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Bacterial incorporation of glucose, leucine, acetate and 4-hydroxybenzoic acid (HBA) was investigated in an artificially divided humic lake (Grosse Fuchskuhle, Germany). Two basins with contrasting influx of allochthonous organic carbon were sampled during late summer stratification (oxic and anoxic layers) and after autumn mixing. High total and cell-specific incorporation rates were observed for glucose and HBA in stratified and mixed waters respectively, but only a small fraction of bacteria visibly incorporated HBA. The oxic layer of the more humic-rich basin featured a significantly lower fraction of glucose incorporating cells and substantially higher proportions of acetate assimilating bacteria. Niche differentiation was observed in two betaproteobacterial populations: cells affiliated with the Polynucleobacter C subcluster efficiently incorporated acetate but little glucose, whereas the opposite was found for members of the R-BT065 clade. By contrast, leucine incorporation was variable in both taxa. Considering the high concentrations and rapid photochemical generation of organic acids in humic waters our results may help to explain the success of the Polynucleobacter C lineage in such habitats. Specific substrate or habitat preferences were also present in three subgroups of the actinobacterial acI lineage: The numerically dominant clade in oxic waters (acI-840-1) was absent in the anoxic zone and did not incorporate acetate. A second group (acI-840-2) was found both in the epi- and hypolimnion, whereas the third one (acI-840-3) only occurred in anoxic waters. Altogether our results suggest a constitutive preference for some substrates versus an adaptive utilization of others in the studied microbial groups. [ABSTRACT FROM AUTHOR]

Abstract: A purple sulfur bacterium, strain SW26, was isolated in pure culture from intertidal sediments from the Sylt-Rømø Basin, German Wadden Sea, sharing many properties with validated Marichromatium species, but differing significantly by possessing a plasmid, by tolerating up to 16mM sulfide, and up to 44°C for growth. Strain SW26 has a DNA base composition of 68.3mol% G+C, a 16S rRNA gene sequence similarity of >99% to those of Marichromatium species, and shows the highest level of genomic relationship with Marichromatium gracile, despite its remarkably different phenotypic characters. Based upon high genomic similarity but different physiological properties of strain SW26 with respect to the type strain of M. gracile, a novel biotype, designated as M. gracile biotype thermosulfidiphilum is described. [Copyright &y& Elsevier]

Abstract: Molecular biological methods were used to investigate the microbial diversity and community structure in intertidal sandy sediments near the island of Sylt (Wadden Sea) at a site which was characterized for transport and mineralization rates in a parallel study (D. de Beer, F. Wenzhöfer, T. Ferdelman, S.E. Boehme, M. Huettel, J.E.E. van Beusekom, M.E. Böttcher, N. Musat, N. Dubilier, Transport and mineralization rates in North Sea sandy intertidal sediments, Sylt-Romo Basin, Wadden Sea, Limnol. Oceanogr. 50 (2005) 113–127). Comparative 16S rRNA sequence analysis revealed a high bacterial diversity. Most sequences retrieved by PCR with a general bacterial primer set were affiliated with Bacteroidetes, Gammaproteobacteria, Deltaproteobacteria and the Pirellula cluster of Planctomycetales. Fluorescence in situ hybridization (FISH) and slot-blot hybridization with group-specific rRNA-targeted oligonucleotide probes were used to characterize the microbial community structure over depth (0–12cm) and seasons (March, July, October). We found high abundances of bacteria with total cell numbers up to 3×109 cellsml−1 and a clear seasonal variation, with higher values in July and October versus March. The microbial community was dominated by members of the Planctomycetes, the Cytophaga/Flavobacterium group, Gammaproteobacteria, and bacteria of the Desulfosarcina/Desulfococcus group. The high abundance (1.5×107–1.8×108 cellsml−1 accounting for 3–19% of all cells) of presumably aerobic heterotrophic polymer-degrading planctomycetes is in line with the high permeability, deep oxygen penetration, and the high rates of aerobic mineralization of algal biomass measured in the sandy sediments by de Beer et al. (2005). The high and stable abundance of members of the Desulfosarcina/Desulfococcus group, both over depth and season, suggests that these bacteria may play a more important role than previously assumed based on low sulfate reduction rates in parallel cores (de Beer et al., 2005). [Copyright &y& Elsevier]

