9 ¥ 106 prokaryotes ml-1) and
Leucate (3.6 ¥ 106 prokaryotes ml-1) were not significantly
different (Tukey Kramer, P > 0.05). Likewise, viral abun-
2 dances in Gruissan (1.7 ¥ 108 viral particles ml-1) were not
significantly different from Leucate (1.3 ¥ 108 viral parti-
cles ml-1) (Tukey Kramer, P > 0.05). The resulting virus to
1
prokaryote ratio (VPR) averaged 38.13 in Gruissan
(range: 9.88–65.88), 40.35 in Leucate (range: 16.44–
0 65.38), and was highest in Bages with 43.60 (range:
Leucate Bages Gruissan 8.76–68.62), but was not significantly different among
lagoons (ANOVA, F = 0.43, P > 0.05).
Lagoon
Using a full factorial analysis of variance, we show
Fig. 1. A and B. Annual dynamics of chlorophyll a (A) and salinity that across all lagoons, viral abundance significantly
(B) concentrations. increased with prokaryote abundance (ANOVA, F = 16.86,
C. Calculated index of annual environmental variability. Chl a:
chlorophyll a; DOC: dissolved organic carbon; TP and TN: total
P < 0.001), but not with Chl a concentrations (ANOVA,
phosphate and nitrogen respectively. F = 2.95, P > 0.05); and the interaction between Chl a and

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475
2466 C. F. Maurice, C. Bouvier, R. Wit and T. Bouvier

Leucate respiring cells both accounted for 20% of the total

Bages prokaryote communities respectively. The variability of the
Prokaryote abundance (106 cells ml -1)
Gruissan
different physiological categories, determined by their
coefficient of variation (CV), was not significantly dif-
9
ferent among the three lagoons (ANOVA, F = 0.78,
P > 0.05 and F = 1.31, P > 0.05, for the CVs of CTC+ cells
and DiBAC+ cells respectively), with CV values ranging
7 from 0.23 to 1.03 for CTC+ cells and from 0.17 to 0.89 for
DiBAC+ cells.

5
Viral replication cycle dynamics
Lysogeny, determined using incubations with mitomycin
3 C, was detected in all three lagoons, although on many
sampling occasions it remained below the detection limit.
Lysogeny was observed on 13% of the sampling dates in
Gruissan (2/15 sampling dates), 27% of the sampling
1
dates in Leucate (4/15) and 47% of the sampling dates in
Bages (7/15; Fig. 3). When detected, the frequency of
lysogenic cells (FLC) was highly variable, ranging from
Viral abundance (108 particles ml -1)

0.5% to over 65%. Lysogeny did not follow any seasonal

pattern in the three lagoons, and its occurrence was
4
dynamic and transient (Fig. 3). The annual average FLC
values were lowest in Leucate (3.9%), intermediate in
Gruissan (5.2%) and highest in Bages (6.4%), but did not
3 differ significantly across lagoons (ANOVA, F = 0.146,
P > 0.05).
In contrast to lysogens, visibly infected cells were
2 detected on almost all sampling occasions, with the
exception of the January 2009 sampling in Leucate. The
frequency of visibly infected cells (FVIC) showed a sea-
sonal pattern, with maximum values observed during
1
spring and autumn. The annual mean FVIC values

