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Microb Ecol (2016) 72:347358

DOI 10.1007/s00248-016-0782-0

ENVIRONMENTAL MICROBIOLOGY

Functional Responses of Bacterioplankton Diversity


and Metabolism to Experimental Bottom-Up and Top-Down
Forcings
A. S. Pradeep Ram 1 & S. Chaibi-Slouma 1 & J. Keshri 1,2 & J. Colombet 1 & T. Sime-Ngando 1

Received: 23 March 2016 / Accepted: 4 May 2016 / Published online: 14 May 2016
# Springer Science+Business Media New York 2016

Abstract We conducted an experimental approach using structure which was examined using 16S ribosomal
microcosms to simultaneously examine the functional re- RNA (rRNA) gene sequencing by Illuminas Miseq plat-
sponse of natural freshwater bacterial assemblages to the form. Chao estimator and Shannon diversity index values
impact of resources (nutrients) and top-down factors (vi- suggested that bacterial species richness was highest in
ruses and grazers) on bacterial physiological state and the presence of both the top-down factors, indicating a
their community structure. Addition of organic and inor- tighter control of bacterioplankton dominants within a rel-
ganic nutrients led to the proliferation of high nucleic acid atively stable bacterial community. The increase in bacte-
content bacterial cells accompanied by high bacterial rial metabolism with nutrient addition followed by subse-
growth efficiency (considered as proxy of bacterial carbon quent lysis of bacterial dominants indicate that both re-
metabolism) estimates, suggesting that this subgroup rep- sources and top-down factors work in concert for the sus-
resented the most active fraction of bacterial community tenance of stable bacterial communities.
and had a high capacity to incorporate carbon into its
biomass. However, their rapid growth induced the pres- Keywords Virusbacteria interaction . Bacterial
sure of viral lytic infection which led to their lysis toward physiological state . Bacterial community . Freshwater lake .
the end of the experiment. In microcosms with flagellates Microcosm
plus viruses, and with viruses alone, the selective removal
of metabolically active high nucleic acid cells through
viral lysis benefitted the less active low nucleic acid con- Introduction
tent cells, perhaps via the use of lysis products for its
growth and survival. Changes in bacterial physiological Bacterioplankton communities harbor a significant frac-
state in microcosms were reflected in their community tion of global genetic diversity in aquatic systems where
they play a vital role in the flow of energy and nutrients
through plankton food web [1]. Due to intrinsic complex-
Electronic supplementary material The online version of this article ity of bacterial diversity, knowledge on the factors con-
(doi:10.1007/s00248-016-0782-0) contains supplementary material,
which is available to authorized users.
trolling their community composition and activity are cen-
tral to the understanding of their ecological and biogeo-
* A. S. Pradeep Ram chemical roles in aquatic systems and therefore have been
Pradeep_Ram.ANGIA_SRIRAM@univ-bpclermont.fr the subject of a large number of studies in recent years [2,
3]. In aquatic systems, homogenous in time and space, at
1
least two extreme mechanisms are known for biodiversity
Laboratoire Microorganismes: Gnome et Environnement, UMR
CNRS 6023, Universit Blaise Pascal, 1 Impasse Amlie Murat,
control of bacteria, which include bottom-up (nature of
63178 Aubire, Cedex, France substrates and water chemistry) and top-down mecha-
2
Present address: Institute of Postharvest and Food Sciences,
nisms (protistan grazing and viral lysis). Studies carried
Agricultural Research Organization, Volcani Centre, Bet out in the past two decades have shown that viral infec-
Dagan 50250, Israel tions are another major mechanisms of bacterial mortality
348 A. S. Pradeep Ram et al.

and can destroy approximately 20 to 40 % of daily bac- Materials and Methods


terial production, comparable with the rates of grazing by
bacterivorous protists in freshwater environments [4, 5]. Laboratory experiment was conducted using water sam-
With viral infection being more specific (Bspecies^ level) ples from Lake Aydat located in the French Massif
than protistan grazing (which is not considered species- Central (45 39 48 N, 02 59 04 E; see Pradeep Ram
selective), the total number of organisms in the commu- et al. [13] for detailed site description and characteristics).
nity would be controlled by grazing, while the loss to lytic For microcosm experiment, triplicate water samples were
viruses would determine the size of each host group and, collected with a Van Dorn bottle from a fixed depth
thus, diversity [6]. Both viruses and protists through their (0.5 m) at the deepest central point of the lake on 2
mortality processes can strongly alter the proportion of February 2015 at 09:00 h by filling 20 l (3 replicates)
active to inactive cells in a natural bacterial assemblage, capacity insulated carboys which had been previously
that can influence two key bacterial metabolic pathways cleaned with 1.2 N HCl and rinsed three times with
namely production and respiration which are controlled Mill-Q and lake water. Water samples were transported
by bacterial growth efficiency (considered as a proxy of to laboratory within 1 h from the time of collection, where
bacterial carbon metabolism). they were processed immediately.
In aquatic systems, both top-down and bottom-up fac-
tors work in concert and are difficult to separate both Experimental Design and Setup
experimentally and conceptually. In a hypothetical envi-
ronment, both control mechanisms can have contrasting For the experiments, natural water samples were pre-
effects and impact bacterial diversity with interspecific filtered through a succession of nylon fiber sieves (150,
competition lowering community diversity and predation 63, and 25 m porosities) to remove larger eukaryotes
sustaining community diversity and richness [7, 8]. So far, and particles. The filtrate represented the Btotal fraction^
microcosms studied in manipulated systems using filtra- which comprised bacteria, viruses, and heterotrophic
tion and dilution experiments have been conducted sepa- nanoflagellates (BVF). The total fraction was sequentially
rately to either determine the impact of viruses on bacte- filtered through two membrane barriers (3 and 1 m pore
rial community composition [9] or on the regulation of size cellulose acetate filters, Sartorius) under attenuated
bacterial carbon metabolism [10]. Experimental investiga- light conditions and low differential pressure (<50 mm
tions on the top-down mechanisms especially viruses reg- Hg), to yield a grazer-free fraction which contained bac-
ulating bacterial metabolism and structure has never been teria and viruses only (BV), confirmed by microscopic
performed. Henceforth, the impact of viruses on the above examination. One part of the grazer-free fraction received
aspects remains unclear. Our previous field investigations combination of organic and inorganic nutrients (BVN)
carried out in a set of freshwater systems has shown the which included carbon (glucose 125 M C), nitrogen (so-
existence of antagonistic relationship between viruses and dium nitrate 25 M N), and phosphate (sodium
bacterial metabolism [11], but direct experimental evi- dihydrogen phosphate 5 M P) sources. The experimental
dence is lacking. We hypothesize that top-down mecha- samples involving three different treatments (i.e., BVF,
nism especially viruses can impact bacterial metabolism BV, and BVN) were distributed in 5-L capacity polycar-
through selective lysis of active cells which subsequently bonate bottles previously washed with 1.2 N HCl and
lead to the alteration of bacterial community composition. rinsed three times with Milli-Q water and with the appro-
To test this hypothesis, experimental verification through priate experimental samples. All treatments were prepared
time series laboratory microcosms experiment was carried out in triplicates (3 treatment 3 replicates). The experimen-
to investigate the impact of top-down and resource factors on tal samples were incubated in the dark at 18 1 C after a
bacterial physiological state and carbon metabolism effectuat- brief acclimatization for a 2-h period. The chosen temper-
ed through alteration in bacterial community structure. In the ature condition corresponds to the summer season of the
present study, we investigated simultaneous impact of preda- lake with an objective to evoke significant variation in
tors and resources on bacterial physiological state, metabo- microbial characteristics. Subsamples from the treatments
lism, and their community structure through time course were taken from each replicates at 0, 24, 48, 72, 96, and
changes in different microcosms with or without addition of 120-h time periods and analyzed for microbial parameters.
nutrients. In order to better evaluate the complex intercommu- Precautionary measures were taken to avoid contamina-
nity and intracommunity response to environmental variables, tion of the experimental samples. All containers, filtering
bacterial composition and diversity at each time point during devices, glassware, and tubing coming into contact with
the experimental study was followed by adopting high- experimental samples had been acid-washed (10 % hydro-
throughput DNA sequencing approach using Illumina chloric acid) and thoroughly rinsed with deionized water
MiSeq Sequencing platform [12]. prior to use.
Functional Responses of Bacterioplankton Diversity and Metabolism 349

