Abstract
Published online ahead of print on 13 April 2004 as DOI 10.1099/ijs.0.02915-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains KMM 3956T, KMM 3958T, KMM 3957T, LMG 13164T and LMG 21435T are AJ575263, AJ575264, AJ575265, AJ575266 and AJ608641, respectively.
A scanning electron micrograph of cells of strain KMM 3956T is available as supplementary material in IJSEM Online.
The genus Algoriphagus was proposed by Bowman et al. (2003) for heterotrophic, Gram-negative, non-motile, strictly aerobic, saccharolytic, rod-like, pink-pigmented and cold-adapted strains isolated from sea ice and from saline lake cyanobacterial mats collected in Antarctica. The genus is phylogenetically distantly related to the genus Cyclobacterium of the phylum Bacteroidetes and was represented by a single species Algoriphagus ratkowskyi with type strain IC025T. A novel genus, Hongiella, closely related to the genus Algoriphagus, was recognized by Yi & Chun (2004) and comprised three species of pinkorange-pigmented, aerobic, non-motile, heterotrophic and mesophilic bacteria from marine sediments of the Sea of Japan. In the present paper, the taxonomic position of four novel bacteria was studied, which were obtained from sea water and algae from the Sea of Japan and which were related to the genera Algoriphagus and Hongiella.
Strains KMM 3956T, KMM 3977 and KMM 3957T were isolated in Troitsa Bay (Gulf of Peter the Great, Sea of Japan), the former two from the green alga Acrosiphonia sonderi and the latter from the brown alga Chorda filum. KMM 3958T was isolated in Amursky Bay (Gulf of Peter the Great) from a sea-water sample. All samples were collected in June 2000. For isolation, 0·1 ml algal homogenates or 0·1 ml sea water was transferred on marine agar 2216 (Difco) at 28 °C, and the isolates were purified and cultivated for further experiments under the same conditions and stored at 80 °C in marine broth (Difco) supplemented with 20 % (v/v) glycerol.
Gram-staining reaction was performed as described by Gerhardt et al. (1994). Cell morphology was observed by scanning electron microscopy according to the method described by Bruns et al. (2001). Strains isolated in this study were Gram-negative, chemo-organotrophic, aerobic, non-motile organisms with short rod-shaped cells, 0·40·6 µm in diameter and 1·21·5 µm in length (an image of cells of strain KMM 3956T is available as supplementary material in IJSEM Online). On marine agar, colonies formed by strains KMM 3956T, KMM 3977 and KMM 3957T were round, convex, 23 mm in diameter, bright-pink coloured and grew into the agar. Colonies of strain KMM 3958T were a pale-pink colour and grew on the agar surface.
The phylogenetic position of three novel isolates, KMM 3956T, KMM 3957T and KMM 3958T, and the reference strains A. ratkowskyi LMG 21435T and Cyclobacterium marinum LMG 13164T was determined by complete 16S rRNA gene sequence analysis. Genomic DNA was prepared according to the protocol of Niemann et al. (1997). 16S rRNA genes were amplified using oligonucleotide primers complementary to highly conserved regions of bacterial 16S rRNA genes. The forward primer was 5'-AGAGTTTGATCCTGGCTCAG-3' (hybridizing at positions 827, according to the Escherichia coli numbering system) and the reverse primer was 5'-AAGGAGGTGATCCAGCCGCA-3' (hybridizing at positions 15411522). PCR products were purified using a QIAquick PCR purification kit (Qiagen), according to the manufacturer's instructions. Purified PCR products were sequenced using the ABI Prism Big Dye Terminator cycle sequencing ready reaction kit and an Applied Biosystems 3100 DNA sequencer, using the protocols of the manufacturer (Applied Biosystems). The eight sequencing primers used are listed in Coenye et al. (1999). Sequence assembly was performed using the program AUTOASSEMBLER (Applied Biosystems).
