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Description of Alcanivorax venustensis sp. nov. and reclassification of Fundibacter jadensis DSM 12178^T (Bruns and Berthe-Corti 1999) as Alcanivorax jadensis comb. nov., members of the emended genus Alcanivorax

Javier Fernández-Martínez¹, María J. Pujalte^2,3, Jesús García-Martínez^1,, Manuel Mata¹, Esperanza Garay^2,3 and Francisco Rodríguez-Valera¹

¹ División de Microbiología, Universidad Miguel Hernández, Campus de San Juan, Ctra. de Valencia km 87, 03550 San Juan, Alicante, Spain
² Departamento de Microbiología, Universitat de València, Campus de Burjassot, 46100 Burjassot, Valencia, Spain
³ Instituto Cavanilles de Biodiversidad y Biologia Evolutiva, Universitat de València, Spain

Correspondence
Jesús García-Martínez
jesus.garcia{at}ua.es

	ABSTRACT

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Two strains of a novel bacterium were isolated independently of each other, from different depths in the Mediterranean Sea, within a time period of 7 months, using two different isolation approaches that were focused on different objectives. Both strains, designated ISO1 and ISO4^T, were halophilic, Gram-negative, strictly aerobic, straight rods that were oxidase- and catalase-positive. Both strains produced mucoid colonies in some defined minimal media and were able to grow with organic acids and some alkanes; they were also able to accumulate intracellular poly--hydroxybutyrate granules. The G+C content of the DNA of strain ISO4^T was 66 mol%. Comparative analysis of 16S rRNA gene sequences showed that the closest described species to the novel strains were Alcanivorax borkumensis and Fundibacter jadensis, both of the -Proteobacteria. Both of these recognized species were originally isolated from North Sea waters and are able to degrade aliphatic compounds, a property shared with strains ISO1 and ISO4^T. However, strains ISO1 and ISO4^T were different from A. borkumensis and F. jadensis, not only in their 16S rDNA sequences but also in the motility of their cells (by polar flagella) and by the presence of C_{19 : 0cyclo} in their cellular fatty acids, among other differential features. On the basis of biochemical and molecular data, it is suggested that strains ISO1 and ISO4^T be recognized as a novel species of the genus Alcanivorax, for which the name Alcanivorax venustensis (ISO4^T =DSM 13974^T =CECT 5388^T) is proposed. On the basis of its high phenotypic similarity and close phylogenetic relatedness to A. borkumensis, it is also proposed that F. jadensis (DSM 12178^T) be reclassified as Alcanivorax jadensis in the genus Alcanivorax, and that the description of the genus Alcanivorax be emended.

The GenBank accession numbers for the nucleotide sequences reported in this study are AF328761 (Alcanivorax venustensis ISO1, 16S rRNA gene), AF328762 (A. venustensis ISO4^T, 16S rRNA gene), AF328763 (ISO1, large ITS), AF328764 (ISO1, small ITS), AF328765 (ISO4^T, large ITS), AF328766 (ISO4^T, small ITS), AF328767 (Fundibacter jadensis T9^T, large ITS) and AF328768 (T9^T, small ITS).

Present address: Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Ctra. de San Vicente s/n. Ap. 99-03080, Alicante, Spain.

	INTRODUCTION

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The so-called PCR approach has shown that marine waters are vast reservoirs of previously unrecognized micro-organisms (Giovannoni et al., 1995). Although the number of novel taxa that have been brought into pure culture has not increased dramatically, the use of molecular techniques to characterize micro-organisms and the development of more sophisticated culture media and retrieval strategies (trying to match natural conditions) have certainly contributed to increasing the success of isolating novel, previously uncultivated prokaryotes from a given environment (Button et al., 1998). Therefore, we have tried to improve our chances of isolating novel marine micro-organisms by using extremely oligotrophic enrichment conditions and size fractionation of samples (i.e. inoculation after filtering the sample through absolute membrane filters of different pore sizes). As a result of two independent sampling campaigns in the Mediterranean, a variety of bacterial strains were isolated using these two different approaches. Of these strains, two (designated ISO1 and ISO4^T) showed marked similarity in their 16S rDNA sequences (99·7 %) and were not related to any other strains/species in the databases above a similarity level of 94 %. Both strains were able to utilize several aliphatic compounds. Hydrocarbon-degrading bacteria are of potential importance in bioremediation processes (Chayabutra & Ju, 2000; Wilson et al., 1999; Banat et al., 2000; Nocentini et al., 2000) and many of these micro-organisms have, in fact, been isolated from polluted areas in the Mediterranean (Bertrand et al., 1983). Here, we characterize strains ISO1 and ISO4^T and, on the basis of their phenotypic and phylogenetic characteristics, propose they that be classified as a novel species within the genus Alcanivorax.

