Distribution of decane within the unit cell of the inverted hexagonal (HII) phase of lipid-water-decane systems determined by neutron diffraction.

The addition of a free alkane such as decane to lipid-water systems is known to promote the formation of a low-temperature inverted hexagonal (HII) phase [Kirk, G. L., & Gruner, S. M. (1985) J. Phys. (Paris) 46, 761]. Kirk et al. [Kirk, G. L., Gruner, S. M., & Stein, D. E. (1984) Biochemistry 23, 1093] have discussed the hydrocarbon packing anisotropy in the HII unit cell and have suggested that free alkane will distribute in a way that reduces this packing anisotropy by allowing the lipid chain environment to become more uniform. By combining neutron and X-ray diffraction data to do a Fourier reconstruction of the HII phase of dioleoylphosphatidylethanolamine (DOPE) + water + deuterated decane, it was found that the decane preferentially partitions into the interstitial regions of the HII unit cell where it should be the most effective in alleviating the hydrocarbon chain packing stress, supporting the suggestion of Kirk et al. Using the distribution of decane within the unit cell, we have calculated the lipid length distribution for the situations with and without added alkane. With a suitable molecular model, this lipid length distribution may eventually be used to calculate the free energy change upon the addition of alkane. Such a measurement is important for a more realistic understanding of the interactions which lead to the formation of the HII phase.     

Ethanenitronate is a peroxide-dependent suicide substrate for catalase

We find ethanenitronate (formula; see text) to be a H2O2- (and peracetic acid-) dependent suicide substrate for bovine liver catalase (E) which converts E to Em, a modified form of the enzyme. The catalytic and suicide pathways are related to E, Em, Compound I, and Compound II according to the following scheme. (formula; see text) The catalytic cycle generates free radical products (EN.) which then participate in an O2-dependent chain reaction. Within experimental error the exclusive target for inactivation by EN- is Compound II. This partitions in the ratio (k4 = 1.2 M-1 s-1)/(k3 = 1.6 M-1 s-1) to generate Em and E, respectively. The species Em acquires 1 eq of 14C/ferriheme from [1-14C]ethanenitronate which is firmly (presumably covalently) affixed to the protein moiety. According to the standard H2O2 assay, Em is 7% as active catalytically as E. We regard inactivation as resulting from that fraction of EN. in the E...EN. complex which fails to diffuse from the complex because it is trapped by reaction with a neighboring amino acid residue to generate Em irreversibly. (formula; see text) This mechanism is identical to that deduced previously for suicide inactivation of horseradish peroxidase by alkane nitronates (Porter, D. J. T., and Bright, H. J. (1983) J. Biol. Chem. 258, 9913-9924) with the exception that EN. is trapped in that case by a methine carbon at the edge of the ferriheme rather than by the apoenzyme. The labeled residue in the catalase apoenzyme probably resides at or near the site of reduction of Compound II.     

Characterization of Yarrowia lipolytica mutants affected in hydrophobic substrate utilization

In order to get deeper insights into oxidative degradation of the hydrophobic substrates (HS) triglycerides and alkanes by yeasts, tagged mutants affected in these pathways were generated by random insertion of a mutagenesis cassette MTC into the genome of Yarrowia lipolytica. About 9.600 Ura+ transformants were screened in plate tests for utilization of alkanes (C10, C16), oleic acid and tributyrin. HS degradation mutants were recovered as unable to grow on alkane or on intermediates of the pathway (AlkA-AlkE phenotype classes). To identify the disrupted genes, insertion points of the MTC were sequenced using convergent and divergent PCR. Sequence analysis evidenced both known and new genes required for HS utilization, e.g. for AlkD/E mutants MTC insertion had occurred in genes of thioredoxin reductase, peroxines PEX14 and PEX20, succinate-fumarate carrier SFC1, and isocitrate lyase ICL1. Several mutants were affected in alkane utilization depending on chain length. Mutant Z110 (AlkAb: C10- C16+) was shown to be disrupted for ANT1 encoding a peroxisomal membrane localized adenine nucleotide transporter protein, providing ATP for the activation of short-chain fatty acids by acyl-CoA synthetase II in peroxisomes. Mutants N046 and B095 (AlkAc: C10+ C16-) were disrupted for the ABC transporter encoded by ABC1 gene, thus providing first evidence for its participation in chain length dependent alkane transport processes.     

