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Int J Syst Evol Microbiol 55 (2005), 345-351; DOI  10.1099/ijs.0.63236-0
© 2005 International Union of Microbiological Societies

Marinilactibacillus piezotolerans sp. nov., a novel marine lactic acid bacterium isolated from deep sub-seafloor sediment of the Nankai Trough

Laurent Toffin1,{dagger}, Klaus Zink2, Chiaki Kato3, Patricia Pignet4, Adeline Bidault1, Nadège Bienvenu1, Jean-Louis Birrien1 and Daniel Prieur1

1 UMR 6539, Centre National de la Recherche Scientifique et Université de Bretagne Occidentale, Technopôle Brest-Iroise, Place Nicolas Copernic, F-29280 Plouzané, France
2 GeoForschungsZentrumPotsdam (GFZ), Telegrafenberg, D-14473 Potsdam, Germany
3 Extremobiosphere Research Centre, Japan Agency for Marine–Earth Science and Technology (JAMSTEC), 2–15 Natsushima-cho, Yokosuka 237-0061, Japan
4 Laboratoire de Microbiologie et de Biotechnologie des Extrêmophiles, Département de Valorisation des Produits, IFREMER, Centre de Brest, BP 70, F-29280 Plouzané, France

Correspondence
Laurent Toffin
toffin{at}jamstec.go.jp


    ABSTRACT
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A piezotolerant, mesophilic, marine lactic acid bacterium (strain LT20T) was isolated from a deep sub-seafloor sediment core collected at Nankai Trough, off the coast of Japan. Cells were Gram-positive, rod-shaped, non-sporulating and non-motile. The NaCl concentration range for growth was 0–120 g l–1, with the optimum at 10–20 g l–1. The temperature range for growth at pH 7·0 was 4–50 °C, with the optimum at 37–40 °C. The optimum pH for growth was 7·0–8·0. The optimum pressure for growth was 0·1 MPa with tolerance up to 30 MPa. The main cellular phospholipids were phosphatidylglycerols (25 %), diphosphatidylglycerols (34 %) and a group of compounds tentatively identified as ammonium-containing phosphatidylserines (32 %); phosphatidylethanolamines (9 %) were minor components. The fatty acid composition was dominated by side chains of 16 : 0, 14 : 0 and 16 : 1. The G+C content of the genomic DNA was 42 mol%. On the basis of 16S rRNA gene sequence analysis and the secondary structure of the V6 region, this organism was found to belong to the genus Marinilactibacillus and was closely related to Marinilactibacillus psychrotolerans M13-2T (99 %), Marinilactibacillus sp. strain MJYP.25.24 (99 %) and Alkalibacterium olivapovliticus strain ww2-SN4C (97 %). Despite the high similarity between their 16S rRNA gene sequences (99 %), the DNA–DNA hybridization levels were less than 20 %. On the basis of physiological and genetic characteristics, it is proposed that this organism be classified as a novel species, Marinilactibacillus piezotolerans sp. nov. The type strain is LT20T (=DSM 16108T=JCM 12337T).


Abbreviations: PLFA, phospholipid fatty acid

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Marinilactibacillus piezotolerans LT20T is AY485792.

Details of the morphology, differential phenotypic characteristics and the effect of pH on glucose fermentation in strain LT20T are available as supplementary material in IJSEM Online.

{dagger}Present address: Extremobiosphere Research Centre, JAMSTEC, 2–15 Natsushima-cho, Yokosuka 237-0061, Japan. Back


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The occurrence of a deep bacterial biosphere in marine sediments to more than 800 m below the sea floor (Parkes et al., 2000Go) has raised questions about what types of bacteria inhabit deeply buried sediments. Populations of viable micro-organisms have been found in deep marine deposits, but studies have focused on sulphate-reducers (Bale et al., 1997Go) and methanogens (Mikucki et al., 2003Go). Recently, however, autotrophic and heterotrophic bacteria belonging to the phyla Firmicutes, Cytophaga, Spirochaetes and Proteobacteria have been enriched from deep sediments of the Nankai Trough south-east of Japan (Toffin et al., 2004aGo, bGo). In near-surface deep marine sediments, fermentative micro-organisms could play a significant role in the transformation of organic matter. At present, little is known about the origin and the adaptation of viable fermentative micro-organisms in such deep environments. To date, only a few types of lactic acid bacteria have been isolated from marine environments (Franzmann et al., 1991Go), such as the two species from Ace Lake in Antarctica, namely Carnobacterium fundicum and Carnobacterium alterfundicum (Spielmeyer et al., 1993Go). Recently, the fermentative marine lactic acid bacterium Marinilactibacillus psychrotolerans was isolated from dead and living marine organisms at Miura Peninsula in Japan (Ishikawa et al., 2003Go). In addition, members of the genus Marinilactibacillus have been isolated from coastal sub-seafloor sediments of the Okhotsk Sea (Inagaki et al., 2003Go) and Carnobacterium-like sequences have been retrieved from Nankai Trough (Newberry et al., 2004Go). At present, the genus Marinilactibacillus comprises only one species, M. psychrotolerans, which has developed some adaptations to salt, temperature and pH. This species is also able to ferment a wide range of organic substrates, producing lactic acid as the main end product of metabolism.

