Christoph Feenders

Subcellular protein localization (cell envelope) in Phaeobacter inhibens DSM 17395.

Phaeobacter inhibens DSM 17395 is a metabolically versatile, secondary metabolite producing and surface colonizing member of the alphaproteobacterial Roseobacter clade. Proteins compartmentalized across the Gram‐negative cell envelope are expected to be relevant for the habitat success of P. inhibens DSM 17395. Subcellular fractionation was followed by gel‐ or nano‐LC‐based separation of proteins and peptides, respectively. Subsequent MS‐based identification of in total 1187 proteins allowed allocation to cytoplasm (303 proteins), cytoplasmic membrane (346), periplasm (325), outer membrane (76), and extracellular milieu (22). Multidimensional scaling was used to visualize the spreading of heuristically allocated proteins across the five different compartments. Experimentally inferred subcellular protein localization was compared with PSORTb prediction of protein secretion and membrane localization. Determined subcellular localizations of identified proteins were interpreted to reconstruct the functional traits of the different cell envelope compartments, in particular protein secretion and sorting, direct effector molecule transit, and cell envelope biogenesis. From a proteogenomic perspective, functional prediction of 74 genes (including 17 coding for proteins of hitherto unknown function) could be refined.

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.

Motif analysis in directed ordered networks and applications to food webs

The analysis of small recurrent substructures, so called network motifs, has become a standard tool of complex network science to unveil the design principles underlying the structure of empirical networks. In many natural systems network nodes are associated with an intrinsic property according to which they can be ordered and compared against each other. Here, we expand standard motif analysis to be able to capture the hierarchical structure in such ordered networks. Our new approach is based on the identification of all ordered 3-node substructures and the visualization of their significance profile. We present a technique to calculate the fine grained motif spectrum by resolving the individual members of isomorphism classes (sets of substructures formed by permuting node-order). We apply this technique to computer generated ensembles of ordered networks and to empirical food web data, demonstrating the importance of considering node order for food-web analysis. Our approach may not only be helpful to identify hierarchical patterns in empirical food webs and other natural networks, it may also provide the base for extending motif analysis to other types of multi-layered networks.

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.

High performance CCD camera system for digitalisation of 2D DIGE gels

An essential step in 2D DIGE‐based analysis of differential proteome profiles is the accurate and sensitive digitalisation of 2D DIGE gels. The performance progress of commercially available charge‐coupled device (CCD) camera‐based systems combined with light emitting diodes (LED) opens up a new possibility for this type of digitalisation. Here, we assessed the performance of a CCD camera system (Intas Advanced 2D Imager) as alternative to a traditionally employed, high‐end laser scanner system (Typhoon 9400) for digitalisation of differential protein profiles from three different environmental bacteria. Overall, the performance of the CCD camera system was comparable to the laser scanner, as evident from very similar protein abundance changes (irrespective of spot position and volume), as well as from linear range and limit of detection.

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

Abstract 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 Liebigs 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.

Evolutionary food web models: effects of an additional resource

Many empirical food webs contain multiple resources, which can lead to the emergence of sub-communities—partitions—in a food web that are weakly connected with each other. These partitions interact and affect the complete food web. However, the fact that food webs can contain multiple resources is often neglected when describing food web assembly theoretically, by considering only a single resource. We present an allometric, evolutionary food web model and include two resources of different sizes. Simulations show that an additional resource can lead to the emergence of partitions, i.e. groups of species that specialise on different resources. For certain arrangements of these partitions, the interactions between them alter the food web properties. First, these interactions increase the variety of emerging network structures, since hierarchical bodysize relationships are weakened. Therefore, they could play an important role in explaining the variety of food web structures that is observed in empirical data. Second, interacting partitions can destabilise the population dynamics by introducing indirect interactions with a certain strength between predator and prey species, leading to biomass oscillations and evolutionary intermittence.

