Diversity and dynamics of free-living and particle-associated
Betaproteobacteria
and
Actinobacteria
in relation to phytoplankton
and zooplankton communities
Bushra Parveen1,2, Jean-Philippe Reveilliez1,2, Isabelle Mary1,2, Viviane Ravet1,2, Gise`le Bronner1,2, Jean-Franc¸ois Mangot1,2,3, Isabelle Domaizon3& Didier Debroas1,2
1
Clermont Universit ´e, Universit ´e Blaise Pascal, Laboratoire ‘Microorganismes: G ´enome et Environnement’, BP 10448, Clermont-Ferrand, France;
2CNRS, UMR 6023, LMGE, Aubiere, France; and3INRA, UMR 42 CARRTEL, Thonon, France
Correspondence: Didier Debroas, Laboratoire de Biologie des Protistes, Universite Blaise Pascal, UMR CNRS 6023, Aubiere 63177, France. Tel.: 133 4 7340 7873; fax: 133 4 7340 7670;
e-mail: didier.debroas@univ-bpclermont.fr Received 21 December 2010; revised 28 April 2011; accepted 4 May 2011.
Final version published online 16 June 2011. DOI:10.1111/j.1574-6941.2011.01130.x Editor: Riks Laanbroek
Keywords
diversity;Actinobacteria; Betaproteobacteria;
free living; attached fraction; 16S rRNA gene.
Abstract
The diversity of attached and free-living Actinobacteria and Betaproteobacteria, based on 16S rRNA gene sequences, was investigated in a mesotrophic lake during two periods of contrasting phytoplankton dominance. Comparison analyses showed a phylogenetic difference between attached and free-living communities for the two bacterial groups. For Betaproteobacteria, the betaI clade was detected at all sampling dates in free-living and attached bacterial communities and was the dominant clade contributing to 57.8% of the total retrieved operational taxonomic units (OTUs). For Actinobacteria, the acIV cluster was found to be dominant, followed by acI contributing to 45% and 25% of the total retrieved OTUs, respectively. This study allows the determination of eight new putative clades among the Betaproteobacteria termed lbI–lbVIII and a new putative clade named acLBI belonging to the Actinobacteria. The seasonal dynamics of phytoplankton and zooplankton communities have been reflected as changes in distinct bacterial phylotypes for both attached and free-living communities. For attached commu-nities, relationships were observed between Actinobacteria and Chrysophyceae, and between Betaproteobacteria and Dinophyceae and Chlorophyceae biomass. On the other hand, within free-living communities, few actinobacterial clades were found to be dependent on either nutrients or phytoplankton communities, whereas Betaproteobacteria were mainly associated with biological parameters (i.e. phyto-plankton and copepod communities).
Introduction
In aquatic ecosystems, microbial metabolism accounts for most of the carbon turnover and plays a key role in the trophic dynamics of aquatic food webs (e.g. Azam et al., 1983). Microorganisms play a major role in primary pro-duction and nutrient cycling of an ecosystem; thus, they are direct and sensitive indicators of the ecosystem status and changes (Paerl et al., 2002). Bacteria usually represent 4 90% of microorganism abundance in aquatic habitats and they are considered the dominant group in terms of their contribution to ecosystem processes (Hahn, 2006).
In aquatic environments, bacterial communities can be divided into two assemblages: free-living bacteria and bac-teria attached to particles, phytoplankton or zooplankton
(Crump et al., 1998; Allgaier & Grossart, 2006a; Bruckner et al., 2008; Grossart et al., 2009). The attached bacteria are morphologically different from the free-living ones (Zhang et al., 2007) and are often larger in size (Acinas et al., 1999). Even though the attached bacteria account for only a small proportion (o 10%) of the total bacterial abundance in coastal marine and freshwater systems (e.g. Unanue et al., 1992; Turley & Stutt, 2000), they could be functionally more active and their specific activities seem to be greater than those of the free-living ones (Simon, 1985). Attached bacteria are responsible for the degradation of a major proportion of the particulate organic matter in lacustrine ecosystems (Richardot et al., 1999, 2000). It has also been reported that attached bacteria from the freshwater column of the Columbia River estuary could account for 90% of
MICR
OBIOLOGY ECOLOGY
heterotrophic bacterial activity (Crump & Baross, 1996; Crump et al., 1998). Furthermore, attached bacteria show higher cell-specific growth rates than their free-living coun-terparts in aquatic ecosystems and more particularly in lakes (Fandino et al., 2001; Lemarchand et al., 2006). Finally, it has been reported that attached bacteria have significant impacts on microbial productivity, nutrient exchange and the sorption and transport of contaminants (Paerl & Pinck-ney, 1996; Neu, 2000).
