Bacterioplankton phylotypes of alpha-Proteobacteria have been detected in various marine regions, but systematic biogeographical studies of their global distribution are missing. Alpha-Proteobacteria comprise one of the largest fractions of heterotrophic marine bacteria and include two clades, SAR11 and Roseobacter, which account for 26 and 16% of 16S ribosomal RNA gene clones retrieved from marine bacterioplankton. The SAR11 clade attracted much interest because related 16S rRNA gene clones were among the first groups of marine bacteria to be identified by cultivation-independent approaches and appear to dominate subtropical surface bacterioplankton communities. Here we report on the global distribution of a newly discovered cluster affiliated to the Roseobacter clade, comprising only as-yet-uncultured phylotypes. Bacteria of this cluster occur from temperate to polar regions with highest abundance in the Southern Ocean, but not in tropical and subtropical regions. Between the south Atlantic subtropical front and Antarctica, we detected two distinct phylotypes, one north and one south of the polar front, indicating that two adjacent but different oceanic provinces allow the persistence of distinct but closely related phylotypes. These results suggest that the global distribution of major marine bacterioplankton components is related to oceanic water masses and controlled by their environmental and biogeochemical properties.

We assessed the composition of the bacterioplankton in the Atlantic sector of the Southern Ocean in austral fall and winter and in New Zealand coastal waters in summer. The various water masses between the subtropics/Agulhas-Benguela boundary region and the Antarctic coastal current exhibited distinct bacterioplankton communities with the highest richness in the polar frontal region, as shown by denaturing gradient gel electrophoresis of 16S rRNA gene fragments. The SAR11 clade and the Roseobacter clade-affiliated (RCA) cluster were quantified by real-time quantitative PCR. SAR11 was detected in all samples analysed from subtropical waters to the coastal current and to depths of > 1000 m. In fall and winter, this clade constituted < 3% to 48% and 4-28% of total bacterial 16S rRNA genes respectively, with highest fractions in subtropical to polar frontal regions. The RCA cluster was only present in New Zealand coastal surface waters not exceeding 17 degrees C, in the Agulhas-Benguela boundary region (visited only during the winter cruise), in subantarctic waters and in the Southern Ocean. In fall, this cluster constituted up to 36% of total bacterial 16S rRNA genes with highest fractions in the Antarctic coastal current and outnumbered the SAR11 clade at most stations in the polar frontal region and further south. In winter, the RCA cluster constituted lower proportions than the SAR11 clade and did not exceed 8% of total bacterial 16S rRNA genes. In fall, the RCA cluster exhibited significant positive correlations with latitude and ammonium concentrations and negative correlations with concentrations of nitrate, phosphate, and for near-surface samples also with chlorophyll a, biomass production of heterotrophic prokaryotes and glucose turnover rates. The findings show that the various water masses between the subtropics and the Antarctic coastal current harbour distinct bacterioplankton communities. They further indicate that the RCA cluster, despite the narrow sequence similarity of > 98% of its 16S rRNA gene, is an abundant component of the heterotrophic bacterioplankton in the Southern Ocean, in particular in its coldest regions.

