bphA Gene Research Articles

The evaluation of Hudson River sediment as a growth substrate – Microbial activity, PCB-degradation potential and risk assessment

The potential use of growth substrates prepared with an admixture of 10% to 75% Hudson River sediments was evaluated by analysis of changes in microbial activity (measured using Biolog Ecoplates) and molecular markers (presence of degradative tceA1 and bphA genes) as well as potential risks toward humans and the environment (health and environmental risk quotients). The highest microbial activity was found in growth substrate with 25% Hudson River sediments compared to unamended control soil. Significant differences were observed between samples amended with lower (0–10%) and higher (25–75%) proportion of sediment. Microbial activity increased with the proportion of sediment amendment (≥25% sediment); however, this increase in microbial activity was not affected by increasing pollutant concentrations (PCBs, Pb, Cr Ni and Zn) nor decreasing TOC content. The growth substrate amended with Hudson River sediments demonstrated a potential for PCB degradation, as evidenced by the presence of tceA1 and bphA genes responsible, respectively, for reductive dehalogenation and oxidation of a range of aromatic organic compounds including PCBs. An assessment of risk quotients showed that the growth substrates containing lower doses of Hudson River sediments (10–50%) meet the international requirements for use in agriculture/horticulture for the production of non-food crops. Nevertheless, due to the elevated content of some toxic metals and PCBs, the growth substrate prepared with the highest proportion of sediments (75%) was not suitable for agricultural/horticultural use.

Predominant Biphenyl Dioxygenase From Legacy Polychlorinated Biphenyl (PCB)-Contaminated Soil Is a Part of Unusual Gene Cluster and Transforms Flavone and Flavanone.

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.

Biphenyl 2,3-Dioxygenase in Pseudomonas alcaliphila JAB1 Is Both Induced by Phenolics and Monoterpenes and Involved in Their Transformation.

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.

HETEROLOGOUS EXPRESSION OF A GENE ENCODING BIPHENYL 2,3-DIOXYGENASE FROM BURKHOLDERIA CEPACIA IN ESCHERICHIA COLI

Biphenyl 2,3-dioxygenase (BphA) is an enzyme that participates in initiation step of the pathway of biphenyl degradation in bacteria. Biphenyl is an aromatic hydrocarbon including two benzene rings that causes a serious environmental pollution. This report presents the preliminary results on the cloning and expression of BphA gene of B. cepacia strains DF2 and DF4 which were isolated from dioxin contaminated soil in Vietnam. The coding DNA sequences of BphA genes are 897 bp long and 99% identical. Recombinant BphA enzyme (33 kDa) has strongly expressed in E. coli M15 after induction of 0.5 mM IPTG for 4 h at 37ºC.

Degradation of BTEX mixture by a new Pseudomonas putida strain: role of the quorum sensing in the modulation of the upper BTEX oxidative pathway.

A new Pseudomonas putida strain (AQ8) was isolated from a decommissioned oil refinery's soil in Italy and characterized for its ability to degrade BTEX. The draft genome of the new strain was sequenced and annotated for genes that encode enzymes putatively involved in BTEX degradation and quorum sensing. The strain was transformed with a plasmid expressing lactonase, which cleaves the autoinducer quorum sensing signal molecule, the acyl-homoserine lactone, to obtain a quorum sensing minus strain. P. putida AQ8 depleted the 40% on average of all the components of the initial BTEX concentration in 36h. The quorum sensing minus strain, in the same time interval, depleted only the 10% of the initial BTEX concentration. The role of quorum sensing in regulating the expression of the annotated benzene/toluene dioxygenase gene (benzA) and biphenyl/toluene/benzene dioxygenase (bphA) genes, which are involved in BTEX degradation, was studied by quantitative RT-real-time quantitative (q)PCR analysis. The qPCR data showed decreased levels of expression of the benzA and bphA genes in the quorum sensing minus strain. Our results showed, for the first time, quorum sensing modulation of the level of transcription of dioxygenase genes in the upper BTEX oxidation pathway.

Biphenyl degradation by recombinant photosynthetic cyanobacterium Synechocystis sp. PCC6803 in an oligotrophic environment using unphysiological electron transfer.