	METHODS

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Isolation and cultivation.
Strain ISO1 was retrieved from a sea-water sample obtained, with a Niskin bottle, off the coast of Alicante (Mediterranean Sea, southeastern Spain; at 38°06·970'N, 0°27·626'W) from a depth of 5 m (bottom depth 45 m) during December 1999. The sample was filtered through 5 µm and 0·22 µm pore-diameter filters (Durapore; Millipore), respectively, to remove larger particles, eukaryotes and larger prokaryotes. Initial enrichments were prepared in duplicate by inoculating 1 ml of the filtered sample into 9 ml of medium containing aged Mediterranean sea water filtered through a 0·22 µm pore-size membane and supplemented with 0·1 g FRV l^-1 [FRV=a mix of equal parts of Spirulina (Sigma), Artemia salina (the kind provided by pet shops for aquaria) and fish-meal (animal-feed grade), resuspended in filtered sea water for 4 h and then passed through filter paper to remove larger particles]. The medium was adjusted to pH 7·4 and autoclaved. Enrichments were incubated in the dark at 14 °C and at room temperature (22–23 °C) under light for 1 week. After this period, enrichments were again filtered through a 0·22 µm pore-size membrane; they were then diluted into aged sea water with 3x10^-3 g FRV l^-1, and incubated again at 14 °C in the dark and at room temperature under light for 1 month. After this incubation, the enrichments were plated onto solid FRV (3x10^-3 g l^-1). This enrichment strategy was intended to select for ultramicrobacteria, which are able to pass through 0·2 µm pore-size filters, or bacteria producing smaller starvation forms under oligotrophic conditions. Single colonies were then transferred serially to obtain pure cultures and to produce biomass for further analyses.

Strain ISO4^T was isolated from a sea-water sample obtained 22 miles off the coast of Santa Pola (Alicante, Spain; at 37° 55·020'N, 0° 17·014'W) at a depth of 200 m (bottom depth 280 m), using a Niskin bottle, during June 1999. The sample was submitted to enrichment in continuous culture at extremely low nutrient concentrations and dilution rates, to retrieve bacteria able to grow under oligotrophic conditions. The sea-water sample was passed through a 5 µm pore-size filter to remove large particles. The small bioreactor (150 ml) was completely filled up with the sample and maintained at 14 °C (in situ temperature). After incubation without any supplement for 24 h, a flow rate was established with a medium made with sterile sea water supplemented with 3x10^-3 g FRV l^-1. The dilution rate was set at 120 h^-1. Aliquots were taken from the bioreactor at regular intervals for up to 3 months after the beginning of the experiment. These aliquots were immediately plated onto medium (sterile sea water supplemented with 3x10^-3 g FRV l^-1) containing 1·5 % agar. After 2 months incubation, the smallest detectable colonies on the agar were selected and re-isolated to obtain pure cultures.

Alcanivorax borkumensis DSM 11573^T (=SK2^T =CECT 5355^T) and Fundibacter jadensis DSM 12178^T (=T9^T =CECT 5356^T) were obtained from the Colección Española de Cultivos Tipo (CECT) (Universitat de València, Valencia, Spain) and grown as recommended (Yakimov et al., 1998; Bruns & Berthe-Corti, 1999).

DNA extraction, amplification and sequencing.
Single colonies from pure cultures were picked up with autoclaved toothpicks, resuspended in 150 µl sterile distilled water and boiled for 10 min. The suspensions were then centrifuged at maximum speed. Five microlitres of the resulting supernatant were used to perform PCRs in a final volume of 50 µl (Taq DNA polymerase, Recombinant; GIBCO). The ribosomal 16S and 23S primers used for amplifications were ANT1 (16S; forward; positions 7–26, Escherichia coli numbering; 5'-AGAGTTTGATCATGGCTCAG-3'; García-Martínez et al., 1999), 16S14F (16S; forward; 1389–1407; 5'-CTTGTACACACCGCCCGTC-3'; García-Martínez et al., 1996) and 23S1R (23S; reverse; 110–130; 5'-GGGTTTCCCCATTCGGAAATC-3'; García-Martínez et al., 1999). The primer pair ANT1/23S1R was used to amplify the complete 16S rRNA genes plus the 16S–23S internal transcribed spacers (ITSs) of strains ISO1 and ISO4^T, whereas primer pair 16S14F/23S1R was used to amplify the ITS region of F. jadensis T9^T. Amplification products were run on 1 % low-melting-point agarose gels (Bio-Rad), and the resulting bands were purified using the GENECLEAN II kit (BIO 101). Sequencing of the fragments was carried out on a model 377 automated DNA sequencer (Applied Biosystems).

