CYP153 Gene Research Articles

The potential of enhanced phytoremediation to clean up multi-contaminated soil – insights from metatranscriptomics

This study aimed to (i) investigate the potential for enhanced phytoremediation to remove contaminants from soil historically co-contaminated with petroleum hydrocarbons (PHs) and heavy metals (HMs) and (ii) analyze the expression of crucial bacterial genes and whole metatranscriptomics profiles for better understanding of soil processes during applied treatment. Phytoremediation was performed using Zea mays and supported by the Pseudomonas qingdaonensis ZCR6 strain and a natural biofertilizer: meat and bone meal (MBM). In previous investigations, mechanisms supporting plant growth and PH degradation were described in the ZCR6 strain. Here, ZCR6 survived in the soil throughout the experiment, but the efficacy of PH removal from all soils fertilized with MBM reached 32 % regardless of the bacterial inoculation. All experimental groups contained 2 % (w/w) MBM. The toxic effect of this amendment on plants was detected 30 days after germination, irrespective of ZCR6 inoculation. Among the 17 genes tested using the qPCR method, only expression of the acdS gene, encoding 1-aminocyclopropane-1-carboxylic acid deaminase, and the CYP153 gene, encoding cytochrome P450-type alkane hydroxylase, was detected in soils. Metatranscriptomic analysis of soils indicated increased expression of methane particulated ammonia monooxygenase subunit A (pmoA-amoA) by Nitrosomonadales bacteria in all soils enriched with MBM compared to the non-fertilized control. We suggest that the addition of 2 % (w/w) MBM caused the toxic effect on plants via the rapid release of ammonia, and this led to high pmoA-amoA expression. In parallel, due to its wide substrate specificity, enhanced bacterial hydrocarbon removal in MBM-treated soils was observed. The metatranscriptomic results indicate that MBM application should be considered to improve bioremediation of soils polluted with PHs rather than phytoremediation. However, lower concentrations of MBM could be considered for phytoremediation enhancement. From a broader perspective, these results indicated the superior capability of metatranscriptomics to investigate the microbial mechanisms driving various bioremediation techniques.

Bioaugmentation with zeolite-immobilized bacterial consortium OPK results in a bacterial community shift and enhances the bioremediation of crude oil-polluted marine sandy soil microcosms

A pyrene-degrading consortium OPK containing Mycolicibacterium strains PO1 and PO2, Novosphingobium pentaromativorans PY1 and Bacillus subtilis FW1 effectively biodegraded medium- and long-chain alkanes as well as mixed hydrocarbons in crude oil. The detection of alkB and CYP153 genes in the genome of OPK members supports its phenotypic ability to effectively degrade a broad range of saturated hydrocarbons in crude oil. Zeolite-immobilized OPK was developed as a ready-to-use bioproduct and it exhibited 74% removal of 1000 mg L−1 crude oil within 96 h in sterilized seawater without nutrient supplementation and maintained high crude oil-removal activity under a broad range of pH values (5.0–9.0), temperatures (30–40 °C) and salinities (20–60‰). In addition, the immobilized OPK retained a high crude oil removal efficacy in semicontinuous experiments and showed reusability for at least 5 cycles. Remarkably, bioaugmentation with zeolite-immobilized OPK in sandy soil microcosms significantly increased crude oil (10,000 mg kg−1 soil) removal from 45% to 80.67% within 21 days compared to biostimulation and natural attenuation. Moreover, bioaugmentation with exogenous immobilized OPK stimulated an increase in the relative abundances of Alcanivorax genus, indigenous hydrocarbon-degrading bacteria, which in turn enhanced removal efficiency of crude oil contamination from sandy soil microcosms. The results indicate positive interactions between the bioaugmented immobilized consortium, harboring Mycolicibacterium as a key player, and indigenous Alcanivorax, which exhibited crucial functions for improving crude oil removal efficacy. The knowledge obtained forms an important basis for further synthesis and handling of a promising bio-based product for enhancing the in situ bioremediation of crude oil-polluted marine environments.

