Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5

Alcanivorax dieselolei strain B-5 is a marine bacterium that can utilize a broad range of n-alkanes (C(5) -C(36) ) as sole carbon source. However, the mechanisms responsible for this trait remain to be established. Here we report on the characterization of four alkane hydroxylases from A. dieselolei, including two homologues of AlkB (AlkB1 and AlkB2), a CYP153 homologue (P450), as well as an AlmA-like (AlmA) alkane hydroxylase. Heterologous expression of alkB1, alkB2, p450 and almA in Pseudomonas putida GPo12 (pGEc47ΔB) or P. fluorescens KOB2Δ1 verified their functions in alkane oxidation. Quantitative real-time RT-PCR analysis showed that these genes could be induced by alkanes ranging from C(8) to C(36) . Notably, the expression of the p450 and almA genes was only upregulated in the presence of medium-chain (C(8) -C(16) ) or long-chain (C(22) -C(36) ) n-alkanes, respectively; while alkB1 and alkB2 responded to both medium- and long-chain n-alkanes (C(12) -C(26) ). Moreover, branched alkanes (pristane and phytane) significantly elevated alkB1 and almA expression levels. Our findings demonstrate that the multiple alkane hydroxylase systems ensure the utilization of substrates of a broad chain length range.     

红灯食烷菌(Alcanivorax hongdengensis)黄素结合单加氧酶(AlmA)的基因克隆及其烷烃诱导表达

【目的】研究海洋烷烃降解菌新种模式菌株Alcanivorax hongdengensis A-11-3降解长链烷烃的分子机制。【方法】PCR克隆编码黄素结合单加氧酶的基因序列,利用生物信息学软件对序列进行分析,运用RT-PCR和实时荧光定量PCR技术分析基因在不同烷烃诱导下的表达水平。【结果】从菌株A-11-3中克隆获得了两个黄素结合单加氧酶基因片段(almA1和almA2)。它们编码的氨基酸序列与菌株Acinetobacter sp.DSM17874的AlmA同源性分别为58.6%和53.2%。实时荧光定量PCR分析表明,almA1基因只在长链烷烃(C28-C32)的诱导下上调表达,而almA2基因中能在更宽范围的长链烷烃(C24-C34)和支链烷烃诱导下上调表达。两者均在C9-C22的烷烃诱导下没有上调表达。【结论】黄素结合单加氧酶可能是A-11-3降解长链烷烃和支链烷烃的关键酶。     

Selective hydroxylation of alkanes by an extracellular fungal peroxygenase

Fungal peroxygenases are novel extracellular heme-thiolate biocatalysts that are capable of catalyzing the selective monooxygenation of diverse organic compounds, using only H(2)O(2) as a cosubstrate. Little is known about the physiological role or the catalytic mechanism of these enzymes. We have found that the peroxygenase secreted by Agrocybe aegerita catalyzes the H(2)O(2)-dependent hydroxylation of linear alkanes at the 2-position and 3-position with high efficiency, as well as the regioselective monooxygenation of branched and cyclic alkanes. Experiments with n-heptane and n-octane showed that the hydroxylation proceeded with complete stereoselectivity for the (R)-enantiomer of the corresponding 3-alcohol. Investigations with a number of model substrates provided information about the route of alkane hydroxylation: (a) the hydroxylation of cyclohexane mediated by H(2)(18)(2) resulted in complete incorporation of (18)O into the hydroxyl group of the product cyclohexanol; (b) the hydroxylation of n-hexane-1,1,1,2,2,3,3-D(7) showed a large intramolecular deuterium isotope effect [(k(H)/k(D))(obs)] of 16.0 ± 1.0 for 2-hexanol and 8.9 ± 0.9 for 3-hexanol; and (c) the hydroxylation of the radical clock norcarane led to an estimated radical lifetime of 9.4 ps and an oxygen rebound rate of 1.06 × 10(11) s(-1). These results point to a hydrogen abstraction and oxygen rebound mechanism for alkane hydroxylation. The peroxygenase appeared to lack activity on long-chain alkanes (> C(16)) and highly branched alkanes (e.g. tetramethylpentane), but otherwise exhibited a broad substrate range. It may accordingly have a role in the bioconversion of natural and anthropogenic alkane-containing structures (including alkyl chains of complex biomaterials) in soils, plant litter, and wood.     

Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase

LadA, a long-chain alkane monooxygenase, utilizes a terminal oxidation pathway for the conversion of long-chain alkanes (up to at least C₃₆) to corresponding primary alcohols in thermophilic bacillus Geobacillus thermodenitrificans NG80-2. Here, we report the first structure of the long-chain alkane hydroxylase, LadA, and its complex with the flavin mononucleotide (FMN) coenzyme. LadA is characterized as a new member of the SsuD subfamily of the bacterial luciferase family via a surprising structural relationship. The LadA:FMN binary complex structure and a LadA:FMN:alkane model reveal a hydrophobic cavity that has dual roles: to provide a hydrogen-bond donor (His138) for catalysis and to create a solvent-free environment in which to stabilize the C4a-hydroperoxyflavin intermediate. Consequently, LadA should catalyze the conversion of long-chain alkanes via the acknowledged flavoprotein monooxygenase mechanism. This finding suggests that the ability of LadA to catalyze the degradation of long-chain alkanes is determined by the binding mode of the long-chain alkane substrates. The LadA structure opens a rational perspective to explore and alter the substrate binding site of LadA, with potential biotechnological applications in areas such as petroleum exploration and treatment of environmental oil pollution.     

Microsomal preparation from an animal tissue catalyzes release of carbon monoxide from a fatty aldehyde to generate an alkane

Alkanes are widely distributed in nature and impaired alkane synthesis was implicated in certain neurological disorders. However, the mechanism of synthesis of alkanes in animals is unknown. Our search to find a convenient animal tissue to study alkane biosynthesis resulted in the finding that the uropygial gland (a modified sebaceous gland) of the eared grebe (Podiceps nigricollis) produces large amounts of alkanes. These alkanes, which constitute 35-41% of the total lipid produced, are mainly C21, C23, C25, and C27 n-alkanes. Cell free homogenates of this tissue synthesized alkanes from both fatty acid and aldehyde in the absence of O2. Differential centrifugation of the homogenates indicated that this activity was located in the microsomal fraction. With isolated microsomes conversion of fatty acid to alkane required CoA, ATP, and NADH whereas conversion of an aldehyde to alkane did not require the addition of cofactors. That the final step in alkane synthesis is a decarbonylation was shown by the stoichiometric production of heptadecane and CO from octadecanal. CO was identified by adsorption to RhCl [(C6H6)3P]3 and oxidation of the trapped CO to CO2 by watergas shift reaction. The enzyme preparation also catalyzed incorporation of 14C from 14CO into octadecanal showing the reversible nature of the decarbonylase. This decarbonylase had a sharp pH optimum at 7.0, a Kapp of 180 microM and a V1/2 of 90 rho mol/min/mg protein for octadecanal. The enzyme was inhibited by the metal chelators EDTA, O-phenanthroline, and 8-hydroxyquinoline, but not by KCN. It was stimulated nearly 3-fold by 5 microM 2-mercaptoethanol and inhibited by the presence of O2. During the conversion of [1-3H]octadecanal to heptadecane, 3H was lost to water and 3H from 3H2O was incorporated into the alkane generated from unlabeled octadecanal. The mechanism of the decarbonylation and the nature of the enzyme remain to be elucidated.     

