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.

Department of Biochemistry and Microbiology Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague Czechia

Faculty of Science Institute for Environmental Studies Charles University Prague Czechia

Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czechia

Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

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Arumugam K., Bağcı C., Bessarab I., Beier S., Buchfink B., Górska A., et al. (2019). Annotated bacterial chromosomes from frame-shift-corrected long-read metagenomic data. PubMed DOI PMC

Austen R., Dunn N. (1977). A comparative study of the NAH and TOL catabolic plasmids in PubMed DOI

Barriault D., Lepine F., Mohammadi M., Milot S., Leberre N., Sylvestre M. (2004). Revisiting the regiospecificity of PubMed DOI

Barriault D., Plante M. M., Sylvestre M. (2002). Family shuffling of a targeted bphA region to engineer biphenyl dioxygenase. PubMed PMC

Barriault D., Sylvestre M. (2004). Evolution of the biphenyl dioxygenase BphA from PubMed

Barriault D., Vedadi M., Powlowski J., Sylvestre M. (1999). Cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase and cis-1,2-dihydro-1,2-dihydroxynaphathalene dehydrogenase catalyze dehydrogenation of the same range of substrates. PubMed DOI

Buchfink B., Xie C., Huson D. H. (2015). Fast and sensitive protein alignment using DIAMOND. PubMed DOI

Callahan B. J., Mcmurdie P. J., Rosen M. J., Han A. W., Johnson A. J. A., Holmes S. P. (2016). DADA2: high-resolution sample inference from Illumina amplicon data. PubMed DOI PMC

Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., et al. (2009). BLAST+: architecture and applications. PubMed DOI PMC

Cesco S., Mimmo T., Tonon G., Tomasi N., Pinton R., Terzano R., et al. (2012). Plant-borne flavonoids released into the rhizosphere: impact on soil bio-activities related to plant nutrition. A review. DOI

Cesco S., Neumann G., Tomasi N., Pinton R., Weisskopf L. (2010). Release of plant-borne flavonoids into the rhizosphere and their role in plant nutrition. DOI

Chebrou H., Hurtubise Y., Barriault D., Sylvestre M. (1999). Heterologous expression and characterization of the purified oxygenase component of PubMed PMC

Chung S.-Y., Maeda M., Song E., Horikoshij K., Kudo T. (1994). A Gram-positive Polychlorinated Biphenyl-degrading Bacterium, DOI

Cortes-Tolalpa L., Norder J., Van Elsas J. D., Falcao Salles J. (2018). Halotolerant microbial consortia able to degrade highly recalcitrant plant biomass substrate. PubMed DOI PMC

Dhindwal S., Gomez-Gil L., Neau D. B., Pham T. T. M., Sylvestre M., Eltis L. D., et al. (2016). Structural basis of the enhanced pollutant-degrading capabilities of an engineered biphenyl dioxygenase. PubMed DOI PMC

Di Tommaso P., Moretti S., Xenarios I., Orobitg M., Montanyola A., Chang J. M., et al. (2011). T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. PubMed DOI PMC

Díaz E., Ferrández A., García J. L. (1998). Characterization of the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid in PubMed DOI PMC

Erickson B. D., Mondello F. J. (1992). Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in PubMed DOI PMC

Focht D. D. (1995). Strategies for the improvement of aerobic metabolism of polychlorinated biphenyls. DOI

Fukuda M., Yasukochi Y., Kikuchi Y., Nagata Y., Kimbara K., Horiuchi H., et al. (1994). Identification of the bphA and bphB genes of PubMed DOI

Furukawa K. (2000). Biochemical and genetic bases of microbial degradation of polychlorinated biphenyls (PCBs). PubMed

Furukawa K., Hayase N., Taira K., Tomizuka N. (1989). Molecular relationship of chromosomal genes encoding biphenyl/polychlorinated biphenyl catabolism: some soil bacteria possess a highly conserved bph operon. PubMed PMC

Furukawa K., Suenaga H., Goto M. (2004). Biphenyl dioxygenases: functional versatilities and directed evolution. PubMed PMC

Garrido-Sanz D., Sansegundo-Lobato P., Redondo-Nieto M., Šuman J., Cajthaml T., Blanco-Romero E., et al. (2020). Analysis of the biodegradative and adaptive potential of the novel polychlorinated biphenyl degrader PubMed DOI PMC