0 Table 2. Prokaryote community physiology in the three lagoons.

Feb Apr Jun Aug Oct Dec Feb
Lagoon CTC+ (%) DiBAC+ (%)
Time (m) Leucate 14.8 (0.7) 21.7 (0.8)
Winter 9.1 ⫾ ND 40.9 ⫾ 32.9
6 -1 8
Fig. 2. Dynamics of prokaryote (¥10 cells ml ) and viral (¥10 Spring 14.3 ⫾ 3.3 17.3 ⫾ 12.1
particles ml-1) abundances in the three lagoons. Error bars Summer 14.4 ⫾ 14.8 10.3 ⫾ 5.1
represent standard errors from triplicate samples. Fall 17.0 ⫾ 13.5 23.0 ⫾ 8.3
Bages 17.3 (1.0) 21.3 (0.9)
Winter 8.2 ⫾ ND 43.3 ⫾ 34.7
prokaryote abundance was not significant either (ANOVA, Spring 29.4 ⫾ 25.5 21.9 ⫾ 13.3
F = 0.13, P > 0.05). Jointly, these data suggest that most Summer 11.2 ⫾ 6.8 12.6 ⫾ 2.1
of the viruses in these systems are bacteriophages. Fall 13.5 ⫾ 11.2 12.9 ⫾ 4.2
Gruissan 15.7 (0.5) 21.7 (0.9)
Collectively, the proxies of prokaryotic activity deter- Winter 6.9 ⫾ ND 42.9 ⫾ 32.0
mined by vital staining and flow cytometry showed coher- Spring 21.1 ⫾ 5.9 16.4 ⫾ 11.5
ent seasonal patterns during the survey. The proportions Summer 17.7 ⫾ 9.7 9.8 ⫾ 3.9
Fall 10.4 ⫾ 5.0 22.9 ⫾ 20.3
of respiring cells (CTC+ cells) were high during spring and
summer in the three lagoons, seasons with relatively low Mean annual values of the proportions of CTC+ and DiBAC+ cells
proportions of depolarized cells (DiBAC+ cells); whereas detected by flow cytometry are in bold, with the annual coefficient of
variation in parenthesis. Within each lagoon, seasonal average pro-
the proportions of depolarized cells were highest during portions of each physiological feature (⫾ SD) are also given. CTC+:
winter (Table 2). On average, fractions of depolarized and respiring cells; DiBAC+: depolarized cells. ND: not determined.

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475
 Bacteriophage replication strategies in lagoons 2467

5
Leucate A Leucate B
Bages Bages
60 Gruissan Gruissan
4

FVIC (%)
3
FLC (%)

40

20
1

0 0
Feb Apr Jun Aug Oct Dec Feb Feb Apr Jun Aug Oct Dec Feb
Time (m) Time (m)

10 10
C D

8 8
FVIC (%)

6 6
FLC (%)

4 4

2 2

0 0
Leucate Bages Gruissan Leucate Bages Gruissan

Fig. 3. Annual dynamics of viral replication cycles (A, B) in the three lagoons. Annual average of viral replication cycles (C, D) in the three
lagoons (n = 15 in each lagoon). Error bars represent standard errors. FLC: frequency of inducible lysogenic cells; FVIC: frequency of visibly
infected cells.

were 0.92% in Leucate, 1.33% in Gruissan and 1.42%
in Bages, and were not statistically different (ANOVA,
F = 2.837, P > 0.05).
We observed two types of patterns between the lytic
and lysogenic replication cycles (Fig. 4). Considering only
the sampling dates with detectable mitomycin C-inducible
lysogens, we report a negative, non-significant trend with
substantial scatter of FLC and FVIC values (Pearson cor-
relation, r = -0.399, P > 0.05), in agreement with a pattern
of replacement between cycles and predominance of one
cycle over the other. When including the sampling dates
where lysogens are below the detection limit, FLC values
are distributed across the entire FVIC range without any
significant correlation between cycles (Pearson correla-
tion, r = -0.040, P > 0.05).
Environmental factors and phage replication strategies
At the annual scale, considering all 15 sampling dates in
the three lagoons, we did not observe any significant
relationship between the FLC or FVIC and the index of
annual environmental variability based on the coefficient
of variation of DOC, Chl a, TN, TP, dissolved O2 concen-
trations and salinity (Model II regression, P > 0.05). There
was a significant relationship between FVIC and the
contact rates between prokaryotes and virus (Model II

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475
2468 C. F. Maurice, C. Bouvier, R. Wit and T. Bouvier

values of FVIC and the seasonal mean values of DOC

and Chl a concentrations (Model II regression, P = 0.015
60
and P = 0.028 respectively; Fig. 5). These regressions
explained 45.9% and 39.2% of the observed variances
respectively. However, the seasonal mean values of FVIC
40 did not show a significant relation with the seasonal coef-
FLC (%)

ficients of variation (CV) of these two variables (Model II

regression, P > 0.05).
In contrast, FLC was neither significantly related to any
20 of the seasonal mean values of environmental param-
eters (P > 0.05), nor significantly related to their seasonal
variability based on the seasonal coefficients of variation
(Model II regression, P > 0.05).
0
Prokaryote physiology and phage replication strategies

