Quantitative Molecular Analysis of the Microbial Community in Marine Arctic Sediments (Svalbard)

Fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI (4′,6′-diamidino-2-phenylindole) cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides δ-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts and up to 6.1% of prokaryotic rRNA. Furthermore, members of the order Planctomycetales accounted for up to 3.9% of total cell counts. In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments. Members of the γ-proteobacteria also constituted a significant fraction in this sediment (6.1% ± 2.5% of total cell counts, 14.4% ± 3.6% of prokaryotic rRNA). A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed. A significant number of cells was detected by this probe (2.1% ± 0.7% of total DAPI cell counts, 13.2% ± 4.6% of prokaryotic rRNA), showing no clear zonation along the vertical profile. Gram-positive bacteria and the β-proteobacteria were near the detection limit in all sediments.     

Optimization of direct cell counting in sediment

This study reports a method for optimizing direct counts of bacteria in sediment, designed to reduce the masking by sediment particles. The protocol was designed to determine appropriate dilution factors by incorporating counting statistics and was used to measure depth-associated changes in microbial abundance in metal-impacted freshwater sediments. We demonstrated a direct method to determine appropriate sample dilution for accurate counting by adding a known amount of cells to the sediment. For accurate counting in our sediment samples, we determined that the average number of bacteria per microscope ocular field must be between 8.5 and 10. This is well below the 30 bacteria/field previously suggested for accurate counting. These results indicate that an optimal dilution rate must be determined before accurate direct counts in sediment can be achieved.     

Utility of Green Fluorescent Nucleic Acid Dyes and Aluminum Oxide Membrane Filters for Rapid Epifluorescence Enumeration of Soil and Sediment Bacteria

High background fluorescence and unspecific staining hampered the epifluorescence enumeration of bacteria in 45% of the tested soil and sediment samples with 4′,6-diamidino-2-phenylindole (DAPI) and polycarbonate membrane filters. These problems of the determination of total cell counts can be circumvented by using green fluorescent high-affinity nucleic acid dyes and aluminum oxide membrane filters. Due to the bright staining of cells, we recommend SYBR Green II as dye.     

Differential Growth Response of Colony-Forming α- and γ-Proteobacteria in Dilution Culture and Nutrient Addition Experiments from Lake Kinneret (Israel), the Eastern Mediterranean Sea, and the Gulf of Eilat

Even though it is widely accepted that bacterioplankton growth in lakes and marine ecosystems is determined by the trophic status of the systems, knowledge of the relationship between nutrient concentrations and growth of particular bacterial species is almost nonexistent. To address this question, we performed a series of culture experiments with water from Lake Kinneret (Israel), the eastern Mediterranean Sea, and the Gulf of Eilat (northern Red Sea). In the initial water samples, the proportion of CFU was typically <0.002% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. During incubation until the early stationary phase, the proportion of CFU increased to 20% of the DAPI counts and to 2 to 15% of the DAPI counts in unenriched lake water and seawater dilution cultures, respectively. Sequencing of the 16S ribosomal DNA of colony-forming bacteria in these cultures consistently revealed an abundance of α-proteobacteria, but notable phylogenetic differences were found at the genus level. Marine dilution cultures were dominated by bacteria in the Roseobacter clade, while lake dilution cultures were dominated by bacteria affiliated with the genera Sphingomonas and Caulobacter. In nutrient (glucose, ammonium, phosphate) addition experiments the CFU comprised 20 to 83% of the newly grown cells. In these incubation experiments fast-growing γ-proteobacteria dominated; in the marine experiments primarily different Vibrio and Alteromonas species appeared, while in the lake water experiments species of the genera Shewanella, Aeromonas, and Rheinheimera grew. These results suggest that major, but different, γ-proteobacterial genera in both freshwater and marine environments have a preference for elevated concentrations of nutrients and easily assimilated organic carbon sources but are selectively outcompeted by α-proteobacteria in the presence of low nutrient concentrations.     

