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1 GBF – German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany
2 BCCM/LMG Bacteria Collection, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
3 Area de Microbiologia, Campus UIB - Edifici Guillem Colom, Universitat de les Illes Balears, Crta. Valldemossa km 7·5, 07122 Palma de Mallorca, Spain
4 Dept of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada
5 The Macaulay Research Institute, Craigiebuckler, Aberdeen AB15 8QH, UK
Correspondence
Wolf-Rainer Abraham
wab{at}gbf.de
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Published online ahead of print on 9 February 2004 as DOI 10.1099/ijs.0.02943-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of strains CM243T and CM251 are AJ578476 and AJ578477, respectively.
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, 1981b). These bacteria were observed to have dimorphic, prosthecate cells in which reproduction takes place by the separation of two cells that are morphologically and behaviourally different from each other. One cell is sessile, fixed by adhesive material to the substrata and possessing one elongated cylindrical appendage – a prostheca (Staley, 1968). The other cell is motile by a single polar flagellum. The mode of reproduction of the dimorphic prosthecate bacteria helps disperse the population at each generation, thereby minimizing competition for resources. It is consistent with their life style that these bacteria possess tolerance of prolonged nutrient scarcity (Poindexter, 1981a).
Caulobacteria are ubiquitous in aquatic and marine ecosystems and are presumed to be responsible for considerable mineralization of dissolved organic material in aquatic environments, where nutrient concentrations and ambient temperatures are low (Staley et al., 1987). It is consistent with this presumption that practically any type of sea water contains caulobacteria (Jannasch & Jones, 1960; Anast & Smit, 1988).
The ubiquity of this type of bacteria led to their discovery more than a century ago, where the first isolation of a Caulobacter sp. was reported by Loeffler (1890). In 1935, the genus Caulobacter was described (Henrici & Johnson, 1935) and, three decades later, Jean Poindexter isolated a large number of caulobacteria from different habitats and described nine species (Poindexter, 1964).
Anast & Smit (1988) were the first to recognize differences between freshwater and marine caulobacteria in a large set of strains. The comparison of 16S rRNA gene sequences of several isolates belonging to Caulobacter revealed that the isolates actually form two different lineages (Stahl et al., 1992). A study of the diversity of more than 100 different freshwater and marine Caulobacter strains including lipid analysis, immunological profiling, 16S rRNA gene sequencing and physiological data led to the reclassification of many Caulobacter species as Brevundimonas species and the proposal of the new genus Maricaulis (Abraham et al., 1999). In this study, marine isolates were identified as belonging to this new genus, and four additional species of Maricaulis have since been proposed (Abraham et al., 2002). However, some of the marine strains isolated by Poindexter and others did not fit into the genus Maricaulis or into the newly described genus Oceanicaulis (Strömpl et al., 2003), and the aim of this communication is to describe two of them and to place them into a new genus.
The strains of this study and the origin of the isolates are listed in Table 1. All strains were grown in marine-Caulobacter medium SPYEM: 30 g sea salts (Sigma), 0·5 g NH4Cl, 1 l Milli-Q water. After autoclaving and cooling, 20 ml 50xPYE (100 g peptone and 50 g yeast extract in 1 l deionized water, autoclaved), 2 ml 50 % glucose (sterile) and 5 ml filter-sterilized riboflavin (0·2 mg ml–1) were added. The strains were incubated in 2 l flasks at 30 °C and 100 r.p.m. and the biomass was harvested in the late exponential phase after 72 h.
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). Resulting sequences were aligned and phylogenetic trees were constructed in the ARB program (http://www.arb-home.de). For analysis of the 16S–23S rDNA interspacer region (ITS1), two PCR primers were used: 16f945, corresponding to positions 927 to 945 of the Escherichia coli 16S rDNA (Brosius et al., 1978), and 23r458, corresponding to positions 458 to 473 of the E. coli 23S rDNA (Brosius et al., 1980). Fingerprint analysis of the ITS1 PCR products after digestion with TaqI and single-strand conformation polymorphism (SSCP) analysis (Orita et al., 1989) of the resulting restriction fragments were performed as described elsewhere (Abraham et al., 2002).