Phylogenetically, there are usually significant differences between free and attached bacterial community composition (BCC) (DeLong et al., 1993; Acinas et al., 1999; Crump et al., 1999; Fandino et al., 2001). However, similar community structures have been reported in coastal marine systems (Hollibaugh et al., 2000) and freshwater microcosms (Riemann & Winding, 2001). These differences in BCC can be attributed to phytoplankton, zooplankton or detrital particles (Biddanda & Pomeroy, 1988; Pinhassi et al., 2004; Grossart et al., 2009). The habitat of phytoplankton-associated bacteria has been referred to as the ‘phycosphere’ (Bell & Mitchell, 1972), which describes the zone of influence or interaction between algae and bacteria (Doucette, 1995). In their study, Grossart et al. (2005) reported that bacterial communities associated with the diatoms Thalassiosira rotula and Skeletonema costatum were found to be phylogenetically different from that of their free-living counterparts. Similarly, the composition of the bacterial community associated with Nodularia sp. in the Baltic Sea seems to be different from that of the unassociated one (Tuomainen et al., 2006). In freshwater ecosystems, Grossart et al. (2009) showed that Betaproteo-bacteria and Bacteroidetes specifically colonize cyclopoid cope-pod by attachment to its body surface. Finally, other particles like detrital and more specifically transparent-exopolymeric and coomassie-stained particles also harbour specific bacterial populations (Lemarchand et al., 2006).
Until now, the attached bacterial community has been mainly studied in estuaries and coastal ecosystems (Delong et al., 1993; Acinas et al., 1999; Crump et al., 1999; Hollibaugh et al., 2000). In contrast, less is known about the comparison of attached and free-living bacterioplankton communities in freshwater ecosystems. However, the typical freshwater bacteria described by Zwart et al. (2002) and reviewed by Hahn (2006) correspond only to free-living bacteria and few studies have reported the analysis of the phylogenetic diversity of particle-associated freshwater eu-bacterial communities (Tang et al., 2009, 2011). Therefore, it is hypothesized that (1) attached-fraction bacteria harbour new putative clades, different from those found in the free-living fraction, and (2) the dynamics of these bacterioplank-ton assemblages are related to changes in phytoplankbacterioplank-ton and zooplankton community composition.
In the present study, we focused on the community composition of attached and free-living Actinobacteria and
Betaproteobacteria, because of their key role in nutrient and energy cycles in freshwater ecosystems (Schweitzer et al., 2001; Elifantz et al., 2005). Moreover, there are evidences showing the usual dominance of these two phylogenetic groups among the bacterial assemblages in lakes of Europe (e.g. Boucher et al., 2006) and particularly in the Lake Bourget (Debroas et al., 2009; Humbert et al., 2009).
Materials and methods
Description of the sampling site and sampling The study was conducted on the mesotrophic Lake Bourget located at the edge of the Alps (France). Lake Bourget is a medium-sized lake (surface area: 45 km2, maximum depth: 145 m) with water residence time 10 years. The lake watershed area (560 km2) encompasses two urban areas (Aix-les-Bains and Chamb´ery) with maximum and average altitudes of 700 and 184 m, respectively.
This lake had been affected by eutrophication in the early 1980s. After restoration programmes, water quality has clearly improved during the last two decades and the phosphorus concentration is about 20 mg P L1 (Lepe`re et al., 2010). Lake Bourget is now considered as meso-trophic; however, this lake has been characterized by a recurrent bloom of the filamentous cyanobacterium Plank-tothrix rubescens since 1998 (Briand et al., 2005). This stratified lake has a thermocline generally located between 15 and 20 m depth. The annual recorded mean for transpar-ency depth is approximately 7 m (Lepe`re et al., 2010); thus, the first 20 m of the water column represents a euphotic zone within which phytoplanktonic species develop them-selves. The homogeneity of free microbial community abundances (virus, bacteria and picophytoplankton) in the water depth zone from 0 to 20 m has been described elsewhere (Personnic et al., 2009).
Water samples were collected from point B (4514305500N,
515200600W) considered as a permanent central reference
station for a water quality survey and located in the deepest zone of the water column. The 0–20 m layer was sampled using an integrating water sampler to carry out sampling right through the water column [sampling system patented by Pelletier & Orand (1978) and adapted from the Schr¨oder (1969) integral sampler]. The final obtained volume (1.2 L) represents an integrated sample from the entire water column (0–20 m). The experimental procedures for the determination of the main limnological parameters (nutri-ents, phytoplankton and zooplankton) are described in Mangot et al. (2009).
Analyses of the Actinobacteria and Betaproteobacteria community compositions were performed on six different dates during the years 2006–2007 (selected dates were characterized by different phytoplanktonic assemblages).
Based on the presence or the absence of Cyanobacteria, these dates could be divided into two periods: September–March, represented by the dominance of Cyanobacteria, and May–August corresponding to the absence or codominance of Cyanobacteria with Diatoms or Chlorophyceae (Support-ing Information, Fig. S1a). Attached and free-liv(Support-ing com-munities were separated by filtration through a 5.0-mm pore-size filter according to the criteria commonly used in previous studies (e.g. Riemann & Winding, 2001; Allgaier & Grossart, 2006a). A subsample of 300 mL was prefiltered through 5.0-mm pore-size polycarbonate filters (Millipore) at a pressure of o 150 103bar. Free-living bacteria pre-sent in the filtrate (o 5.0-mm size fraction) were collected (pressure o 150 103bar) on 0.22-mm pore-size polycar-bonate filters (Millipore) and stored at 80 1C until further nucleic acid extraction.
