Changes in Bacterioplankton Community Structure and Activity with Depth in a Eutrophic Lake as Revealed by 5S rRNA Analysis

The community structure of bacterioplankton was studied at differentdepths (0 to 25 m) of a temperate eutrophic lake (Lake Plußseein northern Germany) by using comparative 5S rRNA analysis.The relative amounts of taxonomic groups were estimated from5S rRNA bands separated by high-resolution electrophoresis.Comparison of partial 5S rRNA sequences enabled detection ofchanges in single taxa over space and during seasons. Overall,the bacterioplankton community was dominated by 3 to 14 abundant(>4% of the total 5S rRNA) taxa. In general, the number of5S rRNA bands (i.e., the number of bacterial taxa) decreasedwith depth. In the fall, when thermal stratification and chemicalstratification were much more pronounced than they were in thespring, the correlation between the depth layers and the communitystructure was more pronounced. Therefore, in the fall each layerhad its own community structure; i.e., there were differentcommunity structures in the epilimnion, the metalimnion, andthe hypolimnion. Only three 5S rRNA bands were detected in thehypolimnion during the fall, and one band accounted for about70% of the total 5S rRNA. The sequences of individual 5S rRNAbands from the spring and fall were different for all size classesanalyzed except two bands, one of which was identified as Comamonasacidivorans. In the overall analysis of the depth profiles,the diversity in the epilimnion contrasted with the reduceddiversity of the bacterioplankton communities in the hypolimnion,and large differences occurred in the composition of the epilimnionat different seasons except for generalists like C. acidivorans.

INTRODUCTION

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Introduction

A fundamental concept in limnology is that the water columnof a lake is divided into three horizontal layers, the epilimnion,the metalimnion, and the hypolimnion (22). These strata areshaped by solar radiation and wind. In temperate regions, theyare characterized by gradients of temperature and oxygen, andthey have different functions for the lake as an ecosystem.The epilimnion is well mixed and acts as a bioreactor for primaryproduction, and the hypolimnion is a sink where biomass accumulatesand is remineralized (43). These two parts are connected bythe metalimnion or thermocline, often characterized by a steepdecrease in temperature. This basic view of the structure ofa lake illustrates that depth is a key factor for all limniclife and that depth profiles are key features for understandingaquatic ecosystems.

The depth dependence of the structure of phyto- and zooplanktoncommunities in lakes and oceans has been studied since the beginningof aquatic ecology and has contributed enormously to the understandingof the functioning of freshwater and marine ecosystems. Despitethe recognition of the important role of bacteria in the microbialfood web and the contribution of bacteria to the functioningof aquatic ecosystems, knowledge about the depth distributionof these organisms is still scarce (1, 5). Pioneering work hasbeen done on the depth-dependent community structure of bacterioplanktonin the oceans by DeLong (6), Giovannoni et al. (15), and Fuhrmanet al. (13). These studies were based on analysis of 16S ribosomalDNA (rDNA) clone libraries from deep ocean samples (500 to 2,000m) and provided insight into the depth distribution of singletaxa. Field et al. (12) obtained higher resolution of the variationin bacterial communities with depth by using group-specific16S rRNA-targeted oligonucleotide probes and environmental bulkDNA-RNA for assessment of the abundance of specific taxa belongingto the ${alpha}$ subgroup of the Proteobacteria in the Sargasso Sea.However, community structure can be assessed most rapidly bycommunity fingerprint methods that are based on electrophoreticseparation of 16S rDNA amplicons or 5S rRNA obtained from naturalmicrobial communities (27, 30, 34, 35). The first communityfingerprinting analysis of bacterioplankton with depth was doneby using amplicons of the V3 region of 16S rRNA and archaealand bacterial primers (29). Øvreås et al. (29)found that the diversity of the bacterial community decreasedfrom the surface to the bottom in an estuary with a strong verticalgradient of salinity and oxygen. However, a major problem ofPCR-based methods is that they can be biased for some phylogeneticgroups, and quantitative assessment of community diversity isdifficult (39). Therefore, we have used 5S rRNA fingerprintingto obtain a direct molecular fingerprint from natural communitiesand have calculated, based on the relative densities of individual5S rRNA bands, the diversity of the bacterioplankton duringthe year (20).

