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Applied and Environmental Microbiology, September 2005, p. 5551-5559, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5551-5559.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Abundances, Identity, and Growth State of Actinobacteria in Mountain Lakes of Different UV Transparency

Falk Warnecke,1,{dagger} Ruben Sommaruga,2 Raju Sekar,1,{ddagger} Julia S. Hofer,2 and Jakob Pernthaler1*

Max-Planck-Institute for Marine Microbiology, Bremen, Germany,1 Laboratory of Aquatic Photobiology and Plankton Ecology, Institute of Zoology and Limnology, University of Innsbruck, 6020 Innsbruck, Austria2

Received 16 February 2005/ Accepted 12 April 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The occurrence, identity, and activity of microbes from the class Actinobacteria was studied in the surface waters of 10 oligo- to mesotrophic mountain lakes located between 913 m and 2,799 m above sea level. Oligonucleotide probes were designed to distinguish between individual lineages within this group by means of fluorescence in situ hybridization (FISH). Bacteria of a single phylogenetic lineage (acI) represented >90% of all Actinobacteria in the studied lakes, and they constituted up to 70% of the total bacterial abundances. In the subset of eight lakes situated above the treeline, the community contribution of bacteria from the acI lineage was significantly correlated with the ambient levels of solar UV radiation (UV transparency, r2 = 0.72; P < 0.01). Three distinct genotypic subpopulations were distinguished within acI that constituted varying fractions of all Actinobacteria in the different lakes. The abundance of growing actinobacterial cells was estimated by FISH and immunocytochemical detection of bromodeoxyuridine (BrdU) incorporation into de novo-synthesized DNA. The percentages of Actinobacteria with visible DNA synthesis approximately corresponded to the average percentages of BrdU-positive cells in the total assemblages. Actinobacteria from different subclades of the acI lineage, therefore, constituted an important autochthonous element of the aquatic microbial communities in many of the studied lakes, potentially also due to their higher UV resistance.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Members of the class Actinobacteria (44) are regarded as typical inhabitants of soil environments (14). Evidence is accumulating that these bacteria are also present in a variety of freshwater habitats (7, 10, 17, 48, 49). Bacteria from this group may in fact constitute a large fraction of the bacterioplankton in oligo- to mesotrophic lakes or in humic lakes (8, 13, 36). In order to understand the role of Actinobacteria in different freshwater habitats, it is important to distinguish between the various phylogenetic groups within this lineage (13, 47, 48) and to determine their population sizes in situ. This can be achieved by fluorescence in situ hybridization (FISH) with specific rRNA-targeted oligonucleotide probes (36).

However, the occurrence of particular phylogenetic groups of Actinobacteria in the plankton of lakes does not necessarily imply that these bacteria are growing in this environment. Many cultivated Actinobacteria perform a life cycle which may involve a vegetative and a resting stage (i.e., spores) (11). Spores are highly resistant to unfavorable environmental conditions and can survive for extended periods of time. This life strategy promotes the survival and dispersal of Actinobacteria in soils. Thus, actinobacterial phylotypes that are found in freshwater might potentially be resting stages of predominently soil-dwelling allochthonous microorganisms. In order to prove that this phylogenetic group is an indigenous component of freshwater planktonic microbial assemblages, it is necessary to demonstrate cell growth in situ, e.g., by visualization of DNA de novo synthesis at the single-cell level (31).

Our knowledge on the environmental conditions that favor the occurrence of Actinobacteria in lakes is scarce. The first sequence types from this group originate from acidified lakes in the Adirondack Mountains (17). Actinobacteria were conspicuously abundant in the water column of an alpine lake that is characterized by high levels of incident solar UV radiation and by high water transparency (13, 42). Solar UV radiation is harmful to various aquatic microorganisms, including viruses, bacteria, and unicellular eukaryotes (19, 40, 45). Furthermore, UV radiation is known to inhibit bacteria-mediated processes (41), and picoplankton organisms from clear-water ecosystems accumulate significant UV-induced DNA damage (6). DNA rich in A+T content can be particularly susceptible due to the higher probability of production of cyclobutane thymine dimers. One typical feature of the Actinobacteria is their high genomic G+C content (5). It is thus conceivable that such bacteria might be favored in the bacterioplankton assemblages of UV-transparent lakes.

In order to test this hypothesis, the relative abundances of Actinobacteria were determined in the bacterial assemblages of 10 mountain lakes. These lakes are located along an altitude gradient, and they cover a wide range of water column UV transparencies. The composition of the microbial assemblages in these lakes was analyzed using a set of available and newly designed FISH probes. Specifically, we attempted to identify the numerically important taxa of freshwater Actinobacteria. In addition, the proportions of growing Actinobacteria were determined by pulse-labeling with bromodeoxyuridine (31).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sampling sites and scheme.
A set of 10 mountain lakes in the Tyrolean Alps (Austria) located at altitudes between 913 and 2,799 m above sea level (a.s.l.) were selected for sampling. Abbreviations of lake names are given in Table 1. Background information on their main limnological characteristics as well as on their UV transparency is found elsewhere (21, 42, 43). Samples of lake surface water were collected between 16 and 22 July 2003 with a 5-liter Schindler-Patalas sampler. Water was stored in the dark and at in situ temperature until further processing (usually within 1 h after sampling). Subsamples for FISH analysis (50 ml) were fixed with an equal volume of ethanol (final concentration, 50%) for 24 h, filtered onto white polycarbonate membrane filters (type GTTP; pore size, 0.2 µm; diameter, 47 mm; Millipore, Eschborn, Germany), and washed with 10 ml of deionized particle-free water (MilliQ; Millipore). Filters were stored at –20°C until further processing.


