Deteksi Virus Bakteri Dengan Metode Plaque
Deteksi Virus Bakteri Dengan Metode Plaque
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correlation, suggesting that continuous induction events lytic infections. However, to our knowledge, there is no in
aliment the pool of visibly infected cells (Bettarel et al., situ evidence linking environmental variability and phage
2008; Maurice et al., 2010); and finally others have replication cycles so far.
reported no correlation between these cycles (Boras Mediterranean lagoons are shallow water bodies
et al., 2009). These results highlight our limited under- subject to major periodic nutrient inputs from human
standing of the natural processes regulating phage repli- activities [agriculture, extensive urbanization (Castel
cation cycles. et al., 1996; Ifremer, 2008)], as well as irregular and
Changes in environmental conditions can alter viral strong periods of rainfall in autumn and spring, followed
dynamics and replication cycles (reviewed by Wommack by long periods of dryness in summer. These systems
and Colwell, 2000; Weinbauer, 2004), but it is unclear thus go through great temporal and spatial environmental
whether this is due to the nature of the environmental changes, differing in their amplitude and nature, and
perturbation, its amplitude and/or frequency, or through its thereby constitute model ecosystems to study the impor-
impact on the prokaryote host. In addition, a given vari- tance of environmental variability in phage replication
able can affect the lytic and lysogenic cycles in dissimilar cycles. Over the course of 1 year, we sampled three
ways. For example, high growth rates generally associ- Mediterranean coastal lagoons characterized by contrast-
ated with lytic replication can also correspond to an induc- ing annual variability in: salinity; dissolved organic carbon
tion signal for the prophage, in order to maximize viral (DOC); total nitrogen (TN) and phosphorus (TP); and
production (Gottesman and Oppenheim, 1994; Paul, chlorophyll a (Chl a) concentrations. These variables are
2008). Similarly, low levels of prokaryote activity usually all recognized for their impact on prokaryote physiology
associated with lysogeny can also lead to the induction of (del Giorgio and Bouvier, 2002; Hewson and Fuhrman,
the prophage before the host’s lysis and loss of the viral 2004) and viral replication cycles (Cochran and Paul,
genome (Clarke, 1998; Maurice et al., 2010). These dis- 1998; Weinbauer, 2004; Bettarel et al., 2009). We aimed
crepancies emphasize that the variables regulating the to determine: (i) the type of pattern (negative/positive
lysogenic and the lytic replication cycles in aquatic correlation or no pattern) occurring between the lytic and
systems remain poorly understood. lysogenic cycles in coastal lagoons, and (ii) how environ-
Observing the dynamics of phage replication cycles in mental variables, the host’s physiology, and their variabil-
natural systems with strong environmental variability ity over short (seasonal) and long (annual) timescales
would provide an initial framework to characterize the might explain the patterns of occurrence and proportions
relationship between environmental variability and phage of both cycles.
replication cycles. These systems harbour prokaryote
communities that are very diverse in their physiology, Results
activity and community structure (as observed in estuar-
Environmental variables and annual variability
ies or coastal waters; del Giorgio and Bouvier, 2002;
Riemann and Middelboe, 2002; Hewson and Fuhrman, Three coastal Mediterranean lagoons, Leucate, Bages
2004). Constant fluctuations in the host physiology and and Gruissan, were sampled every 3 weeks, and the
activity could constitute unfavourable conditions for the means and ranges of values of all environmental
phage communities and promote the establishment of variables measured are reported in Table 1. Mean
lysogeny (Paul, 2008). Alternatively, these environmental annual DOC concentrations were significantly different
fluctuations could promote inducing events and high rates among lagoons (ANOVA, F = 5.060, P = 0.011; Table 1),
of successful viral and host encounters, thereby favouring 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)
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
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
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)
© 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% significant correlation between cycles (Pearson correla-
in Bages, and were not statistically different (ANOVA, tion, r = -0.040, P > 0.05).
F = 2.837, P > 0.05).
