Adaptation of the Hydrocarbonoclastic Bacterium Alcanivoraxborkumensis SK2 to Alkanes and Toxic Organic Compounds: aPhysiological and Transcriptomic Approach

Daniela J. Naether,a* Slavtscho Slawtschew,b Sebastian Stasik,b Maria Engel,b Martin Olzog,b Lukas Y. Wick,c Kenneth N. Timmis,a

Hermann J. Heipieperb

Department of Environmental Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germanya; Department of Environmental Biotechnology,Helmholtz Centre for Environmental Research, Leipzig, Germanyb; Department of Environmental Microbiology, Helmholtz Centre for Environmental Research,Leipzig, Germanyc

The marine hydrocarbonoclastic bacterium Alcanivorax borkumensis is able to degrade mixtures of n-alkanes as they occur inmarine oil spills. However, investigations of growth behavior and physiology of these bacteria when cultivated with n-alkanes ofdifferent chain lengths (C6 to C30) as the substrates are still lacking. Growth rates increased with increasing alkane chain lengthup to a maximum between C12 and C19, with no evident difference between even- and odd-numbered chain lengths, before de-creasing with chain lengths greater than C19. Surface hydrophobicity of alkane-grown cells, assessed by determination of the wa-ter contact angles, showed a similar pattern, with maximum values associated with growth rates on alkanes with chain lengthsbetween C11 and C19 and significantly lower values for cells grown on pyruvate. A. borkumensis was found to incorporate andmodify the fatty acid intermediates generated by the corresponding n-alkane degradation pathway. Cells grown on distinct n-al-kanes proved that A. borkumensis is able to not only incorporate but also modify fatty acid intermediates derived from the al-kane degradation pathway. Comparing cells grown on pyruvate with those cultivated on hexadecane in terms of their tolerancetoward two groups of toxic organic compounds, chlorophenols and alkanols, representing intensely studied organic compounds,revealed similar tolerances toward chlorophenols, whereas the toxicities of different n-alkanols were significantly reduced whenhexadecane was used as a carbon source. As one adaptive mechanism of A. borkumensis to these toxic organic solvents, the activ-ity of cis-trans isomerization of unsaturated fatty acids was proven. These findings could be verified by a detailed transcriptomiccomparison between cultures grown on hexadecane and pyruvate and including solvent stress caused by the addition of 1-octa-nol as the most toxic intermediate of n-alkane degradation.

Aunique group of oil-degrading marine gammaproteobacteria,the so-called marine hydrocarbonoclastic bacteria belonging

to the Oceanospirillales, has drawn attention during the last de-cade, and it is the general opinion that these bacteria are of majorimportance for biodegradation of marine crude oil contaminants(1–3). These bacteria can metabolize only a couple of organicacids (acetate and pyruvate) and instead feed on a variety of ali-phatic hydrocarbons (1). Several of these extraordinary marinebacteria, such as Cycloclasticus, Marinobacter, Thalassolituus, Nep-tunomonas, Oleiphilus, Oleispira, and Alcanivorax species, havebeen discovered in the sea all over the world, always occurring invery small abundances (4).

Alcanivorax borkumensis SK2, the most studied species amongthe marine hydrocarbonoclastic bacteria, has so far been culti-vated only on different hydrocarbons and pyruvate as sole carbonand energy sources (4). Due to its ability to utilize a broad varietyof aliphatic hydrocarbons, A. borkumensis blooms during oil spillsand can represent up to 80 to 90% of the associated bacterialcommunity (5, 6). Because of its outstanding importance to biore-mediation of oil-contaminated marine environments, its genomehas been sequenced (3), and proteome studies have also been per-formed (7).

Schneiker et al. (3) showed that in its genome A. borkumensisencodes different pathways for catalyzing the initial oxidation stepof alkane degradation to the corresponding n-alkanol intermedi-ates. In addition to the alkB-encoded monooxygenase system,which is very common among proteobacteria (3, 5, 8), A. borku-

mensis harbors two other enzymes, a cytochrome P450-like sys-tem and a flavin monooxygenase (7).

In addition to the option of degrading aliphatic hydrocarbons,hydrocarbonoclastic bacteria have also developed strategies to ac-cess their hydrophobic substrates. One strategy is the productionof biosurfactants, such as glucolipids, which emulsify aliphatichydrocarbons and lower their surface tension, thereby increasingtheir bioavailability (9). As aliphatic hydrocarbons are not veryabundant in marine environments, A. borkumensis builds up in-tracellular carbon stocks, such as triacylglycerols or wax esters, inorder to survive periods of starvation (2).

Proteomic analysis of A. borkumensis growing on pyruvate incomparison to hexadecane revealed that when the bacteria is cul-tivated with hexadecane, the genes for 97 cytoplasmic and mem-brane-bound proteins were overexpressed (7). However, an inves-

Received 6 March 2013 Accepted 30 April 2013

Published ahead of print 3 May 2013

Address correspondence to Hermann J. Heipieper, hermann.heipieper@ufz.de.

* Present address: Daniela J. Naether, Department of Molecular Biosciences,Goethe University Frankfurt, Frankfurt, Germany.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00694-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.00694-13

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tigation of the growth behavior on different n-alkanes as theyoccur in raw oil is still lacking, as are studies on the adaptive stressresponses of these bacteria.

In this study, a first systematic analysis of growth rates, surfaceproperties, and membrane lipid composition of A. borkumensisSK2 cultivated on a wide range of n-alkanes was carried out inorder to elucidate cell adaptation to the exclusive n-alkane sub-strate spectrum of these bacteria. Two groups of toxic organiccompounds, chlorophenols and alkanols, representing organiccompounds that have been intensely studied with regard to theireffects on bacteria (i.e., toxicity and adaptive response mecha-nisms) (10–12), were used to compare bacteria cultivated onhexadecane or pyruvate, the only known nonalkane substrate, atboth the transcriptional and biochemical levels. The results re-vealed enormous adaptive properties of A. borkumensis SK2 to itssubstrates and to environmental stress on both physiological andgenetic levels.

MATERIALS AND METHODSStrain and chemicals. Alcanivorax borkumensis SK2 is the reference strainfor hydrocarbonoclastic bacteria and has previously been described. Allchemicals were reagent grade and obtained from commercial suppliers.

Culture conditions. A. borkumensis SK2 (NCIMB 11132) was culti-vated at 30°C in a modified ONR7 medium which contains (per liter)NaCl (22.79 g), Na2SO4 (3.98 g), KCl (0.72 g), NaBr (83 mg), NaHCO3

(31 mg), H3BO3 (27 mg), TAPSO {3-[N-tris(hydroxymethyl)methyl-amino]-2-hydroxypropanesulfonic acid} (1.3 g), MgCl2 · 6H2O (11.18 g),CaCl2 · 2H2O (1.46 g), SrCl2 · 6H2O (24 mg), FeCl2 · 4H2O (40 mg),NH4Cl (5.4 g), Na2HPO4 · 7H2O (1.78 g), and Na2HPO4 (0.94 g). Traceelements were from a 500-times concentrated stock solution (final con-centrations, per liter) of MgSO4 · 7H2O (100 mg), FeSO4 · 7H2O (10 mg),MnSO4 · H2O (5 mg), ZnCl2 (6.4 mg), CaCl2 · 6H2O (1 mg), BaCl2 (0.6mg), CuSO4 · 7H2O (0.36 mg), CuSO4 · 5H2O (0.36 mg), H3BO3 (6.5 mg),and EDTA (10 mg). An n-alkane (0.5%, vol/vol) or pyruvate (20 g) wasadded as the carbon and energy source. The pH was adjusted to 7.6 withNaOH. Cells were grown in 100-ml shake cultures in a shaking water bath(Shaker GFL, Burgwedel, Germany) at 200 rpm.

