In this study, the diversity of bphA genes was assessed in a 13C-enriched metagenome upon stable isotope probing (SIP) of microbial populations in legacy PCB-contaminated soil with 13C-biphenyl (BP). In total, 13 bphA sequence variants (SVs) were identified in the final amplicon dataset. Of these, one SV comprised 59% of all sequences, and when it was translated into a protein sequence, it exhibited 87, 77.4, and 76.7% identity to its homologs from Pseudomonas furukawaii KF707, Cupriavidus sp. WS, and Pseudomonas alcaliphila B-367, respectively. This same BphA sequence also contained unusual amino acid residues, Alanine, Valine, and Serine in region III, which had been reported to be crucial for the substrate specificity of the corresponding biphenyl dioxygenase (BPDO), and was accordingly designated BphA_AVS. The DNA locus of 18 kbp containing the BphA_AVS-coding sequence retrieved from the metagenome was comprised of 16 ORFs and was most likely borne by Paraburkholderia sp. The BPDO corresponding to bphAE_AVS was cloned and heterologously expressed in E. coli, and its substrate specificity toward PCBs and a spectrum of flavonoids was assessed. Although depleting a rather narrow spectrum of PCB congeners, the efficient transformation of flavone and flavanone was demonstrated through dihydroxylation of the B-ring of the molecules. The homology-based functional assignment of the putative proteins encoded by the rest of ORFs in the AVS region suggests their potential involvement in the transformation of aromatic compounds, such as flavonoids. In conclusion, this study contributes to the body of information on the involvement of soil-borne BPDOs in the metabolism of flavonoid compounds, and our paper provides a more advanced context for understanding the interactions between plants, microbes and anthropogenic compounds in the soil.

• Klíčová slova
• aromatic ring-hydroxylating dioxygenases, biphenyl dioxygenase, flavonoids, functional metagenomics, polychlorinated biphenyls, secondary plant metabolites,
• Publikační typ
• časopisecké články MeSH

Aerobic mineralization of PCBs, which are toxic and persistent organic pollutants, involves the upper (biphenyl, BP) and lower (benzoate, BZ) degradation pathways. The activity of different members of the soil microbial community in performing one or both pathways, and their synergistic interactions during PCB biodegradation, are not well understood. This study investigates BP and BZ biodegradation and subsequent carbon flow through the microbial community in PCB-contaminated soil. DNA stable isotope probing (SIP) was used to identify the bacterial guilds involved in utilizing (13)C-biphenyl (unchlorinated analogue of PCBs) and/or (13)C-benzoate (product/intermediate of BP degradation and analogue of chlorobenzoates). By performing SIP with two substrates in parallel, we reveal microbes performing the upper (BP) and/or lower (BZ) degradation pathways, and heterotrophic bacteria involved indirectly in processing carbon derived from these substrates (i.e. through crossfeeding). Substrate mineralization rates and shifts in relative abundance of labeled taxa suggest that BP and BZ biotransformations were performed by microorganisms with different growth strategies: BZ-associated bacteria were fast growing, potentially copiotrophic organisms, while microbes that transform BP were oligotrophic, slower growing, organisms. Our findings provide novel insight into the functional interactions of soil bacteria active in processing biphenyl and related aromatic compounds in soil, revealing how carbon flows through a bacterial community.

• MeSH
• aromatické uhlovodíky metabolismus   MeSH
• Bacteria genetika   metabolismus   MeSH
• benzoáty metabolismus   MeSH
• bifenylové sloučeniny metabolismus   MeSH
• biodegradace MeSH
• látky znečišťující půdu metabolismus   MeSH
• nebezpečný odpad MeSH
• polychlorované bifenyly metabolismus   MeSH
• půda chemie   MeSH
• půdní mikrobiologie * MeSH
• RNA ribozomální 16S genetika   MeSH
• znečištění životního prostředí * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• aromatické uhlovodíky MeSH
• benzoáty MeSH
• bifenylové sloučeniny MeSH
• diphenyl MeSH Prohlížeč
• látky znečišťující půdu MeSH
• nebezpečný odpad MeSH
• polychlorované bifenyly MeSH
• půda MeSH
• RNA ribozomální 16S MeSH

