Phosphorus application enhances alkane hydroxylase gene abundance in the rhizosphere of wild plants grown in petroleum-hydrocarbon-contaminated soil.

This study assessed the ability of phosphorus (P) fertilizer to remediate the rhizosphere of three wild plant species (Banksia seminuda, a tree; Chloris truncata, a grass; and Hakea prostrata, a shrub) growing in a soil contaminated with total (aliphatic) petroleum hydrocarbon (TPH). Plant growth, photosynthesis (via chlorophyll fluorescence), soil microbial activity, alkane hydroxylase AlkB (aliphatic hydrocarbon-degrading) gene abundance, and TPH removal were evaluated 120 days after planting. Overall, although TPH served as an additional carbon source for soil microorganisms, the presence of TPH in soil resulted in decreased plant growth and photosynthesis. However, growth, photosynthesis, microbial activities, and AlkB gene abundance were enhanced by the application of P fertilizer, thereby increasing TPH removal rates, although the extent and optimum P dosage varied among the plant species. The highest TPH removal (64.66%) was observed in soil planted with the Poaceae species, C. truncata, and amended with 100 mg P kg-1 soil, while H. prostrata showed higher TPH removal compared to the plant belonging to the same Proteaceae family, B. seminuda. The presence of plants resulted in higher AlkB gene abundance and TPH removal relative to the unplanted control. The removal of TPH was associated directly with AlkB gene abundance (R2 > 0.9, p < 0.001), which was affected by plant identity and P levels. The results indicated that an integrated approach involving wild plant species and optimum P amendment, which was determined through experimentation using different plant species, was an efficient way to remediate soil contaminated with TPH.

A novel, divergent alkane monooxygenase (alkB) clade involved in crude oil biodegradation.

Alkanes are ubiquitous in marine ecosystems and originate from diverse sources ranging from natural oil seeps to anthropogenic inputs and biogenic production by cyanobacteria. Enzymes that degrade cyanobacterial alkanes (typically C15-C17 compounds) such as the alkane monooxygenase (AlkB) are widespread, but it remains unclear whether or not AlkB variants exist that specialize in degradation of crude oil from natural or accidental spills, a much more complex mixture of long-chain hydrocarbons. In the present study, large-scale analysis of available metagenomic and genomic data from the Gulf of Mexico (GoM) oil spill revealed a novel, divergent AlkB clade recovered from genomes with no cultured representatives that was dramatically increased in abundance in crude-oil impacted ecosystems. In contrast, the AlkB clades associated with biotransformation of cyanobacterial alkanes belonged to 'canonical' or hydrocarbonoclastic clades, and based on metatranscriptomics data and compared to the novel clade, were much more weakly expressed during crude oil biodegradation in laboratory mesocosms. The absence of this divergent AlkB clade in metagenomes of uncontaminated samples from the global ocean survey but not from the GoM as well as its frequent horizontal gene transfer indicated a priming effect of the Gulf for crude oil biodegradation likely driven by natural oil seeps.

Biodegradation of binary mixtures of octane with benzene, toluene, ethylbenzene or xylene (BTEX): insights on the potential of Burkholderia, Pseudomonas and Cupriavidus isolates.

The contamination of the environment by crude oil and its by-products, mainly composed of aliphatic and aromatic hydrocarbons, is a widespread problem. Biodegradation by bacteria is one of the processes responsible for the removal of these pollutants. This study was conducted to determine the abilities of Burkholderia sp. B5, Cupriavidus sp. B1, Pseudomonas sp. T1, and another Cupriavidus sp. X5 to degrade binary mixtures of octane (representing aliphatic hydrocarbons) with benzene, toluene, ethylbenzene, or xylene (BTEX as aromatic hydrocarbons) at a final concentration of 100 ppm under aerobic conditions. These strains were isolated from an enriched bacterial consortium (Yabase or Y consortium) that prefer to degrade aromatic hydrocarbon over aliphatic hydrocarbons. We found that B5 degraded all BTEX compounds more rapidly than octane. In contrast, B1, T1 and X5 utilized more of octane over BTX compounds. B5 also preferred to use benzene over octane with varying concentrations of up to 200 mg/l. B5 possesses alkane hydroxylase (alkB) and catechol 2,3-dioxygenase (C23D) genes, which are responsible for the degradation of alkanes and aromatic hydrocarbons, respectively. This study strongly supports our notion that Burkholderia played a key role in the preferential degradation of aromatic hydrocarbons over aliphatic hydrocarbons in the previously characterized Y consortium. The preferential degradation of more toxic aromatic hydrocarbons over aliphatics is crucial in risk-based bioremediation.