Bacterial and Viral Abundances calculated as differences in oxygen concentration between


initial and final replicate bottles, over the duration of the in-
Samples (1 ml each) for counting abundances of bacteria (BA) cubation. We used a factor of 0.375 to convert from oxygen to
and viruses (VA) was fixed with paraformaldehyde (1 % final carbon units assuming a respiratory quotient of 1.
concentration) and kept in dark at 4 C for 30 min. Bacterial growth efficiency (BGE) was calculated as BP /
Abundances were determined using a FACS Calibur flow (BP + BR) and expressed as percentage.
cytometer (Becton Dickinson, Franklin Lake, NJ, USA)
equipped with an air-cooled laser providing 15 mW at Viral Lytic Infection
488 nm with the standard filter setup as described by
Brussaard et al. [14]. Briefly, extracted samples were diluted Bacterial cells contained in formalin-fixed water samples (fi-
with 0.2-m pre-filtered TE buffer (10 mM TrisHCl and nal conc. 2 % v/v) were collected on triplicate electron micro-
1 mM EDTA, pH 8) and stained with SYBR Green I (10, scope grids (400-mesh, carbon-coated Formvar film) by ultra-
000-fold dilution of commercial stock, Molecular Probes, centrifugation (Optima LE-80K, Beckman Coulter SW40 Ti
Oregon, USA). Mixture was incubated for 5 min, heated for Swing-Out-Rotor at 70,000g for 20 min at 4 C) according to
10 min at 80 C in the dark, and cooled for 5 min prior to Pradeep Ram et al. [13]. Each grid was stained at room tem-
analysis. Bacteria and viruses differing in fluorescence inten- perature (ca. 20 C) for 30 s with uranyl acetate (2 %, pH = 4),
sity were detected by their signature in a side scatter versus rinsed twice with 0.02 m filtered distilled water to remove
green fluorescence (530 nm wavelength, fluorescence channel excess stain, and dried on filter paper. Grids were examined
1 of the instrument) plot. Flow cytometry list modes were using a JEOL 1200Ex transmission electron microscope
analyzed using CellQuest Pro software (BD Biosciences, ver- (TEM) operated at 80 kV and a magnification of 20,000 to
sion 4.0). A blank was routinely examined to control for con- 60,000 to distinguish between bacterial cells with and with-
tamination of the equipment and reagents. out intracellular viruses. A bacterium was considered infected
Based on the differences in the individual cell fluorescence when at least five viruses, identified by shape and size, were
(related to nucleic acid content) and in the side and forward clearly visible inside the host cell. At least 400600 bacterial
light scattering signal on the cytogram, at least two major cells were inspected per grid to determine frequency of visibly
bacterial subgroups were distinguished: cells with low nucleic infected bacterial cells (FVIC). FVIC counts were converted
acid content (LNA cells) and cells with high nucleic acid to the frequency of infected cells (FIC) using the following
content (HNA cells) [15]. Cell abundances for each of these formula: FIC = 9.524FVIC 3.256 [16].
groups and their contributions in the whole bacterial commu-
nity were determined. Metagenomic DNA Extraction

Bacterial Production and Respiration For metagenomic DNA extraction, bacteria from the experi-
mental samples were collected onto 0.2-m white polycar-
Bacterial production (BP) was derived from their specific bonate membrane filters (Sartorius, Germany) by applying
growth rate (). Subsamples for bacterial abundance were low vacuum, and community DNA were extracted using
collected regularly over a 24-h incubation period. The value Fast DNA spin kit for soil (MP Biomedicals, Germany).
of was then calculated using the least-squares method as the Total DNA was extracted from filters using two consecutive
slope of the linear regression analysis of natural logarithmic bead-beating steps, first, for 40 s, followed by a second bead
bacterial abundance. Bacterial production was then calculated beating 60 s to disrupt additional, previously unlysed cells.
as the product initial bacterial abundance. In order to Remaining steps were followed as per manufacturers proto-
obtain bacterial production in carbon equivalents, bacterial col. The extracted genomic DNA was quantified, checked for
carbon content of 20 fg cell1 was used as a constant conver- purity at A260/280 nm by NanoDrop spectrophotometer
sion factor. (Thermo Scientific, USA) and stored at 20 C.
Bacterial respiration (BR) was estimated from the con-
sumption of dissolved oxygen concentration in filtered water PCR Amplification of 16S rRNA Gene and Sequencing
samples as described previously [11]. Water samples (in trip-
licates) from BV and BVN treatments were carefully trans- PCR amplification was carried out to amplify V3V4 con-
ferred to calibrated borosilicate glass bottles (150-ml served regions of bacterial 16S ribosomal RNA (rRNA) gene
capacity) by a sipper system to avoid the formation of air sequences in triplicate using the 16S rRNA gene specific
bubbles. Dissolved oxygen levels were measured by primers (forward primer 5 -CTTTCCCTACACGACGCT
Winklers method based on endpoint detection before (T0) CTTCCGATCTACGGRAGGCAGCAG-3 and reverse 5 -
and after the incubation (T24, T48, T72, T96, and T120) of GGAGTTCAGACGTGTGCTCTTCCGATCTTACCAGG-
the experimental water samples. Respiration rates were GTATCTAATCCT-3 ) [17]. These primers include the
350 A. S. Pradeep Ram et al.