0 1 2 3 As shown in Fig. 6, phage replication cycles were influ-

FVIC (%)
Fig. 4. Patterns between viral replication cycles. Sampling dates
with FLC below the detection limit are represented by clear circles;
sampling dates with mitomycin C inducible cells (FLC > 0) are
represented by dark circles. FLC: frequency of inducible lysogenic
cells; FVIC: frequency of visibly infected cells.
regression, FVIC = 0.23 + 0.11 contact rate; R2 = 0.518;
P = 0.029); but this was not the case for FLC (R2 = 0.080,
P > 0.05).
At the seasonal scale, we observed a positive and
significant relationship between the seasonal mean
enced by both the seasonal means of prokaryote physi-
ology (left panels) and their seasonal variability (right
panels). Model II linear regression analysis showed that
the lytic cycle (represented by the FVIC) significantly
increased with the seasonal mean of the proportion of
respiring cells (CTC+ cells) and decreased with the sea-
sonal mean of the proportion of damaged cells (DiBAC+
cells), explaining 45% of the variance in both cases.
However, there was no significant link between lysogeny
and any of the two measured prokaryote physiology
signals (Model II regression, P > 0.05, Fig. 6). In contrast,
lysogeny significantly increased with the seasonal vari-
ability of prokaryote physiology, both for CTC+ and for

2 2
FVIC = 0.334 + 0.003 DOC, R = 0.459, p = 0.015 FVIC = 0.874 + 0.107 Chl a, R = 0.392, p = 0.028
2.5 2.5
Leucate Leucate
Bages Bages
Gruissan Gruissan
2.0 2.0

1.5 1.5
FVIC (%)
FVIC (%)

1.0 1.0

0.5 0.5

0.0 0.0
0 100 200 300 400 500 600 0 2 4 6 8 10
-1 -1
DOC, μg C l Chl a, μg l
Fig. 5. Relationship between the seasonal means of FVIC plotted against the seasonal means of the dissolved organic carbon and
chlorophyll a concentrations (three lagoons, four seasons: n = 12). Chl a: chlorophyll a; DOC: dissolved organic carbon; FVIC: frequency of
visibly infected cells.

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475
 Bacteriophage replication strategies in lagoons 2469
Fig. 6. Left panels: Seasonal means of
FVIC and FLC plotted against the
seasonal means of the proportion of
CTC+ and DiBAC+ cells (three lagoons,
four seasons, n = 12). Right panels:
Seasonal means of FVIC and FLC plotted
against the coefficient of variation (CV,
n = 3 measurements per season in each
lagoon) of CTC+ and DiBAC+ cells (three
lagoons, four seasons: n = 12). Linear
regressions are shown for relationships
that are statistically significant (P < 0.05).
Circle: Leucate; square: Bages; triangle:
Gruissan. CTC+ cells: respiring cells;
DiBAC+ cells: depolarized cells; CV:
coefficient of variation; FVIC: frequency of
visibly infected cells; FLC: frequency of
inducible lysogenic cells.