Incubation with toxic organic compounds. Compounds were addedto exponentially growing cells. Cultures were incubated in the presence ofdifferent organic solvents for 3 h. Cells were then harvested, washed twicewith potassium phosphate buffer (50 mM; pH 7.0), and stored at �20 °Cprior to use. Toxicity of the organic compounds was quantified by deter-mining the 50% effective concentration (EC50), i.e., the concentrationthat causes a 50% inhibition of bacterial growth, as described earlier byHeipieper et al. (13). Growth inhibition caused by the toxic compoundswas measured by comparing the differences in growth rates (� h�1) be-tween toxin-treated cultures (�toxin) and control cultures (�control). Thegrowth inhibition of different concentrations of the organic compoundswas defined as the percentage of the growth rate of the toxin-treated cul-ture relative to that of a control culture without toxin addition, calculatedas follows: relative growth rate (%) � (�toxin/�control) � 100.

Lipid extraction, transesterification, and fatty acid analysis. Mem-brane lipids were extracted with chloroform-methanol-water as describedby Bligh and Dyer (14). Fatty acid methyl esters (FAME) were prepared byincubation for 15 min at 95°C in boron trifluoride-methanol, applying themethod of Morrison and Smith (15). FAME were extracted with hexane.

Analysis of fatty acid composition by GC-MS. Analysis of FAME inhexane was performed by means of gas chromatography-mass spectrom-etry (GC-MS) (GC7890A and MS5975C instruments; Agilent, Boeblin-gen, Germany) with the following parameters: MS source at 230°C, MSquadrupole at 150°C, inlet with splitless mode at 280°C, HP-5ms column(30 m by 0.25 mm by 0.25 �m) at 50°C for 1 min, 4°C min�1 to 250°C, and20°C min�1 to 300°C for 5 min, followed by a 10-min postrun at 300°C.

The mass spectra were evaluated using a library of previously determinedmass spectra of fatty acid standards.

Analysis of fatty acid composition by GC-FID. Analysis of FAME inhexane was performed using a quadrupole GC system (HP5890; HewlettPackard, Palo Alto, CA) equipped with a split/splitless injector and a flameionization detector (FID). A CP-Sil 88 capillary column (Chrompack,Middelburg, The Netherlands) (length, 50 m; inner diameter, 0.25 mm;0.25-�m film) was used for the separation of the FAME. GC conditionswere as follows. The injector temperature was held at 240°C; the detectortemperature was held at 270°C. The injection was splitless, and the carriergas was He at a flow of 2 ml min�1. The temperature program was asfollows: a 2-min isothermal at 40°C, a rise of 8°C min�1 to 220°C, and a15-min isothermal at 220°C. The peak areas of the FAME were used todetermine their relative amounts. The fatty acids were identified by GCwith coinjection of authentic reference compounds obtained from Su-pelco (Bellefonte, USA). The degree of saturation of the membrane fattyacids was defined as the ratio between the saturated fatty acids and theunsaturated fatty acids present in these bacteria.

Characterization of bacterial cell surface hydrophobicities. Physico-chemical cell surface properties of bacteria were investigated using stan-dard methods as described by other investigators (16). Bacterial lawnsneeded for contact angle (�w) measurements were prepared by collectingcell suspensions in 10 mM KNO3 on 0.45-�m-pore-size Micropore filters(Schleicher & Schuell, Dassel, Germany), mounting the filters on glassslides, and drying them for 2 h at room temperature. Cells exposed to thesolvents were washed six times with 10 mM KNO3 in order to guaranteethe measurement of the cell properties and concomitantly avoid measure-ment interference by the physicochemical effect of the solvents adheringto or accumulating in the cells. Cell surface hydrophobicities were derivedfrom the �w of water drops on the bacterial lawns by using a Krüss dropshape analysis system (DSA 100; Krüss GmbH, Hamburg, Germany) (16).According to an earlier classification, cells exhibiting contact angles ofless than �20°, between 20° and 50°, and greater than 50° are hydrophilic,intermediately hydrophilic, and hydrophobic, respectively (17).

Microarray hybridization and data analysis. Total RNA was isolatedfrom the cells by using a RiboPure Bacteria Kit (Ambion, Kaufungen,Germany). The quality of the RNA was analyzed with an Agilent Technol-ogies 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).Total RNA (1,000 ng) was used for Cy3 labeling with a Message AmpIIBacteria Kit according to the recommendation of the supplier (Ambion,Kaufungen, Germany). The in vitro transcription with aminoallyl-labeledUTP (aaUTP) generated Cy3-coupled antisense RNA (aRNA). The aRNAquality was assessed by verifying intact 16S and 23S rRNAs with an AgilentTechnologies 2100 Bioanalyzer and by quantifying the A260/A280 andA260/A230 ratios using the microarray function on the NanoDrop spectro-photometer. A total of 600 ng of the fluorescently labeled aRNA washybridized to a custom microarray for A. borkumensis (8-plex, 15,000oligonucleotides; Agilent Technologies, Waldbronn, Germany) for 17 h at68°C and scanned afterwards with an Agilent DNA Microarray Scanner.The expression values (raw data) were calculated with the software pack-age Feature Extraction, version 10.5.1.1 (Agilent Technologies, Wald-bronn, Germany), using default values for the extraction protocolGE1_107_Sep07.

The data were normalized (quantile normalization) and then globallyscaled using the Genespring GX software package (version 10; AgilentTechnologies). Welch’s t test with unequal variances was first used tocalculate P values and to test the hypothesis that a gene was differentiallyexpressed under treatment and control conditions. The Benjamini-Hoch-berg procedure was then used to correct the P values for multiple hypoth-esis testing and, finally, convert the P values into false-discovery rates(FDRs) (18). For a gene to be classified as differentially expressed undertwo conditions, the false-discovery rates had to be less than 0.05 and thefold difference in hybridization signal intensities had to be greater than 2.Genes that met these criteria were sorted by hierarchical clustering (19)

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with the Genespring GX software package (version 10; Agilent Technolo-gies) and the Genesis software package (20).

Microarray data accession number. The hybridization signals havebeen deposited in the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) (21) under accession number GPL16725 (samplesGSM1088974 to GSM1089004) according to MIAME (minimum infor-mation about a microarray experiment) standards (22).

RESULTSEffect of growth substrate on growth rates, cell surface proper-ties, and membrane fatty acid composition of A. borkumensis.Alcanivorax borkumensis SK2 was cultivated in ONR7 mineral me-dium using different n-alkanes with chain lengths ranging from C6

to C30 as the sole carbon and energy sources. In addition, pyruvate,as the only known nonalkane substrate on which this bacteriumcan be cultivated, was tested as a reference. The bacteria werecultivated in water shake batch cultures with 0.5% (vol/vol) sub-strate. The exponential-phase growth rate, � (h�1), for each sub-strate was calculated, and values are presented in Fig. 1. The aver-age growth rate on pyruvate (0.212 h�1) corresponds to adoubling time of 3.27 h. Generally, growth rates of the bacteria onn-alkanes increased with increasing chain length of the providedn-alkane substrates up to a chain length of C19. The only excep-tions were n-alkanes with chain lengths between C8 and C10, forwhich growth rates were significantly lower than the trend. Thehighest growth rates have been observed with n-alkanes rangingfrom C14 to C19, and a maximum was observed with n-octadecane(C18) as the sole carbon and energy source (�, 0.360; doublingtime, 1.92 h). When cultivated on n-alkanes with chain lengthslonger than C20, growth rates decreased until the bacteria nearlystopped growing at C30.

In order to investigate the effect of different carbon and energysources on surface hydrophobicity, water contact angles were de-termined. Contact angles of pyruvate-grown cultures were con-siderably lower than those of cultures grown on n-alkanes. Trendswere similar to those seen when growth rates were analyzed. In-creasing contact angles were observed with increasing chainlengths of the n-alkanes used as substrates up to a chain length ofC19. With further increases in chain length, contact angles de-creased again slightly (Fig. 1).

As shown previously (23), typical membrane phospholipidfatty acid patterns of A. borkumensis SK2 grown on pyruvate or

n-hexadecane were detected, consisting of C14:0, C16:0, C16:1�9 trans,C16:1�9 cis, C18:0, C18:1�11 trans, and C18:1�11 cis.

