Anaerobic n-alkane metabolism by a sulfate-reducing bacterium, Desulfatibacillum aliphaticivorans strain CV2803T

The alkane-degrading, sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803T, recently isolated from marine sediments, was investigated for n-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of D. aliphaticivorans on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that D. aliphaticivorans strain CV2803T oxidizes n-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic n-alkane metabolism by D. aliphaticivorans is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids.     

Anaerobic 1-Alkene Metabolism by the Alkane- and Alkene-Degrading Sulfate Reducer Desulfatibacillum aliphaticivorans Strain CV2803T

The alkane- and alkene-degrading, marine sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803^T, known to oxidize n-alkanes anaerobically by fumarate addition at C-2, was investigated for its 1-alkene metabolism. The total cellular fatty acids of this strain were predominantly C-(even number) (C-even) when it was grown on C-even 1-alkenes and predominantly C-(odd number) (C-odd) when it was grown on C-odd 1-alkenes. Detailed analyses of those fatty acids by gas chromatography-mass spectrometry after 6- to 10-week incubations allowed the identification of saturated 2- and 4-ethyl-, 2- and 4-methyl-, and monounsaturated 4-methyl-branched fatty acids with chain lengths that correlated with those of the 1-alkene. The growth of D. aliphaticivorans on (per)deuterated 1-alkenes provided direct evidence of the anaerobic transformation of these alkenes into the corresponding 1-alcohols and into linear as well as 10- and 4-methyl-branched fatty acids. Experiments performed with [¹³C]bicarbonate indicated that the initial activation of 1-alkene by the addition of inorganic carbon does not occur. These results demonstrate that D. aliphaticivorans metabolizes 1-alkene by the oxidation of the double bond at C-1 and by the subterminal addition of organic carbon at both ends of the molecule [C-2 and C-(ω-1)]. The detection of ethyl-branched fatty acids from unlabeled 1-alkenes further suggests that carbon addition also occurs at C-3. Alkylsuccinates were not observed as potential initial intermediates in alkene metabolism. Based on our observations, the first pathways for anaerobic 1-alkene metabolism in an anaerobic bacterium are proposed. Those pathways indicate that diverse initial reactions of 1-alkene activation can occur simultaneously in the same strain of sulfate-reducing bacterium.     

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.     

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 [1,2-¹³C₂]hexadecane or perdeuterated pentadecane was used as the growth substrate, ¹³C-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 ¹³C-labeled 2-, 4-, and 6-methylated fatty acids by mass spectrometry showed that each of them contained two ¹³C atoms, located at the methyl group and the adjacent carbon, thus indicating that the methyl group was the original terminal carbon of the [1,2-¹³C₂]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.     

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.     

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.     

Mono- and Dialkyl Glycerol Ether Lipids in Anaerobic Bacteria: Biosynthetic Insights from the Mesophilic Sulfate Reducer Desulfatibacillum alkenivorans PF2803T

Bacterial glycerol ether lipids (alkylglycerols) have received increasing attention during the last decades, notably due to their potential role in cell resistance or adaptation to adverse environmental conditions. Major uncertainties remain, however, regarding the origin, biosynthesis, and modes of formation of these uncommon bacterial lipids. We report here the preponderance of monoalkyl- and dialkylglycerols (1-O-alkyl-, 2-O-alkyl-, and 1,2-O-dialkylglycerols) among the hydrolyzed lipids of the marine mesophilic sulfate-reducing proteobacterium Desulfatibacillum alkenivorans PF2803^T grown on n-alkenes (pentadec-1-ene or hexadec-1-ene) as the sole carbon and energy source. Alkylglycerols account for one-third to two-thirds of the total cellular lipids (alkylglycerols plus acylglycerols), depending on the growth substrate, with dialkylglycerols contributing to one-fifth to two-fifths of the total ether lipids. The carbon chain distribution of the lipids of D. alkenivorans also depends on that of the substrate, but the chain length and methyl-branching patterns of fatty acids and monoalkyl- and dialkylglycerols are systematically congruent, supporting the idea of a biosynthetic link between the three classes of compounds. Vinyl ethers (1-alken-1′-yl-glycerols, known as plasmalogens) are not detected among the lipids of strain PF2803^T. Cultures grown on different (per)deuterated n-alkene, n-alkanol, and n-fatty acid substrates further demonstrate that saturated alkylglycerols are not formed via the reduction of hypothetic alken-1′-yl intermediates. Our results support an unprecedented biosynthetic pathway to monoalkyl/monoacyl- and dialkylglycerols in anaerobic bacteria and suggest that n-alkyl compounds present in the environment can serve as the substrates for supplying the building blocks of ether phospholipids of heterotrophic bacteria.     

