Alcanivorax Borkurnensis Gen. Now, Sp. Nov., a New, Hydrocarbon-Degrading and Surfactant-Producing Marine Bacterium

International Journal of Systematic Bacteriology (1 998), 48,339-348 Printed in Great Britain

Alcanivorax borkurnensis gen. now, sp. nov., a new, hydrocarbon-degrading and surfactant-producing marine bacterium

Michail M. Yakimov,’ Peter N. Golyshin,’ Siegmund Lang,2 Edward R. B. Moore,’ Wolf-Rainer Abraham,’ Heinrich LUnsdorf’ and Kenneth N. Timmis’

Author for correspondence: Peter N. Golyshin. Tel: +49 531 6181498. Fax: +49 531 6181411. e-mail : [email protected]

1 Division of Microbiology, During screening for biosurfactant-producing, n-alkane-degrading marine GBF National Research bacteria, six heterotrophic bacterial strains were isolated from enriched mixed Centre for Biotechnology, Mascheroder Weg 1, cultures, obtained from sea waterkediment samples collected near the Isle of D-38124 Braunschweig, Borkum (North Sea), using Mihagol-S (C,,,,-n-alkanes) as principal carbon Germany source. These Gram-negative, aerobic, rod-shaped bacteria use a limited 2 Institute for Biochemistry number of organic compounds, including aliphatic hydrocarbons, volatile fatty and Biotechnology, acids, and pyruvate and its methyl ether. During cultivation on n-alkanes as Technical University of Braunschweig, Konstantin- sole source of carbon and energy, all strains produced both extracellular and Uhde Strasse 5, D-38106 cell-bound surface-active glucose lipids which reduced the surface tension of Braunschweig, Germany water from 72 to 29 mN m-’ (16). This novel class of glycolipids was found to be produced only by these strains. The 165 rRNA gene sequence analysis showed that these strains are all members of the y-subclass of the Proteobacteria. Their phospholipid ester-linked fatty acid composition was shown to be similar to that of members of the genus Halomonas, although they did not demonstrate a close phylogenetic relationship to any previously described species. On the basis of the information summarized above, a new genus and species, Akaniworax borkumensis, is described to include these bacteria. Strain SKZT is the type strain of A. borkumensis.

Keywords : Alcanivorax borkumensis, marine bacterium, hydrocarbon degradation, surfactant production

INTRODUCTION part of residual oil forms water-in-oil emulsions changing later to tar balls which tend to sediment. Petroleum hydrocarbons are major pollutants of Little data exist on the detailed characteristics of marine environments as a result of terrestrial and surfactant- and/or emulsifier-producing marine bac- freshwater run-off, refuse from coastal oil refineries, teria, although the emulsification of hydrocarbons by offshore oil production, shipping activities and acci- surface-active agents produced by bacteria is con- dental spillage of fuels and other petroleum products. sidered an essential step in hydrocarbon biodegra- Although evaporation and photo-oxidation play an dation in the marine environment (4, 5, 12, 18, 21). important role in oil detoxification, ultimate and The first description of the best known marine bio- complete degradation is accomplished mainly by surfactant, now exploited commercially as Emulsan, marine microflora, bacteria being dominant in this appeared in 1972 (10). This emulsifier is produced by a function. Unfortunately, natural microbial degra- marine isolate, Acinetobacter calcoaceticus RAG- 1, dation occurs relatively slowly in marine environments wrongly identified originally as Arthrobacter sp., and due to the low temperatures, limited availability of forms a significant and stable dispersion of crude oil in nitrogen and phosphorous salts, and because a large sea water (10). A study conducted along the French Mediterranean coast to analyse the hydrocarbon- Abbreviation: PLFA, phospholipid ester-linked fatty acid. degrading potential of bacterial populations led to the The GenBank accession number for the 165 rDNA sequence of A. isolation of numerous oil-degrading strains from sea borkumensis strain SKZT reported in this paper is Y12579. water and sediments collected in polluted areas. It was

