A yellow-pigmented, Gram-negative, slowly gliding, rod-shaped, strictly aerobic bacterium

(UST040801-001T) was isolated from marine sediment. The DNA G+C content was 39?9 mol%.

The predominant fatty acids were a15 : 0, i15 : 0, i15 : 0 3-OH, i17 : 1v9c, i17 : 0 3-OH and

summed feature 3, comprising i15 : 0 2-OH and/or 16 : 1v7c (altogether representing 76?2% of

the total). MK-6 was the only respiratory quinone. Flexirubin-type pigments were not produced.

Phylogenetic analysis based on 16S rRNA gene sequences indicated that Gramella echinicola

KMM 6050T (the only species in the genus) was the closest relative of UST040801-001T,

sharing 98?0% sequence similarity. The DNA–DNA relatedness between UST040801-001T and

Gramella echinicola KMM 6050T was 13 %. Strain UST040801-001T can be distinguished

from G. echinicola by means of 11 phenotypic traits. The results of molecular and phenotypic

analyses suggested that UST040801-001T represents a novel species of Gramella. The name

Gramella portivictoriae sp. nov. is proposed for this bacterium, with UST040801-001T

(=NRRL 41137T=JCM 13192T) as the type strain

Dobretsov, Sergey V.

Lau, S. C. K.

Li, X. C.

Plakhotnikova, I.

Tsoi, M. M. Y.

Wong, P. K.

