TY - JOUR A1 - Srivastava, Abhishek A1 - McMahon, Katherine D. A1 - Stepanauskas, Ramunas A1 - Großart, Hans-Peter T1 - De novo synthesis and functional analysis of the phosphatase-encoding gene acI-B of uncultured Actinobacteria from Lake Stechlin (NE Germany) JF - International microbiology : official journal of the Spanish Society for Microbiology N2 - The National Center for Biotechnology Information [http://www.ncbi.nlm.nih. gov/guide/taxonomy/] database enlists more than 15,500 bacterial species. But this also includes a plethora of uncultured bacterial representations. Owing to their metabolism, they directly influence biogeochemical cycles, which underscores the the important status of bacteria on our planet. To study the function of a gene from an uncultured bacterium, we have undertaken a de novo gene synthesis approach. Actinobacteria of the acI-B subcluster are important but yet uncultured members of the bacterioplankton in temperate lakes of the northern hemisphere such as oligotrophic Lake Stechlin (NE Germany). This lake is relatively poor in phosphate (P) and harbors on average similar to 1.3 x 10(6) bacterial cells/ml, whereby Actinobacteria of the ac-I lineage can contribute to almost half of the entire bacterial community depending on seasonal variability. Single cell genome analysis of Actinobacterium SCGC AB141-P03, a member of the acI-B tribe in Lake Stechlin has revealed several phosphate-metabolizing genes. The genome of acI-B Actinobacteria indicates potential to degrade polyphosphate compound. To test for this genetic potential, we targeted the exoP-annotated gene potentially encoding polyphosphatase and synthesized it artificially to examine its biochemical role. Heterologous overexpression of the gene in Escherichia coli and protein purification revealed phosphatase activity. Comparative genome analysis suggested that homologs of this gene should be also present in other Actinobacteria of the acI lineages. This strategic retention of specialized genes in their genome provides a metabolic advantage over other members of the aquatic food web in a P-limited ecosystem. KW - acI-B in Actinobacteria KW - phosphatases KW - single cell genomics KW - phosphate limitation KW - Lake Stechlin KW - NE Germany Y1 - 2016 U6 - https://doi.org/10.2436/20.1501.01.262 SN - 1139-6709 SN - 1618-1905 VL - 19 SP - 39 EP - 47 PB - Institut d'Estudis Catalans CY - Barcelona ER - TY - JOUR A1 - Tada, Yuya A1 - Grossart, Hans-Peter T1 - Community shifts of actively growing lake bacteria after N-acetyl-glucosamine addition: improving the BrdU-FACS method JF - The ISME journal : multidisciplinary journal of microbial ecology N2 - In aquatic environments, community dynamics of bacteria, especially actively growing bacteria (AGB), are tightly linked with dissolved organic matter (DOM) quantity and quality. We analyzed the community dynamics of DNA-synthesizing and accordingly AGB by linking an improved bromodeoxyuridine immunocytochemistry approach with fluorescence-activated cell sorting (BrdU-FACS). FACS-sorted cells of even oligotrophic ecosystems in winter were characterized by 16S rRNA gene analysis. In incubation experiments, we examined community shifts of AGB in response to the addition of N-acetyl-glucosamine (NAG), one of the most abundant aminosugars in aquatic systems. Our improved BrdU-FACS analysis revealed that AGB winter communities of oligotrophic Lake Stechlin (northeastern Germany) substantially differ from those of total bacteria and consist of Alpha-, Beta-, Gamma-, Deltaproteobacteria, Actinobacteria, Candidatus OP10 and Chloroflexi. AGB populations with different BrdU-fluorescence intensities and cell sizes represented different phylotypes suggesting that single-cell growth potential varies at the taxon level. NAG incubation experiments demonstrated that a variety of widespread taxa related to Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Planctomycetes, Spirochaetes, Verrucomicrobia and Chloroflexi actively grow in the presence of NAG. The BrdU-FACS approach enables detailed phylogenetic studies of AGB and, thus, to identify those phylotypes which are potential key players in aquatic DOM cycling. KW - actively growing bacteria (AGB) KW - bromodeoxyuridine (BrdU) immunocytochemistry KW - fluorescence-activated cell sorting (FACS) KW - bacterial community composition KW - N-acetyl-glucosamine (NAG) KW - Lake Stechlin Y1 - 2014 U6 - https://doi.org/10.1038/ismej.2013.148 SN - 1751-7362 SN - 1751-7370 VL - 8 IS - 2 SP - 441 EP - 454 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Roesel, Stefan A1 - Grossart, Hans-Peter T1 - Contrasting dynamics in activity and community composition of free-living and particle-associated bacteria in spring JF - Aquatic microbial ecology : international journal N2 - Phytoplankton development affects the community structure and dynamics of freshwater bacteria by changing the availability of nutrients, algal exudates and biological surfaces. To elucidate these effects of phytoplankton development in spring in oligotrophic Lake Stechlin (Germany), we measured limnological and biological parameters, including the bacterial community composition (BCC), at the depth of the highest chlorophyll a concentration. To increase the resolution of BCC measurements, we separated particle-associated (PA) and free-living (FL) bacteria using serial filtration through 5.0 and 0.2 mu m pore size filters, respectively. The BCC of ultramicrobacteria was also determined by collecting the 0.2 mu m filtrate on 0.1 mu m filters. Changes in the community composition of Bacteria and particularly of Actinobacteria, one of the most important bacterial groups in temperate freshwater habitats, were studied via DGGE analysis of PCR-amplified 16S rRNA gene fragments. Patterns in BCC dynamics of FL Bacteria and Actinobacteria remained fairly constant throughout the study period, while patterns of PA Bacteria were more variable over time. At the breakdown of the diatom spring bloom, bacterial production and abundance sharply increased, indicating a close coupling between heterotrophic bacteria and algal detritus. The succession in BCC revealed life-style dependent patterns related to specific environmental variables. Our results indicate independent dynamics of PA and FL Bacteria as well as Actinobacteria during succession of phytoplankton spring blooms. These differences in bacterial lifestyle can only be resolved when the PA and FL fractions of microorganisms are separated. KW - Bacterial community composition (BCC) KW - Spring bloom KW - Bacteria-phytoplankton coupling KW - Pollen KW - Free-living and particle-associated bacteria KW - Lake Stechlin Y1 - 2012 U6 - https://doi.org/10.3354/ame01568 SN - 0948-3055 VL - 66 IS - 2 SP - 169 EP - + PB - Institute of Mathematical Statistics CY - Oldendorf Luhe ER -