Microbial Incorporation of Fatty Acids Derived From n-Alkanes Into Glycerides and Waxes

When n-alkanes with 13 to 20 carbon atoms were fed to a Nocardia closely related to N. salmonicolor, the produced cellular triglycerides and aliphatic waxes invariably contained fatty acids with an even or an odd number of carbon atoms subject to this feature of the n-alkane substrate. Beta-oxidation and C₂ addition are both operative, as evidenced by the spectra of fatty acids incorporated into the cellular lipid components. There is no distinction in the rate of microbial incorporation of the odd-or even-numbered carbon chains. The fatty acids are apparently directly derived from the long chain n-alkanes, rather than synthesized via the classic C₂-condensation route. The alcohol component of waxes produced by the Nocardia is invariably of the same chain length as the n-alkane substrate.     

Growth and cellular fatty-acid composition of a sulphate-reducing bacterium, Desulfatibacillum aliphaticivorans strain CV2803T, grown on n-alkenes

The anaerobic degradation of n-alkenes by a sulphate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803T was investigated. Results suggest that enzymes required for alkene degradation are inducible. Moreover, total cellular fatty acids of strain CV2803T were predominantly C-odd when the strain was grown on C-odd substrates and C-even when grown on C-even substrates. In addition to classical bacterial fatty acids, unusual 4-Me-17:1delta11 and 4-Me-18:1delta11 fatty acids and their saturated homologues were detected when strain CV2803T was grown on 1-pentadecene and 1-hexadecene, respectively. These methyl-branched monounsaturated fatty acids could constitute specific metabolites of n-alkene degradation by sulphate-reducing bacteria.     

Effect of Substrate on the Fatty Acid Composition of Hydrocarbon- and Ketone-utilizing Microorganisms

The fatty acid pattern in hydrocarbon- and ketone-utilizing bacteria after growth on various substrates was examined. The fatty acid composition of one hydrocarbon-utilizing organism (Mycobacterium sp. strain OFS) was investigated in detail after growth on n-alkanes, 1-alkenes, ketones, and n-alcohols. n-Alkanes shorter than C₁₃ or longer than C₁₇ were not incorporated into cellular fatty acids without some degradation. Strain OFS incorporated C₁₄ to C₁₇ 1-alkenes into cellular fatty acids as the ω-monoenoic fatty acid. Methyl ketones were incorporated into strain OFS after removal of one- or two-carbon fragments from the carbonyl end of the molecule. An organism isolated by enrichment on methyl ketones was incapable of n-alkane utilization but could grow on, although not incorporate, ketones or long chain n-alcohols into cellular fatty acids.     

Sequencing and analysis of plasmids pAV1 and pAV2 ofAcinetobacter venetianus VE-C3 involved in diesel fuel degradation

Acinetobacter venetianus strain VE-C3 was isolated in the Venice lagoon (Italy) as a strain able to degrade diesel fuel oil. This strain possesses genes of the alkane monoxygenase complex responsible forn-alkane degradation and carries two plasmids, pAV1 (10820 bp) and pAV2 (15135 bp), which were supposed from the analysis of Alk mutant strains to harbour genetic determinants for hydrocarbon degradation. In this work we determined the nucleotide sequence of both plasmids and showed the presence of a putative aldehyde dehydrogenase gene, essential for hydrocarbon degradation, on plasmid pAV2, and of an ORF similar toalkL gene present on pAV1 plasmid. These data, combined with genetic reports indicating that strains lacking one of the two plasmids or carrying transposon insertion on pAV1, are defective inn-alkane degradation, suggest a complex genomic organisation of genes involved in alkane degradation inA. venetianus VE-C3. In this bacterium these genes are carried by both the chromosome and the plasmids, while inAcinetobacter sp. strain ADP1 and M1 all the genes for alkane monoxygenase complex are located only on the chromosome.     