M. psychrotolerans strain M13-2T (=IAM 14980T) was used as a reference strain. This strain was obtained from the IAM culture collection, Institute of Molecular and Cellular Biosciences (Tokyo University, Japan).

The novel isolate was obtained from a sediment core collected at 4·15 m below the sea floor from a water depth of 4790·7 m in the Pacific Ocean at Nankai Trough, off the coast of Japan (site 1173: 32° 14·7' N 135° 1·5' E) between 28 May and 7 June 2000 (Ocean Drilling Program, Leg 190). All details of the environmental conditions, sampling and sub-sampling procedures have been previously reported (Cragg et al., 1992aGo, bGo; Parkes et al., 1995Go; Moore et al., 2001Go; Toffin et al., 2004aGo).

Cultures were enriched anaerobically in 50 ml vials containing 10 ml medium MM, which consisted of the following (l–1 distilled water): 23 g NaCl, 3 g MgCl2.6H2O, 4 g Na2SO4, 0·7 g KCl, 0·15 g CaCl2, 0·5 g NH4Cl, 0·27 g KH2PO4, 15 ml 1 M NaHCO3, 0·1 g yeast extract (Difco), 1 ml trace elements (Widdel & Bak, 1992Go), 1 ml vitamin solution (Widdel & Bak, 1992Go), 1 ml thiamine (0·01 %, w/v; Widdel & Bak, 1992Go), 1 ml vitamin B12 (0·005 %, w/v; Widdel & Bak, 1992Go), 1·0 g sodium acetate, 2·0 g monomethylamine, 5·0 g sodium formate, 0·5 % (v/v) methanol and 0·5 mg resazurin. The pH of the medium was adjusted to 7·2 at room temperature before autoclaving. Sterile medium was reduced by adding 0·5 g sodium sulphide l–1 and then distributed into serum vials before inoculation. Cultures were incubated at 25 °C in the dark. Growth of lemon-shaped, coccoid, vibrio-shaped, long rod-shaped and short rod-shaped cells was observed in enrichment cultures after 2 days incubation. Positive enrichments were subcultured into the same medium under anaerobic conditions. Once stable enrichment cultures had been established, subsequent enrichment cultures were grown on agar plates (1 %, w/v; Difco), incubated anaerobically at 25 °C. Small, translucent, round colonies around 1 mm in diameter were visible after 3 days and one colony was randomly picked. The dilution to extinction technique, followed by repeated streaking onto plates, was employed to obtain pure culture. The isolate was designated strain LT20T (=DSM 16108T=JCM 12337T) and the purity of the isolate was checked by means of microscopic observations and by cloning and sequencing five independent 16S rRNA gene clones.

Isolate LT20T was routinely grown in YPG medium containing the following (l–1 distilled water): 25 g NaCl, 3 g MgCl2, 0·5 g KCl, 4 g Na2SO4, 2 g glucose, 5 g yeast extract (Difco), 5 g peptone (Difco), 34·6 g PIPES and 0·05 g KH2PO4. The pH of the medium was adjusted to 7·2 before autoclaving. Unless indicated otherwise, cultures were incubated aerobically at 37 °C with shaking. Stock cultures of isolate LT20T were stored in culture medium at 4 °C. For long-term storage, pure cultures were stored at –80 °C in the same medium containing 20 % (w/v) glycerol.