An oligonucleotide probe-based DNA microarray was evaluated for its ability to detect 16S rRNA targets in marine bacterioplankton samples without prior amplification by polymerase chain reaction (PCR). The results obtained were compared with those of quantitative fluorescence in situ hybridization (FISH). For extraction and direct labelling of total RNA, a fast and efficient protocol based on commercially available kits was established. A set of redundant and hierarchically structured probes was applied, and specificity of hybridization was assessed by additional control oligonucleotides comprising single central mismatches. The protocol was initially tested by microarray analysis of bacterial pure cultures. Complete discrimination of all control oligonucleotides was achieved, indicating a high degree of hybridization specificity. In a co-culture, abundant members were detected by microarray analysis, but signal ratios of positive probes did not correlate well with quantitative data from FISH experiments. A marine picoplankton sample from the German Bight was analysed. Bacterial populations with relative abundances of at least 5% were detected by hybridizing 0.1 µg of total RNA extracted from a sample of 375 ml equivalent to 4.1 × 108 cells. Our results demonstrate that major populations of marine bacterioplankton can be identified by microarray analysis in a fast and reliable way, even in relatively low volumes of sea water. [ABSTRACT FROM AUTHOR]

In winter 2009/10, a sudden under-ice bloom of heterotrophic bacteria occurred in the seasonally ice-covered, temperate, deep, oligotrophic Lake Stechlin (Germany). Extraordinarily high bacterial abundance and biomass were fueled by the breakdown of a massive bloom of Aphanizomenon flos-aquae after ice formation. A reduction in light resulting from snow coverage exerted a pronounced physiological stress on the cyanobacteria. Consequently, these were rapidly colonized, leading to a sudden proliferation of attached and subsequently of free-living heterotrophic bacteria. Total bacterial protein production reached 201 µg C L(-1) d(-1), ca. five times higher than spring-peak values that year. Fluorescence in situ hybridization and denaturing gradient gel electrophoresis at high temporal resolution showed pronounced changes in bacterial community structure coinciding with changes in the physiology of the cyanobacteria. Pyrosequencing of 16S rRNA genes revealed that during breakdown of the cyanobacterial population, the diversity of attached and free-living bacterial communities were reduced to a few dominant families. Some of these were not detectable during the early stages of the cyanobacterial bloom indicating that only specific, well adapted bacterial communities can colonize senescent cyanobacteria. Our study suggests that in winter, unlike commonly postulated, carbon rather than temperature is the limiting factor for bacterial growth. Frequent phytoplankton blooms in ice-covered systems highlight the need for year-round studies of aquatic ecosystems including the winter season to correctly understand element and energy cycling through aquatic food webs, particularly the microbial loop. On a global scale, such knowledge is required to determine climate change induced alterations in carbon budgets in polar and temperate aquatic systems.

The degradation of diatoms is mainly catalyzed by Bacteroidetes and this process is of global relevance for the carbon cycle. In this study, a combination of catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) and fluorescent lectin binding analysis (FLBA) was used to identify and map glycoconjugates involved in the specific interactions of Bacteroidetes and diatoms, as well as detritus, at the coastal marine site Helgoland Roads (German Bight, North Sea). The study probed both the presence of lectin-specific extracellular polymeric substances (EPS) of Bacteroidetes for cell attachment and that of glycoconjugates on diatoms with respect to binding sites for Bacteroidetes. Members of the clades Polaribacter and Ulvibacter were shown to form microcolonies within aggregates for which FLBA indicated the presence of galactose containing slime. Polaribacter spp. was shown to bind specifically to the setae of the abundant diatom Chaetoceros spp., and the setae were stained with fucose-specific lectins. In contrast, Ulvibacter spp. attached to diatoms of the genus Asterionella which bound, among others, the mannose-specific lectin PSA. The newly developed CARD-FISH/FLBA protocol was limited to the glycoconjugates that persisted after the initial CARD-FISH procedure. The differential attachment of bacteroidetal clades to diatoms and their discrete staining by FLBA provided evidence for the essential role that formation and recognition of glycoconjugates play in the interaction of bacteria with phytoplankton., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Members of the highly diverse bacterial phylum Verrucomicrobia are globally distributed in various terrestrial and aquatic habitats. They are key players in soils, but little is known about their role in aquatic systems. Here, we report on the design and evaluation of a 16S rRNA-targeted probe set for the identification of Verrucomicrobia and of clades within this phylum. Subsequently, the probe set was applied to a study concerning the seasonal abundance of Verrucomicrobia in waters of the humic lake Grosse Fuchskuhle (Germany) by catalyzed reporter deposition fluorescence in situ hybridization. The lake hosted diverse Verrucomicrobia clades in all seasons. Either Spartobacteria (up to 19%) or Opitutus spp. (up to 7%) dominated the communities, whereas Prosthecobacter spp. were omnipresent in low numbers (<1%). Verrucomicrobial abundance and community composition varied between the seasons, and between more and less humic basins, but were rather stable in oxic and seasonally anoxic waters.

