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1 Division of Microbiology, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany
2 Fisheries Research Services, Marine Laboratory, Victoria Road, Aberdeen AB11 9DB, UK
Correspondence
Carsten Strömpl
cst{at}gbf.de
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ABSTRACT |
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The EMBL accession numbers for the 16S rRNA gene sequences of Oceanicaulis alexandrii C116-3 and C116-18T are AJ309863 and AJ309862, respectively.
A selection of 16S rDNA sequence stretches and helices where the five strains of O. alexandrii exhibit diagnostic differences from related genera (Table I) and characteristics useful for differentiating the genus Oceanicaulis from related genera (Table II) are available in IJSEM Online.
Present address: Department of Medicine and Therapeutics, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, UK. 
Present address: The Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, UK.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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, stalked bacteria from a wide range of sources, including soils, freshwater and marine environments, have been assigned to the genus, mainly on the basis of their characteristic prosthecate (Anast & Smit, 1988; Stahl et al., 1992). Originally thought to be restricted exclusively to oligotrophic habitats, in the last decade an extensive number of isolates exhibiting heterotrophic traits and relatively versatile metabolisms have been isolated from eutrophic environments, such as secondary waste water treatment facilities (Fenton, 1994; MacRae & Smit, 1991), and assigned to the genus Caulobacter. It subsequently became clear, from 16S rRNA gene sequence analyses, that these isolates and the previously described species of Caulobacter were genotypically heterogeneous (Stahl et al., 1992), and other studies (Ariskina, 1995; Moore, 1978; Nikitin et al., 1990) supported these findings. A polyphasic analysis (Abraham et al., 1999) of the validly published Caulobacter species, members of the phylogenetically related genus Brevundimonas and other unpublished caulobacterial isolates yielded a reclassification of many of the described species of Caulobacter and the proposal of a new genus, Maricaulis, to accommodate the strictly halophilic species. Caulobacter sensu stricto is currently composed of only freshwater species and isolates with a specific range of 16S rRNA gene sequence similarity, a characteristic membrane protein antigen and typical polar lipid fingerprints. The amended genus Brevundimonas (Abraham et al., 1999), as defined by the same methods, comprises a range of stalked and non-stalked strains from clinical sources, soils, freshwater and marine habitats. Accordingly, species within this genus collectively possess a wide range of phenotypic traits. The genus Maricaulis consists of halophilic caulobacteria exclusively of marine origin and is more closely related to the genera of budding marine bacteria, Hyphomonas and Hirschia, than to Caulobacter. In this study, five strains of stalked bacteria isolated from a non-toxigenic culture of the dinoflagellate Alexandrium tamarense are described and a new genus, Oceanicaulis, and species, Oceanicaulis alexandrii, are proposed which are distinct from previously described stalked bacteria, as their phylogenetic assignment implies.
Bacterial strains were isolated from a culture of Alexandrium tamarense (Lebour) Balech CCMP 116 (NEPCC C116), which was collected at Ria de Vigo, Spain, under conditions described previously (Hold et al., 2001). Five isolates were chosen for detailed characterization.
The cultures were routinely maintained on marine agar medium (Difco). Standard methods were used for metabolic testing (Smibert & Krieg, 1981). For fatty acid analysis, the bacteria were grown in marine Caulobacter medium (SPYEM), containing 30 g sea salts (Sigma), 0·5 g NH4Cl and 1 l deionized water, and 20 ml of 50xPYE (100 g peptone, 50 g yeast extract in 1 l deionized water); 2 ml of 50 % (w/v) glucose (heat-sterilized) and 5 ml riboflavin (0·2 mg ml-1, filter-sterilized) were added after autoclaving. Testing for oxidation of carbon sources with Biolog GN plates gave poor results. Four strains reacted with Tween 40, two strains were positive for 
-hydroxybutyric acid and glycyl-L-glutamic acid, and single strains tested positive for D-raffinose, methylpyruvate, -ketoglutaric acid, 
-hydroxybutyric acid, propionic acid, inosine and uridine. Sample preparation and tests for paralytic shellfish toxins (mouse neuroblastoma assay, HPLC, capillary electrophoresis-mass spectroscopy, ELISA) were carried out as described previously (Gallacher et al., 1997). All five strains of the novel bacterium tested negative for paralytic shellfish toxins (results not shown). The strains have been deposited at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany): C116-3, DSM 11627; C116-14, DSM 11669; C116-17, DSM 11626; C116-18T, DSM 11625T; C116-20, DSM 11670. Strain C116-18T has been deposited with the NCIMB (National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland, UK) as NCIMB 13905T.