Phylogenetic analysis.
BLAST similarity searches in the databases (Altschul et al., 1997) were conducted with the sequences generated for strains ISO1, ISO4^T and T9^T. The presence/absence of tRNA genes within the ITS regions was confirmed using the tRNAScan-SE search server (Lowe & Eddy, 1997). Alignments were carried out using CLUSTAL W in the MEGALIGN program (DNASTAR), while phylogenetic analyses were carried out using MEGA (version 1.02; Pennsylvania State University, USA).

Determination of G+C content and cellular fatty acid analysis.
DNA G+C content was determined only for strain ISO4^T, whereas analysis of fatty acids was performed on strains ISO1 and ISO4^T. All tests were carried out at the identification service laboratories of the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). Cells were grown as recommended by Yakimov et al. (1998).

Automated ribotyping.
Automated ribotyping was performed using the RiboPrinter microbial characterization system according to the manufacturer's instructions (Qualicon). Restriction enzymes used were EcoRI, PstI and PvuII. A large DNA probe harbouring the genes for both the small- and large-subunit rRNA of E. coli was employed. Each set of data was normalized, using an adjacent standard marker set, by the RiboPrinter integrated software.

Phenotypic characterization.
The strains were tested for growth on marine agar (MA; Difco) and marine broth (MB), both at standard nutrient concentration and as 1/10 dilutions of the standard nutrient concentration, at 13 and 25 °C. Diluted versions of MA and MB were obtained by dissolving 1/10 of the usual amount of MA or MB powder in half-strength artificial sea water (Baumann & Baumann, 1981), to maintain the salinity of the medium, with the addition of purified agar (final concn 1·2 %, w/v; Oxoid) to MA. After obtaining growth on MA and in MB, routine cultivation was done on MA or in MB at 23–25 °C.

Morphology and motility of the cells were examined on wet-mounts by phase-contrast microscopy (Leica; DAS Mikroskop LeiztDMR), using 3-day-old cultures of the strains grown on MA. Flagellar arrangement was observed after staining the same cells used in the phase-contrast microscopy, following the method of Heimbrook et al. (1989). The cells were also examined by scanning and transmission electron microscopy, according to Acinas et al. (1999) and Cole & Popkin (1981). Methods used to determine most of the phenotypic traits of the strains have been described previously (Baumann & Baumann, 1981; Ortigosa et al., 1994), except for sodium, magnesium and calcium requirements and salinity tolerance, which were determined in salt tolerance broth (STB 1/10: 0·5 g tryptone l^-1; 0·2 g yeast extract l^-1; 0–200 g NaCl l^-1; 0–1 g MgCl₂ l^-1; 0–1 g CaCl₂ l^-1; pH 7·2) and MB plus NaCl (from normal to 20 %, w/v, total NaCl). The use of nitrate as sole nitrogen source by the strains was tested on basal medium agar with NaNO₃ instead of NH₄Cl, and 1 g sodium pyruvate l^-1 as the carbon source. Tests for hydrolases, anaerobic growth with glucose or nitrate, growth at 4 °C and growth in the presence of sole carbon sources were incubated for up to 28 days; other tests were determined after 14 days incubation. The presence of intracellular poly--hydroxybutyrate (PHB) accumulation was examined as described by Burdon (1946). Susceptibility to antimicrobial agents was determined by the disc-diffusion test on MA plates, after 7 days incubation.

The filterability of strain ISO1 was examined by plating successive dilutions (from 10^-1 to 10^-6) of a cell suspension from a colony on solid medium onto MA, before and after filtration through 0·22 µm pore-size membrane filters.

	RESULTS AND DISCUSSION

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Isolation and phenotypic characterization
Strains ISO1 and ISO4^T were Gram-negative, straight rods (0·9–1·8x0·3–0·5 µm wide), when observed on wet-mounts by optical microscopy and by scanning electron microscopy (Fig. 1). The fact that they were isolated after two passes through 0·22 µm membrane filters might be an indication of either an extremely flexible cell wall or the hypothetical presence of some smaller, filterable forms. However, no such filterability was observed under laboratory conditions, i.e. no colonies were detected after filtration of as many as 10⁴ cells (c.f.u.) through 0·22 µm membrane filters. Strains ISO1 and ISO4^T were motile by at least one polar flagellum, as observed for a few cells by transmission electron microscopy. Flagella were abundant in mounts prepared by the method of Heimbrook et al. (1989) on actively moving cells, but it was difficult to observe the number of flagella per pole. When grown on glucose-containing media, the cells showed some PHB accumulation (granules).