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475
2470 C. F. Maurice, C. Bouvier, R. Wit and T. Bouvier
+
DiBAC cells (Model II regression explaining 35% and integrases, GTAs, or other transcriptional regulators
33% of the variances of the seasonal CVs of CTC+ and (Paul, 2008; Reyes et al., 2010; Sharon et al., 2011).
DiBAC+ cells respectively); which was not the case for the This would enable a better evaluation of lysogeny and
lytic cycle (Fig. 6). increased comprehension of the underlying mechanisms
regulating viral replication cycles.
Discussion
Environmental factors and viral replication cycles
Patterns between viral replication strategies
The dynamics of phage replication cycles have been
In their spatial study, Weinbauer and colleagues (2003)
scarcely explored in coastal lagoons, which constitute
found that the lysogenic and lytic cycles followed a
ideal systems to explore the impact of fluctuating environ-
replacement pattern with one predominant cycle.
mental conditions. In this annual survey, we have selected
However, evidence that both cycles can increase in
three coastal lagoons differing in their levels of environ-
tandem within the same ecosystem suggests the replace-
mental variability in order to identify how this variability
ment pattern does not always hold true (Bettarel et al.,
affects lysogenic and lytic infections.
2008; Maurice et al., 2010). If we hypothesize that the
Lysogeny is thought to be strongly dependent on
same parameters influence both replication cycles with
changes in environmental parameters, such as tempera-
contrasting results (e.g. high prokaryote activity associ-
ture, salinity or nutrient concentrations, where a strong
ated with lytic replication, as well as the induction of
environmental variation or shock acts as an inducing
lysogenic cells), then the predominance of one cycle over
agent (Cochran and Paul, 1998; Williamson et al., 2002;
the other can be expected, as previously reported
Laybourn-Parry et al., 2007; Shkilnyj and Koudelka,
(Weinbauer et al., 2003). However, when both cycles are
2007). However, our results show that FLC was neither
regulated by distinct parameters, all potentially interacting
significantly affected by any specific environmental
with each other, resulting patterns between the cycles are
parameter, nor significantly affected by their seasonal
expected to become more difficult to model or predict.
variability. Although we cannot exclude the inducing
When excluding all data below the detection limit, we
potential of these environmental factors on lysogenic
observed a negative trend between cycles with substan-
cells, the absence of link reported here indicates that this
tial scatter, suggesting a shift from the lytic cycle to lys-
induction would take place at a smaller timescale than the
ogeny, similar to the one described by Weinbauer and
seasonal one we investigated. Nevertheless it is worth
colleagues (2003). However, this trend was not significant
noting that when we explored this issue at a finer times-
and disappeared altogether when considering the full
cale, considering the mean and variability of these param-
range of measured FLC values (including null values).
eters from the three sampling dates preceding the
Our results therefore strongly suggest that in lagoons,
detection of lysogens in the system, we still did not detect
lytic and lysogenic replication cycles are not regulated by
a significant trend (data not shown). It is possible that
the same environmental factors.
additional environmental variables such as UV light or
The null values of FLC we report here could result from
chemical pollutants (Jiang and Paul, 1996; Maranger
a real and sustained absence of lysogeny in these
et al., 2002) strongly impact the occurrence of lysogeny in
systems, but could alternatively reflect continuous or
these systems.
massive induction events, leading to the recurrent
In contrast to FLC, FVIC was linked to specific environ-
absence of detection of lysogens. This would imply that
mental variables, i.e. FVIC increased with increasing DOC
lysogeny, although not always detectable, remains
and Chl a concentrations (Fig. 5). Lytic infections have
an important replication strategy in these systems
been shown to be nutrient-dependent, with the addition of
(Weinbauer and Suttle, 1996; Cochran and Paul, 1998;
organic and inorganic nutrient increasing lytic viral pro-
Bettarel et al., 2009), and that null values, presumably
duction (Tuomi and Kuuppo, 1999; Williamson and Paul,
resulting from recent induction events, could simply mask
2004). However, it is also possible that higher prokaryote
the true pattern between both cycles. By analogy to the
and viral abundances lead to more successful encounters
conversion equation applied between the ‘frequency of
between phages and their hosts, resulting in higher
visibly infected cells’ and the ‘frequency of infected cells’
success of lytic infection. The positive and significant
(Binder, 1999; Weinbauer et al., 2002), future studies
regressions between FVIC, prokaryote and viral abun-
should attempt to model or quantify the experimental
dances and contact rates all support this scenario.
limitations inherent in the evaluation of lysogeny with
chemical inducers. Metagenomics approaches could be
Prokaryote physiology and viral replication cycles
combined with induction experiments to explore the
prevalence and dynamics of specific genes required for In this study, we determined the fractions of cells actively
lysogeny and the integration of the viral genome, such as respiring or with compromised membranes, representing