PCR amplification of nearly complete 16S rRNA genes (Weisburg et al., 1991) and Taq-polymerase-initiated cycle sequencing, using fluorescent-dye-labelled dideoxynucleotides, were carried out as described previously (Abraham et al., 1999). The methodology for alignment and analysis of the resulting DNA sequences has also been reported previously (Abraham et al., 1999).
Comparative sequence analysis revealed two 16S rRNA gene sequence types, which differed in only three nucleotide positions over the nearly complete gene (Escherichia coli 16S rRNA gene sequence positions 284, 295, 379), and two (C116-3 and C116-14) and three (C116-17, C116-18T and C116-20) strains, respectively, were observed to have identical 16S rRNA gene sequences. Comparison of these sequences with those of reference organisms showed the isolates to be phylogenetically related to species of the proposed family ‘Rhodobacteraceae’ (Garrity et al., 2001) within the ‘Alphaproteobacteria’ and similar to species of the genera Maricaulis, Hyphomonas and Hirschia (Fig. 1). The five strains were phylogenetically most closely related to the unclassified caulobacterial isolate MCS33 (93·8 % sequence similarity), which was isolated from a hydrothermal vent near Vancouver Island, Canada (Abraham et al., 1999). Sequence similarities of strain C116-18T to the type strains of related genera and species were as follows: Hyphomonas polymorpha, 88·1 %; Hyphomonas jannaschiana, 89·0 %; Hirschia baltica, 87·3 %; Maricaulis maris 92·5 %; Maricaulis washingtonensis, 91·7 %; Maricaulis parjimensis, 92·1 %; Caulobacter vibrioides, 86·0 %. Strain and sequence accession numbers used for similarity calculations are given in Fig. 1.
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) where the five strains of O. alexandrii exhibit diagnostic differences from the individual related genera. The sequence patterns listed demonstrate that strains C116-3, C116-4, C-116-17, C116-18T and C-116-20 are related to the species of the genus Maricaulis, although sequence similarity values are lower than what is generally observed between species of a genus. Besides, as the sequence features of Oceanicaulis are not restricted to the highly variable regions within the 16S rRNA gene, this is concluded to be sufficient to place Oceanicaulis in a distinct genus.
Genomic DNA was isolated from 2 ml of cultures grown in marine broth medium according to the protocol of Wilson (1987), followed by treatment with Ribonuclease A (Sigma) at 50 °C for 2 h, and a phenol/chloroform/isoamyl alcohol (50 : 40 : 10) extraction. Appropriate amounts of DNA were enzymically cleaved to yield the nucleosides and the mean G+C contents were determined by HPLC (Tamaoka & Komagata, 1984). Similarity calculations were carried out according to Mesbah et al. (1989), with non-methylated 
-phage DNA (Sigma) as a standard.
Cells harvested from 1 ml of liquid culture of each of the five strains studied were lysed by alkaline treatment, according to Birnboim & Doly (1979); screening for plasmids was carried out by electrophoresis on 0·8 % agarose/TAE gels. Four of the strains, but not C116-17, showed large plasmids which have not been further analysed. Anast & Smit (1988) reported the occurrence of large native plasmids (100–150 kb) in two out of 25 marine caulobacteria which they had isolated and studied for their suitability for genetic experiments.
Lipids were extracted, using a modified Bligh–Dyer procedure (Bligh & Dyer, 1959), and fatty acid methyl esters were generated and analysed by GC, as described previously (Vancanneyt et al., 1996).
Fast atom bombardment MS in the negative mode was performed on the first of two mass spectrometers of a tandem high-resolution instrument of E1B1E2B2 configuration. Negative daughter ion spectra were recorded using all four sectors of the tandem mass spectrometer. High-energy collision-induced dissociation (CID) took place in the third field-free region. Helium served as the collision gas (Abraham et al., 1997).
Two different types of polar lipids could be detected, i.e. phosphatidyl glycerol and sulfo-quinovosyl diacylglycerol (Fig. 2). Molecular ions were observed at m/z 719, 733, 761 and 773, which could be attributed to known phosphatidyl glycerols (Abraham et al., 1997). CID spectra of these ions could not be measured because of their low concentrations in the polar lipid fraction of strain C116-18T. Strain C116-18T possessed mainly sulfolipids, while phosphatidyl glycerols were found only in low amounts. With the aid of CID MS, the structures of most of the sulfolipids were elucidated. Diagnostically important fragments were generated by cleavage of diacylglycerol, leading to the dehydrated sulfo-quinovose, which is observed as the (M-1)- ion at m/z 226 (Abraham et al., 1997). CID of the (M-H)- ion from the sulfolipids yielded neutral losses of the sn-2 and sn-1 substituent as free carboxylic acid, thus, allowing the identification of the fatty acids attached to the different sulfo-quinovoses. Furthermore, the positions of the fatty acids at the glycerol backbone can be determined because, for the fatty acid positioned at sn-2, the neutral loss as free fatty acid is more frequent than for that at sn-1 (Heller et al., 1988; Murphy & Harrison, 1994). With this method, the structure of the sulfolipids was identified (Abraham et al., 1997). Table 1 summarizes the results.