The 16S rRNA gene sequences (continuous stretches of 1473 bp for KMM 3956T, KMM 3957T and KMM 3958T, 1436 bp for A. ratkowskyi LMG 21435T and 1476 bp for C. marinum LMG 13164T) and sequences of strains retrieved from EMBL were aligned and a phylogenetic tree was constructed by the neighbour-joining method using the BIONUMERICS software package, version 3.50 (Applied Maths). Unknown bases were discarded from the calculations. Phylogenetic analysis of nearly complete 16S rRNA gene sequences of strains KMM 3956T, KMM 3957T and KMM 3958T revealed sequence similarities of 98·298·9 %. The marine isolates represent part of the phylum Bacteroidetes. Highest sequence similarities (98·099·1 %) were obtained with the sequence of A. ratkowskyi LMG 21435T, determined in the present study. The latter sequence was significantly different from the originally deposited sequence of A. ratkowskyi IC025T (GenBank/EMBL accession no. U85891), in which multiple errors were found (confirmed by J. Bowman, personal communication). Other closest phylogenetic neighbours were the genera Hongiella, Cyclobacterium and Belliella (Yi & Chun, 2004; Raj & Maloy, 1990; Brettar et al., 2004; Fig. 1). Among these and the marine isolates, significantly high sequence similarities were obtained with the species Hongiella halophila (96·897·5 %), whereas Hongiella mannitolivorans and Hongiella ornithinivorans were more distantly related (93·794·0 and 94·394·6 %, respectively). Sequence similarities with the genera Cyclobacterium and Belliella were in the range 92·893·5 %.
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DNA G+C contents were determined for the three marine isolates KMM 3956T, KMM 3957T and KMM 3958T, A. ratkowskyi LMG 21435T and H. halophila DSM 15292T. DNA was extracted from strains KMM 3956T, KMM 3957T and KMM 3958T using the protocol described by Marmur (1961) for determination of DNA G+C contents by the thermal denaturation method of Marmur & Doty (1962). DNA base contents of the latter isolates, A. ratkowskyi LMG 21435T and H. halophila DSM 15292T were also determined by HPLC (Mesbah et al., 1989) with DNA extracts prepared using the protocol described by Pitcher et al. (1989), modified for large-scale preparation. For the HPLC experiments, the nucleoside mixture was separated by using a Waters SymmetryShield C8 column maintained at a temperature of 37 °C. The solvent was 0·02 M NH4H2PO4 (pH 4·0) with 1·5 % acetonitrile. Non-methylated λ-phage DNA (Sigma) was used as the calibration reference. The DNA G+C contents of the three marine strains KMM 3956T, KMM 3957T and KMM 3958T were 42 (Tm) and 39 mol% (HPLC), 40 (Tm) and 37 mol% (HPLC) and 41 (Tm) and 41 mol% (HPLC), respectively. The values for A. ratkowskyi and H. halophila were 37 mol% (HPLC), similar to the values of 3536 and 37 mol%, respectively, from the literature (Bowman et al., 2003; Yi & Chun, 2004).
DNADNA hybridizations were performed between strains KMM 3956T, KMM 3977, KMM 3957T and KMM 3958T, A. ratkowskyi LMG 21435T and H. halophila DSM 15292T. For hybridizations among strains KMM 3956T, KMM 3977, KMM 3957T and KMM 3958T, DNA was prepared using the protocol of Marmur (1961) and hybridizations were performed spectrophotometrically using the initial renaturation rate method of De Ley et al. (1970). For determination of the binding levels between the marine isolates and A. ratkowskyi LMG 21435T and H. halophila DSM 15292T, DNA was prepared using the modified protocol of Pitcher et al. (1989) (as described above). For the latter experiments, the microplate method was used as described by Ezaki et al. (1989) and Goris et al. (1998), using an HTS7000 Bio Assay Reader (Perkin Elmer) for fluorescence measurements. Biotinylated DNA was hybridized with unlabelled ssDNA, which was bound non-covalently to microplate wells. Hybridizations were performed at 33 °C in hybridization mixture (2x SSC, 5x Denhardt's solution, 2·5 % dextran sulphate, 50 % formamide, 100 µg denatured salmon sperm DNA ml1, 1250 ng biotinylated probe DNA ml1). Experiments carried out between strains KMM 3956T and KMM 3957T, strains KMM 3956T and KMM 3958T and strains KMM 3957T and KMM 3958T showed low binding values of 46, 38 and 33 %, respectively; this indicates that the strains represent different species. DNADNA hybridization between KMM 3956T and KMM 3977 resulted in a binding level of 81 %. Hybridizations performed between A. ratkowskyi LMG 21435T and strains KMM 3956T, KMM 3957T and KMM 3958T revealed binding levels of 14, 18 and 36 %, respectively. Binding levels between H. halophila DSM 15292T and strains KMM 3956T, KMM 3957T and KMM 3958T were 913 %. A. ratkowskyi LMG 21435T and H. halophila DSM 15292T had a binding level of 7 %. All these data indicate a separate species status for each of the three marine strains.