Phylogeny and diversity of alkane-degrading enzyme gene variants in the laurentian great lakes and western atlantic.

Many aquatic environments are at risk for oil contamination and alkanes are one of the primary constituents of oil. The alkane hydroxylase (AlkB) is a common enzyme used by microorganisms to initiate the process of alkane-degradation. While many aspects of alkane bioremediation have been studied, the diversity and evolution of genes involved in hydrocarbon degradation from environmental settings is relatively understudied. The majority of work done to-date has focused on the marine environment. Here we sought to better understand the phylogenetic diversity of alkB genes across marine and freshwater settings using culture-independent methods. We hypothesized that there would be distinct phylogenetic diversity of alkB genes in freshwater relative to the marine environment. Our results confirm that alkB has distinct variants based on environment while our diversity analyses demonstrate that freshwater and marine alkB communities have unique responses to oil amendments. Our results also demonstrate that in the marine environment, depth is a key factor impacting diversity of alkB genes.

Elucidation of multiple alkane hydroxylase systems in biodegradation of crude oil n-alkane pollution by Pseudomonas aeruginosa DN1.

AIMS: The purpose of this study was to elucidate the characteristics of multiple alkane hydroxylase systems in Pseudomonas aeruginosa DN1, including two homologues of AlkB (AlkB1 and AlkB2 ), a CYP153 homologue (P450), and two homologues of Alm-like (AlmA1 and AlmA2 ). METHODS AND RESULTS: DN1 was capable of utilizing diverse n-alkanes with chain lengths from 8 to 40 C atoms as the sole carbon source, and displayed high degradation efficiency (＞85%) of crude oil and a majority of n-alkanes using gas chromatography method. RT-qPCR analysis showed that the five enzyme genes could be induced by n-alkanes ranging from medium-chain length to long-chain length which indicated the dissimilarity of expression between those genes when grown on different n-alkanes. Notably, the expression of alkB2 gene was upregulated in the presence of all of the tested n-alkanes, particularly responded to long-chain n-alkanes like C20 and C32 . Meanwhile, long-chain n-alkanes (C20 -C36 ) significantly elevated cyp153 expression level, and the expression of two almA genes was only upregulated in the presence of n-alkanes with chain lengths of 20C's and longer. Furthermore, the disruption of those genes demonstrated that AlkB2 appeared to play a key role in the biodegradation of substrates of a broad-chain length ranges, besides other alkane hydroxylase systems ensured the utilization of n-alkanes with chain lengths of from 20 to 40 C atoms. CONCLUSION: The five functional alkane hydroxylase genes make DN1 an attractive option for its versatile alkane degradation, which is primarily dependent on the expression of alkB2 . SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggest that P. aeruginosa DN1 is a predominately potential long-chain n-alkane-degrading bacterium with multiple alkane hydroxylase systems in crude oil-contaminated environment.

Low-Abundance Dietzia Inhabiting a Water-Flooding Oil Reservoir and the Application Potential for Oil Recovery.

With the development of molecular ecology, increasing low-abundance microbial populations were detected in oil reservoirs. However, our knowledge about the oil recovery potential of these populations is lacking. In this study, the oil recovery potential of low-abundance Dietzia that accounts for less than 0.5% in microbial communities of a water-flooding oil reservoir was investigated. On the one hand, Dietzia sp. strain ZQ-4 was isolated from the water-flooding reservoir, and the oil recovery potential was evaluated from the perspective of metabolisms and oil-displacing test. On the other hand, the strain has alkane hydroxylase genes alkB and P450 CYP153 and can degrade hydrocarbons and produce surfactants. The core-flooding test indicated that displacing fluid with 2% ZQ-4 fermentation broth increased 18.82% oil displacement efficiency, and in situ fermentation of ZQ-4 increased 1.97% oil displacement efficiency. Furthermore, the responses of Dietzia in the reservoir accompanied by the nutrient stimulation process was investigated and showed that Dietzia in some oil production wells significantly increased in the initial phase of nutrient injection and sharply decreased along with the continuous nutrient injection. Overall, this study indicates that Dietzia sp. strain has application potential for enhancing oil recovery through an ex situ way, yet the ability of oil recovery in situ based on nutrient injection is limited.

Characteristics of crude oil-degrading bacteria Gordonia iterans isolated from marine coastal in Taean sediment.