Growth was monitored by measuring changes in turbidity at 600 nm using a Spectronic 401 spectrophotometer (Spectronic Instruments). Direct cell counts were determined on samples fixed with 2 % (v/v) glutaraldehyde for 1 h at 4 °C by using a counting chamber (depth 0·02 mm; Thoma). An Olympus CX40 microscope was used routinely to observe the cells. For negative staining, 20 µl bacterial suspension fixed with 2 % (w/v) glutaraldehyde was dropped onto a carbon-coated copper grid (Nishin EM). A droplet of a 1 % (w/v) neutral phosphotungstic acid solution (pH 7·6) was added to the carbon grid for 40 s and excess liquid was removed. Specimens were air-dried and electron micrographs taken using a JEOL electron microscope (JEM 1210). Gram staining, catalase, oxidase, gelatinase and amylase (1 %, w/v) reactions were tested with cells grown in YPG medium.

Cells of strain LT20T were straight to slightly curved rod-shaped cells approximately 2–2·2 µm in length and 0·3–0·35 µm wide (see the transmission electron micrograph available as a supplementary figure in IJSEM Online), but were slightly elongated in older cultures. They occurred singly, in pairs or in small chains at 37 °C and stained Gram-positive. The cells were non-motile. Endospores or spores were not observed and cells were catalase-positive and oxidase-negative.

To determine the optimum temperature, pH and NaCl, cells were grown in aerobic tubes (15 ml) containing 5 ml YPG medium with shaking (130 r.p.m.) as reported previously (Toffin et al., 2004bGo). To determine the effect of pH on growth, YPG medium was modified with the following 100 mM buffers (Sigma): for pH 5·0, no buffer; for pH 5·5 and 6·0, MES buffer; for pH 6·5 and 7·0, PIPES buffer; for pH 7·5, HEPES buffer; for pH 8·0 and 8·5, Tris buffer; for pH 9·0, 10·0 and 11·0, no buffer.

The effect of hydrostatic pressure on growth rates of strain LT20T was determined at 0·1, 10, 20, 30, 40 and 50 MPa at 37 °C as previously described (Kato et al., 1995Go).

The ability of strain LT20T to utilize single carbon sources was investigated by adding one of the following organic compounds to the modified YPG medium, from which carbon sources were omitted: formate (30 mM), pyruvate (20 mM), propionate (20 mM), butyrate (10 mM), isobutyrate (5 mM), 2-methyl butyrate (5 mM), 3-methyl butyrate (5 mM), valerate (5 mM), isovalerate (5 mM), glutamate (5 mM), caproate (5 mM), malate (10 mM), succinate (10 mM), heptanoate (5 mM), acetate (15 mM), lactate (20 mM), caprylate (2·5 mM), monomethylamine (0·2 %, w/v), propanol (5 mM), ethanol (0·2 %, w/v) and peptone, tryptone, L-arabinose, cellobiose, fructose, galactose, lactose, maltose, D-mannitol, mannose, L-rhamnose, D-sorbitol, sucrose, xylose, glucose (each at 0·2 %, w/v). Strain LT20T was additionally characterized by using the identification system API 20E (bioMérieux) at 37 °C according to the manufacturer's instructions.

The ability of the isolate to grow in the presence of different electron acceptors was tested on YP medium (Toffin et al., 2004bGo) containing maltose (0·2 %, w/v) and yeast extract (0·01 %, w/v). The following electron acceptors were tested: elemental sulphur and L-cystine, each at 1 % (w/v); thiosulphate, sulphate, nitrate and nitrite, each at 20 mM. The aerobic headspace in each case was replaced with N2 (100 %, 100 kPa). Preparation of amorphous Fe(III) oxide and Fe(II) determination were performed as described by Slobodkin et al. (1999)Go. For nitrite and ammonium analysis, cultures were grown anaerobically in modified YP medium supplemented with 5 mM KNO3 and under an atmosphere of N2.

Fermentation products from the sugars glucose, ribose, xylose, fructose, cellobiose, maltose, sucrose, mannitol, adonitol, L-arabinose, D-arabinose, galactose, lactose, melezitose, glycerol, sorbitol and starch were tested (each at 0·2 %, w/v). Additional analysis of fermentation end products formed from glucose at different pH values was performed in modified YPG medium in which the buffer was replaced by 100 mM HEPES (Sigma) and adjusted to pH 7·0, 8·0 and 9·0 to minimize the pH decrease in media during cultivation.

The organic acid metabolic end products during fermentation and electron-acceptor tests were analysed by using HPLC (Alliance 2690; Waters) as described by Wery et al. (2001)Go.