Illumina adapter overhang nucleotide sequences as well as Physico Chemical Parameters


V3V4-specific sequences producing an amplicon of 460 bp
in length. The PCR library preparation was carried out using Water temperature and dissolved oxygen profiles were deter-
KAPA Hi Fi Hot Start Ready-Mix PCR Kit (KAPA mined in situ using an YSI Pro ODOTM probe (Yellow
BIOSYSTEMS, USA). Briefly, each 25 l of PCR reaction Springs, Ohio, USA). Phytoplankton biomass was estimated
contains 10 ng l1 (1 l) of genomic DNA template, 12.5 l in situ as g equivalent chlorophyll-a l1 (Chl) using a fluo-
2 Mastermix KAPA Hi Fidelity DNA polymerase (1 U), rescence photometer (Fluoroprobe; BBE-Moldaenke, Kiel,
10 M each primer, and nuclease-free water. PCR reactions Germany). The device was calibrated for the studied system,
were carried out with initial denaturation step at 95 C for so as to minimize the likely errors associated with its measure-
3 min followed by 24 cycles of 98 C for 30 s, 65 C for ments. Total organic carbon concentrations were determined
30 s, and 72 C for 30 s and ended with an extension step at by high-temperature catalytic oxidation method (680 C)
72 C for 5 min. The PCR products were confirmed by 1.5 % using a TOC analyzer (Shimadzu TOC-V CPN, Japan) [24]
agarose gel electrophoresis. Amplicons from respective sam- and total dissolved nitrogen using the same analyzer with an
ples were pooled together and sequenced (paired-end at a attached measuring unit [25]. Concentrations of organic car-
length of 300 nucleotides in each direction) on the Illumina bon and nitrogen were obtained from five-point potassium
MiSeq platform at the INRA, Toulouse, France. Sequence hydrogen phthalate and potassium nitrate standard calibration
data was received as.fastq files and was submitted to the curve, respectively. All reported values were corrected for the
National Center for Biotechnology Sequence Read Archive instrument blank, and the CV was <5.0 %.
under Bio Project accession number SRX 1565314 to SRX
1565329.
Statistical Analysis
Sequence Data Analysis
Differences in microbial parameters between treatments were
analyzed by one-way ANOVA. A significant response was
Sequence processing was performed using Mothur software,
considered to be at p < 0.05. Potential relationships among
version 1.36.1 [18]. The paired-end MiSeq Illumina reads
various measured variables were tested by Pearsons correla-
from the 16 studied samples were aligned and converted to
tion analysis. All statistical analyses were performed using
contigs yielding 586,968 reads. Sequence analysis of each
Minitabs software for Windows (Release 17, Minitab).
combined single fasta file was processed using the Mothur
Miseq SOP accessed on the 10 December 2015 [19].
Sequences were quality checked and denoised using the
trim.seqs command. Sequences having ambiguous characters, Results
homopolymers longer than 8 bp, were removed. High-quality
sequences were aligned against the Mothur version of SILVA In Situ Characteristics of the Sampled Site
bacterial reference sequences [20], and the sequences which
were not aligned to a reference alignment of the correct se- The in situ physiochemical and biological characteristics of
quencing region were also removed from the analysis. Unique the environmental samples are summarized in Table 1. The
sequences and their frequency in each sample were identified, within-sample variability for all variables was judged satisfac-
and then, a pre-clustering algorithm was used to further tory with coefficients of variation of <15 % and standard de-
denoise sequences within each sample [21]. Singletons were viations mostly lower than detection limits [26]. The samples
removed from the dataset, and chimera removal was per- for laboratory experiment which originated from Lake Aydat
formed using the UCHIME algorithm [22]. Operational taxo- during the well-mixed winter period were marked by in situ
nomic units (OTUs) was assigned at 97 % identity, and clas- temperature (2.7 0.1 C) and dissolved oxygen concentra-
sification of the high quality 265,304 sequences was done by tion (11.8 0.2 mg l1) that was typical for this time of the
the Mothur version of Bayesian classifier with the RDP train- year. Among the parameters, total organic carbon, total nitro-
ing set version 14 having 10,244 bacterial and 435 archaeal gen, and chlorophyll concentrations were indicative of lake
16S rRNA gene sequences [23]. Rarefaction curve was gen- trophic status. During the study period, VA (4.1
erated to examine the adequacy of the sampling depth for 0.2 107 ml1) was an order of magnitude higher than bac-
OTUs formed at the level of genus. After normalizing the teria (3.4 0.4 106 cells ml1) with a virus to bacteria ratio of
number of sequences in each sample (based on rarefied or 12.1. Among the bacterial subgroups, the LNA cells dominat-
subsampled data. i.e., the minimum number of remaining se- ed HNA cells by 4-fold. Low bacterial production (7.9
quences in any of the samples); alpha diversity was assessed 1.5 g C l1 day1) coinciding with high bacterial respiration
by calculating the richness estimator (Chao1, Ace, Jackknife) (93.0 10.2 g C l1 day1) resulted in low bacterial growth
and the diversity indices (Shannon and Simpson). efficiency estimate (7.8 1.2 %) at in situ conditions.
Functional Responses of Bacterioplankton Diversity and Metabolism 351

Table 1 Mean (SD for triplicates) surface water characteristics of treatments. Their abundance increased from T0 in all the ex-
Lake Aydat during the experimental study
perimental microcosms, but their maxima varied with differ-
Parameters (units) Values ent incubation time period (Fig. 1). VA increased progressive-
ly with time and their values roughly doubled at the end of the
Water temperature (C) 2.7 0.1 experiment when compared to T0 irrespective of the treat-
Dissolved oxygen (mg l1) 11.8 0.2 ments (Fig. 1). Viruses were found to increase sharply follow-
Total organic carbon (mg l1) 6.0 0.4 ing BA maxima suggesting preferential viral lysis in BV and
Total nitrogen (mg l1) 1.6 0.3 BVN and together with flagellates in BVF treatments. The
Chlorophyll a (g l1) 13.5 0.3 decrease in BA after their maxima at T24 and T48 in BVF
6 1
Bacterial abundance (10 cells ml ) 3.4 0.4 and BV respectively could be explained by the presence of the
Alphaproteobacteria (%) 4.5a type of predators in these treatments. Addition of nutrients
Betaproteobacteria (%) 33.7a (carbon, nitrogen, and phosphorus source) provoked a strong
Gammaproteobacteria (%) 14.6a and significant (ANOVA, p < 0.05) increase in BA with a max-
Bacteriodetes (%) 9.9a ima at T96 (8.3 106 cells ml1) in BVN treatment. In this
Actinobacteria (%) 36.5a treatment, BA increased by 258 % compared to T0 which
Bacterial production (g C l1 day1) 7.9 1.8 corresponded to a specific growth rate of 0.24 days1. In the
Bacterial respiration (g C l1 day1) 93.0 10.1
Bacterial growth efficiency (%) 7.8 1.1 9.0 LNA HNA BA VA 2.8
BVF 1
Viral abundance (107 ml1) 4.1 3.2 8.0
2.4
2
Virus to bacteria ratio 12.1 1.8 7.0
2.0
Heterotrophic nanoflagellate abundance (103 cells ml1) 2.9 0.2 6.0
Frequency of infected bacterial cells (%) 13.0 2.2 5.0 1.6

a 4.0 1.2
Percentage of total sequences
3.0
0.8
Effect of Filtrations by Size Fractionation 2.0
0.4
1.0