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475

two distinct levels of prokaryote physiology and activity
(del Giorgio and Gasol, 2008). These fractions remained
within the range of previously published observations
(Gasol et al., 1999; Sherr et al., 1999; Davidson et al.,
2004), irrespective of the environmental fluctuation
observed in each lagoon.
The positive regression observed between the lytic
cycle and the proportion of CTC+ cells (Fig. 6, explaining
45% of the variance) indicates that phage replication is
dependent on host metabolism, with lytic viral infections
increasing with higher host activity. This is further sup-
ported by the negative regression observed with the pro-
portion of DiBAC+ cells (Fig. 6). These results are
consistent with previous findings obtained either at the
community level or with isolated phage host systems
(Maranger et al., 1994; You et al., 2002; Säwström et al.,
2007).
Lysogeny is thought to be predominant in starving
cells, i.e. cells with a physiology that does not allow effi-
cient phage production (Echols, 1972; Levin et al., 1977;
Jiang and Paul, 1998), but our data did not support
a link between lysogeny and prokaryote physiology.
However, lysogeny did increase with increasing sea-
sonal variability in prokaryote physiology, both in
depolarized and in respiring cells (Fig. 6). These results
suggest that lysogeny is not affected directly by the
level of prokaryote activity or membrane damage, but
rather responds to the amplitude of variability in key fea-
tures of prokaryote physiology. This is particularly rel-
evant in transition systems such as coastal lagoons,
where highly fluctuating environmental conditions can
rapidly and significantly modulate prokaryote physiology,
and thus the occurrence of lysogeny. This is also sug-
gested by the transient and acute dynamics of FLC we
observed in all three systems. Whether variability in host
physiology is a signal to enter the lysogenic replication
cycle or to remain in the lysogenic state remains to be
explored.
The objectives of our work were to determine the type
of pattern between the lytic and lysogenic replication
cycles in coastal lagoons, and to explore how environ-
mental factors, host physiology and their variability
affect the occurrence and dynamics of these cycles. This
annual survey indicates that replication cycles are regu-
lated by different signals of prokaryote physiology: the
lytic cycle is directly dependent on prokaryote activity,
whereas the lysogenic cycle increases with the ampli-
tude of change in prokaryote physiology. Using phage-
host systems, one could modulate the level of variability
in the host’s physiology, in order to identify how this
variability translates into a signal for lysogeny at the
molecular level. This would provide further insight into
the dynamics of lysogeny in natural bacterioplankton
communities.
© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475
Bacteriophage replication strategies in lagoons 2471
Experimental procedures
Sampling
Three Mediterranean lagoons, Leucate (+42°48′23.4″N,
+2°59′11.2″E), Bages (+43°03′40.8″N, +3°00′12.6″E) and
Gruissan (+43°06′53.1″N, +3°04′56.5″E), were sampled
every 3 weeks during a yearlong survey, from February 2008
to January 2009. These lagoons were selected according to
their salinity and Chl a concentrations from previous reports
(Bec et al., 2011). The first lagoon, Leucate, generally shows
small seasonal variations of Chl a and salinity (between 1 and
3 mg Chl a l-1, and 33 and 38 respectively). The second
lagoon, Bages, shows strong seasonal variations of Chl a
and low fluctuations of salinity (between 0.8 and 23.6 mg Chl
a l-1, and 29 and 40 respectively), whereas the last lagoon,
Gruissan, shows strong seasonal variations of both Chl a and
salinity (between 0.3 and 24.5 mg Chl a l-1, and 17 and 44
respectively). Water was sampled between 0.5 and 1 m
depth, using 5 l acid-washed dark bottles thoroughly rinsed
with milliQ water. Samples were kept cool and stored for less
than 4 h before processing. Temperature, salinity, pH and
oxygen measurements were measured directly on site with a
calibrated multi-parameter Multi 350i instrument (WTW).
Environmental and chemical variables
Triplicate 50 ml samples were filtered on GF/F filters for
determination of Chl a concentration, according to Yentsch
and Menzel (1963), with a Trilogy fluorometer (Turner). To