00656 0 1998 IUMS 339 M. M. Yakimov and others shown that, during growth in crude oil-containing carboxylase activities, and accumulation of poly-P-hydroxy- medium, a mixed population, EM-4, composed of butyrate were determined using techniques for marine eight distinct strains, produced an emulsifier which bacteria (3). Testing for nitrate reduction activity was generated stable emulsions of oil in sea water (5). A performed after 1 week of incubation in SM1 medium supplemented with pyruvate. Utilization of carbon sources Gram-negative bacterium, originally named Altero- was tested in SM1 medium supplemented with the com- monas strain SP.17, was found to produce large pounds listed in Table 1 and below at a concentration of amounts of a non-dialysable bioemulsifier and to 25 mM. The organic acids tested included formate, acetate, degrade various liquid and solid hydrocarbons (1). propionate, butyrate, benzoate, lactate and p-hydroxy- Further taxonomic verification of this strain showed phenylacetate. that the organism represented a new species within a The effects of salinity and temperature on growth were also new genus rank, Marinobacter hydrocarbonoclasticus examined. To determine the salinity range for growth, SMl (9). Additionally, a Gram-negative, alkane-degrading medium supplemented with pyruvate or hexadecane was marine organism, MM1, has been isolated which prepared by adjusting the concentration of NaCl (0.1- produces another low-molecular-mass cell-bound gly- 2.5 M). The temperature range for growth was determined colipid (16). Using oil-degrading mixed cultures ob- by incubation of cultures on SM1 medium supplemented tained from marine samples around the Isle of Helgo- with pyruvate at 4, 15,20,25, 30,35 and 45 "C. land (North Sea), two Arthrobacter strains producing Electron microscopy surface-active glycolipids were isolated (21). (a) Negative stain preparation. Carbon-coated Formvar grids In this paper, the results of an examination of were placed face down on top of a 70 p1 droplet of mid- phenotypic characteristics of strain MMl and five new exponential-phase cells in the original growth medium. After 30 to 60 s, the grid was picked up, blotted briefly with filter isolates obtained from the same sea water/sediment paper and, without drying, was transferred to 2% (w/v) sample (Isle of Borkum, North Sea) are described, as uranyl acetate, pH 4.5, blotted quickly, air-dried and ex- well as their phylogenetic placement, based upon the amined with the electron microscope. analysis of their 16s rRNA sequences. Considering (b) Embedding and ultrathin sectioning. Mid-exponential-phase their potential as alkane-degrading organisms together cells were washed three times in 5 mM MgC1, to prevent cell with their origin, the name Alcanivorax borkumensis lysis (9). Cells were fixed in 2.5% (v/v) glutardialdehyde, gen. nov., sp. nov. is proposed and strain SK2T buffered with PBS (20 mM sodium phosphate, 150 mM designated as the type strain. NaC1, pH 7-2), at ambient temperature for 30 min. After washing in 0.1 x PBS, the cells were post-fixed with 1 % METHODS (w/v) OsO, in 0.1 M sodium cacodylate, pH 7.0, overnight at 4 "C. Cells were immobilized in 2 Yo (w/v) Difco agar in Bacterial strains. Six isolates, AP1, MM1, SK, SK2, SK4A PBS. Dehydration in an acetone series (30, 50, 70, 90, 100 and SK7, were isolated from sea water/sediment samples and 100 YO,v/v) was done at ambient temperature, requiring collected around the Isle of Borkum (North Sea) using 20min for each dehydration step. Infiltration with an enrichment culture techniques with n-alkanes (Merck) as embedding resin/acetone mixture (1 : 1, v/v) was done at sole carbon source. ambient temperature overnight, followed by pure resin Growth conditions. The bacterial strains were cultivated infiltration overnight again and finally for 4 h (24). Probes aerobically in synthetic sea water medium 1 (SM1) (16) were transferred into a gelatin capsule and filled with resin supplemented with 3 YO Mihagol-S (mixture of C14,15-n- monomer. Polymerization was done for 8 h at 70 "C. alkanes) or with 10 g pyruvate 1-1 and all major cations and Ultrathin sections were cut with a diamond knife using the anions present in concentrations greater than 1 mg 1-1 in sea Leica Ultracut ultramicrotome and were picked up with water (16). Nitrogen was provided in the form of NaNO, Formvar-coated grids (300 mesh, Cu). Sections were post- and phosphorous in the form of Na,HPO,. SM1 contained stained for 10 min with 3 % (w/v) aqueous uranyl acetate (1-1 distilled water) 23 g NaC1, 0.75 g KCl, 1.47 g and for 5 min with 0.5 % (w/v) lead citrate as described by CaCl, .2H,O, 5.08 g MgCl, . 6H,O, 6-16g MgSO, . 7H,O, Reynolds (20). The samples were examined using a trans- 0.89g Na2HPO,.2H,O, 5-Og NaNO, and 0.03g mission electron microscope (Zeiss CEM 902, conventional FeSO, . 7H,O. To prevent precipitation, four separate sol- mode, 30 pm objective aperture, 80 kV acceleration voltage) utions were prepared and then mixed together after auto- at a magnification of x 3000- x 30000 and photographed on claving when the solutions had cooled to room temperature; Agfa Scientia 23D56 P3 AH (Agfa-Gevaert). the first solution contained Na,HPO, and NaNO, (the pH Phospholipid ester-linked fatty acid analysis value of medium was adjusted to 7.5 by the addition of a (a) Extraction of cellular lipids. Total cellular lipids were 10 YO solution of NaOH), the second solution contained extracted from mid-exponential-phase cells grown at 28 "C NaCl, KCl and CaCl,, the third solution contained MgCl, in SM1 medium supplemented with pyruvate using a and MgSO,, and the fourth solution contained FeSO,. modified Bligh-Dyer procedure (6) as described previously Bacto agar (Difco) (15 g 1-l) was added to the first solution (27). All solvents were freshly distilled, all glassware was pre- for the preparation of solid media. Cell growth was rinsed with dichloromethane and gloves were used to reduce monitored as cell dry weight as described previously (16). the risk of contamination of samples. Wet cells (2 g) were Phenotypic analysis. For all phenotypic tests, cultures were resuspended in a mixture of methanol, dichloromethane and grown in SM1 medium supplemented with pyruvate. Rou- 50 mM sodium phosphate buffer, pH 7.4 (52.6, 26.3 and tine tests [Gram stain, and amylase, oxidase, catalase, lipase 21.1 ml, respectively), treated for 15 min with an ultrasonic (tweenase), agarase and gelatinase activities] were carried probe (Labsonic U) and kept overnight at room tempera- out as described by Smibert 8z Krieg (23). Arginine di- ture. Additional methanol, dichloromethane and buffer hydrolase, lysine decarboxylase, urease and ornithine de- (35.4,61 and 57 ml, respectively) were added, followed again