一株不动杆菌降解石油烃的特性及关键烷烃降解基因分析

【背景】Acinetobacter sp. Tust-DM21 (GenBank登录号KX390866)是本实验室前期从渤海湾海洋石油勘探船废油收集区采集的水油混合样中分离出的一株高效石油降解菌,其对短、中、长链烷烃均表现出很强的降解能力,有较好的应用前景。【目的】从应用层面探究其最佳降解条件,同时从生物信息层面探究其降解基因的作用。【方法】将其在不同温度、pH下培养144h,通过GC-MS内标法测定石油烃各组分的变化情况,计算出其最佳降解条件;同时,通过生物信息学手段确定基因组中的降解基因,每个基因分别选择7个同源基因,对它们的蛋白序列进行比较;最后对2个降解基因在0-144 h的表达情况进行了Real-time PCR分析。【结果】Acinetobacter sp. Tust-DM21最佳降解条件为35°C、pH 8.5,该条件下对石油降解率可达97.5%,其中,对长链烷烃降解率达98.5%,对环烃为81%,对芳香烃为87%;同时,研究发现基因组中含有常见烷烃降解基因alk B(GenBank登录号MH368539)和长链烷烃降解基因alm A (GenBank登录号MH357335),2个降解基因的蛋白经比较均与其同源蛋白表现出一定的相似性,同属菌的相似性最高;通过Real-timePCR发现这2个基因在0-144 h的相对表达量随时间逐步提高。【结论】Acinetobacter sp. Tust-DM21在最佳降解条件下对石油各组分都显示出了优良的降解能力,特别对长链烷烃的降解能力尤为突出;将2个降解基因的相对表达量结合该时间段的生长趋势,证明了菌株Acinetobacter sp. Tust-DM21的生长和降解与alk B和alm A基因的上调表达存在关联。     

Determining the identity and roles of oil-metabolizing marine bacteria from the Thames estuary, UK

Crude oil is a complex mixture of different hydrocarbons. While diverse bacterial communities can degrade oil, the specific roles of individual members within such communities remain unclear. To identify the key bacterial taxa involved in aerobic degradation of specific hydrocarbons, microcosm experiments were established using seawater from Stanford le Hope, Thames estuary, UK, adjacent to a major oil refinery. In all microcosms, hydrocarbon degradation was significant within 10 weeks, ranging from > 99% of low-molecular-weight alkanes (C(10)-C(18)), 41-84% of high-molecular-weight alkanes (C(20)-C(32)) and pristane, and 32-88% of polycyclic aromatic hydrocarbons (PAHs). Analysis of 16S rRNA sequences from clone libraries and denaturing gradient gel electrophoresis (DGGE) indicated that, except when incubated with fluorene, PAH-degrading communities were dominated by Cycloclasticus. Moreover, PAH-degrading communities were distinct from those in microcosms containing alkanes. Degradation of the branched alkane, pristane, was carried out almost exclusively by Alcanivorax. Bacteria related to Thalassolituus oleivorans (99-100% identity) were the dominant known alkane degraders in n-alkane (C(12)-C(32)) microcosms, while Roseobacter-related bacteria were also consistently found in these microcosms. However, in contrast to previous studies, Thalassolituus, rather than Alcanivorax, was dominant in crude oil-enriched microcosms. The communities in n-decane microcosms differed from those in microcosms supplemented with less volatile alkanes, with a phylogenetically distinct species of Thalassolituus out-competing T. oleivorans. These data suggest that the diversity and importance of the genus Thalassolituus is greater than previously established. Overall, these experiments demonstrate how degradation of different petroleum hydrocarbons is partitioned between different bacterial taxa, which together as a community can remediate petroleum hydrocarbon-impacted estuarine environments.     

Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases

Selection experiments and protein engineering were used to identify an amino acid position in integral membrane alkane hydroxylases (AHs) that determines whether long-chain-length alkanes can be hydroxylated by these enzymes. First, substrate range mutants of the Pseudomonas putida GPo1 and Alcanivorax borkumensis AP1 medium-chain-length AHs were obtained by selection experiments with a specially constructed host. In all mutants able to oxidize alkanes longer than C13, W55 (in the case of P. putida AlkB) or W58 (in the case of A. borkumensis AlkB1) had changed to a much less bulky amino acid, usually serine or cysteine. The corresponding position in AHs from other bacteria that oxidize alkanes longer than C13 is occupied by a less bulky hydrophobic residue (A, V, L, or I). Site-directed mutagenesis of this position in the Mycobacterium tuberculosis H37Rv AH, which oxidizes C10 to C16 alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C10 and C11 alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.     

Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria

We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47 Delta B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C(6) to C(12)), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C(12) to C(16) alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.     

Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. strain DSM 17874

Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C10H22) to that of tetracontane (C40H82) as a sole carbon source. Two genes encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30 degrees C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C32H66) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.     

