A general approach to explore prokaryotic protein glycosylation reveals the unique surface layer modulation of an anammox bacterium.

The enormous chemical diversity and strain variability of prokaryotic protein glycosylation makes their large-scale exploration exceptionally challenging. Therefore, despite the universal relevance of protein glycosylation across all domains of life, the understanding of their biological significance and the evolutionary forces shaping oligosaccharide structures remains highly limited. Here, we report on a newly established mass binning glycoproteomics approach that establishes the chemical identity of the carbohydrate components and performs untargeted exploration of prokaryotic oligosaccharides from large-scale proteomics data directly. We demonstrate our approach by exploring an enrichment culture of the globally relevant anaerobic ammonium-oxidizing bacterium Ca. Kuenenia stuttgartiensis. By doing so we resolve a remarkable array of oligosaccharides, which are produced by two seemingly unrelated biosynthetic routes, and which modify the same surface-layer protein simultaneously. More intriguingly, the investigated strain also accomplished modulation of highly specialized sugars, supposedly in response to its energy metabolism-the anaerobic oxidation of ammonium-which depends on the acquisition of substrates of opposite charges. Ultimately, we provide a systematic approach for the compositional exploration of prokaryotic protein glycosylation, and reveal a remarkable example for the evolution of complex oligosaccharides in bacteria.

Database-independent de novo metaproteomics of complex microbial communities.

Metaproteomics has emerged as one of the most promising approaches for determining the composition and metabolic functions of complete microbial communities. Conventional metaproteomics approaches rely on the construction of protein sequence databases and efficient peptide-spectrum-matching algorithms, an approach that is intrinsically biased towards the content of the constructed sequence database. Here, we introduce a highly efficient, database-independent de novo metaproteomics approach and systematically evaluate its quantitative performance using synthetic and natural microbial communities comprising dozens of taxonomic families. Our work demonstrates that the de novo sequencing approach can vastly expand many metaproteomics applications by enabling rapid quantitative profiling and by capturing unsequenced community members that otherwise remain inaccessible for further interpretation. Kleikamp et al., describe a novel de novo metaproteomics pipeline (NovoBridge) that enables rapid community profiling without the need for constructing protein sequence databases.

Tackling the chemical diversity of microbial nonulosonic acids - a universal large-scale survey approach.

Nonulosonic acids, commonly referred to as sialic acids, are a highly important group of nine-carbon sugars common to all domains of life. They all share biosynthetic and structural features, but otherwise display a remarkable chemical diversity. In humans, sialic acids cover all cells which makes them important for processes such as cellular protection, immunity and brain development. On the other hand, sialic acids and other nonulosonic acids have been associated with pathological processes including cancer and viral infections. In prokaryotes, nonulosonic acids are commonly associated with pathogens, which developed through molecular mimicry a strategy to circumvent the host's immune response. However, the remarkably large chemical diversity of prokaryotic nonulosonic acids challenges their discovery, and research on molecular characteristics essential for medical applications are often not feasible. Here, we demonstrate a novel, universal large-scale discovery approach that tackles the unmapped diversity of prokaryotic nonulosonic acids. Thereby, we utilize selective chemical labelling combined with a newly established mass spectrometric all-ion-reaction scanning approach to identify nonulosonic acids and other ulosonic acid-like sugars. In doing so, we provide a first molecular-level comparative study on the frequency and diversity across different phyla. We not only illustrate their surprisingly wide-spread occurrence in non-pathogenic species, but also provide evidence of potential higher carbon variants. Many biomedical studies rely on synthetic routes for sialic acids, which are highly demanding and often of low product yields. Our approach enables large-scale exploration for alternative sources of these highly important compounds.

Chemical characterization methods for the analysis of structural extracellular polymeric substances (EPS).

Biofilm structure and functionality depend on extracellular polymeric substances (EPS), but analytical methods for EPS often lack specificity which limits progress of biofilm research. EPS were extracted from aerobic granular sludge and analyzed with frequently applied colorimetric methods. The colorimetric methods were evaluated based on their applicability for EPS analysis. EPS fractions of interest were proteins, sugars, uronic acids and phenolic compounds. The applied methods (Lowry method, bicinchoninic acid assay, phenol sulfuric acid method, carbazole sulfuric acid method) were investigated in terms of their sensitivity towards the selected standard compound. Interference of compounds present in EPS with the colorimetric methods was further evaluated. All methods showed to be highly depending on the choice of standard compound and susceptible towards interference by compounds present in EPS. This study shows that currently used colorimetric methods are not capable of accurately characterizing EPS. More advanced methods are needed to be able to draw conclusions about biofilm composition, structure and functionality.