Cyanobacteria are potentially useful photosynthetic microorganisms for bioremediation under oligotrophic environments. Here, the biphenyl degradation pathway genes of β-proteobacterium Acidovorax sp. strain KKS102 were co-expressed in cyanobacterium Synechocystis sp. PCC6803 cells under control of the photo-inducible psbE promoter. In the KKS102 cells, biphenyl is dioxygenated by bphA1 and bphA2 gene products complex using electrons supplied from NADH via bphA4 and bphA3 gene products (BphA4 and BphA3, respectively), and converted to benzoic acid by bphB, bphC and bphD gene products. Unexpectedly, biphenyl was effectively hydroxylated in oligotrophic BG11 medium by co-expressing the bphA3, bphA1 and bphA2 genes without the bphA4 gene, suggesting that endogenous cyanobacteria-derived protein(s) can supply electrons to reduce BphA3 at the start of the biphenyl degradation pathway. Furthermore, biphenyl was converted to benzoic acid by cyanobacterial cells co-expressing bphA3, bphA1, bphA2, bphB, bphC and bphD. Structural gene-screening using recombinant Escherichia coli cells co-expressing bphA3, bphA1, bphA2, bphB and bphC suggested that petH, which encodes long- and short-type NADP-ferredoxin oxidoreductase isomers (FNRL and FNRS, respectively), and slr0600, which is annotated as an NADPH-thioredoxin reductase gene in CyanoBase, were BphA3-reducible proteins. Purified FNRL and FNRS, and the slr0600 gene product showed BphA3 reductase activity dependent on NADPH and the reduced form of glutathione, respectively, potentially shedding light on the physiological roles of the slr0600 gene product in cyanobacterial cells. Collectively, our results demonstrate the utility of Synechocystis sp. PCC6803 cells as a host for bioremediation of biphenyl compounds under oligotrophic environments without an organic carbon source.

Diversity of the bphA1 Genes in a Microbial Community from Anthropogenically Contaminated Soil and Isolation of New Pseudomonads Degrading Biphenyl/Chlorinated Biphenyls

Molecular biological and cultivation-based approaches were used to investigate the microbial community of tehnogenic soil contaminated with poorly degradable toxic (chlorinated) aromatic compounds. Diversity of the bphA1 genes, the key genes for the degradation of biphenyl/polychlorinated biphenyls (PCB) was assessed, and new bacterial degraders of biphemyl/PCB were isolated. Cloning of the PCR product obtained using the DNA isolated from soil as a template and the primers to the biphenyl 2,3-dioxygenase α-subunit gene (bphA1) revealed two types of the genes of aromatic dioxygenases (DO) with the highest similarity (97.8‒99.5%) to the genes encoding the Rieske cluster of DO α-subunits (bphA1) from uncultured bacteria. Two biphenyl-degrading isolates obtained from an enrichment culture of a soil sample incubated with biphenyl were identified as Pseudomonas (VRP2-6 and VRP2-2). According to their 16S rRNA gene sequences, they exhibited the highest similarity to the type strain of P. taiwanensis (99%) and P. alcaligenes (100%), respectively. Analysis of the bphA1 sequences of strains VRP2-6 and VRP2-2 revealed the similarity to those of the known biphenyl-degrading pseudomonads not exceeding 97.3%. The isolate VRP2-6 efficiently utilized ortho- and para-monochlorinated biphenyls and degraded dichlorinated biphenyl oxidizing both the ortho- and para-chlorinated rings of the biphenyl molecule. New pseudomonad strains may be of interest for development of biotechnologies aimed at monitoring and remediation of biphenyl/PCB-contaminated soils.

Aerobic degradation of 3,3′,4,4′-tetrachlorobiphenyl by a resuscitated strain Castellaniella sp. SPC4: Kinetics model and pathway for biodegradation

Resuscitated strains which were obtained by addition of resuscitation promoting factor (Rpf) could provide a vast majority of microbial source for obtaining highly efficient polychlorinated biphenyl (PCB)-degrading bacteria. In this study, the Castellaniella sp. strain SPC4 which was resuscitated by Rpf addition showed the highest efficiency in degradation of 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) among the resuscitated and non-resuscitated isolates. Further investigations on the PCB 77 degradation capability of the resuscitated strain SPC4 showed that SPC4 could efficiently degrade PCB 77 with maximum degradation rate (qmax) of 0.066/h at about 20 mg/L of PCB 77. The maximum growth rate on PCB 77 was 2.663 × 107 CFU/(mL·h) (0.024/h). The most suitable model of Edward demonstrated that the SPC4 could achieve qmax of 0.9315/h, with substrate-affinity of 11.33 mg/L and substrate-inhibition constants of 11.41 mg/L. Meanwhile, the presence of bphA gene expression and chlorine ions release, together with the identification of metabolites, confirmed that the bph-encoded biphenyl pathway was involved in PCB 77 mineralization by SPC4. This report is the first to demonstrate aerobic degradation of PCB 77 by the resuscitated strain Castellaniella sp. SPC4, indicating excellent potential for PCB bioremediation.