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  		TABLE 1. Characteristics of sampled lakes

 
Physical and chemical parameters.
Water samples were analyzed for basic parameters such as pH, conductivity, alkalinity, and temperature. The chromophoric dissolved organic matter characteristics were determined by measurements of dissolved organic carbon (DOC) concentration and spectral absorptivity of filtered lake water. The DOC-specific absorption at 320 nm was estimated as the absorptivity at 320 nm normalized to the DOC concentration. For these analyses, the lake water was filtered on the same day (within 6 h) through glass fiber filters (Whatman GF/F; precombusted for 1 h at 450°C, rinsed with 20 ml of Milli-Q water and 10 ml of lake water) and stored in clean precombusted glass bottles (Teflon capped) in the dark at 4°C until analysis (within 24 h). The DOC was measured in a model 5000 TOC analyzer (Shimadzu, Duisburg, Germany) equipped with a platinized-quartz catalyst for high sensitivity analysis. Absorption by chromophoric dissolved organic matter was determined by spectrophotometry (scans between 250 and 750 nm; Hitachi U-2000 double-beam spectrophotometer) using an acid-cleaned 10-cm quartz cuvette (Suprasil I) prerinsed two times with the sample. Pigments were extracted with 90% acetone for 24 h in the dark at 4°C. Filters were briefly sonicated with a tip sonicator (1 min, 4 W) and the extracts cleared using an Anodisc filter (pore size, 0.1 µm; Whatman). The extracts were scanned in a spectrophotometer (same as above; scans between 400 and 750 nm) against an acetone reference using 5-cm glass cuvettes. The diffuse vertical attenuation coefficients of UVB radiation at 320 nm (Kd320) were estimated either from direct measurements in the water column (Gossenköllesee [GKS], Piburgersee [PIB], Achensee [ACH]) done with a PUV-501 radiometer (Biospherical Instruments Inc., San Diego, CA) or with an empirical model established for mountain lakes (including the ones investigated here) that uses the absorptivity at 320 nm as an independent variable (21).

Oligonucleotide probe design and testing.
Based on a previously published phylogenetic analysis of 16S rRNA sequences (47) oligonucleotide probes for FISH were designed for four lineages of freshwater Actinobacteria and for subgroups within these lineages. Discriminatory oligonucleotide sequences were discovered using the software packages ARB (22) and Primrose (2). In Primrose, 2,000 sequences from other actinobacterial groups were imported from the ARB database as a non-target-background data set. Subsequently, groups of sequences from the four actinobacterial clusters acI to acIV (47) or from subgroups within these clusters were imported as targets for the automatic probe design. Primrose—as opposed to ARB—is able to design probes that contain "wobbles," i.e., alternative bases at specific positions. Probe candidates were checked for specificity against the ARB database and against sequences in GenBank via a BLAST search. Probes were ranked according to their coverage of target groups, the number and location of mismatching nucleotides to nontarget hits, and the potential in situ accessibility of their respective target sites on the 16S rRNA (4). A list of newly designed (and other, previously published) probes that were used for the analysis of bacterioplankton communities in the studied lakes is given in Table 2.


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  		TABLE 2. Oligonucleotide probes used in this study

 
Due to a lack of cultivated representatives from the actinobacterial acI lineage, the following strategies were applied for the establishment of specific hybridization conditions. For probe AcI-852, a series of hybridizations with increasing formamide concentrations in the hybridization buffer (formamide series, 30 to 70%) were performed on an environmental sample (Schwarzsee ob Sölden [SOS]) and in parallel with cells from a culture of Actinoplanes philippinensis (DSMZ 43019). This organism features a single-base mismatch to the probe at a central location of the probe target site on the 16S rRNA. It was therefore comparable to other database entries of uncultured microbes outside the acI group that feature such a mismatch. Probe AcI-1214 is targeted to the same group as AcI-852, i.e., the acI lineage (Table 2). Stringent hybridization conditions for AcI-1214 were established by comparative analysis of the fraction of hybridized cells in several environmental samples at increasing stringency (i.e., concentrations of formamide in the hybridization buffer). The hybridization conditions for probes AcI-840-1 to AcI-840-3 were derived from those reported by Glöckner et al. (13) for probe HG1-840 and recalculated for hybridization at 35°C (i.e., 30% of formamide instead of 10%).

CARD-FISH.
Fluorescence in situ hybridization with horseradish-peroxidase-labeled probes and tyramide signal amplification was performed as described previously (30) using the modified permeabilization protocol developed for freshwater bacterioplankton (36). Specificity of hybridizations at 35°C was established by the increased addition of formamide to the hybridization buffers (Table 2). Probe AcI-852 and the set of AcI-840-1 to AcI-840-3 probes required the presence of unlabeled helper oligonucleotides to improve the accessibility of the probe target site on the 16S rRNA (12). Helpers were added to the hybridization buffer at the same concentration as the labeled probes (0.5 ng µl–1). Counterstaining of catalyzed reporter deposition (CARD)-FISH preparations with 4',6'-diamidino-2-phenylindole (DAPI; 1 µg ml–1), mounting, and microscopic evaluations were performed as described previously (29). Total bacterial abundances and the fractions of FISH-stained bacteria in at least 1,000 DAPI-stained cells per sample were quantified at a 1,000x magnification on a Zeiss Axioplan II epifluorescence microscope (Carl Zeiss, Jena, Germany).

Leucine and BrdU incorporation.
Bacterial production was estimated via the incorporation of [14C]leucine (Amersham, Little Chalfont, England). Radiolabeled leucine was added at saturating concentrations (20 nM) to triplicate subsamples and to one formaldehyde-killed control (3% final concentration). The treatments were incubated at in situ temperature in the dark for 1 h and fixed by the addition of formaldehyde (3%). Macromolecule extractions were performed as previously described (41). Measurements of leucine incorporation into bacterial biomass were done using a Beckman LS5000TD liquid scintillation counter (Beckman, Fullerton, CA).