assess total nitrogen and phosphate concentrations (TN and
TP respectively), triplicate samples were oxidized with per-
sulfate before being analysed with a Technicon Autoanalyser
AA3 (Menzel and Corwin, 1965; Raimbault and Slawik,
1991).
Viral enumeration
Viral abundance was determined by epifluorescence micros-
copy, following Chen and colleagues (2001). Triplicate fixed
samples (formaldehyde, 1% final concentration) were filtered
onto 0.02-mm-pore-size Anodisc membranes (Whatman) and
stained for 15 min with the SYBR®Gold Nucleic Acid Stain
(Molecular Probes), at 2.5¥ final concentration. Samples
were enumerated using an Olympus Provis-AX70 epifluores-
cence microscope at 488 nm excitation, and a minimum of
300 viruses and 20 fields per filter were counted.
Prokaryote abundance and single-cell analysis
of physiology
The abundance and physiological characteristics of the
prokaryote communities were determined by flow cytometry
(FCM) on fresh, unfixed samples. All analyses were per-
formed using a FACSCalibur Flowcytometer (Becton Dickin-
son) equipped with an air-cooled argon laser providing
15 mW at 488 nm and with standard filter set-up. Total
prokaryote abundance was determined after nucleic acid
staining by the SYBR®GreenI dye (Molecular Probes).
Briefly, 0.5 ml subsamples were incubated with 0.5 ml of
2472 C. F. Maurice, C. Bouvier, R. Wit and T. Bouvier
SYBR®GreenI (10 000¥) for 15 min at room temperature in
the dark (del Giorgio et al., 1996). Stained prokaryotes then
were discriminated and enumerated by using right angle light
scatter (SSC) and green fluorescence (FL1).
We used two different cellular characteristics, membrane
polarity and respiratory enzyme activity, to describe the physi-
ological state of the prokaryote cells and their level of activity.
Depolarized cells were identified using the anionic membrane
potential-sensitive dye DiBAC4 (Molecular Probes, 1 ml in
0.5 ml sample, 10 min incubation). DiBAC4 accumulates
within cells that have lost their membrane potential (Novo
et al., 2000), and will consequently show bright green fluo-
rescence (Jepras et al., 1995; Lopez-Amoros et al., 1995;
Nebe-von-Caron et al., 2000). Cells with high respiratory
enzyme activity were determined after staining with 5-cyano-
2,3-ditolyl tetrazolium chloride (CTC), an indicator of the res-
piratory electron transport system activity (Sherr et al., 1999).
Freshly prepared stock solution of 50 mmol l-1 CTC (tebu-bio)
was filtered through 0.1 mm, added to 0.45 ml of sample
(5 mmol l-1 final concentration) and incubated for 3 h at room
temperature in the dark. Active cells reduce the tetrazolium
salt CTC into its red fluorescent formazan detectable by FCM
(del Giorgio et al., 1997; Sieracki et al., 1999).
The proportions of the different physiological categories
(CTC+ and DiBAC+) were calculated relative to the total
prokaryote counts obtained by SYBR®GreenI staining. Fluo-
rescent latex beads (0.94 mm diameter, Polysciences) were
systematically added to each sample as internal standard for
cell counts, cell fluorescence and light scatter measure-
ments. Bead stock solution concentration was determined by
epifluorescence microscopy prior to every FCM analysis. All
data were obtained and analysed with the CellQuest Pro
software (BD Biosciences).
Frequency of lysogenic cells (FLC)
The proportion of mitomycin C-inducible lysogens, classically
referred to as the frequency of lysogenic cells (FLC), was
obtained with triplicate incubations of untreated samples
(control incubations) and samples treated with the most com-
monly used chemical inducer, mitomycin C (1 mg ml-1 final
concentration; Sigma). All systems sampled were alkaline,
precluding the instability of this antibiotic under acidic condi-
tions (Windholz et al., 1976). Preliminary 24 h time-series
incubations indicated that incubations longer than 17 h were
unnecessary. Three subsamples (6 h, 8 h and 17 h) of these
incubations were fixed with formaldehyde (2% final concen-
tration for prokaryote and viral enumeration, as detailed
previously). Induction took place when we observed simulta-
neously significantly (P < 0.05) lower prokaryote abundance
and significantly higher viral abundance in the mitomycin C
treatment relative to controls (Cochran and Paul, 1998;
Tapper and Hicks, 1998). To calculate the frequency of lys-
ogenic cells (FLC), we used the average published value of
burst size of 24 acquired from studies in marine systems
