Understanding prokaryotic transformation of recalcitrant pollutants and the in-situ metabolic nets require the integration of massive amounts of biological data. Decades of biochemical studies together with novel next-generation sequencing data have exponentially increased information on aerobic aromatic degradation pathways. However, the majority of protein sequences in public databases have not been experimentally characterized and homology-based methods are still the most routinely used approach to assign protein function, allowing the propagation of misannotations. AromaDeg is a web-based resource targeting aerobic degradation of aromatics that comprises recently updated (September 2013) and manually curated databases constructed based on a phylogenomic approach. Grounded in phylogenetic analyses of protein sequences of key catabolic protein families and of proteins of documented function, AromaDeg allows query and data mining of novel genomic, metagenomic or metatranscriptomic data sets. Essentially, each query sequence that match a given protein family of AromaDeg is associated to a specific cluster of a given phylogenetic tree and further function annotation and/or substrate specificity may be inferred from the neighboring cluster members with experimentally validated function. This allows a detailed characterization of individual protein superfamilies as well as high-throughput functional classifications. Thus, AromaDeg addresses the deficiencies of homology-based protein function prediction, combining phylogenetic tree construction and integration of experimental data to obtain more accurate annotations of new biological data related to aerobic aromatic biodegradation pathways. We pursue in future the expansion of AromaDeg to other enzyme families involved in aromatic degradation and its regular update.Database URL: http://aromadeg.siona.helmholtz-hzi.de

The aim of this work was to develop an analytical method to predict total anthocyanins content (TAC) and total phenolic compounds (TPC) in intact wax jambu fruit [Syzygium malaccense (L.) Merryl et Perry] using near-infrared spectroscopy (NIRS) and partial least squares (PLS). The estimation accuracy was based on parameters such as root mean square error of prediction (RMSEP), correlation coefficients [calibration (rc) and prediction (rp) set] and ratio of performance to deviation (RPD). TAC, rp = 0.98, RMSEP = 9.0 mg L(-1) and RPD = 5.19 were attained using second derivative pre-treatment. TPC, rp = 0.94, RMSEP = 22.18 (mg gallic acid equivalents (GAE)/100g) and RPD = 3.27 (excellent accuracy) were also obtained using second derivative pre-treatment. These findings suggest that the NIRS and PLS algorithms can be used to determine TCA and TPC in intact wax jambu fruit.

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.

Current knowledge of the physiology and phylogeny of polycyclic aromatic hydrocarbon (PAH) degrading bacteria often relies on laboratory enrichments and isolations. In the present study, in situ microcosms consisting of activated carbon pellets (BACTRAP®s) were loaded with either (13) C-naphthalene or (13) C-fluorene and were subsequently exposed in the contaminant source and plume fringe region of a PAH-contaminated aquifer. Metaproteomic analysis and protein-stable isotope probing revealed Burkholderiales, Actinomycetales, and Rhizobiales as the most active microorganisms in the groundwater communities. Proteins identified of the naphthalene degradation pathway showed a relative (13) C isotope abundance of approximately 50 atom% demonstrating that the identified naphthalene-degrading bacteria gained at least 80% of their carbon by PAH degradation. Although the microbial community grown on the fluorene-BACTRAPs showed a structure similar to the naphthalene-BACTRAPs, the identification of fluorene degraders and degradation pathways failed in situ. In complementary laboratory microcosms, a clear enrichment in proteins related to Rhodococcus and possible fluorene degradation enzymes was observed. This result demonstrates the impact of laboratory conditions on microbial community structure and activity of certain species and underlines the need on in situ exploration of microbial community functions. In situ microcosms in combination with protein-stable isotope probing may be a significant tool for in situ identification of metabolic key players as well as degradation pathways.

Polycyclic aromatic hydrocarbons (PAH) are widespread and persistent environmental contaminants, especially in oxygen-free environments. The occurrence of anaerobic PAH-degrading bacteria and their underlying metabolic pathways are rarely known. In this study, PAH degraders were enriched in laboratory microcosms under sulfate-reducing conditions using groundwater and sediment samples from four PAH-contaminated aquifers. Five enrichment cultures were obtained showing sulfate-dependent naphthalene degradation. Mineralization of naphthalene was demonstrated by the formation of sulfide concomitant with the depletion of naphthalene and the development of (13)C-labeled CO2 from [(13)C6]-naphthalene. 16S rRNA gene and metaproteome analyses revealed that organisms related to Desulfobacterium str. N47 were the main naphthalene degraders in four enrichment cultures. Protein sequences highly similar to enzymes of the naphthalene degradation pathway of N47 were identified, suggesting that naphthalene was activated by a carboxylase, and that the central metabolite 2-naphthoyl-CoA was further reduced by two reductases. The data indicate an importance of members of the family Desulfobacteraceae for naphthalene degradation under sulfate-reducing conditions in freshwater environments.