Characterization of a novel long-chain n-alkane-degrading strain, Dietzia sp. E1

The newly isolated strain E1, identified as a Dietzia sp., proved to have an excellent ability to degrade n-C12 to n-C38 alkane components of crude oil. The preferred substrate was the very long-chain alkane n-eicosane at an optimal temperature of 37 degrees C and an optimal pH of 8 under aerobic conditions. The growth and substrate uptake kinetics were monitored during the n-alkane fermentation process, and Dietzia sp. E1 cells were found to possess three distinct levels of cell-surface hydrophobicity. Gas chromatographic/mass spectrometric analysis revealed that intracellular substrate mineralization occurred through the conversion of n-alkane to the corresponding n-alkanal. The monoterminal oxidation pathway was presumably initiated by AlkB and CYP153 terminal alkane hydroxylases, both of their partial coding sequences were successfully detected in the genome of strain E1, a novel member of the Dietzia genus.     

Molecular dynamics simulation of the folding of single alkane chains with different lengths on single-walled carbon nanotubes and graphene

Chain folding is an important step during polymer crystallization. In order to study the effects of the surface on chain folding, molecular dynamics simulations of the folding of different alkane chains on three kinds of single-walled carbon nanotubes (SWCNTs) and graphene were performed. The folding behaviors of the single alkane chains on these surfaces were found to be different from their folding behaviors in vacuum. The end-to-end distances of the chains were calculated to explore the chain folding. An increasing tendency to fold into two or more stems with increasing alkane chain length was observed. This result indicates that the occurrence and the stability of chain folding are related to the surface curvature, the diameter of the SWCNT, and surface texture. In addition, the angle between the direction of the alkane chain segment and the direction of the surface texture was measured on different surfaces.     

Synthesis of alkane hydroxylase of Pseudomonas oleovorans increases the iron requirement of alk+ bacterial strains

The alk genes enable Pseudomonas oleovorans to utilize alkanes as sole carbon and energy source. Expression of the alk genes in P. oleovorans and in two Escherichia coli recombinants induced iron limitation in minimal medium cultures. Further investigation showed that the expression of the alkB gene, encoding the integral cytoplasmic membrane protein AlkB, was responsible for the increase of the iron requirement of E. coli W3110 (pGEc47). AlkB is the non-heme iron monooxygenase component of the alkane hydroxylase system, and can be synthesized to levels up to 10% (w/w) of total cell protein in E. coli W3110 (pGEc47). Its synthesis is, however, strictly dependent on the presence of sufficient iron in the medium. Our results show that a glucose-grown E. coli alk+ strain can reach alkane hydroxylase activities of about 25 U/g cdw, and are consistent with the recent finding that catalytically active AlkB contains two, rather than one iron atom per polypeptide chain.     

Biodegradation of Crude Oil by Thermophilic Bacteria Isolated from a Volcano Island

One-hundred and fifty different thermophilic bacteria isolated from a volcanic island were screened for detection of an alkane hydroxylase gene using degenerated primers developed to amplify genes related to the Pseudomonas putida and Pseudomonas oleovorans alkane hydroxylases. Ten isolates carrying the alkJ gene were further characterized by 16s rDNA gene sequencing. Nine out of ten isolates were phylogenetically affiliated with Geobacillus species and one isolate with Bacillus species. These isolates were able to grow in liquid cultures with crude oil as the sole carbon source and were found to degrade long chain crude oil alkanes in a range between 46.64% and 87.68%. Results indicated that indigenous thermophilic hydrocarbon degraders of Bacillus and Geobacillus species are of special significance as they could be efficiently used for bioremediation of oil-polluted soil and composting processes.     

Leaf n‐Alkanes as Characters Differentiating Coastal and Continental Juniperus deltoides Populations from the Balkan Peninsula

The composition of the cuticular n‐alkanes isolated from the leaves of nine populations of Juniperus deltoides R.P.Adams from continental and coastal areas of the Balkan Peninsula was characterized by GC‐FID and GC/MS analyses. In the leaf waxes, 14 n‐alkane homologues with chain‐lengths ranging from C₂₂ to C₃₅ were identified. n‐Tritriacontane (C₃₃) was dominant in the waxes of all populations, but variations between the populations in the contents of all n‐alkanes were observed. Several statistical methods (ANOVA, principal component, discriminant, and cluster analyses) were used to investigate the diversity and variability of the cuticular‐leaf‐n‐alkane patterns of the nine J. deltoides populations. This is the first report on the n‐alkane composition for this species. The multivariate statistical analyses evidenced a high correlation of the leaf‐n‐alkane pattern with the geographical distribution of the investigated samples, differentiating the coastal from the continental populations of this taxon.     