Whole-Cell versus Total RNA Extraction for Analysis of Microbial Community Structure with 16S rRNA-Targeted Oligonucleotide Probes in Salt Marsh Sediments

rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% ± 3.7% [mean ± standard deviation]) relative to direct DAPI (4′,6′-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r² = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.     

Benthic flagellates and ciliates in fine freshwater sediments: Calibration of a live counting procedure and estimation of their abundances

Despite the recognized importance of protozoans (flagellates and ciliates) as predators of bacteria, there are very few estimates of their abundance in fine sediments of freshwater lakes. This is due, in part, to the lack of a standard methodology. Because of the low concentration of protists in relation to particles, epifluorescence counts can not always be used. Instead, dilution followed by live counting was used to solve the masking by sediment particles. One to twenty 1 sample aliquots were diluted with filtered lake water in a Palmer-Maloney counting slide. Four to eight replicates were sufficient to minimize the counting error, while minimizing effort. The method is highly replicable and could potentially be calibrated for different sediment types because sediment masking depends on the mean particle size of the sediment. When this method was applied in a survey of benthic sites in Quebec lakes, flagellate abundances were found to range from 100 to 180,000 cells ml^–1, while ciliate numbers ranged from 26 to 11,000 cells ml^–1. Bacteria are 105 to 10⁷ times more abundant than protists and, thus, the impact of these protists on sediment bacterial dynamics is likely to be minimal.     

A disintegration method for direct counting of bacteria in clay-dominated sediments: dissolving silicates and subsequent fluorescent staining of bacteria

The masking of bacteria by abundant microparticles of the clay and silt fraction and cell losses due to sonication hampered direct enumeration of bacteria in sediments dominated by fine sediments. These problems can be circumvented by dissolving silicate fine particles using hydrofluoric acid and subsequent staining of bacteria by DTAF. The developed disintegration method partly replaces mechanical separation of bacteria from sediment particles by chemical disintegration of the silicates. Recovery efficiency ranged from 90% to 111% for different clays and clay-dominated sediments. Especially for the analysis of fine sediments and clays, this method circumvents both strong dilution of the sediment sample and harsh sonication. The method can also therefore be used in sediments where particle abundance is several orders of magnitude higher than bacterial abundance and simple dilution would not suffice in reliably counting bacteria.     

Large Fraction of Dead and Inactive Bacteria in Coastal Marine Sediments: Comparison of Protocols for Determination and Ecological Significance

It is now universally recognized that only a portion of aquatic bacteria is actively growing, but quantitative information on the fraction of living versus dormant or dead bacteria in marine sediments is completely lacking. We compared different protocols for the determination of the dead, dormant, and active bacterial fractions in two different marine sediments and at different depths into the sediment core. Bacterial counts ranged between (1.5 ± 0.2) × 10⁸ cells g⁻¹ and (53.1 ± 16.0) × 10⁸ cells g⁻¹ in sandy and muddy sediments, respectively. Bacteria displaying intact membrane (live bacterial cells) accounted for 26 to 30% of total bacterial counts, while dead cells represented the most abundant fraction (70 to 74%). Among living bacterial cells, nucleoid-containing cells represented only 4% of total bacterial counts, indicating that only a very limited fraction of bacterial assemblage was actively growing. Nucleoid-containing cells increased with increasing sediment organic content. The number of bacteria responsive to antibiotic treatment (direct viable count; range, 0.3 to 4.8% of the total bacterial number) was significantly lower than nucleoid-containing cell counts. An experiment of nutrient enrichment to stimulate a response of the dormant bacterial fraction determined a significant increase of nucleoid-containing cells. After nutrient enrichment, a large fraction of dormant bacteria (6 to 11% of the total bacterial number) was “reactivated.” Bacterial turnover rates estimated ranged from 0.01 to 0.1 day⁻¹ but were 50 to 80 times higher when only the fraction of active bacteria was considered (on average 3.2 day⁻¹). Our results suggest that the fraction of active bacteria in marine sediments is controlled by nutrient supply and availability and that their turnover rates are at least 1 order of magnitude higher than previously reported.     

