International Journal of Systematic and Evolutionary Microbiology (2004), 54, 1757–1764 DOI 10.1099/ijs.0.02915-0 Description of Algoriphagus aquimarinus sp. nov., Algoriphagus chordae sp. nov. and Algoriphagus winogradskyi sp. nov., from sea water and algae, transfer of Hongiella halophila Yi and Chun 2004 to the genus Algoriphagus as Algoriphagus halophilus comb. nov. and emended descriptions of the genera Algoriphagus Bowman et al. 2003 and Hongiella Yi and Chun 2004 Olga I. Nedashkovskaya,1 Marc Vancanneyt,2 Stefanie Van Trappen,3 Katrien Vandemeulebroecke,2 Anatoly M. Lysenko,4 Manfred Rohde,5 Enevold Falsen,6 Galina M. Frolova,1 Valery V. Mikhailov1 and Jean Swings2,3 Correspondence 1Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy Olga I. Nedashkovskaya of Sciences, Pr. 100 Let Vladivostoku 159, 690022, Vladivostok, Russia [email protected] 2,3BCCM/LMG Bacteria Collection2 and Laboratory of Microbiology3, Ghent University, or Ledeganckstraat 35, B-9000 Ghent, Belgium [email protected] 4Institute of Microbiology of the Russian Academy of Sciences, Pr. 60 Let October 7/2, Moscow, 117811, Russia 5Bereich Mikrobiologie, Abt. Mikrobielle Pathogenitat und Impfstoffforschung, GBF – Gesellschaft fu¨r Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany 6Culture Collection, Department of Clinical Bacteriology, University of Go¨teborg, Guldhedsgatan 10, S-413 46 Go¨teborg, Sweden Four marine heterotrophic, aerobic, pink-pigmented and non-motile bacterial strains were isolated from sea water and algae collected in the Sea of Japan. In a polyphasic taxonomic study, 16S rRNA gene sequence analysis indicated that the strains were phylogenetically highly related to Algoriphagus ratkowskyi LMG 21435T, a member of the phylum Bacteroidetes. Further phenotypic, chemotaxonomic and genomic analyses revealed that the strains represent three novel species of the genus Algoriphagus, for which the following names are proposed: Algoriphagus aquimarinus sp. nov., Algoriphagus chordae sp. nov. and Algoriphagus winogradskyi sp. nov., with type strains KMM 3958T (=LMG 21971T=CCUG 47101T), KMM 3957T (=LMG 21970T=CCUG 47095T) and KMM 3956T (=LMG 21969T=CCUG 47094T), respectively. The species Hongiella halophila Yi and Chun 2004 is transferred to the genus Algoriphagus as Algoriphagus halophilus comb. nov. because of its close phylogenetic relatedness to Algoriphagus species and analogous phenotypic and chemotaxonomic properties. The above-mentioned novel species descriptions and species reclassification justify emended descriptions of the genera Algoriphagus and Hongiella. 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. Downloaded from www.microbiologyresearch.org by 02915 G 2004 IUMS Printed in Great Britain 1757 IP: 193.191.134.1 On: Fri, 21 Oct 2016 10:33:24 O. I. Nedashkovskaya and others The genus Algoriphagus was proposed by Bowman et al. according to the manufacturer’s instructions. Purified PCR (2003) for heterotrophic, Gram-negative, non-motile, products were sequenced using the ABI Prism Big Dye strictly aerobic, saccharolytic, rod-like, pink-pigmented Terminator cycle sequencing ready reaction kit and an and cold-adapted strains isolated from sea ice and from Applied Biosystems 3100 DNA sequencer, using the saline lake cyanobacterial mats collected in Antarctica. The protocols of the manufacturer (Applied Biosystems). The genus is phylogenetically distantly related to the genus eight sequencing primers used are listed in Coenye et al. Cyclobacterium of the phylum Bacteroidetes and was repre- (1999). Sequence assembly was performed using the sented by a single species Algoriphagus ratkowskyi with type program AUTOASSEMBLER (Applied Biosystems). strain IC025T. A novel genus, Hongiella, closely related to the genus Algoriphagus, was recognized by Yi & Chun (2004) The 16S rRNA gene sequences (continuous stretches of T T T and comprised three species of pink–orange-pigmented, 1473 bp for KMM 3956 , KMM 3957 and KMM 3958 , T aerobic, non-motile, heterotrophic and mesophilic bacteria 1436 bp for A. ratkowskyi LMG 21435 and 1476 bp for C. from marine sediments of the Sea of Japan. In the present marinum LMG 13164T) and sequences of strains retrieved paper, the taxonomic position of four novel bacteria was from EMBL were aligned and a phylogenetic tree was studied, which were obtained from sea water and algae from constructed by the neighbour-joining method using the the Sea of Japan and which were related to the genera BIONUMERICS software package, version 3.50 (Applied Algoriphagus and Hongiella. Maths). Unknown