When the fatty acid pattern of cells grown on pyruvate wascompared with that of cells grown on n-hexadecane (Fig. 2), bigdifferences in the relative abundances of certain fatty acids wereobserved. The most prominent ones were the relative increases inpalmitic acid (C16:0), stearic acid (C18:0), and cis-vaccenic acid(C18:1 cis) when A. borkumensis SK2 was cultivated on n-hexade-cane. Thus, the bacteria obviously incorporated the acidic degra-dation intermediates of these alkanes directly into their mem-brane phospholipids. This phenomenon is known to occur inseveral bacteria and was recently described in detail for Rhodococ-cus erythropolis (24).

However, fatty acid patterns completely differed when the bac-teria were cultivated on n-alkanes with chain lengths of C11

to C18. When grown on n-alkanes with odd-numbered chainlengths (hereafter termed odd-numbered n-alkanes) (n-unde-cane, n-tridecane, and n-pentadecane), the obtained patterns ofthe membrane fatty acids showed an even more complex and un-expected result. The occurrence of unknown GC-FID peaks indi-cated the presence of fatty acids that had so far not been reportedfor A. borkumensis. However, analysis of the retention times using

FIG 1 Growth rates (bars) and water contact angles (filled circles) of A. borkumensis grown with pyruvate and n-alkanes with different chain lengths as the solecarbon and energy sources.

FIG 2 Fatty acid patterns of A. borkumensis grown with pyruvate (white bars)or hexadecane (black bars) as the sole carbon and energy source.

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the so-called Kovats retention index (25) indicated the presence ofadditional fatty acids with an odd number of carbon atoms, whichwas proven by GC-MS. Pentadecanoic (15:0) fatty acid was mostdominant when A. borkumensis SK2 was cultivated on n-pentade-cane, while tridecanoic (13:0) fatty acid was detected only in n-tridecane-grown cultures. The detection of the 15:0 and 17:0 fattyacids in all cultures grown on odd-numbered n-alkanes, includingn-undecane, indicated an elongation of these incorporated fattyacids. Furthermore, corresponding unsaturated fatty acids withodd-numbered chain lengths (hereafter termed odd-numberedfatty acids) in both cis and trans configurations were detectedwhen A. borkumensis was cultivated on odd-numbered n-alkanes.This observation proved the ability of A. borkumensis SK2 to fur-ther modify incorporated fatty acids by desaturation. These find-ings are in agreement with the genome of the bacterium, in whichtwo genes of putative desaturases (ABO_2546 and ABO_2585)were identified (3).

It was shown that an increased abundance of C16:0 and C14:0 fattyacids correlated with the use of the growth substrates n-hexadecaneand n-tetradecane. Detection of saturated odd-numbered fatty acids(C13:0, C15:0, and C17:0) occurred when cells were grown on n-tride-cane or n-pentadecane. Along with fatty acids corresponding to theodd-numbered n-alkane substrates, longer odd-numbered fatty ac-ids were also detected. C15:0 and C17:0 fatty acids were also found incultures grown on n-undecane, n-tridecane, or n-pentadecane.

After the integration of saturated odd-numbered fatty acidswas verified, the cis and trans unsaturated fatty acids were alsodetected. Their relative amounts were notably smaller than theamounts of the corresponding saturated fatty acids (Fig. 3 andTable 1).

Effect of different organic compounds on the growth of A.borkumensis cultivated on pyruvate or hexadecane. In order totest the effects of different growth substrates on the ability of thisbacterium to tolerate and adapt to toxic organic compounds, asystematic survey of the effects of n-alkanols and (chloro)phenolson the growth of A. borkumensis was carried out. These two groupsof toxic solvents were chosen because n-alkanols are the mainintermediates of n-alkane degradation (26) whereas chlorophe-nols represent the most intensively investigated group of toxicorganic solvents (11, 13). The toxic compounds were added indifferent concentrations to exponentially growing cell cultures.Relative growth rates in the presence of the toxic compounds werecalculated using optical density (OD) values according to themethod described by Heipieper et al. (13). Figure 4 shows therelative growth rates of cells grown on n-hexadecane or pyruvatein the presence of 1-octanol as the tested toxic organic compound.Cells grown on hexadecane showed a significantly higher toler-ance toward 1-octanol than pyruvate-grown bacteria. In addition,the increasing trans/cis ratios indicate a cis-trans isomerase (CTI)activity toward unsaturated fatty acids, as shown in Fig. 4. Thisunique adaptive mechanism has so far been proven only in Pseu-domonas and Vibrio strains (27, 28) as well as in Methylococcuscapsulatus BATH (11). The presence of a CTI in A. borkumensisSK2 was already predicted in a comparative BLAST study (11), asthe CTI gene is also present in the genome (ABO_1700). In thisstudy, we demonstrated a clear dose-dependent physiological ac-tivity of a cis-trans isomerase. The main function of this enzymecan be described as a fast-response mechanism enabling a rapiddecrease in membrane fluidity when membrane-destructive envi-ronmental factors are present (27).

Sensitivity to the tested alkanols and phenols correlated withtheir chain lengths and hydrophobicities, given as the logarithm ofthe ratio of the partition coefficients of 1-octanol and water[log(Po/w), or simply log(P)]. In the case of the n-alkanols, toxicitywas significantly lower for hexadecane-grown cells than for cellsgrown on pyruvate. The difference ranged from a factor of 2.5 to 3.In contrast, the tested chlorophenols did not show differences intoxicity. The results are summarized in Fig. 5 and Table 2.

Transcriptomics during 1-octanol stress on cells cultivatedon pyruvate or hexadecane. The aim of transcriptomic analysiswas the identification of genes that distinctly respond to the addi-tion of 1-octanol, which is known to be the most toxic compoundaccumulating as a degradation intermediate in bacteria grownwith an n-alkane as the substrate (24, 26). The results were filteredfor short-term or long-term responses of the transcription levelsand analyzed for potential adaptation mechanisms.

The EC50s of 1-octanol for both carbon sources (Table 2) wereused as the reference concentrations for induction of toxic stressduring this study in order to guarantee comparable solvent stresssituations for the different carbon sources. The cells were grown toan OD at 600 nm (OD600) of 0.5 to 0.6 before 1-octanol was added(see Fig. S1 in the supplemental material). A control sample wastaken just before stress induction (time point 0). For systematicanalysis of the corresponding transcripts, samples were harvestedat defined time points of 15 min, 30 min, 60 min, and 90 min afterthe perturbation with 1-octanol. Additionally, controls without1-octanol addition were analyzed.

Hierarchical clustering and principle component analysisshowed that replicates of the tested conditions as well as all sam-ples for pyruvate or hexadecane showed distinct clusters. Testedconditions clustered closer to each other than to the control rep-licates (data not shown).

Fifteen minutes after the addition of 1-octanol to cultures witheither carbon source, a strong response of regulated genes wasevident. A. borkumensis grown on pyruvate exhibited upregulatedexpression of 10% of genes (294 of 2,806 genes) and downregu-lated expression of 17% (470 of 2,806) (Table 3). Also, when thebacterium was grown on hexadecane, after 15 min 7% (199 of2,806) of the genes were upregulated and 6% (171 of 2,806) weredownregulated. In the course of the experiment, the number ofgenes with altered expression levels decreased. Only 60 min afterthe addition of 1-octanol addition, in the cultures grown on py-ruvate, 6% of genes (163 of 2,806 genes) were upregulated and 9%(257 of 2,806 genes) were downregulated. On hexadecane, only asmall number of genes were still regulated, with 22 of 2,806 havingincreased and 34 of 2,806 having decreased expression levels. Togain insight into the mechanisms that control the immediatestress reactions and also highlight putative adaptive mechanisms,the data were filtered for genes that showed a minimum of 2-foldregulation at all tested time points in relation to the controls. Here,expression levels of 7% of the genes (191 of 2,806 genes) werefound to be regulated on pyruvate-grown cells, and expressionlevels of 1% of the genes (31 of 2,806 genes) for A. borkumensiscells grown on hexadecane were regulated a minimum of 2-foldand even more 90 min after the addition of 1-octanol. The regu-lation pattern of the genes of pyruvate-grown cells was balanced,with expression levels increasing for 3.5% and decreasing for3.4%. The same relationship was found for stressed hexadecanecultures, where half of the genes were up- or downregulated (0.7%or 0.5%, respectively).