Isoprenoid quinones were extracted from lyophilized cells of strains KMM 3956T, KMM 3957T and KMM 3958T and analysed as described by Akagawa-Matsushita et al. (1992). Isoprenoid quinone composition was characterized by HPLC (Shimadzu Instruments) using a reverse-phase type Zorbax ODS column (250x4·6 mm) and acetonitrile/2-propanol (65 : 35 v/v) as a mobile phase at a flow rate of 0·5 ml min1. The column was kept at 40 °C. Menaquinones were detected by monitoring at 270 nm and were identified by comparison with known quinones from reference strain C. marinum LMG 13164T. The major lipoquinone is MK-7.
For fatty acid methyl ester analysis, a loopful of well-grown cells of strains KMM 3956T, KMM 3957T and KMM 3958T, A. ratkowskyi LMG 21435T and the type strains of the three Hongiella species were harvested. Fatty acid methyl esters were prepared as described by Vandamme et al. (1992) and separated and identified using the Sherlock Microbial Identification System (version 3.0, MIDI Inc.). Predominant cellular fatty acids for all strains analysed were 15 : 0iso, 16 : 0iso and 17 : 0iso 3-OH and summed feature 3 (Table 1). No characteristic fatty acids were observed that clearly distinguished the three marine isolates, the type strain of A. ratkowskyi and type strains of species of the genus Hongiella. When performing cluster analysis (data not shown), the three marine isolates and the type strains of A. ratkowskyi and H. halophila group more closely together and separate from H. mannitolivorans LMG 22066T and H. ornithinivorans LMG 22068T owing to significantly higher percentages of summed feature 3 and 16 : 1ω5c (Table 1).
Table 1. Fatty acid content (percentage of total) of whole-cell hydrolysates of Algoriphagus species and related taxa Species: 1, A. ratkowskyi LMG 21435T; 2, A. aquimarinus sp. nov. KMM 3958T; 3, A. chordae sp. nov. KMM 3957T; 4, A. winogradskyi sp. nov. KMM 3956T; 5, A. halophilus comb. nov. LMG 22067T; 6, H. mannitolivorans LMG 22066T; 7, H. ornithinivorans LMG 22068T. Only fatty acids comprising more than 1·0 % for one of the strains are indicated. ECL, unidentified fatty acid with equivalent chain-length. , Not detected.
Conventional biochemical tests and carbohydrate fermentation tests were carried out. Spreading growth was observed by cultivation on medium B containing 1 g Bacto peptone l1 (Difco), 1 g yeast extract l1 (Difco), 15 g agar l1 and half-strength natural sea water under high moisture conditions. Gliding motility was determined as described by Bowman (2000). Oxidative or fermentative utilization of glucose was determined on the Hugh & Leifson medium modified for marine bacteria (Lemos et al., 1985). Degradation of agar, starch, casein, gelatin, cellulose (filter paper and CM-cellulose), chitin, DNA, urea and alginic acids, flexirubin production, growth at different pH, production of acid from carbohydrates and susceptibility to antibiotics were tested as described by Nedashkovskaya et al. (2003). Hydrolysis of Tweens 20, 40 and 80, nitrate reduction, production of hydrogen sulphide, acetoin (VogesProskauer reaction) and indole, β-galactosidase, oxidase, catalase and alkaline phosphatase activities were tested according to the methods of Gerhardt et al. (1994). To study the temperature range for growth, bacteria were cultivated on medium A containing (per litre of an equal volume of natural sea water and distilled water): 5 g Bacto peptone (Difco), 2 g Bacto yeast extract (Difco), 1 g glucose, 0·02 g KH2PO4 and 0·05 g MgSO4.7H2O. Growth at different NaCl concentrations was checked in medium A prepared in distilled water with 0, 1, 2, 3, 5, 6, 8, 10 and 12 % (w/v) NaCl. Carbon source utilization was tested using commercial API 20NE identification strips following the manufacturer's instructions, except for the medium, which contained 0·2 g NaNO3, 0·2 g NH4Cl, 0·05 g yeast extract (Difco) and 0·4 % (w/v) carbon source per litre of artificial sea water.