Three types of contaminated soil from three geographically different areas were subjected to a constant supply of benzene or benzene/toluene/ethylbenzene/xylenes (BTEX) for a period of 3 months. Different from the soil from Brazil (BRA) and Switzerland (SUI), the Czech Republic (CZE) soil which was previously subjected to intensive in situ bioremediation displayed only negligible changes in community structure. BRA and SUI soil samples showed a clear succession of phylotypes. A rapid response to benzene stress was observed, whereas the response to BTEX pollution was significantly slower. After extended incubation, actinobacterial phylotypes increased in relative abundance, indicating their superior fitness to pollution stress. Commonalities but also differences in the phylotypes were observed. Catabolic gene surveys confirmed the enrichment of actinobacteria by identifying the increase of actinobacterial genes involved in the degradation of pollutants. Proteobacterial phylotypes increased in relative abundance in SUI microcosms after short-term stress with benzene, and catabolic gene surveys indicated enriched metabolic routes. Interestingly, CZE soil, despite staying constant in community structure, showed a change in the catabolic gene structure. This indicates that a highly adapted community, which had to adjust its gene pool to meet novel challenges, has been enriched.

• MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• benzen metabolismus   MeSH
• benzenové deriváty metabolismus   MeSH
• biodegradace MeSH
• biodiverzita MeSH
• látky znečišťující půdu metabolismus   MeSH
• půda chemie   MeSH
• půdní mikrobiologie * MeSH
• toluen metabolismus   MeSH
• xyleny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Geografické názvy
• Brazílie MeSH
• Česká republika MeSH
• Švýcarsko MeSH
• Názvy látek
• bakteriální proteiny MeSH
• benzen MeSH
• benzenové deriváty MeSH
• ethylbenzene MeSH Prohlížeč
• látky znečišťující půdu MeSH
• půda MeSH
• toluen MeSH
• xyleny MeSH

Degradation of chlorobenzoic acids (e.g., products of microbial degradation of PCB) by strains of microorganisms isolated from PCB contaminated soils was assessed. From seven bulk-soil isolates two strains unique in ability to degrade a wider range of chlorobenzoic acids than others were selected, individually and even in a complex mixture of 11 different chlorobenzoic acids. Such a feature is lacking in most tested degraders. To investigate the influence of vegetation on chlorobenzoic acids degraders, root exudates of two plant species known for supporting PCB degradation in soil were tested. While with individual chlorobenzoic acids the presence of plant exudates leads to a decrease of degradation yield, in case of a mixture of chlorobenzoic acids either a change in bacterial degradation specificity, associated with 3- and 4-chlorobenzoic acid, or an extension of the spectrum of degraded chlorobenzoic acids was observed.

• MeSH
• Arthrobacter izolace a purifikace   metabolismus   MeSH
• biodegradace účinky léků   MeSH
• chlorbenzoáty metabolismus   MeSH
• kořeny rostlin chemie   MeSH
• Pseudomonas izolace a purifikace   metabolismus   MeSH
• rostlinné extrakty farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorbenzoáty MeSH
• rostlinné extrakty MeSH

