Distinct biogeographic patterns of bacterioplankton composition and single-cell activity between the subtropics and Antarctica.

Bacterial biogeography and activity in the Southern Ocean are poorly understood to date. Here, we applied CARD-FISH to quantify bacterial community structure from the subtropics to Antarctica between 10°W and 10°E, covering four biogeographic provinces with distinct environmental properties. In addition, incorporation of radiolabeled glucose, amino acids and leucine via MAR-FISH served to quantify the contribution to substrate turnover by selected bacterial groups. SAR11, Bacteroidetes, Gammaproteobacteria and the Roseobacter group accounted for the majority of the bacterial community (52%-88% of DAPI-stained cells) but showed little distributional variation between provinces. In contrast, taxonomic subclades Polaribacter, NS5, NS2b (Bacteroidetes) as well as RCA (Roseobacter group) featured marked geographic variation, illustrated by NMDS and coefficients of variation. Roseobacter (specifically RCA) and Gammaproteobacteria constituted considerable fractions of cells incorporating glucose and amino acids respectively. Bacteroidetes had generally lower activities, but Polaribacter accounted for a major fraction of biomass production at one station near the Antarctic ice shelf. In conclusion, distributional patterns at finer taxonomic level and highest substrate turnover by less abundant taxa highlight the importance of taxonomic subclades in marine carbon fluxes, contributing to the understanding of functional bacterial biogeography in the Southern Ocean.

Adaptation of an abundant Roseobacter RCA organism to pelagic systems revealed by genomic and transcriptomic analyses.

The RCA (Roseobacter clade affiliated) cluster, with an internal 16S rRNA gene sequence similarity of >98%, is the largest cluster of the marine Roseobacter clade and most abundant in temperate to (sub)polar oceans, constituting up to 35% of total bacterioplankton. The genome analysis of the first described species of the RCA cluster, Planktomarina temperata RCA23, revealed that this phylogenetic lineage is deeply branching within the Roseobacter clade. It shares not >65.7% of homologous genes with any other organism of this clade. The genome is the smallest of all closed genomes of the Roseobacter clade, exhibits various features of genome streamlining and encompasses genes for aerobic anoxygenic photosynthesis (AAP) and CO oxidation. In order to assess the biogeochemical significance of the RCA cluster we investigated a phytoplankton spring bloom in the North Sea. This cluster constituted 5.1% of the total, but 10-31% (mean 18.5%) of the active bacterioplankton. A metatranscriptomic analysis showed that the genome of P. temperata RCA23 was transcribed to 94% in the bloom with some variations during day and night. The genome of P. temperata RCA23 was also retrieved to 84% from metagenomic data sets from a Norwegian fjord and to 82% from stations of the Global Ocean Sampling expedition in the northwestern Atlantic. In this region, up to 6.5% of the total reads mapped on the genome of P. temperata RCA23. This abundant taxon appears to be a major player in ocean biogeochemistry.

Planktomarina temperata gen. nov., sp. nov., belonging to the globally distributed RCA cluster of the marine Roseobacter clade, isolated from the German Wadden Sea.

Four heterotrophic bacterial strains belonging to the globally distributed marine RCA (Roseobacter clade-affiliated) cluster (family Rhodobacteraceae, class Alphaproteobacteria) were obtained from coastal seawater samples. Strain RCA23(T) was isolated from a 10(-7) dilution culture inoculated with seawater from the German Wadden Sea (southern North Sea), reflecting the high abundance of RCA bacteria in this habitat. Strains IMCC1909, IMCC1923 and IMCC1933 were isolated from diluted seawater (10(-3)) of the Yellow Sea, South Korea. Based on 16S rRNA gene sequence comparison, Octadecabacter antarcticus 307(T) is the closest described relative of the RCA strains, with 95.4-95.5 % sequence similarity. Cells of RCA23(T), IMCC1909, IMCC1923 and IMCC1933 are small motile rods requiring sodium ions. Optimal growth of RCA23(T) occurs at 25 °C and within a very narrow pH range (pH 7-8, optimum pH 7.5). The DNA G+C base content of RCA23(T) is 53.67 mol%. The major respiratory lipoquinone is ubiquinone-10 (Q-10) and the dominant fatty acids (>1 %) are 12 : 1 3-OH, 16 : 1ω7c, 16 : 0, 18 : 1ω7c, 18 : 0 and 11-methyl 18 : 1ω7c. The polar lipid pattern indicated the presence of phosphatidylglycerol, two unidentified aminolipids and two unidentified phospholipids. On marine agar, RCA23(T) forms non-pigmented, transparent to light beige, small (<1 mm), circular, convex colonies. Strain RCA23(T) harbours all genes for the production of bacteriochlorophyll a (BChl a). Genes encoding the light-harvesting reaction centre of BChl a (pufM) were identified in all RCA strains. No visible pigmentation was observed for any of the strains under laboratory conditions, but spectrophotometric analysis revealed weak production of BChl a by RCA23(T). Morphological, physiological and genotypic features of strain RCA23(T) suggest that it represents a novel species of a new genus within the Rhodobacteraceae, for which we propose the name Planktomarina temperata gen. nov., sp. nov., described previously by Giebel et al. [ISME J 5 (2011), 8-19] as 'Candidatus Planktomarina temperata'. The type strain of Planktomarina temperata is RCA23(T) ( = DSM 22400(T) = JCM 18269(T)).