Pre-filtration and sequential filtration of experimental samples 0.0 0.0


did not bring about substantial reduction (<10 %) in bacterial 9.0 2.8
BV
or viral variables under study. At the start of the experiment 8.0
2.4
BA,LNA,HNA (x 106 cells ml-1)

(T0), the initial bacterial abundance in the total and grazer-free 7.0
fraction was 3.4 0.2 106 and 3.2 0.1 106 cells ml1, re- 6.0
2.0

VA (x 107 ml-1)
spectively. Filtration did not bring about significant changes in 5.0 1.6
LNA/HNA ratio with former dominating the latter subgroup
4.0 1.2
by 4-fold at the start of the experiment. Initial experimental
3.0
differences in FIC values (11 %) among the treatments were 0.8
2.0
not significant. At the start of the experiment, alpha, beta, and
0.4
gammaproteobacteria belonging to phyla Proteobacteria 1.0

(53 %), Actinobacteria (36.5 %), and Bacteriodetes (9.9 %) 0.0 0.0

were the dominant bacterial groups in the experimental sam- 9.0 2.8
BVN
ples. Overall at T0, our experimental manipulations yielded 8.0
2.4
no marked bias apparent at our level on the variables under 7.0
2.0
study. 6.0
5.0 1.6

Effects of Top-Down Factors and Resources on Bacterial 4.0 1.2


Parameters 3.0
0.8
2.0
0.4
The results presented here correspond to mean value 1.0
(triplicates) of different incubation times (T0, T24, T48, 0.0 0.0
0 24 48 72 96 120
T72, T96, and T120). Increased temperature evoked positive Time (h)
response in bacterial and viral parameters in experimental Fig. 1 Time course changes in the abundances of bacteria (BA), viruses
treatments. BA was more dynamic than VA and showed pro- (VA), high nucleic acid (HNA), and low nucleic acid content cells (LNA)
nounced variability with respect to different experimental in different experimental treatments. Error bars represent SE (n = 3)
352 A. S. Pradeep Ram et al.

latter part of the time period, viruses continued to increase was not found to limit bacterial growth and activity till the end
despite in the decrease in BA which resulted in higher VBR of the experiment. In this treatment, LNA cells were more
values when compared to BVN (Fig. 2). conservative and did not respond to nutrient addition. The
Among the bacterial subgroups, LNA cells generally ex- positive response of HNA to nutrient addition with a specific
plained for the fluctuations in BA in BVF and BV treatments. growth rate of 0.6 day1 suggested that this fraction of bacte-
LNA dominated HNA cells throughout the experimental time rial community was more active than LNA fraction and that
period with the ratio LNA/HNA ranging from 2.8 to 4.5 there could exist two different communities with varying
(Fig. 1). Although LNA cells exerted its dominance, the peak physiological characteristics.
in BA in BVF and BV coincided with the slight increase in Because of the interference of flagellates in respiration
HNA cells. The peak in VA resulted in the decrease in LNA measurements, BGE was calculated only for BV and BVN
cells by 50 % without any changes in HNA. In sharp contrast treatments. BP calculated from specific growth rate () using
to the above, the highest proportion of HNA cells was found the least-squares method as the slope of the linear regression
in BVN which increased exponentially and dominated LNA analysis of natural logarithmic bacterial abundance was sig-
cells at T48 (HNA/LNA = 1.2) to T96 (HNA/LNA = 1.9) nificantly higher (p < 0.001) in BVN (25.9 g C l1 day1)
which was followed by drastic decrease toward the end of than BV (14.7 g C l1 day1) treatments. Overall, BR was
the experiment (i.e., T120) (Fig. 1). The concentration of significantly higher (p < 0.001) in BV than BVN treatments
added nutrients in BVN treatment was sufficient and therefore and overall ranged from 63 to 267g C l1 day1 (Fig. 3).
BGE was significantly lower (p < 0.001) in BV (6.4 %) than
35.0 FIC VBR 12.0 BVN (28.4 %). Differences in BGE were explained by the
BVF
30.0 10.0
dominance of LNA and HNA cells in BV and BVN treat-
ments, respectively.
25.0
8.0
20.0
6.0
15.0 Viral Lytic Infection
4.0
10.0
Unlike VA, viral lytic infection showed significant trends and
2.0
5.0 marked changes in experimental treatments with progress in
0.0 0.0 incubation time period (Fig. 2). The percentage of viral infect-
ed cells (FIC) was generally high toward latter part of the
35.0 14.0
BV experiment (i.e., T96 and T120) in all the treatments, but the
Frequency of infected cells (%)

30.0 12.0 trend during the progression of experiment differed among


treatments. While FIC increased gradually from T0 to T120
Virus to Baceria ratio

25.0 10.0
in BVF treatment, FIC rose sharply after T72 in both BV (2-
20.0 8.0 fold) and BVN (3-fold) treatments. However, in BVN, signif-
15.0 6.0 icant increase in FIC (314 %) coincided with concomitant
decrease in HNA cells (Figs. 2 and 3). In treatments with no
10.0 4.0
nutrient additions (i.e., BVF and BV), the increase in FIC was
5.0 2.0 positively related to VA (p < 0.001) and VBR (p < 0.001) ratio
0.0 0.0
35.0 12.0 BP BR BGE
BVN 300 35
30.0 10.0
250 30
25.0
BP, BR (g C l-1 d-1)

8.0
25
20.0 200
BGE (%)

6.0 20
15.0 150
4.0 15
10.0
100
5.0 2.0 10
50 5
0.0 0.0
0 24 48 72 96 120
0 0
Time (h) BV BVN
Fig. 2 Time course changes in the virus to bacteria ratio (VBR) and Fig. 3 Bacterial production (BP), respiration (BR), and bacterial growth
frequency of viral infected bacterial cells (FIC) in different efficiency (BGE) estimates in different experimental treatments. Error
experimental treatments. Error bars represent SE (n = 3) bars represent SE (n = 3)
Functional Responses of Bacterioplankton Diversity and Metabolism 353