Phytoremediation of soil contaminated with PCBs using different plants and their associated microbial communities

In this work, we evaluate the abilities of the plants Brassica juncea, Avena sativa, Brachiaria decumbens, and Medicago sativa to uptake polychlorinated biphenyls (PCBs) and induce degradation of soil microorganisms from contaminated soil. Removal of PCBs 44, 66, 118, 153, 170, and 180 was evaluated in both rhizospheric and nonrhizospheric soils. Microbial and bphA1 gene quantifications were performed by real-time PCR. The PCB concentrations in plant tissues and soil were determined, and a fluorescein diacetate (FDA) hydrolysis assay was used to measure microbial activity in soil. The removal percentages for all PCB congeners in planted soil versus unplanted control soil were statistically significant and varied between 45% and 63%. PCBs 118, 153, 138, and 170 were detected in Brachiaria decumbens roots at different concentrations. In planted soil, an increase in the concentration of bacteria was observed compared to the initial concentration and the concentration in unplanted control soil; however, no significant differences were identified between plants. The number of copies of the bphA1 gene was higher in rhizospheric versus non- rhizospheric soil for all plants at the end of the experiment. However, alfalfa and oat rhizospheric soil showed significant differences in the copy number of the bphA1 gene. In general, the concentration of fluorescein in the rhizospheric soil was greater than that in the nonrhizospheric soil. Although the plants had a positive effect on PCB removal, this effect varied depending on the type of PCB, the plant, and the soil.

Biphenyl-Metabolizing Microbial Community and a Functional Operon Revealed in E-Waste-Contaminated Soil.

Primitive electronic waste (e-waste) recycling activities release massive amounts of persistent organic pollutants (POPs) and heavy metals into surrounding soils, posing a major threat to the ecosystem and human health. Microbes capable of metabolizing POPs play important roles in POPs remediation in soils, but their phylotypes and functions remain unclear. Polychlorinated biphenyls (PCBs), one of the main pollutants in e-waste contaminated soils, have drawn increasing attention due to their high persistence, toxicity, and bioaccumulation. In the present study, we employed the culture-independent method of DNA stable-isotope probing to identify active biphenyl and PCB degraders in e-waste-contaminated soil. A total of 19 rare operational taxonomic units and three dominant bacterial genera ( Ralstonia, Cupriavidus, and uncultured bacterium DA101) were enriched in the 13C heavy DNA fraction, confirming their functions in PCBs metabolism. Additionally, a 13.8 kb bph operon was amplified, containing a bphA gene labeled by 13C that was concentrated in the heavy DNA fraction. The tetranucleotide signature characteristics of the bph operon suggest that it originated from Ralstonia. The bph operon may be shared by horizontal gene transfer because it contains a transposon gene and is found in various bacterial species. This study gives us a deeper understanding of PCB-degrading mechanisms and provides a potential resource for the bioremediation of PCBs-contaminated soils.

Identification of biphenyl-metabolising microbes in activated biosludge using cultivation-independent and -dependent approaches

Microbes have important roles in removing organic pollutants in wastewater treatment plants (WWTPs), especially in mineralising recalcitrant persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs). However, the majority of the microorganisms that metabolise these pollutants in situ remain elusive owing to barriers of traditional techniques in unravel yet-to-be-cultivated microbes. In this study, DNA stable-isotope probing (SIP) coupled with high-throughput sequencing was applied to identify the microbes responsible for PCB degradation in the activated biosludge of a WWTP using 13C-labelled biphenyl (BP). Results of time-course SIP revealed different bacteria and archaea involved in BP metabolism, which dominated the BP-degrading community at different time points. BP degradation by the genera Spartobacteria, Alicyclobacillus, Flavobacterium and the order Cenarchaeales has not been reported previously. The abundance of biphenyl dioxygenase (bphA) genes increased over time and a novel bphA gene was identified from the 13C-heavy DNA fraction. In addition, three cultivable BP degraders were isolated, but did not participate in BP degradation in situ or contain the identified bphA genes. Taken together, these data reveal the huge potential and important roles of yet-to-be-cultivated microbes responsible for PCB degradation in activated biosludge, providing fundamental knowledge on WWTP management to remove POPs.