The goals of the present study were to compare changes in physical,chemical, and biological parameters with depth at a high spatialresolution (i.e., every meter) with changes in the structureof the bacterioplankton community and to extend the previousseasonal study in terms of depth (20). The ecosystem chosenwas Lake Plußsee, a naturally eutrophic lake in northernGermany which has been examined extensively (28). We analyzedthe 5S rRNA fraction of environmental rRNA obtained directly(i.e., without PCR) from the bacterioplankton. The structureof the bacterioplankton community as a function of depth wasanalyzed based on high-resolution 5S rRNA community fingerprintsand partial or full sequences of individual 5S rRNA.

MATERIALS AND METHODS

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Materials and Methods

Sampling.
Water samples were collected from the central part of Lake Plußsee(Schleswig-Holstein, Germany; 54°10.0'N, 10°0.23'E)on 25 April and 19 September 1994 by using a 2.5-liter Ruttnersampler at the following depths: 0, 1, 2, 3, 4, 5, 6, 7, 8,10, 15, 20, and 25 m. The bacterioplankton fraction was filteredonto a sandwich consisting of a glass fiber filter (type GF/F;diameter, 90 mm; Whatman Corp.) on top of a polycarbonate filter(pore size, 0.2 µm; Nuclepore Corp.) and was stored frozen(-70°C) for later RNA analysis. The samples had been prefilteredthrough a polycarbonate filter (Nuclepore Corp.) with a poresize of 3 µm to eliminate larger organisms.

Biological background parameters, such as total bacterial counts,total heterotrophic nanoflagellate (HNF) counts, and total particulatechlorophyll a concentrations, were measured by standard techniques;details are given elsewhere (4).

5S rRNA fingerprints of bacterioplankton.
5S rRNA analysis of bacterioplankton started with extractionof the total RNA directly from the frozen filter sandwiches.This extraction was based on mechanical extraction with glassbeads in a bead beater in combination with chemical extractionwith phenol and sodium dodecyl sulfate (SDS) (8, 16). In short,total environmental RNA was extracted from the sandwiches byusing 4 g of 2- and 3-mm-diameter glass beads, a high-speedcell disrupter (Microdismembrator; Braun-Diessel Corp.) witha Teflon extraction cell, and a mixture of 5 ml of RNA extractionbuffer and 5 ml of buffer-saturated phenol (17). Extractionbuffer consisted of 50 mM sodium acetate, 10 mM EDTA, and 1%SDS (pH 4.2). Phenol was saturated with extraction buffer lacking1% SDS until a final pH of 4.2 to 4.4 was reached. A thin slurry(total volume, about 12 ml) of the two filters and the extractionmixture was generated by vibration for 2 min with the cell disrupter.This slurry was then centrifuged for 10 min at 8,500 x g. Afterrepeated phenol (pH 4.2) extraction of the pellet, the supernatantswere pooled and treated twice with 5 ml of chloroform. TotalRNA was precipitated from the pooled supernatant by additionof ethanol (2.5 times the volume of the supernatant) and storageat -20°C overnight.

The total RNA obtained by the filter extraction procedure was3' end labeled with cytidine 3',5'-[5'-³²P]bisphosphate (specificactivity, 3,000 Ci/mmol) by using RNA ligase with an efficiencyof about 62% (7, 10). The radioactively labeled RNA was subjectedto denaturing high-resolution electrophoresis on a 10% polyacrylamidegel (Sequicel; 0.4 mm by 38 cm by 80 cm; Bio-Rad) by using runningconditions consisting of a stepwise increase over a 5-h periodfrom 100 to 300 W. After electrophoresis, the gel was exposedto X-ray film or a storage phosphor screen (Molecular ProbesCorp.) for 30 min to several hours. More details concerningRNA extraction and electrophoretic analysis are given elsewhere(8, 16, 17, 19, 40).

Comparative analysis of 5S rRNA fingerprints.
The 5S rRNA fraction was evaluated quantitatively by scanningthe autoradiograms with an optical gel scanner (Hirschmann Corp.)and by using a phosphorimager (Molecular Probes Corp.). In thisway the abundance of single 5S rRNA bands was quantified interms of baseline corrected peak areas and in terms of numberof pixels per band, respectively. These arbitrary units wereused to calculate the relative abundance of every individual5S rRNA band and its contribution to the total 5S rRNA.

The diversity of the community was determined by determiningthe number of 5S rRNA bands per sample and by calculating theShannon diversity index (H) with the following equation:

, where n_i is the relative abundance of a singletaxon (single 5S RNA band), N is the total abundance of alltaxa, and S is the total number of abundant species. Only bandsaccounting for more than 4% of the total 5S rRNA were includedin the calculation of the diversity index and the total numberof bands. This 4% threshold is not based on the detection limitof the 5S rRNA analysis, which is less than 0.1%. Rather, itis an arbitrary threshold set to make our data analysis comparablewith analyses in earlier studies (18).

Sequence analysis of single environmental 5S rRNA.
After high-resolution electrophoresis, ³²P-labeled 5S rRNA bandswere excised from the gel, eluted with 150 µl of elutionbuffer (0.5 M sodium acetate, 0.1% SDS, 1 mM magnesium chloride,100 µM EDTA; pH 7.8), and ethanol precipitated at -20°Covernight. Before application to the sequencing gel, 5S rRNAwas enzymatically digested with base-specific RNases (9, 16).The cleaved 5S rRNA of a single excised band was run on a high-resolutionsequencing gel (80 by 40 cm; thickness, 0.4 mm;10% acrylamideLongRanger [FMC Corp.] prepared according to the protocol ofthe manufacturer) to determine the positions of the bases incomparison to the bases of totally hydrolyzed 5S rRNA of Escherichiacoli, which was used as the molecular size marker. This sequencinggel provided information about the positions of almost all basesof the 5S rRNA from the 5' end. Only around 2% of the molecule(i.e., two or three nucleotides at the 3' end) could not beread. These sequences could lead to classification or identificationof the taxon represented by a single band provided that thereference sequence of a known bacterial species is availablein the 5S rRNA sequence database (33). Also, this sequence informationwas used to determine the homogeneity of a band (i.e., if oneor more taxa formed the band). Furthermore, it allowed comparisonof 5S rRNA bands of the same length for samples from differentdepths and times of the year. Such comparisons were also possiblewith partial sequences (e.g., by using only RNase T₁ to examinethe positions of all guanine residues within a 5S rRNA bandof a specific size).

RESULTS

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Results

Environmental conditions in Lake Plußsee.
Physical, chemical, and biological background data for spring1994 for Lake Plußsee are given in Fig. 1. The temperaturesteadily decreased with depth from 1 to 10 m, and thus therewas not an extended epilimnion. The oxygen content peaked at2 m, corresponding to the top of the chlorophyll a maximum (Fig.1b), indicating that the maximum primary production occurredat this depth. The phosphate concentrations decreased to about50 µg/liter in the upper 5 m, indicating that the phytoplanktonwas most active in this layer. Below 5 m, the phosphate andinorganic nitrogen contents (7; data not shown) increased rapidlyto the high hypolimnic concentrations. The chlorophyll a concentrationsreached 65 µg/liter, indicating that there was a well-developedspring phytoplankton bloom and that the lake was eutrophic.The phytoplankton community during this spring bloom was composedof a variety of diatoms and green algae (28). The total bacterialnumbers (Fig. 1b) in the upper 5 m were rather constant (morethan 1.2 x 10⁷ cells/ml) and decreased steadily with depth.The concentration of HNF peaked at 3 m at about 1 x 10⁵ cells/ml;the concentration then dropped rapidly to less than 4 x 10³cells/ml. These background data indicate that there was a fairlynormal spring situation for a temperate eutrophic lake witha small, unstable epilimnion (0 to 1 m), an extended metalimnion(1 to 8 m) with maximum primary production close to the surface(2 m), and a hypolimnion below 8 m which was still fully oxic.