The phylogeny, abundance, and biogeography of the NOR5/OM60 clade was investigated. This clade includes "Congregibacter litoralis" strain KT71, the first cultured representative of marine aerobic anoxygenic phototrophic Gammaproteobacteria. More than 500 16S rRNA sequences affiliated with this clade were retrieved from public databases. By comparative sequence analysis, 13 subclades could be identified, some of which are currently restricted to discrete habitat types. Almost all sequences in the largest subclade NOR5-1 and related subclade NOR5-4 originated from marine surface water samples. Overall, most of the NOR5/OM60 sequences were retrieved from marine coastal settings, whereas there were fewer from open-ocean surface waters, deep-sea sediment, freshwater, saline lakes and soil. The abundance of members of the NOR5/OM60 clade in various marine sites was determined by fluorescence in situ hybridization using a newly designed and optimized probe set. Relative abundances in coastal marine waters off Namibia and the Yangtze estuary were up to 3% of the total 4',6-diamidino-2-phenylindole (DAPI) counts, and in the German Bight off Helgoland the abundance was even up to 7%. In an open-ocean North Atlantic transect, between Iceland and the Azores, the NOR5/OM60 group was much less abundant (0.1-0.5%). Interestingly, the surface layer of North Sea intertidal sediments was very rich in NOR5/OM60, with absolute numbers >10(8) cells cm(-3) (or 4% of the total DAPI). An analysis of the frequencies of NOR5/OM60 16S rRNA genes in the Global Ocean Survey datasets provided further support for a marine cosmopolitan occurrence of NOR5/OM60, and a clear preference for coastal marine waters.

Three rod-shaped, Gram-negative, chemo-organotrophic, heterotrophic, strictly aerobic, gliding bacterial strains, KT02ds18-4, KT02ds18-5 and KT02ds18-6T, were isolated from North Sea surface waters near the island of Helgoland, Germany. Their taxonomic position was investigated by a polyphasic approach. The three strains were light yellow, oxidase- and catalase-positive, and grew optimally at 25 degrees C, at pH 7.5, and in the presence of 2.5 % (w/v) NaCl. The Chargaff's coefficient was 34.2-34.4 mol%. The three strains shared >90 % DNA-DNA relatedness and an identical 16S rRNA gene sequence. Comparative 16S rRNA gene sequence analysis allocated the three strains to the genus Maribacter in the family Flavobacteriaceae, with similarities of 97.0-97.4 % to five of the recognized Maribacter species. Their low level of DNA-DNA relatedness (<20 %) with these species and differentiating phenotypic characteristics demonstrated that they constitute a new Maribacter species for which the name Maribacter forsetii sp. nov. is proposed. Strain KT02ds18-6T (=CIP 109504T=DSM 18668T) is the type strain. An emended description of the genus Maribacter is also proposed.

In situ identification of prokaryotic cells in subsurface sediments is hampered by the low cellular rRNA contents of the target organisms. Fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) has the potential to overcome this limitation, and was therefore optimized for a 40 cm deep sediment core sampled from a tidal sandy flat of the German Wadden Sea. Treatment with methanol and H(2)O(2) inactivated endogenous peroxidases and effectively reduced the background signal. Percentage of DAPI stained cells detected with the probe combination EUB(I-III), targeting nearly all the Bacteria, were comparable for CARD-FISH with a horseradish peroxidase (HRP)-labeled probe and FISH with a fluorescently monolabeled probe in the 2-3 cm depth interval (92% and 82%, respectively), but significantly higher with the HRP-labeled probe at 35-40 cm, the deepest layer sampled (63% with HRP vs. 26% with monolabeled probe). With CARD-FISH Alphaproteobacteria and the Desulfobulbaceae group of sulfate-reducing bacteria were detected only in the upper layers. In contrast, Desulfosarcinales, the Bacteroidetes group, Planctomycetes, Betaproteobacteria, and Gammaproteobacteria were found at all depths. Archaea were detectable with ARCH915-HRP after achromopeptidase treatment. Surprisingly, aggregates of Bacteria and Archaea were found, below 12 cm depth, that strongly resemble consortia involved in anoxic oxidation of methane that have previously been found in sediments near methane hydrate deposits. With the optimized CARD-FISH protocol, microbial populations could also be detected in deeper sediment horizons. Furthermore, the intensity of the CARD-FISH signals improved detection of rare organisms such as Archaea.