Evaluation of quantitative recovery of bacterial cells and DNA from different lake sediments by Nycodenz density gradient centrifugation

While purified bacterial cells and DNA – the signature of life – from soil and sediment matrices have been extensively studied in a wide range of environments and in different microbial ecosystems, the paucity of data on DNA extraction from contaminated sediments emphasizes the need for further research on the isolation and quantification of bacterial cells and DNA in sediments. Consequently, the Nycondez gradient centrifugation method was applied to extract bacterial cells from contaminated and uncontaminated sediments. Quantitative estimates of recovered bacterial cells were obtained from direct counts performed using DAPI (4′,6′-diamino-2-phenylindole hypochloride) staining couples with fluorescence microscopy and indirect counts (colony-forming units). The estimation was improved by using an efficient method of comparing sediment types composed of quantifying bacterial densities in three steps: S₁ the initial freshwater sediments; S₂ the first supernatant recovered after mixing the sediments with sodium hexametaphosphate solution followed by centrifugation; and S₃ the extracted cells. Total and extracellular DNA were extracted and quantified in each of the three steps. Additional analysis of faecal indicator bacteria (FIB) including E. coli and Enterococcus (ENT) was also performed in each step. The results display considerable variability in the quantity of bacteria cells depending on sediment type, ranging from 1.2 × 10⁵ to 6.2 × 10⁹ cell g^?1 dry sediments. The treatment with sodium hexametaphosphate solution (2%) leads to the desorption of bacterial populations which were firmly adsorbed on contaminated sediment surfaces resulting in more than 90% of the FIB being recovered. The Nycondez density gradient centrifugation method makes it possible to extract bacterial cells from freshwater sediments without extracellular DNA so it is ideal for metagenomic analysis of bacteria.     

Spatial Heterogeneity of Bacterial Populations along an Environmental Gradient at a Shallow Submarine Hydrothermal Vent near Milos Island (Greece)

The spatial heterogeneity of bacterial populations at a shallow-water hydrothermal vent in the Aegean Sea close to the island of Milos (Greece) was examined at two different times by using acridine orange staining for total cell counts, cultivation-based techniques, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments. Concurrent with measurements of geochemical parameters, samples were taken along a transect from the center of the vent to the surrounding area. Most-probable-number (MPN) counts of metabolically defined subpopulations generally constituted a minor fraction of the total cell counts; both counting procedures revealed the highest cell numbers in a transition zone from the strongly hydrothermally influenced sediments to normal sedimentary conditions. Total cell counts ranged from 3.2 × 10⁵ cells ml⁻¹ in the water overlying the sediments to 6.4 × 10⁸ cells g (wet weight) of sediment⁻¹. MPN counts of chemolithoautotrophic sulfur-oxidizing bacteria varied between undetectable and 1.4 × 10⁶ cells g⁻¹. MPN counts for sulfate-reducing bacteria and dissimilatory iron-reducing bacteria ranged from 8 to 1.4 × 10⁵ cells g⁻¹ and from undetectable to 1.4 × 10⁶ cells g⁻¹, respectively. DGGE revealed a trend from a diverse range of bacterial populations which were present in approximately equal abundance in the transition zone to a community dominated by few populations close to the center of the vent. Temperature was found to be an important parameter in determining this trend. However, at one sampling time this trend was not discernible, possibly due to storm-induced disturbance of the upper sediment layers.     

Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342

We investigated the accuracy and precision of flow cytometric (FCM) estimates of bacterial abundances using 4′, 6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 (HO342, a bisbenzamide derivative) on paraformaldehyde-fixed seawater samples collected from two stations near Oahu, Hawaii. The accuracy of FCM estimates was assessed against direct counts by using epifluorescence microscopy. DAPI and HO342 differ in two aspects of their chemistry that make HO342 better suited for staining marine heterotrophic bacteria for FCM analysis. These differences are most important in studies of open-ocean ecosystems that require dual-beam FCM analysis to clearly separate heterotrophic bacterial populations from populations of photosynthetic Prochlorococcus spp. Bacterial populations were easier to distinguish from background fluorescence when stained with HO342 than when stained with DAPI, because HO342 has a higher relative fluorescence quantum yield. A substantially higher coefficient of variation of blue fluorescence, which was probably due to fluorescent complexes formed by DAPI with double-stranded RNA, was observed for DAPI-stained populations. FCM estimates averaged 2.0 and 12% higher than corresponding epifluorescence microscopy direct counts for HO342 and DAPI-stained samples, respectively. A paired-sample t test between FCM estimates and direct counts found no significant difference for HO342-stained samples but a significant difference for DAPI-stained samples. Coefficients of variation of replicate FCM abundance estimates ranged from 0.63 to 2.9% (average, 1.5%) for natural bacterial concentrations of 6 × 10⁵ to 15 × 10⁵ cells ml^-1.     

Some special problems in the determination of viable counts of groundwater microorganisms

Factors affecting viable cell counts in groundwater or sediments were studied with samples from the Segeberg Forest test area in northern Germany. There was very little variation in results with the season (April, August, November) or depth of sampling; generally there were 10³–10⁴ aerobic cells per ml or g sediment. Long incubation times resulted in higher cell counts; groundwater samples required 4–5 weeks, and sediment extracts had to be cultured for 7 weeks. Total cell counts in sediment were 10²–10⁴ cell/g higher than viable cell counts of aerobes. This was explained partly by the additional presence of anaerobes and partly by the observation that some morphotypes may not have grown under our conditions. Viable cell counts were not influenced by cell extraction from the sediment with either Na-pyrophosphate or groundwater extracts. However, iron-precipitating or manganese-oxidizing bacteria were better extracted with sterile groundwater. The microflora of wells was more numerous than that of the free aquifer; consequently it was better to pump off all well water before aquifer water was sampled. The diameter of the well was also important; thinner tubes had higher cell counts than those with wider diameter. For sampling, wells should be at least 1 year old, since young wells contain higher numbers of microorganisms due to underground disturbances from the drilling. Turbid water samples could be clarified by filtration, but this reduced the viable counts by 1–2 orders of magnitude. Two different media inoculated with a sample dilution resulted in the same cell counts, but their microbial diversity was different. Storage of groundwater samples before processing resulted in up to 17-fold increases in cell counts and loss of diversity in the first 24 hours. Cell numbers decreased slowly during longer storage.     

Direct in situ detection of cells in deep-sea sediment cores from the Peru Margin (ODP Leg 201, Site 1229)

Microbiological investigations of deep‐sea sediments recovered from the Peru Margin during the ODP Leg 201 (Hole 1229A, 1–110 mbsf) demonstrated that microoganisms were a consistent component throughout the profile. Optimization of the dilution factor and DAPI‐staining procedures for direct cell counts allowed the determination of the abundance of the entire microbial community, which was about 10⁸ cells per g dry sediment. Microbial diversity in discrete samples taken from the 110‐m profile was analysed using horseradish‐peroxydase‐rRNA‐probes. In general, the majority of the detected cells belonged to the Eubacteria kingdom with a dominance of sulphate‐reducing bacteria. The composition of the suflate‐reducing community varied with depth. Desulfobacteriaceae were dominant in the uppermost sulphate‐reducing zone and Desulfovibrionaceae at deeper depths in the upward diffusing sulphate‐rich brines. Both sulphate‐reducing groups were also detected in the methanogenic zone. Similarly, Archaea were detected throughout the profile, not only in the methanogenic zone but also in the upper and lower sulphate‐reducing zones.     