DNA extraction, PCR amplification and clone library construction
Nucleic acids were extracted following the protocol described previously by Debroas et al. (2009). For the amplification of 16S rRNA gene, specific primers 27f (50-GTTTGATCCTGGCT
CAG-30), 1165r (50-ACCTTCCTCCGAGTTRAC-30) for Acti-nobacteria (Lane, 1991; L¨udemann & Conrad, 2000) and the 680f (50-CRCGTGTAGCAGTGA-30), 1492r (50-GGTTACCT TGTTACGACTT-30) (Lane, 1991; Overmann et al., 1999) for Betaproteobacteria were used. The PCR mixture (25 mL) was prepared with buffer NH41 (Bioline) and contained 200 mM
of dNTP (Bioline), 2 mM MgCl2(Bioline), 1.5 U of Taq DNA
polymerase (Bioline), 25 pmol of each primer, 50 ng of genomic DNA and 0.12 mg mL1of bovine serum albumin (Fermentas). PCR amplification of the 16S rRNA gene was carried out in an automated thermocycler (VWR Unocycler) using the following program: initial denaturation at 94 1C for 5 min; 30 standard cycles of: denaturation (at 94 1C for 30 s); annealing (65 and 57 1C for 1 min, for Actinobacteria and Betaproteobacteria, respectively); extension (72 1C for 1 min); and a final extension at 72 1C for 10 min.
The clone libraries were constructed using PCR vector 2.1 and the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA) following the recommendations of the manufacturer. For each library, 96 clones were randomly picked from different plates and the presence of the target small-subunit rRNA gene insert in positive colonies was checked by PCR amplification using flanking vector primers (M13f and M13r). Amplicons of the expected size were subsequently digested with restric-tion enzyme HaeIII and the resulting restricrestric-tion fragment length polymorphism (RFLP) products were separated by electrophoresis on a 2.5% w/v low-melting-point agarose gel (NuSieve) (Lepe`re et al., 2008). Clones from each library that produced the same RFLP pattern were grouped together and considered members of the same operational taxonomic unit
(OTU). At least one clone of each OTUs family was selected for sequencing. Sequencing reactions were performed by GATC Biotech, Germany (http://www.gatc-biotech.com/fr/ home.html). Clone sequences have been deposited in Gen-Bank (HQ453605–HQ453960).
Phylogenetic analysis and statistical analysis The sequences aligned with ‘Greengenes’ (http://greengenes. lbl.gov) were used for phylogenetic analyses usingARB
soft-ware (Ludwig et al., 2004). OTUs defined by their RFLP profiles were included in theARBdatabase downloaded from
Greengenes site. These sequences were added to a back-bone tree built with the main bacteria phyla found in aquatic ecosystems and typical freshwater clades defined previously (Zwart et al., 2002) using the parsimony tool of ARB and
neighbour-joining methods. The resulting presented trees were pruned to retain representative OTUs and those belonging to the closest typical freshwater bacterial clades for both phylogenetic groups (Betaproteobacteria and Actinobacteria). Representative OTUs were obtained by clus-tering OTUs with a cut-off level of 97% withMOTHUR(Schloss
et al., 2009). New putative clades were identified based on the following criteria: (1) each contains sequences from at least two different sampling dates; (2) each is a monophyletic group supported by the two tree constructions; and (3) the bootstrap values are higher than 60%. Using these criteria, the percentage of similarity among environmental sequences belonging to the same cluster was 4 90%.
Unifrac (http://bmf2.colorado.edu/unifrac/index.psp) metrics was used for comparing microbial communities based on phylogenetic information (Lozupone & Knight, 2005). Canonical correspondence analysis (CCA) was used after forward selection (Borcard et al., 1992) of the para-meters describing the environmental and biological vari-ables likely to explain the most significant part of the changes in the abundance (OTUs) of the defined clades for Actinobacteria and Betaproteobacteria in the free-living and attached fractions.
Richness was estimated according to the nonparametric model of Chao (1984) to predict the number of OTUs in each sampling date of attached and free-living fractions for both phylogenetic groups.
The physicochemical environmental variables used were the concentrations of nitrogen (N-NH4mg N L1,
N-NO3mg N L1), phosphorus (P-PO4mg P L1), silica
(SiO2mg Si L1) and chlorophyll a (Chl a) (mg L1). The
biological variables were biomass of the phytoplankton groups (mg L1) (Cyanobacteria, Dinophyceae, Cryptophyceae, Chrysophyceae, Diatoms, Chlorophyceae, Desmidiaceae and Zygnemataceae), abundance of the zooplankton groups (ind. m2) (rotifers, copepods, cladocerans), phytoplankton richness and diversity (Shannon index).
Results
Chemical variables
The mean total phosphorus (P-PO4) concentration ranged
from 0 to 0.005 mg P L1; high values were recorded in the summer as compared with winter. Of the two forms of inorganic nitrogen analysed, N-NO3was dominant, with an
average concentration ranging from 0.180 to 0.50 mg N L1, whereas the mean concentration of N-NH4 varied from
0.001 to 0.044 mg N L1. For inorganic nitrogen, higher values were recorded in summer, compared with winter and spring. The mean values for SiO2concentration ranged
from 0.057 to 2.420 mg L1, the highest values being re-corded in March (Table S1).
Phytoplankton and zooplankton community dynamics
During the study period, 124 phytoplankton taxa were identified at the species level from the first 20 m depth of lake. Out of these, Chlorophyceae showed the highest diver-sity (39 species), followed by Diatoms (27 species). While other classes were comparatively less diverse, Cyanobacteria (12 species), Dinophyceae (nine species), Chrysophyceae (11 species), Cryptophyceae (five species) and Desmidiaceae/ Zygnemataceae (11 species). While other classes were com-paratively less diverse in species number as: Cyanobacteria (12 species), Dinophyceae (nine species), Chrysophyceae (11 species), Cryptophyceae (five species) and Desmidiaceae/ Zygnemataceae (11 species). Quantitatively, in terms of abundance and biomass, the dominant groups were Cyano-bacteria and Diatoms (Fig. S1a).
