Isolation and characterization of Rhodococcus sp. strains TMP2 and T12 that degrade 2,6,10,14-tetramethylpentadecane (pristane) at moderately low temperatures

Branched alkanes including 2,6,10,14-tetramethylpentadecane (pristane) are more resistant to biological degradation than straight-chain alkanes especially under low-temperature conditions, such as 10 degrees C. Two bacterial strains, TMP2 and T12, that are capable of degrading pristane at 10 degrees C were isolated and characterized. Both strains grew optimally at 30 degrees C and were identified as Rhodococcus sp. based on the 16S rRNA gene sequences. Strain T12 degraded comparable amounts of pristane in a range of temperatures from 10 to 30 degrees C and strain TMP2 degraded pristane similarly at 10 and 20 degrees C but did not degrade it at 30 degrees C. These data suggest that the strains have adapted their pristane degradation system to moderately low-temperature conditions.     

The fluidity of DOPC bilayers and membrane fractions prepared from murine plasmacytoma cells is unchanged after incorporation of pristane (2,6,10,14-tetramethylpentadecane) as assessed by fluorescence polarization analysis.

The nature of the plasmacytomagenic activity of pristane (2,6,10,14-tetramethylpentadecane) is poorly defined. However, evidence for tumor promoting properties of pristane has recently come forward that includes direct cellular effects on B lymphocytes; i.e., the plasmacytoma precursor cell. Bly et al. (Cancer Biochem. Biophys. 11, 1990, 145-154) observed changed membrane fluidities in lymphocytes after administration of pristane in vivo. We measured steady-state fluorescence polarization using DPH (1,6-diphenyl-1,3,5-hexatriene) and APCL (1-acyl-2-[12-(9-anthryl)-11-trans-dodecenoyl]-sn-glycero-3- phosphocholine) as probes in DOPC (L-alpha-dioleoylphosphatidylcholin) model membranes and membrane fractions derived from plasmacytoma cells after incorporation of pristane in vitro. In a previous investigation, we verified the in vitro uptake of pristane into DOPC bilayers under the conditions employed here (Gawrisch and Janz, Biochim. Biophys. Acta 1070, 1991, 409-418). However, neither in DOPC bilayers nor in plasmacytoma membrane fractions could we detect changes in fluorescence polarization after in vitro incorporation of pristane within reasonable error limits. Therefore, we suggest that the observed alterations in membrane fluidity in lymphocytes from pristane-treated animals are the indirect result of the in vivo treatment but not a direct effect of pristane on membrane fluidity.     

Isolation and functional characterization of hydrocarbon emulsifying and solubilizing factors produced by a Pseudomonas species

Pseudomonas PG-1 cultivated on pristane produced in good amount a heat-stable polymeric substance which showed strong hydrocarbon emulsifying and solubilizing properties. The substance was isolated in crude form and was found to contain 34% protein, 16% carbohydrate, and 40% lipid. The hydrocarbon solubilizing activity of the isolate was strongly inhibited by EDTA but the chelating agent had no effect on the hydrocarbon emulsifying activity. Both activities of the isolate were strongly inhibited by chymotrypsin treatment indicating the importance of the protein moiety for its activity. Hydrocarbon solubilization by the isolate showed a certain degree of specificity to pristane in modest agitation generally used in microbial cultivation, but this specificity was lost by vigorous agitation in a Waring blender. It was proposed that in the first case, solubilization was effected by a solubilizing factor specific to pristane, whereas in the latter case, nonspecific solubilization occurred due to the action of the emulsifying factor. The rate of pristane solubilization by heat-treated culture broth under the conditions of agitation used in cultivation (rotary shaker, 120 rpm) was found to be ca. 750 mg L(-1) h(-1) which was much larger than the maximal pristane uptake rate of 170 mg L(-1) h(-1) observed during microbial growth on the substrate. It was concluded that hydrocarbon solubilization could satisfactorily account for the substrate uptake and growth.     

Pristane induced gene activation.

Studies were performed to examine the effects of 2,6,10,14-tetramethyl pentadecane (pristane) versus 12-O-tetradecanoylphorbol 13-acetate (TPA) on the activation of the CAT gene under the regulatory control of viral promoter/enhancer elements transfected into NIH-3T3, CV-1 and COS-7 cells. The results of these studies demonstrated that (1) pristane or TPA induced trans-activation of SV2cat, HIVcat, RSVcat and MMTVcat in cells transfected with each respective plasmid construct, (2) only pristane induced activation of pA10cat and pOSP/11 and (3) neither TPA nor pristane trans-activated pSV0cat. Furthermore, treatment with either pristane or TPA elicited changes in the morphology of each of the cell lines. Collectively these results indicate that pristane is a potent inducer of gene expression and exhibits similar characteristics as the tumor promoter, TPA.     

