Enhancing metaproteomics--The value of models and defined environmental microbial systems.

Metaproteomics--the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time--has provided new features to study complex microbial communities in order to unravel these "black boxes." New technical challenges arose that were not an issue for classical proteome analytics before that could be tackled by the application of different model systems. Here, we review different current and future model systems for metaproteome analysis. Following a short introduction to microbial communities and metaproteomics, we introduce model systems for clinical and biotechnological research questions including acid mine drainage, anaerobic digesters, and activated sludge. Model systems are useful to evaluate the challenges encountered within (but not limited to) metaproteomics, including species complexity and coverage, biomass availability, or reliable protein extraction. The implementation of model systems can be considered as a step forward to better understand microbial community responses and ecological functions of single member organisms. In the future, improvements are necessary to fully explore complex environmental systems by metaproteomics.

In situ†protein-SIP highlights Burkholderiaceae as key players degrading toluene by para ring hydroxylation in a constructed wetland model.

In constructed wetlands, organic pollutants are mainly degraded via microbial processes. Helophytes, plants that are commonly used in these systems, provide oxygen and root exudates to the rhizosphere, stimulating microbial degradation. While the treatment performance of constructed wetlands can be remarkable, a mechanistic understanding of microbial degradation processes in the rhizosphere is still limited. We investigated microbial toluene removal in a constructed wetland model system combining 16S rRNA gene sequencing, metaproteomics and (13) C-toluene in situ protein-based stable isotope probing (protein-SIP). The rhizospheric bacterial community was dominated by Burkholderiales and Rhizobiales, each contributing about 20% to total taxon abundance. Protein-SIP data revealed that the members of Burkholderiaceae, the proteins of which showed about 73% of (13) C-incorporation, were the main degraders of toluene in the planted system, while the members of Comamonadaceae were involved to a lesser extent in degradation (about 64% (13) C-incorporation). Among the Burkholderiaceae, one of the key players of toluene degradation could be assigned to Ralstonia pickettii. We observed that the main pathway of toluene degradation occurred via two subsequent monooxygenations of the aromatic ring. Our study provides a suitable approach to assess the key processes and microbes that are involved in the degradation of organic pollutants in complex rhizospheric ecosystems.

Aerobic Toluene Degraders in the Rhizosphere of a Constructed Wetland Model Show Diurnal Polyhydroxyalkanoate Metabolism.

UNLABELLED: Constructed wetlands (CWs) are successfully applied for the treatment of waters contaminated with aromatic compounds. In these systems, plants provide oxygen and root exudates to the rhizosphere and thereby stimulate microbial degradation processes. Root exudation of oxygen and organic compounds depends on photosynthetic activity and thus may show day-night fluctuations. While diurnal changes in CW effluent composition have been observed, information on respective fluctuations of bacterial activity are scarce. We investigated microbial processes in a CW model system treating toluene-contaminated water which showed diurnal oscillations of oxygen concentrations using metaproteomics. Quantitative real-time PCR was applied to assess diurnal expression patterns of genes involved in aerobic and anaerobic toluene degradation. We observed stable aerobic toluene turnover by Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis was upregulated in these bacteria during the day, suggesting that they additionally feed on organic root exudates while reutilizing the stored carbon compounds during the night via the glyoxylate cycle. Although mRNA copies encoding the anaerobic enzyme benzylsuccinate synthase (bssA) were relatively abundant and increased slightly at night, the corresponding protein could not be detected in the CW model system. Our study provides insights into diurnal patterns of microbial processes occurring in the rhizosphere of an aquatic ecosystem. IMPORTANCE: Constructed wetlands are a well-established and cost-efficient option for the bioremediation of contaminated waters. While it is commonly accepted knowledge that the function of CWs is determined by the interplay of plants and microorganisms, the detailed molecular processes are considered a black box. Here, we used a well-characterized CW model system treating toluene-contaminated water to investigate the microbial processes influenced by diurnal plant root exudation. Our results indicated stable aerobic toluene degradation by members of the Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis in these bacteria was higher during the day, suggesting that they additionally fed on organic root exudates and reutilized the stored carbon compounds during the night. Our study illuminates microbial processes occurring in the rhizosphere of an aquatic ecosystem.

Benzylsuccinate Synthase is Post-Transcriptionally Regulated in the Toluene-Degrading Denitrifier Magnetospirillum sp. Strain 15-1.