Hydrocarbon-degrading genes in root endophytic communities on oil sands reclamation covers

In response to environmental regulations, the Canadian oil sands industry aims to reclaim all disturbed areas to equivalent land capability prior to mining operations. However, tailing sands used in reclamation contain residual hydrocarbons and plants growing in these areas may rely on hydrocarbon-degrading endophytic bacteria to survive. This study assessed the hydrocarbon-degrading potential (genes: CYP153, alkB and nah) of culturable and unculturable endophytic bacteria associated with annual barley (Hordeum vulgare) and sweet clover (Melilotus albus) plants in an oil sands reclamation area. Our results suggest higher CYP153 gene copy numbers in sweet clover when compared to barley. Yet, no significant differences were detected in 16S rRNA, alkB and nah genes. In addition, total hydrocarbons, pH, total soil carbon, organic carbon and total nitrogen play an important role in determining hydrocarbon-degrading potential in these communities. The assessment of culturable hydrocarbon-degrading bacteria revealed 42 isolates (total of 316) that were positive for at least one hydrocarbon-degrading gene. Most of these isolates were positive for alkB, and closely match the database for Pantoea, Pseudomonas and Enterobacter spp. Thus, to improve oil sands reclamation strategies, plant inoculation with select hydrocarbon-degrading endophytes could be used to increase plant tolerance and hydrocarbon degradation in these areas.

Cyclohexane, naphthalene, and diesel fuel increase oxidative stress, CYP153, sodA, and recA gene expression in Rhodococcus erythropolis.

In this study, we compared the expression of CYP153, sodA, sodC, and recA genes and ROS generation in hydrocarbon‐degrading Rhodococcus erythropolis in the presence of cyclohexane, naphthalene, and diesel fuel. The expression of cytochrome P450, sodA (encoding Fe/Mn superoxide dismutase), recA, and superoxide anion radical generation rate increased after the addition of all studied hydrocarbons. The peak of CYP153, sodA, and recA gene expression was registered in the presence of naphthalene. The same substrate upregulated the Cu/Zn superoxide dismutase gene, sodC. Cyclohexane generated the highest level of superoxide anion radical production. Hydrogen peroxide accumulated in the medium enriched with diesel fuel. Taken together, hydrocarbon biotransformation leads to oxidative stress and upregulation of antioxidant enzymes and CYP153 genes, and increases DNA reparation levels in R. erythropolis cells.

CoxR Directly Represses Alkane Hydroxylase Gene Expression in Caulobacter crescentus

Alkanes are commonly found in nature and many gram positive and gram negative bacteria have developed enzymes for degrading them. The alphaproteobacteria Caulobacter crescentus has the CYP153 gene which codes for a medium‐chain alkane hydroxylase in the cytochrome P450 family. However, the regulatory mechanism controlling this gene has not been well‐characterized. A reverse genetics screen found that a deletion in the TetR family coxR gene results in a strain that is sensitive to oxidative stress. We used RNA‐seq and EMSA with Ni‐NTA purified CoxR to determine which genes CoxR directly regulates. Expression of the CYP153, ferredoxin and ferredoxin reductase genes were shown to be upregulated in the coxR deletion strain. Furthermore, CoxR was shown to directly bind to a 35bp region of the CYP153 promoter. In summary, we have shown that the transcription factor CoxR directly represses CYP153 gene expression.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effect of bioaugmentation of petroleum-contaminated soil with Rhodococcus erythropolis strains on removal of petroleum from soil