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FIG. 1. Physical, chemical, and biological background data for depth profiles of Lake Plußsee during the spring (25 April 1994). (a) Temperature ( ${square}$ ), oxygen content (), and phosphorus content ( ${blacktriangleup}$ ); (b) chlorophyll a content ( ${diamondsuit}$ ), total bacterial number ( ${circ}$ ), and number of HNF ( ${triangleup}$ ).

The fall data (Fig. 2) revealed a homogenous epilimnion (0 to5 m), a metalimnion with very steep decreases in oxygen concentrationand temperature (5 to 8 m), and a fully anoxic hypolimnion atdepths below 8 m. The phosphate concentrations were below thedetection limit in the epilimnion and increased to rather highlevels in the metalimnion. The chlorophyll a concentration waslow in the epilimnion and peaked at 8 m (i.e., in the anoxichypolimnion). The bacterial concentration did not vary significantlywith depth, but the concentration of HNF peaked together withthe chlorophyll a concentration, as it did in the spring. Asin the spring, in the fall of 1994 Lake Plußsee had thevery stable and well-structured depth profile of a naturallyeutrophic lake during autumn (22).

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FIG. 2. Physical, chemical, and biological background data for depth profiles of Lake Plußsee during the fall (19 September 1994). (a) Temperature ( ${square}$ ), oxygen content (), and phosphorus content ( ${blacktriangleup}$ ); (b) chlorophyll a content ( ${diamondsuit}$ ), total bacterial number ( ${circ}$ ), and number of HNF ( ${triangleup}$ ).

Changes in the overall structure of the bacterioplankton community with depth.
In the spring, the community structure for the depth profileshowed three different banding patterns of the 5S rRNA thatcorresponded with the three layers of the lake (Fig. 3a). Theepilimnion (0 to 3 m) had six or seven abundant bands rangingin size from 116 to 123 nucleotides (nt), the metalimnion (4to 8 m) had the highest number of bands with additional bandsin the size range from 109 to 115 nt, and the hypolimnion (10to 25 m) had the lowest number of bands, with one band comprisingmore than 50% of the total 5S rRNA. At greater depths the communitystructure changed the most. For example, at 15 to 20 m the twomost abundant bands comprised more than 70% of the total 5SrRNA and the 5S rRNA bands at 122 and 123 nt had disappeared.The amount of small 5S rRNA (111 to 113 nt), which comprisedabout 20% of the total 5S rRNA, increased in the deepest water(25 m).

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FIG. 3. Autoradiograms of 5S rRNA from bacterioplankton obtained at different depths. Samples of the water column were taken at different depths. For detection the 5S rRNA was 3' end labeled with ³²P. (a) Depth profile for the spring (25 April 1994). Lanes M contained a molecular weight standard consisting of hydrolyzed 5S rRNA from E. coli. The intensity of the bands in the boxed area was electronically reduced for better visibility of the single bands. Each lane is labeled with the depth (in meters) at which the sample examined was obtained. (b) Depth profile for the autumn (19 September 1994): Lanes M contained a molecular weight standard consisting of hydrolyzed 5S rRNA from E. coli. Each lane is labeled with the depth (in meters) at which the sample examined was obtained.