negatively related to BA (p < 0.01) and LNA (p < 0.01) cells. demonstrates species richness and evenness was highest for
Addition of nutrients to experiment samples resulted in larger BVF (mean = 3.83) and lowest for BVN (mean = 3.22) thus
scatter between variables which ultimately led to weaker or indicating that bacterial diversity is sustained in the presence
insignificant relation among them. High proportion of HNA of both the predators whereas the dominance of specific pop-
cells in BVN enhanced viral lytic pressure which resulted in ulations which are more efficient in exploiting nutrients
higher lysis leading to significant reduction of HNA cell abun- thrived in nutrient-amended treatments (Supplementary
dance toward the end of the experiment. The dominance of Material, Table S1).
less active LNA over active HNA cells in the BVF and BVand
vice versa in the BVN treatment could directly be related to
the impact of lytic viruses. These findings indicated that vi- Bacterial Community Composition
ruses through lytic infection had its marked influence on the
overall physiological state of bacterial community in experi- Incubations of the resident bacterial assemblage in different
mental treatments. treatments yielded significant changes in their composition
with distinct signatures in bacterial community composition
observed at order level (Fig. 4).
Bacterial Species Richness and Diversity in Experimental The bacterial community in the microcosms comprised of
Treatments 16 different phyla along with unclassified bacterial members
with varying abundance (Supplementary Material, Table S2,
Paired-end sequencing of 16S rRNA gene produced a total of S3, and S4). Three phyla (Proteobacteria, Bacteriodetes, and
586,968 raw sequences which after quality filtering, single- Actinobacteria) represented more than 96 % of sequences
tons and chimera removal yielded 265,304 effective se- with the relative abundance of these dominant phyla varying
quences (BVF 97,332; BV 86,854; BVN 81,118) at different at different time points in microcosms. Proteobacteria was the
time points in experimental microcosms. These effective se- most dominant phyla covering 29.9 to 78.4 % (mean = 53.2 %)
quences were normalized for the further analysis of diversity of the sequences followed by Actinobacteria (20.7 %) and
indices (Supplementary Material, Table S1). All of the rare- Bacteriodetes (20.2 %). The values of Chao estimator (151
faction curves which reached saturation and sequencing depth 544) and Shannon index (2.724.38) at different time points in
was considerable enough to cover the whole bacterial diversi- microcosms indicated a marked variability in diversity which
ty (Supplementary Material, Fig. S1). The highest number of resulted in distinct pattern in the occurrence of different taxo-
OTUs was determined by Chao estimator in BV (1995) com- nomical groups (Supplementary Material, Fig. S2). Among
pared to BVF (1969), and BVN (1222) indicated that the BV the treatments, the total number of effective sequences was
had the greatest richness. The Shannon diversity index which higher in BVF than in BV and BVN microcosms. Class

100%

90%

80%
Relative abundance

70%

60%

50%

40%

30%

20%

10%

0%
BVF_0h BVF_24h BVF_48h BVF_72h BVF_96h BVF_108h BV_24h BV_48h BV_72h BV_96h BV_108h BVN_24h BVN_48h BVN_72h BVN_96h BVN_108h

Others Acidimicrobiales Actinomycetales Unclassified Actinobacteria


Bacteroidales Cytophagales Flavobacteriales Sphingobacteriales
Caulobacterales Rhizobiales Rhodobacterales Rhodospirillales
Rickettsiales SAR11 Sphingomonadales Unclassified Bacteriodetes
Burkholderiales Gallionellales Methylophilales Neisseriales
Nitrosomonadales Unclassified Betaproteobacteria Bdellovibrionales Desulfobacterales
Desulfuromonadales Myxococcales Syntrophobacterales Unclassified Deltaproteobacteria
Campylobacterales Unclassified Epsilonproteobacteria Aeromonadales Alteromonadales
Chromatiales Enterobacteriales Gammaproteobacteria_incertae_sedis Legionellales
Methylococcales Pseudomonadales Xanthomonadales Unclassified Gammaproteobacteria

Fig. 4 Time course changes in the relative abundance of dominant bacterial orders in different experimental treatments
354 A. S. Pradeep Ram et al.

Alphaproteobacteria was better represented in BVF with a relative to in situ values [30]. In addition, filtration methods
peak at T48 followed by gradual decline toward the end of which we used to size fractionate the community can obvious-
the experiment. On the other hand, BV and BVN treatments ly alter preypredator interactions through the rupture of mi-
did not show any significant pattern. Among the sequences crobial cells which can eventually alter the concentration and
belonging to class Alphaproteobacteria, the order composition of dissolved organic matter (DOM). In spite of its
Sphingomonadales (5.4 %) was well represented followed limitations, these experimental tools are useful for investigat-
by Caulobacterales (2.6 %) and Rhizobiales (1.3 %). ing how environmental processes induce temporal variations
Actinobacteria showed conspicuous pattern in the presence in bacterial structure, diversity, and activity [9, 31]. In order to
of predators with a peak at T72 and T48 in BVF and BV mechanically understand microbial communities, we chose to
treatment respectively. In BVN, this phylum failed to respond reduce their complexity by excluding larger eukaryotes
to nutrients henceforth gradually declined toward the end of through filtration (size fractionation) and to suppress primary
the experiment. In BV, the late decline in the population of production by dark incubation. Since temperature is known to
Actinobacteria coincided with high infection rates observed limit the microbial growth and its metabolic activity, water
during the same time period. The variation of this class in samples were incubated at chosen temperature corresponding
microcosms was larger attributed to order Acidimicrobiales to the summer season of the lake to evoke significant patterns
and Actinomycetales which accounted to 8.2 and 9.1 % of and variations in microbial characteristics; as a consequence,
the sequences, respectively. Both Betaproteobacteria and we could have introduced a potential source of bias in our
Bacteriodetes showed similar variations with greater response microcosms. In the present study, incubation time (up to
in BVF and BVN than in BV treatments. The class 120 h) coupled with the volume of microcosms (i.e., 5 l)
Betaproteobacteria which represented 29.0 % of total se- was likely to limit confinement effects. This incubation time
quences was largely dominated by the order was realistic compared to the generation time of microorgan-
Burkholderiales. Bacteriodetes represented 20.2 % and was isms and considered sufficient to obtain significant changes in
dominated by order Bacteroidales. Gammaproteobacteria did bacterial and viral parameters, as observed in natural systems.
not show any clear pattern among the microcosms, and over-
all, this class covered 18.4 % of total sequences with order Impact of Top-Down Factors on Bacterial Characteristics
Pseudomonadales accounting 15.5 % of the total sequences.
In experimental treatments, contrasting patterns in bacterial
community characteristics (abundance, physiology, and activ-
Discussion ity) were observed. The nucleic acid content of bacterial cells
(high or low) are often taken as an indicative of physiology
Our study is one among the few to demonstrate the effects of and activity of bacteria, with HNA cells presumably
viral-induced mortality and nutrient enrichment on bacterial representing the more active fraction of the community than
physiological state, community metabolism, and their com- LNA cells [15]. The relative contribution of HNA cells to total
munity structure and strikingly differs from other previous abundance has been used as a proxy for bacterial activity [32]
investigations that have examined the above aspects separate- and is known to contribute for largest share of bacterial pro-
ly in aquatic environments [27, 28]. By adopting a unifying duction in aquatic environments [33]. In our experiment, the
simple experimental approach using microcosms, we were domination of LNA cells in microcosms receiving no nutrient
able to show the strong and significant influences of viral lysis additions (namely BVF and BV) and HNA cells in micro-
on active high nucleic acid content bacterial cells which con- cosms receiving nutrient additions strongly suggests the phys-
tributed to high carbon metabolism, accompanied by changes iology and activity of bacterial assemblage differed between
in their community structure in freshwater microcosms. treatments. The strong increase in bacterial abundance and
Results from our experimental investigations are consistent their growth rate together with high proportion and dominance
with the hypothesis that viral production depends on the of HNA over LNA cells (from 48 to 96-h time period) in BVN
growth of hosts especially the active members which, in turn, treatment suggest that this subgroup was the clear winner in
is regulated by the availability of labile nutrients. competing for resources which are known to have high cell-
specific activity [10]. Such a transition (i.e., LNA to HNA)
Experimental Approach suggests that HNA cells perhaps represent the most metabol-
ically active fraction of the bacterial community, similar to
Experimental manipulations such as those conducted in the previous observations made from freshwater environments
present study are not without its share of problems with re- [34, 35]. Batch culture experiments carried out using estuarine
spect to interpretation of the data and inferences made about in bacterial communities has suggested HNA cell growth dy-
situ processes. Confining microbial populations in containers namics followed the same pattern as the bacterial abundances
can alter both bacterial diversity [29] and cell-specific activity and growth rate thereby proving that %HNA to be an excellent
Functional Responses of Bacterioplankton Diversity and Metabolism 355

