Members of the prokaryotic picoplankton are the main drivers of the biogeochemical cycles over large areas of the world's oceans. In order to ascertain changes in picoplankton composition in the euphotic and twilight zones at an ocean basin scale we determined the distribution of 11 marine bacterial and archaeal phyla in three different water layers along a transect across the Atlantic Ocean from South Africa (32.9 degrees S) to the UK (46.4 degrees N) during boreal spring. Depth profiles down to 500 m at 65 stations were analysed by catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and automated epifluorescence microscopy. There was no obvious overall difference in microbial community composition between the surface water layer and the deep chlorophyll maximum (DCM) layer. There were, however, significant differences between the two photic water layers and the mesopelagic zone. SAR11 (35 +/- 9%) and Prochlorococcus (12 +/- 8%) together dominated the surface waters, whereas SAR11 and Crenarchaeota of the marine group I formed equal proportions of the picoplankton community below the DCM (both approximately 15%). However, due to their small cell sizes Crenarchaeota contributed distinctly less to total microbial biomass than SAR11 in this mesopelagic water layer. Bacteria from the uncultured Chloroflexi-related clade SAR202 occurred preferentially below the DCM (4-6%). Distinct latitudinal distribution patterns were found both in the photic zone and in the mesopelagic waters: in the photic zone, SAR11 was more abundant in the Northern Atlantic Ocean (up to 45%) than in the Southern Atlantic gyre (approximately 25%), the biomass of Prochlorococcus peaked in the tropical Atlantic Ocean, and Bacteroidetes and Gammaproteobacteria bloomed in the nutrient-rich northern temperate waters and in the Benguela upwelling. In mesopelagic waters, higher proportions of SAR202 were present in both central gyre regions, whereas Crenarchaeota were clearly more abundant in the upwelling regions and in higher latitudes. Other phylogenetic groups such as the Planctomycetes, marine group II Euryarchaeota and the uncultured clades SAR406, SAR324 and SAR86 rarely exceeded more than 5% of relative abundance.

We describe a method for microscopic identification of DNA-synthesizing cells in bacterioplankton samples. After incubation with the halogenated thymidine analogue bromodeoxyuridine (BrdU), environmental bacteria were identified by fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-linked oligonucleotide probes. Tyramide signal amplification was used to preserve the FISH staining during the subsequent immunocytochemical detection of BrdU incorporation. DNA-synthesizing cells were visualized by means of an HRP-labeled antibody Fab fragment and a second tyramide signal amplification step. We applied our protocol to samples of prefiltered (pore size, 1.2 m) North Sea surface water collected during early autumn. After 4 h of incubation, BrdU incorporation was detected in 3% of all bacterial cells. Within 20 h the detectable DNA-synthesizing fraction increased to >14%. During this period, the cell numbers of members of the Roseobacter lineage remained constant, but the fraction of BrdU-incorporating Roseobacter sp. cells doubled, from 24 to 42%. In Alteromonas sp. high BrdU labeling rates after 4 to 8 h were followed by a 10-fold increase in abundance. Rapid BrdU incorporation was also observed in members of the SAR86 lineage. After 4 h of incubation, cells affiliated with this clade constituted 8% of the total bacteria but almost 50% of the visibly DNA-synthesizing bacterial fraction. Thus, this clade might be an important contributor to total bacterioplankton activity in coastal North Sea water during periods of low phytoplankton primary production. The small size and low ribosome content of SAR86 cells are probably not indications of inactivity or dormancy.As awareness of the importance, diversity, and complexity of marine microbial communities develops, it is increasingly recognized that an understanding of the ecology of the various bacterial (and archaeal) populations requires simultaneous information about both the identity and the activity of individual cells in environmental samples (8,27,49). Consequently, a considerable amount of recent research effort has focused on the development of techniques that permit estimation of various activities of single bacterial cells (16,35). In addition, cultivation-independent approaches have been developed for quantification of the sizes of populations of individual bacterial taxa, such as fluorescence in situ hybridization (FISH) with oligonucleotide or polynucleotide rRNA-targeted probes (1,9,10,17). FISH is an increasingly popular tool for microscopic visualization of individual groups of bacteria and archaea in the marine environment (7,9,14,22,29,31). Furthermore, recent technical advances have greatly increased the sensitivity of this staining technique (9, 29, 30), so that the low ribosome content of many planktonic bacteria no longer limits the applicability of FISH to productive or coastal regions (30). One of the challenges of present-day microbial ecology is to combine FISH with techniques that allow researchers to determine defined mi...