~~ ~ 340 International journal of Systematic Bacteriology 48 Alcanivorax borkumensis gen. nov., sp. nov.

Fig. 7. Ultrastructure of A. borkumensis SKZT cells. (a) Negatively stained mid-exponential-phase cells appear as short rods. Because of osmotic sensitivity the periplasmic space at the apex of the cells has dilated and appears electron- translucent (asterisk). (b) Ultrathin section of SKZT after binary fission, showing typical cytoplasmic granularity. An individual electron-dense granule of polyphosphate is circled. The Gram-negativecell wall construction is shown in detail in the insert. Outer membrane (om), peptidoglycan (pg) and cytoplasmic membrane (cm) are indicated.

International Journal of Systematic Bacteriology 48 34 1 M. M. Yakimov and others by 5 min of ultrasonic treatment. The samples were centri- operated at 6 kV with xenon. In the positive mode 3- fuged at 5860g for 15 min to separate the phases. The nitrobenzyl alcohol was used as matrix and in the negative dichloromethane phase was filtered through dry sodium mode a mixture of triethanolamine and tetramethylurea sulphate and a hydrophobic filter. The methanol/phosphate (2 :1, v/v) was used. buffer phase was re-extracted by adding 25ml dichloromethane followed by centrifugation and filtration. The G+C content. The G+C content of the isolates was resulting fraction, containing the total lipids (TL), was determined by using an HPLC method described previously reduced in volume using a rotary evaporator at 40 "C and (14,25). Purified non-methylated il phage DNA (Sigma) was further fractionated using a chromatographic column (B&J used as a control. Inert SPE, Silica, Burdick & Jackson). The column was conditioned by heating overnight at 100°C and 10ml 165 rRNA gene sequence determination and analysis of dichloromethane was added after cooling to room tem- phylogenetic relationships. Total genomic DNA was iso- perature. The loaded total lipids were fractionated by lated from 20 ml of late-exponential-phase cells in SMl sequential elution with dichloromethane, acetone and meth- medium, supplemented with 2 % (v/v) hexadecane, using anol resulting in three polarity classes : neutral- (NL), glyco- the CTAB miniprep protocol for bacterial genomic DNA (GL) and phospholipids (PL). The eluates were collected in preparations (29). PCR amplification of 16s rRNA genes separate tubes and dried under nitrogen. The PL fraction was obtained using the forward primer 16F27 (5' AGAGTT- was further dissolved in 1 ml dichloromethane/methanol TGATCMTGGCTCAG 3') and the reverse primer 16R1492 (1 : 1, v/v) and subjected to a mild alkaline hydrolysis (5' TACGGYTACCTTGTTACGACTT 3'). Direct se- procedure (0.5 ml 1 M potassium hydroxide, 2 ml methanol) quence determination of PCR-amplified DNA was carried at 40 "C overnight. The free fatty acids were washed with out using an automated DNA sequencer and Taq cycle hexane to remove hydrophobic impurities. After removing sequencing reactions according to the protocols of the the organic phase, dichloromethane, buffer and 6 M HC1 manufacturer (Perkin-Elmer, Applied Biosystems). Se- were added to the aqueous phase. The organic phase was quence data were aligned initially with 16s rRNA and separated, dried and the free fatty acids were methylated as rDNA sequences using the e-mail servers at the Ribosomal described previously (27). n-Octane (1 ml) containing the Database Project (RDP) (13) and FASTA searches of the internal standard (n-hexadecane and n-tetracosane, 10 and DNA sequence database, version 3.0t7 1 (17). Phylogenetic 12 ng ml-', respectively) was added to the dried fatty acid relationships were