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Mortality processes such as grazing and viral lysis can alter both phylogenetic diversity and cell-specific activity of bacterioplankton. We conducted experiments to examine the effect of reduction in grazer or viral abundance on metabolically active bacterioplankton at a eutrophic shelf station and an oligotrophic basin station. Leucine assimilation was used as a proxy to characterize metabolically active bacterial cells. The phylogenetic affiliation of marine bacterioplankton assimilating leucine was identified with fluorescence in situ hybridization and microautoradiography. Flow cytometric sorting of leucine-labeled cells quantified cell-specific activity of high nucleic acid (HNA) and low nucleic acid cells. The activity and diversity of the bacterial community at the oligotrophic station was more responsive to a reduction in mortality compared to the community at the eutrophic station. HNA cells at the oligotrophic station showed a 4-fold increase in cell-specific leucine incorporation when the abundance of flagellates was reduced and an 8-fold increase when the abundance of flagellates, viruses, and bacterial cells was reduced. The abundance of active Alphaproteobacteria increased at the oligotrophic station when either grazer or viral abundance was reduced. Activity responses were less striking at the eutrophic station, and the abundance of active Gammaproteobacteria showed a greater increase with reduced flagellates compared to the treatment with reduced flagellates, viruses, and bacterioplankton. Our results indicate both the presence and type of mortality process play a key role in structuring a bacterial community, and this effect varies in ecosystems of differing trophic state.KEY WORDS: Marine bacterioplankton · Top-down control · Leucine incorporation · Flow cytometry · Microautoradiography · Fluorescence in situ hybridization · Grazing · Viral lysis Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 58: [153][154][155][156][157][158][159][160][161][162][163][164][165] 2010 Protocols involving a combination of fluorescence in situ hybridization (FISH) with microautoradiography allow identification of metabolically active phylogenetic groups within marine bacterioplankton (e.g. Ouverney & Fuhrman 1999, Cottrell & Kirchman 2000. This approach has provided important insights into both spatial (Elifantz et al. 2007, Varela et al. 2008) and temporal (Alonso-Sáez & Gasol 2007) variability in the diversity of active bacterial cells. In previous work in the Oregon upwelling system, we found little spatial variability in the diversity of active cells (Longnecker et al. 2005), although the abundance of active cells increased with ecosystem trophic state (Longnecker et al. 2006a). These findings raised the question of the relative importance of top-down (mortality) versus bottom-up (substrate availability) processes in eutrophic compared to oligotrophic marine ecosystems.In addition to the qualitative information derived from FISH analysis, flow cyto...

The spacing of peptides away from a hydrogel matrix dramatically impacts their availability and subsequent interactions with cells. Peptides were synthesized with monodisperse poly(ethylene glycol) spacers of different lengths that separate the peptide from the monomeric functionality which reacts during hydrogel polymerization. Specifically, bioactive RGD ligands were conjugated to PEG5, PEG11 or PEG27 spacers via solid phase techniques and then functionalized with an acryloyl end group. These acryloyl-PEGx-RGD conjugates were then copolymerized with PEGDA to form an inert hydrogel network decorated with RGD ligands for cell interactions. As the PEG spacer length increases, the RGD concentration required to support cell attachment and spreading decreases. The competitive detachment of hTCEpi cells in the presence of soluble linear RGD also shows non-linear dependence on the PEG spacer length, as more cells remained attached and spread on gels functionalized with longer PEG-RGD conjugates in comparison to the shorter PEG-RGD conjugates. The strategy and synthetic techniques developed here allow for reproducible control over peptide-hydrogel spacing and peptide concentration, and may be extended for incorporation of multiple peptides and to other hydrogel platforms.

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