Initial reactions in anaerobic alkane degradation by a sulfate reducer, strain AK-01

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, isolated from a petroleum-contaminated sediment was studied to elucidate its mechanism of alkane metabolism. Total cellular fatty acids of AK-01 were predominantly C even when it was grown on C-even alkanes and were predominantly C odd when grown on C-odd alkanes, suggesting that the bacterium anaerobically oxidizes alkanes to fatty acids. Among these fatty acids, some 2-, 4-, and 6-methylated fatty acids were specifically found only when AK-01 was grown on alkanes, and their chain lengths always correlated with those of the alkanes. When [1,2-(13)C(2)]hexadecane or perdeuterated pentadecane was used as the growth substrate, (13)C-labeled 2-Me-16:0, 4-Me-18:0, and 6-Me-20:0 fatty acids or deuterated 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0 fatty acids were recovered, respectively, confirming that these monomethylated fatty acids were alkane derived. Examination of the (13)C-labeled 2-, 4-, and 6-methylated fatty acids by mass spectrometry showed that each of them contained two (13)C atoms, located at the methyl group and the adjacent carbon, thus indicating that the methyl group was the original terminal carbon of the [1, 2-(13)C(2)]hexadecane. For perdeuterated pentadecane, the presence of three deuterium atoms, on the methyl group and its adjacent carbon, in each of the deuterated 2-, 4-, and 6-methylated fatty acids further supported the hypothesis that the methyl group was the terminal carbon of the alkane. Thus, exogenous carbon appears to be initially added to an alkane subterminally at the C-2 position such that the original terminal carbon of the alkane becomes a methyl group on the subsequently formed fatty acid. The carbon addition reaction, however, does not appear to be a direct carboxylation of inorganic bicarbonate. A pathway for anaerobic metabolism of alkanes by strain AK-01 is proposed.     

The potential use of long-chain alcohols and fatty acids as diet composition markers: factors influencing faecal recovery rates and diet composition estimates in sheep

The limitations of the alkane technique in estimating the diet components of herbivores call for the introduction of new diet composition markers. Recently, long-chain alcohols (alcohols) and long-chain fatty acids (acids) have received the most attention and show great potential, when combined with alkanes, to estimate composition of complex diets. In the current study, faecal recoveries of alcohols and acids were determined in sheep in four different live weight groups fed three herbage species, either Leymus chinensis, L. dasystachys or Elymus sibiricum. Analysis of variance (ANOVA) was used to examine the effects of herbage species and live weight of sheep on faecal recoveries of individual alcohols and acids. Further, an indoor experiment with six sheep fed a diet of equal proportions, on dry matter (DM) basis, of three herbages was performed, allowing to assess the accuracy of alcohols and/or acids in combination with alkanes, to estimate diet composition. A one-sample t-test was carried out to test the accuracy of these estimates. Results of the first experiment indicated that the faecal recoveries of alcohols and acids were significantly affected by herbage species (P < 0.05). While the effects were significant or near significant for the faecal recoveries of some alcohols (C24-ol, C30-ol and C26-ol) (P 0.05), no effect of live weight on faecal recoveries of acids was observed (P > 0.05). Therefore, adjustments based on diet-specific faecal recoveries might improve diet composition estimates. This was illustrated by the results of the second experiment. The diet composition estimated from alcohols or all combinations of alcohols with other marker types, after diet-specific correction of faecal recoveries, did not significantly differ from the actual composition (P > 0.05). However, using acids as additional markers resulted in poorer diet composition estimates. This study confirmed the utility of alcohols, combined with alkanes, as markers to estimate composition of complex diets. Although corrections based on mean faecal recoveries, average over animals and diets, resulted in some accuracy loss, results were still satisfactory and better than without recovery correction.     

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.     

Effects of leaf water evaporative 2H‐enrichment and biosynthetic fractionation on leaf wax n‐alkane δ2H values in C3 and C4 grasses