We studied the spatio-temporal dynamics and community composition of the free-living and particle-associated bacterial community in the salinity gradient of the Weser estuary, Germany, between March and December 1999 and in May 2000. Bacterial numbers covaried with temperature and those of the particle-associated fraction with the turbidity, exhibiting highest proportions in the turbidity maximum between July and December. The analysis of the composition of the particle-associated bacterial community by fluorescence in situ hybridization (FISH) with group-specific oligonucleotide probes showed that Cytophaga/Flavobacteria (CF) comprised the highest proportions in the freshwater section (mean: 28 ± 8.9% of DAPI cell counts) and decreased towards the marine section to 14.0 ± 3.7%. α-, β-and γ-Proteobacteria constituted around 10% without pronounced variations among the various sections. The community analysis based on PCR-amplified fragments of the 16S rRNA gene, along with denaturing gradient gel electrophoresis (DGGE) and a cluster analysis of the banding patterns, exhibited pronounced differences along the salinity gradient and well-separated communities within the freshwater, brackish and marine sections. Seasonal differences within the separate communities and between the particle-associated and free-living bacterial communities were less pronounced. The sequence analysis of prominent bands revealed that the communities consisted of bacteria affiliated to Proteobacteria, CF and Actinobacteria. Clones of the CF cluster were rather distantly related to phylotypes from other aquatic environments, whereas clones related to Actinobacteria clustered closely together with phylotypes from other aquatic systems. Clones belonging to α-and β-Proteobacteria also affiliated closely to phylotypes from other aquatic systems but more closely to isolated strains than the CF and Actinobacteria clones.KEY WORDS: Bacteria · DGGE · Cluster analysis · 16S rDNA sequences · Fluorescence in situ hybridization · Estuaries · Turbidity maximum Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 30: [221][222][223][224][225][226][227][228][229][230][231][232][233][234][235][236][237] 2003 exist in estuaries consisting of organisms with a wider salt tolerance than most other marine and freshwater biota (Nybakken 2001). Whether typical prokaryotic estuarine communities also exist is basically unknown, mainly because there have been fewer studies of the composition of estuarine bacterial communities than of freshwater and marine bacterial communities, which are quite different. Certain clusters within the α-and β-subclass Proteobacteria are typical for freshwater environments but are basically absent in marine environments where other bacterial groups typically prosper (Rheinheimer 1991, Zwart et al. 1998, Glöckner et al. 1999. There are some indications of a distinct estuarine bacterial community from the Columbia River estuary, which consists of selected freshwater and marine compon...

The composition of the bacterioplankton community in mesotrophic Lake Constance was studied from April 1996 to December 1997 by fluorescence in situ hybridization (FISH) with group specific oligonucleotide probes and denaturing gradient gel electrophoresis (DGGE). Between 10 and 59% of the DAPI-stained cells were detected as Bacteria with means of 38 ± 12, 27 ± 10 and 23 ± 6% at 3, 20, and 50 m depth, respectively. In the euphotic zone at 3 m depth, β-Proteobacteria always constituted highest proportions of the bacterial community detected by FISH and constituted 6 to 23% of the DAPIstained cells with a mean of 12%. Proportions of bacteria belonging to the Cytophaga/Flavobacteria (CF) cluster and the α-Proteobacteria at this depth were 5.4 ± 2.5 and 4.1 ± 3.5%, respectively. Proportions of β-Proteobacteria decreased at 20 and 50 m, whereas that of α-Proteobacteria remained constant and that of the CF-cluster increased to 7.4%, such that they comprised similar proportions as β-Proteobacteria at 50 m depth. γ-Proteobacteria were always of minor importance and constituted only 2.2 ± 1.3% of the DAPI cell counts. Cumulative proportions of all detected bacterial groups constituted 75% of the Bacteria counts at 3 m, 84% at 20 m and 99% at 50 m. The DGGE analysis of the bacterioplankton community showed seasonal and vertical variations of the banding patterns even though several bands occurred throughout the study period and at all depths. A cluster analysis revealed distinct bacterial clusters of the phytoplankton spring bloom and the later period and of epilimnetic and hypolimnetic waters. The sequence analysis of the excised bands indicated that all groups which were detected by FISH were represented in the patterns. Sequence similarities of the retrieved sequences to known isolates were < 90 to 96.1%, and to other phylotypes as close as 99%. Phylotypes of β-Proteobacteria and Actinobacteria of the typical banding patterns affiliated closely with recently described clusters of typical freshwater bacteria. In addition, we identified phylotypes of the Holophaga/Acidobacterium and Nitrospira phylum during the stratified period in samples from 50 m and during mixing from all 3 depths. KEY WORDS: Bacteria · DGGE · Fluorescence in situ hybridization · Lake Constance · Actinobacteria · Nitrospira · Holophaga Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 31: [211][212][213][214][215][216][217][218][219][220][221][222][223][224][225] 2003