Phenotypic characteristics of the strains are given in Table 2 and summarized below in the species descriptions. Strains KMM 3956T and KMM 3977 were similar to each other and different from the other two marine strains studied by the ability to reduce nitrates to nitrites, growth at 39 °C, absence of growth above 6 % NaCl and hydrolysis of starch. Strain KMM 3957T was distinguished from other strains studied by hydrolysis of gelatin, requirement of NaCl for growth, absence of acid production from N-acetylglucosamine and resistance to carbenicillin and oleandomycin. Strain KMM 3958T was distinguished from the above-mentioned strains by the hydrolysis of casein, DNA and Tween 80 and the inability to produce acid from galactose and maltose and to hydrolyse agar. The genomic data in combination with differentiating phenotypic data (Table 2) confirm that KMM 3956T, KMM 3957T and KMM 3958T represent three separate species.
Table 2. Phenotypic characteristics of Algoriphagus strains and related taxa Species: 1, A. ratkowskyi; 2, A. aquimarinus sp. nov.; 3, A. chordae sp. nov.; 4, A. winogradskyi sp. nov.; 5, A. halophilus comb. nov.; 6, H. mannitolivorans; 7, H. ornithinivorans. Data were obtained from Bowman et al. (2003), Yi & Chun (2004) and this study. All strains were positive for: respiratory metabolism; oxidase, catalase, β-galactosidase and alkaline phosphatase activities; growth at 1032 °C and 16 % NaCl; hydrolysis of alginate; utilization of L-arabinose, D-glucose, D-lactose, D-mannose and D-sucrose. All strains were negative for: gliding motility; flexirubin pigments; H2S, indole and acetoin production; hydrolysis of cellulose (filter paper, CM-cellulose) and urea; oxidation of L-sorbose, adonitol, glycerol and mannitol; utilization of citrate; susceptibility to gentamicin, kanamycin, neomycin and polymyxin B. +, Positive; , negative; V, variable; ND, not determined.
We conclude that the data presented indicate that marine bacteria KMM 3956T, KMM 3957T and KMM 3958T represent three novel Algoriphagus species, for which we propose the names Algoriphagus winogradskyi sp. nov., Algoriphagus chordae sp. nov. and Algoriphagus aquimarinus sp. nov., respectively. Based on phylogenetic and chemotaxonomic data, we further propose that Hongiella halophila be reclassified as Algoriphagus halophilus comb. nov. The new species descriptions and species reclassification justify emendation of the descriptions of the genera Algoriphagus and Hongiella.
Emended description of the genus Hongiella Yi and Chun 2004
Hongiella (Hong.i.el'la. N.L. dim. fem. n. Hongiella named after Soon-Woo Hong, a Korean microbiologist who devoted his life to the study of soil micro-organisms).
Rod-shaped cells, non-motile. Gram-negative. Does not form endospores. May require Na+ ions for growth. Strictly aerobic. Produces non-diffusible pink pigments. No flexirubins are formed. Chemo-organotroph. Cytochrome oxidase-, catalase- and alkaline phosphatase-positive. Can reduce nitrates to nitrites. Main cellular fatty acids are 15 : 0iso, 16 : 0iso, 17 : 1ω6c, 17 : 0iso 3-OH and 17 : 1isoω9c. The major isoprenoid quinone is MK-7. The DNA G+C content is 3843 mol%. As determined by 16S rRNA gene sequence analysis, the genus Hongiella is a member of the phylum Bacteroidetes.