The concentration of Chl a was measured as an approx-imation of the total phytoplankton biomass. The summer peak in Chl a concentration (9.8 mg L1) was observed in May 2006, when the abundance of Cyanobacteria and Diatoms was high (Fig. S1a). The highest value for the Chl a concentration in winter (6.6 mg L1) was measured in December 2006, when only Cyanobacteria were dominant, particularly P. rubescens.
Twenty-one species of metazooplankton were identified from the collected samples. Copepods, as cladocerans, were represented by eight species, whereas four species belonged to rotifers and one to bivalve larvae. Copepods were the dominant group in terms of abundance for all sampling dates, accounting for 66.2% (September) to 90.5% (March) of the total zooplankton community. The two dominant species were Eudiaptomus gracilis and Cyclops prealpinus. Cladocerans were also observed throughout the study period and were second to the copepods in terms of density. Daphnids (primarily Daphnia hyalina) accounted for the most abundant group among cladocerans, with peak abundance in June 2006 (103 579 ind. m2) during the clear
water phase of the lake. Finally, rotifers were the least prevalent group throughout the study period (Fig. S1b).
Betaproteobacteriain the free -living and
attached fractions
Among the 171 Betaproteobacteria sequences, 98 OTUs (n = 345 clones) were identified for attached bacteria and 73 OTUs (n = 419 clones) for the free-living form.
In total, 128 OTUs were found to be affiliated with previously reported phylotypes from the freshwater Beta-proteobacteria clusters betaI, betaII, betaIII and betaIV, defined by Zwart et al. (2002) (Fig. 1a and b). These phylotypes are clearly separate from Betaproteobacteria lineages of soil and marine habitats. In contrast, 43 OTUs could not be associated with any environmental reference clade for Betaproteobacteria; out of these, 36 OTUs (n = 140 clones) were placed in eight novel putative clades, namely lbI, lbII, lbIII, lbIV, lbV, lbVI, lbVII and lbVIII, whereas the remaining 7 OTUs were left unclassified (Fig. 2, Table S2a).
The clade betaI, represented by 99 OTUs, consisted of 62.6% (n = 214 clones) attached bacteria and 37.4% (n = 264 clones) free-living bacteria. Similarly, the clade betaIV
0 5 10 15 20 25 30 35 (a) (b)
May-06 Jun-06 Aug-06 Sept-06 Dec-06 Mar-07
No. of OTUs lbVIII lbVI lbV lbIII betaIII lbII lbI betaII betaIV betaI 21 27 142 12 44.3 19 0 5 10 15 20 25
May-06 Jun-06 Aug-06 Sept-06 Dec-06 Mar-07
No. of OTUs lbVIII lbVII lbIV betaIII lbII lbI betaIV betaI 40.2 8 22 17.2 9.5 5 Sampling dates
Fig. 1. Temporal diversity of (a) attached and (b) free-living Betaproteo-bacteria (values on bar = Schao1).
included OTUs from both free-living and attached fractions: out of the 22 OTUs, 77.3% (n = 78 clones) were from free-living and 22.7% (n = 13 clones) were from attached bacter-ia. On the contrary, the clade betaII was exclusively repre-sented by attached bacteria (3 OTUs) and betaIII by free-living bacteria (4 OTUs). Three new putative clades, termed as lbIII, lbV and lbVI, seemed to be specific to the attached bacterial community (Fig. 2, Table S2a).
Unifrac was used to compare the attached and free-living Betaproteobacteria assemblages. Phylogenetically, both bac-terial communities (attached and free-living) were found to be highly significantly different from each other (Po 0.001). Principal coordinate analysis (PCA) using Unifrac metrics was performed to discriminate the general patterns of distribution between attached and free-living Betaproteobacteria for each sampling date (Fig. 3a). The first
(a)
F C
C C
C F
Fig. 2. 16S rRNA gene phylogenetic tree of partial sequences for Betaproteobacteria showing the representative OTUs in the proposed clusters: (a) clade betaI; (b) clades betaII, betaIV and lbI–VIII. The sequences obtained in this study are depicted in the form ‘Beta_sn_sup/inf_n’, where Beta, Betaproteobacteria; sn, sampling number; sup/inf, attached/free-living; and n, name of the clone.
two principal coordinate axes, i.e. PC1 and PC2 explained 19.1% and 16.5%, respectively, of the total variation for the previously defined clades presented in Fig. 2. The distribu-tion patterns for all attached and free-living assemblages showed three distinct groups on PCA. This analysis allowed therefore to separate attached from free-living bacteria, with the exception of the free-living fraction from June and September that was associated with the attached
communities from May, June and December. However, among this Betaproteobacteria, only the bacterial commu-nity sampled in June seemed to be similar between both the fractions studied (Fig. 3a).