OceanRep

Downloaded from www.microbiologyresearch.org byIP:  134.245.215.185On: Wed, 24 May 2017 11:55:17Gramella portivictoriae sp. nov., a novel member ofthe family Flavobacteriaceae isolated from marinesedimentStanley C. K. Lau,1 Mandy M. Y. Tsoi,1 Xiancui Li,1 Ioulia Plakhotnikova,1Sergey Dobretsov,1 Po-Keung Wong2 and Pei-Yuan Qian1CorrespondencePei-Yuan Qianboqianpy@ust.hk1Coastal Marine Laboratory/Department of Biology, The Hong Kong University of Science andTechnology, Clear Water Bay, Kowloon, Hong Kong SAR2Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories,Hong Kong SARA yellow-pigmented, Gram-negative, slowly gliding, rod-shaped, strictly aerobic bacterium(UST040801-001T) was isolated from marine sediment. The DNA G+C content was 39?9 mol%.The predominant fatty acids were a15 : 0, i15 : 0, i15 : 0 3-OH, i17 : 1v9c, i17 : 0 3-OH andsummed feature 3, comprising i15 : 0 2-OH and/or 16 : 1v7c (altogether representing 76?2 % ofthe total). MK-6 was the only respiratory quinone. Flexirubin-type pigments were not produced.Phylogenetic analysis based on 16S rRNA gene sequences indicated that Gramella echinicolaKMM 6050T (the only species in the genus) was the closest relative of UST040801-001T,sharing 98?0 % sequence similarity. The DNA–DNA relatedness between UST040801-001T andGramella echinicola KMM 6050T was 13 %. Strain UST040801-001T can be distinguishedfrom G. echinicola by means of 11 phenotypic traits. The results of molecular and phenotypicanalyses suggested that UST040801-001T represents a novel species of Gramella. The nameGramella portivictoriae sp. nov. is proposed for this bacterium, with UST040801-001T(=NRRL 41137T=JCM 13192T) as the type strain.The genus Gramella, belonging to the family Flavo-bacteriaceae (Bernardet et al., 1996), was created recentlyby Nedashkovskaya et al. (2005) and currently consists of asingle species, Gramella echinicola, originating from the seaurchin Strongylocentrotus intermedius in the Sea of Japan.In this study, we propose a novel species of Gramellaoriginating from marine sediment.During the characterization of a microbial community inmarine sediment collected from Victoria Harbour, HongKong, the bacterial strain UST040801-001T was isolatedon an agar medium composed of 5 g peptone l21, 3 g yeastextract l21 (both Oxoid) and 0?22-mm-filtered sea water(referred to as marine agar hereafter). After 48 h incubationat 30 uC on marine agar, UST040801-001T appeared asyellow, convex, circular colonies (2–4 mm in diameter)with smooth surfaces and entire translucent margins. Nodiffusible pigment was observed. Unless otherwise specified,all characteristics described hereafter were based on culturesgrown on marine agar for 48 h at 30 uC.The nearly-complete 16S rRNA gene sequence ofUST040801-001T (1468 bp) was obtained using a dye termi-nator method, as described elsewhere (Lau et al., 2004).Fragments of DNA sequence obtained from individualprimers with at least six replicates were each assembledusing the SEQUENCHER software package (Gene Codes).Comparison of the nearly-complete 16S rRNA genesequence of UST040801-001T with those available fromGenBank revealed that UST040801-001T was a member ofthe family Flavobacteriaceae and was closely related to G.echinicola KMM 6050T (98?0% sequence similarity) and tothe uncharacterized marine bacteria KT0803 and KMM6048 (97?8 and 98?4% sequence similarity, respectively). Aneighbour-joining phylogenetic tree constructed using theARB software package (Ludwig et al., 2004) showed that thefour bacteria formed a distinctive clade (Fig. 1). Within thisclade, UST040801-001T formed a branch with the marinebacterium KMM 6048 without significant bootstrap sup-port. Trees based on maximum-parsimony and maximum-likelihood methods in the ARB software package showedidentical topology (Fig. 1). The level of DNA–DNA related-ness between UST040801-001T and G. echinicola KMMPublished online ahead of print on 29 July 2005 as DOI 10.1099/ijs.0.63824-0.The GenBank/EMBL/DDBJ accession number for the 16S rRNA genesequence of strain UST040801-001T is DQ002871.An electron micrograph of cells of strain UST040801-001T, the resultsof API 20E, API 20NE and API ZYM tests and details of the carbonsources tested are available as supplementary material in IJSEM Online.63824 G 2005 IUMS Printed in Great Britain 2497International Journal of Systematic and Evolutionary Microbiology (2005), 55, 2497–2500 DOI 10.1099/ijs.0.63824-0Downloaded from www.microbiologyresearch.org byIP:  134.245.215.185On: Wed, 24 May 2017 11:55:176050T was 13%, which clearly indicated that UST040801-001T should belong to a novel species of the genus Gramella.DNA–DNA hybridization experiments were performed bythe BCCM/LMG Bacteria Collection, Laboratorium voorMicrobiologie, Universiteit Gent (Gent, Belgium) usingphotobiotin-labelled probes as described by Ezaki et al.(1989).The DNA G+C content of UST040801-001T, determinedusing an HPLC method (Mesbah et al., 1989), was 39?9±0?2 mol% (three replicates). This value was similar to thatfor G. echinicola KMM 6050T (39?6 mol%). The dominantcellular fatty acids of UST040801-001T were a15 : 0, i15 : 0,i15 : 0 3-OH, i17 : 1v9c, i17 : 0 3-OH and summed feature 3,comprising i15 : 0 2-OH and/or 16 : 1v7c (altogether re-presenting 76?2% of the total), as determined using theSherlock Microbial Identification System (MIDI) accordingto themanufacturer’s protocol (Table 1).MK-6was the onlyrespiratory quinone, as determined using an HPLC methodaccording to Collins (1994). Menaquinones extracted fromCellulophaga lytica (Johansen et al., 1999) and Pedobacterheparinus (Steyn et al., 1998) served as references for MK-6and MK-7, respectively.Anaerobic growth was examined using the Oxoid AnaerobicSystem. The requirement for NaCl was tested in a mediumcontaining (l21) 5 g MgCl2, 2 g MgSO4, 0?5 g CaCl2, 1 gKCl, 5 g peptone and various amounts of NaCl, adjustedto pH 7?5 using KOH (Isnansetyo & Kamei, 2003). Cellmorphology was examined using scanning electron micro-scopy (7600F; JEOL) according to Neu et al. (2001) (seeSupplementary Fig. S1 available in IJSEM Online). Thereaction to Gram stain was determined using light micro-scopy according to Smibert & Krieg (1994). Gliding motilitywas determined using phase-contrast light microscopy(Olympia) after growth on quarter-strength marine 2216medium (Oxoid) solidified with 1% agar according toBowman (2000). Susceptibility to antibiotics was testedas described by Acar (1980). Flexirubin-type pigmentproduction and carboxymethylcellulose hydrolysis weredetermined as described by Bernardet et al. (2002). CaseinFig. 