View larger version (132K): [in this window] [in a new window]   Fig. 1. Scanning electron micrographs of cells of (A) strain ISO1 and (B) strain ISO4T. Bars, 1 µm.

Strains ISO1 and ISO4^T were able to grow at temperatures ranging from 4 to 40 °C, but not at 45 °C. Both strains were strictly halophilic, since no growth was observed without the addition of marine salts to the media. Furthermore, media containing NaCl alone or a combination of NaCl, MgCl₂ and CaCl₂ did not support growth, but good growth was observed in normal MB and in MB with NaCl added up to 10 %. Weak growth of the strains was also observed in MB supplemented with NaCl up to 15 % and even up to 20 %, although growth on the latter was very weak. This suggests that strains ISO1 and ISO4^T have more complex ionic requirements than other marine bacteria, which often only require the addition of NaCl to media.

Strains ISO1 and ISO4^T were aerobic chemoheterotrophs that were unable to ferment glucose or to develop with nitrate in anaerobic conditions. They did not reduce nitrate to nitrite or gas. They used both ammonium and nitrate as a nitrogen source in basal medium containing pyruvate. No extracellular hydrolysis was detected on casein, gelatin, starch, agar, alginate or DNA. Tween 80 was readily hydrolysed by the strains. Both strains were oxidase- and catalase-positive, but no activities were observed for arginine dihydrolase (in both Thornley and Moeller's media) or lysine decarboxylase. Neither strain produced indole from tryptophan.

The spectrum of carbon sources used by the bacteria was narrow. Out of the 62 compounds tested with strains ISO1 and ISO4^T, good growth was obtained only with some organic acids (propionate, pyruvate, acetate and 3-hydroxybutyrate) and the alkane tetramethylpentadecane. The strains also grew, to a lesser extent, on hexadecane and tetradecane. Variable results were obtained on DL-lactate, D-mannose and glycerol. Some carbohydrates rendered slight growth of the strains (D-ribose, D-glucose, D-fructose, D-trehalose, sucrose, N-acetylglucosamine and D-mannitol), but these results were not reproducible. None of the following substrates was used by either strain: L-arabinose; D-xylose; D-galactose; L-rhamnose; maltose; D-cellobiose; lactose; D-melibiose; raffinose; salicin; amygdalin; D-gluconate; D-glucuronate; D-galacturonate; glucosamine; D-sorbitol; m-inositol; D-glycerate; saccharate; citrate; trans-aconitate; 2-oxoglutarate; succinate; fumarate; malate; p-hydroxybenzoate; n-eicosane; phenanthrene; glycine; L-leucine; L-serine; L-threonine; L-arginine; L-tyrosine; L-glutamate; L-alanine; GABA; L-ornithine; L-citrulline; L-aspartate; L-glutamine; L-lysine; L-histidine; sarcosine; putrescine.

Antimicrobial susceptibility testing revealed that strain ISO1 was sensitive to augmentin (30 µg ml^-1), cefotaxime (30 µg ml^-1), cefuroxime (30 µg ml^-1), cephalotin (30 µg ml^-1), ciprofloxacin (1 µg ml^-1) and rifampicin (2 µg ml^-1), whereas strain ISO4^T was only sensitive to cefotaxime (30 µg ml^-1) and cephalotin (30 µg ml^-1).

Table 1 shows characteristics useful for distinguishing strains ISO1 and ISO4^T from related marine bacterial taxa.

View this table: [in this window] [in a new window]   Table 1. Characteristics useful for distinguishing strains ISO1 and ISO4T from related marine -Proteobacteria Strains/species: 1, strains ISO1/ISO4T (this study); 2, A. borkumensis (Yakimov et al., 1998); 3, F. jadensis (Bruns & Berthe-Corti, 1999); 4, Marinobacter spp. (Gauthier et al., 1992; Huu et al., 1999); 5, Neptunomonas naphthovorans (Hedlund et al., 1999); 6, Halomonas spp. (Vreeland, 1992; Kersters, 1992); 7, Oceanospirillum spp. (Krieg, 1984). ND, No data; +, positive reaction or growth; -, no reaction or growth; W, weak reaction; V, variable reaction.