To assess the composition and abundant members of the bacterial community in the Weser estuary, Germany, we applied a PCR-dependent approach simultaneously with a PCRindependent approach, the dilution culture technique. Dilution cultures growing in autoclaved ambient water without any nutrient addition from the limnetic, brackish and marine sections of the Weser estuary from May, August and November 1999 yielded growth to a dilution of 10 -6 to 10 -9, equivalent to a cultivation efficiency of 1.5 to 66%. Bacteria enriched in the highest dilution steps were analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. The results show that with increasing dilution steps, fewer (in 7 cases only 1) DGGE bands occurred that matched prominent bands in the DGGE patterns of the natural bacterial community. In the 7 cases with only 1 band, almost complete 16S rRNA gene sequences were retrieved, thus extending the limited sequence information provided by the DGGE approach. The dominant phylotypes included bacteria of clusters within α-and β-Proteobacteria, the Flavobacteria/Sphingobacteria group of the Bacteroidetes phylum and Actinobacteria, comprising cultivated strains but also clusters of as yet uncultured phylotypes. Closely related bacteria and phylotypes in most of these clusters are well known from other limnetic and marine systems. We also obtained isolates from the high dilution steps. More than 30% of them, belonging to various clusters within α-and β-Proteobacteria and the Flavobacteria/Sphingobacteria group, had a sequence similarity of < 96% to described species. α-Proteobacteria constituted the most abundant phylotypes of the DGGE bands in the high dilution steps and of isolates, whereas Flavobacteria/Sphingobacteria constituted only low proportions. There were pronounced differences in the composition of bacterial communities on solid-and liquid-phase media in enrichment cultures with algal extracts and a preferential growth of γ-Proteobacteria that did not occur in the natural samples and the high dilution steps.

One aim of the EC Framework V project, 'Rumen-up' (QLK5-CT-2001-00 992), was to find plants or plant extracts that would inhibit the nutritionally wasteful degradation of protein in the rumen. A total of 500 samples were screened in vitro using 14 C-labelled casein in a 30-min incubation with ruminal digesta. Eight were selected for further investigation using a batch fermentation system and soya protein and bovine serum albumin as proteolysis substrates; proteolysis was monitored over 12 h by the disappearance of soluble protein and the production of branched SCFA and NH 3 . Freeze-dried, ground foliage of Peltiphyllum peltatum, Helianthemum canum, Arbutus unedo, Arctostaphylos uva-ursi and Knautia arvensis inhibited proteolysis (P, 0·05), while Daucus carota, Clematis vitalba and Erica arborea had little effect. Inhibition by the first four samples appeared to be caused by the formation of insoluble tannin -protein complexes. The samples were rich in phenolics and inhibition was reversed by polyethyleneglycol. In contrast, K. arvensis contained low concentrations of phenolics and no tannins, had no effect in the 30-min assay, yet inhibited the degradation rate of soluble protein (by 14 %, P, 0·0001) and the production of branched SCFA (by 17 %, P , 0·05) without precipitating protein in the 12-h batch fermentation. The effects showed some resemblance to those obtained in parallel incubations containing 3 mM-monensin, suggesting that K. arvensis may be a plant-derived feed additive that can suppress growth and activity of key proteolytic ruminal micro-organisms in a manner similar to that already well known for monensin.

To examine the adaptation of bacterioplankton communities in the Southern Ocean to various biopolymeric substrates, we carried out experiments at the Polar Front and in the Antarctic Coastal Current (CC) in which samples were enriched with agarose, starch, peptone and extracts of the green alga Scenedesmus acutus and diatoms from the Polar Front. Growth and metabolic activity were assessed based on leucine incorporation and turnover rates of glucose, dissolved free amino acids and protein. In both experiments, growth was highest on peptone and the diatom extract and lowest on agarose. There was a general trend of decreasing proportions of Alphaproteobacteria and increasing proportions of Flavobacteria/Sphingobacteria and Gammaproteobacteria. The growth response and shift to Flavobacteria/Sphingobacteria was more pronounced in the CC experiment. A cluster analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns showed that bacterial communities in each treatment of both experiments differed and that those in the treatments with peptone and the Scenedesmus and diatom extracts clustered together. Sequencing of excised bands revealed that Gammaproteobacteria comprised members of Pseudoalteromonadaceae, the Colwellia cluster of Alteromonadaceae and 1 other cluster. Alphaproteobacteria comprised only members of the Roseobacter clade and the Bacteroidetes phylum only of Flavobacteria and Cytophagales. The results show that the bacterioplankton communities in the Southern Ocean are able to adapt to various biopolymeric substrates. The most pronounced response to the additions of peptone and the diatom extract rich in protein is in line with previous observations that proteins are preferred bacterial substrates in this oceanic region.KEY WORDS: Bacteria · Fluorescence in situ hybridization · DGGE · Cluster analysis · Biopolymers · Southern OceanResale or republication not permitted without written consent of the publisher