Members of the gammaproteobacterial clade NOR5/OM60 regularly form an abundant part, up to 11%, of the bacterioplankton community in coastal systems during the summer months. Here, we report the nearly complete genome sequence of one cultured representative, Congregibacter litoralis strain KT71, isolated from North Sea surface water. Unexpectedly, a complete photosynthesis superoperon, including genes for accessory pigments, was discov- from a surface water sample taken near the North Sea island Helgoland, by direct plating on complex low-nutrient media. Phylogenetic analysis showed that KT71 was the first cultured representative of a cosmopolitan gammaproteobacterial lineage, which we in the following refer to as the NOR5/OM60 clade (Fig. 1). The first indication for this clade dates back to 1997, when Rappe et al. retrieved two 16S rRNA clones, OM60 and OM241, from the continental shelf off Cape Hatteras, NC (2). In the following years, many sequences have been retrieved that were related to the clone OM60 (e.g.,. By the end of 2005, Ͼ180 partial and full length 16S rRNA sequences available within the public databases were related to KT71 and OM60.KT71 is a Gram-negative, pleomorphic, strictly aerobic, and motile bacterium. It is of an average size of 2 ϫ 0.5 m, has a generation time of 4.5 h, and often grows in flocs. Based on this conspicuous trait and the site of isolation, the name Congregibacter litoralis has been proposed. A full taxonomic description of strain KT71 is currently ongoing (B.M.F., S.S., and R.A., unpublished work). Several more strains belonging to the NOR5/ OM60 clade were isolated off the coast of Oregon, in sterilized seawater, using a high-throughput dilution-to-extinction technique (9, 10). Meanwhile, representatives of the NOR5/OM60 clade were also isolated from Arctic sea ice (11) and coastal sediments (12,13).FISH with rRNA-targeted oligonucleotide probes for NOR5/ OM60 confirmed this clade as an abundant component of the bacterioplankton community in the North Sea around the island Helgoland (1). By the end of July 1998, up to 8% of the total bacterioplankton community comprised members of the NOR5/ OM60 clade (1). A second peak of NOR5/OM60 cells was visible in mid-June (6%). However, NOR5/OM60 was not detected by FISH during the winter months, October to March, suggesting a marked seasonality. The fraction of DNA-synthesizing NOR5/ OM60 cells seems to be quite variable. Active DNA synthesis could be detected in August but not in May 2002, even though NOR5/OM60 was present in high numbers in both samples (6% and 11% of total bacterioplankton cell, respectively) (14).In 2004, KT71 was selected for whole-genome sequencing as part of the Gordon and Betty Moore Foundation (GBMF) Marine Microbiology Initiative. Here, we present data derived from the analysis of the genome of strain KT71 and from ecophysiological experiments addressing some of the predictions derived from genome annotation. Results and Discussion Structure and Phylogenetic Analysis of the Photosynthesis (PS)Operon. The...

No abstract

In the age of ever-increasing "-omics" studies, the accurate and statistically robust determination of microbial cell numbers within often-complex samples remains a key task in microbial ecology. Microscopic quantification is still the only method to enumerate specific subgroups of microbial clades within complex communities by, for example, fluorescence in situ hybridization (FISH). In this study, we improved an existing automatic image acquisition and cell enumeration system and adapted it for usage at high seas on board an oceanographic research ship. The system was evaluated by testing settings such as minimal pixel area and image exposure times ashore under stable laboratory conditions before being brought on board and tested under various wind and wave conditions. The system was robust enough to produce high-quality images even with ship heaves of up to 3 m and pitch and roll angles of up to 6.3°. On board the research ship, on average, 25% of the images acquired from plankton samples on filter membranes could be used for cell enumeration. Automated enumeration was highly correlated with manual counts (r 2 > 0.9). Even the smallest of microbial cells in the open ocean, members of the alphaproteobacterial SAR11 clade, could be confidently detected and enumerated. The automated image acquisition and cell enumeration system developed here enables an accurate and reproducible determination of microbial cell counts in planktonic samples and allows insight into the abundance and distribution of specific microorganisms already on board within a few hours. IMPORTANCEIn this research article, we report on a new system and software pipeline, which allows for an easy and quick image acquisition and the subsequent enumeration of cells in the acquired images. We put this pipeline through vigorous testing and compared it to manual microscopy counts of microbial cells on membrane filters. Furthermore, we tested this system at sea on board a marine research vessel and counted bacteria on board within a few hours after the retrieval of water samples. The imaging and counting system described here has been successfully applied to a number of laboratory-based studies and allowed the quantifi- T he exact quantification of cells is fundamental to microbial ecology and hence for understanding the interaction of microorganisms with biotic and abiotic factors. Still, the counting of microbial cells on membrane filters using an epifluorescence microscope remains the method of choice (1-3) for the enumeration of picoplankton cells, although it is rather time-consuming and relies on the experience of the individual person counting. Additionally, manual counting of an entire membrane filter is not practical within a given time frame, and therefore, only a small part is analyzed (usually 12 to 20 fields of view [FOVs]) (3, 4). Consequently, several tools have been developed over the past 2 decades to automatically enumerate microbial cells by means of image acquisition and subsequent image analysis (e.g., references 5-13). Mos...