Bacteria Associated to Plants Naturally Selected in a Historical PCB Polluted Soil Show Potential to Sustain Natural Attenuation.

The exploitation of the association between plants and microorganisms is a promising approach able to boost natural attenuation processes for soil clean-up in vast polluted areas characterized by mixed chemical contamination. We aimed to explore the selection of root-associated bacterial communities driven by different plant species spontaneously established in abandoned agricultural soils within a historical polluted site in north Italy. The site is highly contaminated by chlorinated persistent organic pollutants, mainly constituted by polychlorobiphenyls (PCBs), together with heavy metals and metalloids, in variable concentrations and uneven distribution. The overall structure of the non-vegetated and root-associated soil fractions bacterial communities was described by high-throughput sequencing of the 16S rRNA gene, and a collection of 165 rhizobacterial isolates able to use biphenyl as unique carbon source was assayed for plant growth promotion (PGP) traits and bioremediation potential. The results showed that the recruitment of specific bacterial communities in the root-associated soil fractions was driven by both soil fractions and plant species, explaining 21 and 18% of the total bacterial microbiome variation, respectively. PCR-based detection in the soil metagenome of bacterial bphA gene, encoding for the biphenyl dioxygenase α subunit, indicated that the soil in the site possesses metabolic traits linked to PCB degradation. Biphenyl-utilizing bacteria isolated from the rhizosphere of the three different plant species showed low phylogenetic diversity and well represented functional traits, in terms of PGP and bioremediation potential. On average, 72% of the strains harbored the bphA gene and/or displayed catechol 2,3-dioxygenase activity, involved in aromatic ring cleavage. PGP traits, including 1-aminocyclopropane-1-carboxylic acid deaminase activity potentially associated to plant stress tolerance induction, were widely distributed among the isolates according to in vitro assays. PGP tested in vivo on tomato plants using eleven selected bacterial isolates, confirmed the promotion and protection potential of the rhizosphere bacteria. Different spontaneous plant species naturally selected in a historical chronically polluted site showed to determine the enrichment of peculiar bacterial communities in the soil fractions associated to the roots. All the rhizosphere communities, nevertheless, hosted bacteria with degradation/detoxification and PGP potential, putatively sustaining the natural attenuation process.

Spatial Patterns of bphA Gene Diversity Reveal Local Adaptation of Microbial Communities to PCB and PAH Contaminants.

Biphenyl dioxygenases, encoded by the bphA gene, initiate the oxidation of polychlorinated biphenyls (PCBs) and specify the substrate range of PCB congeners metabolized by bacteria. Increased bphA gene diversity within microbial communities may allow a broader range of PCB congeners to be catabolized, thus resulting in greater PCB degradation. To assess the role of PCBs in modulating bphA gene diversity, 16S ribosomal RNA (rRNA) gene and bphA environmental DNA libraries were generated from bacterial communities in sediments with a steep gradient of PCB contamination. Multiple measures of sequence diversity revealed greater heterogeneity of bphA sequences in polluted compared to unpolluted locations. Codon-based signatures of selection in bphA sequences provided evidence of purifying selection. Unifrac analysis of 16S rRNA sequences revealed independent taxonomic lineages from polluted and unpolluted locations, consistent with the presence of locally adapted bacterial communities. Phylogenetic analysis of bphA sequences indicated that dioxygenases from sediments were closely related to previously characterized dioxygenases that metabolize PCBs and polynuclear aromatic hydrocarbons (PAHs), consistent with high levels of these contaminants within the studied sediments. Structural analyses indicated that the BphA protein of Rhodococcus jostii, capable of metabolizing both PCBs and PAHs, provided a more optimal modeling template for bphA sequences reported in this study than a BphA homologue with more restricted substrate specificity. Results from this study suggest that PCBs and PAHs may drive local adaptation of microbial communities by acting as strong selective agents for biphenyl dioxygenases capable of metabolizing a wide range of congeners.

A benzoate-activated promoter from Aspergillus niger and regulation of its activity.