The aim of the study was to assess the impact of inoculation of petroleum-contaminated soil with the hydrocarbon-degrading bacterial strains Rhodococcus erythropolis CD 130 and CD 167 or their consortium on the removal of hydrocarbons from the soil. Additionally, changes in the activity and structure of soil autochthonous bacterial communities were studied. At the end of the experiment, the fastest hydrocarbon removal was seen in the soil treated with the CD 167 strain (38.40%) and was statistically higher compared to the removal of total petroleum hydrocarbons (TPH) observed in soils inoculated with strain CD 130 (29.8%) or bacterial consortium CD 130 + CD 167 (29.72%). The rifampicin-resistant CD 130 and CD 167 strains, introduced as single strains or a consortium, survived in the soil for 42 days. The introduction of gram-positive strains of R. erythropolis primarily caused an increase in the biomass of branched phospholipid fatty acids (PLFAs), characteristic for gram-positive bacteria. Nevertheless, changes in the concentrations of gram-positive and gram-negative PLFA markers were periodic, and at the end of the experiment, significant changes were observed only in the case of the soil bioaugmented with the CD 167 strain. After the bioaugmentation, higher values of substrate-induced respiration (SIR) were observed in all the inoculated soils compared to the non-inoculated control. Nonetheless, after 91 days of incubation, a significant decrease in soil respiration was observed in the soil treated with single CD 130 or CD 167 strains or with their consortium. The number of transcripts of the CYP153 gene obtained on days 91 and 182 reflected the results of the hydrocarbon loss. The level of expression of the alkH gene in experimental soil was estimated and found to be higher than the level of expression of the CYP153 gene but did not coincide with the loss of hydrocarbons. The introduction of strains CD 130, CD 167, or CD 130 + CD 167 caused temporary changes in the composition of the soil autochthonous bacterial community, but it seems that these changes were needed for the enhanced removal of hydrocarbons from this soil.

High efficiency degradation of alkanes and crude oil by a salt-tolerant bacterium Dietzia species CN-3

A bacterial strain, CN-3, was isolated from petroleum-contaminated sediment in Bohai Bay, China. Morphological, physiological and phylogenetic analyses indicated that the strain belonged to the genus Dietzia. It was capable of utilizing a wide range of alkanes (C14–C31), aromatic compounds and crude oil as the sole carbon and energy sources. The bacterium showed high degradation rate (>80%) of the saturated hydrocarbons of crude oil in the pH range of 6–9. The degradation rate was hardly affected at a NaCl concentration up to 85 g L−1, indicating that the strain can tolerate high salinities. The real-time quantitative Polymerase Chain Reaction (PCR) results showed that the CYP153 genes could be significantly induced by C14, C15, C16, C26 and pristane, whereas the transcription level of alkB gene was increased moderately with C14, C15, C16, C26 and pristane induction condition. A high degradation extent, wide substrate range and facile conditions enhanced the potential for this bacterial strain to be used in the bioremediation of crude oil pollution.

Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b.

CYP153, one of the most common medium-chain n-alkane hydroxylases belonging to the cytochrome P450 superfamily, is widely expressed in n-alkane-degrading bacteria. CYP153 is also thought to cooperate with AlkB in degrading various n-alkanes. However, the mechanisms regulating the expression of the protein remain largely unknown. In this paper, we studied CYP153 gene transcription regulation by the potential AraC family regulator (CypR) located upstream of the CYP153 gene cluster in a broad-spectrum n-alkane-degrading Gram-positive bacterium, Dietzia sp. strain DQ12-45-1b. We first identified the transcriptional start site and the promoter of the CYP153 gene cluster. Sequence alignment of upstream regions of CYP153 gene clusters revealed high conservation in the -10 and -35 regions in Actinobacteria. Further analysis of the β-galactosidase activity in the CYP153 gene promoter-lacZ fusion cell indicated that the CYP153 gene promoter was induced by n-alkanes comprised of 8 to 14 carbon atoms, but not by derived decanol and decanic acid. Moreover, we constructed a cypR mutant strain and found that the CYP153 gene promoter activities and CYP153 gene transcriptional levels in the mutant strain were depressed compared with those in the wild-type strain in the presence of n-alkanes, suggesting that CypR served as an activator for the CYP153 gene promoter. By comparing CYP153 gene arrangements in Actinobacteria and Proteobacteria, we found that the AraC family regulator is ubiquitously located upstream of the CYP153 gene, suggesting its universal regulatory role in CYP153 gene transcription. We further hypothesize that the observed mode of CYP153 gene regulation is shared by many Actinobacteria.