For the determination of the fraction of cells with DNA de novo synthesis (31), subsamples of 50 ml were incubated in triplicate at in situ temperature with 5-bromo-2'-deoxyuridine (BrdU; final concentration, 20 µM; Roche Diagnostics, Mannheim, Germany) and thymidine (final concentration, 33 nM) for 4 h in the dark. Subsequently, the samples were fixed with equal volumes of ethanol (final concentration, 50%) and filtered onto polycarbonate membrane filters (type GTTP; diameter, 47 mm) as described above for CARD-FISH. One sample that was fixed prior to BrdU and thymidine addition served as the control. The immunocytochemical detection of incorporated BrdU in bacterial cells and in members of the Actinobacteria was performed using an improved version of the original protocol (28). Subsequently, samples were hybridized with probe EUBI-III (9) and HGC69a (35) by the modified CARD-FISH procedure of Sekar et al. (36). The fractions of BrdU-incorporating cells were determined in at least 1,000 probe-positive cells per sample on an Axioplan II epifluorescence microscope (Carl Zeiss) at a 1,000x magnification.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sampled lakes.
The physical and chemical characteristics of the sampled lakes reflected a wide range of environmental conditions (Table 1). For example, the surface water temperature ranged from 11.7 to 23.4°C, the pH from 6.25 to 8.41, and the DOC concentrations from 0.5 to 2.5 mg liter–1. The highest DOC concentrations were measured in lakes situated <1,000 m a.s.l. within a forested catchment area (ACH, PIB). Bacterial cell numbers varied over 1 order of magnitude (Table 1). The highest cell densities were found in mesotrophic PIB and the lowest ones in Drachensee (DRA) and GKS. The FISH detection rate of bacteria as determined with the probe EUBI-III ranged from 63% to 95% and showed a significant trend to increase in lakes situated at higher altitudes (r2 = 0.87; P < 0.01) (Table 1).

Design of FISH probes targeting freshwater Actinobacteria.
Two probes targeting the whole acI lineage (47) were designed and tested during this study (Table 2). Both probes were found using the Primrose software. Cells from the A. philippinensis culture hybridized with probe AcI-852 were no longer visible at 55% formamide or above in the hybridization buffer (data not shown). This concentration was therefore used to determine the abundance of members of the acI lineage in environmental samples (Fig. 1A). Since the counts with both probes for the acI lineage, AcI-852 and AcI-1214, were very similar, it is likely that equivalent populations were targeted (i.e., that specific hybridization conditions were correctly established). The observed differences between probes (±2.5%) were within the typical error margin of the FISH counting method (33).



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  		FIG. 1. (A) Relative abundances of members of the class Actinobacteria and of the acI lineage in the surface waters of 10 mountain lakes. (B) Relative abundances of different subgroups of Actinobacteria from the acI lineage. A detailed description of the different probes is given in Table 1.

 
The previously published probe HG1-840 is targeted to many but not all sequences of the acI lineage of Actinobacteria (13). It was originally designed with a wobble at position 6 (G or C). Since we strived for a higher phylogenetic resolution of the actinobacterial community structure, these two sequence variants were applied separately (probes AcI-840-1 and AcI-840-2) (Table 2). The two probes approximately cover the subclusters acI-A and acI-B as defined by Warnecke et al. (47). A third variant (AcI-840-3) was designed by manual sequence comparison. It is targeted to a few sequences inside the acI-B subcluster. In order to depict the phylogenetic affiliations of the targets for the AcI-840 probe set, a phylogenetic tree from all currently available nearly complete 16S rRNA gene sequences from the acI clade was calculated as described previously (47).

Evidence for the high specificity of these three probes was provided by the contrasting counting results found in the environmental samples (Fig. 1B). For example, 23% of total bacterial counts were detected with probe AcI-840-1 in Oberer Plenderlesee (OPL), but only 2% with probe AcI-840-2 (Fig. 1B). In contrast, the majority of bacterial cells in SOS (53%) was detected with probe AcI-840-2, but only 5% with AcI-840-1. These observations suggest that most probably, there was no unspecific cross-hybridization between the two probes. Similar unequivocal differences between the fractions of hybridized cells were also obtained with the probe pair AcI-840-1 and AcI-840-3 (e.g., in Rotfelssee [ROT] and DRA) (Fig. 1B).

Application of probe AcI-852 and the AcI-840-1 to AcI-840-3 probe set was hampered by a low in situ accessibility of the target site, as indicated by their respective positions in the lowest 16S rRNA accessibility classes (IV and V, respectively), as defined by Behrens et al. (4). Consequently, two helper oligonucleotides (Table 2) were required to enhance the signal intensities of probe AcI-852. The three helper oligonucleotides designed by Glöckner et al. (13) were sufficient to perform FISH with the AcI-840-1 to AcI-840-3 probe set.

Abundances of Actinobacteria.
The contribution of Actinobacteria to the bacterioplankton communities varied from <5% in Mittlerer Plenderlesee (MPL) to 70% in SOS, as determined with the general actinobacterial probe HGC69a (Fig. 1A). FISH with two specific probes for the same subgroup (AcI-852, AcI-1214) revealed that the vast majority of actinobacterial phylotypes in the studied habitats belonged to the acI lineage (Fig. 1A). This was also supported by low counts obtained with other probes targeting subclades A through C of the acII lineage, the acII-D clade, or "Luna" cluster (16), and the A and B subclusters of the acIV clade (data not shown).