Identification of alkane hydroxylase genes in Rhodococcus sp. strain TMP2 that degrades a branched alkane

Rhodococcus sp. TMP2 is an alkane-degrading strain that can grow with a branched alkane as a sole carbon source. TMP2 degrades considerable amounts of pristane at 20 degrees C but not at 30 degrees C. In order to gain insights into microbial alkane degradation, we characterized one of the key enzymes for alkane degradation. TMP2 contains at least five genes for membrane-bound, non-heme iron, alkane hydroxylase, known as AlkB (alkB1-5). Phylogenetical analysis using bacterial alkB genes indicates that TMP2 is a close relative of the alkane-degrading bacteria, such as Rhodococcus erythropolis NRRL B-16531 and Q15. RT-PCR analysis showed that expressions of the genes for AlkB1 and AlkB2 were apparently induced by the addition of pristane at a low temperature. The results suggest that TMP2 recruits certain alkane hydroxylase systems to utilize a branched alkane under low temperature conditions.     

On the origin of pristane in marine organisms

Phytol-U-(14)C was adsorbed on algae and ingested in this form by zooplanktonic copepods (two species of Calanus). The lipids of these animals were analyzed after 48 hr and found to contain radioactive pristane and radioactive phytanic acid. The conversion of phytol to pristane by the copepods is interpreted as a likely biological source of pristane in nature.     

Alcanivorax which prevails in oil-contaminated seawater exhibits broad substrate specificity for alkane degradation

Alcanivorax is an alkane-degrading marine bacterium which propagates and becomes predominant in crude-oil-containing seawater when nitrogen and phosphorus nutrients are supplemented. In order to understand why Alcanivorax overcomes other bacteria under such cultural conditions, competition experiments between Alcanivorax indigenous to seawater and the exogenous alkane-degrading marine bacterium, Acinetobacter venetianus strain T4, were conducted. When oil-containing seawater supplemented with nitrogen and phosphorus nutrients was inoculated with A. venetianus strain T4, this bacterium was the dominant population at the early stage of culture. However, its density began to decrease after day 6, and Alcanivorax predominated in the culture after day 20. The crude-oil-degrading profiles of both bacteria were therefore investigated. Alcanivorax borkumensis strain ST-T1 isolated from the Sea of Japan exhibited higher ability to degrade branched alkanes (pristane and phytane) than A. venetianus strain T4. It seems that this higher ability of Alcanivorax to degrade branched alkanes allowed this bacterium to predominate in oil-containing seawater. It is known that some marine zooplanktons produce pristane and Alcanivorax may play a major role in the biodegradation of pristane in seawater.     

Pristane induced changes in rat lymphocyte membrane fluidity

The ability of pristane (2,6,10,14-tetramethylpentadecane) to act as a membrane perturbant was examined. Data obtained from rats treated with pristane by either intraperitoneal injection or the diet indicated there were significant increases over normal in the amount of pristane in lymphoid cells; 50-89% was incorporated into the plasma membranes. Fluorescence polarization analyses, using 1,6-diphenyl-1,3,5-hexatriene, of normal plasma membrane isolates demonstrated that splenic and Peyer's patch lymphocytic membranes were more viscous than those of the thymus, mesenteric lymph nodes or peripheral blood. Studies to assess the effects of pristane on membrane viscosity demonstrated that there were significant differences in the viscosities of plasma membrane isolates from lymphocytes of normal versus pristane treated rats. The observed changes were dependent on route of administration, length of exposure and the lymphoid organ examined.     

The uptake of pristane (2,6,10,14-tetramethylpentadecane) into phospholipid bilayers as assessed by NMR, DSC, and tritium labeling methods.

Unilamellar dioleoylphosphatidylcholine (DOPC) liposomes (250 microM) incorporated 2 mol% of [3H]pristane at 37 degrees C after addition of 50 microM pristane solubilized with beta-cyclodextrin. Conventional solubilization in dimethyl sulphoxide resulted in much lower uptake. Premixing of perdeuterated pristane with DOPC and dipalmitoylphosphatidylcholine (DPPC) prior to the formation of multilamellar liposomes resulted in homogeneous incorporation of up to 5 mol% pristane at 22 degrees C and 50 degrees C, respectively, as observed by 2H-NMR. Lipid order parameters measured by 31P and 2H-NMR remained unchanged after pristane uptake. Pristane induced the transformation of part of the dioleoylphosphatidylethanolamine (DOPE)/DOPC (3:1, mol/mol) liquid crystalline lamellar phase into an inverse hexagonal phase. 5 mol% pristane in DPPC bilayers decreased the midpoint of the main phase transition temperature of DPPC from 41.5 degrees C to 40.9 degrees C. Upon cooling in the temperature range from 41 degrees C to 36 degrees C, pristane was either displaced from the DPPC bilayer or the mode of incorporation changed. These results may aid in defining the mechanisms whereby pristane, an isoprenoid C19-isoalkane, induces plasmacytomagenesis in mice.     