As highlighted by this PCA, the structure of Betaproteo-bacteria community composition in the two fractions stu-died showed temporal variations. The extent of these variations in terms of OTUs richness for each sampling date
(b) L C C C C C L B C C C F U Fig. 2. Continued.
was estimated by Chao1. The values of Chao1 varied from 12 to 142 for attached and from 5 to 40 for free-living bacterial communities, respectively (Fig. 1a and b). As indicated by Chao1, the highest richness was observed in September, where 36 OTUs (n = 123 clones) were retrieved for attached and free-living Betaproteobacteria. Out of these, 21 OTUs (n = 53 clones) belonged to attached bacteria and were grouped into nine distinct clades. Relatively few clades were observed for free-living counterparts for which 15 retrieved OTUs (n = 70 clones) fell into six distinct clades. The maximum abundance and the lowest number of clades for Betaproteobacteria were observed in the month of June 2006, where all 32 OTUs (n = 65 clones) for attached communities were grouped into a single clade, betaI, and 21 OTUs (n = 51 clones) for free-living bacterial commu-nity fell into four distinct clades. The rest of the sampling dates showed intermediate OTUs richness and clade diversity for both attached and free-living bacterial communities. How-ever, betaI was ubiquitously present in attached as well as in free-living bacterial assemblages for all sampling dates, with some seasonal variation in its proportion in libraries.
The possible relationship between attached and free-living clades of Betaproteobacteria, and environmental variables was investigated using CCA. The analysis applied to the Betapro-teobacteria for the attached fraction showed that the first two axes explained 80.5% of the cumulative variance (Fig. 3b). CCA1, characterized by 40.5% of the total variations, showed that clades lbI, lbIII, lbVI and betaIII were associated with phytoplankton richness and the biomass of Chlorophyceae. Similarly, for the free-living fraction, the first two axes accounted for 65.6% of the total variations, which provided an explanation for the temporal variations of lbI, lbIV and lbVIII (Fig. S2a). The clades lbIV and lbVIII seemed to be linked to copepod dynamics, whereas lbI was associated with the highest value of phytoplankton diversity (Shannon index).
Actinobacteriain the free -living and attached
fractions
In total, 193 OTUs were retrieved. One hundred and twelve OTUs (n = 465 clones) were found to belong to the attached
(a)
(b)
Fig. 3. (a) PCA computed from UniFrac distance metric from Betaproteobacteria phylogeny for attached and free-living bacterial communities. (b) CCA plot linking attached Betaproteobacteria clades and environmental parameters.
(a)
Fig. 4. 16S rRNA gene phylogenetic tree of partial sequences for Actinobacteria showing representative OTUs in the proposed clusters: (a) clades acI, acII, acSTL, Corynebacteriaceae, Propionibacteriaceae, Micrococcus, Sporichtya and Nocardiaceae; (b) clades acIV, acV, Microthrix and Nakamurella. The sequences obtained in this study are depicted in the form ‘Act_sn_sup/inf_n,’ where Act, Actinobacteria; sn, sampling number; sup/inf, attached/free-living; and n, name of clone.
and 81 (n = 439 clones) to the free-living Actinobacteria. According to the clade definition used in the present study, 29 OTUs (n = 94 clones) were affiliated to actinobacterial lineages, namely: Sporichthya, Microthrix, Corynebacteria-ceae, PropionibacteriaCorynebacteria-ceae, Micrococcus, Nocardiaceae and Nakamurella. In contrast, a large number of OTUs (n = 155
clones) were related to previously reported specific clades of freshwater Actinobacteria, namely acI-A, acI-B, acII-B, acIV and acSTL, as defined by Warnecke et al. (2004) and Allgaier & Grossart (2006b). In this study, a novel putative cluster acLBI has been proposed for freshwater Actinobacteria. The 3 OTUs, retrieved in acLBI, belong exclusively to the
(b)
Fig. 4. Continued.
attached bacterial community. Nine OTUs could not be affiliated with any of the previously proposed environmental clades of freshwater Actinobacteria (Fig. 4, Table S2b).
For free-living Actinobacteria, the highest number of retrieved OTUs (n = 41) belongs to acIV, whereas the rest of the OTUs (n = 40) were affiliated with other distinct clades of Actinobacteria: acI-A (n = 27), acSTL (n = 3), acI-B (n = 2), acII-B (n = 1) and Micrococcus (n = 3). The phyloge-netic affiliation shows that attached Actinobacteria clustered in more clades than their free-living counterparts (13 vs. 6). Therefore, the OTUs retrieved from the attached Actinobac-teria were affiliated to different lineages as follows: acIV (n = 45), acI-A (n = 16), Microthrix (n = 14), acI-B (n = 8), Sporichyta (n = 7), acSTL (n = 5), acII-B (n = 2), Corynebac-teriaceae (n = 2), PropionibacCorynebac-teriaceae (n = 1), Micrococcus (n = 1), Nakamurella (n = 1), Nocardiaceae (n = 2) and acLBI (n = 3) (Fig. 5a and b).
Temporal variability was clearly observed in the propor-tion of different actinobacterial lineages detected in the clone libraries, as well as in Chao1 values, which varied from 13 (June 2006) to 31.1 (September 2006) for the attached bacterial community (Fig. 5a) and from 10.5 (August 2006) to 30 (September 2006) for the free-living community (Fig. 5b). Within the attached Actinobacteria community, the clade acIV was ubiquitously present as a dominant repre-sentative on all sampling dates, with variations in the proportion of retrieved OTUs. The highest number of clades for attached actinobacterial communities as observed in December 2006, where 24 retrieved OTUs (n = 87 clones)
clustered into nine distinct clades. In the free-living com-munity, clade acIV and subclade acI-A were reported ubi-quitously, whereas OTUs for the other lineages showed different contributions for all sampling dates.
