Mountain pine beetle (Dendroctonus ponderosae) CYP4Gs convert long and short chain alcohols and aldehydes to hydrocarbons.

Hydrocarbon biosynthesis in insects involves the elongation of fatty acyl-CoAs to very-long chain fatty acyl-CoAs that are then reduced and converted to hydrocarbon, with the last step involving the oxidative decarbonylation of an aldehyde to hydrocarbon and carbon dioxide. Cytochromes P450 in the 4G family decarbonylate aldehydes to hydrocarbon. All insect acyl-CoA reductases studied to date reduce fatty acyl-CoAs to alcohols. The results of the work reported herein demonstrate that CYP4G55 and CYP4G56 from the mountain pine beetle, Dendroctonus ponderosae, expressed as fusion proteins with house fly cytochrome P450 reductase (CPR), convert both long chain aldehydes and long chain alcohols to hydrocarbons. CYP4G55 and CYP4G56 appear to prefer primary alcohols to aldehydes as substrates. These data strongly suggest that hydrocarbon biosynthesis in insects occurs by the two-step reduction of very long chain fatty acyl-CoAs to alcohols, which are then oxidized to aldehydes and then oxidatively decarbonylated to hydrocarbon by CYP4G enzymes. In addition, both CYP4G55 and CYP4G56 fusion proteins convert C10 alcohols and aldehydes to hydrocarbons, including the conversion of (Z)-7-decenal, a putative intermediate in the exo-brevicomin pheromone biosynthetic pathway, to (Z)-3-nonene. These data demonstrate that the highly conserved CYP4G enzymes accept a broad range of carbon chain lengths, including C10 and C18, and have evolved to function in cuticular hydrocarbon biosynthesis and pheromone production.

Accelerated methanogenesis from aliphatic and aromatic hydrocarbons under iron- and sulfate-reducing conditions.

The impact of four electron acceptors on hydrocarbon-induced methanogenesis was studied. Methanogenesis from residual hydrocarbons may enhance the exploitation of oil reservoirs and may improve bioremediation. The conditions to drive the rate-limiting first hydrocarbon-oxidizing steps for the conversion of hydrocarbons into methanogenic substrates are crucial. Thus, the electron acceptors ferrihydrite, manganese dioxide, nitrate or sulfate were added to sediment microcosms acquired from two brackish water locations. Hexadecane, ethylbenzene or 1-(13)C-naphthalene were used as model hydrocarbons. Methane was released most rapidly from incubations amended with ferrihydrite and hexadecane. Ferrihydrite enhanced only hexadecane-dependent methanogenesis. The rates of methanogenesis were negatively affected by sulfate and nitrate at concentrations of more than 5 and 1 mM, respectively. Metal-reducing Geobacteraceae and potential sulfate reducers as well as Methanosarcina were present in situ and in vitro. Ferrihydrite addition triggered the growth of Methanosarcina-related methanogens. Additionally, methane was removed concomitantly by anaerobic methanotrophy. ANME-1 and -2 methyl coenzyme M reductase genes were detected, indicating anaerobic methanotrophy as an accompanying process [Correction added 16 December after online publication: 'methyl coenzyme A' changed to 'methyl coenzyme M' in this sentence]. The experiments presented here demonstrate the feasibility of enhancing methanogenic alkane degradation by ferrihydrite or sulfate addition in different geological settings.

Oxidation of aliphatic, branched chain, and aromatic hydrocarbons by Nocardia cyriacigeorgica isolated from oil-polluted sand samples collected in the Saudi Arabian Desert.

A soil bacterium isolated from oil-polluted sand samples collected in the Saudi Arabian Desert has been determined as Nocardia cyriacigeorgica, which has a high capacity of degrading and utilizing a broad range of hydrocarbons. The metabolic pathways of three classes of hydrocarbons were elucidated by identifying metabolites in cell-free extracts analyzed by GC/MS and HPLC/UV-Vis in comparison with standard compounds. During tetradecane oxidation, tetradecanol; tetradecanoic acid; dodecanoic acid; decanoic acid could be found as metabolites, indicating a monoterminal degradation pathway of n -alkanes. The oxidation of pristane resulted in the presence of pristanoic acid; 2-methylglutaric acid; 4,8-dimethylnonanoic acid; and 2,6-dimethylheptanoic acid, which give rise to a possible mono- and di-terminal oxidation. In case of sec -octylbenzene, eight metabolites were detected including 5-phenylhexanoic acid; 3-phenylbutyric acid; 2-phenylpropionic acid; beta -methylcinnamic acid; acetophenone; beta -hydroxy acetophenone; 2,3-dihydroxy benzoic acid and succinic acid. From these intermediates a new degradation pathway for sec -octylbenzene was investigated. Our results indicate that N. cyriacigeorgica has the ability to degrade aliphatic and branched chain alkanes as well as alkylbenzene effectively and, therefore, N. cyriacigeorgica is probably a suitable bacterium for biodegradation of oil or petroleum products in contaminated soils.