Bacteria were identified associated with biodegradation of aromatic pollutants biphenyl, benzoate, and naphthalene in a long-term polychlorinated biphenyl- and polyaromatic hydrocarbon-contaminated soil. In order to avoid biases of culture-based approaches, stable isotope probing was applied in combination with sequence analysis of 16 S rRNA gene pyrotags amplified from (13)C-enriched DNA fractions. Special attention was paid to pyrosequencing data analysis in order to eliminate the errors caused by either generation of amplicons (random errors caused by DNA polymerase, formation of chimeric sequences) or sequencing itself. Therefore, sample DNA was amplified, sequenced, and analyzed along with the DNA of a mock community constructed out of 8 bacterial strains. This warranted that appropriate tools and parameters were chosen for sequence data processing. (13)C-labeled metagenomes isolated after the incubation of soil samples with all three studied aromatics were largely dominated by Proteobacteria, namely sequences clustering with the genera Rhodanobacter Burkholderia, Pandoraea, Dyella as well as some Rudaea- and Skermanella-related ones. Pseudomonads were mostly labeled by (13)C from naphthalene and benzoate. The results of this study show that many biphenyl/benzoate-assimilating bacteria derive carbon also from naphthalene, pointing out broader biodegradation abilities of some soil microbiota. The results also demonstrate that, in addition to traditionally isolated genera of degradative bacteria, yet-to-be cultured bacteria are important players in bioremediation. Overall, the study contributes to our understanding of biodegradation processes in contaminated soil. At the same time our results show the importance of sequencing and analyzing a mock community in order to more correctly process and analyze sequence data.

• MeSH
• Bacteria genetika   izolace a purifikace   metabolismus   MeSH
• benzoáty metabolismus   MeSH
• bifenylové sloučeniny metabolismus   MeSH
• biodegradace MeSH
• DNA bakterií metabolismus   MeSH
• fylogeneze MeSH
• izotopové značení MeSH
• izotopy uhlíku MeSH
• látky znečišťující půdu analýza   MeSH
• naftaleny metabolismus   MeSH
• půdní mikrobiologie * MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzoáty MeSH
• bifenylové sloučeniny MeSH
• diphenyl MeSH Prohlížeč
• DNA bakterií MeSH
• izotopy uhlíku MeSH
• látky znečišťující půdu MeSH
• naftaleny MeSH
• naphthalene MeSH Prohlížeč

The extradiol dioxygenase diversity of a site highly contaminated with aliphatic and aromatic hydrocarbons under air-sparging treatment was assessed by functional screening of a fosmid library in Escherichia coli with catechol as substrate. The 235 positive clones from inserts of DNA extracted from contaminated soil were equivalent to one extradiol dioxygenase-encoding gene per 3.6 Mb of DNA screened, indicating a strong selection for genes encoding this function. Three subfamilies were identified as being predominant, with 72, 55 and 43 fosmid inserts carrying genes, related to those encoding TbuE of Ralstonia pickettii PK01 (EXDO-D), IpbC of Pseudomonas sp. JR1 (EXDO-K2) or DbtC of Burkholderia sp. DBT1 (EXDO-Dbt), respectively, whereas genes encoding enzymes related to XylE of Pseudomonas putida mt-2 were not observed. Genes encoding oxygenases related to isopropylbenzene dioxygenases were usually colocalized with genes encoding EXDO-K2 dioxygenases. Functional analysis of representative proteins indicated a subcluster of EXDO-D proteins to show exceptional high affinity towards different catecholic substrates. Based on V(max)/K(m) specificity constants, a task-sharing between different extradiol dioxygenases in the community of the contaminated site can be supposed, attaining a complementary and community-balanced catalytic power against diverse catecholic derivatives, as necessary for effective degradation of mixtures of aromatics.

• MeSH
• Bacteria enzymologie   genetika   izolace a purifikace   MeSH
• bakteriální geny MeSH
• biodegradace MeSH
• DNA bakterií izolace a purifikace   MeSH
• fylogeneze MeSH
• genetická variace MeSH
• katechol-2,3-dioxygenasa genetika   izolace a purifikace   metabolismus   MeSH
• látky znečišťující půdu metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• monitorování životního prostředí MeSH
• oxygenasy genetika   izolace a purifikace   metabolismus   MeSH
• půdní mikrobiologie MeSH
• ropa metabolismus   MeSH
• sekvence nukleotidů MeSH
• uhlovodíky metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• extradiol dioxygenase MeSH Prohlížeč
• katechol-2,3-dioxygenasa MeSH
• látky znečišťující půdu MeSH
• oxygenasy MeSH
• ropa MeSH
• uhlovodíky MeSH