A new pathway of molinate mineralization has recently been described. Among the five members of the mixed culture able to promote such a process, Gulosibacter molinativorax ON4 T has been observed to promote the initial breakdown of the herbicide into ethanethiol and azepane-1-carboxylate. In the current study, the gene encoding the enzyme responsible for molinate hydrolysis was identified and heterologously expressed, and the resultant active protein was purified and characterized. Nucleotide sequence analysis revealed that the gene encodes a 465-amino-acid protein of the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily with a predicted molecular mass of 50.9 kDa. Molinate hydrolase shares the highest amino acid sequence identity (48 to 50%) with phenylurea hydrolases of Arthrobacter globiformis and Mycobacterium brisbanense. However, in contrast to previously described members of the metal-dependent hydrolase A subfamily, molinate hydrolase contains cobalt as the only active-site metal.Molinate is a herbicide which is extensively applied to rice fields worldwide. When this thiocarbamate herbicide is applied to the flooded paddies, it dissipates into the environment largely through volatilization. However, (photo)chemical and microbiological molinate transformation also occurs (28), resulting in accumulation of oxidized metabolites, such as oxomolinate and molinate sulfoxide (11, 13), which have increased toxicity (5). The only biological system described so far as being able to mineralize molinate and use the herbicide as the sole source of carbon, energy, and nitrogen is a five-membered bacterial mixed culture (2, 6). Among the five community members, Gulosibacter molinativorax ON4 T (gen. nov., sp. nov.), the only representative of this genus thus far (21), is responsible for the initial breakdown of molinate. In contrast to previously described molinate transformation reactions (13), G. molinativorax ON4T cleaves the thioester bond of molinate, releasing ethanethiol and azepane-1-carboxylate (ACA) (Fig. 1) (1). While this hydrolysis proceeds in the absence of oxygen, the further metabolism and mineralization of ACA by G. molinativorax ON4 T necessitate oxygen. Ethanethiol is not transformed by this organism but is spontaneously oxidized to diethyl disulfide. At molinate concentrations of Ͼ2 mM, the accumulating sulfur compounds are toxic for G. molinativorax ON4T and molinate mineralization is achieved only when other mixed-culture members able to degrade the sulfurcontaining metabolites are present (1, 2).In the present study, the molinate hydrolase (MolA) from G. molinativorax ON4T responsible for the key step of molinate breakdown was purified and characterized. The encoding gene was identified and expressed in Escherichia coli. This enzyme belongs to the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily; however, in contrast to previously characterized members, it contains cobalt as a cofactor. To the best of our knowledge, this is the first description of an...

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A culture-independent function-based screening approach was used to assess the microbial aerobic catabolome for polycyclic aromatic hydrocarbons degradation of a soil subjected to 12 years of in situ bioremediation. A total of 422 750 fosmid clones were screened for key aromatic ring-cleavage activities using 2,3-dihydroxybiphenyl as substrate. Most of the genes encoding ring-cleavage enzymes on the 768 retrieved positive fosmids could not be identified using primer-based approaches and, thus, 205 fosmid inserts were sequenced. Nearly two hundred extradiol dioxygenase encoding genes of three different superfamilies could be identified. Additional key genes of aromatic metabolic pathways were identified, including a high abundance of Rieske non-heme iron oxygenases that provided detailed information on enzymes activating aromatic compounds and enzymes involved in activation of the side chain of methylsubstituted aromatics. The gained insights indicated a complex microbial network acting at the site under study, which comprises organisms similar to recently identified Immundisolibacter cernigliae TR3.2 and Rugosibacter aromaticivorans Ca6 and underlined the great potential of an approach that combines an activity-screening, a cost-effective high-throughput sequencing of fosmid clones and a phylogenomic-routed and manually curated database to carefully identify key proteins dedicated to aerobic degradation of aromatic compounds.