Viriobenthos Production and Virioplankton Sorptive Scavenging by Suspended Sediment Particles in Coastal and Pelagic Waters

Virus production in oxic surface sediments and virioplankton sorption to suspended particles was estimated across three stations in the Southern California region (33.4°N, 118.6°W). Viriobenthos production was estimated using a sterile sediment and filtered porewater dilution technique that targeted production from both attached bacteria and bacteria living free in the porewater, and attached bacteria alone. Potential virus production rates by bacteria free in the porewater ranged from 1.7 to 4.6 × 10⁸ VLP cm^–3 h^–1, while attached bacteria had slower potential production rates of between 0.4 and 1.1 × 10⁸ VLP cm^–3 h^–1, suggesting turnover rates of viruses in sediments (1–5 h) which are significantly higher than those of virioplankton (~24–48 h). Virioplankton adsorbed to small (<150 µm) suspended sediments at stations with high ambient suspended solid concentrations. Virioplankton scavenging rates combined with published sedimentation rates demonstrate that this mechanism of virus arrival could only account for 0.01% of daily benthic virus production. Calculated mortality rates of benthic bacteria (4–14% h^–1) suggest viruses may play an important role in sediment carbon cycling.     

The effects of sediment slurrying on microbial processes,and the role of amino acids as substrates for sulfate reduction in anoxic marine sediments

In sediment slurry experiments with anoxic marine sediments collected in Cape Lookout Bight, NC, and a site in mid-Chesapeake Bay, the rates of sulfate reduction and ammonium production decrease with increasing dilution of sediment with oxygen-free sea-water. The effect of sediment dilution on the rates of these processes can be described by a simple mathematical relationship, and when these rates are corrected for sediment dilution they yield values which agree well with direct measurements of these processes.In sediment slurry studies of amino acid utilization in Cape Lookout Bight sediments, the fermentative decarboxylation of glutamic acid (to -aminobutyric acid) or aspartic acid (to alanine or -alanine) did not occur when either of these amino acids were added to Cape Lookout Bight slurries. The addition of glutamic acid did however lead to a small (1) transient build-up of -aminoglutaric acid. Measured rates of glutamic acid uptake in these slurries also decreased with increasing sediment dilution.Molybdate inhibition experiments demonstrated that dissolved free amino acids represent 1–3% of the carbon sources/electron donors used for sulfate reduction in Cape Lookout Bight sediments. The direct oxidation of amino acids by sulfate reducing bacteria also accounts for 13–20% of the total ammonium produced. Glutamic acid, alanine, -aminoglutaric acid, aspartic acid and asparagine are the major amino acids oxidized by sulfate reducing bacteria in Cape Lookout Bight sediments.     

Decomposition Studies in Two Central Ontario Lakes Having Surficial pHs of 4.6 and 6.6

The rates of cellulose breakdown, composition of detrital microflora, and density of bacterial populations were determined in the epilimnetic sediments and water columns of two poorly buffered, oligotrophic, Canadian Shield lakes having mean surficial pHs of 4.6 (Bat Lake) and 6.6 (Harp Lake). The decomposition rate was significantly lower in oxic sediment of the acidified lake than of the circumneutral lake, but water column rates were almost identical in the two lakes. These results are explained in terms of the groups of cellulolytic microorganisms which were observed by phase-contrast microscopy as being active at the different sites: fungi in Bat Lake water and Cytophaga-like bacteria in the water and sediment of Harp Lake. Cytophaga-like bacteria were also the main decomposers in Bat Lake sediment, but their activity was restricted at porewater pHs of <5.0. Acridine orange direct counts of bacteria in the top centimeter of sediment ranged from 3.7 × 10⁸ to 1.0 × 10⁹ per g, and counts in planktonic water samples ranged from 4.9 × 10⁵ to 1.2 × 10⁶ per ml. Bacterial densities at most sites decreased significantly (P < 0.001) from August to late October, but did not show a consistent pattern of differences related to pH.     