Oceanobacter-related bacteria are important for the degradation of petroleum aliphatic hydrocarbons in the tropical marine environment.

Petroleum-hydrocarbon-degrading bacteria were obtained after enrichment on crude oil (as a 'chocolate mousse') in a continuous supply of Indonesian seawater amended with nitrogen, phosphorus and iron nutrients. They were related to Alcanivorax and Marinobacter strains, which are ubiquitous petroleum-hydrocarbon-degrading bacteria in marine environments, and to Oceanobacter kriegii (96.4-96.5 % similarities in almost full-length 16S rRNA gene sequences). The Oceanobacter-related bacteria showed high n-alkane-degrading activity, comparable to that of Alcanivorax borkumensis strain SK2. On the other hand, Alcanivorax strains exhibited high activity for branched-alkane degradation and thus could be key bacteria for branched-alkane biodegradation in tropical seas. Oceanobacter-related bacteria became most dominant in microcosms that simulated a crude oil spill event with Indonesian seawater. The dominance was observed in microcosms that were unamended or amended with fertilizer, suggesting that the Oceanobacter-related strains could become dominant in the natural tropical marine environment after an accidental oil spill, and would continue to dominate in the environment after biostimulation. These results suggest that Oceanobacter-related bacteria could be major degraders of petroleum n-alkanes spilt in the tropical sea.

Screening of Australian native grasses for rhizoremediation of aliphatic hydrocarbon-contaminated soil.

Rhizoremediation involves the breakdown of contaminants in soil resulting from microbial activity that is enhanced in the plant root zone. The objective of this study was to identify Australian native grass species as suitable candidates for rhizoremediation application. Seeds of nine perennial Australian native grasses were sown in soil from a mine site and artificially contaminated with a 60:40 diesel/oil mixture at concentrations of 1% (w/w), 0.5% (w/w), and 0% (control). Seedling emergence was not adversely affected by the presence of hydrocarbon contamination for all but one grass species. Three promising species (Brachiaria decumbens, Cymbopogon ambiguus, and Microlaena stipoides var. Griffin) were assessed for growth characterization in contaminated and uncontaminated soils. The evaluated species survived for 120 days in the contaminated soil and, in some instances, produced considerably more root biomass in the presence of contamination. C. ambiguus showed growth stimulation in the presence of contamination (1% and 0.5% w/w) with significantly increased root biomass production compared with the control (p = 0.0001). B. decumbens and M. stipoides showed tolerance, without adverse growth effects in the presence of diesel/oil at the exposed concentrations. Stimulation of the rhizosphere microbial population that is capable of degrading diesel/oil was found for all of the species tested, using a most probable number method for enumeration. This investigation has identified suitable candidates for further investigation of their rhizoremediation potential.

Efficient preparation of an acyclic permutant of kalata B1 from a recombinant fusion protein with thioredoxin.

A new approach to prepare an acyclic permutant of kalata B1, a cysteine-rich plant cyclopeptide with uterotonic activity, is described. The synthetic codon-optimized cDNA sequence encoding this 29-residue peptide was cloned and fused in-frame to the His(6)-tagged thioredoxin gene in the bacterial expression vector pET-32a. The fusion protein was overexpressed in the bacterial host, Escherichia coli strain BL21 (DE3), and isolated by affinity chromatography on a metal-chelating Sepharose column. An enterokinase recognition sequence incorporated immediately upstream of the target peptide allowed the 29-residue peptide to be released without any unwanted residues upon treatment with enterokinase. This peptide was subsequently separated from the larger thioredoxin moiety by ultracentrifugation through a semipermeable membrane. Further purification was achieved using reversed-phase HPLC. Hydrogen peroxide was found to enhance the rate of enterokinase cleavage in a concentration-dependent manner. Thermal stability studies demonstrated that the recombinant acyclic kalata B1 (ac kalata) was exceptionally stable against thermal denaturation. Mass spectrometric analysis revealed that the recombinant ac kalata was obtained in a fully oxidized form, indicating a high reducing potential and a strong tendency of the 29-residue peptide to form a tightly folded structure.

Bioaccumulation of anthropogenic contaminants by detritivorous fish in the Río de la Plata estuary: 1-aliphatic hydrocarbons.