Gómez-Gil L., Kumar P., Barriault D., Bolin J. T., Sylvestre M., Eltis L. D. (2007). Characterization of biphenyl dioxygenase of PubMed DOI PMC

Han J., Kim S.-Y., Jung J., Lim Y., Ahn J.-H., Kim S.-I., et al. (2005). Epoxide formation on the aromatic B Ring of Flavanone by Biphenyl Dioxygenase of PubMed DOI PMC

Hernández M., Neufeld J. D., Dumont M. G. (2017). “Enhancing functional metagenomics of complex microbial communities using stable isotopes,” in

Hu J., Wu J., Qu X. (2018). Decomposition characteristics of organic materials and their effects on labile and recalcitrant organic carbon fractions in a semi-arid soil under plastic mulch and drip irrigation. DOI

Iwai S., Chai B., Sul W. J., Cole J. R., Hashsham S. A., Tiedje J. M. (2010). Gene-targeted-metagenomics reveals extensive diversity of aromatic dioxygenase genes in the environment. PubMed PMC

Iwashina T. (2015). Contribution to flower colors of flavonoids including anthocyanins: a review. PubMed

Jones D. T., Taylor W. R., Thornton J. M. (1992). The rapid generation of mutation data matrices from protein sequences. PubMed DOI

Kagami O., Shindo K., Kyojima A., Takeda K., Ikenaga H., Furukawa K., et al. (2008). Protein engineering on biphenyl dioxygenase for conferring activity to convert 7-hydroxyflavone and 5,7-dihydroxyflavone (chrysin). PubMed DOI

Kim S. Y., Jung J., Lim Y., Ahn J. H., Kim S. I., Hur H. G. (2003). Cis-2′, 3′-dihydrodiol production on flavone B-ring by biphenyl dioxygenase from PubMed DOI

Kraus T. E. C., Dahlgren R. A., Zasoski R. J. (2003). Tannins in nutrient dynamics of forest ecosystems - a review.

Kumar S., Stecher G., Li M., Knyaz C., Tamura K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. PubMed DOI PMC

Kumamaru T., Suenaga H., Mitsuoka M., Watanabe T., Furukawa K. (1998). Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase. PubMed DOI

Kweon O., Kim S. J., Baek S., Chae J. C., Adjei M. D., Baek D. H., et al. (2008). A new classification system for bacterial Rieske non-heme iron aromatic ring-hydroxylating oxygenases. PubMed DOI PMC

Lee N., Kwon D. Y. (2016). Characteristics of a recombinant 2,3-dihydroxybiphenyl 1,2-dioxygenase from PubMed DOI PMC

Li A., Qu Y., Zhou J., Ma F. (2009). Enzyme–substrate interaction and characterization of a 2,3-dihydroxybiphenyl 1,2-dioxygenase from PubMed DOI

Lin J. J., Smith M., Jessee J., Bloom F. (1992). DH11S: an PubMed

Liu C.-W., Murray J. D. (2016). The role of flavonoids in nodulation host-range specificity: an update. PubMed PMC

Mackova M., Dowling D., Macek T. (eds) (2006).

Marin A. M., De La Torre J., Ricardo Marques, Oliveira A., Barison A., Satie Chubatsu L., et al. (2016). Genetic and functional characterization of a novel meta-pathway for degradation of naringenin in PubMed DOI

Marin A. M., Souza E. M., Pedrosa F. O., Souza L. M., Sassaki G. L., Baura V. A., et al. (2013). Naringenin degradation by the endophytic diazotroph PubMed DOI

Martin M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. DOI

Masai E., Yamada A., Healy J. M., Hatta T., Kimbara K., Fukuda M., et al. (1995). Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader PubMed PMC

Mason J. R., Cammack R. (1992). The electron-transport proteins of hydroxylating bacterial dioxygenases. PubMed DOI

Master E. R., Mohn W. W. (2001). Induction of bphA, encoding biphenyl dioxygenase, in two polychlorinated biphenyl-degrading bacteria, psychrotolerant PubMed DOI PMC

Misawa N., Shindo K., Takahashi H., Suenaga H., Iguchi K., Okazaki H., et al. (2002). Hydroxylation of various molecules including heterocyclic aromatics using recombinant DOI

Mohammadi M., Sylvestre M. (2005). Resolving the profile of metabolites generated during oxidation of Dibenzofuran and Chlorodibenzofurans by the Biphenyl Catabolic Pathway Enzymes. PubMed DOI