After an incubation period of 48 h on SPYEM agar plates at 28 °C, a loopful of biomass was harvested for whole-cell fatty acid analysis and fatty acid methyl esters were prepared as described previously (Abraham et al., 2002). Fatty acid methyl esters were separated and identified using the Microbial Identification System (MIDI). Lipids were extracted using a modified Bligh–Dyer procedure (Bligh & Dyer, 1959) as described previously (Vancanneyt et al., 1996). This total lipid fraction was fractionated by column chromatography and the phospholipid fraction was analysed by electrospray ionization mass spectrometry (ESI-MS). ESI-MS in the negative mode was performed in a QTOF-MS. Neon served as the collision gas for high-energy collision-induced dissociation (CID). 1H NMR spectra were recorded in 7 : 3 d-chloroform/d3-methanol at 300 K on a Bruker ARX-400 NMR spectrometer relative to internal tetramethylsilane.
Strains were grown with different concentrations of NaCl and at different temperatures, for phenotypic characterizations, as described elsewhere (Abraham et al., 1999). Enzyme activity tests were conducted with the use of API ZYM test strips (bioMérieux), according to the protocol supplied by the manufacturer. Substrate specificity tests were conducted with the use of API 20NE test strips (bioMérieux) using a protocol supplied by the manufacturer. The test strips were incubated at 30 °C for 7 days and monitored after 1, 2 and 7 days. A test was considered positive when the interface between sample well and air was visibly turbid due to bacterial growth after a 7 day incubation period.
Morphology and ultrastructure of mid-exponentially growing cells of CM243T were analysed as negatively stained and shadow-cast samples with the transmission electron microscope as described previously (Golyshina et al., 2000; Yakimov et al., 1998). As seen in Fig. 1, these cells show the typical features of Caulobacter-type Gram-negative bacteria. Both morphologies can be recognized (Fig. 1). Bacteria with a characteristic stalk, ranging from 2·5 µm up to more than 12 µm in length, a median diameter of 135 nm (±13 nm; n=43), and terminated by a distinct holdfast, are often found. They are associated with planktonic motile variants (Fig. 1), which are monotrichously and monopolarly flagellated (Fig. 1a; fl). Mid-exponentially growing cells show cell lengths from 1·51 to 5·4 µm and a mean cell diameter of 690 nm (±13 nm; n=30).
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C exchange at base position 306. Results from initial FASTA database searches indicated affiliation of the two strains with the ‘Alphaproteobacteria’ (Garrity et al., 2001). Detailed alignments in ARB showed that strains CM243T and CM251 clustered within the Rhodobacteraceae and were most closely related to the genera of marine caulobacteria, Maricaulis and Oceanicaulis (Fig. 2). Estimated evolutionary distances of strain CM243T from the type strains of closely related species and relevant taxa were: 7·48 % to Maricaulis maris, 7·79 % to Maricaulis washingtonensis, 7·87 % to Maricaulis salignorans, 6·55 % to Maricaulis parjimensis, 6·32 % to Maricaulis virginensis, 7·16 % to Oceanicaulis alexandrii, 10·58 % to Hyphomonas polymorpha, 11·60 % to Hirschia baltica and 13·76 % to Caulobacter vibrioides.
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) (data not shown). Identification of distinguishing sequence patterns, together with the evolutionary distances estimated from sequence similarity values, and the dendrogram topologies, suggest that strains CM243T and CM251 are equally distant from Maricaulis and Oceanicaulis.