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The genes with differential expression levels for the testedstressed samples in relation to the control were classified into clus-ters of orthologous groups (COGs) (29) according to their pre-dicted or respective/known functions (Fig. 6). About 35%(pyruvate cultures) and 38% (hexadecane cultures) of the regu-

lated genes represent genes that are not classified into COGs or aredescribed with a general or unknown function. A complete list forthe two tested carbon sources and the corresponding expressionlevels of the genes in relation to the control can be found in TablesS1 and S2 in the supplemental material. In the following, the

FIG 3 Gas chromatography patterns of fatty acids obtained from cultures of A. borkumensis grown on pyruvate, n-tridecane, n-tetradecane, n-pentadecane, orn-hexadecane as the sole carbon and energy source. Approximate retention times for C13:0, C15:0, and C17:0 fatty acids are shown in yellow. Approximate retentiontimes for C15:1 and C17:1 fatty acids are shown in red. The peak for myristic acid in n-tetradecane-grown cultures is shown in green. Regular even-numbered fattyacid peaks are labeled only in pyruvate- or n-hexadecane-grown samples. Detailed quantification of the data is presented in Table 1.

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COGs with significant regulation of the expression levels of theirgenes will be discussed for each carbon source.

In pyruvate cultures that were stressed with 1-octanol, thestrongest regulation pattern is due to the downregulation of mo-

tility genes and genes related to signal transduction mechanisms.Seventeen percent of the genes that were assigned by COG analysisto motility functions and 10% of the genes assigned to signaltransduction functions were regulated. In particular, pil genesneeded for pilus and fimbria formation, like pilB (ABO_0614),pilT (ABO_2670 and ABO_2671), pilP (ABO_2234), pilI(ABO_0107), and pilH (ABO_0108), were downregulated. Also,genes coding for proteins related to motility, like a chemotaxisprotein methyltransferase (ABO_1308) and a methyl-acceptingchemotaxis protein (ABO_0106), were downregulated.

Nearly all genes that are necessary and known in A. borkumen-sis to be relevant for the degradation of alkanes were upregulated.These genes can be found in the categories lipid transport, inor-ganic transport and metabolism, and amino acid transport/me-tabolism. Genes for alkane oxidation and �-oxidation and en-zymes from the tricarboxylic acid cycle (TCA) cycle wereupregulated. In accordance with previously described proteomicand other transcriptomics studies (7, 30), the expression level ofthe alkane monooxygenase alkB1 (ABO_2707) was increased.Also, the genes for rubredoxin (ABO_2708), two aldehyde dehy-drogenases (ABO_2414 and ABO_2709 [alkH]), and the alcoholdehydrogenase alkJ2 (ABO_0202) were found to be upregulatedin stressed cells growing on pyruvate.

Furthermore, genes for all necessary enzymes that support

TABLE 2 Relationship between hydrophobicity of different n-alkanolsand chlorophenols and their toxicity to A. borkumensis cultivated withpyruvate or hexadecane as the sole carbon and energy source

Organic compoundHydrophobicity[log(Po/w)]

EC50 (mM)

Pyruvate Hexadecane

1-Butanol 0.88 25.0 60.01-Hexanol 1.87 3.4 9.61-Octanol 2.92 0.66 1.641-Decanol 3.97 0.08 0.17Phenol 1.45 5.31 7.444-Chlorophenol 2.40 0.66 0.702,4-Dichlorophenol 3.20 0.13 0.18

TABLE 1 Effect of pyruvate and n-alkane carbon sources with differentchain lengths on the composition of phospholipid fatty acids in cells ofAlcanivorax borkumensis SK2 at 30°C

Fatty acid

Abundance (%) by carbon sourcea

Pyruvate C13 C14 C15 C16

13:0 ND 11.0 ND ND ND14:0 2.6 0.1 34.6 1.6 4.115:0 ND 12.3 ND 28.9 ND15:1 trans ND 2.3 ND 2.3 ND15:1 cis ND 7.6 ND 11.7 ND16:0 32.6 10.4 20.8 9.8 43.016:1 trans 0.2 0.7 7.9 1.5 1.916:1 cis 28.2 11.1 17.9 10.3 25.817:0 ND 19.9 ND 13.3 ND17:1 trans ND 2.1 ND 0.9 ND17:1 cis ND 14.7 ND 8.6 ND18:0 2.4 0.6 1.9 0.7 15.418:1 trans 0.3 0.1 0.9 0.2 0.418:1 cis 33.7 7.1 15.9 10.1 9.5a ND, not detected. The growth rate, � (h�1), on each carbon source was as follows:pyruvate, 0.21; C13, 0.24; C14, 0.30; C15, 0.31; and C16, 0.29.

FIG 4 Effect of 1-decanol on growth rate (�) and trans/cis ratio of unsatu-rated fatty acids (�) of A. borkumensis grown with pyruvate (A) or hexadecane(B) as the sole carbon and energy source.

FIG 5 Correlation between hydrophobicity, given as the log(P) value, andgrowth inhibition caused by chlorophenols (triangles) and n-alkanols (circles)for A. borkumensis grown with pyruvate (open symbols) or hexadecane (filledsymbols) as the sole carbon and energy source. Growth inhibition is presentedas the EC50s.

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the terminal breakdown of lipids in �-oxidation were upregu-lated. Several enzymes coding for acyl-coenzyme A (CoA) de-hydrogenases that were not described before to be relevant forterminal oxidation were upregulated (ABO_0571, ABO_2102,and ABO_2223). Genes coding for the enoyl-CoA hydratasefadAB (ABO_2452), two hydroxyacyl-CoA dehydrogenases(ABO_0632 and ABO_1713), and a 3-ketoacyl-CoA thiolase(ABO_1567) were upregulated.

The expression levels of several genes coding for heat shockproteins (HSPs) and chaperones were also increased, for example,the heat shock proteins GrpE (ABO_0313), DnaK (ABO_0314),HtpG (ABO_1489), and IbpA (ABO_1777) and two chaperonesubunits (ABO_0633 and ABO_0634).

In addition, the gene for the heat shock protein HtpX(ABO_1180) was found to be upregulated in pyruvate-grown aswell as in hexadecane-grown cells. The same pattern was found for

the alkyl hydroperoxide reductase (ABO_2431), whereas in pyru-vate-grown cells the upregulation was much stronger (87- to 189-fold) than in the hexadecane cultures (highest regulation at 4-foldafter 60 min). Other than these two genes, no genes were found tobe regulated during growth on either of these carbon sources un-der the given parameters (constant regulation of the expressionlevels at more than 2-fold for all tested time points, relative to thecontrol).

In hexadecane cultures, the strongest upregulation could befound in the categories cell cycle control (3% of genes), posttrans-lational control (2%), and replication/recombination (2%). Asdescribed before, the general level of gene regulation is not veryhigh, and there are only some single genes distributed in severalCOGs that are regulated. Only 29 genes are regulated duringgrowth on hexadecane as a carbon source 90 min after perturba-tion with 1-octanol. The highest expression ratio was found forthe genes encoding one hypothetical protein (ABO_1620), anRNA methyltransferase (ABO_2271), and the cell division proteinFtsL (ABO_0591). The genes for alkane oxidation were also up-regulated directly after the addition of 1-octanol, but only after 15and 30 min. Finally, 60 min after the addition of 1-octanol, mostof the genes related to alkane oxidation were no longer regulated(Fig. 7).

DISCUSSION

Bacterial bioremediation of marine oil spills is still present in thepublic consciousness due to the ongoing discussions about com-pensation and responsibilities connected to the environmental ca-

TABLE 3 Changes in expression levels of genes in relation tocategorized open reading frames

Time after 1-octanolperturbation (min)

% of genes regulated during growth on:

Hexadecane Pyruvate

Up Down Up Down

15 7 6 10 1730 2 2 7 1260 1 1 6 990 0.7 0.5 4 3

FIG 6 Classification of differentially expressed genes of A. borkumensis in response to 1-octanol (15 min after perturbation) in cells grown on pyruvate (A) orhexadecane (B) as the carbon source. Gray bars show upregulated and black bars show downregulated genes. Values represent the percentages of open readingframes that were assigned to each COG category.