Large cell populations were detected by the subgroup-specific probes AcI-840-1 and AcI-840-2 in several of the investigated lakes (Fig. 1B). For example, in SOS, the AcI-840-2 sequence type was dominant. In contrast, large cell populations were detected by probe AcI-840-1 in GKS and OPL, whereas Actinobacteria of sequence type AcI-840-2 were rare. In some lakes (e.g., ROT and DRA), a substantial proportion of the acI lineage, as determined with AcI-852, could not be detected with either of the two probes (Fig. 1B). This led to a reinvestigation of sequence data within the acI lineage. Only three of the nearly complete sequences in this cluster were not detected by either probe AcI-840-1 or AcI-840-2 because of a single-base mismatch with probe AcI-840-2 (Fig. 2). Additionally, 17 partial sequences in the data set carried the same single-nucleotide exchange. These sequences originate from three independent studies from the River Weser (37), the eutrophic Lake Zwischenahner Meer (7), and a Siberian reservoir (46). Thus, a sequencing artifact appeared unlikely. Counts with the corresponding probe AcI-840-3 (Table 2) revealed that this sequence type represented, e.g., 15% of total cell numbers in ROT and 10% in DRA and SOS (Fig. 1B).



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  		FIG. 2. Phylogenetic tree of all full (>1400 nucleotides) and selected partial (>1,000 nucleotides) actinobacterial 16S rRNA sequences from the acI lineage. Sequences depicted in boldface represent the targets of the three probes AcI-840-1 (underlined), AcI-840-2 (italics), and AcI-840-3 (framed in box). Partial sequences are marked by asterisks. The bar represents 10% of the estimated sequence divergence.

 
Abundances of ß-proteobacteria.
ß-Proteobacteria also represented an abundant lineage in some of the bacterial communities (Fig. 3). Counts with the general probe Bet42a varied between 7% and 38%, with the lowest contribution found in mesotrophic PIB and the highest in oligotrophic Seebensee (SEE). Only a relatively low fraction of ß-proteobacteria could be assigned to two freshwater subgroups (13) with specific probes targeted to the beta I (38) and beta II lineages (8).



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  		FIG. 3. Relative abundances of members of the ß-proteobacteria and freshwater beta I (probe R-Bt065) and beta II (probe Bet2-870) lineages in the surface waters of 10 mountain lakes.

 
Growth state of the bacterial populations.
Bulk incorporation of [14C]leucine varied by about 1 order of magnitude across the studied lakes (Fig. 5). Bacteria from the mesotrophic PIB showed the highest uptake rate (ca. 330 pmol liter–1 h–1), whereas the lowest values were found in the oligotrophic DRA (ca. 33 pmol liter–1 h–1). DNA de novo synthesis was detected in freshwater Actinobacteria via BrdU incorporation (Fig. 4). The incubation experiments revealed total densities of ca. 0.2 x 106 to 0.8 x 106 DNA-synthesizing bacteria ml–1 in lakes Geirneggsee (GEI), MPL, and PIB (Fig. 5). The smallest population of cells with visible DNA de novo synthesis was again detected in DRA. There was no significant correlation between the incorporation of radiolabeled leucine and the total abundances of BrdU-active bacteria (r2 = 0.35; P > 0.05) (Fig. 5).



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  		FIG. 5. Incorporation rate of [14C]leucine into bacterial biomass and fractions of cells with detectable DNA synthesis (BrdU-positive cells).

 


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  		FIG. 4. Photomicrographs of hybridized Actinobacteria (A) and of all cells with incorporated BrdU (B) (same microscopic field) in the surface waters of GEI. Arrows highlight examples of double-stained cells. Bar, 10 µm.

 
A comparison of the fractions of Actinobacteria with visible DNA de novo synthesis with all BrdU-positive bacteria revealed that there was no significant deviation from the 1:1 line in the majority of studied lakes (Fig. 6). In SEE and ACH, a fraction of dividing Actinobacteria significantly higher than the community average was observed. A smaller than average amount of active Actinobacteria was found in the surface water microbial assemblage of PIB.



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  		FIG. 6. Comparison of the relative abundances of BrdU-positive Actinobacteria with the total fraction of BrdU-positive bacteria in the different lakes. Lakes with significant deviation from a 1:1 relationship (broken line) are labeled.

 
Actinobacterial abundances and water UV transparency.
In 3 of the 10 lakes (GKS, ACH, and PIB), the Kd320 was measured in situ. The measured values were very similar to those estimated using the empirical model of Laurion et al. (21) (for GKS, modeled, 0.22 m–1, and measured, 0.25 m–1; for ACH, modeled, 1.71 m–1, and measured, 1.72 m–1; for PIB, modeled, 2.98 m–1, and measured, 3.05 m–1). The relative contribution of Actinobacteria from the acI lineage to the total bacterial abundance was negatively correlated with the estimated Kd320 in lakes above the treeline. Seventy-two percent of the observed variation in community contribution could be explained by this single parameter if the lakes from <1,000 m a.s.l. (ACH and PIB) were excluded from the data set (Fig. 7A). A significant correlation was also observed with the DOC-specific UV absorption across the whole data set (Fig. 7B). No correlations were found with other measured environmental parameters, e.g., altitude or the ratio of lake-to-catchment area (a proxy for the importance of the watershed as a source of Actinobacteria).



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  		FIG. 7. Relationships between the fraction of Actinobacteria from the acI lineage (as determined by probe AcI-852) and the modeled diffuse UV-B attenuation coefficient at 320 nm (A) or the UV absorption at 320 nm normalized to the respective concentrations of DOC (B). In panel A, the two lakes situated below the timberline (open symbols) were excluded from the regression.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bacterioplankton populations in mountain lakes.
As illustrated in earlier investigations, oligotrophic alpine lakes are habitats that can be readily studied by FISH (13, 32) or CARD-FISH (36). In our investigation, the detection rates of hybridized bacteria (i.e., the fraction of cells hybridized by probe EUBI-III) were positively correlated with lake altitude (r2 = 0.87; P < 0.001) (Table 1). At present, we can only speculate about this result. It is, for example, conceivable that high mountain lakes contain smaller fractions of objects that typically lead to the overestimation of bacterial abundances when using DNA staining and epifluorescence microscopy. Such objects could be dead bacterial cells (50), bacteria-sized DNA-containing detrital particles (26), or large viruses (39).