The facultative denitrifying alphaproteobacterium Magnetospirillum sp. strain 15-1 had been isolated from the hypoxic rhizosphere of a constructed wetland model fed with toluene. This bacterium can catabolize toluene anaerobically but not aerobically. Here, we used strain 15-1 to investigate regulation of expression of the highly oxygen-sensitive glycyl radical enzyme benzylsuccinate synthase, which catalyzes the first step in anaerobic toluene degradation. In cells growing aerobically with benzoate, the addition of toluene resulted in a ~20-fold increased transcription of bssA, encoding for the catalytically active subunit of the enzyme. Under anoxic conditions, bssA mRNA copy numbers were up to 129-fold higher in cells growing with toluene as compared to cells growing with benzoate. Proteomics showed that abundance of benzylsuccinate synthase increased in cells growing anaerobically with toluene. In contrast, peptides of this enzyme were never detected in oxic conditions. These findings show that synthesis of benzylsuccinate synthase was under stringent post-transcriptional control in the presence of oxygen, which is a novel level of regulation for glycyl radical enzymes.

Insight into the modulation of intestinal proteome of juvenile common carp (Cyprinus carpio L.) after dietary exposure to ZnO nanoparticles.

ZnO nanoparticles (NPs) are widely used in industrial and consumer products. Therefore understanding their interaction with biological systems is key to their safe application. Proteomics was applied to assess the sub-lethal effects of dietary ZnO NPs on two parts of carp intestine, the intestinal folds and the muscular parts. A commercial carp feed containing 500mgkg-1 of ZnO NPs was fed to fish for six weeks. The abundances of 32 proteins in the treated intestinal folds were significantly changed and in addition, 28 proteins were significantly changed in the muscular parts. Pathways analysis revealed downregulation of pathways attributed to protein synthesis in both parts of the treated intestine. Remodelling of actin cytoskeleton pathways were regulated positively and negatively in intestinal folds and muscular parts, respectively, albeit via different mechanisms. Apoptosis response was indicated in exposed intestinal folds, whereas elevated levels of protein associated with cancerous cell survival were observed in the muscular parts. Results showed that ZnO NPs affected the protein abundances associated with cell motility, immune system response, oxidative stress response, as well as cell metabolism. Data are available via ProteomeXchange with identifier PXD006867.

Effects of chronic dietary exposure of zinc oxide nanoparticles on the serum protein profile of juvenile common carp (Cyprinus carpio L.).

Zinc oxide (ZnO) nanoparticles (NPs) have been dramatically used in industry, biology, and medicine. Despite their interesting physico-chemical properties for application in various industrial, medical, and consumer products, safe use of ZnO NPs are under challenges due to the inadequate information related to their toxicological endpoints. Proteomics was applied to evaluate the sub-lethal effects of dietary exposure to ZnO NPs on serum proteome profile of juvenile common carp, (Cyprinus carpio). Therefore, ZnO NPs solution (500mgkg-1 of feed) was added to a commercial carp feed for six weeks. We compared the serum proteome profile from 7 controls and 7 treated fish. In addition, zinc accumulation were measured in intestine, liver, gill and brain. In total, we were able to identify 326 proteins from 6845 distinct peptides. As a result of the data analysis, the abundance levels of four proteins were significantly altered (fold change (fc) ≥2 and p<0.05) after dietary exposure to ZnO NPs. The protein levels of the complement component C4-2 (fc 2.5) and the uncharacterised protein encoded by kng1 (fc 5.8) were increased and major histocompatibility class I (fc 4.9) and the uncharacterised protein encoded by lum (fc 3.5) were decreased (fc 2.5). Molecular pathway analysis revealed four canonical pathways including acute-phase response signalling, liver and retinoid X receptors activation, and intrinsic and extrinsic prothrombin activation pathways as significantly regulated in the treated fish. No significant difference was observed for zinc accumulation in exposed fish compared to controls. In summary, despite no apparent accumulation, ZnO NPs exposure to common carp probably disturbs the fish homeostasis by affecting proteins of the haematological and the immune systems.

Protein-SIP in environmental studies.

Metaproteomics coupled to stable isotope probing (SIP) was established to detect metabolically active key players in microbial communities. Here, we discuss the current state of protein-based stable isotope probing (protein-SIP) and the perspectives of using different stable isotope atoms (i.e. 13C, 15N, 18O, 34/36S), multiple isotope labelling, the utilisation of substrates of major abundance and micro-pollutants [pesticides, herbicides and pharmaceuticals present in the environment at very low concentrations (ngµg/L)], and applications in complex model systems and in situ studies in the environment.