Polychlorinated biphenyl (PCB)-contaminated soils represent a major treat for ecosystems health. Plant biostimulation of autochthonous microbial PCB degraders is a way to restore polluted sites where traditional remediation techniques are not sustainable, though its success requires the understanding of site-specific plant-microbe interactions. In an historical PCB contaminated soil, we applied DNA stable isotope probing (SIP) using 13C-labeled 4-chlorobiphenyl (4-CB) and 16S rRNA MiSeq amplicon sequencing to determine how the structure of total and PCB-degrading bacterial populations were affected by different treatments: biostimulation with Phalaris arundinacea subjected (PhalRed) or not (Phal) to a redox cycle and the non-planted controls (Bulk and BulkRed). Phal soils hosted the most diverse community and plant biostimulation induced an enrichment of Actinobacteria. Mineralization of 4-CB in SIP microcosms varied between 10% in Bulk and 39% in PhalRed soil. The most abundant taxa deriving carbon from PCB were Betaproteobacteria and Actinobacteria. Comamonadaceae was the family most represented in Phal soils, Rhodocyclaceae and Nocardiaceae in non-planted soils. Planted soils subjected to redox cycle enriched PCB degraders affiliated to Pseudonocardiaceae, Micromonosporaceae and Nocardioidaceae. Overall, we demonstrated different responses of soil bacterial taxa to specific rhizoremediation treatments and we provided new insights into the populations active in PCB biodegradation.

• MeSH
• Actinomycetales * genetika   MeSH
• Bacteria MeSH
• biodegradace MeSH
• DNA bakterií genetika   metabolismus   MeSH
• DNA metabolismus   MeSH
• ekosystém MeSH
• izotopy metabolismus   MeSH
• látky znečišťující půdu * metabolismus   MeSH
• polychlorované bifenyly * metabolismus   MeSH
• půda chemie   MeSH
• půdní mikrobiologie MeSH
• RNA ribozomální 16S genetika   metabolismus   MeSH
• rostliny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• DNA MeSH
• izotopy MeSH
• látky znečišťující půdu * MeSH
• polychlorované bifenyly * MeSH
• půda MeSH
• RNA ribozomální 16S 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.

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

A bacterial species is best characterized after its isolation in a pure culture. This is an arduous endeavor for many soil microorganisms, but it can be simplified by several techniques for improving culturability: for example, by using growth-promoting factors. We investigated the potential of a Micrococcus luteus culture supernatant containing resuscitation-promoting factor (SRpf) to increase the number and diversity of cultured bacterial taxa from a nutrient-rich compost soil. Phosphate-buffered saline and inactivated SRpf were included as controls. After agitation with SRpf at 28°C for 1 day, the soil suspension was diluted and plated on two different solid, oligotrophic media: tenfold diluted Reasoner's 2A agar (R2A) and soil extract-based agar (SA). Colonies were collected from the plates to assess the differences in diversity between different treatments and cultivation media. The diversity on both R2A and SA was higher in the SRpf-amended extracts than the controls, but the differences on R2A were higher. Importantly, 51 potentially novel bacterial species were isolated on R2A and SA after SRpf treatment. Diversity in the soil extracts was also determined by high-throughput 16S rRNA amplicon sequencing, which showed an increase in the abundance of specific taxa before their successful cultivation. Conclusively, SRpf can effectively enhance the growth of soil bacterial species, including those hitherto uncultured.

• Klíčová slova
• Micrococcus luteus, extracellular organic matter, increased culturability, non-cultured bacteria, oligotrophic medium, resuscitation-promoting factor, viable but non-culturable state,
• Publikační typ
• časopisecké články MeSH