estimated using the Phylogeny Inference methyl esters, which were analysed by GC and MS. Capillary Package (PHYLIP version 3.4). Jukes-Cantor evolutionary GC analyses were performed on a Hewlett Packard 5890 distances were calculated using the DNADIST program and Series I1 gas chromatograph equipped with a capillary dendrograms depicting phylogenetic relationships were de- column, HP Ultra 2 (50m by 0.2mm; film thickness rived using the FITCH program (Fitch-Margoliash method, 0.1 1 mm) and a flame ionization detector (FID). Hydrogen version 3.572~)with random order input of sequences and served as the carrier gas. The injector temperature was set to 250 "C and the detector temperature was 300 "C. The oven the global rearrangement option (8). program was 90 "C for 2 min and an increase from 90 to 280 "C at 4 "C min-l followed by an isothermal period of 10 min. RESULTS (b) GC-MS and electron impact (El)-MS. GC-MS analyses were Phenotypic characteristics performed with a gas chromatograph similar to that described above (same column and conditions but with helium Six strains, APl, MM1, SK, SK2T, SK4A and SK7, as carrier gas) connected to a HP 5989A quadropole mass spectrometer. The electron impact ion source was main- were isolated by enrichment culture techniques using tained at 200°C while the quadropole temperature was n-alkanes as sole carbon source from sea water/ 100 "C. Electron energy was 70 eV. Polar lipid fatty acid sediment samples collected at a site located close to the methyl esters were identified by comparison of peak profiles Isle of Borkum (North Sea). All strains produced a with standards or GC-MS analysis. The determination of cell-bound and extracellular surface-active glucose double bond positions in monounsaturated fatty acids of the lipid representing a novel class of glycolipid (1 6). FAB- standards was achieved by the use of the dimethyldisulfide MS analysis of this surfactant, generated in the (DMDS) derivatives. These were prepared as previously negative and positive modes, led to the detection of a described (15). The positions of hydroxy groups were mixture of glucose lipids differentiated in their fatty indicated through silylated derivatives by their fragmen- acid compositions forming lipophilic moiety in the tation pattern in MS (2). Polar lipid fatty acid methyl esters were identified by GC-MS analyses using FAME adducts surfactant molecule (data not shown). The structure of described above for the determination of the hydroxyl group a glucose lipid consisting of four 3-hydroxydecanoic and double bond positions. cis-trans isomers were differ- acids linked together by ester bonds and coupled entiated by comparison of their retention times with auth- glycosidically with C-1 of glucose has been published entic standards. (12, 16). The structural elucidation of additional glucose lipids present in the mixture is under study. Fast atom bombardment (FAB)-MS. The structure of the surface-active glucose lipid, isolated using the procedure In general, all six isolates produced identical results described by Passeri et al. (1 6), was confirmed by FAB-MS. FAB-MS in the positive and negative mode was performed from phenotypic characterization tests. The isolates on the first of two mass spectrometers of a tandem high- are aerobic, Gram-negative, non-spore-forming, cata- resolution instrument of El B1 E2B2 configuration (JMS- lase- and oxidase-positive, non-mo tile rods. Cells HX/HXllOA, JEOL) at 10 kV accelerating voltage. The actively growing in SMl supplemented with pyruvate resolution was set to 1:2000. The JEOL FAB gun was at 28 "C were 2.0-3.0 pm in length and 04-0.7 pm in