We observed two types of patterns between the lytic
Environmental factors and phage replication strategies
and lysogenic replication cycles (Fig. 4). Considering only
the sampling dates with detectable mitomycin C-inducible At the annual scale, considering all 15 sampling dates in
lysogens, we report a negative, non-significant trend with the three lagoons, we did not observe any significant
substantial scatter of FLC and FVIC values (Pearson cor- relationship between the FLC or FVIC and the index of
relation, r = -0.399, P > 0.05), in agreement with a pattern annual environmental variability based on the coefficient
of replacement between cycles and predominance of one of variation of DOC, Chl a, TN, TP, dissolved O2 concen-
cycle over the other. When including the sampling dates trations and salinity (Model II regression, P > 0.05). There
where lysogens are below the detection limit, FLC values was a significant relationship between FVIC and the
are distributed across the entire FVIC range without any 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
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
Bacteriophage replication strategies in lagoons 2471
© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475
2472 C. F. Maurice, C. Bouvier, R. Wit and T. Bouvier
SYBR®GreenI (10 000¥) for 15 min at room temperature in of fixed sample (formaldehyde, 2% final concentration) were
the dark (del Giorgio et al., 1996). Stained prokaryotes then centrifuged using an AirFuge® Ultracentrifuge (Beckman
were discriminated and enumerated by using right angle light Coulter) at 30 000 g for 10 min at 20°C on duplicate 400-
scatter (SSC) and green fluorescence (FL1). mesh copper formvar, carbon-coated electron microscope
We used two different cellular characteristics, membrane grids (Delta Microscopies). Samples were then stained with
polarity and respiratory enzyme activity, to describe the physi- uranyl acetate (final concentration 0.5%) for 20 s and rinsed
ological state of the prokaryote cells and their level of activity. twice in distilled water. At least 1000 prokaryote cells per
Depolarized cells were identified using the anionic membrane grid were observed at 40 000¥ magnification, and at an
potential-sensitive dye DiBAC4 (Molecular Probes, 1 ml in acceleration voltage of 120 kV. A cell was considered visibly
0.5 ml sample, 10 min incubation). DiBAC4 accumulates infected when five virus-like particles could be seen in its
within cells that have lost their membrane potential (Novo cytoplasm (Weinbauer and Hofle, 1998; Bettarel et al.,
et al., 2000), and will consequently show bright green fluo- 2004).
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- Data treatment and statistical analyses
2,3-ditolyl tetrazolium chloride (CTC), an indicator of the res-
The contact rate between the prokaryotic hosts and viruses
piratory electron transport system activity (Sherr et al., 1999).
was calculated using the mathematical model from Murray
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 and Jackson (1992): R = (Sh 2p d Dv) ¥ VP; where R is the
(5 mmol l-1 final concentration) and incubated for 3 h at room number of virus attached ml-1 s-1, Sh is the Sherwood number
temperature in the dark. Active cells reduce the tetrazolium (1), d is the prokaryote cell diameter (0.42 mm), Dv is the
salt CTC into its red fluorescent formazan detectable by FCM diffusivity of virus (eqn 9 in Murray and Jackson, 1992), and
(del Giorgio et al., 1997; Sieracki et al., 1999). V and P are the virus and prokaryote abundance respectively.
The proportions of the different physiological categories We used the average prokaryote cell size determined by
(CTC+ and DiBAC+) were calculated relative to the total epifluorescence microscopy in Thau lagoon (n = 1800 cells;
prokaryote counts obtained by SYBR®GreenI staining. Fluo- measured in January, April and August); whereas the average
rescent latex beads (0.94 mm diameter, Polysciences) were viral diameter was set to 45 nm, based on data from coastal
systematically added to each sample as internal standard for systems (Weinbauer, 2004).