The involvement of bacterial aromatic ring-hydroxylating dioxygenases (ARHDs) in the degradation of aromatic pollutants, such as polychlorinated biphenyls (PCBs), has been well studied. However, there is considerable speculation as to the origin of this ability. One hypothesis is centered on a connection between the ability to degrade aromatic pollutants and the necessity of soil bacteria to cope with and/or utilize secondary plant metabolites (SPMs). To investigate this connection, we researched the involvement of biphenyl 2,3-dioxygenase (BPDO), an ARHD essential for the degradation of PCBs, in the metabolism of SPMs in the soil bacterium Pseudomonas alcaliphila JAB1, a versatile degrader of PCBs. We demonstrated the ability of the strain JAB1 to transform a variety of SPMs, namely the flavonoids apigenin, flavone, flavanone, naringenin, fisetin, quercetin, morin, and catechin, caffeic acid, trans-cinnamic acid, and the monoterpenes (S)-limonene and (R)-carvone. Of those, the transformation of flavone, flavanone, and (S)-limonene was conditioned by the activity of JAB1-borne BPDO and thus was researched in more detail, and we found evidence for the limonene monooxygenase activity of the BPDO. Furthermore, the bphA gene in the strain JAB1 was demonstrated to be induced by a wide range of SPMs, with monoterpenes being the strongest inducers of the SPMs tested. Thus, our findings contribute to the growing body of evidence that ARHDs not only play a role in the catabolism of aromatic pollutants, but also of natural plant-derived aromatics, and this study supports the hypothesis that ARHDs participate in ecological processes mediated by SPMs.

• Klíčová slova
• aromatic ring-hydroxylating dioxygenases, biphenyl dioxygenase, monoterpenes, phenolics, secondary plant metabolites,
• Publikační typ
• časopisecké články MeSH

Certain industrial chemicals accumulate in the environment due to their recalcitrant properties. Bioremediation uses the capability of some environmental bacteria to break down these chemicals and attenuate the pollution. One such bacterial strain, designated Pvy, was isolated from sediment samples from a lagoon in Romania located near an oil refinery due to its capacity to degrade dibenzofuran (DF). The genome sequence of the Pvy strain was obtained using an Oxford Nanopore MiniION platform. According to the consensus 16S rRNA gene sequence that was compiled from six 16S rRNA gene copies contained in the genome and orthologous average nucleotide identity (OrthoANI) calculation, the Pvy strain was identified as Pseudomonas veronii, which confirmed the identification obtained with the aid of MALDI-TOF mass spectrometry and MALDI BioTyper. The genome was analyzed with respect to enzymes responsible for the overall biodegradative versatility of the strain. The Pvy strain was able to derive carbon from naphthalene (NP) and several aromatic compounds of natural origin, including salicylic, protocatechuic, p-hydroxybenzoic, trans-cinnamic, vanillic, and indoleacetic acids or vanillin, and was shown to degrade but not utilize DF. In total seven loci were found in the Pvy genome, which enables the strain to participate in the degradation of these aromatic compounds. Our experimental data also indicate that the transcription of the NP-dioxygenase α-subunit gene (ndoB), carried by the plasmid of the Pvy strain, is inducible by DF. These features make the Pvy strain a potential candidate for various bioremediation applications.

• Klíčová slova
• Pseudomonas veronii strain Pvy, biodegradation, denitrification, dibenzofuran, dioxygenase, heavy-metal tolerance, nanopore technology, organic phosphate mineralization, whole-genome sequencing,
• MeSH
• biodegradace MeSH
• dibenzofurany * MeSH
• genomika * MeSH
• Pseudomonas MeSH
• RNA ribozomální 16S MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dibenzofurany * MeSH
• RNA ribozomální 16S MeSH