~~ ~~~~ ~ 342 International Journal of Systematic Bacteriology 48 Alcanivorax borkumensis gen. nov., sp. nov.

Table 1. Comparison of key phenotypic characteristics of A. borkumensis and other heterotrophic marine halophilic bacteria belonging to the y-subclass of the Proteobacteria ...... + , Positive reaction or growth; & , variable reaction; -, negative reaction or growth; NR, not reported.

Characteristic Marinomonas Marinobacter Oceanospirillum Halomonas Alcanivorax SPP*" hy drocarbonoclasticus~ SPPmS elongara§ borkumensis Typical Gram-negative cell + + + + wall profile Morphology Rods Rods Helical cells Rods No. and arrangement of 1, polar 1, polar, or none Bipolar tufts Peritrichous flagella Optimal temperature ("C) 20-25 32 25-32 30 for growth Growth at 4 "C - - k + Growth at 45 "C + + - NR Optimal NaCl concn (%) for 0.7-3.5 3-6 0.5-8 3-8 growth Maximal NaCl concn (YO) NR 20 9.75 32.5 at which growth occurs Nitrate reduced to nitrite - + - + Oxidase test (Kovacs) - + + + Arginine dihydrolase - - NR - activity Ornithine decarboxylase - NR + Gelatine liquefaction - - k Starch hydrolysis - - - Lysine decarboxylase - NR + Urease activity - - -+ Utilization of sole carbon source : D-Glucose + - + D-Fructose - + D-Mannose + - + Sucrose - - + Cellobiose -+ - + D-Mannitol + - + Glycerol + - + D-Gluconate + NR + Succinate + + + L-Glutamate + k + Aspartate NR - + L-Serine -+ - + DL-Afanine + rk + L-Arginine -I - + Hexadecane + NR + p-Hydroxybenzoate + rk - Accumulation of poly-,& - + + hydroxybutyrate DNA G+C (mol%) 44-48 42-5 1 60-61 * Data from Baumann et al. (3). ?Data from Gauthier et al. (9). 3 Data from Krieg (1 1). 8 Data from Vreeland (28). diameter. They were shorter (1.0-1.5 pm) in SMl (Fig. la). Cells are osmotically sensitive, as shown by supplemented with n-alkanes. The morphology of mid- the intense positive staining of the cell body and the exponential-phasecells was shown by negative staining electron-transparent dilatation of the periplasmic

International Journal of Systematic Bacteriology 48 343 M. M. Yakimov and others

Table 2. Comparison of PLFA composition among the strains of A. borkumensis and two halotolerant Halomonas strains ...... ,...... * ...... Values are means of GC data obtained from three independent series of PLFA isolation. Values are given as a percentage of the total amount of fatty acids isolated from phospholipids.