During thermal stratification in the autumn the number of 5SrRNA bands was high in the epilimnion and was greatly reducedbelow the thermocline (Fig. 3b). Also, in the upper 5 m thenumber of bands, their relative positions, and their relativeamounts were very stable. Only at 1 m was an additional bandat 117 nt observed. The bacterioplankton community of the anoxichypolimnion (7 to 25 m) was dominated by a single 5S rRNA bandat 121 nt (on average, this band accounted for 63% of the total5S rRNA from 10 m and below).

The results of a detailed quantitative analysis of the amountof 5S rRNA per abundant band in the autoradiograms, obtainedby using a phosphorimager for quantification, are shown in Fig.4. In both depth profiles the number of abundant bands was ratherlimited, ranging from three to seven. Also, these abundant bands(i.e., bands that accounted for more than 4% of the total 5SrRNA) comprised more than 90% of the total 5S rRNA in most samples(Fig. 4B). The spring profile was slightly more variable inthis respect than the fall profile due to the larger numberof less abundant bands occurring in the metalimnion. Anothergeneral trend for both depth profiles was that in the epilimnionno single band exceeded a relative abundance of 34% and a smallnumber of bands (three to five bands), each comprising 10 to30% of the total, represented the majority of the bacterioplanktoncommunity. With increasing depth one band became dominant inboth depth profiles; i.e., it had a relative abundance of morethan 50%. Furthermore, in both profiles for depths below 15m the dominant band was the same size (121 nt) despite the anoxicconditions in the fall. The two depth profiles did differ, however;when anoxic conditions were reached at depths below 6 m, the123-nt band disappeared and a 113-nt band appeared. In general,changes in the community composition were more gradual in thespring profile than in the fall profile.

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FIG. 4. (A) Relative amounts of the single abundant 5S rRNA bands for the spring shown in Fig. 3a. Different colors indicate 5S rRNA bands of different sizes that account for more than 4% of the total 5S rRNA calculated as detailed in Materials and Methods. (B) Relative amounts of single abundant 5S rRNA bands for the fall shown in Fig. 3b.

To examine overall diversity, the Shannon indices were calculatedfor all samples based on the total number of 5S rRNA bands,not just the number of abundant 5S rRNA bands, and their relativeamounts (Fig. 5). The total number of bands was determined bycounting every band that had a relative abundance of more than0.1% of the total 5S rRNA. This threshold reflects the levelof resolution of the 5S rRNA fingerprinting technique when aphosphorimager is used. In contrast, the 4% threshold for theabundant 5S rRNA is an arbitrary threshold that was introducedearlier to make community fingerprints more comprehensible (18).Additionally, the following assumptions for calculation of theShannon diversity index based on the relative amounts of individual5S rRNA bands were made: (i) RNA was extracted with comparableefficiencies from all taxa, (ii) 5S rRNA was ³²P labeled withcomparable efficiencies, and (iii) sequence analysis of individual5S rRNA shows homogeneity. The validity of these assumptionswas demonstrated in calibration experiments with reference cultures(7, 8, 40) or direct sequencing (see below). During the spring,the overall diversity increased until 5 m and then stayed maximaluntil 10 m. Below 10 m, the diversity decreased by a factorof two until 25 m (Fig. 5a). During the fall, the diversitywas highest in the epilimnion and then declined uniformly inthe anoxic part of the water column (Fig. 5b). In both profilesthe Shannon index generally followed the total number of 5SrRNA bands. The most striking difference was that the maximumdiversity occurred in the spring below the maximum chlorophylla concentration, whereas in the fall the maximum diversity wasfound above the maximum chlorophyll a concentration.

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FIG. 5. (a) Total number of bands () (bands containing more than 0.1% of the total 5S rRNA) and Shannon diversity index ( ${square}$ ) calculated from all 5S rRNA bands at each depth during the spring. The chlorophyll a content ( ${diamondsuit}$ ) is shown for comparison. (b) Total number of bands () and Shannon diversity index ( ${square}$ ) calculated from all 5S rRNA bands at each depth during the fall. The chlorophyll a content ( ${diamondsuit}$ ) is shown for comparison.