Automated ribotyping
The ribotype patterns of strains ISO1 and ISO4^T were always identical, whatever the enzyme used, and were different from the ones produced from A. borkumensis DSM 11573^T and F. jadensis DSM 12178^T. A. borkumensis DSM 11573^T and F. jadensis DSM 12178^T also produced identical patterns to each other when digested with the three enzymes. Fig. 2 shows the profiles obtained with EcoRI.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Ribotype patterns obtained with EcoRI for strains ISO1 and ISO4T (lanes 3 and 4, respectively), and A. borkumensis DSM 11573T (lane 1) and F. jadensis DSM 12178T (lane 2). M, Molecular size markers.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Phylogenetic tree showing relationship of strains ISO1 and ISO4T with other -Proteobacteria, as inferred from a comparison of aligned 16S rDNA sequences (1197 bp; Kimura two-correction parameter, neighbour-joining method). Only significant bootstrap values are shown at the nodes.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Genetic relationships inferred using the alignable regions of the 16S–23S rRNA ITS sequences (large and small, 225 bp in total) from two strains of A. borkumensis, SK2T and LE4, F. jadensis DSM 12178T and strains ISO1 and ISO4T (Kimura two-correction parameter, neighbour-joining method). Bootstrap values are shown at the nodes.

-Proteobacteria

However, the assignment of the novel species to a genus is problematic. The two recognized species most closely related to the novel strains in the phylogenetic analysis were A. borkumensis and F. jadensis, two species that share several key physiological characteristics with strains ISO1 and ISO4^T, such as their ability to use alkanes as carbon and energy sources, their requirement for marine salts and their major fatty acids. The phylogenetic distance, based on 16S rDNA sequence analysis, of strains ISO1 and ISO4^T from both recognized species was on the borderline of the 95 % level suggested to define genera (Rosselló-Mora & Amann, 2000), and analyses of differences in ITS sequences were also compatible with the inclusion of the novel species in the genus Alcanivorax or Fundibacter: the differences were within the mean differences characteristic for a single and well-defined genus (García-Martínez et al., 1999; García-Martínez & Rodríguez-Valera, 2000). In addition, the 16S rRNA gene sequences of A. borkumensis DSM 11573^T and F. jadensis DSM 12178^T displayed 97·2 % similarity, which was higher than the 16S rRNA gene sequence similarity observed between the ISO strains and these recognized species. The ribotypes of A. borkumensis and F. jadensis were always coincident and few phenotypic traits allowed the discrimination of these supposedly different genera. For example, PHB accumulation, exopolymer production and fimbriae synthesis are traits that are usually considered to be strain- or culture-dependent. The only feature that allowed discrimination of the two recognized genera was DNA G+C content. From our analyses, it appears that A. borkumensis DSM 11573^T and F. jadensis DSM 12178^T are, in fact, more similar to each other than to strains ISO1 and ISO4^T, with the exception of G+C content and PHB accumulation, which were more similar between F. jadensis DSM 12178^T and the ISO strains. The fatty acid pattern of strains ISO1 and ISO4^T showed the same principal components as A. borkumensis DSM 11573^T and F. jadensis DSM 12178^T, but it also included a specific component, C_{10 : 0cyclo} (Table 2). Nevertheless, the unusually high percentage of undetermined fatty acids of F. jadensis DSM 12178^T (Bruns & Berthe-Corti, 1999) makes it difficult to establish any definitive conclusion. In contrast to A. borkumensis (DSM 11573^T) and F. jadensis (DSM 12178^T), which were non-motile, strains ISO1 and ISO4^T were motile by polar flagella.

Thus, from the above data, it is our opinion that the maintenance of the genera Alcanivorax and Fundibacter and the description of a novel genus to accommodate strains ISO1 and ISO4^T is unjustified. The global phenotypic similarity of the three species and the monophyly of the group in the phylogenetic analyses done here (16S rRNA and ITS) have led us to emend the description of the genus Alcanivorax to accommodate the transfer of F. jadensis DSM 12178^T to the genus, and to describe a novel species, represented by strains ISO1 and ISO4^T, for which the name Alcanivorax venustensis is proposed.