Comparison of bacteria from deep subsurface sediment and adjacent groundwater

Samples of groundwater and the enclosing sediments were compared for densities of bacteria using direct (acridine orange direct staining) and viable (growth on 1% PTYG medium) count methodology. Sediments to a depth of 550 m were collected from boreholes at three sites on the Savannah River Site near Aiken, South Carolina, using techniques to insure a minimum of surface contamination. Clusters of wells screened at discreet intervals were established at each site. Bacterial densities in sediment were higher, by both direct and viable count, than in groundwater samples. Differences between direct and viable counts were much greater for groundwater samples than for sediment samples. Densities of bacteria in sediment ranged from less than 1.00×10⁶ bacteria/g dry weight (gdw) up to 5.01 ×10⁸ bacteria/gdw for direct counts, while viable counts were less than 1.00×10³ CFU/gdw to 4.07×10⁷ CFU/gdw. Bacteria densities in groundwater were 1.00×10³–6.31×10⁴ bacteria/ml and 5.75–4.57×10² CFU/ml for direct and viable counts, respectively. Isolates from sediment were also found to assimilate a wider variety of carbon compounds than groundwater bacteria. The data suggest that oligotrophic aquifer sediments have unique and dense bacterial communities that are attached and not reflected in groundwater found in the strata. Effective in situ bioremediation of contaimination in these aquifers may require sampling and characterization of sediment communities.     

Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present.

Understanding the role of bacteria in microbial food webs is intimately connected to the methods applied in the direct enumeration of bacteria. We have examined over 220 papers describing studies in which fluorochrome staining followed by epifluorescent microscopic direct counts was used to estimate total bacterial abundances. In this review, we summarize patterns in the use of 3,6-bis[dimethylamino]acridinium chloride (acridine orange) and 4',6-diamidino-2-phenylindole (DAPI), the two stains most frequently used in bacterial enumeration. The staining of samples with these fluorochromes, followed by filtration and direct counting of bacterial cells on filter surfaces, has become routine over the past 10 years. We examine trends in features of the standard direct count methods, such as sample preservation and preparation techniques, membrane filter types used, applied stain concentrations, duration of staining, and counting strategies, in relation to the types of samples being examined. The high variability in bacterial counts observed within similar sample types may be partially accounted for by differences in methods. Synthesizing review findings, we include a recommended method for the direct enumeration of bacteria in environmental samples.     

Fluorescent heterochromatin staining in primate chromosomes

Recently, in addition to quinacrine staining, fluorochrome techniques have been developed which brilliantly stain other heterochromatic regions. Two of these staining techniques are Distamycin/DAPI (DA/DAPI) and D287/170. We stained the chromosomes of all species of great apes and 14 species of primates (48 individuals) using these three fluorochrome techniques. Only african apes and man show brilliant quinacrine staining while, man and all the great apes show brilliant DA/DAPI staining and only species belonging to the hominoidea (including the siamang) showed bright D287/170 staining. In the lower primates a medium level of DA/DAPI fluorescence was found in some species with large amount of pericentromeric heterochromatin. Brilliant DA/DAPI staining could represent a derived trait linking all great apes and humans, while D287/170 may link all hominoidea. Fluorochrome staining is believed to be correlated with some satellite DNA sequences. However, data available on the chromosome location of satellite DNAs in non-human primates were derived from buoyant density fractions resulting in cross hybridization and now are not considered reliable. Before making any correlation between fluorochrome staining and satellite DNAs in non human primates there is need of data onin situ hybridization with cloned DNA sequences on primate chromosomes. These data would help clarify the evolution and relationship of satellite DNAs and heterochromatin in primates.     

α- and β-Proteobacteria Control the Consumption and Release of Amino Acids on Lake Snow Aggregates

We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4′,6′-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% ± 7.9% and 14.2% ± 10.2% of the DAPI cell counts were detected by probes specific for α- and β-Proteobacteria. These proportions increased to 12.0% ± 3.3% and 54.0% ± 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% ± 1.4% and 41.1% ± 8.4%, indicating a clear dominance of β-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. γ-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the α-Proteobacteria. In addition, with three probes highly specific for close relatives of the β-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the β-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of β-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of α- and β-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.