cell counts, cell fluorescence and light scatter measure- To determine the effect of the annual ecosystem environ-
ments. Bead stock solution concentration was determined by mental variability on phage replication cycles, we calculated
epifluorescence microscopy prior to every FCM analysis. All an index of annual variability encountered within each lagoon
data were obtained and analysed with the CellQuest Pro by summing the coefficients of variation (CV) of the DOC, Chl
software (BD Biosciences). 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
Frequency of lysogenic cells (FLC)
lagoons, this global annual index of variability could mask the
The proportion of mitomycin C-inducible lysogens, classically variability of environmental variables occurring at seasonal
referred to as the frequency of lysogenic cells (FLC), was scales (Winter: February 2008–March 2008 and December
obtained with triplicate incubations of untreated samples 2009–January 2009; Spring: March 2008–June 2008;
(control incubations) and samples treated with the most com- Summer: June 2008–September 2008; Fall: September
monly used chemical inducer, mitomycin C (1 mg ml-1 final 2008–December 2008). Thus, we also calculated the sea-
concentration; Sigma). All systems sampled were alkaline, sonal coefficients of variation of the DOC, Chl a, TN, TP,
precluding the instability of this antibiotic under acidic condi- dissolved O2 concentration and salinity, as well as the sea-
tions (Windholz et al., 1976). Preliminary 24 h time-series sonal coefficients of variation of the prokaryote physiological
incubations indicated that incubations longer than 17 h were categories measured (CTC+ and DiBAC+ cells), and com-
unnecessary. Three subsamples (6 h, 8 h and 17 h) of these pared this seasonal level of variability to the seasonal mean
incubations were fixed with formaldehyde (2% final concen- values of FLC and FVIC for the lysogenic and lytic cycles
tration for prokaryote and viral enumeration, as detailed respectively (see above).
previously). Induction took place when we observed simulta- Statistical analyses were made using the JMP 5.01 soft-
neously significantly (P < 0.05) lower prokaryote abundance ware (SAS institute). All data were checked for normality.
and significantly higher viral abundance in the mitomycin C The homoscedasticity of the variances of the abiotic and
treatment relative to controls (Cochran and Paul, 1998; biotic variables among lagoons were tested with Bartlett’s
Tapper and Hicks, 1998). To calculate the frequency of lys- test, and one-way ANOVAs were performed. A posteriori
ogenic cells (FLC), we used the average published value of pairwise comparisons of means between lagoons were
burst size of 24 acquired from studies in marine systems made using Tukey-Kramer’s post-hoc analysis (alpha
(Parada et al., 2006). When induction was not detected, we level = 0.05). One-way ANOVAs were performed between
considered that FLC = 0. control and mitomycin C treatment samples at each time
point to determine whether induction took place. We also
Frequency of visibly infected cells (FVIC) used a full factorial analysis of variance (ANOVA) to identify
the combined effects of prokaryote abundance and Chl a
The frequency of visibly infected cells FVIC was determined concentration on viral abundance. We used correlation
by transmission electron microscopy (TEM). Briefly, 500 ml analyses to test the links between FVIC and FLC, and
© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 15, 2463–2475
Bacteriophage replication strategies in lagoons 2473
performed regression analyses in order to test whether the etry to enumerate marine viruses stained with SYBR gold.
FLC and FVIC were dependent of abiotic and biotic factors. Appl Environ Microbiol 67: 539–545.
Because both variables were not fixed but scattered ran- Chibani-Chennoufi, S., Bruttin, A., Dillmann, M.L., and
domly, we applied Model II reduced major axis regression Brüssow, H. (2004) Phage-host interaction: an ecological
(Sokal and Rohlf, 1995). perspective. J Bacteriol 186: 3677–3686.
Clarke, K.J. (1998) Virus particle production in lysogenic bac-
teria exposed to protozoan grazing. FEMS Microbiol Lett
Acknowledgements 166: 177–180.
Cochran, P.K., and Paul, J.H. (1998) Seasonal abundance of
The authors are grateful to Dr E. Rochelle-Newall for the lysogenic bacteria in a subtropical estuary. Appl Environ
DOC measurements and the technical support on Chl a Microbiol 64: 2308–2312.
analyses. Total nitrogen and phosphorus analyses were Colombet, J., Sime Ngando, T., Cauchie, H.M., Fonty, G.,
made with the help of Dr D. Munaron and G. Messiaen from Hoffman, L., and Demeure, G. (2006) Depth-related gradi-
the Ifremer laboratory (Sète). We thank Dr M. Agis for the ents of viral activity in Lake Pavin. Appl Environ Microbiol
preparation of TEM grids, and the Service de Microscopie 72: 4440–4445.
électronique et analytique (UM2) for technical support. We Davidson, A.T., Thomson, P.G., Westwood, K., and van den
acknowledge HJH and RC for editing earlier versions of this Enden, R. (2004) Estimation of bacterioplankton activity in
manuscript. This work was supported by the French ANR Tasmanian coastal waters and between Tasmania and Ant-
Aquaphage 07 BDIV 015-02. arctica using stains. Aquat Microb Ecol 37: 33–45.
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