Diversity of alkane hydroxylase genes on the rhizoplane of grasses planted in petroleum-contaminated soils.

The study investigated the diversity and genotypic features of alkane hydroxylase genes on rhizoplanes of grasses planted in artificial petroleum-contaminated soils to acquire new insights into the bacterial communities responsible for petroleum degradation in phytoremediation. Four types of grass (Cynodon dactylon, two phenotypes of Zoysia japonica, and Z. matrella) were used. The concentrations of total petroleum hydrocarbon effectively decreased in the grass-planted systems compared with the unplanted system. Among the representative alkane hydroxylase genes alkB, CYP153, almA and ladA, the first two were detected in this study, and the genotypes of both genes were apparently different among the systems studied. Their diversity was also higher on the rhizoplanes of the grasses than in unplanted oil-contaminated soils. Actinobacteria-related genes in particular were among the most diverse alkane hydroxylase genes on the rhizoplane in this study, indicating that they are one of the main contributors to degrading alkanes in oil-contaminated soils during phytoremediation. Actinobacteria-related alkB genes and CYP153 genes close to the genera Parvibaculum and Aeromicrobium were found in significant numbers on the rhizoplanes of grasses. These results suggest that the increase in diversity and genotype differences of the alkB and CYP153 genes are important factors affecting petroleum hydrocarbon-degrading ability during phytoremediation.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1312-0) contains supplementary material, which is available to authorized users.

Characteristics of hydrocarbon hydroxylase genes in a thermophilic aerobic biological system treating oily produced wastewater.

Alkane and aromatic hydroxylase genes in a full-scale aerobic system treating oily produced wastewater under thermophilic condition (45-50 °C) in the Jidong oilfield, China, were investigated using clone library and quantitative polymerase chain reaction methods. Rather than the normally encountered integral-membrane non-haem iron monooxygenase (alkB) genes, only CYP153-type P450 hydroxylase genes were detected for the alkane activation, indicating that the terminal oxidation of alkanes might be mainly mediated by the CYP153-type alkane hydroxylases in the thermophilic aerobic process. Most of the obtained CYP153 gene clones showed distant homology with the reference sequences, which might represent novel alkane hydroxylases. For the aromatic activation, the polycyclic aromatic hydrocarbon-ring hydroxylating dioxygenase (PAH-RHD) gene was derived from Gram-negative PAH-degraders belonging to the Burkholderiales order, with a 0.72% relative abundance of PAH-RHD gene to 16S rRNA gene. This was consistent with the result of 16S rRNA gene analysis, indicating that Burkholderiales bacteria might play a key role in the full-scale process of thermophilic hydrocarbon degradation.

Nutrients can enhance the abundance and expression of alkane hydroxylase CYP153 gene in the rhizosphere of ryegrass planted in hydrocarbon-polluted soil.

Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K) in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum) was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination.

Diverse alkane hydroxylase genes in microorganisms and environments.

AlkB and CYP153 are important alkane hydroxylases responsible for aerobic alkane degradation in bioremediation of oil-polluted environments and microbial enhanced oil recovery. Since their distribution in nature is not clear, we made the investigation among thus-far sequenced 3,979 microbial genomes and 137 metagenomes from terrestrial, freshwater, and marine environments. Hundreds of diverse alkB and CYP153 genes including many novel ones were found in bacterial genomes, whereas none were found in archaeal genomes. Moreover, these genes were detected with different distributional patterns in the terrestrial, freshwater, and marine metagenomes. Hints for horizontal gene transfer, gene duplication, and gene fusion were found, which together are likely responsible for diversifying the alkB and CYP153 genes adapt to the ubiquitous distribution of different alkanes in nature. In addition, different distributions of these genes between bacterial genomes and metagenomes suggested the potentially important roles of unknown or less common alkane degraders in nature.