bases were discarded from the calcula- tions. Phylogenetic analysis of nearly complete 16S rRNA T T Strains KMM 3956 , KMM 3977 and KMM 3957 were gene sequences of strains KMM 3956T, KMM 3957T and isolated in Troitsa Bay (Gulf of Peter the Great, Sea of KMM 3958T revealed sequence similarities of 98?2–98?9%. Japan), the former two from the green alga Acrosiphonia The marine isolates represent part of the phylum Bac- sonderi and the latter from the brown alga Chorda filum. ? ? T teroidetes. Highest sequence similarities (98 0–99 1 %) were KMM 3958 was isolated in Amursky Bay (Gulf of Peter the obtained with the sequence of A. ratkowskyi LMG 21435T, Great) from a sea-water sample. All samples were collected determined in the present study. The latter sequence was in June 2000. For isolation, 0?1 ml algal homogenates or significantly different from the originally deposited 0?1 ml sea water was transferred on marine agar 2216 sequence of A. ratkowskyi IC025T (GenBank/EMBL acces- (Difco) at 28 uC, and the isolates were purified and sion no. U85891), in which multiple errors were found cultivated for further experiments under the same condi- (confirmed by J. Bowman, personal communication). Other tions and stored at 280 uC in marine broth (Difco) closest phylogenetic neighbours were the genera Hongiella, supplemented with 20 % (v/v) glycerol. Cyclobacterium and Belliella (Yi & Chun, 2004; Raj & Maloy, Gram-staining reaction was performed as described by 1990; Brettar et al., 2004; Fig. 1). Among these and the Gerhardt et al. (1994). Cell morphology was observed by marine isolates, significantly high sequence similarities scanning electron microscopy according to the method were obtained with the species Hongiella halophila (96?8– described by Bruns et al. (2001). Strains isolated in this 97?5 %), whereas Hongiella mannitolivorans and Hongiella study were Gram-negative, chemo-organotrophic, aerobic, ornithinivorans were more distantly related (93?7–94?0 and non-motile organisms with short rod-shaped cells, 0?4– 94?3–94?6 %, respectively). Sequence similarities with the 0?6 mm in diameter and 1?2–1?5 mm in length (an image of genera Cyclobacterium and Belliella were in the range cells of strain KMM 3956T is available as supplementary 92?8–93?5%. material in IJSEM Online). On marine agar, colonies formed DNA G+C contents were determined for the three marine by strains KMM 3956T, KMM 3977 and KMM 3957T were isolates KMM 3956T, KMM 3957T and KMM 3958T, A. round, convex, 2–3 mm in diameter, bright-pink coloured ratkowskyi LMG 21435T and H. halophila DSM 15292T. and grew into the agar. Colonies of strain KMM 3958T were DNA was extracted from strains KMM 3956T, KMM 3957T a pale-pink colour and grew on the agar surface. and KMM 3958T using the protocol described by Marmur The phylogenetic position of three novel isolates, KMM (1961) for determination of DNA G+C contents by the 3956T, KMM 3957T and KMM 3958T, and the reference thermal denaturation method of Marmur & Doty (1962). strains A. ratkowskyi LMG 21435T and Cyclobacterium DNA base contents of the latter isolates, A. ratkowskyi T T marinum LMG 13164T was determined by complete 16S LMG 21435 and H. halophila DSM 15292 were also rRNA gene sequence analysis. Genomic DNA was prepared determined by HPLC (Mesbah et al., 1989) with DNA according to the protocol of Niemann et al. (1997). 16S extracts prepared using the protocol described by Pitcher rRNA genes were amplified using oligonucleotide primers et al. (1989), modified for large-scale preparation. For complementary to highly conserved regions of bacterial 16S the HPLC experiments, the nucleoside mixture was rRNA genes. The forward primer was 59-AGAGTTT- separated by using a Waters SymmetryShield C8 column GATCCTGGCTCAG-39 (hybridizing at positions 8–27, maintained at a temperature of 37 uC. The solvent was according to the Escherichia coli numbering system) and 0?02 M NH4H2PO4 (pH 4?0) with 1?5 % acetonitrile. Non- the reverse primer was 59-AAGGAGGTGATCCAGCCGCA- methylated l-phage DNA (Sigma) was used as the 39 (hybridizing at positions 1541–1522). PCR products were calibration reference. The DNA G+C contents of the purified using a QIAquick PCR purification kit (Qiagen), three marine strains KMM 3956T, KMM 3957T and KMM Downloaded from www.microbiologyresearch.org by 1758 International Journal of Systematic and Evolutionary Microbiology 54 IP: 193.191.134.1 On: Fri, 21 Oct 2016 10:33:24 Description of three novel Algoriphagus species Fig. 1. Distance matrix tree showing the phylogenetic relationships of the genus