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tastrophe of the Deepwater Horizon oil spill in the Gulf of Mexico3 years ago (31–33). Here and in other marine environments pol-luted by crude oil, marine hydrocarbonoclastic bacteria such as A.borkumensis were detected as key players in bioremediation (34–37). A. borkumensis had therefore already been intensively studiedwith respect to genetics and metabolic adaptation (2, 3, 7). How-ever, detailed investigations of the growth behavior and adaptiveresponses of these bacteria to their exclusive n-alkane diet werestill lacking.

When the bacteria are grown with different n-alkanes, an in-crease in the growth rate with increasing carbon chain length ofthe n-alkanes used as substrates was observed. It is known thatshort-chain (�11) alkanols, as environmental degradation inter-mediates of n-alkanes, have a toxic effect on most bacteria (10, 12,26, 38, 39). The main reason for the reduced growth rates is thefact that alcohol intermediates of the n-alkane degradation path-way are generated in n-alkane-degrading bacteria, and these inter-mediates are extremely toxic to the cells (40, 41). This explainswhy A. borkumensis showed a massive drop in growth rate when itwas cultivated on n-octane or n-decane. The degradation interme-diates 1-octanol and 1-decanol accumulate in the membrane andcause a drastic increase in the fluidity of the cytoplasmic mem-brane, which then leads to growth inhibition (24, 26).

As n-alkanes are very hydrophobic, their bioavailability be-comes a problem when they are the sole sources of carbon andenergy (24). It was observed that the surfaces of several bacteriabecome more hydrophobic so that these microbes can access hy-drophobic substrates more easily (42). It is a common bacterialstrategy to create a more lipophilic surface to better reach hydro-phobic substrates (24, 42). This adaptation is also important be-cause bacterial n-alkane oxygenase systems are located in the

membrane and need direct contact with their substrates (40, 41,43). As also reported for various other oil-degrading bacteria (44),A. borkumensis produces a biosurfactant on its surface in order toachieve the effect of increased surface hydrophobicity (1).

The n-alkanes used as substrates in this study get more hydro-phobic with increasing carbon chain length. A. borkumensisadapts to this trend by making its surface more lipophilic thelonger the supplied n-alkane carbon chain gets.

This bacterium showed an increase in growth rate with increas-ing chain length of the n-alkane used as the substrate, up to amaximum between C12 and C19. A logical explanation could bethe incorporation of the corresponding n-alkane acidic degrada-tion intermediates into the membrane lipids of A. borkumensis.The membrane accounts for about 10% of the bacterial biomass,and de novo synthesis of fatty acids from acetyl-CoA is extremelyenergy consuming. Therefore, it is reasonable to assume that cul-tures incorporating their fatty acids instead of synthesizing themde novo have an advantage and are able to grow faster. Only fattyacids with a chain length of C14 to C18 are expected to be incorpo-rated into the membrane, and palmitic acid (C16:0) is one of thecommonly dominant fatty acids in A. borkumensis (23). Hence, itwas expected that growth on n-hexadecane would be faster thangrowth on pyruvate and actually also faster than growth on allother supplied substrates. This was the case for similar experi-ments with Rhodococcus erythropolis (24).

In contrast to this assumption, growth on n-hexadecane didnot exceed growth on n-tridecane, n-tetradecane, or n-pentade-cane. This finding contradicted previous observations with R.erythropolis, where cultures grown on odd-numbered n-alkaneswere considerably smaller than those grown on even-numberedn-alkanes of similar carbon chain length (24). Hence, there was no

FIG 7 Hierarchical cluster of A. borkumensis genes involved in n-alkane degradation. The RNA samples were taken at the given time points after the addition of1-octanol. Expression ratios of the pyruvate samples are shown in the left-hand columns, and those of the hexadecane samples are shown in the right-handcolumns; up- and downregulation as indicated are shown in the color legend at the top of the figure.

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huge difference in energy savings between cultures grown on thesesubstrates. Therefore, growth rates were similar for cultures inwhich the carbon chain length of n-alkane substrates exceeded 13.Thus, it can be assumed that all of these substrates can be incor-porated directly into the membrane. A. borkumensis, as a hydro-carbonoclastic bacterium, is able to catalyze the initial oxidationstep for n-alkane degradation, forming the corresponding alkanolwith three different monooxygenases (3, 7). These alkanol inter-mediates were detected in very small amounts by GC-MS (datanot shown). As an intermediate after a second oxidation step, thecorresponding fatty acid is formed (41).

It was shown for R. erythropolis that it is possible to incorporateoxidized n-alkanes directly into the membrane (24). Similar re-sults for A. borkumensis were shown in the present work. Clearlyincreased amounts of myristic acid and palmitic acid were de-tected when n-tetradecane and n-hexadecane, respectively, wereused as the carbon sources. Hence, we state that the energy savingsassociated with not having to synthesize all fatty acids de novoresult in differences in the growth rates of A. borkumensis cultures.

Previously, for studies of membrane fatty acid composition, A.borkumensis was grown only on pyruvate or n-hexadecane (23).As discovered earlier and also seen in the results presented here,the membrane fatty acid pattern of A. borkumensis normally con-sists of mainly seven fatty acids (14:0, 16:0, 16:1�9 cis, 16:1�9trans, 18:0, 18:1�11 cis, and 18:1�11 trans).

Therefore, the most exciting discoveries of this work were theadditional peaks found in the chromatography of fatty acidmethyl esters (FAME) of bacteria grown on odd-numbered n-alkanes. The occurrence of these peaks revealed the great ability ofthis bacterium to incorporate and modify n-alkanes into themembrane fatty acid composition. The first indications of thepresence of these unusual fatty acids in the membrane were foundusing the Kovats retention index (25). This could be verified byadditional mass spectrometry (GC-MS). Thus, in the presence ofodd-numbered n-alkanes, the bacteria were obviously able to ox-idize and additionally convert them to 13:0, 15:0, 15:1 cis, 15:1trans, 17:0, 17:1 cis, and 17:1 trans fatty acids.

We could also clearly show that when the bacteria were grownon n-tridecane or n-pentadecane, tridecanoic acid or pentade-canoic acid, respectively, was the most dominant fatty acid. Thecorrelation between the incorporation of odd-numbered fatty ac-ids into the membrane and the use of odd-numbered n-alkanesubstrates was reported also for the sedimentary marine bacte-rium Marinobacter hydrocarbonoclasticus (45). Hence, it can bestated that A. borkumensis is capable of incorporating fatty acidsobtained by n-alkane oxidation into their corresponding fattyacids.

As shown by Doumenq et al. (45), Marinobacter hydrocarbono-clasticus not only can incorporate odd-numbered fatty acids intothe membrane but also is capable of shortening them before in-corporation. For example, an increased amount of heptadecanoicacid was observed when M. hydrocarbonoclasticus grew on non-adecane (C19:0). In contrast, an extension was not observed inMarinobacter hydrocarbonoclasticus. For Rhodococcus erythropolis,neither incorporation, nor extension, nor a shortening of incor-porated odd-numbered fatty acids was reported (24). We wereable to show that A. borkumensis is capable of extending fatty acidsincorporated after n-alkane oxidation. Thus, this bacterium musthave an enzyme system that allows fatty acid elongation, as de-scribed, e.g., for Bacillus subtilis (46). Another indication for this

elongation of fatty acids withdrawn from the alkane degradationpathway is the presence of pentadecanoic acid in cultures grownon n-undecane or n-tridecane, along with the observed heptade-canoic acid in all cultures grown on odd-numbered n-alkanes. So,we assume that it is possible not only to insert the degradationintermediates unchanged in the membrane under suitable condi-tions but also to elongate the n-alkane-oxidized fatty acids by ad-dition of C2 units if needed.