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge.

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na2CO3) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca2+). From the six extraction methods used, only the Na2CO3 extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca2+. The Ca2+-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.

[Cu2+ biosorption by bacterial alginate extracted from aerobic granules and its mechanism investigation].

The biosorption characteristic of copper ions onto dried Ca-alginate made of bacterial alginate extracted from aerobic granules was investigated in batch system. The biosorption rate of Cu2+ onto the alginate, effects of pH and alginate dosage on Cu2+ biosorption capacity and adsorption isotherm were analyzed. The biosorption of Cu2+ onto the dried Ca-alginate was a rapid process. The maximum Cu2+ uptake was 67.67 mg/g at pH 4, initial Cu2+ concentration of 100 mg/L, dried Ca-alginate dosage 0.7 g/L. Accumulation of Cu2+ followed Langmuir and Freundlich adsorption isotherm. Cu2+ adsorption was accompanied by Ca2+ releasing from the dried Ca-alginate and H+ uptake, indicating that the ion exchange between Cu2+ and Ca2+ requires H+ for charge balance. FT-IR secondary derivative spectra and atomic force microscopy analysis gave proof that, the reaction of MG blocks in dried Ca-alginate with Cu2+ and Ca+ were different. Cu2+ may form complex with MG blocks, leading to a much more ordered structure on the surface of the dried Ca-alginate. The dried Ca-alginate bio-adsorbent was regenerated by 100 mmol/L HCI with 91% Cu2+ recovery.

Biosorption of copper by calcium alginate from excess activated sludge.

To evaluate the potential use of the bacterial alginate extracted from excess activated sludge as a sustainable copper bioadsorbent, dried alginate-Ca made from municipal excess activated sludge was characterized by atomic force microscopy, and tested in a batch system. The alginate-Ca yield from the activated sludge was 103 +/- 15 mg g(-1). The maximum Cu2+ uptake was 41.96 mg g(-1). Accumulation of Cu2+ reached equilibrium within 50 min and followed the Langmuir adsorption isotherm. The solution pH and initial Cu2+ concentration were critical for Cu2+ biosorption. The sludge alginate-Ca bioadsorbent could be regenerated by 100 mmol L(-1) HCl with 82% Cu2+ recovery. Calcium alginate from excess activated sludge was proved to be a good bioadsorbent for heavy metal elimination.

[Self-aggregation property of bacterial alginates extracted from aerobic granules].

To explore bacterial alginates role in aerobic granules' formation, the alginate was extracted from aerobic granules and identified. Its aggregation property in 50 mg x L(-1) CaCl2 solution was investigated by the atomic force microscopy (AFM). Bacterial alginates amounted to 35.1% +/- 1.9% of granules' dry mass. With the concentration increased from 10 mg x L(-1) to 500 mg x L(-1) in 50 mg x L(-1) CaCl2, the extracted alginates tended to form ordered aggregations, with the shape changed from randomly distributed globules, to rod-like and flower-shaped aggregations, and finally to weblike networks due to their supramolecular self-assembly property. The three dimensional alginate-metal gel is the structural polymer of aerobic granules, and the alginates-Ca2+ gel formation plays an important role in granules' formation and structure stabilization.

Kinetic responses of activated sludge microorganisms to individual and joint copper and zinc.

The individual and joint effects of copper and zinc on the growth kinetics of activated sludge microorganisms were studied in batch reactors fed with glucose as sole carbon source. The observed specific growth rate, substrate utilization rate and removal efficiency were determined at different concentrations of individual and joint Cu and Zn. Activated sludge microorganisms showed different kinetic responses to Cu and Zn, and the Haldane model seemed applicable for the inhibitory effect of Zn, while a first-order kinetics for Cu. Compared to Zn, Cu exhibited high toxicity to activated sludge microorganisms even at a very low concentration of 1.0 mg/dm3. However, Zn concentrations less than 40 mg/dm3 would stimulate the growth of activated sludge microorganism, and Zn acted as inhibitory chemical only at concentrations above this value. The combined effect of Cu and Zn could be regarded as neither synergistic nor antagonistic action.

A thermodynamic interpretation of the Monod equation.

The Monod equation for microbial growth is purely empirical, and the theoretical basis of this model stays unclear. Similar to any chemical reactions, overall microbial growth process is dependent upon the changes in free energy. This study showed that the Monod equation could be interpreted in a thermodynamic sense very well. It was probably for the first time demonstrated that the Monod constant ( K(s)) was inversely related to the equilibrium constant of the overall microbial growth process.