Molecular typing analysis of the 16S–23S rDNA ITS1 has been applied as a rapid bacterial identification tool for strains CM243T and CM251 in order to confirm the close relationship between the strains. The use of the ITS1 region has allowed discrimination at, approximately, the species level, with correlation to DNA–DNA relatedness data (Guasp et al., 2000). For both strains, equivalent single, 1·6 kb ITS1 PCR products were obtained (data not shown). These PCR fragments contained approximately 0·6 kb of the 5'-region of the 16S rDNA and nearby 0·5 kb of the 3'-region of the 23S rDNA, with 0·5 kb corresponding to the ITS1 region. TaqI restriction fingerprints obtained from the PCR products of CM243T and CM251 were identical, consisting of six bands of between 155 and 396 bp. Furthermore, no differences between the strains were detected in the resulting single-strand profiles analysed by SSCP. Consequently, significant sequence heterogeneities that would result in different conformations and, consequently, differences in the mobilities of single strands were assumed to be absent. These results confirmed unequivocally a close phylogenetic association between the two strains.
The mass spectra of the polar lipids of strains CM243T and CM251 showed surprisingly few peaks. The spectrum of strain CM243T was dominated by two peaks at m/z 797 and 847, with two minor peaks at 795 and 845. A fifth peak was detected at m/z 904 (Fig. 3). With the aid of CID MS, the main lipid compounds were elucidated. The ions at m/z 795 and 797 were identified as 
-D-glucopyranosyl diacylglycerol and -D-glucopyranuronosyl diacylglycerols known from many other Caulobacter, Brevundimonas, Maricaulis and Hyphomonas strains (Abraham et al., 1997, 1999). The ions at m/z 845 and 847 were identified as sulfoquinovosyl diacylglycerols and the ion at m/z 904 as -D-glucuronopyranosyl diacylglycerol taurine amide also described in Maricaulis and Hyphomonas strains. The fatty acids and their position on the glycerol backbone can be determined by the more frequent loss of those fatty acids positioned at sn-2 as free fatty acid as well as substituted ketene (Murphy & Harrison, 1994). With this method, the structures of the anions and hence the structure of the glyco- and sulfolipids were identified (Table 2). For the ions at m/z 795 and 904, because of their low intensities, no CID spectra were obtained. Instead, their compositions were assumed to be similar to identical ions observed in the MS spectra of Maricaulis and Hyphomonas species (Abraham et al., 1997).
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-D-glucuronopyranosyl-sn-glycerol taurineamide, also found in CM243T and CM251, is known from Hyphomonas species but was found only in those Maricaulis strains that are closely related to M. maris (Abraham et al., 2002) (Table 3). Strains CM243T and CM251 showed a low diversity of fatty acids and phospholipids, in marked contrast to the phylogenetically related genera Maricaulis and Hyphomonas. Such a low diversity of fatty acids was recently reported, as well, for Parvularcula bermudensis, a marine bacterium that comprises a deep phylogenetic branch in the ‘Alphaproteobacteria’ (Cho & Giovannoni, 2003).
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). The predominant fatty acids present in both strains were 12 : 0 3-OH, 16 : 0, 17 : 0, 18 : 0, summed feature 3, 18 : 1
7c and the unidentified fatty acid ECL 15·275. Strains CM243T and CM251 differed from Maricaulis species in their fatty acid profiles by the presence of 12 : 0 3-OH, summed feature 3 and the unidentified fatty acid ECL 15·275, and by the absence of 11 : 0 iso 3-OH, 17 : 16c, 17 : 18c, iso-17 : 0, iso-17 : 19c, 18 : 19c, 11-Me 18 : 15t, summed feature 4 and the unidentified fatty acids ECL 18·424 and ECL 18·797 (Table 3
). Hyphomonas species were observed to differ from strains CM243T and CM251 by the absence of summed feature 3 and the unidentified fatty acid ECL 15·275, and by the presence of 12 : 1 3-OH, 15 : 0, 17 : 16c, 17 : 18c and the unidentified fatty acid ECL 18·797 (Table 3). Poly-
-hydroxybutyrate was detected in the 1H NMR spectra of total lipid extracts of CM243T and CM251.