Detailed community structure as revealed by 5S rRNA sequence analysis.
A sequence analysis of single 5S rRNA bands was performed byusing base-specific RNases in order to compare 5S rRNA bandsfrom different depths that were the same size and to identifythe closest taxonomic relatives by comparison with the internationalsequence database (33). To examine 5S rRNA bands with depth,only partial sequences were determined by using RNase T₁ andsometimes RNase U₂ digests to obtain a G and/or A ladder ofthe 5S rRNA band for about 80% of the molecule, as describedpreviously (17, 20). In the spring, 5S rRNA bands at 116, 118,120, 121, and 123 nt were compared. All of the bands analyzedhad the same partial sequences from 0 to 8 m. Below 8 m, onlya few bands could be sequenced due to the much lower amountof radioactivity per band (Fig. 3a). The partial sequence ofthe 121-nt band from 25 m, which dominated the community atthis depth, was different from the sequence of the 121-nt bandobtained from the upper 8 m.

In the fall, 5S rRNA bands at 116, 120, 121, 122, and 123 ntwere compared for the upper 5 m. In addition, the 121-nt bandwas sequenced from all samples of the depth profile. There wereno sequence differences detected in any of the fall 5S rRNAbands with depth; i.e., all bands sequenced in the upper 5 mand the 121-nt band found throughout the water column were thesame. A comparison of the sequences from the spring with thesequences from the fall showed significant differences for allof the single size classes analyzed except the 116- and 122-ntbands. These bands had identical partial sequences; i.e., allAs and Gs were at the same position in the 5S rRNA for bothseasons. The 121-nt band, which was the most abundant 5S rRNAin the hypolimnion in both depth profiles, had different sequencesin the two seasons, despite the identical sizes. These sequencedata indicate that different taxa dominated the hypolimnionin the spring and in the fall.

The only 5S rRNA for which a definite phylogenetic relativecould be found was the 116-nt band, which had a sequence identicalto a Comamonas acidovorans sequence (accession number AJ131594).The presence of C. acidovorans has been documented in samplesfrom Lake Plußsee before (11, 20). For all other bandsexcept the 123-nt band either the sequence information obtainedwas not complete enough for alignment with sequences from theinternational database or no close relative could be found;the closest phylogenetic neighbor of the 123-nt band was inthe genera Thiovolum and Campylobacter of the ${epsilon}$ subgroup of theProteobacteria.

DISCUSSION

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Discussion

Overall community structure and activity of bacterioplankton at different depths and in different seasons.
In contrast to PCR-driven community fingerprints, 5S rRNA fingerprintsof bacterioplankton reflect directly the amounts of different5S rRNA from different members of the bacterial community. Therefore,we have defined a single 5S rRNA band with a homogenous sequenceas an operational taxonomic unit (OTU) (20). This OTU definitionenables us to calculate the Shannon diversity index for eachcommunity based on the relative abundance of each 5S rRNA incomparison to the total amount of 5S rRNA for each depth. Twotechnical prerequisites for assessment of the relative amountsof 5S rRNA have to be fulfilled; the efficiencies of extractingRNA from natural bacterioplankton should be similar for alltaxa, and the efficiencies of ³²P labeling the 3' ends of the5S rRNA should be similar. It has been demonstrated that bothprerequisites are fulfilled with the methodology used here (7,8, 20, 40). Another theoretical and technical considerationfor calculation of the Shannon index is to set a lower limitfor the relative abundance of a single OTU. Such a lower limithas been set at a relative abundance of 4% for clarity (Fig.4) and comparability with past studies (18, 20). On the otherhand, the detection limit for single ³²P-labeled 5S rRNA bandsis about 0.1% if adjacent bands are not so large that they completelycover the weak bands next to them in the autoradiogram. Thisanalytical problem can be overcome by using a phosphorimagerthat enables detection of bands next to each other whose bandintensities are more than 4 orders of magnitude different. A0.1% detection limit means that we were able to detect taxawhose concentrations were as low as 0.5 x 10⁶ to 1 x 10⁶ cellsper liter in the 5S rRNA fingerprints of bacterioplankton fromLake Plußsee with the described methodology if an averagecell density of 5 x 10⁹ to 10 x 10⁹ cells per liter was assumed(Fig. 1b and 2b). This detection limit is about 1 order of magnitudelower than the 1% limit commonly reported for PCR-based fingerprintmethods, such as denaturing gradient gel electrophoresis orsingle-strand conformation polymorphism analysis (27, 31). Inaddition, not only does the presence of a 5S rRNA band reflectthe abundance of an OTU, but the band also represents an activemember of the bacterioplankton community since the amount ofrRNA is correlated with the growth of bacteria (3).