The filamentous fungus Aspergillus niger is able to use benzoic acid as a sole carbon source by conversion to protocatechuic acid and subsequent metabolism. Synthesis of the first enzyme in this metabolic pathway, benzoate p-hydroxylase, is encoded by the bphA gene and positively regulated at the transcriptional level by benzoic acid. Methyl benzoate and para-aminobenzoate also act as inducers of the bphA gene. We show that bphA expression in A. niger in response to benzoate is confined to a 530-bp fragment from the bphA promoter region from -787 to -509bp from the transcriptional start site. Electrophoretic mobility-shift assays show that a benzoate-response element, consisting of a single 6-bp sequence (5'-TAGTCA-3') within a 51-bp sequence in this region, is most likely to be involved in binding of one or more proteins that modulate the activity of the promoter in response to benzoic acid. We show through fusion of promoter fragments with the green fluorescent protein that the active sequences are located within a 200-bp sequence containing the TAGTCA benzoate-response element. Identification of the benzoate-response element in the bphA promoter region constitutes the first step in the development of a benzoate-inducible promoter system that could be used to control gene expression in fungi, and possibly in other organisms, such as plant and animal cells.

Characterization of the metabolic pathway and catabolic gene expression in biphenyl degrading marine bacterium Pseudomonas aeruginosa JP-11.

Metabolic pathway of biphenyl assimilation and the catabolic gene expression in a marine bacterium Pseudomonas aeruginosa JP-11, isolated from the coastal sediments of Odisha, India have been studied. This strain utilized 98.86% ± 2.29% of biphenyl within 72 h when supplied as the sole source of carbon, however, preferential utilization of glucose was observed over catechol and biphenyl when grown in a complex medium. Combination of chromatographic and spectrophotometric techniques confirmed the catechol pathway and identified 2-Hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate as the intermediate metabolic product. Assimilation of biphenyl was initiated by its dioxygenation, forming cis-2, 3-dihydro-2, 3-dihydroxybiphenyl subsequently transformed to 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate. In the lower pathway, cis-1, 6-dihydroxy-2, 4-cyclohexadiene-1-carboxylic acid was detected which formed catechol before entering into the Krebs cycle. Detection of key enzyme catechol-1, 2-dioxygenase in the cell-free extract of P. aeruginosa JP-11 supported the proposed degradation pathway. The primary enzyme for biphenyl assimilation, biphenyl dioxygenase encoded by bphA gene was found in the genome of the isolate. On increasing biphenyl stress (50, 100, 150 and 200 mg L(-1)), bphA gene showed a significant (P < 0.01) up-regulation upto 43.5 folds. Production of biosurfactant was confirmed and the rhamnolipid synthesizing gene rhlAB was amplified. This gene also showed a significant (P < 0.01) up-regulation upto 258 folds on increasing biphenyl stress.

Diversity of key biphenyl destruction genes in the microbial community of the Anadyr Bay coastal sediments

Biphenyl 2,3-dioxygenase is the key enzyme involved in the bacterial destruction of biphenyl and polychlo- rinated biphenyls (PCBs), which are highly stable toxic compounds. The diversity of bphA1 genes encoding the biphenyl 2,3-dioxygenase a subunit of biphenyl-decomposing bacteria from the microbial community of the Bering Sea coastal sediments (the Anadyr port area) was studied. The enrichment culture was obtained by the incubation of bottom sediments samples with biphenyl as the only carbon source. It was followed by total DNA extraction and PCR analysis with degenerate primers specific to the bacterial biphenyl 2,3-dioxygenase a subunit genes. Subsequent cloning of the PCR products led to the identification of three types of aromatic dioxygenase genes, which appeared to be phylogenetically close to the genes of the biphenyl/toluene dioxygenase and 3-phenylpropionate dioxygenase subfamilies of the Actinomycetales bacteria.

Polymorphism of the bphA genes in bacteria destructing biphenyl/chlorinated biphenils

Polymorphism of the bphA genes in bacteria destructing biphenyl/chlorinated biphenils

Optimizing Polychlorinated Biphenyl Degradation by Flavonoid-Induced Cells of the Rhizobacterium Rhodococcus erythropolis U23A.