Identification of different alkane hydroxylase systems in Rhodococcus ruber strain SP2B,an hexane‐degrading actinomycete

Aims: To investigate the alkane‐hydroxylating system of isolate SP2B, closely related to Rhodococcus ruber DSM 43338^T and uncharacterized so far for its alkane degradation genes. Methods and Results: Although isolate SP2B and reference strain can grow on by‐products from hexane degradation, the type strain R. ruber was unable, unlike SP2B isolate, to use short‐chain alkanes, as assessed by gas chromatography. Using PCR with specific or degenerated primers, inverse PCR and Southern blot, two alkane hydroxylase encoding genes (alkB) were detected in both bacteria, which is in agreement with their alkane range. The first AlkB was related to Rhodococcus AlkB7 enzymes and contains a nonbulky residue at a specific position, suggesting it might be involved in medium‐ and long‐chain alkane oxidation. The second partial alkB gene potentially belongs to alkB5‐type, which was found in bacteria unable to use hexane. Moreover, a partial P450 cytochrome alkane hydroxylase, thought to be responsible for the hexane degradation, was detected only in the isolated strain. Conclusions: Rhodococcus ruber SP2B should prove to be a promising candidate for bioremediation studies of contaminated sites because of its large degradation range of alkanes. Significance and Impact of the Study: This is the first thorough study on R.ruber alkane degradation systems.     

The effect of iron overload on urinary excretion of immunoreactive prostaglandin E2

The effect of in vivo lipid peroxidation on the excretion of immunoreactive prostaglandin E2 (PGE2) in the urine of rats was studied. Weanling, male Sprague-Dawley rats were fed a vitamin E-deficient diet containing 10% tocopherol-stripped corn oil (CO) or 5% cod liver oil (CLO) with or without 40 mg dl-alpha-tocopheryl acetate/kg. To induce a high, sustained level of lipid peroxidation, some rats were injected intraperitoneally with 100 mg of iron as iron dextran after 10 days of feeding. Iron overload stimulated in vivo lipid peroxidation in rats, as measured by the increase in expired ethane and pentane. Dietary vitamin E reversed this effect. Rats fed the CLO diet excreted 9.5-fold more urinary thiobarbituric acid-reactive substances (TBARS) than did rats fed the CO diet. Iron overload increased the excretion of TBARS in the urine of rats fed the CO diet, but not in urine of rats fed the CLO diet. Dietary vitamin E decreased TBARS in the urine of rats fed either the CO or the CLO diet. Iron overload decreased by 40% the urinary excretion of PGE2 by rats fed the CO diet, and dietary vitamin E did not reverse this effect. Iron overload had no statistically significant effect on urinary excretion of PGE2 by rats fed the CLO diet. A high level of lipid peroxidation occurred in iron-treated rats, as evidenced by an increase in alkane production and in TBARS in urine in this study, and by an increase in alkane production by slices of kidney from iron-treated rats in a previous study [V. C. Gavino, C. J. Dillard, and A. L. Tappel (1984) Arch. Biochem. Biophys. 233, 741-747]. Since PGE2 excretion in urine was not correlated with these effects, lipid peroxidation appears not to be a major factor in renal PGE2 flux.     

Anaerobic transformation of alkanes to fatty acids by a sulfate-reducing bacterium,strain Hxd3

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and (13)C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and (13)C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-(13)C(2)]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [(13)C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a (13)C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-(13)C(2)]hexadecane-derived fatty acids contained either two (13)C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.     

Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli

The OCT plasmid encodes enzymes for alkane hydroxylation and alkanol dehydrogenation. Structural components are encoded on the 7.5-kilobase pair alkBAC operon, whereas positive regulatory components are encoded by alkR. We have constructed plasmids containing fusions of cloned alkBAC and alkR DNA and used these fusion plasmids to study the functional expression of the alkBAC operon and the regulatory locus alkR in Pseudomonas putida and in Escherichia coli. Growth on alkanes requires a functional chromosomally encoded fatty acid degradation system in addition to the plasmid-borne alk system. While such a system is active in P. putida, it is active in E. coli only in fadR mutants in which fatty acid degradation enzymes are expressed constitutively. Using such mutants, we found that E. coli as well as P. putida grew on octane as the sole source of carbon and energy when they were supplied with the cloned complete alk system. The alkR locus was strictly necessary in E. coli as well as in P. putida for expression of the alkBAC operon. The alkBAC operon could, however, be further reduced to a 5-kilobase pair operon without affecting the Alk phenotype in either species to a significant extent. Although with this reduction the plasmid-encoded alkanol dehydrogenase activity was lost, chromosomally encoded alkanol dehydrogenases in P. putida and E. coli compensated for this loss. The induction kinetics of the alk system was studied in detail in P. putida and E. coli. We used specific antibodies raised against alkane hydroxylase to follow the appearance of this protein following induction with octane. We found the induction kinetics of alkane hydroxylase to be similar in both species. A steady-state level was reached after about 2 h of induction in which time the alkane hydroxylase accounted for about 1.5% of total newly synthesized protein. Thus, alkBAC expression is very efficient and strictly regulated to both P. putida and E. coli.     

Biogeographic Variation of Foliar n‐Alkanes of Juniperus communis var. saxatilis Pallas from the Balkans

The composition of the epicuticular n‐alkanes isolated from the leaves of ten populations of Juniperus communis L. var. saxatilis Pallas from central (continental) and western (coastal) areas of the Balkan Peninsula was characterized by GC‐FID and GC/MS analyses. In the leaf waxes, 14 n‐alkane homologues with chain‐lengths ranging from C₂₂ to C₃₅ were identified. All samples were dominated by n‐tritriacontane (C₃₃), but differences in two other dominant n‐alkanes allowed separating the coastal from the continental populations. Several statistical methods (ANOVA, principal component, discriminant, and cluster analyses as well as the Mantel test) were deployed to analyze the diversity and variability of the epicuticular‐leaf‐n‐alkane patterns of the ten natural populations of J. communis var. saxatilis and their relation to different geographic and bioclimatic parameters. Cluster analysis showed a high correlation of the leaf‐n‐alkane patterns with the geographical distribution of the investigated samples, differentiating the coastal from the continental populations of this taxon. Several bioclimatic parameters related to aridity were highly correlated with this differentiation.     

Breath alkanes as a marker of oxidative stress in different clinical conditions

We assessed oxidative stress in three different clinical conditions: smoking, human immunodeficiency virus (HIV) infection, and inflammatory bowel disease, using breath alkane output and other lipid peroxidation parameters such as plasma lipid peroxides (LPO) and malondialdehyde (MDA). Antioxidant micronutrients such as selenium, vitamin E, C, beta-carotene and carotenoids were also measured. Lipid peroxidation was significantly higher and antioxidant vitamins significantly lower in smokers compared to nonsmokers. Beta-carotene or vitamin E supplementation significantly reduced lipid peroxidation in that population. However, vitamin C supplementation had no effect. In HIV-infected subjects, lipid peroxidation parameters were also elevated and antioxidant vitamins reduced compared to seronegative controls. Vitamin E and C supplementation resulted in a significant decrease in lipid peroxidation with a trend toward a reduction in viral load. In patients with inflammatory bowel disease, breath alkane output was also significantly elevated when compared to healthy controls. A trial with vitamin E and C is underway. In conclusion, breath alkane output, plasma LPO and MDA are elevated in certain clinical conditions such as smoking, HIV infection, and inflammatory bowel disease. This is associated with lower levels of antioxidant micronutrients. Supplementation with antioxidant vitamins significantly reduced these lipid peroxidation parameters. The results suggest that these measures are good markers for lipid peroxidation.     