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) was not detected in C116-18T. Furthermore, 1,2-diacyl-3--D-glucuronopyranosyl-sn-glycerol taurineamide, reported for Hyphomonas species (Batrakov et al., 1996) and found in about half of Maricaulis strains, was absent.
Strain C116-18T differed from Maricaulis strains by a very low content of C16 : 0 and higher amounts of 7-Me-C18 : 1
6, C19 : 0 and C19 : 18. The branched C17 : 0i found in 4–14 % of Maricaulis strains is missing (Table 2).
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a, b), as described in detail by Yakimov et al. (1998). Negatively stained cells were embedded in epoxy resin (Spurr, 1969) and ultra-thin sections (Fig. 3c) were analysed using an energy-filtered transmission electron microscope (CEM 902; Zeiss), as described in detail by Lünsdorf et al. (2001). Upon light and electron microscopy examination, strain C116-18T was seen as straight or curved motile rods. Further details are given in Fig. 3.
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have already noted that MCS24 is tolerant of freshwater conditions, and a phylogenetic analysis (Abraham et al., 1999) revealed it to be a member of the genus Brevundimonas and not of Maricaulis, a genus of exclusively marine origin. For the first time, stalk cross walls have been observed in a caulobacterium which is phylogenetically affiliated with the caulobacteria of marine origin in the genus Maricaulis. This is a morphological feature which separates the C116- strains from all species of Maricaulis examined so far.
Most caulobacteria require complex organic additives, such as peptone, in their growth media (Poindexter, 1964), which complicates their nutritional characterization. Therefore, the description of the five new strains as a new genus and species given below is mainly based on their characteristic lipid composition, their distinct 16S rDNA sequences and morphological differences to related genera. Characteristics useful for differentiating the genus Oceanicaulis from related genera can be found in Supplementary Table II (http://ijs.sgmjournals.org).
Description of Oceanicaulis gen. nov.
Oceanicaulis [O.ce.an.i.cau'lis. L. masc. n. oceanus the ocean; L. masc. n. caulis stalk, referring to a prostheca; N.L. masc. n. Oceanicaulis stalk(ed organism) from the ocean].
Gram-negative, non-spore-forming. Cells are rod-shaped or vibrioid. Cultivated cells are 1 µm by 3 µm. Cells possess one prostheca, which extends centrally along the long cell axis from one pole. Adhesive material is present at the distal end of the prostheca. During binary fission, at the point of separation, one cell possesses a prostheca and the other a single polar flagellum. Colonies are round, convex and non-pigmented. Aerobic and chemo-organotrophic. Nitrate is reduced to nitrite by most strains. All strains grow with 20–100 g NaCl l-1. Mesophilic. Polar lipids are phosphatidyl glycerol and sulfo-quinovosyl diacylglycerol. Major fatty acids are C18 : 17, C18 : 0, 7-Me-C18 : 16 and C19 : 0. The G+C content of the DNA is 61–62 mol%. Phylogenetically, the genus is related to the prosthecate bacteria of marine origin, namely, Maricaulis, Hyphomonas and Hirschia, within the order ‘Rhodobacterales’.
The type species is Oceanicaulis alexandrii.
Description of Oceanicaulis alexandrii sp. nov.
Oceanicaulis alexandrii [a.lex.an.dri'i. L. n. Alexandrium the (dinoflagellate) from Alexandria; L. masc. gen. alexandrii of Alexandrium, the source of isolation and postulated natural habitat].
The stalk of prosthecate cells possesses a characteristic constriction. In addition to the major fatty acids typical for the genus, the following minor fatty acids are present: C14 : 0, C16 : 0, C17 : 16, C17 : 0, C18 : 16, C19 : 18, C19 : 0d8,9. Optimal growth occurs at 30 °C, with a growth range from 4 to 37 °C. Cells grow in marine broth at full strength and at 1/10 strength. Strains are catalase- and oxidase-positive. Strains tested negative for lecithinase, starch, xanthine, indole, o-nitrophenylgalactoside and Voges–Proskauer, and most tested negative for aesculin, elastinase, gelatinase and lipase. Most strains contain large plasmids.
The type strain is C116-18T (=DSM 11625T=NCIMB 13905T). The G+C content of its genomic DNA is 61·8 mol%.
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ACKNOWLEDGEMENTS |
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