Aromatic ring-hydroxylating dioxygenases (ARHDs) play a crucial role in the aerobic biodegradation of both natural and anthropogenic aromatic compounds. Although their ability to process contaminants is not entirely understood, it is thought to have evolved from the transformation of structurally similar secondary plant metabolites (SPMs). Hence, to investigate this connection, we tested a variety of SPMs from the monoterpene and flavonoid classes as carbon sources and transcriptional effectors of several phylogenetically distant ARHD genes involved in the degradation of aromatic pollutants. Specifically, we focused on bphA1, nahA1 and phtA1 in Rhodococcus opacus C1, whose genomic analysis is also presented hereinafter, and bphA1a, nahA1-bphA1b and etbA1ab in Rhodococcus sp. WAY2. Whilst induction was only observed with (R)-carvone for bphA1a and nahA1-bphA1b of strain WAY2, and with p-cymene for nahA1 and nahA1-bphA1b of strains C1 and WAY2, respectively, an extensive inhibition by flavonoids was observed for most of the genes in both strains. To the best of our knowledge, our study is the first to report the effect of flavonoids and monoterpenes on the transcription of nahA1, etbA1 and phtA1 genes. In addition, we show that, in contrast to pseudomonads, many flavonoids inhibit the transcription of the ARHD genes in rhodococci. Thus, our work provides a new perspective on flavonoids as the transcriptional effectors of ARHDs, highlighting the significant variability of these enzymes and the divergent responses that they elicit. Moreover, our results contribute to understanding the complex interactions between microorganisms and SPMs and provide insights into the molecular basis of a number of them.

• Keywords
• Rhodococcus, aromatic pollutants, aromatic ring-hydroxylating dioxygenases, biodegradation, flavonoids, monoterpenes,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Biodegradation, Environmental MeSH
• Dioxygenases * genetics   metabolism   MeSH
• Flavonoids * pharmacology   metabolism   MeSH
• Phylogeny MeSH
• Transcription, Genetic drug effects   MeSH
• Monoterpenes * pharmacology   metabolism   MeSH
• Gene Expression Regulation, Bacterial drug effects   MeSH
• Rhodococcus * genetics   drug effects   enzymology   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Dioxygenases * MeSH
• Flavonoids * MeSH
• Monoterpenes * MeSH

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.

• Keywords
• aromatic ring-hydroxylating dioxygenases, biphenyl dioxygenase, flavonoids, functional metagenomics, polychlorinated biphenyls, secondary plant metabolites,
• Publication type
• Journal Article MeSH

Extended soil contamination by polychlorinated biphenyls (PCBs) represents a global environmental issue that can hardly be addressed with the conventional remediation treatments. Rhizoremediation is a sustainable alternative, exploiting plants to stimulate in situ the degradative bacterial communities naturally occurring in historically polluted areas. This approach can be enhanced by the use of bacterial strains that combine PCB degradation potential with the ability to promote plant and root development. With this aim, we established a collection of aerobic bacteria isolated from the soil of the highly PCB-polluted site "SIN Brescia-Caffaro" (Italy) biostimulated by the plant Phalaris arundinacea. The strains, selected on biphenyl and plant secondary metabolites provided as unique carbon source, were largely dominated by Actinobacteria and a significant number showed traits of interest for remediation, harbouring genes homologous to bphA, involved in the PCB oxidation pathway, and displaying 2,3-catechol dioxygenase activity and emulsification properties. Several strains also showed the potential to alleviate plant stress through 1-aminocyclopropane-1-carboxylate deaminase activity. In particular, we identified three Rhodococcus strains able to degrade in vitro several PCB congeners and to promote lateral root emergence in the model plant Arabidopsis thaliana in vivo. In addition, these strains showed the capacity to colonize the root system and to increase the plant biomass in PCB contaminated soil, making them ideal candidates to sustain microbial-assisted PCB rhizoremediation through a bioaugmentation approach.

• MeSH
• Arabidopsis growth & development   microbiology   MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Biodegradation, Environmental MeSH
• Gene Expression MeSH
• Catechol 2,3-Dioxygenase genetics   metabolism   MeSH
• Plant Roots growth & development   microbiology   MeSH
• Soil Pollutants metabolism   MeSH
• Carbon-Carbon Lyases genetics   metabolism   MeSH
• Oxidation-Reduction MeSH
• Phalaris growth & development   microbiology   MeSH
• Polychlorinated Biphenyls metabolism   MeSH
• Soil chemistry   MeSH
• Soil Microbiology MeSH
• Rhodococcus enzymology   genetics   MeSH
• Secondary Metabolism genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-aminocyclopropane-1-carboxylate deaminase MeSH Browser
• Bacterial Proteins MeSH
• Catechol 2,3-Dioxygenase MeSH
• Soil Pollutants MeSH
• Carbon-Carbon Lyases MeSH
• Polychlorinated Biphenyls MeSH
• Soil 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
• Hydrocarbons, Aromatic metabolism   MeSH
• Bacteria genetics   metabolism   MeSH
• Benzoates metabolism   MeSH
• Biphenyl Compounds metabolism   MeSH
• Biodegradation, Environmental MeSH
• Soil Pollutants metabolism   MeSH
• Hazardous Waste MeSH
• Polychlorinated Biphenyls metabolism   MeSH
• Soil chemistry   MeSH
• Soil Microbiology * MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Environmental Pollution * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Hydrocarbons, Aromatic MeSH
• Benzoates MeSH
• Biphenyl Compounds MeSH
• biphenyl MeSH Browser
• Soil Pollutants MeSH
• Hazardous Waste MeSH
• Polychlorinated Biphenyls MeSH
• Soil MeSH
• RNA, Ribosomal, 16S MeSH

Functional gene ecological analyses using amplicon sequencing can be challenging as translated sequences are often burdened with shifted reading frames. The aim of this work was to evaluate several bioinformatics tools designed to correct errors which arise during sequencing in an effort to reduce the number of frameshifts (FS). Genes encoding for alpha subunits of biphenyl (bphA) and benzoate (benA) dioxygenases were used as model sequences. FrameBot, a FS correction tool, was able to reduce the number of detected FS to zero. However, up to 44% of sequences were discarded by FrameBot as non-specific targets. Therefore, we proposed a de novo mode of FrameBot for FS correction, which works on a similar basis as common chimera identifying platforms and is not dependent on reference sequences. By nature of FrameBot de novo design, it is crucial to provide it with data as error free as possible. We tested the ability of several publicly available correction tools to decrease the number of errors in the data sets. The combination of maximum expected error filtering and single linkage pre-clustering proved to be the most efficient read processing approach. Applying FrameBot de novo on the processed data enabled analysis of BphA sequences with minimal losses of potentially functional sequences not homologous to those previously known. This experiment also demonstrated the extensive diversity of dioxygenases in soil. A script which performs FrameBot de novo is presented in the supplementary material to the study or available at https://github.com/strejcem/FBdenovo. The tool was also implemented into FunGene Pipeline available at http://fungene.cme.msu.edu/FunGenePipeline/.

• Keywords
• FrameBot, Frameshift, amplicon sequencing, benzoate dioxygenase, biphenyl dioxygenase, functional genes,
• Publication type
• Journal Article MeSH