The type species is Hongiella mannitolivorans.
Emended description of the genus Algoriphagus Bowman et al. 2003
Algoriphagus (Al.go.ri.pha'gus. L. masc. n. algor cold; Gr. masc. n. phagos glutton; N.L. masc. n. Algoriphagus the cold eater).
Rod-shaped cells, non-motile. Gram-negative. Does not form endospores. May require Na+ ions for growth. Strictly aerobic. Produces non-diffusible pink pigments. No flexirubins are formed. Chemo-organotroph. Cytochrome oxidase-, catalase- and alkaline phosphatase-positive. Can hydrolyse agar, gelatin, starch and DNA. Can reduce nitrates to nitrites. The main cellular fatty acids are 15 : 0iso, 17 : 0iso 3-OH and summed feature 3. The major isoprenoid quinone is MK-7. The DNA G+C content is 3542 mol%. As determined by 16S rRNA gene sequence analysis, the genus Algoriphagus is a member of the phylum Bacteroidetes.
The type species is Algoriphagus ratkowskyi. Inhabits sea ice and sea water, algae, marine sediments.
Description of Algoriphagus aquimarinus sp. nov.
Algoriphagus aquimarinus (a.qui.ma.ri'nus. L. fem. n. aqua water, L. adj. marinus-, -a, -um marine, of the sea; N.L. masc. adj. aquimarinus of sea water).
Main characteristics are the same as those given for the genus. In addition, cells range from 0·5 to 0·7 µm in width and 2 to 10 µm in length. On marine agar, colonies are 23 mm in diameter, circular, shiny with entire edges and are pale-pink pigmented. Growth occurs at 434 °C (optimum 2325 °C). Growth occurs at 010 % NaCl. Decomposes casein, gelatin, aesculin, alginate, DNA and Tweens 20, 40 and 80. Does not hydrolyse agar, starch, cellulose (CM-cellulose and filter paper) or chitin. Forms acid from D-cellobiose, L-fucose, D-glucose, D-lactose, D-melibiose, L-rhamnose, D-sucrose, DL-xylose and N-acetylglucosamine, but not from L-arabinose, D-galactose, D-maltose, L-raffinose, L-sorbose, adonitol, glycerol or mannitol. Utilizes L-arabinose, D-glucose, D-lactose, D-mannose, D-sucrose and mannitol, but not inositol, sorbitol, malonate or citrate. Produces β-galactosidase. Nitrate is not reduced. H2S, indole and acetoin (VogesProskauer reaction) production are negative. Susceptible to carbenicillin, lincomycin, oleandomycin and tetracycline; resistant to ampicillin, benzylpenicillin, gentamicin, kanamycin, neomycin, polymyxin B and streptomycin. The predominant fatty acids are 15 : 0iso, 16 : 1ω5c, 17 : 0iso 3-OH, 17 : 1isoω9c and summed feature 3. The G+C content of the DNA is 41 mol%.
The type strain is KMM 3958T (=LMG 21971T=CCUG 47101T). Isolated from a sea-water sample.
Description of Algoriphagus chordae sp. nov.
Algoriphagus chordae (chor'dae. N.L. gen. n. chordae of Chorda, the generic name of the brown alga Chorda filum, from which the type strain was isolated).