Members of the acI clade (47) were the most prominent phylogenetic group of Actinobacteria in all studied mountain lakes (Fig. 1A). This was confirmed by two newly designed probes (AcI-1214, AcI-852). At the selected hybridization conditions, AcI-852 could readily discriminate a single mismatching base in A. philippinensis. Since a similarly powerful testing system was not available for AcI-1214, we recommend using probe AcI-852 to quantify bacteria affiliated with the acI clade in freshwater by FISH.

The newly designed probes covered the acI clade much more completely than the previously published probe HG1-840 (13) (Fig. 1B). The original probe for this lineage designed by Glöckner et al. (13) features a G-C wobble at probe position 6 (5'-3'). If the probe was split into its two components (AcI-840-1, AcI-840-2) (Table 1), then the sums of cells that hybridized with these two probe variants were significantly smaller than the total fraction of acI-related bacteria (as detected by probe AcI-852) in 8 out of the 10 lakes studied (Fig. 1B). We therefore constructed a third probe modification, AcI-840-3 (G at position 8) (Table 1). In several of the lakes studied, a prominent fraction of cells was hybridized by this probe (Fig. 1B). For example, in ROT, >15% of hybridized cells belonged to this group, which corresponded to more than one-third of all Actinobacteria in this lake. Therefore, at least three closely related subpopulations of the cosmopolitan acI clade may coexist in the water column of lakes. Interestingly, a varying fraction of members of the acI clade (as determined by FISH with two cluster-specific probes, AcI-852 and AcI-1214) (Fig. 1A) could not be detected by any of the AcI-840 probe variants (Fig. 1B). The three AcI-840 probes cover all but two of the published acI sequence types consisting of >1,000 nucleotides (Fig. 2). Our FISH data thus hint at additional 16S rRNA diversity within the actinobacterial acI group in mountain lakes.

Cells from the actinobacterial lineages acII to acIV were rare or absent in the studied lakes, as determined by other specific FISH probes (data not shown). Specifically, we found no or only very few bacteria related to the Luna cluster (16) (the D subclade) of the acII Actinobacteria. Recently, several isolates from this clade have been described (16), and in situ studies have been called for "to reveal the contributions of these bacteria to the bacterioplankton of different freshwater ecosystems" (16). In view of our results, it is likely that members of the acII clade play a minor role in the plankton of oligo- to mesotrophic mountain lakes (Fig. 1A).

Since our study was focused mainly on Actinobacteria, we did not attempt an exhaustive community analysis by FISH. Nevertheless, we also determined the abundances of two groups of ß-proteobacteria known to form prominent populations in a eutrophic reservoir (probe R-Bt065 for the freshwater beta I clade) (38) and in an acidified forest lake (probe BET2-870 for the freshwater beta II clade) (8). We found evidence for the occurrence of both groups in several lakes. For example, 5% and >10% of all bacteria were members of the freshwater beta I lineage in GKS and OPL, respectively (Fig. 3). In a previous study, bacterial populations affiliated with the beta I clade were abundant in the water column of GKS over periods of several months (32). Interestingly, sequences of the puf operon have been found in an isolate from the beta I clade that is closely related to a 16S rRNA gene sequence type from GKS (27). We thus speculate that some freshwater ß-proteobacteria in oligotrophic mountain lakes might be capable of photosynthesis. Currently, it is unknown if bacteria from the culturable beta II (Polynucleobacter necessarius) lineage (15) might also be capable of photosynthesis. In contrast to the predominance of members of the acI lineage within the freshwater Actinobacteria (Fig. 1A), bacteria of the beta I and beta II subgroups typically represented only a minor fraction of all ß-proteobacteria in the studied systems (Fig. 2). At present, it remains unknown which are the numerically important phylogenetic groups of ß-proteobacteria in these mountain lakes.

Bacterial growth state and activity.
One major goal of this study was to determine whether freshwater Actinobacteria represent an active component within the picoplankton assemblages of mountain lakes. Soil is considered to be a typical habitat of many actinobacterial groups (14), so theoretically, such bacteria might be introduced to lakes from the catchment as dormant cells. Moreover, some gram-positive freshwater Actinobacteria are highly resistant to protistan grazing (16). If such bacteria thus experience significantly lower mortality rates than representatives from other bacterial lineages, they might not require rapid growth to maintain high densities in the water column (34). We therefore hypothesized that the fraction of growing Actinobacteria would be significantly lower than the community average (i.e., the total fraction of growing bacteria). However, this was not the case. Between 14 and 33% of bacterial cells (mean, 20%) exhibited DNA de novo synthesis (Fig. 6), as detected by the visible incorporation of BrdU (31) (Fig. 4). The percentage of BrdU-positive Actinobacteria was statistically indistinguishable from the total fraction of DNA-synthesizing bacteria across the set of studied lakes, and it was even significantly higher for two lakes (ACH and SEE) (Fig. 6). Therefore, it cannot be concluded that Actinobacteria in the studied lakes were dormant or that they were more slowly growing than the community average.

There was no significant correlation between the incorporation of radiolabeled leucine and the total abundances of BrdU-active bacteria. These two parameters address conceptually very different aspects of microbial activity. Leucine uptake is a proxy for the rate of total biomass production, whereas the number of BrdU-positive cells reflects the fraction of cells that are in a particular phase of their cell cycle (DNA synthesis). Interestingly, the correlation between the two parameters drastically improved (r2 = 0.90; P < 0.001) if the two lakes that exhibited the highest chlorophyll a concentrations (GEI and MPL) were excluded (Table 1). Substantially high rates of leucine incorporation per BrdU-positive (i.e., growing) cell were observed in these two lakes, compared to all other study sites (for GEI, 1.3 fmol liter–1 h–1, and for MPL, 3.8 fmol liter–1 h–1; average for other eight lakes, 0.6 fmol liter–1 h–1). One might speculate that the presence of fewer, but more highly active, microbial cells in GEI and MPL is related to the higher levels of phytoplankton in these lakes (Table 1).