Mohammadi M., Viger J.-F., Kumar P., Barriault D., Bolin J. T., Sylvestre M. (2011). Retuning Rieske-type Oxygenases to expand substrate range. PubMed DOI PMC

Mondello F. J. (1989). Cloning and expression in PubMed DOI PMC

Mondello F. J., Turcich M. P., Lobos J. H., Erickson B. D. (1997). Identification and modification of biphenyl dioxygenase sequences that determine the specificity of polychlorinated biphenyl degradation. PubMed PMC

Nurk S., Meleshko D., Korobeynikov A., Pevzner P. A. (2017). metaSPAdes: a new versatile metagenomic assembler. PubMed DOI PMC

O’Leary N. A., Wright M. W., Brister J. R., Ciufo S., Haddad D., Mcveigh R., et al. (2016). Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. PubMed DOI PMC

Parks D. H., Chuvochina M., Chaumeil P. A., Rinke C., Mussig A. J., Hugenholtz P. (2020). A complete domain-to-species taxonomy for Bacteria and Archaea. PubMed DOI

Pham T. T., Pino Rodriguez N. J., Hijri M., Sylvestre M. (2015). Optimizing Polychlorinated Biphenyl Degradation by Flavonoid-Induced Cells of the Rhizobacterium PubMed DOI PMC

Pham T. T., Sylvestre M. (2013). Has the bacterial biphenyl catabolic pathway evolved primarily to degrade biphenyl? The diphenylmethane case. PubMed DOI PMC

Pham T. T. M., Tu Y., Sylvestre M. (2012). Remarkable ability of PubMed DOI PMC

Pieper D. H. (2005). Aerobic degradation of polychlorinated biphenyls. PubMed

Polivkova M., Suman J., Strejcek M., Kracmarova M., Hradilova M., Filipova A., et al. (2018). Diversity of root-associated microbial populations of DOI

R Core Team (2017).

Rausher M. D. (2001). Co-evolution and plant resistance to natural enemies. PubMed

Ridl J., Suman J., Fraraccio S., Hradilova M., Strejcek M., Cajthaml T., et al. (2018). Complete genome sequence of PubMed DOI PMC

Robrock K. R., Mohn W. W., Eltis L. D., Alvarez-Cohen L. (2011). Biphenyl and ethylbenzene dioxygenases of PubMed DOI

Sala-Trepat J. M., Evans W. C. (1971). The meta cleavage of catechol by PubMed DOI

San-Miguel T., Perez-Bermudez P., Gavidia I. (2013). Production of soluble eukaryotic recombinant proteins in PubMed DOI PMC

Seeger M., González M., Cámara B., Muñoz L., Ponce E., Mejías L., et al. (2003). Biotransformation of natural and synthetic isoflavonoids by two recombinant microbial enzymes. PubMed DOI PMC

Seemann T. (2014). Prokka: rapid prokaryotic genome annotation. PubMed DOI

Seo J., Kang S. I., Ryu J. Y., Lee Y. J., Park K. D., Kim M., et al. (2010). Location of flavone B-ring controls regioselectivity and stereoselectivity of naphthalene dioxygenase from PubMed DOI

Shaw L. J., Morris P., Hooker J. E. (2006). Perception and modification of plant flavonoid signals by rhizosphere microorganisms. PubMed DOI

Shimada T., Nagayoshi H., Murayama N., Takenaka S., Katahira J., Kim V., et al. (2021). Liquid chromatography-tandem mass spectrometry analysis of oxidation of 2′-, 3′-, 4′- and 6-hydroxyflavanones by human cytochrome P450 enzymes. PubMed DOI PMC

Shindo K., Kagiyama Y., Nakamura R., Hara A., Ikenaga H., Furukawa K., et al. (2003). Enzymatic synthesis of novel antioxidant flavonoids by DOI

Singer A. C. (2006). “The chemical ecology of pollutant biodegradation: bioremediation and Phytoremediation from mechanistic and ecological perspectives,” in

Singer A. C., Crowley D. E., Thompson I. P. (2003). Secondary plant metabolites in phytoremediation and biotransformation. PubMed DOI

Spence E. L., Kawamukai M., Sanvoisin J., Braven H., Bugg T. D. (1996). Catechol dioxygenases from PubMed DOI PMC

Strejcek M., Wang Q., Ridl J., Uhlik O. (2015). Hunting down frame shifts: ecological analysis of diverse functional gene sequences. PubMed DOI PMC