Anaerobic oxidation of long-chain n-alkanes by the hyperthermophilic sulfate-reducing archaeon,Archaeoglobus fulgidus

The thermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain VC-16 (DSM 4304), which is known to oxidize fatty acids and n-alkenes, was shown to oxidize saturated hydrocarbons (n-alkanes in the range C₁₀–C₂₁) with thiosulfate or sulfate as a terminal electron acceptor. The amount of n-hexadecane degradation observed was in stoichiometric agreement with the theoretically expected amount of thiosulfate reduction. One of the pathways used by anaerobic microorganisms to activate alkanes is addition to fumarate that involves alkylsuccinate synthase as a key enzyme. A search for genes encoding homologous enzymes in A. fulgidus identified the pflD gene (locus-tag AF1449) that was previously annotated as a pyruvate formate lyase. A phylogenetic analysis revealed that this gene is of bacterial origin and was likely acquired by A. fulgidus from a bacterial donor through a horizontal gene transfer. Based on three-dimensional modeling of the corresponding protein and molecular dynamic simulations, we hypothesize an alkylsuccinate synthase activity for this gene product. The pflD gene expression was upregulated during the growth of A. fulgidus on an n-alkane (C₁₆) compared with growth on a fatty acid. Our results suggest that anaerobic alkane degradation in A. fulgidus may involve the gene pflD in alkane activation through addition to fumarate. These findings highlight the possible importance of hydrocarbon oxidation at high temperatures by A. fulgidus in hydrothermal vents and the deep biosphere.     

Regulation of Enzyme Synthesis of the Glyoxylate,the Citric Acid,and the Methylcitric Acid Cycles in Candida lipolytica

Syntheses of the key enzymes of the glyoxylate cycle, in Candida lipolytica, were highly repressed by glucose. Syntheses of the key enzymes of the methylcitric acid cycle were also slightly repressed by glucose but the degrees of repression in the syntheses of these enzymes were nearly equal to those of repression in the syntheses of several enzymes of the citric acid cycle. All enzyme syntheses repressed by glucose were derepressed during incubation with succinate as well as with n-alkanes: enzyme syntheses of the methylcitric acid cycle did not necessitate the addition of propionate or odd-carbon n-alkanes. The enzymes of the methylcitric acid cycle seem to be constitutive, similarly as those of the citric acid cycle.

In the parent strain, the respective enzyme levels of the cells grown on an odd-numbered n-alkane were similar to those of the cells grown on an even-numbered n-alkane. But in the mutant strain lacking 2-methylisocitrate lyase, the cells grown on the odd-numbered alkane contained aconitate hydratase, NADP-Iinked isocitrate dehydrogenase, isocitrate lyase, 2- methylcitrate synthase and 2-methylaconitate hydratase all at higher levels than the cells grown on the even-numbered alkane. Both the parent cells and the mutant cells grown on the same carbon source contained at individually similar levels of the following six enzymes; citrate synthase, NAD-linked isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase, and malate synthase. The pleiotropic changes of enzyme activities in the mutant cells grown on the odd-numbered alkane seem to be ascribable to direct or indirect stimulation caused by threo-d_s-2-methylisocitric acid accumulation.     

Fatty acid composition in the classification of Saccharopolyspora hirsuta

The long-chain fatty acids of 4 Saccharopolyspora hirsuta strains were examined as their methyl and picolinyl esters using gas liquid chromatography (GLC) and GLC-mass spectrometry (GLC-MS). All the strains had similar fatty acid profiles composed mainly of isoanteiso-, and 10-methyl-branched components. Three new substituted 10-methyl-branched fatty acids were also detected and the major component identified as 10,15-dimethylhexadecanoic acid. The implications of these data for Saccharopolyspora systematics are discussed.     