The temporal variability and bioaccumulation dynamics of C(12-25)n-alkanes, isoprenoids and unresolved aliphatic hydrocarbons (UCM) were studied in a detritivorous fish (Sábalo: Prochilodus lineatus) collected from 1999 to 2005 in the sewage impacted Buenos Aires coastal area. Fish muscles contain huge amounts of n-C(12-25) (165+/-93, 70+/-48 or 280+/-134 microg g(-1), dry, fresh and lipid weight, respectively) and UCM (931+/-560, 399+/-288 and 1,567+/-802 microg g(-1)) reflecting the chronic bioaccumulation of fossil fuels from sewage particulates. On a temporal basis, lipid normalized aliphatic concentrations peaked by the end of 2001-2002 during the rainiest period over the last four decades (1,750 vs. 1,083+/-4.6mm in 1999, 2004 and 2005), reflecting an enhanced exposition due to massive anthropogenic fluxes from Metropolitan Buenos Aires in wet years. The hydrocarbon composition in fish muscles is enriched in n-C(15-17) and isoprenoids relative to a fresh crude oil and settling particulates, with fresher signatures during the 2001-2002 maxima. Fish/settling material bioaccumulation factors (BAFs: 0.4-6.4 dry weight or 0.07-0.94 lipid-organic carbon) plotted against K(ow) showed a parabolic pattern maximizing at n-C(14-18) and isoprenoids. The optimal bioaccumulation window corresponds to highly hydrophobic (log K(ow): 7.2-9.9), intermediate-size C(14-18)n-alkanes and C(15-20) isoprenoids (MW: 198-282; length: 17.9 to 25.4A) with melting points ranging from -19.8 to 28 degrees C. The uptake efficiency is inversely correlated to melting points and increased from 75% for n-C(25) to above 90% for n-C(14-15) and isoprenoids.

Translesion DNA synthesis across non-DNA segments in cultured human cells.

DNA lesions that have escaped DNA repair are tolerated via translesion DNA synthesis (TLS), carried out by specialized error-prone DNA polymerases. To evaluate the robustness of the TLS system in human cells, we examined its ability to cope with foreign non-DNA stretches of 3 or 12 methylene residues, using a gap-lesion plasmid assay system. We found that both the trimethylene and dodecamethylene inserts were bypassed with significant efficiencies in human cells, using both misinsertion and misalignment mechanisms. TLS across these non-DNA segments was aphidicolin-sensitive, and did not require poleta. In vitro primer extension assays showed that purified poleta, polkappa and poliota were each capable of inserting each of the four nucleotides opposite the trimethylene chain, but only poleta and polkappa could fully bypass it. Poleta and poliota, but not polkappa, could also insert each of the four nucleotides opposite the dodecamethylene chain, but all three polymerases were severely blocked by this lesion. The ability of TLS polymerases to insert nucleotides opposite a hydrocarbon chain, despite the lack of any similarity to DNA, suggests that they may act via a mode of transient and local template-independent polymerase activity, and highlights the robustness of the TLS system in human cells.

Nestmate recognition cues in the honey bee: differential importance of cuticular alkanes and alkenes.

In social insects, recognition of nestmates from aliens is based on olfactory cues, and many studies have demonstrated that such cues are contained within the lipid layer covering the insect cuticle. These lipids are usually a complex mixture of tens of compounds in which aliphatic hydrocarbons are generally the major components. The experiments described here tested whether artificial changes in the cuticular profile through supplementation of naturally occurring alkanes and alkenes in honeybees affect the behaviour of nestmate guards. Compounds were applied to live foragers in microgram quantities and the bees returned to their hive entrance where the behaviour of the guard bees was observed. In this fashion we compared the effect of single alkenes with that of single alkanes; the effect of mixtures of alkenes versus that of mixtures of alkanes and the whole alkane fraction separated from the cuticular lipids versus the alkene fraction. With only one exception (the comparison between n-C(19) and (Z)9-C(19)), in all the experiments bees treated with alkenes were attacked more intensively than bees treated with alkanes. This leads us to conclude that modification of the natural chemical profile with the two different classes of compounds has a different effect on acceptance and suggests that this may correspond to a differential importance in the recognition signature.

Biodegradation of Maya crude oil fractions by bacterial strains and a defined mixed culture isolated from Cyperus laxus rhizosphere soil in a contaminated site.

Ten bacterial strains were isolated by enrichment culture, using as carbon sources either aliphatics or an aromatic-polar mixture. Oxygen uptake rate was used as a criterion to determine culture transfer timing at each enrichment stage. Biodegradation of aliphatics (10,000 mg L(-1)) and an aromatic-polar mixture (5000 mg L(-1), 2:1) was evaluated for each of the bacterial strains and for a defined culture made up with a standardized mixture of the isolated strains. Degradation of total hydrocarbons (10,000 mg L(-1)) was also determined for the defined mixed culture. Five bacterial strains were able to degrade more than 50% of the aliphatic fraction. The most extensive biodegradation (74%) was obtained with strain Bs 9A, while strains Ps 2AP and UAM 10AP were able to degrade up to 15% of the aromatic-polar mixture. The defined mixed culture degraded 47% of the aliphatics and 6% of the aromatic-polar mixture. The defined mixed culture was able to degrade about 40% of the aliphatic fraction and 26% of the aromatic fraction when grown in the presence of total hydrocarbons, while these microorganisms did not consume the polar hydrocarbons fraction. The proposed strategy that combines enrichment culture together with oxygen uptake rate allowed the isolation of bacterial strains that are able to degrade specific hydrocarbons fractions at high consumption rates.