Emended description of the genus Alcanivorax (Yakimov et al. 1998)
Species are Gram-negative, aerobic, straight rods. Non-motile and non-flagellated, or motile by polar flagella. Strictly respiratory type of metabolism. Do not ferment carbohydrates. Some species may use nitrate as an alternate electron acceptor, but none denitrify nitrogen compounds. Oxidase- and catalase-positive. Halophilic, requiring (at least) Na⁺ ions for growth: some species have more complex ionic requirements. Able to grow in the presence of up to 12 % NaCl. Chemo-organotrophic, using short-chain fatty acids and some alkanes as sole or principal carbon sources. Acetate, pyruvate and hexadecane are used as carbon sources. Principal fatty acids are C_{16 : 0}, C_{18 : 17c} and C_{16 : 17c}. DNA G+C content of species ranges from 53 to 66 mol%. Isolated from marine habitats. On the basis of 16S-rDNA-based phylogenetic analyses, the genus belongs to the -Proteobacteria. Type species is Alcanivorax borkumensis (SK2^T =DSM 11573^T =CECT 5355^T =ATCC 700651^T =CIP 105606^T) (Yakimov et al., 1998).

Description of Alcanivorax jadensis comb. nov. (basonym Fundibacter jadensis Bruns and Berthe-Corti 1999)
The description is identical to the one given by Bruns & Berthe-Corti (1999). The type strain of the species is T9^T (=DSM 12178^T =CECT 5356^T =ATCC 700854^T).

Description of Alcanivorax venustensis sp. nov.
Alcanivorax venustensis (ve.nus.ten'sis. M.L. adj. venustensis from ‘Portus Venustus’, Elegant Port, one of the ancient Latin names of Santa Pola in Roman times, a coastal town south of Alicante, the nearest town to the sites from which the two strains of the species were retrieved. It might also refer to the elegant and slender aspects of the rods).

Aerobic. Gram-negative, straight rods (0·9–1·8x0·3–0·5 µm). Motile by means of polar flagella. Grows well on MA (Difco), as small, regular-shaped, non-pigmented colonies. Growth at extremely low nutrient concentrations is also possible (facultative oligotroph). Strain ISO1 was isolated from filtered sea water (0·22 µm pore size), but no other filterable forms have been detected in the laboratory. Temperature range for growth is 4–40 °C. Marine salts are required for growth and are not replaceable by NaCl at an equivalent concentration. Grows in the presence of up to 15 % (w/v) NaCl in the medium. Degrades Tween 80, but casein, gelatin, starch, alginate, agar and DNA are not hydrolysed. Nitrate is not reduced to nitrite or gas. Sugars are not fermented. Catalase- and oxidase-positive. Carbon sources that support good growth are pyruvate, propionate, acetate, 3-hydroxybutyrate and tetramethylpentadecane. Amino acids are not used. Growth on minimal media with organic acids is accompanied by the formation of mucous colonies. Principal fatty acids are C_{16 : 0}, C_{16 : 1w7c}, C_{18 : 1w7c} and C_{19 : 0cyclo}, with minor amounts of 3OH-C_{12 : 0}, C_{12 : 0} and C_{10 : 0}. DNA G+C content of the type strain is 66 mol%. Isolated from sea water. The type strain is ISO4^T (=DSM 13974^T =CECT 5388^T). Strain ISO1 has also been deposited in the CECT as CECT 5389.

Distribution and ecological aspects
Although both strains of A. venustensis were obtained from two independent samples and enrichment approaches, the sampling sites were geographically quite close (within a few miles of each other). However, it is very likely that related organisms might also be retrieved from rather widespread locations (as was the case with A. borkumensis DSM 11573^T). Analysis of the complete 16S rDNA sequences of strains ISO1 (1530 bp) and ISO4^T (1532 bp) showed that both strains had more than 99 % similarity with the 16S rDNA sequences of unnamed strains isolated in Japan and the North Sea [1302 bp (Yoshinaga et al., 1999) and 1331 bp (Eilers et al., 2000), respectively]. However, the Japanese strain was primarily characterized as being obligately oligotrophic, a trait that is not shared with the proposed novel species, a facultative oligotroph and psychrophile. The most closely related recognized species to A. venustensis, based on 16S rDNA sequence analysis, were A. borkumensis and F. jadensis (Yakimov et al., 1998; Bruns & Berthe-Corti, 1999). The global distribution of A. borkumensis and F. jadensis and the fact that they are essentially non-fastidious micro-organisms that are relatively easy to culture raises some questions regarding their late isolation. One possibility is, of course, that the recent use and development of molecular techniques for large-scale screening of environmental samples is only now revealing the full scale of potentially novel micro-organisms. Another possibility is the likely limited numbers of these species within the total microflora of their respective habitats, making their isolation from mixed populations more difficult. It is possible that the contribution of the oligotrophic conditions in the FRV medium used for the enrichments favoured the growth of strains ISO1 and ISO4^T, even in the case of ISO1 after two filtration steps. Some authors have suggested for hydrocarbon-degrading bacteria such as F. jadensis and Marinobacter hydrocarbonoclasticus (Bruns & Berthe-Corti, 1999) that their abundance in certain locations might be associated with polluted waters. However, strains ISO1 and ISO4^T were isolated from open, apparently pristine, sea waters, with one of the sample sites being 20 miles off the coast and at a depth of 280 m (ISO4^T), with no evidence of hydrocarbon pollution. Nevertheless, it should be noted that the pick-up coordinates for strain ISO4^T were close to a busy shipping route, and occasional oil spillages might occur at this site. If this were the case, members of the genus Alcanivorax and other closely related -Proteobacteria could become useful micro-organisms as bio-indicators of waters contaminated with low to high levels of long-chain hydrocarbons.