Unifrac analysis showed a phylogenetically significant difference (Po 0.01) between the attached and the free-living actinobacterial communities. PC1 and PC2 together explained 69.2% of the total genetic distances (Fig. 6a). The PC1, characterized by 47% of the explained variation, allowed separating mainly attached and free-living actino-bacterial communities for all considered sampling dates. Free-living actinobacterial communities were mainly located on the negative side of this axis, whereas attached bacterial communities of four sampling dates (December 06, June 06, March 07, May 06) and one free-living bacterial community (June 06) were on the positive side. Hence, therefore, this pattern of distribution may be attributed to observed significant phylogenetic differences among the attached and free-living actinobacterial communities.
In order to investigate the relationship between actino-bacterial community composition and the environmental variables, CCA was performed. For the attached actinobac-terial communities, 59.9% of the total variation for clade composition was associated with the environmental vari-ables selected by forward analysis. Chrysophyceae and N-NO3were highly associated with the first axis, whereas the
second axis was found to be dependent on N-NO3and the
Shannon index, computed with phytoplankton biomass (Fig. 6b). The acII-B and Nakamurella groups appeared in association with Chrysophyceae and N-NO3, whereas
Propionibacteriaceae and acLBI groups were related to the Shannon index of phytoplankton diversity and N-NO3
concentration. For the analysis of free-living Actinobacteria (Fig. S2b), CCA1 and CCA2 were characterized by 56.2% and 27% of the total variation, respectively. The temporal changes of the Micrococcus and acII-B clades were mainly linked with Dinophyceae, whereas acSTL was associated with the N-NH4concentration.
Discussion
The link between phytoplankton and bacterioplankton community composition dynamics has been evidenced previously (Brussaard et al., 2005; Rooney-Varga et al., 2005), but is still poorly understood. This study was carried out to explore the seasonal succession of attached and free-living bacterioplankton communities in relation to the dynamics of the physicochemical and biological parameters in a freshwater ecosystem. In this study, we focused on two major groups, Actinobacteria and Betaproteobacteria, based on previous findings revealing their dominance in the Lake Bourget (Debroas et al., 2009; Humbert et al., 2009). More-over, in freshwater ecosystems, Betaproteobacteria has
0 5 10 15 20 25 30 (a) (b)
May-06 Jun-06 Aug-06 Sept-06 Dec-06 Mar-07
No. of OTUs 31.1 30.3 14.8 13 16 21 0 5 10 15 20
May-06 Jun-06 Aug-06 Sept-06 Dec-06 Mar-07
No. of OTUs 11 20 10.5 30 28.3 12.7 Sampling dates
Fig. 5. Temporal diversity of (a) attached and (b) free-living Actinobacteria (values on bar = Schao1).
appeared as a dominant group in bacterial clone libraries (Gl¨ockner et al., 1999, 2000; Wu & Hahn, 2006) and, similarly, Actinobacteria represents a large fraction of fresh-water bacterioplankton (Gl¨ockner et al., 2000; Warnecke et al., 2005; Allgaier & Grossart, 2006b). To our knowledge, this is the first study providing a phylogenetic comparison between attached and free-living freshwater Betaproteobacteria, whereas such research on the freshwater Actinobacteria is scarce (Allgaier et al., 2007; Tang et al., 2009).
Methodological aspects
Group-specific primers used for Actinobacteria (27f/1165r: Lane, 1991; L¨udemann & Conrad, 2000) resulted in the successful amplification of the 16S rRNA gene of the target taxa and sequences exclusively belonging to the desired group were obtained. However, for the Betaproteo-bacteria 16S rRNA gene clones library, the primers used (680f/1492r: Lane, 1991; Overmann et al., 1999) revealed
some nonspecific amplification, with 12.9% (27 OTUs) for Verrucomicrobia and 4.8% (10 OTUs) for Cyanobacteria. Nontarget OTUs were not taken into account for further analyses. Thus, PCR-RFLP was used for a more precise enumeration of desired OTUs, thus reducing the number of nontarget sequences.
Based on previous evidence, the attached bacterial frac-tion was considered as comprising bacteria associated with particles, zooplankton and phytoplankton (Crump et al., 1998; Rooney-Varga et al., 2005; Allgaier & Grossart, 2006a; Grossart et al., 2009). However, the filtration procedure used may be subject to limitations that may bias the analysis. For example, some free-living filamentous bacterial can be larger than 5.0 mm and cannot pass through these filters; conse-quently, they are considered in the pool of attached bacteria. Alternatively, some attached bacteria could be dislodged during the filtration process, subsequently leading to an underestimation of the attached bacterial communities OTUs and vice versa for the free-living communities.
(a)
(b)
Fig. 6. (a) PCA computed from UniFrac distance metric from Actinobacteria phylogeny for attached and free-living bacterial communities. (b) CCA plot linking attached Actinobacteria clades and environmental parameters.