Conformational changes in the DNA of hybridoma cells from pristane treated mice

The effects of pristane on the DNA of hybridoma cells propagated as ascitic tumors in pristane-primed BALB/c mice were determined using flow cytometric analyses. Hybridoma cells maintained in vitro or cell isolates from solid tumors which developed in unprimed mice injected with hybridoma cells exhibited similar propidium iodide (PI) staining characteristics. In contrast, PI stained cells isolated from ascites which developed in pristane-primed mice injected with the hybridoma cells displayed significant decreases in fluorescence intensity. Diphenylamine studies and analyses of pH 10 treated cells indicated that the actual DNA content of the hybridoma cells was not altered by exposure to pristane. Furthermore, the altered staining characteristics of the ascitic tumor cells were reversible in that the fluorescence intensity after serial in vitro passage of the ascites cells was similar to that of the parent cell line which had not been exposed to pristane. In addition, there was a direct correlation between the altered PI staining characteristics and the presence of cell-associated pristane as determined by gas-liquid chromatography analyses of cell extracts. Collectively these results suggest that pristane may have a direct effect on the DNA conformation of hybridoma cells which may in turn enhance their growth as ascitic tumors. The possible role of such an altered DNA conformation in hybridoma cells on the in vivo development of ascites is discussed.     

Carbon source-induced modifications in the mycolic acid content and cell wall permeability of Rhodococcus erythropolis E1

The influence of the carbon source on cell wall properties was analyzed in an efficient alkane-degrading strain of Rhodococcus erythropolis (strain E1), with particular focus on the mycolic acid content. A clear correlation was observed between the carbon source and the mycolic acid profiles as estimated by high-performance liquid chromatography and mass spectrometry. Two types of mycolic acid patterns were observed after growth either on saturated linear alkanes or on short-chain alkanoates. One type of pattern was characterized by the lack of odd-numbered carbon chains and resulted from growth on linear alkanes with even numbers of carbon atoms. The second type of pattern was characterized by mycolic acids with both even- and odd-numbered carbon chains and resulted from growth on compounds with odd-numbered carbon chains, on branched alkanes, or on mixtures of different compounds. Cellular short-chain fatty acids were twice as abundant during growth on a branched alkane (pristane) as during growth on acetate, while equal amounts of mycolic acids were found under both conditions. More hydrocarbon-like compounds and less polysaccharide were exposed at the cell wall surface during growth on alkanes. Whatever the substrate, the cells had the same affinity for aqueous-nonaqueous solvent interfaces. By contrast, bacteria displayed completely opposite susceptibilities to hydrophilic and hydrophobic antibiotics and were found to be strongly stained by hydrophobic dyes after growth on pristane but not after growth on acetate. Taken together, these data show that the cell wall composition of R. erythropolis E1 is influenced by the nutritional regimen and that the most marked effect is a radical change in cell wall permeability.     

Carbon Source-Induced Modifications in the Mycolic Acid Content and Cell Wall Permeability of Rhodococcus erythropolis E1

The influence of the carbon source on cell wall properties was analyzed in an efficient alkane-degrading strain of Rhodococcus erythropolis (strain E1), with particular focus on the mycolic acid content. A clear correlation was observed between the carbon source and the mycolic acid profiles as estimated by high-performance liquid chromatography and mass spectrometry. Two types of mycolic acid patterns were observed after growth either on saturated linear alkanes or on short-chain alkanoates. One type of pattern was characterized by the lack of odd-numbered carbon chains and resulted from growth on linear alkanes with even numbers of carbon atoms. The second type of pattern was characterized by mycolic acids with both even- and odd-numbered carbon chains and resulted from growth on compounds with odd-numbered carbon chains, on branched alkanes, or on mixtures of different compounds. Cellular short-chain fatty acids were twice as abundant during growth on a branched alkane (pristane) as during growth on acetate, while equal amounts of mycolic acids were found under both conditions. More hydrocarbon-like compounds and less polysaccharide were exposed at the cell wall surface during growth on alkanes. Whatever the substrate, the cells had the same affinity for aqueous-nonaqueous solvent interfaces. By contrast, bacteria displayed completely opposite susceptibilities to hydrophilic and hydrophobic antibiotics and were found to be strongly stained by hydrophobic dyes after growth on pristane but not after growth on acetate. Taken together, these data show that the cell wall composition of R. erythropolis E1 is influenced by the nutritional regimen and that the most marked effect is a radical change in cell wall permeability.     