Vertical gradients of physical and chemical parameters stronglyaffect the biology and ecology of all pelagic ecosystems (22).This universal finding is also reflected in the structure ofthe bacterioplankton community. The most striking feature ofboth depth profiles was the much lower diversity in the hypolimnionthan in the upper layers. The decrease can be seen in the reductionof the Shannon index by about one-half, as well as in the numberof bands, which decreased from about 14 to 7 in both depth profiles(Fig. 5). One exception to this overall trend was the 25-m samplecollected in the spring, for which the number of bands increased.Conceivably, the diatoms which dominate the phytoplankton ofLake Plußsee in early spring were still being degradedin the deep hypolimnion after they sank in late March (28, 32).

When the diversity data for the depth profiles were comparedwith the data from the seasonal study of bacterioplankton inLake Plußsee, it was obvious that the low diversity, asindicated by a Shannon index of around 0.4, in the hypolimnionwas encountered only during the three minima occurring in theepilimnion at special times of the year (20). During most ofthe year the Shannon index remained in the range found for theepilimnion for both depth profiles. The first minimum for thediversity index in the seasonal study could be explained bystrong grazing by cladocerans, whereas grazing by HNF did nothave a significant effect on diversity. Grazing by HNF did notdirectly affect the high diversity in the upper layers of depthprofiles, where HNF grazing is the major mortality factor inthe epilimnion (41). On the other hand, viral lysis was demonstratedto be the key mortality factor in the anoxic hypolimnion ofLake Plußsee, where all grazing was strongly reduced (41).This result corroborates the finding that only very few membersof the bacterioplankton community were active in the hypolimnionbecause low-diversity bacterial communities can be controlledmore easily by viral lysis than high-diversity communities (37).The major reason for the low diversity in the hypolimnion forboth depth profiles might be the decrease in the dissolved organiccarbon content, which not only decreased by about 50% with depthbut also accounted for a far higher proportion of refractoryorganic compounds in the hypolimnion than in the epilimnionof Lake Plußsee (26).

Depth-dependent and seasonal changes in detailed structure of the bacterioplankton community.
The presence and depth distribution of C. acidovorans in LakePlußsee have been demonstrated previously by immunofluorescencemicroscopy (11, 42). Additionally, a seasonal study in which5S rRNA profiling was used showed that C. acidovorans is abundantat a depth of 1 m during all seasons (20). Both depth profilesobtained by using 5S rRNA analysis confirmed the presence ofC. acidovorans in the whole profile, with an average abundanceof about 6.0%. (The levels in deeper samples were around 3%and therefore are not shown in Fig. 4). This finding is in contrastto the results obtained by immunofluorescence microscopy thatindicated that C. acidovorans cells disappeared at depths below10 m in the spring of 1993 and that there was a significantlack of C. acidovorans cells in the metalimnion during the fallof 1996 (11, 42). The most likely explanation for this findingis that the monoclonal antibodies used for detection of C. acidovoranscells are specific for a certain subspecies type, whereas the5S rRNA bands represent the whole taxon. Therefore, C. acidovoranswas present in abundance in Lake Plußsee not only duringall seasons but also at all depths, including the anoxic hypolimnion.Furthermore, the presence of significant amounts of rRNA indicatedthat this group of bacteria was metabolically active at alldepths year round. This supports the hypothesis, put forwardearlier (42), that C. acidovorans is a K-strategist well adaptedto the pelagic environment of a eutrophic lake because of itsrelative resistance to protozoan grazing and its ability toutilize refractory dissolved organic matter.