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). The growth requirements and enzyme activities observed are given in detail in the species description. In Table 5, the enzymic activities of strains CM243T and CM251 are compared with those of Maricaulis, Oceanicaulis and Hyphomonas species.
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Description of Woodsholea gen. nov.
Woodsholea (Woods.hol'e.a. N.L. fem. n. Woodsholea named in honour of the Woods Hole Oceanographic Institution, Massachusetts, USA).
Gram-negative cells, rod-shaped, vibriod. Cells possess a stalk, varying in length depending on the strain and environmental conditions, extending from one pole as a continuation of the long axis of the cell. Adhesive material is present at the distal end of the stalk. Occur singly. Multiplication by binary fission. Colonies circular, convex, colourless. Chemo-organotrophic, aerobes, cells can store carbon as poly--hydroxybutyric acid. Requirement for organic growth factors is complex and not satisfied by mixtures of B vitamins and amino acids. Grows on peptone/yeast extract media with 40 g NaCl l–1. Growth is inhibited or cells become deformed in media containing 1 % (w/v) or more organic material. Growth temperature range is 20–40 °C and optimal pH for growth is approximately neutral. Do not reduce nitrate, oxidize tryptophan to indole or hydrolyse arginine, urea, aesculin, gelatin or p-nitrophenyl-3-D-galactopyranoside. Do not use glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate or phenylacetate as carbon sources. Cells show no catalase activity, are positive for alkaline phosphatase, naphthol-AS-BI-phosphohydrolase, leucine arylamidase, acid phosphatase, esterase (C4), esterase lipase (C8), oxidase and trypsin but negative for - and -galactosidase, -glucuronidase, - and -glucosidase, -mannosidase and -fucosidase. The genus is characterized by two major fatty acids, 18 : 0 and 18 : 17c, and minor amounts of 12 : 0 3-OH, 16 : 0, 17 : 0, summed feature 3 and the unidentified fatty acid ECL 15·275. Polar lipids are -D-glucopyranosyl diacylglycerol, -D-glucopyranuronosyl diacylglycerol, sulfoquinovosyl diacylglycerol and -D-glucuronopyranosyl diacylglycerol taurine amide. Isolated from sea water. The G+C content is 65 mol%. The type species is Woodsholea maritima.
Description of Woodsholea maritima sp. nov.
Woodsholea maritima (L. fem. adj. maritima marine).
The description is as that of the genus with the following additions. Cells are 1·5–5·4x0·7 µm with a 2·5–12x0·14 µm stalk; optimal growth occurs between 5 and 100 g NaCl l–1. No growth without salt. Optimal growth temperature is 20–40 °C; some growth is observed at 10 °C. pH range for growth is 6·0–8·0. Main polar lipids are 1-nonadecanoyl-2-octadecenoyl-3-O--D-glucopyranosylglycerol, 1-octadecenoyl-2-octadecanoyl-3-O--D-glucopyranuronosylglycerol and 1,2-di-octadecenoyl-3-O-sulfoquinovosylglycerol. Strains have no to weak -chymotrypsin and N-acetyl--glucosaminidase activities; some isolates including the type strain have very weak lipase activity. Isolates have been obtained from sea water at Woodshole, USA. The G+C content of the type strain is 65·2 mol%.
The type strain is CM243T (=VKM B-1512T=LMG 21817T); a further strain is CM251 (=LMG 21818).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Abraham, W.-R., Strömpl, C., Meyer, H. & 8 other authors (1999). Phylogeny and polyphasic taxonomy of Caulobacter species. Proposal of Maricaulis gen. nov. with Maricaulis maris (Poindexter) comb. nov. as the type species, and emended description of the genera Brevundimonas and Caulobacter. Int J Syst Bacteriol 49, 1053–1073.
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