Changes in iso- and n-alkane distribution during biodegradation of crude oil under nitrate and sulphate reducing conditions

Crude oil consists of a large number of hydrocarbons with different susceptibility to microbial degradation. The influence of hydrocarbon structure and molecular weight on hydrocarbon biodegradation under anaerobic conditions is not fully explored. In this study oxygen, nitrate and sulphate served as terminal electron acceptors (TEAs) for the microbial degradation of a paraffin-rich crude oil in a freshly contaminated soil. During 185 days of incubation, alkanes from n-C11 to n-C39, three n- to iso-alkane ratios commonly used as weathering indicators and the unresolved complex mixture (UCM) were quantified and statistically analyzed. The use of different TEAs for hydrocarbon degradation resulted in dissimilar degradative patterns for n- and iso-alkanes. While n-alkane biodegradation followed well-established patterns under aerobic conditions, lower molecular weight alkanes were found to be more recalcitrant than mid- to high-molecular weight alkanes under nitrate-reducing conditions. Biodegradation with sulphate as the TEA was most pronounced for long-chain (n-C32 to n-C39) alkanes. The observation of increasing ratios of n-C17 to pristane and of n-C18 to phytane provides first evidence of the preferential degradation of branched over normal alkanes under sulphate reducing conditions. The formation of distinctly different n- and iso-alkane biodegradation fingerprints under different electron accepting conditions may be used to assess the occurrence of specific degradation processes at a contaminated site. The use of n- to iso-alkane ratios for this purpose may require adjustment if applied for anaerobic sites.     

Degradation of the multiple branched alkane 2,6,10,14-tetramethyl-pentadecane (pristane) in Rhodococcus ruber and Mycobacterium neoaurum

Pristane, a highly branched hydrocarbon that also contains iso-branched termini, was used as a substrate for several alkane-metabolizing bacteria. Rhodococcus ruber and Mycobacterium neoaurum were able to utilize pristane for growth effectively. The intermediates produced by these bacteria during incubation with pristane were analyzed by gas chromatography (GC) and gas chromatography/mass spectra (GC/MS). The products revealed as products of 4-methyl pentanoic acid; methyl butanedioic acid; 2-methyl pentadioic acid; methyl propanedioic acid; 4-methyl heptanedioic acid; and 2,6,10,14-tetramethyl-pentadecan-3-one were detected in M. neoaurum cultures. In R. ruber, methyl butanedioic acid; 2-methyl pentadioic acid; 4,8-dimethylnonanoic acid, 4-methyl heptanedioic acid; 2,6,10-trimethylundecanoic acid; 3,7-dimethyl decanedioic acid; and 2,6,10,14-tetramethyl-pentadecan-3-one were detected. The occurrence of these intermediates showed that pristane could be catabolized not only via mono- but also by a di-terminal oxidation pathway. Furthermore, the presence of 2,6,10,14-tetramethyl-pentadecan-3-one; 3,7-dimethyldecandioate; and 2-methylbutandioate established a third pathway initiated by sub-terminal oxidation at the third carbon atom of pristane. Novel intermediates detected suggest simultaneous sub-terminal and di-terminal oxidation pathways.     

Pristane induced effects on chromatin of rat lymphoid cells

The effects of pristane on the conformation of chromatin in cells isolated from the lymphoid tissues of pristane-treated Copenhagen rats were examined by flow cytometry, thermal denaturation, sensitivity to enzymatic digestion, and histone protein analyses. Decreases were observed in the fluorescent intensities of propidium iodide (PI) stained nuclei isolated from lymphoid cells of pristane-treated rats when compared with normal rat lymphoid nuclei. Studies to address the possible basis for the pristane-induced changes in the DNA staining characteristics of lymphocytes demonstrated that 1) there were no decreases in the amount of DNA present in the nuclei, 2) nuclei isolated from pristane treated rats were less sensitive to thermal denaturation, as well as DNase I enzymatic digestion, and 3) there were apparent increases in the expression of the H1 histone proteins. Collectively, these results suggest that pristane elicits a conformational change in the chromatin which may be mediated by altered expression of nuclear-associated histone proteins.     