Reduction of nitrate and nitrite was determined by using the indophenol blue method of Koroleff (1969)Go and Solorzano (1969)Go. H2S production was evaluated as described elsewhere (Jeanthon et al., 2002Go).

Strain LT20T is a facultatively anaerobic and obligately heterotrophic bacterium. The isolate was able to grow on various substrates tested as sole carbon sources with O2 as electron acceptor, as revealed by lactic acid production. Peptone, tryptone, cellobiose, fructose, galactose, lactose, maltose, mannitol, sorbitol, sucrose, xylose and glucose were used as sole carbon sources, but formate, pyruvate, propionate, butyrate, isobutyrate, 2-methyl butyrate, 3-methyl butyrate, valerate, isovalerate, glutamate, caproate, malate, succinate, heptanoate, caprylate, acetate, lactate, monomethylamine, propanol, ethanol, L-arabinose, mannose and L-rhamnose were not.

The end products of a wide range of sugar fermentations were determined: lactic acid was produced as an end product with acetate, formate and ethanol (see the phenotypic characterization available as a supplementary table in IJSEM Online); at pH 7·0, lactate was strongly produced from glucose, D-xylose, D-fructose and D-mannitol, with some traces of formate, acetate and ethanol produced at various molar ratios depending on the substrate. The effect of pH on glucose fermentation was also investigated (see the data available as a supplementary table in IJSEM Online) and, despite the use of buffered media, lactic acid production correlated with pH decrease. During growth, pH values decreased by 0·3–0·5 pH units from the initial pH with buffered media. During the course of glucose fermentation at pH 9·0 using buffered medium, the pH decreased and 0·96 mol lactate was produced per mol glucose consumed (see the data available as a supplementary table in IJSEM Online). When the initial pH increased from 7·0 to 9·0, production of lactate increased, while formate, acetate and ethanol decreased, as reported previously for other lactic acid bacteria (Rhee & Pack, 1980Go; Franzmann et al., 1991Go; Janssen et al., 1995Go; Ishikawa et al., 2003Go).

Growth was observed between 0·1 and 30 MPa at 37 °C, but no growth was observed at 40 MPa. The growth rates of the isolate under high-pressure conditions were comparable with those at atmospheric pressure (1/td=0·3 at 0·1 MPa, 0·28 at 10 MPa, 0·26 at 20 MPa and 0·25 at 30 MPa).

Respiratory quinones were extracted using methanol/hexane (Toffin et al., 2004bGo). It was observed that respiratory quinones could not be detected in strain LT20T.

For phospholipid and phospholipid fatty acid (PLFA) analysis of strains LT20T and M13-2T, a modified method of Zink et al. (2003)Go was used. The lipid content of culture samples was extracted by flow-blending (Radke et al., 1978Go) using methanol/dichloromethane (1 : 2, v/v). Distearoyl-D70-phosphatidylcholine (molecular mass 860·6 Da) and palmitoyl-D31-lysophosphatidylcholine (526·82 Da) were added as internal standards. The total extract was rinsed with water to remove water-soluble compounds; therefore, methanol and water (dichloromethane/methanol/water; 1 : 1: 0·9 by vol.) were added for phase separation. Total lipids were obtained from the lower organic phase.

The extract was separated to give four fractions of different polarity modified according to Zink et al. (2003)Go. Half of the fraction containing the intact phospholipids was analysed by LC-MS. The second aliquot was used for mild alkaline hydrolysis, as described by White et al. (1979)Go, to obtain PLFAs, which were analysed, as their methyl esters, by using GC-MS.

Strain LT20T contains four different classes of phospholipids: phosphatidyldiacylglycerols (25 % of total), diphosphatidyldiacylglycerols (34 %), tentatively identified phosphatidyldiacylserines (32 %) and phosphatidyldiacylethanolamines (9 %). It is noteworthy that the phosphatidyldiacylserines had an additional ammonium group linked to the head group, this being corroborated by LC-MS-MS analysis. The PLFAs obtained from GC-MS had chain lengths in the range C12–C19, the most abundant being saturated C16 (44·72 %), saturated C14 (31·45 %) and unsaturated C16 (17·11 %) fatty acids (Table 1Go). Unsaturated fatty acids only occurred as monounsaturated moieties (Table 1Go).


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Table 1. Relative distribution of PLFAs of M. piezotolerans sp. nov. LT20T and M. psychrotolerans M13-2T

Fatty acids are listed in order of elution and values are percentages of total fatty acids, measured as fatty acid methyl esters by using GC-MS. –, Not detected.