Distinct seasonal growth patterns of the bacterium Planktotalea frisia in the North Sea and specific interaction with phytoplankton algae.

We investigated the occurrence of Planktotalea frisia strain SH6-1(T), a member of the Roseobacter clade, in the North Sea, and interactions with phytoplankton algae with a special emphasis on the carbohydrate metabolisms. This bacterium was present in May 2006 throughout the North Sea. Planktotalea frisia SH6-1 was further present in the German Bight between February and early July, with distinct peaks during and after phytoplankton blooms. The highest abundances, as detected by quantitative PCR, were 0.5-0.9% of total bacterial abundance. Comparison by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) with a set of highly specific probes confirmed the high values in one sample. Between mid-July and October, P. frisia SH6-1 was not detected throughout the North Sea. Experimental studies in which P. frisia SH6-1 was grown in the presence of axenic cultures of the algae Phaeocystis globosa, Leptocylindrus danicus and Thalassiosira rotula exhibited distinctly different responses, with the best growth together with P. globosa and T. rotula and very low growth together with L. danicus. The algae greatly differed in the composition of their exuded carbohydrates and in the fact that P. frisia SH6-1 was rather selective in consumption of algae, suggesting that the distinct carbohydrate metabolisms are a key feature to explain the seasonal occurrence of this bacterium in the North Sea.

Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria.

Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2 m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this cluster was only detected on aggregates up to a few meters above the sediment surface, but never in the fraction of free-living bacteria. A quantitative real-time PCR approach revealed that the MMC constituted up to 13% of total bacterial 16S rRNA genes in surface sediments of the North Sea. In a global survey, including sediments from the Mediterranean Sea, the Atlantic, Pacific and Indian Ocean and various climatic regions, the MMC was detected in most samples and to a water depth of 4300 m. Two fosmids of a library from sediment of the southern North Sea containing 16S rRNA genes affiliated with the MMC were sequenced. Both fosmids have a single unlinked 16S rRNA gene and no complete rRNA operon as found in most bacteria. No synteny to other myxobacterial genomes was found. The highest numbers of orthologues for both fosmids were assigned to Sorangium cellulosum and Haliangium ochraceum. Our results show that the MMC is an important and widely distributed but largely unknown component of marine sediment-associated bacterial communities.

Quantitative role of shrimp fecal bacteria in organic matter fluxes in a recirculating shrimp aquaculture system.

Microorganisms play integral roles in the cycling of carbon (C) and nitrogen (N) in recirculating aquaculture systems (RAS) for fish and shellfish production. We quantified the pathways of shrimp fecal bacterial activities and their role in C- and N-flux partitioning relevant to culturing Pacific white shrimp, Penaeus (Litopenaeus) vannamei, in RAS. Freshly produced feces from P. vannamei contained 0.6-7 × 10(10) bacteria g(-1) dry wt belonging to Bacteroidetes (7%), Alphaproteobacteria (4%), and, within the Gammaproteobacteria, almost exclusively to the genus Vibrio (61%). Because of partial disintegration of the feces (up to 27% within 12 h), the experimental seawater became inoculated with fecal bacteria. Bacteria grew rapidly in the feces and in the seawater, and exhibited high levels of aminopeptidase, chitinase, chitobiase, alkaline phosphatase, α- and ß-glucosidase, and lipase activities. Moreover, fecal bacteria enriched the protein content of the feces within 12 h, potentially enriching the feces for the coprophagous shrimp. The bacterial turnover time was much faster in feces (1-10 h) than in mature RAS water (350 h). Thus, shrimp fecal bacteria not only inoculate RAS water but also contribute to bacterial abundance and productivity, and regulate system processes important for shrimp health.

Microfabrication of patterns of adherent marine bacterium Phaeobacter inhibens using soft lithography and scanning probe lithography.

Two lithographic approaches have been explored for the microfabrication of cellular patterns based on the attachment of marine bacterium Phaeobacter inhibens strain T5. Strain T5 produces a new antibiotic that makes this bacterium potentially interesting for the pharmaceutical market and as a probiotic organism in aquacultures and in controlling biofouling. The microcontact printing (microCP) method is based on the micropatterning of self-assembled monolayers (SAMs) terminated with adhesive end groups such as CH(3) and COOH and nonadhesive groups (e.g., short oligomers of ethylene glycol (OEG)) to form micropatterned substrates for the adhesion of strain T5. The scanning probe lithographic method is based on the surface modification of OEG SAM by using a microelectrode, the probe of a scanning electrochemical microscope (SECM). Oxidizing agents (e.g., Br(2)) were electrogenerated in situ at the microelectrodes from Br(-) in aqueous solution to remove OEG SAMs locally, which allows the subsequent adsorption of bacteria. Various micropatterns of bacteria could be formed in situ on the substrate without a prefabricated template. The fabricated cellular patterns may be applied to a variety of marine biological studies that require the analysis of biofilm formation, cell-cell and cell-surface interactions, and cell-based biosensors and bioelectronics.

Detection of ingested bacteria in benthic ciliates using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.

Are readily culturable bacteria in coastal North Sea waters suppressed by selective grazing mortality?

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.