(Parada et al., 2006). When induction was not detected, we
considered that FLC = 0.
Frequency of visibly infected cells (FVIC)
The frequency of visibly infected cells FVIC was determined
by transmission electron microscopy (TEM). Briefly, 500 ml
© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475
of fixed sample (formaldehyde, 2% final concentration) were
centrifuged using an AirFuge® Ultracentrifuge (Beckman
Coulter) at 30 000 g for 10 min at 20°C on duplicate 400-
mesh copper formvar, carbon-coated electron microscope
grids (Delta Microscopies). Samples were then stained with
uranyl acetate (final concentration 0.5%) for 20 s and rinsed
twice in distilled water. At least 1000 prokaryote cells per
grid were observed at 40 000¥ magnification, and at an
acceleration voltage of 120 kV. A cell was considered visibly
infected when five virus-like particles could be seen in its
cytoplasm (Weinbauer and Hofle, 1998; Bettarel et al.,
2004).
Data treatment and statistical analyses
The contact rate between the prokaryotic hosts and viruses
was calculated using the mathematical model from Murray
and Jackson (1992): R = (Sh 2p d Dv) ¥ VP; where R is the
number of virus attached ml-1 s-1, Sh is the Sherwood number
(1), d is the prokaryote cell diameter (0.42 mm), Dv is the
diffusivity of virus (eqn 9 in Murray and Jackson, 1992), and
V and P are the virus and prokaryote abundance respectively.
We used the average prokaryote cell size determined by
epifluorescence microscopy in Thau lagoon (n = 1800 cells;
measured in January, April and August); whereas the average
viral diameter was set to 45 nm, based on data from coastal
systems (Weinbauer, 2004).
To determine the effect of the annual ecosystem environ-
mental variability on phage replication cycles, we calculated
an index of annual variability encountered within each lagoon
by summing the coefficients of variation (CV) of the DOC, Chl
a, TN, TP, dissolved O2 concentration and salinity variables
measured over the year (n = 15 points per lagoon). However,
because of the very dynamic nature of Mediterranean
lagoons, this global annual index of variability could mask the
variability of environmental variables occurring at seasonal
scales (Winter: February 2008–March 2008 and December
2009–January 2009; Spring: March 2008–June 2008;
Summer: June 2008–September 2008; Fall: September
2008–December 2008). Thus, we also calculated the sea-
sonal coefficients of variation of the DOC, Chl a, TN, TP,
dissolved O2 concentration and salinity, as well as the sea-
sonal coefficients of variation of the prokaryote physiological
categories measured (CTC+ and DiBAC+ cells), and com-
pared this seasonal level of variability to the seasonal mean
values of FLC and FVIC for the lysogenic and lytic cycles
respectively (see above).
Statistical analyses were made using the JMP 5.01 soft-
ware (SAS institute). All data were checked for normality.
The homoscedasticity of the variances of the abiotic and
biotic variables among lagoons were tested with Bartlett’s
test, and one-way ANOVAs were performed. A posteriori
pairwise comparisons of means between lagoons were
made using Tukey-Kramer’s post-hoc analysis (alpha
level = 0.05). One-way ANOVAs were performed between
control and mitomycin C treatment samples at each time
point to determine whether induction took place. We also
used a full factorial analysis of variance (ANOVA) to identify
the combined effects of prokaryote abundance and Chl a
concentration on viral abundance. We used correlation
analyses to test the links between FVIC and FLC, and

performed regression analyses in order to test whether the
FLC and FVIC were dependent of abiotic and biotic factors.
Because both variables were not fixed but scattered ran-
domly, we applied Model II reduced major axis regression
(Sokal and Rohlf, 1995).
Acknowledgements
The authors are grateful to Dr E. Rochelle-Newall for the
DOC measurements and the technical support on Chl a
analyses. Total nitrogen and phosphorus analyses were
made with the help of Dr D. Munaron and G. Messiaen from
the Ifremer laboratory (Sète). We thank Dr M. Agis for the
preparation of TEM grids, and the Service de Microscopie
électronique et analytique (UM2) for technical support. We
acknowledge HJH and RC for editing earlier versions of this
manuscript. This work was supported by the French ANR
Aquaphage 07 BDIV 015-02.
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