Richness and diversity ofActinobacteriaand
Betaproteobacteriain attached and free -living
fractions
High richness, in terms of OTUs and clade diversity, was found for attached communities (Betaproteobacteria and Actinobacteria) compared with their free-living counterparts. This observation is consistent with previous studies on marine ecosystems (DeLong et al., 1993; Rath et al., 1998). Although contrasting results have been reported from studies on the Southern Californian coast (Fandino et al., 2001) and Medi-terranean offshore waters (Acinas et al., 1999), showing less phylotype diversity in the attached fraction compared with the free-living one, in the present study, some of the clades (e.g. betaII) and four proposed new putative clades (lbIII, lbV, lbVI, acLBI) were exclusively represented by attached bacteria, suggesting that the structure of attached bacterial commu-nities in freshwater lakes has been overlooked in the past and there is still much to be explored.
The majority of clades for Betaproteobacteria as well as Actinobacteria co-occurred at a detectable level in both attached and free-living assemblages for each sampling date; however, the phylogenetic comparison analysis (Unifrac metrics) showed that these phylotypes were phylogenetically different from each other. These findings are consistent with previous reports from various marine environments (DeLong et al., 1993; Acinas et al., 1999; Crump et al., 1999), indicating that attached microbial consortia differ phylogenetically from those of their free-living counterparts. In our study, OTUs for Actinobacteria belonged to previously reported freshwater Actinobacteria clades and subclades (Warnecke et al., 2004; Allgaier & Grossart, 2006b). The majority of retrieved OTUs belonged to acIV and acI clusters. These results are in agreement with previous reports showing acIV and acI as major freshwater (Warnecke et al., 2004; Allgaier & Grossart, 2006b) as well as marine (Holmfeldt et al., 2009) Actinobacteria clusters. The co-occurrence of free-living and attached Actinobacteria OTUs has been observed in all known clusters, except Sporichyta, Microthrix, Corynebacteriaceae, Propionibacter-iaceae, Nocardiaceae and Nakamurella, where the retrieved OTUs belonged exclusively to the attached fraction, as reported previously by Allgaier et al. (2007). On the contrary, for the Micrococcus group, a major proportion of the retrieved sequences belonged to free-living bacteria.
In this study, we propose a new putative clade, termed acLBI Actinobacteria from freshwater, consisting exclusively of OTUs from the attached fraction. Moreover, these sequences are phylogenetically distinct from those contained in three new putative clades ‘acIB scB5’, ‘acIVE’ and ‘acV’ defined in a study on the Baltic Sea (Holmfeldt et al., 2009). Four phylogenetic clades for freshwater Betaproteobacteria, termed betaI, betaII, betaIII and betaIV, were detected, which
is consistent with the previous findings for the freshwater Betaproteobacteria clusters (Gl¨ockner et al., 2000; Zwart et al., 2002; Salcher et al., 2008). In addition, this study also highlighted eight new putative clades (i.e. lbI to lbVIII). The most dominant clade, betaI, was present at all sampling dates in both free-living and attached bacterial commu-nities. These results are consistent with a previous study conducted by Gl¨ockner et al. (2000), on three lakes (Gos-senk¨ollesee, Fuchskuhle and Baikal), reporting the domi-nance of this clade in the free-living fraction. However, some subclades considered as important in lake ecosystems were not detected or were found to be less represented compared with previous studies. For example, none of the sequences retrieved in this study, for both the attached and the free-living Betaproteobacteria communities, were found to be phylogenetically similar to the subclade R-BT065 of betaI (Salcher et al., 2008; Sˇimek et al., 2010). This subclade could have been expected to be dominant in the phycosphere (attached fraction in this study), as a strong link between the dynamics of the R-BT065 cluster and phytoplankton-derived organic material has been reported (Sˇimek et al., 2008).
Similarly, in our study, another important Betaproteobacteria clade, betaII (including subclades Pnec C, Pnec B and Poly-nucleobacter necessarius), is represented by only three retrieved sequences belonging exclusively to the attached Betaproteobac-teria fraction, whereas previous studies have reported the presence of this clade in the free-living size fraction of various lakes. These studies cover the entire range of trophic statuses: oligotrophic Toolik Lake (Crump et al., 2003); oligomeso-trophic Lake Mondsee (Hahn, 2003); and euoligomeso-trophic Lake Loosdrecht (Zwart et al., 2002), as well as geographical range (Hahn, 2003).
Temporal changes inActinobacteriaand
Betaproteobacteriaclades in relation to
environmental variables
The seasonal dynamics of phytoplankton and zooplankton communities has been reflected as the change in Actinobacteria and Betaproteobacteria community composition. These changes are observed in terms of presence or absence, and relative abundance or richness of distinct bacterial clades for both attached and free-living bacterial communities. How-ever, other factors, not measured in this study, could also explain the temporal variations of bacterial communities. For example, in a previous study, it was reported that proteinaceous and transparent exopolymer particles (4 5.0 mm) harboured different bacterial populations (Lemarchand et al., 2006). It is also known that the availability and quality of organic matter and exudates are strong regulatory factors of attached BCC (Sapp et al., 2007; Tang et al., 2009).
For free-living bacterial assemblages, the CCA revealed that few actinobacterial clades showed an association with nutrients or phytoplankton communities, which is in agree-ment with previous reports supporting the earlier notion that Actinobacteria do not react strongly to inorganic nutrient sources (Haukka et al., 2006). However, a few clusters such as acSTL showed an association with nitrate concentration; likewise, the acII-B cluster and Micrococcus group were found to be linked to the biomass of Dinophyceae. Such associations, therefore, may indicate the potential effect of nutrients or DOC in structuring the BCC.