Crude oil is a major pollutant of marine and coastal ecosystems, and it causes environmental problems more seriously. It is believed ultimate and complete degradation is accomplished mainly by microorganisms. In this study, we aim to search out for bacterial strains with high ability in degrading crude oil. From sediments contaminated by the petroleum spilled in 2007, an accident in Taean, South Korea, we isolated thirty-one bacterial strains in total with potential application in crude oil contamination remediation. In terms of removal percentage after 7 days, one of the strains, Co17, showed the highest removal efficiency with 84.2% of crude oil in Bushnell-Haas media. The Co17 strain even exhibited outstanding ability removing crude oil at a high salt concentration. Through the whole genome sequencing annotation results, many genes related with n-alkane degradation in the genome of Gordonia sp. Co17, revealed alkane-1-monooxygenase, alcohol dehydrogenase, and Baeyer-Villiger monooxygenase. Specially, for confirmation of gene-level, alkB gene encoding alkane hydroxylase (alkane-1-monooxygenase) was found in the strain Co17. The expression of alkB upregulated 125-fold after 18 hr accompany with the removal of n-alkanes of 48.9%. We therefore propose the strain Gordonia iterans Co17, isolated from crude oil-contaminated marine sediment, could be used to offer a new strategy for bioremediation with high efficiency.

An assessment of the microbial community in an urban fringing tidal marsh with an emphasis on petroleum hydrocarbon degradative genes.

Small fringing marshes are ecologically important habitats often impacted by petroleum. We characterized the phylogenetic structure (16S rRNA) and petroleum hydrocarbon degrading alkane hydroxylase genes (alkB and CYP 153A1) in a sediment microbial community from a New Hampshire fringing marsh, using alkane-exposed dilution cultures to enrich for petroleum degrading bacteria. 16S rRNA and alkB analysis demonstrated that the initial sediment community was dominated by Betaproteobacteria (mainly Comamonadaceae) and Gammaproteobacteria (mainly Pseudomonas), while CYP 153A1 sequences predominantly matched Rhizobiales. 24 h of exposure to n-hexane, gasoline, dodecane, or dilution culture alone reduced functional and phylogenetic diversity, enriching for Gammaproteobacteria, especially Pseudomonas. Gammaproteobacteria continued to dominate for 10 days in the n-hexane and no alkane exposed samples, while dodecane and gasoline exposure selected for gram-positive bacteria. The data demonstrate that small fringing marshes in New England harbor petroleum-degrading bacteria, suggesting that petroleum degradation may be an important fringing marsh ecosystem function.

Diversity of crude oil-degrading bacteria and alkane hydroxylase (alkB) genes from the Qinghai-Tibet Plateau.

The aim of this study was to survey the response of the microbial community to crude oil and the diversity of alkane hydroxylase (alkB) genes in soil samples from the Qinghai-Tibet Plateau (QTP). The enrichment cultures and clone libraries were used. Finally, 53 isolates and 94 alkB sequences were obtained from 10 pristine soil samples after enrichment at 10 °C with crude oil as sole carbon source. The isolates fell into the phyla Proteobacteria, Actinobacteria, and Bacteroidetes, with the dominance of Pseudomonas and Acinetobacter. The composition of degraders was different from polar habitats where Acinetobacter sp. is not a predominant responder of alkane degradative microbial communities. Phylogenetic analysis showed that the alkB genes from isolates and enrichment communities formed eight clusters and mainly related with alkB genes of Pseudomonas, Rhodococcus, and Acinetobacter. The alkB gene diversity in the QTP was lower than marine environments and polar soil samples. In particular, a total of 10 isolates exhibiting vigorous growth with crude oil could detect no crude oil degradation-related gene sequences, such as alkB, P450, almA, ndoB, and xylE genes. The Shannon-Wiener index of the alkB clone libraries from the QTP ranged from 1.00 to 2.24 which is similar with polar pristine soil samples but lower than that of contaminated soils. These results indicated that the Pseudomonas, Acinetobacter, and Rhodococcus genera are the candidate for in situ bioremediation, and the environment of QTP may be still relatively uncontaminated by crude oil.

Fatty acid ω-hydroxylases from Solanum tuberosum.