~~ ~ Fatty acids Total PLFA (YO)in:

AP1* MM1* SK* SK2=* SK4A* SK7* H. halophila H. elongata ACAM 68t ACAM 246t

C14: 1 - 0.2 - - - - C14:O 1-5 0-5 0.7 1.7 0.2 0.2 C15:Oa 0.1 0.2 0.1 0.2 - - Sum 1$ 16-2 10-9 14.1 17.1 16.5 10-2 C16: 105 0.6 - - - 0.1 0.1 C16:O 34.3 24.2 36.9 34.5 25.4 23.3 C17: li - - - - 0.4 0.4 C17:O 0.2 - 0.1 - - 0.2 Sum 29 - - 2-4 - 05 0.5 Sum 311 44-9 48.3 37.4 44.2 52.2 61.5 C18: lcu5 0.2 - 0.3 0.1 02 0.2 C18:O 1.9 1.5 0.9 1.4 0.4 0.5 C19: 1cu8 - 2.5 0.1 - - - C 19 :Ocyclo - - 2.1 - 2.7 2.4 C20 : 109 - 3.6 1.3 - 0.2 0-3 Total PLFA detected (YO) 99-9 91.9 96.4 99.2 988 99-8 *A. borkumensis strains. Data from Skerratt et al. (22). $Sum 1, cC16: 107/tC16: 109. 9 Sum 2, C17:Ocyclo/cC17: 106. I( Sum 3, cC18: lo7/tC18: lw7/tC18: 109/C18: 1012. space at the apex of the cell. As shown in Fig. l(b), tivities were not detected. In addition to the nutritional mid-exponential-phase cells demonstrated normal profile shown in Table 1, A. borkurnensis strains were granularity of the cytoplasm with one or more able to utilize formate, acetate, propionate, methyl- electron-dense spheres that may be polyphosphate pyruvate and a-ketoglutarate but were unable to utilize granules. The cell wall is of the Gram-negative type, as L-arabinose, ribose, lactose, N-acetyl-D-galactosam- recognized from the presence of the outer membrane ine, N-acetyl-D-glucosamine, glycolate, citrate, DL- and the peptidoglycan layer (see inset in Fig. lb for lactate, 2,3-butanediol, D-sorbitol, L-asparagine, L- detail). The width of the cell wall ranged from 31 to ornithine and L-proline as single source of carbon and 38 nm. The cultures grew at temperatures ranging energy. The strains grew slowly on a-, b- and y- from 4 to 35 "C with an optimal temperature between hydroxybutyrates, although no accumulation of their 25 and 30 "C.The growth of all strains occurred in the polymers was detected. presence of NaCl concentrations ranging from 1-0 to 12.5 % with an optimum observed between 3 and 10 %. The phospholipid ester-linked fatty acid (PLFA) The strains required Na+ cations, as no growth was patterns of all strains were essentially similar and observed in an osmotically equivalent medium lacking contained features indicative of members of the p- or y- Na+ (data not shown). The divalent cations present in subclasses of the Proteobacteria (Table 2). Some minor SM1 medium were also required for growth and Mg2+ differences were observed in the PLFA compositons of cations were found to be particularly important for these strains (Table 2). AP1, SK2T, SK4A and SK7 preventing cell lysis. These results are in agreement lacked summed feature 2 (17 :0 cyclo/ 17 : lm6), with observations reported for other marine bacteria 19 : 108, 19 :0 cyclo and 20 : la19 fatty acids found in (9, 19). Sodium nitrate could serve as sole nitrogen the PLFA composition of MM1 and SK. The pre- source. Nitrate was reduced to nitrite under facultative dominant PLFAs detected in all six strains were 16 :0, anaerobic conditions. All A. borkurn'knsis strains ex- summed feature 1 (1 6 : 1017416 : 1019t) and summed hibited tweenase activity, although agarase, amylase, feature 3 (18: 107c,t/18: 109t/18: 1012). These fatty arginine dihydrolase, gelatinase and esculinase ac- acids accounted for greater than 95% of the total

~ ~ ~~ 344 lnterna tional Journal of Systematic Bacteriology 48 Alcanivorax borkumensis gen. nov., sp. nov.

Ch. marismortui

Bacterium WB4

A. borkumensis SKZT- Marinobacter sp. CAB Mb. hydrocarbon oclasticus

I? aeruginosa \‘ f! putida I? fluorescens ....., ...... ,...... co. co~igetii’// \Mm. vaga Fig.evolutionary 2. Phylogenetic distances analysis tree derivedof the 165 from rRNA agene Jukes-Cantor sequences of A. borkumensis SKZT and representative members of the \ y-subclass of the Proteobacteria. Alt., Alteromonas; Ch., Chromohalobacter; Co., Codakia; Cy., Cycloclasticus; E., Escherichia; H., Halomonas; Mm., Marinomonas; Mb., Alt. haloplanktis Marinobacter; O., Oceanospirillum; P., Pseudomonas; T., Alt. macleodii Thyasira; Z., Zymobacter; Bacterium 25B1, environmental isolate 25B1 from Lake Bogoria (accession no. X92139); E. coii Bacterium WB4, environmental isolate WB4 from Lake Bogoria 0-1 (X92145).