Biosynthesis of fatty acids and triacylglycerols by 2,6,10,14-tetramethyl pentadecane-grown cells of Nocardia globerula 432

Nocardia globerula strain 432 was able to synthesize triacylglycerols (TAG) during cultivation on 2,6,10,14-tetramethyl pentadecane (pristane) under nitrogen-limiting conditions. Within these cells, 4,8,12-trimethyl tridecanoic acid was the major fatty acid detected. Fatty acids with an odd number of carbon atoms and minor amounts of even-numbered fatty acids were also observed. Experiments carried out with acrylic acid, an inhibitor of beta-oxidation, suggested that odd-numbered fatty acids such as C15:0, C17:0 and 10-methyl C17:0 were synthesized de novo using propionyl-CoA, the beta-oxidation product, as precursor. Although N. globerula 432 incorporated mainly straight chain fatty acids into TAG, the branched fatty acid 4,8,12-trimethyl tridecanoic acid also appeared, to some extent, in the acylglycerols. The importance of TAG biosynthesis by pristane-grown cells of N. globerula strain 432 is discussed.     

On-Site Treatment of Contaminated Soils: An Approach to Bioremediation of Weathered Petroleum Compounds

A bench-scale investigation was conducted prior to on-site bioremediation of 52,000 cubic yards of contaminated soil containing weathered, structurally complex petroleum compounds from an inactive oil refinery. Addition of bulking agents was required to improve soil physical properties. A supplemental study was also conducted to evaluate the effectiveness of bio-enhancement products. Loss of n-alkanes was rapid in soil mixtures containing a high nitrogen sludge compost, but very slow in mixtures containing wood products as bulking agents. By completion of the study at day 110, the isoprenoids pristane and phytane had nearly disappeared from mixtures containing sludge compost. Clearly, pristane and phytane are inadequate biomarkers when conditions favor an advanced stage of biodegradation. Nearly half the complex branched and cyclic alkanes in the unresolved complex mixture also degraded. After 70 days, depletion of dibenzo-thiophenes and phenan-threnes was 75 and 90%, respectively. The most stable PAHs within each group were the highly methylated homologues. Because of their complex structures, both steranes and hopanes were stable in all soil mixtures. Data were normalized to hopanes as a conserved internal standard or biomarker. Use of hopane-normalized data successfully eliminated much of the data variability and permitted a more accurate assessment of biodegradation. A relatively slow decline in total hydrocarbons occurred later in the study. This slowing tendency of microbial utilization is caused not only by substrate depletion, but also because remaining hydrocarbons are structurally more complex and persistent. Because of this, it is important to avoid using kinetic data from early stages of bioremediation to predict later hydrocarbon losses, such as the time required to attain a cleanup standard. In the supplemental study, an oleophilic fertilizer product accelerated hydrocarbon degradation when compared with a conventional fertilizer. This product will be tested in combination with organic bulking agents under field conditions to determine its cost effectiveness.     

Differential Effects of TPA and Pristane on Gene Expression and Transformation in Mouse Epidermal Cells

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) or 2,6,10,14-tetramethylpentadecane (pristane) on gene expression and transformation were examined using two clones (P⁺, TPA transformation sensitive and P^-, TPA resistant) of the mouse epidermal cell line JB6. Results from transformation studies indicated pristane was more efficient, i.e., lower concentrations were required to elicit an equivalent response, in transforming the P⁺, but not the P^-, clone of JB6 compared to TPA. Furthermore, results from these studies demonstrated either TPA or pristane was effective in the transactivation of the chloramphenicol acetyltransferase gene under the regulatory control of most viral promoter/enhancer elements transfected into the P⁺, but not the P^-, clone of JB6. However, if a consensus cAMP response element was linked to the simian virus 40 early promoter, pristane activation was observed in both P⁺ and P^- cells. The differential effects of these two compounds suggest that while they have similar characteristics, they may utilize different pathways to elicit their effects.     

Estimation of pristane levels in murine ascites fluid and in purified monoclonal antibody preparations

A simple method for the rapid analysis of pristane has been designed using a heptane extract of the aqueous sample followed by separation and detection by gas chromatography. Ascites fluid lots from three monoclonal antibodies (MAbs) and their purified preparations were analysed using the procedure. The highest detected level of pristane was 600 μg/mL in a single lot of ascites fluid with other values less than 140 μg/ml. For the purified samples, no pristane was detected down to the demonstrated limit of 4 μg/mg of MAb.