Partial sequencing revealed differences in all bands exceptthe 116- and 122-nt bands in the different seasons. The strongseasonal differences make sense considering the fact that thephytoplankton communities during the two seasons were completelydifferent and that in the fall the hypolimnion was anoxic. Detectionof members of the ${epsilon}$ subgroup of the Proteobacteria in the anaerobicwater column is unusual but has been observed with clone librariesfrom coastal marine water samples obtained during an algal bloomand from the chemocline of a meromictic lake in the Swiss Alps(2, 21).

Comparison of bacterioplankton community structure with other depth distributions.
A variety of studies have been performed with different molecularmethods to understand the depth distribution of the bacterioplanktoncommunity (14, 25). Most of these studies were based on clonelibraries of 16S rDNA genes (see reference 14 for a review),followed by hybridization studies and community fingerprinting.While clone library studies provide insight into community compositionand diversity, fingerprinting studies reveal the overall structureof the whole community and enable direct comparisons of a varietyof samples. Fingerprinting studies of the bacterioplankton ofa brackish meromictic lake and the central Baltic Sea indicatedthat there was higher diversity in the upper layer than in theanoxic bottom layer, as found for Lake Plußsee (18, 29).Recent studies of bacterioplankton in a deep ultraoligotrophiclake and the eastern Mediterranean Sea showed that there weredistinct changes with depth but did not reveal substantial decreasesin diversity (24, 38). Several studies performed with group-specificrRNA-targeted probes revealed the depth dependence of thesegroups in the water column of the oceans (12, 23). The amountsof some of these groups correlated well with microbial processes,such as the intensity of primary production (36). In general,the following picture appears to emerge for the structure ofthe bacterioplankton community with depth. Pelagic environmentshave specific bacterioplankton community structures at specificdepths, and the overall diversity and composition might varyfrom layer to layer depending on the trophic status and theavailability of electron acceptors.

Conclusions.
We demonstrated that there were changes in bacterioplanktoncommunity structure with depth in a eutrophic lake. These changescorresponded well with the three layers, epilimnion, metalimnion,and hypolimnion, of the lake. The more the lake was stratified,the better the three different communities were separated. Whereasthe community composition generally varied with the season,some members of the community, such as C. acidovorans, wereabundant at all depths during both seasons. Another common featurewas the reduction in the overall diversity of the bacterioplanktonwith depth. This decrease in diversity means that the diversityof the bacterioplankton community in the hypolimnion was quitelow compared to the diversity of the community in the epilimnion,where the diversity is low only two or three times during theyear (20).

ACKNOWLEDGMENTS

We thank D. Albrecht from the MPI of Limnology in Plönfor providing background data. The valuable comments of IngridBrettar and Markus G. Weinbauer on an earlier version of themanuscript are greatly acknowledged. Improvement of the Englishby David Kirchman is greatly appreciated.

This project was supported by funds from Bundesministerium fürBildung, Wissenschaft, Forschung und Technologie grant BEO-0319433Band Deutsche Forschungsgemeinschaft grant Ho 930/2-1.

FOOTNOTES

* Corresponding author. Mailing address: GBF-German Research Center for Biotechnology, Department of Environmental Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. Phone: 49-531-6181-419. Fax: 49-531-6181-411. E-mail: mho{at}gbf.de.

REFERENCES

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