Nutrient release could also explain the relationship be-tween clades of free-living Betaproteobacteria community and copepods (Vrede & Vrede, 2005). An interaction between this bacterial class and nitrate also seems to be consistent with the work of Gao et al. (2005), which showed a positive relationship among Betaproteobacteria, nitrate and DOC.
Our study also provides some understanding of the environmental parameters controlling the Actinobacteria and Betaproteobacteria in the attached fraction. With the exception of nitrate, these attached bacterial groups were linked to phytoplankton or metazooplankton, in terms of their composition, richness or diversity. This pattern is therefore in contrast to the parameters interacting with free-living bacteria. The attached actinobacterial clades were highly correlated with the Chrysophyceae biomass, whereas the attached Betaproteobacteria clades were associated with the biomass of Dinophyceae and Chlorophyceae (Figs 3b and 6b). Based on the results of the CCA, the major proportion of the new putative clades for attached Betaproteobacteria, termed lbI, lbVI and lbIII, appeared to be linked to the biomass and diversity of phytoplankton. Similarly, the newly proposed lineage for Actinobacteria, acLBI (exclusively attached bacteria), was correlated with the Shannon diver-sity index of phytoplankton.
Our results support previous reports of the existence of specific interactions between phytoplankton and their attached bacterial communities, where phytoplankton com-munity composition dynamics accounted for significant changes in freshwater attached bacterial assemblages (Riemann & Winding, 2001; Peng et al., 2007) as well as in coastal marine systems (Rooney-Varga et al., 2005).
As shown by CCA analyses, phytoplankton diversity is an important contributing factor in structuring the attached Actinobacteria community. It seems that among phyto-plankton, Cyanobacteria could affect the community com-position of attached Actinobacteria, as Chao1 richness values of attached Actinobacteria were greater when Cyanobacteria were dominant. Even if it is difficult to understand the association between Actinobacteria and Cyanobacteria, there is evidence of such bacteria–Cyanobacteria associations from the study of lake Erkin (Sweden) (Eiler et al., 2006). In this
study, lower affinity for arginine was observed in bacteria associated with the cyanobacterial phycosphere compared with other planktonic counterparts, suggesting that these cyanobacterial-associated bacteria took advantage of phyco-sphere ambient organic compounds compared with free-living counterparts. In another study, from Lake Taihu (China), Actinobacteria appeared as the most significant lineage, with dominant numbers of OTUs during the period of cyanobacterial bloom (Wu et al., 2007).
The possible explanation for such interactions between phytoplankton and bacterioplankton seems to be source mediated, as the release of exudates by phytoplankton and their uptake by bacteria is well documented in algal–bacterial interactions (Baines & Pace, 1991). This in turn may explain the temporal shifts in various bacterial phylotypes with seasonal dynamics of phytoplankton, as previously evi-denced, that changes in phytoplankton community compo-sition can produce a response in bacterioplankton species composition (Pinhassi et al., 2004).
In conclusion, this study revealed new putative lineages and showed that attached Actinobacteria and Betaproteobacteria communities differ from free-living ones, providing a possi-ble explanation for the physiological differences that have been observed previously between attached and free-living bacterial communities. The discovery of the proposed new putative clades for Actinobacteria (acLBI) and Betaproteo-bacteria (lbI–lbVIII) illustrates that microbial diversity in freshwater ecosystems still needs to be explored. This study also showed a link between biological parameters (phyto-plankton, zooplankton) and attached Actinobacteria and Betaproteobacteria communities, indicating that specific interactions between phytoplankton and attached bacterial communities may occur and play an important role in structuring the microbial community composition in fresh-water ecosystems. Nevertheless, our knowledge of the func-tion and metabolism of the attached bacteria, especially within the phycosphere, is still poor. Thus, to better under-stand the interactions occurring within the phycosphere, it is important to further characterize the functional signifi-cance and metabolic activities of the attached prokaryotic communities.
Acknowledgements
We thank G. Paolini and P. Perney for technical contribu-tions to the sampling and analysis of physical and chemical parameters. The chemical parameter analysis, microphyto-plankton and metazoomicrophyto-plankton counts were performed at the INRA’s Thonon station (France). We especially thank J.C. Druart and L. Lain´e for the microphytoplankton and metazooplankton counts. Physicochemical, zooplankton and phytoplankton data were obtained by the observatory on perialpine lakes (UMR CARRTEL, INRA, Thonon). We
would like to extend our special thanks to Higher Educa-tion commission (HEC) Pakistan, for financing this research work.
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Supporting Information
Additional supporting information can be found in the online version of this article:
Fig. S1. Temporal diversity in (a) the biomass of the phytoplankton [values on bar = Chl a (mg L1)] and (b) the abundance of metazooplankton: cladocerans, copepods, and rotifers (ind. m2).
Fig. S2. CCA plot linking (a) free-living Betaproteobacteria clades and environmental parameters and (b) free-living Actinobacteria clades and environmental parameters. Table S1. Physicochemical parameters for all sampling dates.
Table S2. Representative OTUs and OTUs associated deter-mined by RFLP patterns for both phylogenetic groups: (a) Betaproteobacteria and (b) Actinobacteria.
Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials sup-plied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