Subramanian S., Stacey G., Yu O. (2007). Distinct, crucial roles of flavonoids during legume nodulation. PubMed DOI

Sul W. J., Park J., Quensen J. F., Iii, Rodrigues J. L., Seliger L., et al. (2009). DNA-stable isotope probing integrated with metagenomics for retrieval of biphenyl dioxygenase genes from polychlorinated biphenyl-contaminated river sediment. PubMed PMC

Sylvestre M., Sirois M., Hurtubise Y., Bergeron J., Ahmad D., Shareck F., et al. (1996). Sequencing of Comamonas testosteroni strain B-356-biphenyl/chlorobiphenyl dioxygenase genes: evolutionary relationships among Gram-negative bacterial biphenyl dioxygenases. PubMed

Taira K., Hirose J., Hayashida S., Furukawa K. (1992). Analysis of bph operon from the polychlorinated biphenyl-degrading strain of PubMed

Tan H.-M., Tang H.-Y., Joannou C., Abdel-Wahab N., Mason J. (1993). The PubMed DOI

Tellinghuisen J. (2001). Statistical error propagation. DOI

The UniProt Consortium (2018). UniProt: a worldwide hub of protein knowledge. PubMed DOI PMC

Thompson J. R., Marcelino L. A., Polz M. F. (2002). Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR’. PubMed PMC

Toussaint J.-P., Pham T., Barriault D., Sylvestre M. (2012). Plant exudates promote PCB degradation by a rhodococcal rhizobacteria. PubMed DOI

Uhlik O., Jecna K., Mackova M., Vlcek C., Hroudova M., Demnerova K., et al. (2009). Biphenyl-metabolizing bacteria in the rhizosphere of horseradish and bulk soil contaminated by polychlorinated biphenyls as revealed by stable isotope probing. PubMed PMC

Uhlik O., Leewis M. C., Strejcek M., Musilova L., Mackova M., Leigh M. B., et al. (2013). Stable isotope probing in the metagenomics era: a bridge towards improved bioremediation. PubMed DOI PMC

Uhlik O., Wald J., Strejcek M., Musilova L., Ridl J., Hroudova M., et al. (2012). Identification of bacteria utilizing biphenyl, benzoate, and naphthalene in long-term contaminated soil. PubMed DOI PMC

Van der Geize R., Yam K., Heuser T., Wilbrink M. H., Hara H., Anderton M. C., et al. (2007). A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages. PubMed PMC

Vergani L., Mapelli F., Suman J., Cajthaml T., Uhlik O., Borin S. (2019). Novel PCB-degrading Rhodococcus strains able to promote plant growth for assisted rhizoremediation of historically polluted soils. PubMed DOI PMC

Vézina J., Barriault D., Sylvestre M. (2007). Family shuffling of soil DNA to change the regiospecificity of PubMed PMC

Vézina J., Barriault D., Sylvestre M. (2008). Diversity of the C-terminal portion of the biphenyl dioxygenase large subunit. PubMed

Wald J., Hroudova M., Jansa J., Vrchotova B., Macek T., Uhlik O. (2015). Pseudomonads rule degradation of polyaromatic hydrocarbons in aerated sediment. PubMed DOI PMC

Wang Y., Garnon J., Labbe D., Bergeron H., Lau P. C. (1995). Sequence and expression of the bpdC1C2BADE genes involved in the initial steps of biphenyl/chlorobiphenyl degradation by PubMed DOI

Yang X., Liu X., Song L., Xie F., Zhang G., Qian S. (2007). Characterization and functional analysis of a novel gene cluster involved in biphenyl degradation in PubMed DOI

Yang X., Xie F., Zhang G., Shi Y., Qian S. (2008). Purification, characterization, and substrate specificity of two 2,3-dihydroxybiphenyl 1,2-dioxygenase from PubMed DOI

Zhu L., Zhou J., Zhang R., Tang X., Wang J., Li Y., et al. (2020). Degradation mechanism of biphenyl and 4-4′-dichlorobiphenyl cis-dihydroxylation by non-heme 2,3 dioxygenases BphA: a QM/MM approach. PubMed DOI

Zubrova A., Michalikova K., Semerad J., Strejcek M., Cajthaml T., Suman J., et al. (2021). Biphenyl 2,3-dioxygenase in PubMed DOI PMC

Differential effect of monoterpenes and flavonoids on the transcription of aromatic ring-hydroxylating dioxygenase genes in Rhodococcus opacus C1 and Rhodococcus sp. WAY2