Non-Redfield, nutrient synergy and flexible internal elemental stoichiometry in a marine bacterium.

The stoichiometric constraints of algal growth are well understood, whereas there is less knowledge for heterotrophic bacterioplankton. Growth of the marine bacterium Phaeobacter inhibens DSM 17395, belonging to the globally distributed Roseobacter group, was studied across a wide concentration range of NH4+ and PO43-. The unique dataset covers 415 different concentration pairs, corresponding to 207 different molar N:P ratios (from 10-2 to 105). Maximal growth (by growth rate and biomass yield) was observed within a restricted concentration range at N:P ratios (∼50-120) markedly above Redfield. Experimentally determined growth parameters deviated to a large part from model predictions based on Liebig's law of the minimum, thus implicating synergistic co-limitation due to biochemical dependence of resources. Internal elemental ratios of P. inhibens varied with external nutrient supply within physiological constraints, thus adding to the growing evidence that aquatic bacteria can be flexible in their internal elemental composition. Taken together, the findings reported here revealed that P. inhibens is well adapted to fluctuating availability of inorganic N and P, expected to occur in its natural habitat (e.g. colonized algae, coastal areas). Moreover, this study suggests that elemental variability in bacterioplankton needs to be considered in the ecological stoichiometry of the oceans.

Complementary Metaproteomic Approaches to Assess the Bacterioplankton Response toward a Phytoplankton Spring Bloom in the Southern North Sea.

Annually recurring phytoplankton spring blooms are characteristic of temperate coastal shelf seas. During these blooms, environmental conditions, including nutrient availability, differ considerably from non-bloom conditions, affecting the entire ecosystem including the bacterioplankton. Accordingly, the emerging ecological niches during bloom transition are occupied by different bacterial populations, with Roseobacter RCA cluster and SAR92 clade members exhibiting high metabolic activity during bloom events. In this study, the functional response of the ambient bacterial community toward a Phaeocystis globosa bloom in the southern North Sea was studied using metaproteomic approaches. In contrast to other metaproteomic studies of marine bacterial communities, this is the first study comparing two different cell lysis and protein preparation methods [using trifluoroethanol (TFE) and in-solution digest as well as bead beating and SDS-based solubilization and in-gel digest (BB GeLC)]. In addition, two different mass spectrometric techniques (ESI-iontrap MS and MALDI-TOF MS) were used for peptide analysis. A total of 585 different proteins were identified, 296 of which were only detected using the TFE and 191 by the BB GeLC method, demonstrating the complementarity of these sample preparation methods. Furthermore, 158 proteins of the TFE cell lysis samples were exclusively detected by ESI-iontrap MS while 105 were only detected using MALDI-TOF MS, underpinning the value of using two different ionization and mass analysis methods. Notably, 12% of the detected proteins represent predicted integral membrane proteins, including the difficult to detect rhodopsin, indicating a considerable coverage of membrane proteins by this approach. This comprehensive approach verified previous metaproteomic studies of marine bacterioplankton, e.g., detection of many transport-related proteins (17% of the detected proteins). In addition, new insights into e.g., carbon and nitrogen metabolism were obtained. For instance, the C1 pathway was more prominent outside the bloom and different strategies for glucose metabolism seem to be applied under the studied conditions. Furthermore, a higher number of nitrogen assimilating proteins were present under non-bloom conditions, reflecting the competition for this limited macro nutrient under oligotrophic conditions. Overall, application of different sample preparation techniques as well as MS methods facilitated a more holistic picture of the marine bacterioplankton response to changing environmental conditions.

Photometric Determination of Ammonium and Phosphate in Seawater Medium Using a Microplate Reader.

To more efficiently process the large sample numbers for quantitative determination of ammonium (NH4+) and phosphate (orthophosphate, PO43-) generated during comprehensive growth experiments with the marine Roseobacter group member Phaeobacter inhibens DSM 17395, specific colorimetric assays employing a microplate reader (MPR) were established. The NH4+ assay is based on the reaction of NH4+ with hypochlorite and salicylate, yielding a limit of detection of 14 µM, a limit of quantitation of 36 µM, and a linear range for quantitative determination up to 200 µM. The PO43-assay is based on the complex formation of PO43- with ammonium molybdate in the presence of ascorbate and zinc acetate, yielding a limit of detection of 13 µM, a limit of quantitation of 50 µM, and a linear range for quantitative determination up to 1 mM. Both MPR-based assays allowed for fast (significantly lower than 1 h) analysis of 21 samples plus standards for calibration (all measured in triplicates) and showed only low variation across a large collection of biological samples.

Analysis of membrane-protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native-PAGE complexome profiling and 2D blue native-/SDS-PAGE.

Sulfate-reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS-reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone-interacting membrane-bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native-PAGE complexome profiling and 2D BN-/SDS-PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN-/SDS-PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na(+) -based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese-like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP.