Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1

Vibrio furnissii M1 was recently reported to biosynthesize n-alkanes when grown on biopolymers, sugars, or organic acids (M. O. Park, J. Bacteriol. 187:1426-1429, 2005). In the present study, V. furnissii M1 was subjected to genomic analysis and studied biochemically. The sequence of the 16S rRNA gene and repetitive PCR showed that V. furnissii M1 was not identical to other V. furnissii strains tested, but the level of relatedness was consistent with its assignment as a V. furnissii strain. Pulsed-field gel electrophoresis showed chromosomal bands at approximately 3.2 and 1.8 Mb, similar to other Vibrio strains. Complete genomic DNA from V. furnissii M1 was sequenced with 21-fold coverage. Alkane biosynthetic and degradation genes could not be identified. Moreover, V. furnissii M1 did not produce demonstrable levels of n-alkanes in vivo or in vitro. In vivo experiments were conducted by growing V. furnissii M1 under different conditions, extracting with solvent, and analyzing extracts by gas chromatography-mass spectrometry. A highly sensitive assay was used for in vitro experiments with cell extracts and [(14)C]hexadecanol. The data are consistent with the present strain being a V. furnissii with properties similar to those previously described but lacking the alkane-producing phenotype. V. furnissii ATCC 35016, also reported to biosynthesize alkanes, was found in the present study not to produce alkanes.     

Interactions between diacylglycerophosphoethanolamines and n-alkanes in monolayers and bilayers

Surface pressure-area isotherms of l-diacylglycerophosphoethanolamines were measured at the n-alkane/water interfaces for alkanes ranging from n-hexane to n-hexadecane. Transition pressures from the expanded film to the condensed state varied largely depending on the chain length of the n-alkane in the oil phase. The phase transition temperature and the entropy were studied by differential scanning calorimetry in presence of n-alkanes for the same lipid. The temperatures of gel-to-liquid crystalline phase transitions were changed in the same way as the monolayers reflecting the chain lengths of the n-alkanes present. The effects of the n-alkanes on monolayers and bilayers were entirely parallel; they are discussed taking the mixing of the lipids and the n-alkanes into account in the condensed films for the former and in the gel phase for the latter.     

Stable Isotopic Studies of n-Alkane Metabolism by a Sulfate-Reducing Bacterial Enrichment Culture

Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C₆ to C₁₂) and 1-¹³C-labeled n-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d₁₄-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-¹³C-labeled n-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that ¹³C nuclei from 1-¹³C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that ¹³C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177:235-243, 2002) for metabolism of deuterated n-hexane by a denitrifying bacterium.     

Biosynthetic and environmental effects on the stable carbon isotopic compositions of anteiso- (3-methyl) and iso- (2-methyl) alkanes in tobacco leaves

Nicotiana tabacum is the only plant known to synthesise large quantities of anteiso- (3-methyl) alkanes and iso- (2-methyl) alkanes. We investigated the carbon isotope ratios of individual long-chain n-alkanes, anteiso- and iso-alkanes (in the C₂₉-C₃₃ carbon number range) extracted from tobacco grown in chambers under controlled conditions to confirm the pathway used by the tobacco plant to synthesise these particular lipids and to examine whether environmental data are recorded in these compounds. Tobacco was grown under differing temperatures, water availabilities and light intensities in order to control its stable carbon isotope ratios and evaluate isotopic fractionations associated with the synthesis of these particular lipids. The anteiso-alkanes were found to have a predominant even-carbon number distribution (maximising at C₃₂), whereas the iso-alkanes exhibit an odd-carbon number distribution (maximising at C₃₁). Iso-alkanes were relatively more abundant than the anteiso-alkanes and only two anteiso-alkanes (C₃₀ and C₃₂) were observed.The anteiso-alkanes and iso-alkanes were found to be enriched in ¹³C by 2.8-4.3‰ and 0-1.8‰ compared to the n-alkanes, respectively, consistent with different biosynthetic precursors. The assumed precursor for the odd-carbon-numbered iso-alkanes is iso-butyryl-CoA (a C₄ unit derived from valine) followed by subsequent elongation of C₂ units and then decarboxylation. The assumed precursor for even-carbon-numbered anteiso-alkanes is α-methylbutyryl-CoA (a C₅ unit derived from isoleucine) and subsequent elongation by C₂ units followed by decarboxylation. The ratio of carbon atoms derived from α-methylbutyryl-CoA and subsequent C₂ units (from malonyl-CoA) is 1:5 for the biosynthesis of a C₃₀anteiso-alkane. The ratio of carbon atoms derived from iso-butyryl-CoA and subsequent C₂ units (from malonyl-CoA) is 4:25 for the synthesis of a C₂₉iso-alkane. An order of ¹³C depletion n-alkanes > iso-alkanes > anteiso-alkanes is evident from compound specific isotope data. This trend can probably be attributed to the ratio of the two different sources of carbon atoms in the final wax components.Higher water availability generally results in more depleted stable carbon isotope ratios due to maximised discrimination during carboxylation, associated with less diffusional limitation. This was confirmed in the present study by compound specific isotope analyses of iso-alkanes, anteiso-alkanes and n-alkane lipids extracted from the tobacco leaves. Likewise, light intensity has been shown to influence plant bulk δ¹³C in previous studies. The carbon isotope ratios of n-alkanes in tobacco grown under low-light conditions were about 2‰ more depleted in ¹³C than those of lipids extracted from tobacco grown under elevated light conditions. A similar order of difference is observed for the iso-alkanes and anteiso-alkanes (1.8‰ and 1.9‰, respectively). A negligible depletion in carbon isotope ratios was observed for the iso-alkanes and anteiso-alkanes extracted from tobacco grown under elevated temperatures. These results are consistent with the work of Farquhar [Farquhar, G.D., 1980. Carbon isotope discrimination by plants: effects of carbon dioxide concentration and temperature via the ratio of intercellular and atmospheric CO₂ concentrations. In: Pearman, G.I. (Ed.), Carbon Dioxide and Climate: Australian Research. Springer, Berlin, pp. 105-110] where temperature appears to have only a minor effect on plant bulk δ¹³C.     

Structure and Biosynthesis of Cuticular Lipids: Hydroxylation of Palmitic Acid and Decarboxylation of C(28), C(30), and C(32) Acids in Vicia faba Flowers

The structure and composition of the cutin monomers from the flower petals of Vicia faba were determined by hydrogenolysis (LiAlH₄) or deuterolysis (LiAlD₄) followed by thin layer chromatography and combined gas-liquid chromatography and mass spectrometry. The major components were 10, 16-dihydroxyhexadecanoic acid (79.8%), 9, 16-dihydroxyhexadecanoic acid (4.2%), 16-hydroxyhexadecanoic acid (4.2%), 18-hydroxyoctadecanoic acid (1.6%), and hexadecanoic acid (2.4%). These results show that flower petal cutin is very similar to leaf cutin of V. faba. Developing petals readily incorporated exogenous [1-¹⁴C]palmitic acid into cutin. Direct conversion of the exogeneous acid into 16-hydroxyhexadecanoic acid, 10, 16-dihydroxy-, and 9, 16-dihydroxyhexadecanoic acid was demonstrated by radio gas-liquid chromatography of their chemical degradation products. About 1% of the exogenous [1-¹⁴C]palmitic acid was incorporated into C₂₇, C₂₉, and C₃₁n-alkanes, which were identified by combined gas-liquid chromatography and mass spectrometry as the major components of the hydrocarbons of V. faba flowers. The radioactivity distribution among these three alkanes (C₂₇, 15%; C₂₉, 48%; C₃₁, 38%) was similar to the per cent composition of the alkanes (C₂₇, 12%; C₂₉, 43%; C₃₁, 44%). [1-¹⁴C]Stearic acid was also incorporated into C₂₇, C₂₉, and C₃₁n-alkanes in good yield (3%). Trichloroacetate, which has been postulated to be an inhibitor of fatty acid elongation, inhibited the conversion of [1-¹⁴C]stearic acid to alkanes, and the inhibition was greatest for the longer alkanes. Developing flower petals also incorporated exogenous C₂₈, C₃₀, and C₃₂ acids into alkanes in 0.5% to 5% yields. [G-³H]n-octacosanoic acid (C₂₈) was incorporated into C₂₇, C₂₉, and C₃₁n-alkanes. [G-³H]n-triacontanoic acid (C₃₀) was incorporated mainly into C₂₉ and C₃₁ alkanes, whereas [9, 10, 11-³H]n-dotriacontanoic acid (C₃₂) was converted mainly to C₃₁ alkane. Trichloroacetate inhibited the conversion of the exogenous acids into alkanes with carbon chains longer than the exogenous acid, and at the same time increased the amount of the direct decarboxylation product formed. These results clearly demonstrate direct decarboxylation as well as elongation and decarboxylation of exogenous fatty acids, and thus constitute the most direct evidence thus far obtained for an elongation-decarboxylation mechanism for the biosynthesis of alkanes.     

Assimilation of Alkanes and Alkenes by Fungi

A group of filamentous fungi were assayed for their ability to utilize a series of n-alkanes and 1-alkenes as the sole source of carbon. Although strains of Cunninghamella exhibited profuse growth on most of the hydrocarbons tested, the majority of fungi tested were able to produce definite growth on one or more of the compounds. The hydrocarbons with a 14-carbon chain length appeared to be more consistently utilized than any other. Strains of Aspergillus appeared to differ in their capacity to utilize individual members of the hydrocarbon series. Thin-layer chromatographic analyses of ether extracts from C. blakesleeana grown on n-tetradecane and 1-tetradecene were similar and revealed the presence of a monocarboxylic acid, a primary alcohol, and a secondary alcohol.     

Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.     

Taxonomic significance of the distribution of constituents of leaf cuticular waxes of Croton species (Euphorbiaceae)

Foliar epicuticular waxes of specimens of 13 Croton species native in Brazil were extracted. The fractions containing alkanes and primary alcohols were isolated by preparative thin layer chromatography. Derivatized n-primary alcohols were identified by gas chromatography (GC) coupled with mass spectrometry and n-alkanes by GC and comparison with known standards. Relative abundances were estimated by GC coupled with flame ionization detector. The distribution of constituents of both classes was analyzed by cluster analysis, using the UPGMA method and Euclidean distances. The chemical affinities among species were compared with published data of molecular phylogenetic relationships. The distribution of n-alkanes and primary alcohols were shown to be useful markers of Croton species. Primary alcohols were more consistent than n-alkanes for species fingerprinting.     

Microbial Oxidation of Gaseous Hydrocarbons: Production of Secondary Alcohols from Corresponding n-Alkanes by Methane-Utilizing Bacteria

Over 20 new strains of methane-utilizing bacteria were isolated from lake water and soil samples. Cell suspensions of these and of other known strains of methane-utilizing bacteria oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol). The product secondary alcohols accumulated extracellularly. The rate of production of secondary alcohols varied with the organism used for oxidation. The average rate of 2-propanol, 2-butanol, 2-pentanol, and 2-hexanol production was 1.5, 1.0, 0.15, and 0.08 μmol/h per 5.0 mg of protein in cell suspensions, respectively. Secondary alcohols were slowly oxidized further to the corresponding methylketones. Primary alcohols and aldehydes were also detected in low amounts (rate of production were 0.05 to 0.08 μmol/h per 5.0 mg of protein in cell suspensions) as products of n-alkane (propane and butane) oxidation. However, primary alcohols and aldehydes were rapidly metabolized further by cell suspensions. Methanol-grown cells of methane-utilizing bacteria did not oxidize n-alkanes to their corresponding secondary alcohols, indicating that the enzymatic system required for oxidation of n-alkanes was induced only during growth on methane. The optimal conditions for in vivo secondary alcohol formation from n-alkanes were investigated in Methylosinus sp. (CRL-15). The rate of 2-propanol and 2-butanol production was linear for the 40-min incubation period and increased directly with cell protein concentration up to 12 mg/ml. The optimal temperature and pH for the production of 2-propanol and 2-butanol were 40°C and pH 7.0. Metalchelating agents inhibited the production of secondary alcohols. The activities for the hydroxylation of n-alkanes in various methylotrophic bacteria were localized in the cell-free particulate fractions precipitated by centrifugation between 10,000 and 40,000 × g. Both oxygen and reduced nicotinamide adenine dinucleotide were required for hydroxylation activity. The metal-chelating agents inhibited hydroxylation of n-alkanes by the particulate fraction, indicating the involvement of a metal-containing enzyme system in the oxidation of n-alkanes. The production of 2-propanol from the corresponding n-alkane by the particulate fraction was inhibited in the presence of methane, suggesting that the subterminal hydroxylation of n-alkanes may be catalyzed by methane monooxygenase.     

Biosynthetic origin of the saw-toothed profile in δC and δΗ of n-alkanes and systematic isotopic differences between n-, iso- and anteiso-alkanes in leaf waxes of land plants

The n-fatty acids containing an even number of carbons (ECN-n-FAs) in higher plants are biosynthesised by repetitive addition of a two carbon unit from malonyl-ACP. The n-alkanes containing an odd number of carbon atoms (OCN-n-alkanes) are generally formed by the decarboxylation of ECN-n-FAs, but it is unknown how the less abundant even-carbon-numbered alkanes (ECN-n-alkanes) are biosynthesised in higher plants.There is a distinctive compositional pattern of incorporation of stable carbon (¹³C) and hydrogen (²H) isotopes in co-existing ECN- and OCN-n-alkanes in leaves of higher plants, such that the OCN n-alkanes are relatively enriched in ¹³C but relatively depleted in ²H against the ECN-n-alkanes. This is consistent with the OCN-n-fatty acids having a propionate precursor which is derived from reduction of pyruvate. A tentative pathway is presented with propionate produced by enzymatic reduction of pyruvate which is then thio-esterified with CoSH (coenzyme A thiol) in the chloroplast to form the terminal precursor molecule propionyl-CoA. This is then repetitively extended/elongated with the 2-carbon unit from malonyl-ACP to form the long chain OCN-n-fatty acids.The anteiso- and iso-alkanes in Nicotiana tabacum leaf waxes have previously been found to be systematically enriched in ¹³C compared with the n-alkanes by Grice et al. (2008). This is consistent with the isotopic composition of their putative respective precursors (pyruvate as precursor for n-alkanes, valine for iso-alkanes and isoleucine for anteiso-alkanes). The current study complements that of Grice et al. (2008) and looks at the distribution of hydrogen isotopes. The n-alkanes were found to be more enriched in deuterium (²H) than the iso-alkanes which in turn were more enriched than the anteiso-alkanes. We propose therefore that the depletion of ²H in the iso-alkanes, relative to the n-alkanes is the consequence of accepting highly ²H-depleted hydrogen atoms from NADPH during their biosynthesis. The anteiso-alkanes are further depleted again because there are three NADPH-derived hydrogen atoms in their precursor isoleucine, as compared with only one NADPH-derived hydrogen in valine, the precursor of the iso-alkanes.     

Alkanes of the essential oil of Cannabis sativa

A waxy fraction obtained by column chromatography of the essential oil of Cannabis sativa consists of n-alkanes ranging from C₉ to C₃₉, 2-methyl and 3-methyl alkanes and some dimethyl alkanes. The qualitative and quantitative composition of this fraction has been compared with the alkane fraction obtained by extraction of the herb.     

The Rheology of 6-Propyl Duodecane and 5-Dibutyl Nonane by Non-Equilibrium Molecular Dynamics Simulations

Abstract

In recent papers, we reported non-equilibrium molecular dynamics (NEMD) simulations of planar Couette flow for liquid n- and i-butane, and liquid n-decane and 4-propyl heptane, using two collapsed atom models and an atomistically detailed model. It was found that the collapsed atom models predict the viscosities of the n-butane and n-decane quite well, and that the atomistically detailed model does not yield quantitative agreement with the viscosity of the n-alkanes or the branched alkanes, but it does have the one positive feature that the calculated viscosities of the branched alkanes are higher than these of the n-alkanes. In the present paper, we report results of NEMD simulations of planar Couette flow for liquid 6-propyl duodecane and 5-dibutyl nonane at 296 K and 0.782 g/cc, using an expanded collapsed atom model for simplicity. The strain rate dependent viscosity shows three different regions—two shear thinning ones and a Newtonian one. The slopes of the log-log plot for the branched-chain alkanes at high strain rate are different from those at intermediate strain rate, which is characterized as a rheological behavior of branched-chain alkanes. The Newtonian viscosity of the branched-chain alkanes can be extrapolated from the plateau value of the strain rate dependent viscosity at low strain rate as for straight-chain alkanes [J. Chem. Phys., 105, 1214 (1996)]. The results indicate that more-branched alkanes have a larger viscosity than less-branched C₁₇ alkanes.     

Degradation of Car Engine Base Oil by Rhodococcus sp. NDKK48 and Gordonia sp. NDKY76A

Two microorganisms (NDKK48 and NDKY76A) that degrade long-chain cyclic alkanes (c-alkanes) were isolated from soil samples. Strains NDKK48 and NDKY76A were identified as Rhodococcus sp. and Gordonia sp., respectively. Both strains used not only normal alkane (n-alkane) but also c-alkane as a sole carbon and energy source, and the strains degraded more than 27% of car engine base oil (1% addition).     

The direct role of aerobic heterotrophic bacteria associated with cyanobacteria in the degradation of oil compounds

This study aimed at evaluating the role of cyanobacteria and their associated aerobic heterotrophic bacteria in biodegradation of petroleum compounds. We investigated the potential of ten non-axenic typical mat-forming cyanobacterial strains to degrade phenanthrene, pristane, n-octadecane, and dibenzothiophene. Five strains (Aphanothece halophyletica, Dactyolococcopsis salina, Halothece strain EPUS, Oscillatoria strain OSC, and Synechocystis strain UNIGA) were able to degrade n-alkanes. In case of the other five strains (Microcoleus chthonoplastes, Oscillatoria sp. MPI 95 OS 01, Halothece strain EPUG, Halomicronema exentricum, and Phormidium strain UNITF) alkanes were not significantly affected. Moderate changes in the concentration of the aromatic compounds were observed for three isolates only. In follow-up experiments with Oscillatoria strain OSC, we demonstrated that the cyanobacteria-associated aerobic heterotrophic bacteria were responsible for the observed biodegradation. The cyanobacteria themselves apparently do not degrade petroleum compounds, but more likely play a significant, indirect role in biodegradation by supporting the growth and activity of the actual degraders.     

Analysis of alkane-dependent methanogenic community derived from production water of a high-temperature petroleum reservoir

Microbial assemblage in an n-alkanes-dependent thermophilic methanogenic enrichment cultures derived from production waters of a high-temperature petroleum reservoir was investigated in this study. Substantially higher amounts of methane were generated from the enrichment cultures incubated at 55 °C for 528 days with a mixture of long-chain n-alkanes (C₁₅–C₂₀). Stoichiometric estimation showed that alkanes-dependent methanogenesis accounted for about 19.8% of the total amount of methane expected. Hydrogen was occasionally detected together with methane in the gas phase of the cultures. Chemical analysis of the liquid cultures resulted only in low concentrations of acetate and formate. Phylogenetic analysis of the enrichment revealed the presence of several bacterial taxa related to Firmicutes, Thermodesulfobiaceae, Thermotogaceae, Nitrospiraceae, Dictyoglomaceae, Candidate division OP8 and others without close cultured representatives, and Archaea predominantly related to uncultured members in the order Archaeoglobales and CO₂-reducing methanogens. Screening of genomic DNA retrieved from the alkanes-amended enrichment cultures also suggested the presence of new alkylsuccinate synthase alpha-subunit (assA) homologues. These findings suggest the presence of poorly characterized (putative) anaerobic n-alkanes degraders in the thermophilic methanogenic enrichment cultures. Our results indicate that methanogenesis of alkanes under thermophilic condition is likely to proceed via syntrophic acetate and/or formate oxidation linked with hydrogenotrophic methanogenesis.     

Biodegradation of Variable-Chain-Length Alkanes at Low Temperatures by a Psychrotrophic Rhodococcus sp.

The psychrotroph Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5°C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C₁₀ to C₂₁ alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5°C. Mineralization of hexadecane at 5°C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction–gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis thcA gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25°C.     

Degradation of Hydrocarbons by Members of the Genus Candida II. Oxidation of n-Alkanes and 1-Alkenes by Candida lipolytica

Candida lipolytica ATCC 8661 was grown in a mineral-salts hydrocarbon medium. n-Alkanes and 1-alkenes with 14 through 18 carbon atoms were used as substrates. Ether extracts of culture fluids and cells obtained from cultures grown on the various substrates were analyzed by thin-layer and gas-liquid chromatography. Analyses of fluids from cultures grown on n-alkanes indicated a predominance of fatty acids and alcohols of the same chain length as the substrate. In addition, numerous other fatty acids and alcohols were present. Analyses of saponifiable and nonsaponifiable material obtained from the cells revealed essentially the same products. The presence of primary and secondary alcohols, as well as fatty acids, of the same chain length as the n-alkane substrate suggested that attack on both the methyl and α-methylene group was occurring. The significance of these two mechanisms in the degradation of n-alkanes by this organism was not evident from the data presented. Analyses of fluids from cultures grown on 1-alkenes indicated the presence of 1,2-diols, as well as ω-unsaturated fatty acids, of the same chain length as the substrate. Alcohols present were all unsaturated. Saponifiable and nonsaponifiable material obtained from cells contained essentially the same products. The presence of 1,2-diols and ω-unsaturated fatty acids of the same chain length as the substrate from cultures grown on 1-alkenes indicated that both the terminal methyl group and the terminal double bond were being attacked.     

<Emphasis Type="Italic">n</Emphasis>-Alkane assimilation and <Emphasis Type="Italic">tert</Emphasis>-butyl alcohol (TBA) oxidation capacity in <Emphasis Type="Italic">Mycobacterium austroafricanum</Emphasis> strains

Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C₂ to C₁₆). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C₁ to C₁₆) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism.     

Effect of cuticular waxes compounds from table grapes on growth,germination and gene expression in <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

Botrytis cinerea attacks a broad range of host causing significant economic losses in the worldwide fruit export industry. Hitherto, many studies have focused on the penetration mechanisms used by this phytopathogen, but little is known about the early stages of infection, especially those such as adhesion and germination. The aim of this work was to evaluate the effect of cuticular waxes compounds from table grapes on growth, germination and gene expression of B. cinerea. To accomplish this, growth was analyzed using as substrate n-alkanes extracted from waxes of fresh fruit (table grapes, blueberries and apricots). Subsequently, the main compounds of table grape waxes, oleanolic acid (OA) and n-fatty alcohols, were mixed to generate a matrix on which conidia of B. cinerea were added to assess their effect on germination and expression of bctub, bchtr and bchex genes. B. cinerea B05.10, isolated from grapes, increased its growth on a matrix composed by table grapes n-alkanes in comparison to a matrix made with n-alkanes from apricot or blueberries. Moreover, at 2.5 h, B05.10 germination increased 17 and 33 % in presence of n-alkanes from table grape, in comparison to conditions without alkanes or with blueberries alkanes, respectively. Finally, expression of bchtr and bchex showed a significant increase during the first hour after contact with n-fatty alcohols and OA. In conclusion, B. cinerea displays selectivity towards certain compounds found in host waxes, mainly n-fatty alcohols, which could be a good candidate to control this phytopathogen in early stages of infection.