KEY MESSAGE: Potato StCYP86A33 complements the Arabidopsis AtCYP86A1 mutant, horst - 1. Suberin is a cell-wall polymer that comprises both phenolic and aliphatic components found in specialized plant cells. Aliphatic suberin is characterized by bi-functional fatty acids, typically ω-hydroxy fatty acids and α,ω-dioic acids, which are linked via glycerol to form a three-dimensional polymer network. In potato (Solanum tuberosum L.), over 65 % of aliphatics are either ω-hydroxy fatty acids or α,ω-dioic acids. Since the biosynthesis of α,ω-dioic acids proceeds sequentially through ω-hydroxy fatty acids, the formation of ω-hydroxy fatty acids represents a significant metabolic commitment during suberin deposition. Four different plant cytochrome P450 subfamilies catalyze ω-hydroxylation, namely, 86A, 86B, 94A, and 704B; though to date, only a few members have been functionally characterized. In potato, CYP86A33 has been identified and implicated in suberin biosynthesis through reverse genetics (RNAi); however, attempts to express the CYP86A33 protein and characterize its catalytic function have been unsuccessful. Herein, we describe eight fatty acid ω-hydroxylase genes (three CYP86As, one CYP86B, three CYP94As, and a CYP704B) from potato and demonstrate their tissue expression. We also complement the Arabidopsis cyp86A1 mutant horst-1 using StCYP86A33 under the control of the Arabidopsis AtCYP86A1 promoter. Furthermore, we provide preliminary analysis of the StCYP86A33 promoter using a hairy root transformation system to monitor pStCYP86A33::GUS expression constructs. These data confirm the functional role of StCYP86A33 as a fatty acid ω-hydroxylase, and demonstrate the utility of hairy roots in the study of root-specific genes.

In situ detection of alkB2 gene involved in Alcanivorax borkumensis SK2(T) hydrocarbon biodegradation.

This study aimed to develop a new assay based on the whole cell hybridization in order to monitor alkane hydroxylase genes (alkB system) of the marine bacterium Alcanivorax borkumensis SK2(T) commonly reported as the predominant microorganism responsible for the biodegradation of n-alkanes which are the major fraction of petroleum hydrocarbons. The assay based on the whole cell hybridization targeting alkB2 gene was successfully developed and calibrated on a pure culture of Alcanivorax borkumensis SK2(T) with a detection efficiency up to 80%. The approach was further successfully validated on hydrocarbon-contaminated seawater and provided cells abundance (6.74E+04alkB2-carryingcellsmL(-1)) higher of about one order of magnitude than those obtained by qPCR (4.96E+03alkB2genecopiesmL(-1)). This study highlights the validity of the assay for the detection at single cell level of key-functional genes involved in the biodegradation of n-alkanes.

Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains.

Bioremediation potential of microorganisms derived from petroleum reservoirs.

Bacterial strains and metagenomic clones, both obtained from petroleum reservoirs, were evaluated for petroleum degradation abilities either individually or in pools using seawater microcosms for 21 days. Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) analyses were carried out to evaluate crude oil degradation. The results showed that metagenomic clones 1A and 2B were able to biodegrade n-alkanes (C14 to C33) and isoprenoids (phytane and pristane), with rates ranging from 31% to 47%, respectively. The bacteria Dietzia maris CBMAI 705 and Micrococcus sp. CBMAI 636 showed higher rates reaching 99% after 21 days. The metagenomic clone pool biodegraded these compounds at rates ranging from 11% to 45%. Regarding aromatic compound biodegradation, metagenomic clones 2B and 10A were able to biodegrade up to 94% of phenanthrene and methylphenanthrenes (3-MP, 2-MP, 9-MP and 1-MP) with rates ranging from 55% to 70% after 21 days, while the bacteria Dietzia maris CBMAI 705 and Micrococcus sp. CBMAI 636 were able to biodegrade 63% and up to 99% of phenanthrene, respectively, and methylphenanthrenes (3-MP, 2-MP, 9-MP and 1-MP) with rates ranging from 23% to 99% after 21 days. In this work, isolated strains as well as metagenomic clones were capable of degrading several petroleum compounds, revealing an innovative strategy and a great potential for further biotechnological and bioremediation applications.

Isolation and characterization of alkane degrading bacteria from petroleum reservoir waste water in Iran (Kerman and Tehran provenances).