predictor of bacterial production [27]. The response of HNA dominant group [44]. Studies have also indicated that the
cells to fluctuations in nutrient conditions may be linked to its fastest growing bacterial group (superior in nutrient competi-
general adaptive process, as this subgroup is shown to appar- tion) was the most susceptible to viral-induced mortality, sug-
ently depend more on labile substrates for its growth and me- gesting the existence of a trade-off between nutrient uptake
tabolism [36, 37]. In contrast to the above, both flagellates and and resistance to viral infection [2, 45]. Our findings are con-
viruses, and viruses alone in BVF and BV treatments. respec- sistent with Bkilling the winner hypotheses^ where the viruses
tively, had a check on the metabolically active members of regulate the bacterial community by allowing the survival of
bacterial communities by preferential selective grazing/lyses less competitive species by repressing the metabolically active
which allowed the growth of slow growing LNA cells to bacteria [46]. It has been reported that in seawater cultures
dominate. without grazers, the fast-growing opportunistic bacteria (e.g.,
Our finding suggests that top-down factors through their Vibrio) could grow explosively upon substrate additions, more
mortality process can strongly control the proportion, occur- quickly than the viruses that may infect them [47]. Such
rence, and distribution of metabolically active component of growth enhancement of HNA cells due to nutrient additions
bacterioplankton. Studies conducted in marine ecosystems might partly explain differential responses of bacteria and vi-
have shown that top-down control can have greater effect on ruses to substrate additions in our study. However, this situa-
the cell-specific activity of substrate active bacterioplankton, tion could still reflect realistic natural conditions, when there
i.e., HNA cells which are known to incorporate leucine into its is a burst of organic matter (e.g., during a sudden algal bloom).
biomass at faster rate and increase in cell size [38]. The dom- The results from our experimental investigation agrees with
inance of LNA cells and limited response of HNA cells in field studies carried out in intertidal mudflats (French Atlantic
microcosms with predators indicate that both grazers and viral Coast) where nutrient resuspension from the sediment to water
infection had a greater impact on HNA than on LNA cells. column during high tide regime supported metabolically ac-
The interaction between viruses and HNA cells in particular tive bacteria which, in turn, enhanced efficient viral turnover
has received less attention compared to interactions between through lytic viral production [28]. Microcosm experiments
HNA cells and protozoan grazers [39]. In our experiment, the conducted in marine bacterial communities have also sug-
significant negative correlation between HNA cell abundance gested that viral production tend to increase with enhancement
and viral infection further supports and strengthens the hy- of productivity of host community with substrate addition
pothesis of our previous field investigation from temperate [48]. Therefore nutritional conditions especially the quality
lakes that the active HNA cells were the principle target for rather than quantity of DOM can alter the existing microbial
viral lysis [35]. processes (bacteriavirus interactions) [10].
Low estimates of bacterial growth efficiency in BV in con-
trast to high estimates in BVN treatment coincided with the Top-Down Impact on Bacterial Diversity
dominance of LNA cells in the former which could be as
consequence of high preferential viral lysis of active HNA Changes in the bacterial physiological state and carbon me-
cells which have high capability for incorporating carbon into tabolism due to viral activity can strongly evoke changes in
its biomass. Similar conclusion has been reported from marine bacterial community structure at different time period in out
[40] and freshwater systems [11, 34] where viruses have experimental treatments. In this study, we applied high-
shown to depress bacterial growth efficiency at community throughput sequencing techniques to sequence 16S rRNA
level. Selective removal of HNA cells can benefit the LNA amplicons using Illumina MiSeq platform to characterize
cell fraction by reducing competition and perhaps via the use and obtain information of greater resolution on microbial
of lysis products for growth. Although we were not able to communities in the microcosms. In the experimental samples,
measure BGE in the presence of grazers due to their interfer- O T U s b e l o n g e d m a i n l y t o A l p h a p ro t e o b a c t e r i a
ence in respiration measurements, it could be speculated from (Sphingomonadales, Caulobacterales, Rhizobiales),
previous published reports that flagellates can also play its role Betaproteobacteria (Burkholderiales), Gammapro
in reducing BGE through selective elimination of active bac- teobacteria (Pseudomonadales), Actinobacteria
terial communities [41, 42]. (Acidimicrobiales, Actinomycetales), and Bacteriodetes
In contrast to other treatments, addition of labile nutrients (Bacteriodales) which are typically observed in freshwater
in BVN resulted in higher BGE estimate which was explained systems [49, 50].
by the proliferation of HNA cells. However, such proliferation Members of Betaproteobacteria, Gammaproteobacteria,
of HNA cells eventually enhanced virus-induced mortality as and Bacteriodetes which responded to nutrient additions indi-
these cells are reportedly known to have high intrinsic rates of cated that these phylotypes may share functional and metabol-
growth and thus more susceptible to viral infection [10, 43]. ic traits such as patterns of substrate utilization or intrinsic
Our results agree with the reports from previous studies where levels of cellular activity and can together contribute for sig-
the presence of viruses resulted in declining abundance of the nificant proportion of active cells [2]. However, these groups
356 A. S. Pradeep Ram et al.