Carotenoid production from n-alkanes with a broad range of chain lengths by the novel species Gordonia ajoucoccus A2T

A novel diesel-degrading bacterial strain, A2^T, was isolated from soil that was heavily contaminated with oil. Based on phenotypic, phylogenetic, and DNA analyses, strain A2^T was identified as a novel species of the genus Gordonia and named Gordonia ajoucoccus A2^T (KCTC 11900BP and CECT8382). G. ajoucoccus A2^T is able to synthesize carotenoids and produces mainly γ-carotene and keto-γ-carotene. G. ajoucoccus A2^T is also capable of assimilating n-alkanes with a broad range of chain lengths (C6, C8–C25). Batch culture of G. ajoucoccus A2^T in a bioreactor containing 1 % (v/v) hexadecane or 1 % (v/v) commercial diesel yielded 25 mg L^?1 and 2.6 mg L^?1 of carotenoids, respectively. Gas chromatography/mass spectrometry (GC-MS) analysis of hexadecane and hexane degradation metabolites suggested that G. ajoucoccus A2^T may possess a terminal oxidation pathway that allows it to utilize n-alkanes and hexane as carbon and energy sources. G. ajoucoccus A2^T could therefore serve as a good model system for understanding microbial n-alkane degradation pathways. Additionally, the metabolic capabilities of G. ajoucoccus A2^T suggest potential biotechnological applications, such as the bioproduction of carotenoids from industrial discharge or other sources of n-alkanes.     

The Genome of the Moderate Halophile Amycolicicoccus subflavus DQS3-9A1T Reveals Four Alkane Hydroxylation Systems and Provides Some Clues on the Genetic Basis for Its Adaptation to a Petroleum Environment

The moderate halophile Amycolicicoccus subflavus DQS3-9A1^T is the type strain of a novel species in the recently described novel genus Amycolicicoccus, which was isolated from oil mud precipitated from oil produced water. The complete genome of A. subflavus DQS3-9A1^T has been sequenced and is characteristic of harboring the genes for adaption to the harsh petroleum environment with salinity, high osmotic pressure, and poor nutrient levels. Firstly, it characteristically contains four types of alkane hydroxylases, including the integral-membrane non-heme iron monooxygenase (AlkB) and cytochrome P450 CYP153, a long-chain alkane monooxygenase (LadA) and propane monooxygenase. It also accommodates complete pathways for the response to osmotic pressure. Physiological tests proved that the strain could grow on n-alkanes ranging from C10 to C36 and propane as the sole carbon sources, with the differential induction of four kinds of alkane hydroxylase coding genes. In addition, the strain could grow in 1–12% NaCl with the putative genes responsible for osmotic stresses induced as expected. These results reveal the effective adaptation of the strain DQS3-9A1^T to harsh oil environment and provide a genome platform to investigate the global regulation of different alkane metabolisms in bacteria that are crucially important for petroleum degradation. To our knowledge, this is the first report to describe the co-existence of such four types of alkane hydroxylases in a bacterial strain.     