Leaf wax n‐alkane δ²H values carry important information about environmental and ecophysiological processes in plants. However, the physiological and biochemical drivers that shape leaf wax n‐alkane δ²H values are not completely understood. It is particularly unclear why n‐alkanes in grasses are typically ²H‐depleted compared with plants from other taxonomic groups such as dicotyledonous plants and why C3 grasses are ²H‐depleted compared with C4 grasses. To resolve these uncertainties, we quantified the effects of leaf water evaporative ²H‐enrichment and biosynthetic hydrogen isotope fractionation on n‐alkane δ²H values for a range of C3 and C4 grasses grown in climate‐controlled chambers. We found that only a fraction of leaf water evaporative ²H‐enrichment is imprinted on the leaf wax n‐alkane δ²H values in grasses. This is interesting, as previous studies have shown in dicotyledonous plants a nearly complete transfer of this ²H‐enrichment to the n‐alkane δ²H values. We thus infer that the typically observed ²H‐depletion of n‐alkanes in grasses (as opposed to dicots) is because only a fraction of the leaf water evaporative ²H‐enrichment is imprinted on the δ²H values. Our experiments also show that differences in n‐alkane δ²H values between C3 and C4 grasses are largely the result of systematic differences in biosynthetic fractionation between these two plant groups, which was on average ?198‰ and?159‰ for C3 and C4 grasses, respectively.     

长链烷烃降解菌的降解特性

对长链烷烃降解菌的降解能力和摄取模式进行了研究。评价14株烃降解菌利用中长链烃生长的能力,发现只有少数烃降解菌能够获得良好生长,其中Mycobacterium fortuitum514,Pseudomonas aeruginosa1785和Pseudomonas marginata766等3株菌能够高效降解C20到C33的长链烷烃。辛烷不能支持这些长链烷烃降解菌的生长,说明其烃氧化酶与Pseudomonas oleovorans的OCT质粒编码的单氧酶不同。此外,M.fortuitum不产胞外表面活性剂,而P.aeruginosa和P.marginata则是表面活性剂产生菌,然而三者在以烃为碳源生长时均显示出很高的细胞表面疏水性。根据生长现象分析3株菌采用了不同的烷烃摄取模式。     

Metabolism of alkyl glyceryl ethers and their noninvolvement in alkane biosynthesis in plants

[1-¹⁴C]Octadecyl glyceryl ether did not label alkanes in the leaves of Brassica oleracea and Pisum sativum while [1-¹⁴C]octadecanol and [1-¹⁴C]octadecanoic acid readily labeled the alkanes. About 40% of the exogenous-labeled glyceryl ether was incorporated intact into choline phosphatide while 10–20% was converted into fatty acids and alcohols. [1-¹⁴C]octadecanol was not converted into alkyl glyceryl ether, but it was oxidized to the corresponding acid and then incorporated into alkanes. These results show that alkyl ether is not an intermediate in alkane biosynthesis. When [1-¹⁴C-1-³H]-octadecanol was fed to the leaves of B. oleracea and P. sativum, only the ¹⁴C and no ³H was incorporated into alkanes, ketones, and secondary alcohols. These results show that fatty alcohols are first oxidized to the acid before being incorporated into alkanes, ruling out fatty alcohol, alkyl ether, and alk-1-enyl ether as intermediates in alkane biosynthesis. The exogenous alcohols were also readily esterified into wax esters in both tissues.     

Oxidation of long-chain alkanes by Acetobacter rancens

Summary The degradation of hexadecane and tetradecane by Acetobacter rancens CCM 1774 was investigated. It was found that this strain is able to grow to a limited extent on hexadecane as a carbon source. The occurrence of n-alkanoic acids and alcohols among the reaction products of growing as well as resting cells indicates a monoterminal degradation of long-chain alkanes. Both alkane-grown and glucose-grown resting cells exhibited alkane oxidizing activities which were not influenced by chloramphenicol. This suggested a constitutive nature of the appropriate enzymes.     

Alkanes are not innocuous vehicles for hydrophobic reagents in membrane studies

Alkanes (C6-C16) are often used as vehicles for hydrophobic reagents, e.g. long-chain ceramides, in cell biology studies. It is generally understood that they are inert solvents, particularly when added in small volumes. However, simple calculations show that, under standard experimental conditions in cell studies, alkane: phospholipid molar ratios in excess of 1000:1 may be found. Even at much smaller ratios (close to 1:1) our studies with liposomes show that alkanes induce vesicle aggregation. Differential scanning calorimetry shows marked changes in both the gel-fluid and the lamellar-hexagonal transitions. Alkanes inhibit bacterial sphingomyelinase when acting on large unilamellar vesicles, and activate bacterial phospholipase C under the same conditions. Thus, the use of alkanes in cell studies requires strict control experiments to avoid artefactual results.