	ACKNOWLEDGEMENTS

 
We thank Stuart Ingham for graphics and image processing, Carmela Belloch from CECT for her technical assistance in the ribotyping and H.-J. Busse for his help with fatty acid data interpretation. This work was supported by MIDAS (European Commission MAST Grant MAS3-CT-97-0154).

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Acinas, S. G., Antón, J. & Rodríguez-Valera, F. (1999). Diversity of free-living and attached bacteria in offshore Western Mediterranean waters as depicted by analysis of genes encoding 16S rDNA. Appl Environ Microbiol 65, 514–522.[Abstract/Free Full Text]

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[Abstract/Free Full Text]

Banat, I. M., Makkar, R. S. & Cameotra, S. S. (2000). Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol 53, 495–508.[CrossRef][Medline]

Baumann, P. & Baumann, L. (1981). The marine gram-negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas and Alcaligenes. In The Prokaryotes, vol. 1, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balows & H. Schlegel. Berlin: Springer-Verlag.

Bertrand, J. C., Rambeloarisoa, E., Rontani, J. F., Giusi, G. & Mattei, G. (1983). Microbial degradation of crude oil in sea water in continuous culture. Biotechnol Lett 5, 567–572.[CrossRef]

Bruns, A. & Berthe-Corti, L. (1999). Fundibacter jadensis gen. nov., sp. nov., a new slightly halophilic bacterium, isolated from intertidal sediment. Int J Syst Bacteriol 49, 441–448.[Abstract/Free Full Text]

Burdon, K. L. (1946). Fatty material in bacterial and fungi revealed by staining dried, fixed slide preparations. J Bacteriol 52, 665–678.[Free Full Text]

Button, D. K., Robertson, B. R., Lepp, P. W. & Schmidt, T. M. (1998). A small, dilute-cytoplasm, high-affinity, novel bacterium isolated by extinction culture and having kinetic constants compatible with growth at ambient concentrations of dissolved nutrients in seawater. Appl Environ Microbiol 64, 4467–4476.[Abstract/Free Full Text]

Chayabutra, C. & Ju, L. K. (2000). Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions. Appl Environ Microbiol 66, 493–498.[Abstract/Free Full Text]

Chun, J., Huq, A. & Colwell, R. R. (1999). Analysis of 16S-23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus. Appl Environ Microbiol 65, 2202–2208.[Abstract/Free Full Text]

Cole, R. M. & Popkin, T. J. (1981). Electron Microscopy. In Manual of Methods for General Bacteriology, pp. 34–51. Edited by P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, DC: American Society for Microbiology.

Eilers, H., Pernthaler, J., Glockner, F. O. & Amann, R. (2000). Culturability and in situ abundance of pelagic bacteria from the North Sea. Appl Environ Microbiol 66, 3044–3051.[Abstract/Free Full Text]

García-Martínez, J. & Rodríguez-Valera, F. (2000). Microdiversity of uncultured marine prokaryotes: the SAR11 cluster and the marine Archaea of Group I. Mol Ecol 9, 935–948.[CrossRef][Medline]

García-Martínez, J., Martínez-Murcia, A. J., Rodríguez-Valera, F. & Zorraquino, A. (1996). Molecular evidence supporting the existence of two major groups in uropathogenic Escherichia coli. FEMS Immunol Med Microbiol 14, 231–244.[Medline]

García-Martínez, J., Acinas, S. G., Antón, A. I. & Rodríguez-Valera, F. (1999). Use of the 16S-23S ribosomal genes spacer region in studies of prokaryotic diversity. J Microbiol Methods 36, 55–64.[CrossRef][Medline]

Gauthier, M. J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M., Bonin, P. & Bertrand, J.-C. (1992). Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium. Int J Syst Bacteriol 42, 568–576.[Abstract/Free Full Text]

Giovannoni, S. J., Mullins, T. D. & Field, K. G. (1995). Microbial diversity in oceanic systems: rRNA approaches to the study of unculturable microbes. In Molecular Ecology of Aquatic Microbes, NATO ASI Series, vol. G 38, pp. 217–248. Edited by I. Joint. Berlin: Springer-Verlag.