Functional analysis of long-chain n-alkane degradation by Dietzia spp

The genetic background of long-chain n-alkane degradation was investigated in detail in strain E1, a member of the genetically unexplored Dietzia genus. A suicide vector carrying a 518-bp alkB fragment was site-specifically integrated into the E1 chromosome, and the full alkB, as well as its chromosomal environment was sequenced after plasmid rescue experiments. Four out of the nine putative genes were strongly induced by long-chain n-alkanes in wild-type E1. ORF4 encoded a natural fusion protein consisting of an integral membrane alkane hydroxylase and a rubredoxin domain. The significance of the alkB-rub gene in n-alkane degradation was investigated in phenotypic tests, and the disruption mutant strain exhibited severely impaired growth on n-C(20) alkane carbon source. The mutation was successfully complemented with the expression of intact AlkB-Rub protein, the full-length form of which was detected by simultaneous immunoblotting. The presented data furnish the first experimental evidence of the in vivo existence of an AlkB-Rub natural fusion protein, which plays a major role in long-chain n-alkane degradation.     

The effect of benzyl alcohol and cholesterol on the acyl chain order and alkane solubility of bimolecular phosphatidylcholine membranes

An investigation was made of the effects of cholesterol and benzyl alcohol on the partitioning of n-alkanes between lipid bilayer membranes and bulk lipid/alkane solutions (in the torus). Bilayers were formed from solutions containing alkanes of different chain lengths, together with phosphatidylcholine and cholesterol in varying proportions. The partitioning of the alkanes was determined from measurements of the very low frequency (1 Hz) capacitance of the membranes. Perturbation of the internal membrane structure by the inclusion of cholesterol and benzyl alcohol produced very significant changes in the n-alkane partition coefficient, cholesterol causing a decrease and benzyl alcohol an increase in the alkane partitioning into the bilayer. A correlation exists between the effects of these compounds on the alkane partitioning and their effect on the segmental chain order of the acyl chains in the bilayer and this correlation is consistent with a statistical-mechanical model of the lipid/alkane bilayers in the liquid crystalline state. The perturbation by cholesterol and benzyl alcohol of the internal structure of membranes bears on the conflicting reports of the effects of these substances on artificial lipid bilayers and could also be relevant to their known physiological effects.     

Anaerobic Transformation of Alkanes to Fatty Acids by a Sulfate-Reducing Bacterium, Strain Hxd3

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and ¹³C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and ¹³C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-¹³C₂]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [¹³C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a ¹³C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-¹³C₂]hexadecane-derived fatty acids contained either two ¹³C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.     

Characterization of polychlorinated alkane mixtures—a Monte Carlo modeling approach

A Monte Carlo model was developed to characterize the molecular composition of polychlorinated alkane mixtures. The model is based upon a simulation of the free-radical chlorination process by which polychlorinated alkane mixtures are produced industrially from n-alkanes. In the model, the free-radical chlorination reaction was simulated by randomly selecting a position on a partially converted alkane molecule for target by chlorine free-radical attack. The relative reactivities of the hydrogen atoms on the alkane chain towards chlorine free-radical substitution were either determined experimentally or extrapolated from experimental results and incorporated into the model. The result of the simulation is the prediction of the detailed molecular composition of any PCA mixture. Good agreement was found when comparing the distribution of molecules predicted by the model to analytically determined distributions of real PCA mixtures. Results from the model were then coupled with rules describing the action of biological enzymes to estimate the upper limit possible for the aerobic biodegradation of PCA mixtures.     

Monitoring of alkane-degrading bacteria in a sea-water microcosm during crude oil degradation by polymerase chain reaction based on alkane-catabolic genes

Behaviour of microbial populations responsible for degrading n-alkanes, a major component of crude oil, was monitored during crude oil degradation in a sea-water microcosm by both traditional colony culturing and molecular techniques. A DNA extraction method applicable to crude oil-amended sea-water samples was developed to obtain DNA applicable to most probable number (MPN) polymerase chain reaction (PCR). The population of alkane-degrading bacteria responsible for degradation of n-alkanes in a crude oil-amended microcosm altered, so that shorter alkanes were degraded first by alkane-degrading bacteria possessing alkane hydroxylase genes from group I (Kohno et al., 2002, Microb Environ 17: 114-121) and longer ones afterwards by those possessing alkane hydroxylase genes from group II. Thus, the degradation mechanism of the n-alkanes can be clarified during crude oil degradation. Application of the method of detecting different types of alkane-catabolic genes, as shown in the present study, enabled bacterial groups preferring alkanes of either shorter or longer chain lengths to be enumerated selectively.