Microbial assimilation of hydrocarbons: cellular distribution of fatty acids

The distribution of cellular fatty acids in defined lipid classes was analyzed in Micrococcus cerificans after growth on specified hydrocarbons. Neutral lipid, phospholipid, and cell residue fatty acids were qualitatively and quantitatively determined for M. cerificans grown on nutrient broth, tetradecane (C(14)), pentadecane (C(15)), hexadecane (C(16)), and heptadecane (C(17)), respectively. Percentage of total cellular fatty acid localized in defined lipid classes from cells grown on the above growth substrates was (i) neutral lipid-11.8, 1.81, 7.74, 23.1, and 2%; (ii) phospholipid-74.5, 65, 66.43, 62.1, and 86%; (iii) cell residue lipid-13.5, 33.29, 25.82, 14.78, and 11.9%. Phospholipid fatty acid chain length directly reflected the carbon number of the alkane substrate, with 40, 84, 98, and 77% of the fatty acids being 14, 15, 16, and 17 carbons when cells were grown on C(14), C(15), C(16), and C(17)n-alkanes, respectively. The bound lipids of the cell residue after chloroform-methanol extraction were characterized by 2-hydroxydodecanoic and 2-hydroxytetradecanoic acids plus a broad spectrum of fatty acids ranging from C(10) to C(17) chain length. An increase in total unsaturated fatty acid localized in the phospholipids was noted from cells grown on alkanes greater than 15 carbons long. An extracellular accumulation of free fatty acid (FFA) was demonstrated in hexadecane-grown cultures that was not apparent in non-hydrocarbon-grown cultures. Identification of extracellular FFA demonstrated direct derivation from hexadecane oxidation. Studies supporting inhibition of de novo fatty acid biosynthesis in relationship to extracellular FFA and hexadecane oxidation are described. The ability to alter the fatty acid composition of membrane polar lipids in a predictable manner by the alkane carbon source provides an excellent model system for the investigation of membrane structure-function relationships in M. cerificans.     

Desulfovibrio oceani subsp. oceani sp. nov., subsp. nov. and Desulfovibrio oceani subsp. galateae subsp. nov., novel sulfate-reducing bacteria isolated from the oxygen minimum zone off the coast of Peru

Two deltaproteobacterial sulfate reducers, designated strain I.8.1^T and I.9.1^T, were isolated from the oxygen minimum zone water column off the coast of Peru at 400 and 500 m water depth. The strains were Gram-negative, vibrio-shaped and motile. Both strains were psychrotolerant, grew optimally at 20°C at pH 7.0–8.0 and at 2.5–3.5% NaCl (w/v). The strains grew by utilizing hydrogen/acetate, C_3–4 fatty acids, amino acids and glycerol as electron acceptors for sulfate reduction. Fumarate, lactate and pyruvate supported fermentative growth. Sulfate, sulfite, thiosulfate and taurin supported growth as electron acceptors. Both strains were catalase-positive and highly oxygen-tolerant, surviving 24 days of exposure to atmospheric concentrations. MK6 was the only respiratory quinone. The most prominent cellular fatty acid was iso-17:1-ω9c (18%) for strain I.8.1^T and iso-17:0-ω9c (14%) for strain I.9.1^T. The G+C contents of their genomic DNA were 45–46 mol%. Phylogenetic analysis of 16S rRNA and dsrAB gene sequences showed that both strains belong to the genus Desulfovibrio. Desulfovibrio acrylicus DSM 10141^T and Desulfovibrio marinisediminis JCM 14577^T represented their closest validly described relatives with pairwise 16S rRNA gene sequence identities of 98–99%. The level of DNA-DNA hybridization between strains I.8.1^T and I.9.1^T was 30–38%. The two strains shared 10–26% DNA-DNA relatedness with D. acrylicus. Based on a polyphasic investigation it is proposed that strains I.8.1^T and I.9.1^T represent a novel species for which the name Desulfovibrio oceani sp. nov. is proposed with the two subspecies D. oceani subsp. oceani (type strain, I.8.1^T = DSM 21390^T = JCM 15970^T) and D. oceani subsp. galateae (type strain, I.9.1^T = DSM 21391^T = JCM 15971^T).     

Cellular lipid accumulation by Pseudomonas aeruginosa 44T1

Summary Growth of Pseudomonas aeruginosa strain 44T1 on glucose, an n-alkane mixture or olive oil was characterized by the formation of intracellular lipid inclusions and extracellular accumulation of rhamnolipids. Maximum values of cellular lipid accumulation were obtained in olive-oil-grown cells and reached up to 38% w/w of its dry biomass. The principal fatty acids of cellular lipids drived from P. aeruginosa cultures varied with the carbon source employed. The major fatty acids detected were palmitic and trans-oleic acids. Arachidonic acid was only found in medium containing glucose or the n-alkane mixture. Offprint requests to: A. Manresa