It was shown here that A. borkumensis can incorporate andelongate fatty acids withdrawn from the n-alkane degradationpathway. Something completely new is the presence of unsatu-rated odd-numbered fatty acids in A. borkumensis. As alreadystated, odd-numbered fatty acids are unusual for the membranefatty acid pattern of A. borkumensis. So, de novo synthesis can againbe excluded. We therefore state that A. borkumensis also desatu-rates the fatty acid intermediates withdrawn from the n-alkanedegradation pathway. Normally, the synthesis of unsaturated fattyacids is connected to either the so-called anaerobic pathway or theaerobic pathway. Most gammaproteobacteria are capable of usingonly the anaerobic pathway, which does not allow elongation anddesaturation. However, the presence of both pathways was alreadyshown for a marine Vibrio strain (47). A. borkumensis uses bothfatty acid synthesis pathways as well, which was also confirmed bythe presence of genes of both pathways in the genome of the bac-terium (ABO_2546, ABO_2585, ABO_0148, ABO_0526, andABO_0835.). Desaturases always lead to cis-unsaturated fatty ac-ids (48). The fact that we could also detect the correspondingtrans-unsaturated species in our experiments once more confirmsthe presence of a gene encoding an active cis-trans isomerase(ABO_1700) in the genome. The identification of this mechanismfor immediate membrane modification is another indicator of theenormous adaptive toolbox A. borkumensis has available to dealwith alkane substrates and environmental stress conditions.

The adaptability of this bacterium was also shown on a moreglobal level in the studies of adaptive responses toward 1-octanolexposure using global transcriptome profiling of A. borkumensiscells grown on either hexadecane or pyruvate. Toxic organic com-pounds are known to interfere with bacterial cell membrane in-tegrity due to interactions at the lipid-water interface. It influencesthe order of membrane lipids as well as bilayer stability and affects,therefore, membrane characteristics such as fluidity and permea-bility and the activity of membrane-embedded enzymes (49). Thegenome-wide transcriptional analysis of A. borkumensis gene ex-pression in the presence of 1-octanol during growth on two dif-ferent carbon sources revealed an immediate response to the per-turbation with the solvent. Only 15 min after the addition of1-octanol to the cultures, the expression levels of 27% of the genesin pyruvate-grown cells and of 13.1% of the genes in hexadecane-grown cells were changed. After another 15 min (time point 30min), those numbers dropped to overall changes of 18.8% (pyru-vate cultures) and 4.1% (hexadecane cultures). Finally, 90 minafter perturbation with the solvent, only 6.8% (pyruvate) and1.1% (hexadecane) of all genes remained at altered expressionlevels. Several studies have shown that microorganisms feeding onhydrocarbons, polycyclic aromatic hydrocarbon (PAHs), or toxiccompounds in crude oil mixtures very often show adaptation ortolerance mechanisms in relation to the degraded substance ordegradation intermediates (50, 51). A. borkumensis cells grown onhexadecane show a higher tolerance toward the solvent 1-octanolthan cells grown on pyruvate because they are already preadapted

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to alkanol stress, and, therefore, only a very small number of genesneed to be regulated. These findings were additionally verified bythe systematic investigation performed with n-alkanols and chlo-rophenols, where hexadecane-grown cells showed significantlyhigher tolerance toward n-alkanols than pyruvate-grown bacteria.The fact that these tolerance differences could not be found withchlorophenols clearly reveals the specificity of this preadaptationto n-alkanols as toxic degradation intermediates of these exclu-sively n-alkane-degrading bacteria.

Also, the types of regulated genes indicate that A. borkumensisoperates with adaptive mechanisms. The strongest regulation wasfound for the decreased expression levels of motility genes, such asthe pilus and fimbria system, a chemotaxis protein methyltrans-ferase, and a methyl-accepting chemotaxis protein. It is not clearwhy these genes are downregulated exclusively in pyruvate-growncells and not in cells grown on hexadecane. One might speculatethat the energy requirement might be too high to be maintainedunder unfavorable conditions such as a stress situation.

The cellular stress response system utilizes heat shock proteins(HSPs), also called molecular chaperones. When 1-octanol wasadded, genes for a great number of HSPs are strongly upregulated,such as those encoding GrpE, DnaK, HtpG, IbpA, and HtpX andtwo chaperone subunits (ABO_0633 and ABO_0634). HSPs andchaperones play an essential role in the synthesis, transport, fold-ing, and degradation of proteins. The expression of the heat shockproteins is induced by a huge range of stressors, including alsosolvents or toxic chemicals, in almost all organisms that have beenexamined so far with omics approaches (50–59). Under stressconditions, they help to prevent unfavorable aggregation and as-sist in proper refolding of damaged proteins (60).

The influence of energy-dependent processes for dealing withthe toxicity of solvents, such as the mentioned HSPs, would sug-gest that energy generation is an important process needed fordealing with solvent and other chemical stresses. Additionally, thesolvent induces damage to cellular molecules, which therefore re-quire repair or resynthesis. These actions require increased expen-ditures of energy in response to solvent toxicity. The genes neededfor alkane oxidation and �-oxidation are strongly upregulated incells grown on pyruvate but were also upregulated as an immedi-ate response in hexadecane-grown Alcanivorax cells. Energeticprocesses involved in solvent tolerance in Pseudomonas putidahave been recently reported (39, 61). A proteomic study of P.putida demonstrated that under toluene stress, the energy supplywas heavily affected as a result of the disruption of the protonmotive force. To countervail the decreased regulation of the ATPsynthase, genes involved in NAD(P)H metabolism were upregu-lated (62). This suggests that the HSP response uses the cellularresources available from growth for stress management.

Furthermore, the comparison of the transcriptome data dem-onstrates that cells that were grown on hexadecane, despite allother factors, such as the higher hydrophobicity of the membraneand an altered membrane composition, also show a clear adaptivemechanism at the transcriptomic level. After 90 min of exposureto 1-octanol, the expression levels of 192 genes were altered morethan 2-fold (97 genes up and 95 genes down) on pyruvate, butthose of only 32 genes were altered with hexadecane (0.7% up and0.5% down). A comparison of these results to the regulation pat-tern after 30 min (490 genes changed on pyruvate and 116 geneson hexadecane) shows, clearly, that transcriptional expression of asmaller fraction of genes is changed in hexadecane-grown cells.

No new categories of affected COGs were found to be affectedafter 90 min that were not present at 15 min or 30 min. Thissuggests that Alcanivorax borkumensis has overcome the immedi-ate consequences of 1-octanol stress and that a long-term mecha-nism of adaptation to 1-octanol is present.

All of these adaptations and abilities to assimilate n-alkanesexplain why A. borkumensis is so important for the bioremediationof oil-polluted marine environments. Therefore, it is important tofurther increase knowledge about the hydrocarbonoclastic bacte-ria in order to achieve an understanding of how to enhance biore-mediation.

ACKNOWLEDGMENTS

This work was partially supported by a collaborative project (BACSIN,contract number 211684) of the European Commission within its SeventhFramework Programme.

We thank Robert Geffers for helpful advice with the microarray designand the Array Facility at the Helmholtz Centre of Infection Research,Braunschweig, Germany, for assistance with microarray analyses.

REFERENCES1. Golyshin PN, Dos Santos V, Kaiser O, Ferrer M, Sabirova YS, Lunsdorf

H, Chernikova TN, Golyshina OV, Yakimov MM, Puhler A, TimmisKN. 2003. Genome sequence completed of Alcanivorax borkumensis, ahydrocarbon-degrading bacterium that plays a global role in oil removalfrom marine systems. J. Biotechnol. 106:215–220.

2. Kalscheuer R, Stoveken T, Malkus U, Reichelt R, Golyshin PN, Sabi-rova JS, Ferrer M, Timmis KN, Steinbüchel A. 2007. Analysis of storagelivid accumulation in Alcanivorax borkumensis: evidence for alternativetriacylglycerol biosynthesis routes in bacteria. J. Bacteriol. 189:918 –928.

3. Schneiker S, dos Santos V, Bartels D, Bekel T, Brecht M, Buhrmester J,Chernikova TN, Denaro R, Ferrer M, Gertler C, Goesmann A, Goly-shina OV, Kaminski F, Khachane AN, Lang S, Linke B, McHardy AC,Meyer F, Nechitaylo T, Puhler A, Regenhardt D, Rupp O, Sabirova JS,Selbitschka W, Yakimov MM, Timmis KN, Vorholter FJ, Weidner S,Kaiser O, Golyshin PN. 2006. Genome sequence of the ubiquitous hy-drocarbon-degrading marine bacterium Alcanivorax borkumensis. Nat.Biotechnol. 24:997–1004.