Characterization of a CYP153 alkane hydroxylase gene in a Gram-positive Dietzia sp. DQ12-45-1b and its “team role” with alkW1 in alkane degradation

CYP153 and AlkB-like hydroxylases were recently discovered in Gram-positive alkane-degrading bacteria. However, it is unclear whether they cooperate with each other in alkane degradation as they do in Gram-negative bacteria. In this paper, we cloned the CYP153 gene from a representative Gram-positive alkane-degrading bacterium, Dietzia sp. DQ12-45-1b. The CYP153 gene transcription in Dietzia sp. DQ12-45-1b and heterologous expression in alkB gene knockout mutant strain Pseudomonas fluorescens KOB2∆1 both confirmed the functions of CYP153 on C6-C10 n-alkanes degradation, but not on longer chain-length n-alkanes. In addition, substrate-binding analysis of the purified CYP153 protein revealed different substrate affinities to C6-C16 n-alkanes, confirming n-alkanes binding to CYP153 protein. Along with AlkW1, an AlkB-like alkane hydroxylase in Dietzia sp. DQ12-45-1b, a teamwork pattern was found in n-alkane degradation, i.e. CYP153 was responsible for hydroxylating n-alkanes shorter than C10 while AlkW1 was responsible for those longer than C14. Further sequence analysis suggested that the high horizontal gene transfer (HGT) potential of CYP153 genes may be universal in Gram-positive alkane-degrading actinomycetes that contain both alkB and CYP153 genes.

Alkane-degrading properties of Dietzia sp. H0B, a key player in the Prestige oil spill biodegradation (NW Spain)

Investigation of the alkane-degrading properties of Dietzia sp. H0B, one of the isolated Corynebacterineae strains that became dominant after the Prestige oil spill. Using molecular and chemical analyses, the alkane-degrading properties of strain Dietzia sp. H0B were analysed. This Grampositive isolate was able to grow on n-alkanes ranging from C₁₂ to C₃₈ and branched alkanes (pristane and phytane). 8-Hexadecene was detected as an intermediate of hexadecane degradation by Dietzia H0B, suggesting a novel alkane-degrading pathway in this strain. Three putative alkane hydroxylase genes (one alkB homologue and two CYP153 gene homologues of cytochrome P450 family) were PCR-amplified from Dietzia H0B and differed from previously known hydroxylase genes, which might be related to the novel degrading activity observed on Dietzia H0B. The alkane degradation activity and the alkB and CYP153 gene expression were observed constitutively regardless of the presence of the substrate, suggesting additional, novel pathways for alkane degradation. The results from this study suggest novel alkane-degrading pathways in Dietzia H0B and a genetic background coding for two different putative oil-degrading enzymes, which is mostly unexplored and worth to be subject of further functional analysis. This study increases the scarce information available about the genetic background of alkane degradation in genus Dietzia and suggests new pathways and novel expression mechanisms of alkane degradation.

Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants.

Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.

Molecular Characterization of the 56-kDa CYP153 from Acinetobacter sp. EB104

CYP153 a cytochrome P450 from Acinetobacter sp. EB104 catalyzes the hydroxylation of unsubstituted n-alkanes. We have decided to use the CYP153 system as a model for mechanistic studies on regioselective n-alkane oxidation and the interaction of hydrophobic substrates with soluble enzymes. Here the molecular cloning of the CYP153 gene is reported. Single specific primer PCR was applied to yield the whole gene sequence via chromosomal walks. CYP153 consists of 497 amino acids (Mr = 56 kDa) and thus represents an unusually long bacterial P450, containing all P450 typical structural elements. It constitutes the new P450 family CYP153. The prolonged N-terminus of about 90 amino acids does not contain a so far known membrane-anchoring sequence but a 28-amino acid long amphipathic helix. The relevance of the remarkably long N-terminus and of other sequence motives like the hydrophobic F-G loop is discussed with respect to substrate binding and recognition.