The marine bacterium Phaeobacter inhibens secures external ammonium by rapid buildup of intracellular nitrogen stocks

ABSTRACT Reduced nitrogen species are key nutrients for biological productivity in the oceans. Ammonium is often present in low and growth-limiting concentrations, albeit peaks occur during collapse of algal blooms or via input from deep sea upwelling and riverine inflow. Autotrophic phytoplankton exploit ammonium peaks by storing nitrogen intracellularly. In contrast, the strategy of heterotrophic bacterioplankton to acquire ammonium is less well understood. This study revealed the marine bacterium Phaeobacter inhibens DSM 17395, a Roseobacter group member, to have already depleted the external ammonium when only ∼⅓ of the ultimately attained biomass is formed. This was paralleled by a three-fold increase in cellular nitrogen levels and rapid buildup of various nitrogen-containing intracellular metabolites (and enzymes for their biosynthesis) and biopolymers (DNA, RNA and proteins). Moreover, nitrogen-rich cells secreted potential RTX proteins and the antibiotic tropodithietic acid, perhaps to competitively secure pulses of external ammonium and to protect themselves from predation. This complex response may ensure growing cells and their descendants exclusive provision with internal nitrogen stocks. This nutritional strategy appears prevalent also in other roseobacters from distant geographical provenances and could provide a new perspective on the distribution of reduced nitrogen in marine environments, i.e. temporary accumulation in bacterioplankton cells.

Impact of Extraction Methods on the Detectable Protein Complement of Metaproteomic Analyses of Marine Sediments

Metaproteomic analysis targets proteins, the catalytic entities in the habitat, thereby providing direct insights into the metabolic activity of the community studied. A major challenge still remaining for metaproteomics is the effective and comprehensive extraction of proteins from environmental samples, due to their high complexity with respect to organismic diversity and abundance range. Moreover, in certain habitats, the inherent matrix may interfere with protein extraction. In recent years, several studies reported different protein extraction methods for soils known for their complex geochemistry, but only three analyzed marine sediments that generally comprise different though similarly complex geochemistry. In this study, the impact of four different extraction methods was investigated for coastal North Sea and deep sea Pacific Ocean sediments. The extraction methods comprised (i) phenol, (ii) SDS, (iii) a mixture of SDS and phenol, and (iv) urea and thiourea. Prior to extraction, a cell and protein standard (CPS) was added to the sediment samples to trace recovery of proteins from different subcellular locations as well as dissolved BSA. While each extraction method detected distinct peptide complements, SDS‐phenol extraction generally achieved highest protein yield and most comprehensive CPS protein identification. Application of two different methods was shown to further improve proteome coverage.

Influence of NanoLC Column and Gradient Length as well as MS/MS Frequency and Sample Complexity on Shotgun Protein Identification of Marine Bacteria

Protein identification by shotgun proteomics, i.e., nano-liquid chromatography (nanoLC) peptide separation online coupled to electrospray ionization (ESI) mass spectrometry (MS)/MS, is the most widely used gel-free approach in proteome research. While the mass spectrometer accounts for mass accuracy and MS/MS frequency, the nanoLC setup and gradient time influence the number of peptides available for MS analysis, which ultimately determine the number of proteins identifiable. Here, we report on the influence of (i) analytical column length (15, 25, or 50 cm) coupled to (ii) the applied gradient length (120, 240, 360, 480, or 600 min), as well as (iii) MS/MS frequency on peptide/protein identification by shotgun proteomics of (iv) 2 marine bacteria. Longer gradients increased the number of peptides/proteins identified as well as the reproducibility of identification. Furthermore, longer analytical columns strictly enlarge the covered proteome complement. Notably, the proteome complement identified with a short column and applying a long gradient is also covered when using longer columns with shorter gradients. Coverage of the proteome complement further increases with higher MS/MS frequency. Compilation of peptide lists of replicate analyses (same gradient length) improves protein identification, while compilation of analyses with different gradient lengths yields a similar or even higher number of proteins using comparable or even less total analysis time.