Are Actinobacteria favored in UV-transparent lakes?
Another aim of this study was to explore whether there exists a relationship between the fraction of Actinobacteria in the surface bacterioplankton assemblages of mountain lakes and the ambient levels of solar UV irradiation in the water column. The sites under investigation were therefore selected to cover a wide range of incident UV radiation and UV transparency conditions, two factors that increase with altitude (21).

The highly significant correlation between the community contribution of Actinobacteria and the UV transparency in lakes above the treeline (Fig. 7A) suggest that this group may be particularly well adapted to the UV stress that is characteristic of clear, high-altitude mountain lakes. However, the observed relationship is correlative rather than causal. Other, unknown parameters that covary with UV transparency might be ultimately responsible for the contrasting abundances of Actinobacteria in the different lakes. In addition, it is unknown if actinobacterial population sizes in the study sites are stable or if they are subject to rapid fluctuations. Thus, our results need to be extended by more direct experimental evidence, such as in situ incubation experiments in the presence and absence of UV radiation (40, 41).

Many of the genetic mechanisms involved in DNA repair, e.g., recA, are virtually ubiquitous in bacteria, and there is extensive regulatory overlap of DNA repair and other stress-induced responses (25). Nevertheless, there is a wide variation in the phenotypic expression of UV radiation survival among different bacterial species and strains. Some pigmented bacteria tolerate significantly higher levels of UV radiation. Pigment production is widespread within Actinobacteria, e.g., yellow-pigmented ultramicrobacteria from the acII-D (Luna) cluster have recently been isolated from various lakes (16). The majority of UV-resistant bacterial isolates obtained from surface and subsurface soil habitats were gram positive (1). The higher resistance of such bacteria has also been assigned to components in gram-positive cell walls that may help deflect UV photons (18). Enhanced UV resistence is furthermore observed in species with unusually efficient DNA repair mechanisms, such as Deinococcus radiodurans (3). D. radiodurans or the anoxygenic phototroph Rhodobacter sphaeroides has a 65% G+C genomic base composition (3, 23). The genomes of these bacteria feature a significantly higher frequency of CC (rather than TT) dimers than Escherichia coli, which has been interpreted as one possible cause of higher UV resistance (23). The high genomic G+C content of Actinobacteria (20) may therefore potentially also play a role in enhanced UV resistance.

Unfortunately, there are currently no isolated representatives of Actinobacteria from the acI clade. The group is phylogenetically rather distanced from the most closely related genera of cultured representatives (Sporichthya, Kineosporia, Cellulomonas) (see Fig. 3 in reference 47). Thus, it is currently impossible to draw conclusions about the physiological properties of acI Actinobacteria that might promote higher UV resistance.

In any case, a higher UV resistance of Actinobacteria would likely be only one among several reasons that explain the apparent success of this group in freshwater bacterioplankton. This is suggested by the significant correlation between acI Actinobacteria and the DOC-specific absorption at 320 nm (Fig. 7B), a proxy for DOC quality. Moreover, high abundances of such bacteria have also been found, e.g., in a humic forest lake (8). Since Actinobacteria appear to be an autochthonous component of these lakes (Fig. 6), it is moreover likely that their fate is related to organic carbon released by nutrient-limited primary producers. However, the importance of such interactions cannot be assessed from our data set, e.g., we could not find a statistical relationship between chlorophyll a concentrations and actinobacterial abundance.

In summary, our data provide evidence that Actinobacteria from different lineages of the acI clade (47) are active members of the microbial assemblages in mountain lakes. Moreover, there appears to be a relationship between their community contribution in lakes above the treeline and water transparency (Fig. 7). Currently, it remains unresolved whether acI is indeed the only numerically dominant freshwater actinobacterial lineage or if groups such as the acII-D (Luna) cluster (16) might be abundant in other types of lakes.


   ACKNOWLEDGMENTS
 
We thank Claudia Piccini for carrying enormous amounts of water, and we are grateful to Josef Franzoi and Eike Stübner for their help during sample evaluation. Rudolf Amann is gratefully acknowledged for continuous support and discussion.

This study was supported by the German Ministry of Education and Research (BMBF 01 LC0021/TP4), by the Max Planck Society, and by the Austrian Science Foundation (P14153-BIO to R.S.).


   FOOTNOTES
 
* Corresponding author. Present address: Limnological Station, Institute of Plant Biology, University of Zurich, Seestr. 187, CH-8802 Kilchberg, Switzerland. Phone: 41 1 716 1210. Fax: 41 1 716 1225. E-mail: pernthaler{at}limnol.unizh.ch. Back

{dagger} Present address: Microbial Ecology Program, DOE Joint Genome Institute, Walnut Creek, Calif. Back

{ddagger} Present address: Department of Biological Sciences, Florida International University, Miami, Fla. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Arrage, A. A., T. J. Phelps, R. E. Benoit, and D. C. White. 1993. Survival of subsurface microorganisms exposed to UV radiation and hydrogen peroxide. Appl. Environ. Microbiol. 59:3545-3550.[Abstract/Free Full Text]
  2. Ashelford, K. E., A. J. Weightman, and J. C. Fry. 2002. PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database. Nucleic Acids Res. 30:3481-3489.[Abstract/Free Full Text]
  3. Battista, J. R. 1997. Against all odds: the survival strategies of Deinococcus radiodurans. Annu. Rev. Microbiol. 51:203-224.[CrossRef][Medline]
  4. Behrens, S., C. Ruhland, J. Inácio, H. Huber, Á. Fonseca, I. Spencer-Martins, B. M. Fuchs, and R. Amann. 2003. In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes. Appl. Environ. Microbiol. 69:1748-1758.[Abstract/Free Full Text]
  5. Bentley, S. D., and J. Parkhill. 2004. Comparative genomic structure of prokaryotes. Annu. Rev. Genet. 38:771-792.[CrossRef][Medline]
  6. Boelen, P., M. J. W. Veldhuis, and A. G. J. Buma. 2001. Accumulation and removal of UVBR-induced DNA damage in marine tropical plankton subjected to mixed and simulated non-mixed conditions. Aquat. Microb. Ecol. 24:265-274.
  7. Bruns, A., U. Nübel, H. Cypionka, and J. Overmann. 2003. Effect of signal compounds and incubation conditions on the culturability of freshwater bacterioplankton. Appl. Environ. Microbiol. 69:1980-1989.[Abstract/Free Full Text]
  8. Burkert, U., F. Warnecke, D. Babenzien, E. Zwirnmann, and J. Pernthaler. 2003. Members of a readily enriched ß-proteobacterial clade are common in the surface waters of a humic lake. Appl. Environ. Microbiol. 69:6550-6559.[Abstract/Free Full Text]
  9. Daims, H., A. Bruhl, R. Amann, K. H. Schleifer, and M. Wagner. 1999. The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22:434-444.[Medline]
  10. Eiler, A., and S. Bertilsson. 2004. Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes. Environ. Microbiol. 6:1228-1243.[CrossRef][Medline]
  11. Ensign, J. C. 1978. Formation, properties, and germination of actinomycete spores. Annu. Rev. Microbiol. 32:185-219.[CrossRef][Medline]
  12. Fuchs, B. M., F. O. Glöckner, J. Wulf, and R. Amann. 2000. Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 66:3603-3607.[Abstract/Free Full Text]
  13. Glöckner, F.-O., E. Zaichikov, N. Belkova, L. Denissova, J. Pernthaler, A. Pernthaler, and R. Amann. 2000. Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an abundant group of actinobacteria. Appl. Environ. Microbiol. 66:5053-5065.[Abstract/Free Full Text]
  14. Goodfellow, M., and S. T. Williams. 1983. Ecology of actinomycetes. Annu. Rev. Microbiol. 37:189-216.[CrossRef][Medline]
  15. Hahn, M. W. 2003. Isolation of strains belonging to the cosmopolitan Polynucleobacter necessarius cluster from freshwater habitats located in three climatic zones. Appl. Environ. Microbiol. 69:5248-5254.[Abstract/Free Full Text]
  16. Hahn, M. W., H. Lünsdorf, Q. Wu, M. Schauer, M. G. Höfle, J. Boenigk, and P. Stadler. 2003. Isolation of novel ultramicrobacteria classified as Actinobacteria from five freshwater habitats in Europe and Asia. Appl. Environ. Microbiol. 69:1442-1451.[Abstract/Free Full Text]
  17. Hiorns, W. D., B. A. Methe, S. A. Nierzwicki-Bauer, and J. P. Zehr. 1997. Bacterial diversity in Adirondack mountain lakes as revealed by 16S rRNA gene sequences. Appl. Environ. Microbiol. 63:2957-2960.[Abstract]
  18. Jagger, J. 1983. Physiological effects of near-ultraviolet radiation on bacteria. Photochem. Photobiol. Rev. 7:1-75.
  19. Jeffrey, W. H., R. J. Pledger, P. Aas, S. Hager, R. B. Coffin, R. VonHaven, and D. L. Mitchell. 1996. Diel and depth profiles of DNA photodamage in bacterioplankton exposed to ambient solar ultraviolet radiation. Mar. Ecol. Prog. Ser. 137:283-291.
  20. Kunisawa, T. 2003. Gene arrangements and branching orders of gram-positive bacteria. J. Theor. Biol. 222:495-503.[Medline]
  21. Laurion, I., M. Ventura, J. Catalan, R. Psenner, and R. Sommaruga. 2000. Attenuation of ultraviolet radiation in mountain lakes: factors controlling the among- and within-lake variability. Limnol. Oceanogr. 45:1274-1288.
  22. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, Yadhukumar, A. Buchner, T. Lai, S. Steppi, G. Jobb, W. Forster, I. Brettske, S. Gerber, A. W. Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. Konig, T. Liss, R. Lussmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, N. Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode, and K. H. Schleifer. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 32:1363-1371.[Abstract/Free Full Text]
  23. Mackenzie, C., M. Chidambaram, E. J. Sodergren, S. Kaplan, and G. M. Weinstock. 1995. DNA repair mutants of Rhodobacter sphaeroides. J. Bacteriol. 177:3027-3035.[Abstract/Free Full Text]
  24. Manz, W., R. Amann, W. Ludwig, M. Wagner, and K.-H. Schleifer. 1992. Phylogenetic oligodeoxynucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15:593-600.
  25. Miller, R. V., and T. A. Kokjohn. 1990. General microbiology of RecA— environmental and evolutionary significance. Annu. Rev. Microbiol. 44:365-394.[Medline]
  26. Nagata, T., and D. L. Kirchman. 1997. Roles of submicron particles and colloids in microbial food webs and biogeochemical cycles within marine environments, p. 81-103. In J. G. Jones (ed.), Advances in microbial ecology, vol. 15. Plenum Press, New York, N.Y.