4. Yakimov MM, Timmis KN, Golyshin PN. 2007. Obligate oil-degradingmarine bacteria. Curr. Opin. Biotechnol. 18:257–266.

5. Hara A, Baik S, Syutsubo K, Misawa N, Smits THM, van Beilen JB,Harayama S. 2004. Cloning and functional analysis of alkB genes inAlcanivorax borkumensis SK2. Environ. Microbiol. 6:191–197.

6. Yakimov MM, Denaro R, Genovese M, Cappello S, D’Auria G,Chernikova TN, Timmis KN, Golyshin PN, Giluliano L. 2005. Naturalmicrobial diversity in superficial sediments of Milazzo Harbor (Sicily) andcommunity successions during microcosm enrichment with various hy-drocarbons. Environ. Microbiol. 7:1426 –1441.

7. Sabirova JS, Ferrer M, Regenhardt D, Timmis KN, Golyshin PN. 2006.Proteomic insights into metabolic adaptations in Alcanivorax borkumensisinduced by alkane utilization. J. Bacteriol. 188:3763–3773.

8. Hara A, Syutsubo K, Harayama S. 2003. Alcanivorax which prevails inoil-contaminated seawater exhibits broad substrate specificity for alkanedegradation. Environ. Microbiol. 5:746 –753.

9. Abraham WR, Meyer H, Yakimov M. 1998. Novel glycine containingglucolipids from the alkane using bacterium Alcanivorax borkumensis.Biochim. Biophys. Acta 1393:57– 62.

10. Kabelitz N, Santos PM, Heipieper HJ. 2003. Effect of aliphatic alcoholson growth and degree of saturation of membrane lipids in Acinetobactercalcoaceticus. FEMS Microbiol. Lett. 220:223–227.

11. Loffler C, Eberlein C, Mausezahl I, Kappelmeyer U, Heipieper HJ. 2010.Physiological evidence for the presence of a cis-trans isomerase of unsat-urated fatty acids in Methylococcus capsulatus Bath to adapt to the presenceof toxic organic compounds. FEMS Microbiol. Lett. 308:68 –75.

12. Neumann G, Kabelitz N, Zehnsdorf A, Miltner A, Lippold H, Meyer D,Schmid A, Heipieper HJ. 2005. Prediction of the adaptability of Pseu-domonas putida DOT-T1E to a second phase of a solvent for economicallysound two-phase biotransformations. Appl. Environ. Microbiol. 71:6606 – 6612.

Adaptation of Alcanivorax borkumensis SK2

July 2013 Volume 79 Number 14 aem.asm.org 4291

on March 13, 2021 by guest

http://aem.asm

.org/D

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13. Heipieper HJ, Loffeld B, Keweloh H, de Bont JAM. 1995. The cis/transisomerization of unsaturated fatty acids in Pseudomonas putida S12: anindicator for environmental stress due to organic compounds. Chemo-sphere 30:1041–1051.

14. Bligh EG, Dyer WJ. 1959. A rapid method of total lipid extraction andpurification. Can. J. Biochem. Physiol. 37:911–917.

15. Morrison WR, Smith LM. 1964. Preparation of fatty acid methyl estersand dimethylacetals from lipids with boron fluoride-methanol. J. LipidRes. 5:600 – 608.

16. van Loosdrecht MCM, Lyklema J, Norde W, Schraa G, Zehnder AJB.1987. Electrophoretic mobility and hydrophobicity as a measure to pre-dict the initial steps of bacterial adhesion. Appl. Environ. Microbiol. 53:1898 –1901.

17. Rijnaarts HHM, Norde W, Lyklema J, Zehnder AJB. 1995. The isoelec-tric point of bacteria as indicator for the presence of cell surface polymersthat inhibit adhesion. Colloids Surf. B 4:191–197.

18. Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate—apractical and powerful approach to multiple testing. J. R. Stat. Soc. SeriesB Stat. Methodol. 57:289 –300.

19. Eisen MB, Spellman PT, Brown PO, Botstein D. 1998. Cluster analysisand display of genome-wide expression patterns. Proc. Natl. Acad. Sci.U. S. A. 95:14863–14868.

20. Sturn A, Quackenbush J, Trajanoski Z. 2002. Genesis: cluster analysis ofmicroarray data. Bioinformatics 18:207–208.

21. Edgar R, Domrachev M, Lash AE. 2002. Gene Expression Omnibus:NCBI gene expression and hybridization array data repository. NucleicAcids Res. 30:207–210.

22. Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P,Stoeckert C, Aach J, Ansorge W, Ball CA, Causton HC, Gaasterland T,Glenisson P, Holstege FCP, Kim IF, Markowitz V, Matese JC, Parkin-son H, Robinson A, Sarkans U, Schulze-Kremer S, Stewart J, Taylor R,Vilo J, Vingron M. 2001. Minimum information about a microarrayexperiment (MIAME)—toward standards for microarray data. Nat.Genet. 29:365–371.

23. Yakimov MM, Golyshin PN, Lang S, Moore ERB, Abraham WR,Lunsdorf H, Timmis KN. 1998. Alcanivorax borkumensis gen. nov., sp.nov., a new, hydrocarbon-degrading and surfactant-producing marinebacterium. Int. J. Syst. Bacteriol. 48:339 –348.

24. de Carvalho C, Wick LY, Heipieper HJ. 2009. Cell wall adaptations ofplanktonic and biofilm Rhodococcus erythropolis cells to growth on C5 toC16 n-alkane hydrocarbons. Appl. Microbiol. Biotechnol. 82:311–320.

25. Kovats E. 1958. Gas-Chromatographische Charakterisierung organischerVerbindungen. 1. Retentionsindices aliphatischer Halogenide, Alkohole,Aldehyde und Ketone. Helvet. Chim. Acta 41:1915–1932.

26. Chen Q, Janssen DB, Witholt B. 1995. Growth on octane alters themembrane lipid fatty acids of Pseudomonas oleovorans due to the induc-tion of alkB and synthesis of octanol. J. Bacteriol. 177:6894 – 6901.

27. Heipieper HJ, Meinhardt F, Segura A. 2003. The cis-trans isomerase ofunsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molec-ular biology and physiological function of a unique stress adaptive mech-anism. FEMS Microbiol. Lett. 229:1–7.

28. Keweloh H, Heipieper HJ. 1996. Trans unsaturated fatty acids in bacteria.Lipids 31:129 –137.

29. Tatusov RL, Koonin EV, Lipman DJ. 1997. A genomic perspective onprotein families. Science 278:631– 637.

30. Sabirova JS, Chernikova TN, Timmis KN, Golyshin PN. 2008. Niche-specificity factors of a marine oil-degrading bacterium Alcanivorax borku-mensis SK2. FEMS Microbiol. Lett. 285:89 –96.

31. Baelum J, Borglin S, Chakraborty R, Fortney JL, Lamendella R, MasonOU, Auer M, Zemla M, Bill M, Conrad ME, Malfatti SA, Tringe SG,Holman HY, Hazen TC, Jansson JK. 2012. Deep-sea bacteria enriched byoil and dispersant from the Deepwater Horizon spill. Environ. Microbiol.14:2405–2416.

32. Beazley MJ, Martinez RJ, Rajan S, Powell J, Piceno YM, Tom LM,Andersen GL, Hazen TC, Van Nostrand JD, Zhou JZ, Mortazavi B,Sobecky PA. 2012. Microbial community analysis of a coastal salt marshaffected by the Deepwater Horizon oil spill. PLoS One 7:e41305. doi:10.1371/journal.pone.0041305.

33. Hazen TC, Dubinsky EA, DeSantis TZ, Andersen GL, Piceno YM, SinghN, Jansson JK, Probst A, Borglin SE, Fortney JL, Stringfellow WT, BillM, Conrad ME, Tom LM, Chavarria KL, Alusi TR, Lamendella R,Joyner DC, Spier C, Baelum J, Auer M, Zemla ML, Chakraborty R,Sonnenthal EL, D’Haeseleer P, Holman HYN, Osman S, Lu ZM, Van

Nostrand JD, Deng Y, Zhou JZ, Mason OU. 2010. Deep-sea oil plumeenriches indigenous oil-degrading bacteria. Science 330:204 –208.

