Abstract
Published online ahead of print on 13 June 2003 as DOI 10.1099/ijs.0.02701-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of L2-TRT is AF288370.
Details of the growth of Idiomarina loihiensis sp. nov. in different NaCl concentrations and its full fatty acid composition are available as supplementary material in IJSEM Online.
Footnotes
†Present address: Institute for Exploration, WHOI, Mail Stop #7, Woods Hole, MA 02543, USA.Steep thermal and chemical gradients at active, submarine hydrothermal vents affect the composition and metabolic activities of bacterial communities at these sites (Karl et al., 1989). Early perceptions of microbial community structure at such vents were that Archaea would dominate under in situ conditions of high pressure and temperature. Widespread occurrence of Bacteria at hydrothermal vents has since been demonstrated (Ruby et al., 1981; Harwood et al., 1982; Moyer et al., 1994, 1995; Miroschnichenko et al., 1999) and there is compelling evidence that they, rather than Archaea, dominate vent microbial communities (Guezennec et al., 1996; Sievert et al., 2000).
The Lihi Seamount, located 35 km off the south-east coast of the island of Hawaii, covers approximately 40 km2 and rises 3500 m from the sea floor to within 1300 m of the surface of the Pacific Ocean. The area is volcanically active, with localized venting of hydrothermal fluids, lava ejections, earthquakes and landslides (Klein, 1982; Malahoff, 1987; Karl et al., 1988). Karl et al. (1989) detected metabolically active bacteria in vent fluids at Lihi. Moyer et al. (1994, 1995) subsequently described microbial community structure in microbial mats at Lihi by using amplified partial 16S rRNA gene sequences, but only recently has a strain cultivated from Lihi been described (Emerson & Moyer, 2002). As molecular methods such as 16S rDNA clone libraries do not fully describe microbial diversity (Palleroni, 1997; Suzuki et al., 1997; Donachie et al., 2002), nor do they elucidate physiological features that might reveal biogeochemical potential, we cultivated aerobic heterotrophic bacteria from Lihi in order to provide the first insight into these bacteria at the site. Here, we describe the first novel micro-organism isolated from the Lihi Seamount.
During dive no. PV421 of DSRV Pisces V (10 September 1999) into Pele's Pit, Lihi Seamount, we collected hydrothermal fluids (163 °C) venting into sea water (4 °C) at a depth of 1296 m by using a suction bucket. Combined fluids were returned to the RV Ka'imikai-o-Kanaloa within 2 h, during which time the container passed through sea water that ranged from 4 to 26 °C. A subsample (1 l) was transferred aboard ship into a sterile 1 l Nalgene bottle and stored at 4 °C until delivery to a shore laboratory (72 h).
In the laboratory, water was centrifuged (30 min, 4500 g) in an ethanol-rinsed, autoclaved and UV-irradiated 1 l bottle in a KA-9 high-speed composite rotor assembly (Composite Rotor). Spread plates were prepared with 200 µl of the pellet on marine agar (MA; Difco). A translucent beige colony, designated L2-TRT, which arose after 24 h at 30 °C was transferred to MA for isolation and incubated at 30 °C. We selected this incubation temperature because many bacteria cultivated from permanently cold marine waters are in fact mesophilic (Donachie, 1996). Strain purity was checked after 24 h by Gram-stain and further transfers to ensure colony uniformity. L2-TRT was thereafter maintained on MA or in marine broth (MB; Difco). Stock cultures were stored in MB with 30 % glycerol (final concentration) at -80 °C.
Tolerance or requirement of NaCl by L2-TRT was tested on tryptic soy agar (TSA; BBL) with 0·520·0 % (w/v) NaCl at 30 °C for 10 days. Optimum salinity for growth was determined by changes in turbidity with time in 50 % strength MB with a range of NaCl concentrations from 1 to 20 % (w/v). Temperature range for growth was determined on MA plates that were incubated at 450 °C. Anaerobic growth was checked on MA in the BBL GasPak Pouch system, with oxygen and carbon dioxide concentrations of <2 and >4 %, respectively.
Motility was observed in a hanging-drop preparation under a 1000x objective lens with oil immersion after 24 h incubation in MB. Single colonies removed from MA were tested for catalase and cytochrome c oxidase activities with 3 % hydrogen peroxide (Sigma) and tetramethyl p-phenylenediamine (BBL), respectively. The presence of nitrate reductase was tested in nitrate broth that contained 0, 3·2 and 7·5 % (w/v) NaCl. Amylase activity was tested on starch medium (Difco) with a range of NaCl concentrations from 0 to 7·5 % (w/v) by flooding plates with iodine after 7 days growth at 30 °C. DNA hydrolysis was determined on DNase test agar with methyl green (Difco) and gelatinase activity was checked in gelatinase nutrient medium (Difco), both with 2 and 7 % (w/v) NaCl.