There is evidence that many plant secondary metabolites may act as signal molecules to trigger the bacterial ability to metabolize polychlorinated biphenyls (PCBs) during the rhizoremediation process. However, the bases for the PCB rhizoremediation process are still largely unknown. The rhizobacterium Rhodococcus erythropolis U23A is unable to use flavanone as a growth substrate. However, on the basis of an assay that monitors the amount of 4-chlorobenzoate produced from 4-chlorobiphenyl by cells grown co-metabolically on flavanone plus sodium acetate, this flavonoid was previously found to be a potential inducer of the U23A biphenyl catabolic pathway. In this work, and using the same assay, we identified ten other flavonoids that did not support growth, but that acted as inducers of the U23A biphenyl pathway, and we confirmed flavonoid induction of the biphenyl catabolic pathway using quantitative real-time polymerase chain reaction (RT-qPCR) on the bphA gene. We also examined the effect of the growth co-substrate on flavonoid induction. Sodium acetate was replaced by glucose, mannose, sucrose, or mannitol, which are sugars found in plant root exudates. The data showed that the level of induction of strain U23A biphenyl-degrading enzymes was significantly influenced by the nature and concentration of the flavonoid in the growth medium, as well as by the substrate used for growth. Sucrose allowed for an optimal induction response for most flavonoids. Some flavonoids, such as flavone and isoflavone, were better inducers of the biphenyl catabolic enzymes than biphenyl itself. We also found that all flavonoids tested in this work were metabolized by strain U23A during co-metabolic growth, but that the metabolite profiles, as well as the level of efficiency of degradation, differed for each flavonoid. To obtain insight into how flavonoids interact with strain U23A to promote polychlorinated biphenyl (PCB) degradation, we determined the concentration of flavanone at which optimal PCB-degrading performance of strain U23A was achieved. We showed that it corresponded to the concentration required to fully induce the biphenyl catabolic pathway of the strain. Together, our data demonstrate that optimal PCB degradation during the rhizoremediation process will require the adjustment of several parameters, including the presence of the appropriate flavonoids at the proper concentrations and the presence of proper growth substrates that positively influence the ability of flavonoids to induce the pathway.

Polymorphism of the bphA genes in bacteria destructing biphenyl/chlorinated biphenils

Polychlorinated biphenyls (PCBs) are persistent organic pollutants. Biphenyl 2,3-dioxygenase (BDO) is a key enzyme that determines the range of PCBs oxidized by a bacterial strain. BDO subunit α (BphA1) plays an essential role in substrate recognition and binding. The genes for dioxygenases that hydroxylate aromatic rings were screened and analyzed phylogenetically. Genes found in biphenyl-oxidizing Rhodococcus erythropolis strains G12a, B7b, and B106a proved to be similar to the published nucleotide sequences of the Rhodococcus sp. HA99 and R04 and Novosphingobium aromaticivorans F199 bphA1 genes, which code for the α-subunits that do not belong to the biphenyl/toluene dioxygenase (B/TDO) family. PCB-destructing R. ruber P25 was found to possess a unique bphA1 gene, which clusters together with the phenylpropionate dioxygenase (PPDO) α-subunits of Mycobacterium vanbaalenii PYR-1 and Frankia sp. EuI1c. The deduced amino acid sequences of the genes were analyzed. The amino acids of the BDO active site in R. wratislaviensis P1, P12, P13, and P20 (bphA1 genes of the B/TDO family) were identical to those of the active PCB degrader R. jostii RHA1. The Rhodococcus strains in question were shown to be active toward both orthoand parachlorinated ring of 2,4'-dichlorobiphenyl. The α-subunit amino acids responsible for the substrate specificity of the enzyme in Pseudomonas sp. S9, S13, S210, S211, and S212 (B/TDO family) were the same as in P. pseudoalcaligenes KF707. The Pseudomonas strains were active toward the para-chlorinated ring of 2,4'-dichlorobiphenyl. The results of screening bacterial strains for bphA1 can be used to identify the biotechnologically promising PCB destructors.

Organochlorinated pesticide degrading microorganisms isolated from contaminated soil

Degradation of selected organochlorinated pesticides (γ-hexachlorocyclohexane - γ-HCH, dichlorodiphenyltrichloroethane - DDT, hexachlorobenzene - HCB) by soil microorganisms was studied. Bacterial strains isolated from contaminated soil from Klatovy-Luby, Hajek and Neratovice, Czech Republic, capable of growth on the selected pesticides were isolated and characterised. These isolates were subjected to characterisation and identification by MS MALDI-TOF of whole cells and sequence analysis of 16S rRNA genes. The isolates were screened by gas chromatography for their ability to degrade the selected pesticides. Some isolates were able to degrade pesticides, and the formation of degradation products (γ-pentachlorocyclohexane (γ-PCCH), dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD)) observed in liquid culture confirmed their degradation capability. The isolates and DNA samples isolated from the contaminated soil were also screened for the bphA1 gene (encoding biphenyl-2,3-dioxygenase, the first enzyme in the PCB degradation pathway) and its occurrence was demonstrated. The isolates were also screened for the presence of linA, encoding dehydrochlorinase, the first enzyme of the HCH degradation pathway. The linA gene could not be found in any of the tested isolates, possibly due to the high specificity of the primers used. The isolates with the most effective degradation abilities could be used for further in situ bioremediation experiments with contaminated soil.