Guasp, C., Moore, E. R. B., Lalucat, J. & Bennasar, A. (2000). Utility of internally transcribed 16S–23S rDNA spacer regions for the definition of Pseudomonas stutzeri genomovars and other Pseudomonas species. Int J Syst Evol Microbiol 50, 1629–1639.[Abstract]

Hedlund, B. P., Geiselbrecht, A. D., Bair, T. J. & Staley, J. T. (1999). Polycyclic aromatic hydrocarbon degradation by a new marine bacterium, Neptunomonas naphthovorans gen. nov., sp. nov. Appl Environ Microbiol 65, 251–259.[Abstract/Free Full Text]

Heidelberg, J. F., Eisen, J. A., Nelson, W. C. & 23 other authors (2000). DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406, 477–483.[CrossRef][Medline]

Heimbrook, M. E., Wang, W. L. L. & Campbell, G. (1989). Staining bacterial flagella easily. J Clin Microbiol 27, 2612–2615.[Abstract/Free Full Text]

Huu, N. B., Denner, E. B. M., Ha Dang, T. C., Wanner, G. & Stan-Lotter, H. (1999). Marinobacter aquaeolei sp. nov., a halophilic bacterium isolated from Vietnamese oil-producing well. Int J Syst Bacteriol 49, 367–375.[Abstract/Free Full Text]

Kersters, K. (1992). The genus Deleya. In The Prokaryotes, vol. 4, pp. 3189–3197. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer-Verlag.

Koike, S. T., Barak, J. D., Henderson, D. M. & Gilbertson, R. L. (1999). Bacterial blight of leek: a new disease in California caused by Pseudomonas syringae. Plant Dis 83, 165–170.

Krieg, N. R. (1984). Genus Oceanospirillum Hylemon, Wells, Krieg and Jannasch 1973, 361^AL. In Bergey's Manual of Systematic Bacteriology, vol 1, pp. 104–110. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.

Lowe, T. & Eddy, S. R. (1997). tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25, 955–964.[Abstract/Free Full Text]

Nocentini, M., Pinelli, D. & Fava, F. (2000). Bioremediation of a soil contaminated by hydrocarbon mixtures: the residual concentration problem. Chemosphere 41, 1115–1123.[Medline]

Ortigosa, M., Garay, E. & Pujalte, M. J. (1994). Numerical taxonomy of aerobic, Gram-negative bacteria associated to oysters and surrounding seawater of the Mediterranean coast. Syst Appl Microbiol 17, 589–600.

Rosselló-Mora, R. & Amann, R. (2000). The species concept for prokaryotes. FEMS Microbiol Rev 25, 39–67.

Sakane, T. & Yokota, A. (1994). Chemotaxonomic investigation of heterotrophic, aerobic and microaerophilic spirilla, the genera Aquaspirillum, Magnetospirillum and Oceanospirillum. Syst Appl Microbiol 17, 128–134.

Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.[Abstract/Free Full Text]

Vreeland, R. H. (1992). The family Halomonadaceae. In The Prokaryotes, vol. 4, pp. 3181–3188. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer-Verlag.

Wilson, V. L., Tatford, B. C., Yin, X., Rajki, S. C., Walsh, M. M. & LaRock, P. (1999). Species-specific detection of hydrocarbon-utilizing bacteria. J Microbiol Methods 39, 59–78.[CrossRef][Medline]

Yakimov, M. M., Golyshin, P. N., Lang, S., Moore, E. R., Abraham, W. R., Lünsdorf, H. & Timmis, K. N. (1998). Alcanivorax borkumensis gen. nov., sp. nov., a new, hydrocarbon-degrading and surfactant-producing marine bacterium. Int J Syst Bacteriol 48, 339–348.[Abstract/Free Full Text]

Yoshinaga, I., Katanozaka, N. & Ueno, Y. (1999). VBNC (viable but nonculturable) marine bacteria on the molecular aspects. Microb Environ 14, 123–129.

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