Growth and acidification of carbohydrates were determined in API 50CH (bioMérieux Vitek) after 5 days incubation in CHB/E medium prepared according to the manufacturer's instructions, except that SL-8 trace elements solution (Atlas, 1997) was used instead of Cohen-Bazire mineral base and salinity was adjusted to 2·5 % (w/v) NaCl. Constitutive enzyme activities were assayed by using the API ZYM system (bioMérieux Vitek). The Biolog GN system was used to determine oxidation by L2-TRT of carbohydrates, alcohols, organic acids, amino acids and nucleosides presented as single carbon sources. Assimilation and enzyme activity tests were each conducted at least three times. Fatty acids in whole cells grown on MA (48 h, 30 °C) were determined by using the MIDI system (Sasser, 1997). Cells of L2-TRT in MB (48 h, 30 °C) were prepared for scanning electron microscopy as described previously (Donachie et al., 2002).
Genomic DNA was extracted from MB cultures (after 48 h) by using the GNOME DNA Isolation kit (Qbiogene). A ∼1·4 kbp fragment of the 16S rRNA gene was amplified by PCR with Pfu DNA polymerase and primers 27F and 1492R (Mullis & Faloona, 1987; Lane, 1991). The PCR product was purified by using a QIAquick PCR Purification kit (Qiagen) and used as the template in dye terminator sequencing PCRs (Beckman Instruments). PCR products were sequenced by using a Beckman CEQ 2000 DNA analyser. rDNA sequences were assembled and edited in Seqman (DNASTAR). DNADNA hybridization was carried out by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); genomic DNA was isolated in a French pressure cell (Thermo Spectronic) from ∼3 g (wet wt) of cells grown in MB at 30 °C. DNA was then purified by chromatography on hydroxyapatite (Cashion et al., 1977) and hybridized with DNA extracted in the same manner from I. abyssalis ATCC BAA-312T (Ivanova et al., 2000), the closest cultivated neighbour of L2-TRT on the basis of 16S rRNA gene sequence similarity. Hybridization procedures followed De Ley et al. (1970), with modifications after Huß et al. (1983) and Escara & Hutton (1980). A model 2600 spectrophotometer equipped with a model 2527-R thermoprogrammer and plotter (Gilford Instrument Laboratories) was used in the analysis. Renaturation rates were computed with the TRANSFER.BAS program (Jahnke, 1992). DNA G+C content was calculated directly from the ∼2·8 Mbp we have sequenced of the estimated 3 Mbp genome (data not shown). The relationship of L2-TRT with its nearest cultivated and uncultured neighbours and representative members of the family Alteromonadaceae was visualized in a phylogenetic tree based on a CLUSTALX alignment (Thompson et al., 1997) of the respective 16S rRNA gene nucleotide sequences downloaded from GenBank (Altschul et al., 1997).
Colonies of L2-TRT on MA were translucent beige to yellow, 2 mm in diameter, circular, low convex to raised, smooth, shiny and entire. Older colonies (>72 h) were sticky to butyrous. Cells stained Gram-negative and presented as straight to slightly curved rods of 0·35 µm wide and 0·71·8 µm in length, which were exceptionally up to several tens of micrometres long, after 2 days in MB at 30 °C (Fig. 1). L2-TRT grew on TSA that contained 0·5 and 20 % (w/v) NaCl. Optimum salinity for growth in 50 % MB was 10 % (w/v) (see supplementary material in IJSEM Online). Colonies appeared on MA after 23 days at 4 °C and overnight at 43 °C. Growth at 46 °C was very weak; there was no growth at 50 °C. The strain did not grow in a CO2-enriched, anoxic atmosphere. L2-TRT was motile, catalase-positive and cytochrome c oxidase-positive. Nitrate was not reduced either in the absence of NaCl or in the presence of 3·2 % (w/v) NaCl; however, nitrate reduction did proceed with 7·5 % (w/v) NaCl in the medium. DNA and gelatin were hydrolysed, but starch was not. Constitutive enzymes expressed by L2-TRT were alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and phosphohydrolase. Several carbon sources were oxidized in the Biolog GN system (Table 1). The major fatty acid was 13-methyl tetradecanoic acid (iso-C15 : 0) (Table 2).
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Table 1. Selected phenotypic characteristics for the differentiation of Idiomarina loihiensis L2-TRT from type strains of other species of the genus Idiomarina Species: 1, I. loihiensis; 2, I. abyssalis [data from Ivanova et al. (2000)]; 3, I. zobellii [data from Ivanova et al. (2000)]; 4, I. baltica [data from Brettar et al. (2003)].