PLFAs detected in APl, SK2T, SK4A and SK7 and ation. According to the method of analysis, SK2Twas 88.4 and 83.4% of the total PLFA detected in SK found to be phylogenetically most closely related to a and MM 1, respectively. Such a PLFA profile is typical number of marine bacteria that included two species of for members of the halophilic genera Halomonas (22) Marinobacter, gill symbiont Codakia costata, Halo- and no clear distinction based upon PLFA com- monas elongata and recently isolated lignin-degrading position was observed between strains of A. borku- bacterium Mycrobulbifer hydrolyticus (7). A similarity mensis and reference strains of two species of Halo- matrix derived from the sequences most similar to that monas (Table 2). of the SK2T 16s rDNA sequence is shown in Table 3. None of the reference sequences exhibited a level of G + C content of DNA similarity with the SK2T sequence greater than 0.900. The highest similarity values were found for Marino- The G + C content of the DNA of SK2Twas shown to bacter sp. CAB (0*900), Marinobacter hydrocarbono- be 53.4 mol%. This is lower than the typical G+ C clasticus (0-895) and Deleya marina (0.893). Inter- content of the DNA of the genus Halomonas but is estingly, the results of additional analysis of the SK2T comparable with the G+C content of Marinobacter sequence in which the RDP program CHECKCHIMERA hydrocarbonoclasticus SP. 17T (Table 1). was used, demonstrated that one fragment of the SK2T Molecular phylogenetic analysis 16s rDNA sequence (approx. sequence positions 1-670) was more similar to Marinobacter hydro- The 16s rDNA sequences of all six isolates were carbonoclasticus 16s rRNA (S-ab = 0601) and a almost identical. Only one strain, APl, had two second fragment (approx. sequence positions 67 1- replacements, G instead of A and A instead of G at 1497) was more similar to gill symbionts Codakia positions 840 and 1138, respectively (Escherichia coli costata and Thyasira jlexuosa 16s rRNA sequences numbering). The sequences determined indicated that (S-ab = 0-654 and 0.653, respectively). the organisms are members of the y-subclass of the Proteobacteria. The 16s rDNA sequence of strain DISCUSSION SK2T was compared with that of all organisms clustering in the Proteobacteria y-subclass (13,26) and On the basis of the results of phenotypic analyses such the relative phylogenetic position was estimated (Fig. as isolation from marine sources, a restricted nu- 2). Strain SK2T showed a rather deep branching, with tritional profile, high halotolerance, absence of fer- uncertainty concerning its closest phylogenetic affili- mentative metabolism and low G + C content, AP1,

International Journal of Systematic Bacteriology 40 345 M. M. Yakimov and others

Table 3. Similarity levels observed by comparison of 165 rRNA sequence of A. borkurnensis SK2T with those of some other members of the y-subclass of the Proteobacteria

Organism EMBL No. of Similarity to A. accession no. nucleotides borkumensis SK2T

A. borkumensis SK2T Y12579 1503 1*ooo Marinobacter sp. CAB U6 1848 1534 0-900 Marinobacter hydrocarbonoclasticus X67022 1483 0.895 Deleya marina X87217 1478 0.893 Codakia costata (gill symbiont) L25712 1493 0.892 Microbulbifer hydrolyticus U58338 1393 0.892 Halomomas elongata M93355 1479 0.890 0cean ospir illum linum M22365 542 0.888 Cycloclasticus puge tii L34955 494 0.886 Marinomonas vaga X67025 467 0.884 Marinobacterium georgiense U58339 435 0.879 Pseudomonas mendocina M59154 490 0.877 Pseudomonas aeruginosa X06684 537 0.874 Aeromonas caviae X60409 502 0.872 Nitrosococcus oceanus M96395 397 0.870 Shewanella putrefaciens X8 1623 536 0.867 Chroma tium vinosum M26629 1494 0.865 Vibrio pelagus X74722 1464 0.860 VibriofulnLficus X76333 1536 0857 Alteromonas macleodii X82 145 1432 0.850 Escherichia coli 501695 1539 0.849 Comamonas testosteroni M11224 1536 0.806