Petroleum products spill and leakage have become two major environmental challenges in Iran. Sampling was performed in the petroleum reservoir waste water of Tehran and Kerman Provinces of Iran. Alkane degrading bacteria were isolated by enrichment in a Bushnel-Hass medium, with hexadecane as sole source of carbon and energy. The isolated strains were identified by amplification of 16S rDNA gene and sequencing. Specific primers were used for identification of alkane hydroxylase gene. Fifteen alkane degrading bacteria were isolated and 8 strains were selected as powerful degradative bacteria. These 8 strains relate to Rhodococcus jostii, Stenotrophomonas maltophilia, Achromobacter piechaudii, Tsukamurella tyrosinosolvens, Pseudomonas fluorescens, Rhodococcus erythropolis, Stenotrophomonas maltophilia, Pseudomonas aeruginosa genera. The optimum concentration of hexadecane that allowed high growth was 2.5%. Gas chromatography results show that all strains can degrade approximately half of hexadecane in one week of incubation. All of the strains have alkane hydroxylase gene which are important for biodegradation. As a result, this study indicates that there is a high diversity of degradative bacteria in petroleum reservoir waste water in Iran.

Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil, shale oil or diesel fuel.

The coastal waters of the Baltic Sea are constantly threatened by oil spills, due to the extensive transportation of oil products across the sea. To characterise the hydrocarbon-degrading bacterial community of this marine area, microcosm experiments on diesel fuel, crude oil and shale oil were performed. Analysis of these microcosms, using alkane monooxygenase (alkB) and 16S rRNA marker genes in PCR-DGGE experiments, demonstrated that substrate type and concentration strongly influence species composition and the occurrence of alkB genes in respective oil degrading bacterial communities. Gammaproteobacteria (particularly the genus Pseudomonas) and Alphaproteobacteria were dominant in all microcosms treated with oils. All alkB genes carried by bacterial isolates (40 strains), and 8 of the 11 major DGGE bands from the microcosms, had more than 95% sequence identity with the alkB genes of Pseudomonas fluorescens. However, the closest relatives of the majority of sequences (54 sequences from 79) of the alkB gene library from initially collected seawater DNA were Actinobacteria. alkB gene expression, induced by hexadecane, was recorded in isolated bacterial strains. Thus, complementary culture dependent and independent methods provided a more accurate picture about the complex seawater microbial communities of the Baltic Sea.

Chlorella pyrenoidosa ameliorated L-NAME-induced hypertension and cardiorenal remodeling in rats.

PURPOSE: Hypertension is one of the main factors causing cardiovascular diseases. The aim of the study is to investigate the effects of Chlorella pyrenoidosa on blood pressure and cardiorenal remodeling in rats with N (ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced endothelial dysfunction. METHODS: Rats were fed a diet containing L-NAME (40 mg/kg) with or without chlorella (4 or 8 %) for 5 weeks. We found that chlorella retarded the development of hypertension and cardiorenal remodeling during the 5-week experimental period. RESULTS: Although there was no difference in NO( x ) levels or plasma arginine concentrations, plasma and tissues ACE activities were significantly lower in the chlorella groups than in the L-NAME group. Moreover, tissue tumor necrosis factor-α concentrations and renal CYP4A expression were also lower in the chlorella group. CONCLUSION: These results suggest that chlorella might ameliorate the elevation of blood pressure and show cardiorenal-protective effects in nitric oxide-deficient rats, and one possible mechanism might be mediated by its ACE inhibitory activity.

Alkane biodegradation genes from chronically polluted subantarctic coastal sediments and their shifts in response to oil exposure.

Although sediments are the natural hydrocarbon sink in the marine environment, the ecology of hydrocarbon-degrading bacteria in sediments is poorly understood, especially in cold regions. We studied the diversity of alkane-degrading bacterial populations and their response to oil exposure in sediments of a chronically polluted Subantarctic coastal environment, by analyzing alkane monooxygenase (alkB) gene libraries. Sequences from the sediment clone libraries were affiliated with genes described in Proteobacteria and Actinobacteria, with 67 % amino acid identity in average to sequences from isolated microorganisms. The majority of the sequences were most closely related to uncultured microorganisms from cold marine sediments or soils from high latitude regions, highlighting the role of temperature in the structuring of this bacterial guild. The distribution of alkB sequences among samples of different sites and years, and selection after experimental oil exposure allowed us to identify ecologically relevant alkB genes in Subantarctic sediments, which could be used as biomarkers for alkane biodegradation in this environment. 16 S rRNA amplicon pyrosequencing indicated the abundance of several genera for which no alkB genes have yet been described (Oleispira, Thalassospira) or that have not been previously associated with oil biodegradation (Spongiibacter-formerly Melitea-, Maribius, Robiginitomaculum, Bizionia and Gillisia). These genera constitute candidates for future work involving identification of hydrocarbon biodegradation pathway genes.

Isolation and characterization of crude-oil-degrading bacteria from the Persian Gulf and the Caspian Sea.