 
The PLFA distribution determined by GC-MS analysis corresponds with the fatty acyl side-chain composition of intact phospholipids determined by LC-MS.

The distribution of the PLFAs of strain LT20T reflects a common pattern for bacterial organisms with C16 : 0 and C14 : 0 as the predominant fatty acids. Thus, no significant adaptation to environmental conditions can be deduced from the PLFA composition alone. The PLFA distribution of M. psychrotolerans M13-2T, with C18 : 1, C16 : 0, C16 : 1 and C18 : 0 as the dominant fatty acids, differs significantly from the PLFA pattern of strain LT20T (Table 1Go) but corresponds with the findings of Ishikawa et al. (2003)Go.

For determination of the G+C content, DNA was isolated after disruption of cells using a French pressure cell and then purified on hydroxyapatite (Cashion et al., 1977Go). The DNA was hydrolysed with P1 nuclease and the nucleotides were dephosphorylated with bovine alkaline phosphatase (Mesbah et al., 1989Go). The G+C content of the genomic DNA of strain LT20T was determined by using HPLC according to the method described by Tamaoka & Komagata (1984)Go as 42·0 mol%.

Genomic DNA of strain LT20T for 16S rRNA gene sequence analysis and DNA–DNA hybridization was isolated by using the procedure described by Erauso et al. (1992)Go. The complete 16S rRNA gene sequence of strain LT20T was determined as previously reported by Toffin et al. (2004b)Go. Phylogenetic analysis was performed using the software package ARB (Ludwig et al., 2004Go). The 16S rRNA gene sequence was aligned with FastAligner version 3.0 and aligned manually with sequences of representative related genera obtained from the Ribosomal Database Project (Maidak et al., 2001Go) or from recent GenBank releases. The secondary structure was used as a guide to ensure that only homologous regions were compared. Phylogenetic trees were constructed using the neighbour-joining method (Saitou & Nei, 1987Go) with the Jukes–Cantor correction factor (Jukes & Cantor, 1969Go). The topologies of the trees were evaluated by using maximum parsimony (Lake, 1987Go) and maximum likelihood (Felsenstein, 1981Go). A bootstrap test of 1000 replicates was used to identify solid branches and establish a confidence level for each node (Felsenstein, 1985Go).

The 16S rRNA gene sequence analysis placed strain LT20T as a close relative of M. psychrotolerans M13-2T (Fig. 1Go). According to 16S rRNA gene sequences, a similarity matrix generated using the correction of Jukes & Cantor (1969)Go revealed that strain LT20T was 99 % similar to M. psychrotolerans strain M13-2T isolated from living and decomposing marine organisms collected from temperate and subtropical areas in Japan (Ishikawa et al., 2003Go), 99 % similar to Marinilactibacillus sp. strain OHKMJYP.25.24, isolated from sub-seafloor sediment of the Okhotsk Sea (Inagaki et al., 2003Go), and 96·5 % similar to Alkalibacterium olivapovliticus, isolated from edible-olive wash-waters (Ntougias & Russell, 2001Go). Bootstrap values using distance matrices placed strain LT20T with M. psychrotolerans M13-2T with 99 % bootstrap resampling (Fig. 1Go). The predicted secondary structure of the V6 region of the 16S rRNA gene of the novel isolate was compared with those of M. psychrotolerans and other related genera and was found to belong to the genus Marinilactibacillus and to differ from the others as suggested by Ishikawa et al. (2003)Go.



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Fig. 1. Phylogenetic relationships of M. piezotolerans sp. nov. LT20T and other lactic acid bacteria produced by neighbour-joining. The sequence of the small-subunit RNA of strain LT20T was aligned with other 16S rRNA sequences from the RDP and GenBank. Numbers at branch nodes are bootstrap values based on 1000 bootstrap resamplings. This topology was confirmed by maximum-parsimony and maximum-likelihood methods. Bar, 0·05 expected changes per sequence position.

 
Levels of genetic relatedness were determined by performing DNA–DNA dot-blot hybridization experiments, as previously reported, with modifications (Marteinsson et al., 1995Go). Probe DNA was labelled by using an Enhanced ChemiFluorescence random prime labelling kit (fluorescein-labelled DNA probe; Amersham International). The level of DNA–DNA hybridization is the mean level of binding for at least four replicates. No significant relatedness (<20 %) was obtained between bulk cellular DNA of isolate LT20T and M. psychrotolerans M13-2T.