Main characteristics are the same as those given for the genus. In addition, cells range from 0·5 to 0·7 µm in width and 2 to 10 µm in length. On marine agar, colonies are 23 mm in diameter, circular, shiny with entire edges, bright-pink pigmented, compressed into the agar. Growth occurs at 432 °C (optimum 2325 °C). Growth occurs at 110 % NaCl. Decomposes agar, alginate, Tween 20 and Tween 40. Does not hydrolyse casein, gelatin, starch, DNA, Tween 80, cellulose (CM-cellulose and filter paper) or chitin. Forms acid from D-cellobiose, L-fucose, D-galactose, D-glucose, D-lactose, D-maltose, D-melibiose, L-raffinose, L-rhamnose, DL-xylose and D-sucrose, but not from L-arabinose, L-sorbose, N-acetylglucosamine, adonitol, glycerol or mannitol. Utilizes arabinose, glucose, lactose, mannose, sucrose, but not mannitol, inositol, sorbitol, malonate or citrate. Produces β-galactosidase. Nitrate is not reduced. H2S, indole and acetoin (VogesProskauer reaction) production are negative. Susceptible to lincomycin; resistant to ampicillin, benzylpenicillin, carbenicillin, gentamicin, kanamycin, neomycin, polymyxin B, oleandomycin, tetracycline and streptomycin. The predominant fatty acids are 15 : 0iso, 16 : 0iso, 17 : 0iso 3-OH and summed feature 3. The G+C content of the DNA is 3740 mol%.
The type strain is KMM 3957T (=LMG 21970T=CCUG 47095T). Isolated from the brown alga Chorda filum.
Description of Algoriphagus winogradskyi sp. nov.
Algoriphagus winogradskyi (wi.no.grad'sky.i. N.L. gen. n. winogradskyi of Winogradsky, to honour Sergey N. Winogradsky, for his contributions to the study of Cytophaga-like bacteria).
Main characteristics are the same as those given for the genus. In addition, cells range from 0·5 to 0·7 µm in width and 2 to 10 µm in length. On marine agar, colonies are 24 mm in diameter, circular, shiny with entire edges, bright-pink pigmented, weakly compressed into the agar. Growth occurs at 439 °C (optimum 2528 °C). Growth occurs at 06 % NaCl. Decomposes agar, gelatin, aesculin, starch, alginate, Tween 20 and Tween 40. Does not hydrolyse casein, DNA, Tween 80, cellulose (CM-cellulose and filter paper) or chitin. Forms acid from D-cellobiose, D-galactose, D-glucose, D-lactose, D-maltose, D-melibiose, L-raffinose, L-rhamnose, D-sucrose, DL-xylose and N-acetylglucosamine, but not from L-arabinose, L-fucose, L-sorbose, adonitol, glycerol or mannitol. Utilizes L-arabinose, D-glucose, D-lactose, D-mannose, D-sucrose, but not mannitol, inositol, sorbitol, malonate or citrate. Produces β-galactosidase. Nitrate is reduced. H2S, indole and acetoin (VogesProskauer reaction) production are negative. Susceptible to carbenicillin, lincomycin, oleandomycin and tetracycline; resistant to ampicillin, benzylpenicillin, gentamicin, kanamycin, neomycin, polymyxin B and streptomycin. The predominant fatty acids are 15 : 0iso, 17 : 0iso 3-OH and summed feature 3. The G+C content of the DNA is 3942 mol%.
The type strain is KMM 3956T (=LMG 21969T=CCUG 47094T). Isolated from the green alga Acrosiphonia sonderi.
Description of Algoriphagus halophilus (Yi and Chun 2004) comb. nov.
Algoriphagus halophilus (ha.lo.phi'lus. Gr. n. halos salt; Gr. adj. philos loving; N.L. masc. adj. halophilus salt-loving).
Basonym: Hongiella halophila Yi and Chun 2004.
The description is as given by Yi & Chun (2004) with the addition that the organism grows at 1041 °C and at 08 % NaCl. Colonies are bright pink. Hydrolyses starch. Forms acid from D-glucose, D-maltose, DL-xylose and N-acetylglucosamine, but not from L-arabinose, D-cellobiose, L-fucose, D-galactose, D-lactose, D-melibiose, L-raffinose, L-rhamnose, L-sorbose, adonitol, glycerol or mannitol. Utilizes L-arabinose, but not malonate. Susceptible to carbenicillin, lincomycin, oleandomycin and tetracycline; resistant to ampicillin, benzylpenicillin, gentamicin, kanamycin, neomycin, streptomycin and polymyxin B. The predominant fatty acids are 15 : 0iso, 16 : 0iso, 17 : 1isoω9c, 17 : 0iso 3-OH and summed feature 3.
The type strain is JC 2051T (=KCTC 12051T=DSM 15292T).
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