Twenty-five crude-oil-degrading bacteria were isolated from oil-contaminated sites in the Persian Gulf and the Caspian Sea. Based on a high growth rate on crude oil and on hydrocarbon degradation ability, 11 strains were selected from the 25 isolated strains for further study. Determination of the nucleotide sequence of the 16S rRNA gene showed that these isolated strains belonged to genera Acinetobacter, Pseudomonas, Gordonia, Rhodococcus, Cobetia, Halomonas, Alcanivorax, Marinobacter and Microbacterium. Among the 11 isolates, strains BS (Acinetobacter calcoaceticus, 98%) and PG-12 (Alcanivorax dieselolei, 98%) were the most effective in degrading crude oil. Rate of crude-oil degradation of 82% (isolate BS) and 71% (isolate PG-12) were observed after 1 week of cultivation in mineral medium. These strains had high emulsification activity and biosurfactant production. GC-MS analysis showed that A. dieselolei PG-12 can degrade different alkanes in crude oil. Screening of the distribution of the alkane hydroxylase gene in 25 isolates in relation to the source of isolation indicated that the group (II) alkane hydroxylase is prevalent in the Caspian Sea, but in the Persian Gulf, the frequency of the group (III) alkane hydroxylase gene is greater than that of the group (II) alkane hydroxylase gene.

Two novel alkane hydroxylase-rubredoxin fusion genes isolated from a Dietzia bacterium and the functions of fused rubredoxin domains in long-chain n-alkane degradation.

Two alkane hydroxylase-rubredoxin fusion gene homologs (alkW1 and alkW2) were cloned from a Dietzia strain, designated DQ12-45-1b, which can grow on crude oil and n-alkanes ranging in length from 6 to 40 carbon atoms as sole carbon sources. Both AlkW1 and AlkW2 have an integral-membrane alkane monooxygenase (AlkB) conserved domain and a rubredoxin (Rd) conserved domain which are fused together. Phylogenetic analysis showed that these two AlkB-fused Rd domains formed a novel third cluster with all the Rds from the alkane hydroxylase-rubredoxin fusion gene clusters in Gram-positive bacteria and that this third cluster was distant from the known AlkG1- and AlkG2-type Rds. Expression of the alkW1 gene in DQ12-45-1b was induced when cells were grown on C(8) to C(32) n-alkanes as sole carbon sources, but expression of the alkW2 gene was not detected. Functional heterologous expression in an alkB deletion mutant of Pseudomonas fluorescens KOB2Δ1 suggested the alkW1 could restore the growth of KOB2Δ1 on C(14) and C(16) n-alkanes and induce faster growth on C(18) to C(32) n-alkanes than alkW1ΔRd, the Rd domain deletion mutant gene of alkW1, which also caused faster growth than KOB2Δ1 itself. In addition, the artificial fusion of AlkB from the Gram-negative P. fluorescens CHA0 and the Rds from both Gram-negative P. fluorescens CHA0 and Gram-positive Dietzia sp. DQ12-45-1b significantly increased the degradation of C(32) alkane compared to that seen with AlkB itself. In conclusion, the alkW1 gene cloned from Dietzia species encoded an alkane hydroxylase which increased growth on and degradation of n-alkanes up to C(32) in length, with its fused rubredoxin domain being necessary to maintain the functions. In addition, the fusion of alkane hydroxylase and rubredoxin genes from both Gram-positive and -negative bacteria can increase the degradation of long-chain n-alkanes (such as C(32)) in the Gram-negative bacterium.

An improved procedure for the purification of catalytically active alkane hydroxylase from Pseudomonas putida GPo1.

Bacterial alkane hydroxylases are of high interest for bioremediation applications as they allow some bacteria to grow in oil-contaminated environments. Furthermore, they have tremendous biotechnological potential as they catalyse the stereo- and regio-specific hydroxylation of chemically inert alkanes, which can then be used in the synthesis of pharmaceuticals and other high-cost chemicals. Despite their potential, progress on the detailed characterization of these systems has so far been slow mainly due to the lack of a robust procedure to purify its membrane protein component, monooxygenase AlkB, in a stable and active form. This study reports a new method for isolating milligramme amounts of recombinant Pseudomonas putida GPo1 AlkB in a folded, catalytically active form to purity levels above 90%. AlkB solubilised and purified in the detergent lauryldimethylamine oxide was demonstrated to be active in catalysing the epoxidation reaction of 1-octene with an estimated K (m) value of 0.2 mM.