The novel marine, heterofermentative, lactic-acid strain belongs to the genus Marinilactibacillus on the basis of its 16S rRNA gene sequence (V6 region) and some phenotypic characteristics. However, DNA–DNA hybridization studies showed that its closest phylogenetic relative, M. psychrotolerans, could not be assigned to the same species (<20 %) (Stackebrandt & Goebel, 1994Go).

Strain LT20T differs from M. psychrotolerans in its optimum ranges for pH and NaCl concentration. Strain LT20T can also be distinguished from M. psychrotolerans by the positive catalase reaction and {beta}-galactosidase activity and by acetoin production. Tests for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, gelatinase, urease, tryptophan deaminase and H2S production from thiosulfate were negative and indole was not produced. Fermentation of sugars by strain LT20T was also slightly different from that of M. psychrotolerans (see the supplementary tables available in IJSEM Online). Strain LT20T differs from strain M13-2T in the PLFA distribution pattern.

Most obvious is the shift from chain lengths of 14 and 16 carbon atoms (LT20T) to 16 and 18 carbon atoms (M13-2T), and especially the high abundance of C18 : 1 in M13-2T (Table 1Go). At first sight, this difference implies different responses by these species to high pressure and to low temperature in order to maintain membrane fluidity with regard to the phase transition temperature.

From the above results, we conclude that strain LT20T represents a novel Marinilactibacillus species. We propose to give it the name Marinilactibacillus piezotolerans sp. nov. to reflect its deep marine origin and its growth under a range of hydrostatic pressures.

Description of Marinilactibacillus piezotolerans sp. nov.
Marinilactibacillus piezotolerans (pie.zo.to'le.rans. Gr. v. piezo to press; L. part. adj. tolerans tolerating, N.L. part. adj. piezotolerans tolerating high hydrostatic pressure).

Gram-positive. Cells are non-spore-forming, non-motile rods, 2–2·2x0·3–0·35 µm. Facultative anaerobe. Grows between 4 and 50 °C, with optimum growth at around 37–40 °C; no growth is detected above 50 °C. Grows in NaCl concentrations ranging from 0 to 120 g l–1, with the optimum at approximately 10–20 g l–1; no growth detected at or above 130 g NaCl l–1. Grows at pH values between 5·5 and 10·0, with the optimum at around pH 7·0–8·0. The optimum hydrostatic pressure for growth is 0·1 MPa, with tolerance up to 30 MPa. Under optimal growth conditions (YPG medium, 37 °C, pH 7·0 and 20 g NaCl l–1), the doubling time is approximately 57 min. Sulphate, thiosulphate, elemental sulphur, L-cystine, iron oxide, nitrate and nitrite are not reduced. The main components of the lipid complex of the cells are phosphatidylglycerols (25 %), diphosphatidylglycerols (34 %) and tentatively identified ammonium-containing phosphatidylserines (32 %); phosphatidylethanolamines are minor compounds, accounting for 9 %. The most abundant fatty acyl side chains (PLFAs) of these phospholipids are 16 : 0 and 14 : 0 and 16 : 1, while 14 : 1, cyclic 17 : 0 or 17 : 1, cyclic 19 : 0 or 19 : 1, 12 : 0, 18 : 0, 18 : 1 and 15 : 0 are minor components (in decreasing intensity). Quinones are not detected. The G+C content of the DNA of the type strain is 42·0 mol% (as determined by HPLC).

The type strain is LT20T (=DSM 16108T=JCM 12337T), which was isolated from deep sub-seafloor sediment.


    ACKNOWLEDGEMENTS
 
The authors would like to thank the Ocean Drilling Program, Professor John Parkes and Dr Barry Cragg from Cardiff University for the Leg 190 samples. We acknowledge Dr Julian Marchesi (University of Cork) and Dr Jens Kallmeyer (Max Planck Institut für Marine Mikrobiologie, Bremen) for their help with sample handling and preparation. We also thank Dr Gordon Webster (Cardiff University) for helpful comments during preparation of this manuscript. L. T. would like to thank Dr Alexander Slobodkin for iron-reducing tests and Dr Katsuyuki Uematsu for assistance with preparation of electron micrographs. This work was funded by the European Union project DeepBUG (contract number EVK3-CT-1999-017). This research used samples and data provided by the Ocean Drilling Program, which is sponsored by the US National Science Foundation and participating countries under the management of Joint Oceanographic Institutions, Inc.


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