  27. Page, K. A., S. A. Connon, and S. J. Giovannoni. 2004. Representative freshwater bacterioplankton isolated from Crater Lake, Oregon. Appl. Environ. Microbiol. 70:6542-6550.[Abstract/Free Full Text]
  28. Pernthaler, A., and J. Pernthaler. 2005. Diurnal variation of cell proliferation in three bacterial taxa from coastal North Sea waters. Appl. Environ. Microbiol., 71:4638-4644.
  29. Pernthaler, A., J. Pernthaler, and R. Amann. 2002. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol. 68:3094-3101.[Abstract/Free Full Text]
  30. Pernthaler, A., J. Pernthaler, and R. Amann. 2004. Sensitive multicolour fluorescence in situ hybridization for the identification of environmental organisms, p. 711-726. In G. A. Kowalchuk, F. J. De Bruijn, I. M. Head, A. D. L. Akkermans, and J. D. van Elsas (ed.), Molecular microbial ecology manual, 2nd ed. Kluwer Academic Publishers, Dordecht, The Netherlands.
  31. Pernthaler, A., J. Pernthaler, M. Schattenhofer, and R. Amann. 2002. Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton. Appl. Environ. Microbiol. 68:5728-5736.[Abstract/Free Full Text]
  32. Pernthaler, J., F. O. Glöckner, S. Unterholzner, A. Alfreider, R. Psenner, and R. Amann. 1998. Seasonal community and population dynamics of pelagic Bacteria and Archaea in a high mountain lake. Appl. Environ. Microbiol. 64:4299-4306.[Abstract/Free Full Text]
  33. Pernthaler, J., A. Pernthaler, and R. Amann. 2003. Automated enumeration of groups of marine picoplankton after fluorescence in situ hybridization. Appl. Environ. Microbiol. 69:2631-2637.[Abstract/Free Full Text]
  34. Pernthaler, J., T. Posch, K. Simek, J. Vrba, A. Pernthaler, F. O. Glöckner, U. Nübel, R. Psenner, and R. Amann. 2001. Predator-specific enrichment of actinobacteria from a cosmopolitan freshwater clade in mixed continuous culture. Appl. Environ. Microbiol. 67:2145-2155.[Abstract/Free Full Text]
  35. Roller, C., M. Wagner, R. Amann, W. Ludwig, and K.-H. Schleifer. 1994. In situ probing of gram-positive bacteria with high DNA G+C content using 23S rRNA-targeted oligonucleotides. Microbiology 140:2849-2858.[Abstract]
  36. Sekar, R., A. Pernthaler, J. Pernthaler, F. Warnecke, T. Posch, and R. Amann. 2003. An improved protocol for the quantification of freshwater actinobacteria by fluorescence in situ hybridization. Appl. Environ. Microbiol. 69:2928-2935.[Abstract/Free Full Text]
  37. Selje, N., and M. Simon. 2003. Composition and dynamics of particle-associated and free-living bacterial communities in the Weser estuary, Germany. Aquat. Microb. Ecol. 30:221-237.[CrossRef]
  38. Simek, K., J. Pernthaler, M. G. Weinbauer, K. Hornák, J. R. Dolan, J. Nedoma, M. Masín, and R. Amann. 2001. Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir. Appl. Environ. Microbiol. 67:2723-2733.[Abstract/Free Full Text]
  39. Sommaruga, R., M. Krössbacher, W. Salvenmoser, J. Catalan, and R. Psenner. 1995. Presence of large virus-like particles in a eutrophic reservoir. Aquat. Microb. Ecol. 9:305-308.
  40. Sommaruga, R., A. Oberleiter, and R. Psenner. 1996. Effect of UV radiation on the bacterivory of a heterotrophic nanoflagellate. Appl. Environ. Microbiol. 62:4395-4400.[Abstract]
  41. Sommaruga, R., I. Obernosterer, G. J. Herndl, and R. Psenner. 1997. Inhibitory effect of solar radiation on thymidine and leucine incorporation by freshwater and marine bacterioplankton. Appl. Environ. Microbiol. 63:4178-4184.[Abstract]
  42. Sommaruga, R., and R. Psenner. 1997. Ultraviolet radiation in a high mountain lake of the Austrian Alps: air and underwater measurements. Photochem. Photobiol. 65:957-963.
  43. Sommaruga, R., R. Psenner, E. Schafferer, K. A. Koinig, and S. Sommaruga-Wögrath. 1999. Dissolved organic carbon concentration and phytoplankton biomass in high-mountain lakes of the Austrian Alps: potential effect of climatic warming an UV underwater attenuation. Arct. Antarct. Alp. Res. 31:247-253.[CrossRef]
  44. Stackebrandt, E., F. A. Rainey, and N. L. Wardrainey. 1997. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int. J. Syst. Bacteriol. 47:479-491.[CrossRef]
  45. Suttle, C. A., and C. Feng. 1992. Mechanisms and rates of decay of marine viruses in seawater. Appl. Environ. Microbiol. 58:3721-3729.[Abstract/Free Full Text]
  46. Trusova, M. Y., and M. I. Gladyshev. 2002. Phylogenetic diversity of winter bacterioplankton of eutrophic Siberian reservoirs as revealed by 16S rRNA gene sequences. Microb. Ecol. 44:252-259.[CrossRef][Medline]
  47. Warnecke, F., J. Pernthaler, and R. Amann. 2004. Actinobacterial 16S rRNA genes from freshwater habitats cluster in four distinct lineages. Environ. Microbiol. 6:242-253.[CrossRef][Medline]
  48. Zwart, G., B. C. Crump, M. Agterveld, F. Hagen, and S. K. Han. 2002. Typical freshwater bacteria: an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers. Aquat. Microb. Ecol. 28:141-155.[CrossRef]
  49. Zwart, G., E. J. van Hannen, M. P. Kamst-van Agterveld, K. Van der Gucht, E. S. Lindstrom, J. Van Wichelen, T. Lauridsen, B. C. Crump, S. K. Han, and S. Declerck. 2003. Rapid screening for freshwater bacterial groups by using reverse line blot hybridization. Appl. Environ. Microbiol. 69:5875-5883.[Abstract/Free Full Text]
  50. Zweifel, U. L., and Å. Hagström. 1995. Total counts of marine bacteria include a large fraction of non-nucleoid-containing bacteria (ghosts). Appl. Environ. Microbiol. 61:2180-2185.[Abstract]

Applied and Environmental Microbiology, September 2005, p. 5551-5559, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5551-5559.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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