We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the beta-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of beta-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the beta-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.

Almost complete 23S rRNA gene sequences were obtained from 11 Alphaproteobacteria isolated from marine surface water of the German Bight. Five of the strains belong to the "marine alpha" group, a phylogenetic cluster which encompasses members of the genus Roseobacter and closely related bacteria. Phylogenetic sequence analysis based on 52 published as well as unpublished complete 23S rDNA sequences from Alphaproteobacteria including the newly obtained was in general consistent with the 16S rRNA gene sequence-derived phylogeny. 16S and 23S rRNA based phylogenies both showed a distinct cluster for strains associated with the "marine alpha" group. The suitability of both markers for the design of oligonucleotide probes targeting selected groups of Alphaproteobacteria was systematically evaluated and compared in silico. Six clusters of sequences covering different phylogenetic levels as well as two strains were selected in a case study. To compensate for the quantitative difference in the two data sets, the 16S rRNA dataset was truncated to sequences with an equivalent in the 23S rRNA data set. Our results show, that the overall number of phylogenetically redundant probes available could be more than doubled by extending probe design to the 23S rRNA. For small clusters of high sequence similarity and single strains, up to 8 times more discriminating binding sites were provided by the 23S rRNA.

We studied the growth of six culturable bacterial lineages from coastal North Sea picoplankton in environmental samples under different incubation conditions. The grazing pressure of heterotrophic nanoflagellates (HNF) was reduced either by double prefiltration through 0.8- micro m-pore-size filters or by 10-fold dilutions with 0.2- micro m (pore-size) prefiltered seawater. We hypothesized that those gamma-proteobacterial genera that are rapidly enriched would also be most strongly affected by HNF regrowth. In the absence of HNF, the mean protein content per bacterial cell increased in both treatments compared to environmental samples, whereas the opposite trend was found in incubations of unaltered seawater. Significant responses to the experimental manipulations were observed in Alteromonas, Pseudoalteromonas, and Vibrio populations. No treatment-specific effects could be detected for members of the Roseobacter group, the Cytophaga latercula-C. marinoflava lineage, or the NOR5 clade. Statistical analysis confirmed a transient increase in the proportions of Alteromonas, Pseudoalteromonas, and Vibrio cells at reduced HNF densities only, followed by an overproportional decline during the phase of HNF regrowth. Cells from these genera were significantly larger than the community average in the dilution treatments, and changes in their relative abundances were negatively correlated with HNF densities. Our findings suggest that bacteria affiliated with frequently isolated genera such as Alteromonas, Pseudoalteromonas, and Vibrio might be rare in coastal North Sea picoplankton because their rapid growth response to changing environmental conditions is counterbalanced by a higher grazing mortality.

We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters. The application integrates an epifluorescence microscope equipped with motorized z-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software. The relative abundances of specific microbial taxa are quantified in samples of marine picoplankton, as detected by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition. Pairs of microscopic images are automatically acquired from numerous positions at two wavelengths, and microbial cells with both general DNA and FISH staining are counted after object edge detection and signal-to-background ratio thresholding. Microscopic fields that are inappropriate for cell counting are automatically excluded prior to measurements. Two nested walk paths guide the device across a series of triangular preparations until a user-defined number of total cells has been analyzed per sample. A backup autofocusing routine at incident light allows automated refocusing between individual samples and can reestablish the focal plane after fatal focusing errors at epifluorescence illumination. The system was calibrated to produce relative abundances of FISH-stained cells in North Sea samples that were comparable to results obtained by manual evaluation. Up to 28 preparations could be analyzed within 4 h without operator interference. The device was subsequently applied for the counting of different microbial populations in incubation series of North Sea waters. Automated digital microscopy greatly facilitates the processing of numerous FISH-stained samples and might thus open new perspectives for bacterioplankton population ecology.