Many ecological experiments are based on the extraction and downstream analyses of microorganisms from different environmental samples. Due to its high throughput, cost-effectiveness and rapid performance, Matrix Assisted Laser Desorption/Ionization Mass Spectrometry with Time-of-Flight detector (MALDI-TOF MS), which has been proposed as a promising tool for bacterial identification and classification, could be advantageously used for dereplication of recurrent bacterial isolates. In this study, we compared whole-cell MALDI-TOF MS-based analyses of 49 bacterial cultures to two well-established bacterial identification and classification methods based on nearly complete 16S rRNA gene sequence analyses: a phylotype-based approach, using a closest type strain assignment, and a sequence similarity-based approach involving a 98.65% sequence similarity threshold, which has been found to best delineate bacterial species. Culture classification using reference-based MALDI-TOF MS was comparable to that yielded by phylotype assignment up to the genus level. At the species level, agreement between 16S rRNA gene analysis and MALDI-TOF MS was found to be limited, potentially indicating that spectral reference databases need to be improved. We also evaluated the mass spectral similarity technique for species-level delineation which can be used independently of reference databases. We established optimal mass spectral similarity thresholds which group MALDI-TOF mass spectra of common environmental isolates analogically to phylotype- and sequence similarity-based approaches. When using a mass spectrum similarity approach, we recommend a mass range of 4-10 kDa for analysis, which is populated with stable mass signals and contains the majority of phylotype-determining peaks. We show that a cosine similarity (CS) threshold of 0.79 differentiate mass spectra analogously to 98.65% species-level delineation sequence similarity threshold, with corresponding precision and recall values of 0.70 and 0.73, respectively. When matched to species-level phylotype assignment, an optimal CS threshold of 0.92 was calculated, with associated precision and recall values of 0.83 and 0.64, respectively. Overall, our research indicates that a similarity-based MALDI-TOF MS approach can be routinely used for efficient dereplication of isolates for downstream analyses, with minimal loss of unique organisms. In addition, MALDI-TOF MS analysis has further improvement potential unlike 16S rRNA gene analysis, whose methodological limits have reached a plateau.

• Klíčová slova
• 16S rRNA gene, MALDI BioTyper, MALDI-TOF mass spectrometry (MS), bacterial identification, bacterial isolation, dereplication of isolates, species delineation,
• Publikační typ
• časopisecké články MeSH

In this study, following its isolation from contaminated soil, the genomic sequence of Pseudomonas alcaliphila strain JAB1 (=DSM 26533), a biphenyl-degrading bacterium, is reported and analyzed in relation to its extensive degradative capabilities. The P. alcaliphila JAB1 genome (GenBank accession no. CP016162) consists of a single 5.34 Mbp-long chromosome with a GC content of 62.5%. Gene function was assigned to 3816 of the 4908 predicted genes. The genome harbors a bph gene cluster, permitting degradation of biphenyl and many congeners of polychlorinated biphenyls (PCBs), a ben gene cluster, enabling benzoate and its derivatives to be degraded, and phe gene cluster, which permits phenol degradation. In addition, P. alcaliphila JAB1 is capable of cometabolically degrading cis-1,2-dichloroethylene (cDCE) when grown on phenol. The strain carries both catechol and protocatechuate branches of the β-ketoadipate pathway, which is used to funnel the pollutants to the central metabolism. Furthermore, we propose that clustering of MALDI-TOF MS spectra with closest phylogenetic relatives should be used when taxonomically classifying the isolated bacterium; this, together with 16S rRNA gene sequence and chemotaxonomic data analyses, enables more precise identification of the culture at the species level.

• Klíčová slova
• Aromatic compounds, Biodegradation, Bioremediation, Biphenyl, Chlorobenzoic acids (CBAs), Dioxygenase, Genome, MALDI-TOF MS, Monooxygenase, Phenol, Polychlorinated biphenyls (PCBs), Pseudomonadaceae, Pseudomonas alcaliphila JAB1, ben genes, bph genes, cis-1,2-dichloroethylene (cDCE), phe genes,
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

Despite decades of research there is limited understanding of how vegetation impacts the ability of microbial communities to process organic contaminants in soil. Using a combination of traditional and molecular assays, we examined how phytoremediation with willow and/or fertilization affected the microbial community present and active in the transformation of diesel contaminants. In a pot study, willow had a significant role in structuring the total bacterial community and resulted in significant decreases in diesel range organics (DRO). However, stable isotope probing (SIP) indicated that fertilizer drove the differences seen in community structure and function. Finally, analysis of the total variance in both pot and SIP experiments indicated an interactive effect between willow and fertilizer on the bacterial communities. This study clearly demonstrates that a willow native to Alaska accelerates DRO degradation, and together with fertilizer, increases aromatic degradation by shifting microbial community structure and the identity of active naphthalene degraders.

• Klíčová slova
• Salix alaxensis, bioremediation, diesel range organics, fertilizer, microbial community structure, naphthalene degradation, phytoremediation, stable isotope probing,
• 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
• biphenyl MeSH Prohlížeč
• látky znečišťující půdu MeSH
• nebezpečný odpad MeSH
• polychlorované bifenyly MeSH
• půda MeSH
• RNA ribozomální 16S MeSH