MMl, SK, SK2T, SK4A and SK7 share many traits be 53.4 mol%. Analysis of the 16s rRNA gene with the genera Halomonas, Marinomonas, Marino- sequence enabled an estimation of the phylogenetic bacter and Oceanospirillum (Table I), all of which positions of the isolated strains. These bacteria are represent a group of Gram-negative marine species affiliated with the y-subclass of the Proteobacteria. The with respiratory metabolism. The PLFA profile of closest relatives of these organisms are Marinobacter these isolates clearly indicated their similarity to sp. CAB, Marinobacter hydrocarbonoclasticus and members of the halophile and halotolerant genus Deleya marina. A similarity matrix revealed that, Halomonas. However, Halomonas, Marinomonas and although the isolated strains exhibited levels of nucleo- Oceanospirillum species have greater nutritional ver- tide base homology to one another of more than satility, are motile and exhibit lower halotolerance 99.9 %, they are less than 90 % similar to any pre- (Marin om onas and Oceanospirillum) than the isolated viously described organism for which a 16s rRNA strains. A phenotypic distinction from Marinobacter sequence is known. It is apparent from the phenotypic hydrocarbonoclasticus,described by Gauthier et al. (9), properties and the differences in 16s rRNA gene also includes significant differences from the isolated sequences that the hydrocarbon-degrading, sur- strains : motility, higher halotolerance, denitrification factant-producing strains isolated from marine and utilization of succinate, L-glutamate and p- samples from the Isle of Borkum (North Sea) cannot hydroxybenzoate. However, other phenotypic charac- be assigned to any previously recognized bacterial teristics of the isolated strains conformed to those genus and can be described as a new species within a described for the genus Marinobacter (9): rods, gen- new genus under the epithet Alcanivorax borkumensis. erally 0.648 pm wide and 16-23 pm long; chemo- organotrophs capable of respiratory but not fermenta- Description of Alcanivorax gen. nov. tive metabolism; able to grow anaerobically on organic substrates in the presence of nitrate; able to degrade Alcanivorax (Al’.ca.ni.vo’.rax. M.L. masc. n. alcanum alkane, aliphatic hydrocarbon ; adj. vorax vor- aliphatic hydrocarbons ; halotolerant and able to grow L. in a large range of NaCl concentrations; absence of acious, gluttonous ; M.L. masc. n. Alcanivorax alkane- anaerobic growth on glucose ; unable to utilize carbo- devouring). hydrates and amino acids as sole carbon source; able Gram-negative non-motile aerobic rods that are gen- to produce biosurfactants or bioemulsifiers during erally 06-0.8 pm wide and 16-25 pm long when growth on n-alkanes. growing in pyruvate-supplemented medium. They are shorter (1.0-1-5 pm) when n-alkanes are used as single The G + C content of Alcanivorax DNA was found to carbon and energy source. Oxidase- and catalase-

346 International Journal of Systematic Bacteriology 48 Alcanivorax borkumensis gen. nov., sp. nov. positive. Aliphatic hydrocarbons are used as sole or REFERENCES principal carbon sources for growth. A mixture of 1. Al-Mallah, M., Goutx, M., Mille, G. & Bertrand, J. C. (1990). active biosurfactants, glucose lipids, are produced, one Production of emulsifying agents during growth of a marine of which consists of four 3-hydroxydecanoic acids Alteromonas in seawater with eicosane as carbon source, a linked together by ester bonds and coupled glyco- solid hydrocarbon. Oil Chem Pollut 6, 289-305. sidically with C-l of glucose. The G C content of the + 2 Attygalle, A. B. & Morgan, E. D. (1988). Pheromones in DNA isolated from six strains of the single species is nanogram quantities : structure determination by combined 53-54 mol% . The type and only species of the genus is microchemical and gas chromatographic methods. Angew Alcanivorax borkumensis. 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