Table 2. Fatty acid composition of Idiomarina loihiensis L2-TRT, I. abyssalis, I. zobellii and I. baltica Species: 1, I. loihiensis; 2, I. abyssalis [data from Ivanova et al. (2000)]; 3, I. zobellii [data from Ivanova et al. (2000)]; 4, I. baltica [data from Brettar et al. (2003)]. Only those fatty acids detected in amounts >1 % of the total in each strain are shown. Fatty acids that comprised <1 % each are available in a supplementary table in IJSEM Online. , Not detected.
The nucleotide sequence of the 16S rRNA gene in L2-TRT shared 98·9 % similarity over 1415 nt with that of I. abyssalis ATCC BAA-312T, its nearest cultivated neighbour in this respect. L2-TRT fell firmly in the genus Idiomarina in the family Alteromonadaceae in the domain Bacteria (Fig. 2). The 16S rRNA gene sequence of L2-TRT did not share 97 % similarity with any others reported from Lihi (Moyer et al., 1994, 1995), although it was related to cultured and uncultured deep-sea sediment and/or hydrothermal vent bacteria (cf. Fig. 2). As DNADNA hybridization showed that reassociation of genomic DNA from L2-TRT and I. abyssalis ATCC BAA-312T was only 47·3 %, L2-TRT does not belong to the species I. abyssalis when the recommendation of Wayne et al. (1987) is considered. Placement of L2-TRT in the genus Idiomarina, however, was supported by the DNA G+C content of 47·4 mol%, which is in the range reported for other members of this genus.
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L2-TRT differed phenotypically and genotypically from the two species for which the genus Idiomarina was first established, I. abyssalis and Idiomarina zobellii. Phenotypic differences included the maximum growth temperature, with L2-TRT growing above 40 °C compared to a maximum of 30 °C reported for these two species (Ivanova et al., 2000). Significant phenotypic differences extended to cell morphology, carbon sources utilized and NaCl tolerance (Tables 1 and 2). The response of L2-TRT to salinity differed markedly from those of the three described Idiomarina species (Ivanova et al., 2000; Brettar et al., 2003), each of which has an optimum salinity range for growth of 36 % (w/v). Although Idiomarina baltica was tested only to 10 % (w/v) NaCl (Brettar et al., 2003), strain L2-TRT appeared to have the highest optimum salinity for growth and to be the species in this genus with the broadest salinity range for growth.
The fatty acid profile of L2-TRT displayed the same dominance by iso-branched fatty acids that characterizes other members of the genus Idiomarina. Indeed, anteiso-branched fatty acids comprised <1 % of the total fatty acid pool. The taxonomic significance of the fatty acid composition of Idiomarina species is discussed by Brettar et al. (2003). L2-TRT appears to be unique in this genus, however, with twice the percentage of saturated fatty acids that has been reported for I. zobellii and I. baltica and ∼50 % more than that reported for I. abyssalis (Table 2).
Much discussion has centred on which parameters to consider when designating novel species (Stackebrandt & Goebel, 1994). Limited consensus exists on what level of 16S rRNA gene sequence similarity might distinguish one species from another. Stackebrandt & Pukall (1999), however, advised that even a 16S rRNA similarity level of >99·5 % is insufficient evidence to affiliate an isolate to a particular species. We have demonstrated that two cultures that share almost 99 % 16S rRNA gene sequence similarity, L2-TRT and I. abyssalis, can be distinguished at the species level by phenotypic characteristics and DNA hybridization. In light of the differences described above between L2-TRT and other members of the genus Idiomarina, we propose that L2-TRT is a novel species within this genus and that it be assigned the designation Idiomarina loihiensis sp. nov., of which L2-TRT (=ATCC BAA-735T=DSM 15497T) is the type strain.
Description of Idiomarina loihiensis sp. nov.
Idiomarina loihiensis (lo.i.hi.en'sis. N.L. fem. adj. loihiensis originating from Lihi, the site of isolation of the type strain).
Gram-negative rods, 0·35 µm wide and 0·71·0 µm long, which are occasionally up to 1·8 µm in length. Cells are motile by a single polar or subpolar flagellum. Growth occurs at 446 °C. Optimum salinity for growth is 7·510·0 % (w/v). Growth occurs aerobically on the following single carbon sources: methyl pyruvate, acetic acid, α-ketobutyric acid, propionic acid, L-alanine, L-alanylglycine, glycyl L-glutamic acid, L-proline, glycerol, alaninamide, Tween 40, Tween 80 and inositol. The following constitutive enzyme activities are expressed: alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and phosphohydrolase. Major fatty acid is 13-methyl tetradecanoic acid (iso-C15 : 0); most fatty acids are iso-branched. DNA G+C content is 47·4 mol%. Phylogenetic placement based on 16S rRNA gene sequence affiliates the strain to the genus Idiomarina, but there is evidence for divergence from the three previously recognized species in this genus, I. abyssalis ATCC BAA-312T, I. zobellii ATCC BAA-313T and I. baltica DSM 15154T.
The type strain is L2-TRT (=ATCC BAA-735T=DSM 15497T).
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