were strongly kept in check in the presence of viruses and DOM quality are crucial in regulating bacterial biomass and
flagellates. The Betaproteobacteria which comprised mainly their community composition as previously hypothesized by
of order Bukholderiales are known to have a broad spectrum Auguet and colleagues [27]. In ecosystems such as lakes and
of lifestyles and ability to outgrow other bacterial groups un- coastal waters, which receive large inputs of DOC and nutri-
der reduced abundance or absence of predators. However, ents, the microbial processes (bacteriavirus interactions)
their vulnerability to this group to predation effects which could be significantly altered through the increase in bacterial
was observed in our experiment has been similarly reported metabolic capability but at the same time enhance virus-
in other freshwater systems [51, 52]. The actinobacterial induced mortality of bacteria. Therefore, both control mecha-
group, which represents an autochthonous and phylogeneti- nisms, i.e., resources and lysis, tend to operate simultaneously
cally highly diverse component of freshwater and work in concert in controlling bacterial community and
bacterioplankton, increased in the presence of viruses and fla- function. It should be noted that relative roles of both re-
gellates, possibly due to changing substrate conditions be- sources and mortality factors in regulating bacterial metabo-
cause growth and activity of this group are essentially driven lism can change over a period of time within a system spatio-
by the availability of organic carbon [53]. Similarly, temporally or among lakes of differing trophy. Caution should
Alphaproteobacteria cells which are characterized by its small be exercised while applying the result of such highly simpli-
size were less vulnerable to top-down pressures, thereby have fied experiments to complex natural ecosystems, since con-
competitive advantage in harnessing regenerated nutrients for founding factors such as fluctuating resources, multiple spe-
its growth and survival. The number of sequences belonging cies interactions, grazing effects, allelopathy, and the cost of
to Gammaproteobacteria which are generally well represent- resistance may also be very important. The results of such a
ed in marine waters accounted for sizeable fraction in our simplified and short-term experiment cannot be generalized
samples. However, their presence in all the treatments with and therefore should be extrapolated to nature with care.
no distinct patterns among treatments was intriguing, possibly
due to the fact that different genera within this group can have Acknowledgments JK was supported by a postdoctoral fellowship
different responses to substrate additions [54]. from the Universit Blaise Pascal, Clermont-Ferrand. SC was supported
The total number of sequences which led to wide variation in by a student fellowship from Universit Blaise Pascal. We thank F.
Perriere for her technical assistance in nutrient analysis. We appreciate
the Chao estimates (151145) and Shannon diversity indexes
three anonymous reviewers for their time, effort, and valuable contribu-
(2.414.39) suggested marked variability in species diversity in tions to this manuscript.
experimental treatments. Shannon diversity index revealed
higher bacterial diversity in the presence of both the predators
and low in experiments which received nutrient additions. References
Microcosm investigation conducted using estuarine bacterial
communities has suggested a positive relationship between vi- 1. Azam F, Malfatti F (2007) Microbial structuring of marine ecosys-
ral abundance and bacterial diversity thereby reiterating the tems. Nat Rev Microbiol 5:782791
importance of viruses in maintaining bacterial species richness 2. Bouvier T, del Giorgio PA (2007) Key role of selective viral-
[27]. Our results suggested that both viruses and protists has induced mortality in determining marine bacterial community com-
position. Environ Microbiol 9:287297
check on bacterial growth by selecting slower growing phylo-
3. Graham EB et al (2016) Microbes as engines of ecosystem func-
types or phenotypes to sustain bacterial richness [7, 8], thereby tion: when does community structure enhance prediction of ecosys-
corroborating the idea of stable microbial communities as hy- tem processes? Front Microbiol 7:214
pothesized by Simek and colleagues [55]. The predator control 4. Weinbauer MG (2004) Ecology of prokaryotic viruses. FEMS
of the winners in nutrient acquisition, i.e., the dominant phylo- Microbiol Rev 28:127181
5. Sime-Ngando T (2014) Environmental bacteriophages: viruses of
types in nutrient allocation, could cause a reduction of produc-
microbes in aquatic systems. Front Microbiol 5:114
tion and at the same time allow for the coexistence of less 6. Thingstad TF (2000) Elements of a theory for the mechanisms
competitive but more resistant phylotypes [7]. controlling abundance, diversity, and biogeochemical role of lytic
Our experiments followed a community-level approach, bacterial viruses in aquatic systems. Limnol Oceanogr 45:1320
and the results represent average responses by bacteria and 1328
7. Zhang R, Weinbauer MG, Qian PY (2007) Viruses and flagellate
viruses. Despite limitations and pitfalls associated with such
sustain apparent richness and reduce biomass accumulation of
microcosm experiment, we were able to show that viral lysis bacterioplankton in coastal marine waters. Environ Microbiol 9:
and nutrient conditions can have contrasting effects on bacte- 30083018
rial metabolism through alteration of host physiological state. 8. Berdjeb L, Pollet T, Domaizon I, Jacquet S (2011) Effect of grazers
Nutrient uptake significantly stimulate bacterial metabolism and viruses on bacterial community structure and production in two
contrasting trophic lakes. BMC Microbiol 11:88
through the proliferation of highly active HNA cells which 9. Jardillier L, Boucher D, Personnic S, Jacquet S, Thnot A, Sargos
was consequently repressed by viruses. Overall, our results D, Amblard C, Debroas D (2005) Relative importance of nutrients
emphasize that the combined action of viruses and associated and mortality factors on prokaryotic community composition in two
Functional Responses of Bacterioplankton Diversity and Metabolism 357