34. Kasai Y, Kishira H, Sasaki T, Syutsubo K, Watanabe K, Harayama S.2002. Predominant growth of Alcanivorax strains in oil-contaminated andnutrient-supplemented sea water. Environ. Microbiol. 4:141–147.

35. Kostka JE, Prakash O, Overholt WA, Green SJ, Freyer G, Canion A,Delgardio J, Norton N, Hazen TC, Huettel M. 2011. Hydrocarbon-degrading bacteria and the bacterial community response in Gulf of Mex-ico beach sands impacted by the Deepwater Horizon oil spill. Appl. Envi-ron. Microbiol. 77:7962–7974.

36. Lu ZM, Deng Y, Van Nostrand JD, He ZL, Voordeckers J, Zhou AF, LeeYJ, Mason OU, Dubinsky EA, Chavarria KL, Tom LM, Fortney JL,Lamendella R, Jansson JK, D’Haeseleer P, Hazen TC, Zhou JZ. 2012.Microbial gene functions enriched in the Deepwater Horizon deep-sea oilplume. ISME J. 6:451– 460.

37. Mason OU, Hazen TC, Borglin S, Chain PSG, Dubinsky EA, FortneyJL, Han J, Holman HYN, Hultman J, Lamendella R, Mackelprang R,Malfatti S, Tom LM, Tringe SG, Woyke T, Zhou JH, Rubin EM,Jansson JK. 2012. Metagenome, metatranscriptome and single-cell se-quencing reveal microbial response to Deepwater Horizon oil spill. ISMEJ. 6:1715–1727.

38. Ingram LO. 1976. Adaptation of membrane lipids to alcohols. J. Bacteriol.125:670 – 678.

39. Neumann G, Cornelissen S, van Breukelen F, Hunger S, Lippold H,Loffhagen N, Wick LY, Heipieper HJ. 2006. Energetics and surfaceproperties of Pseudomonas putida DOT-T1E in a two-phase fermentationsystem with 1-decanol as second phase. Appl. Environ. Microbiol. 72:4232– 4238.

40. van Beilen JB, Funhoff EG. 2007. Alkane hydroxylases involved in mi-crobial alkane degradation. Appl. Microbiol. Biotechnol. 74:13–21.

41. Wentzel A, Ellingsen TE, Kotlar HK, Zotchev SB, Throne-Holst M.2007. Bacterial metabolism of long-chain n-alkanes. Appl. Microbiol. Bio-technol. 76:1209 –1221.

42. Wick LY, de Munain AR, Springael D, Harms H. 2002. Responses ofMycobacterium sp LB501T to the low bioavailability of solid anthracene.Appl. Microbiol. Biotechnol. 58:378 –385.

43. van Beilen JB, Marin MM, Smits THM, Rothlisberger M, Franchini AG,Witholt B, Rojo F. 2004. Characterization of two alkane hydroxylasegenes from the marine hydrocarbonoclastic bacterium Alcanivorax borku-mensis. Environ. Microbiol. 6:264 –273.

44. Ron EZ, Rosenberg E. 2002. Biosurfactants and oil bioremediation. Curr.Opin. Biotechnol. 13:249 –252.

45. Doumenq P, Aries E, Asia L, Acquaviva M, Artaud J, Gilewicz M, MilleG, Bertrand JC. 2001. Influence of n-alkanes and petroleum on fatty acidcomposition of a hydrocarbonoclastic bacterium: Marinobacter hydrocar-bonoclasticus strain 617. Chemosphere 44:519 –528.

46. Mansilla MC, de Mendoza D. 2005. The Bacillus subtilis desaturase: amodel to understand phospholipid modification and temperature sens-ing. Arch. Microbiol. 183:229 –235.

47. Morita N, Gotoh M, Okajima N, Okuyama H, Hayashi H, Higashi S,Murata N. 1992. Both the anaerobic pathway and aerobic desaturation areinvolved in the synthesis of unsaturated fatty acids in Vibrio sp. strainABE-1. FEBS Lett. 297:9 –12.

48. Hartig C, Loffhagen N, Babel W. 1999. Glucose stimulates a decrease ofthe fatty acid saturation degree in Acinetobacter calcoaceticus. Arch. Micro-biol. 171:166 –172.

49. Weber FJ, de Bont JAM. 1996. Adaptation mechanisms of microorgan-isms to the toxic effects of organic solvents on membranes. Biochim. Bio-phys. Acta 1286:225–245.

50. Kang A, Chang MW. 2012. Identification and reconstitution of geneticregulatory networks for improved microbial tolerance to isooctane. Mol.Biosyst. 8:1350 –1358.

51. Fida TT, Breugelmans P, Lavigne R, Coronado E, Johnson DR, van derMeer JR, Mayer AP, Heipieper HJ, Hofkens J, Springael D. 2012.Exposure to solute stress affects genome-wide expression but not the poly-cyclic aromatic hydrocarbon-degrading activity of Sphingomonas sp.strain. LH128 in biofilms. Appl. Environ. Microbiol. 78:8311– 8320.

52. Alsaker KV, Paredes C, Papoutsakis ET. 2010. Metabolite stress and toler-ance in the production of biofuels and chemicals: gene-expression-based sys-tems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clos-tridium acetobutylicum. Biotechnol. Bioeng. 105:1131–1147.

53. Brynildsen MP, Liao JC. 2009. An integrated network approach identifies

Naether et al.

4292 aem.asm.org Applied and Environmental Microbiology

on March 13, 2021 by guest

http://aem.asm

.org/D

ownloaded from

the isobutanol response network of Escherichia coli. Mol. Syst. Biol. 5:277.doi:10.1038/msb.2009.34.

54. Janssen H, Grimmler C, Ehrenreich A, Bahl H, Fischer RJ. 2012. A transcrip-tional study of acidogenic chemostat cells of Clostridium acetobutylicum—solventstress caused by a transient n-butanol pulse. J. Biotechnol. 161:354–365.

55. Johnson DR, Coronado E, Moreno-Forero SK, Heipieper HJ, van derMeer JR. 2011. Transcriptome and membrane fatty acid analyses revealdifferent strategies for responding to permeating and non-permeatingsolutes in the bacterium Sphingomonas wittichii. BMC Microbiol. 11:250.doi:10.1186/1471-2180-11-250.

56. Polen T, Rittmann D, Wendisch VF, Sahm H. 2003. DNA microarrayanalyses of the long-term adaptive response of Escherichia coli to acetateand propionate. Appl. Environ. Microbiol. 69:1759 –1774.

57. Rutherford BJ, Dahl RH, Price RE, Szmidt HL, Benke PI, Mukhopad-hyay A, Keasling JD. 2010. Functional genomic study of exogenous n-butanol stress in Escherichia coli. Appl. Environ. Microbiol. 76:1935–1945.

58. Tomas CA, Beamish J, Papoutsakis ET. 2004. Transcriptional analysis ofbutanol stress and tolerance in Clostridium acetobutylicum. J. Bacteriol.186:2006 –2018.

59. van Bokhorst-van de Veen H, Abee T, Tempelaars M, Bron PA, Kleere-bezem M, Marco ML. 2011. Short- and long-term adaptation to ethanolstress and its cross-protective consequences in Lactobacillus plantarum.Appl. Environ. Microbiol. 77:5247–5256.

60. Tomas CA, Welker NE, Papoutsakis ET. 2003. Overexpression ofgroESL in Clostridium acetobutylicum results in increased solvent pro-duction and tolerance, prolonged metabolism, and changes in the cell’stranscriptional program. Appl. Environ. Microbiol. 69:4951– 4965.

61. Ray S, Peters CA. 2008. Changes in microbiological metabolism underchemical stress. Chemosphere 71:474 – 483.

62. Volkers RJM, de Jong AL, Hulst AG, van Baar BLM, de Bont JAM,Wery J. 2006. Chemostat-based proteomic analysis of toluene-affectedPseudomonas putida S12. Environ. Microbiol. 8:1674 –1679.

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