lakes of different trophic status: microcosm experiments. FEMS the structure and activity of bacterial communities in the Marennes-
Microb Ecol 53:429443 Olron Bay (France). Microb Ecol 57:295306
10. Xu J, Jing H, Sun M, Harrison PJ, Liu H (2013) Regulation of 28. Montani H, De Crignis MG, Lavaud J (2015) Viral impact on
bacterial metabolic activity by dissolved organic carbon and virus- prokaryotic and microalgal activities in the microphytobenthic bio-
es. J Geophys Res: Biogeosciences 118:15731583 film of an intertidal mudflat (French Atlantic Coast). Front
11. Pradeep Ram AS, Colombet J, Perriere F, Thouvenot A, Sime- Microbiol 6:1214
Ngando T (2015) Viral and grazer regulation of prokaryotic growth 29. Agis M, Granda A, Dolan JR (2007) A cautionary note: examples
efficiency in temperate freshwater pelagic environments. FEMS of possible microbial community dynamics in dilution grazing ex-
Microb Ecol 91:112 periments. J Exp Mar Biol Ecol 341:176183
12. Birtel J, Walser J-C, Pinchon S, Brgmann H, Matthews B (2015) 30. Sherr EB, Sherr BF, Sigmon CT (1999) Activity of marine bacteria
Estimating bacterial diversity for ecological studies: methods, met- under incubated and in situ conditions. Aquat Microb Ecol 20:213223
rics and assumptions. PLoS One 10:e0125356 31. Pradeep Ram AS, Sime-Ngando (2014) Distinctive patterns in prokaryotic
13. Pradeep Ram AS, Rasconi S, Jobard M, Palesse S, Colombet J, community composition in response to viral lysis and flagellate grazing in
Sime-Ngando T (2011) High lytic infection rates but low abun- freshwater microcosms. Freshw Biol 59:19451955
dances of prokaryote viruses in a humic lake (Vassivire, Massif 32. Lebaron P, Servais P, Agogu H, Courties C, Joux F (2001) Does
Central, France). Appl Environ Microbiol 77:56105618 the high nucleic acid content of individual bacterial cells allow us to
14. Brussaard C, Payet JP, Winter C, Weinbauer MG (2010) discriminate between active cells and inactive cells in aquatic sys-
Quantification of aquatic viruses by flow cytometry. In: Wilhelm tems? Appl Environ Microbiol 67:17751782
SW, Weinbauer MG and Suttle C (eds). Manual of Aquatic Viral 33. Morn XAG, Ducklow HW, Erickson M (2011) Single-cell physi-
Ecology, Chapter 11, pp 102109, ASLO ological structure and growth rates of heterotrophic bacteria in a
15. del Giorgio PA, Gasol JM. (2008). Physiological structure and temperate estuary (Waquoit Bay, Massachusetts). Limnol
single-cell activity in marine bacterioplankton. In:Microb Ecol Oceanogr 56:3748
Oceans, John Wiley & Sons, Inc., pp. 243298 34. Maurice CF, Bouvier T, Comte J, Guillemette F, del Giorgio PA
16. Weinbauer MG, Winter C, Hofle MG (2002) Reconsidering trans- (2010) Seasonal variations of phage life strategies and bacterial
mission electron microscopy based estimates of viral infection of physiological states in three northern temperate lakes. Environ
bacterioplankton using conversion factors derived from natural Microbiol 12:628641
communities. Aquat Microb Ecol 27:103110 35. Pradeep Ram AS, Palesse S, Colombet J, Thouvenot A, Sime-
17. Liu Z, Lozupone C, Hamady M, Bushman FD, Knight R (2007) Ngando T (2014) The relative importance of viral lysis and
Short pyrosequencing reads suffice for accurate microbial commu- nanoflagellate grazing for prokaryotic mortality in temperate lakes.
nity analysis. Nucleic Acids Res 35:e120 Freshw Biol 59:300311
18. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister 36. Morn XAG, Bode A, Surez LA, Nogueira E (2007) Assessing the
EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, relevance of nucleic acid content as an indicator of marine bacterial
Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) activity. Aquat Microb Ecol 46:141152
Introducing mothur: open-source, platform-independent, 37. Cuevas LA, Egge JK, Thingstad TF, Topper B (2011) Organic
community-supported software for describing and comparing mi- carbon and nutrient mineral limitation of oxygen consumption,
crobial communities. Appl Environ Microbiol 75:75377541 bacterial growth and efficiency in the Norwegian Sea. Polar Biol
19. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD 34:871882
(2013) Development of a dual-index sequencing strategy and 38. Longnecker K, Sherr BF, Sherr EB (2005) Activity and phyloge-
curation pipeline for analyzing amplicon sequence data on the netic diversity of high and low nucleic acid content, and ETS-ac-
MiSeq Illumina sequencing platform. Appl Environ Microbiol tive, bacterial cells in an upwelling ecosystem. Appl Environ
79(17):511220 Microbiol 71:77377749
20. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, 39. Tadonlk RD, Planas D, Lucotte A (2005) Microbial food webs in
Glckner FO (2007) SILVA, a comprehensive online resource for boreal humic lakes and reservoirs: ciliates as a major factor related to
quality checked and aligned ribosomal RNA sequence data com- the dynamics of the most active bacteria. Microb Ecol 49:325341
patible with ARB. Nucleic Acids Res 35:71887196 40. Motegi C, Nagata T, Miki T, Weinbauer MG, Legendre L,
21. Schloss PD, Gevers D, Westcott SL (2011) Reducing the effects of Rassoulzadegan F (2009) Viral control of bacterial growth efficiency
PCR amplification and sequencing artifacts on 16SrRNA-based in marine pelagic environments. Limnol Oceanogr 54:19011910
studies. PloS One 6:e27310 41. Pernthaler J (2005) Predation of prokaryotes in the water column
22. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) and its ecological implications. Nat Rev Microbiol 3:537546
UCHIME improves sensitivity and speed of chimera detection. 42. Bonilla-Findji O, Herndl G, Gattuso JP, Weinbauer MG (2009)
Bioinformatics 27:21942200 Viral and flagellate control of prokaryotic production and commu-
23. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam- nity structure in offshore Mediterranean waters. Appl Environ
Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje Microbiol 75:48014812
JM (2009) The Ribosomal Database Project: improved alignments 43. Campbell B, Yu L, Heidelberg JF, Kirchman DL (2011) Activity of
and new tools for rRNA analysis. Nucleic Acids Res 37:D141D145 abundant and rare bacteria in a coastal ocean. Proc Natl Acad Sci U
24. Lnborg C, Sndergaard M (2009) Microbial availability and deg- S A 108:1277612781
radation of dissolved organic carbon and nitrogen in two coastal 44. Fuhrman JA, Schwalbach MS (2003) Viral influence on aquatic
areas. Estuar Coast Shelf Sci 81:513520 bacterial communities. The Biol Bull 204:192195
25. Mahaffey C, Benitez-Nelson CR, Bidigare RR, Rii Y, Karl DM 45. Winter C, Bouvier T, Weinbauer MG, Thingstad TF (2010) Trade-
(2008) Nitrogen dynamics within a wind-driven eddy. Deep Sea offs between competition and defense specialists among unicellular
Res II 55:13981411 planktonic organisms: the BKilling the Winner^ hypothesis
26. Wetzel RG, Likens GE (1995) Limnological analysis, 2nd edn. revisited. Microbiol Mol Biol Rev 74:4257
Springer-Verlag, New York 46. Thingstad TF, Lignell R (1997) Theoretical models for the control
27. Auguet JC, Montani H, Hartmann HJ, Lebaron P, Casamayor EO, of bacterial growth rate, abundance, diversity and carbon demand.
Catala P, Delmas D (2009) Potential effect of freshwater virus on Aquat Microb Ecol 13:1927
358 A. S. Pradeep Ram et al.

47. Motegi C, Nagata T (2009) Addition of monomeric and polymeric accelerates development of Flectobacillus populations in a fresh-
organic substrates alleviates lytic pressure on bacterial communities water community. Environ Microbiol 9:789800
in coastal seawaters. Aquat Microb Ecol 57:343350 52. Hornk K, Jezbera J, imek K (2008) Effects of a Microcystis
48. Motegi C, Nagata T (2007) Enhancement of viral production by aeruginosa bloom and bacterivory on bacterial abundance and ac-
addition of nitrogen or nitrogen plus carbon in subtropical surface tivity in a eutrophic reservoir. Aquat Microb Ecol 52:107117
waters of the South Pacific. Aquat Microb Ecol 48:2734 53. Warnecke F, Sommaruga R, Sekar R, Hofer JS, Pernthaler J (2005)
49. Lindstrm ES, Kamst-Van Agtervald MP, Zwart G (2005) Abundances, identity and grow state of Actinobacteria in mountain
Distribution of typical freshwater bacterial groups is associated lakes of different UV transparency. Appl Environ Microbiol 71:
with pH, temperature, and lake water retention time. Appl 55515559
Environ Microbiol 71:82018206 54. Pernthaler A, Pernthaler J, Eilers H, Amann R (2001) Growth pat-
50. Newton RJ, Jones SE, Eiler A, Mcmahon KD, Bertilsson S (2011) terns of two marine isolates: adaptations to substrate patchiness.
A guide to the natural history of freshwater lake bacteria. Microbiol Appl Environ Microbiol 67:40774083
Mol Biol Rev 75:1449 55. imek K, Nedoma J, Penthaler J, Posch T, Dolan JR (2002)
51. imek K, Weinbauer MG, Hornak K, Jezbera J, Nedoma J, Dolan Altering the balance between bacterial production and protistan
JR (2007) Grazer and virus induced mortality of bacterioplankton bacterivory triggers shifts in freshwater bacterial community com-
position. Antonie Van Leeuwenhoek 81:453463

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