1. Neighbour-joining tree showing the estimated phylogenetic relationships among UST040801-001T and relatedspecies, on the basis of 16S rRNA gene sequences. Strains belonging to the genus Flavobacterium served as outgroups. Thetopologies of the trees constructed using maximum-parsimony and maximum-likelihood methods were identical to thetopology in the neighbour-joining tree (not shown). Bootstrap values above 50 % (500 replicates) are indicated at nodes.GenBank accession numbers are shown in parentheses. Bar, 1 nt substitution per 100 nt.Table 1. Cellular fatty acid profiles of UST040801-001Tand G. echinicola KMM 6050TValues are percentages of total fatty acids. Data for UST040801-001Tare means±SD (three replicates). Data for G. echinicola KMM 6050Tare from Nedashkovskaya et al. (2005). Strain UST040801-001T wasgrown in marine agar at 30 uC for 48 h, whereas G. echinicola KMM6050T was grown in marine agar 2216 at 25 uC for 48 h.Fatty acid UST040801-001T Gramella echinicolaKMM 6050Ti13 : 0 0?8±0?1 –i14 : 0 0?5±0?1 1?415 : 0 – 7?115 : 1v6c 1?8±0?3 1?9a15 : 0 6?2±0?7 7?6i15 : 0 38?1±0?5 14?4i15 : 1 – 1?2i16 : 0 1?9±0?4 13?1i16 : 1 – 5?8i16 : 1 H 0?9±0?1 –15 : 0 2-OH 1?0±0?2 2?015 : 0 3-OH 2?5±0?1 –i15 : 0 3-OH 6?2±0?3 1?3i16 : 0 3-OH 3?4±0?3 5?917 : 0 2-OH 1?5±0?2 2?617 : 1v6c 1?7±0?3 3?6a17 : 1v9c – 2?0i17 : 1v9c 5?7±1?3 3?5i17 : 0 3-OH 11?2±0?3 6?718 : 1v5c 1?0±0?1 –Summed feature 3* 8?8±1?2 11?4Unknown 6?3±1?6 4?6*Comprises i15 : 0 2-OH and/or 16 : 1v7c.2498 International Journal of Systematic and Evolutionary Microbiology 55S. C. K. Lau and othersDownloaded from www.microbiologyresearch.org byIP:  134.245.215.185On: Wed, 24 May 2017 11:55:17hydrolysis was determined as described by Norris et al.(1985); Tween 20, 40 and 80 hydrolysis and chitin weretested as described by Baumann & Baumann (1988). Sporeformation, oxidase and catalase activities and the hydrolysisof agar, DNA and starch were tested as described by Smibert& Krieg (1994). Other enzymic activities, the substrate-utilization profile, nitrate reduction and the production ofH2S, indole and acetoin were tested using the commercialsystems API 20E, API 20NE, API 50 CH and API ZYM(all from bioMe´rieux) and MicroLog 3 (Biolog). Cells forinoculation to the API systems were suspended in a sterilesolution of sea-water mix at 22% salinity (MacDonell et al.,1982). Growth on D-arabinose, D-galactose, D-glucose, gly-cerol, D-mannitol, D-melibiose, D-sorbitol, starch and suc-rose as sole carbon sources was additionally tested using amedium that contained 0?2 g NaNO3 l21, 0?2 g NH4Cl l21,0?05 g yeast extract l21 and 4% (w/v) carbon source in asolution of sea-water mix at 35% salinity (Nedashkovskayaet al., 2003). The phenotypic characteristics of UST040801-001T are given in the species description (and in Supple-mentary Table S1, available in IJSEM Online).Strain UST040801-001T can be distinguished from G.echinicola by (i) the absence of b-galactosidase activity;(ii) the ability to hydrolyse Tween 20, to produce acetoinand to utilize D-mannitol and D-sorbitol; and (iii) theinability to grow in 15% NaCl and to hydrolyse casein andDNA (Table 2). The results of molecular analysis, togetherwith the phenotypic characteristics, suggest that strainUST040801-001T represents a novel species of the genusGramella.Description of Gramella portivictoriae sp. nov.Gramella portivictoriae (por9ti.vic.to9ri.ae. L. n. portusharbour; L. gen. fem. n. victoriae of Victoria; N.L. gen.fem. n. portivictoriae from/of Victoria Harbour, Hong Kong,the source of isolation of the type strain).Cells are Gram-negative, rod-shaped (0?6–3?6 mm), glideslowly and do not form spores. Colonies on marine agarare yellow in colour without diffusible pigment, circular,2?0–4?0 mm in diameter and convex with smooth surfacesand entire translucent margins after 48 h at 30?0 uC. Growthis strictly aerobic and occurs between 4?0 and 36?0 uC(28?0–30?0 uC optimum), at pH 6?0–10?0 (pH 7?0–8?0optimum) and at 1?0–6?0% NaCl. The DNA G+C contentis 39?9 mol%. The predominant cellular fatty acids area15 : 0, i15 : 0, i15 : 0 3-OH, i17 : 1v9c, i17 : 0 3-OH andsummed feature 3, comprising i15 : 0 2-OH and/or 16 : 1v7c(altogether representing 76?2% of the total). The majorquinone is MK-6. Flexirubin-type pigments are not pro-duced. Susceptible to ampicillin, chloramphenicol, penicil-lin and tetracycline, but not to kanamycin or streptomycin.Acetoin is produced, but indole and H2S are not. Citrate isnot utilized. Nitrate is not reduced. Aesculin ferric citrate,gelatin, starch and Tweens 20, 40 and 80 are hydrolysed, butagar, casein, carboxymethylcellulose, chitin andDNAare nothydrolysed. Positive for acid phosphatase, alkaline phos-phatase, a-chymotrypsin, catalase, cystine arylamidase, leucinearylamidase, valine arylamidase, esterase (C4), esterase lipase(C8), lipase (C14), oxidase, a-galactosidase, a-glucosidase, b-glucosidase, trypsin and naphthol-AS-BI-phosphohydrolaseactivities. Negative for N-acetyl-b-glucosaminidase, argininedihydrolase, a-fucosidase, b-galactosidase, b-glucuronidase,a-mannosidase, lysine decarboxylase, ornithine decarboxylase,tryptophan deaminase and urease activities. Utilization ofD-arabinose, D-galactose, D-glucose, glycerol, D-mannitol,D-melibiose, D-sorbitol, starch and sucrose as sole carbonsources is observed on agar medium supplemented with 4%(w/v) carbon source. Utilization of c-hydroxybutyric acid isobserved with the MicroLog 3 system. However, no growthor acid production is observed from the carbon sources inthe API 50 CH, API 20E and API 20NE test systems (lists ofcarbon sources included in the API and MicroLog 3 testsystems are available in Supplementary Tables S2 and S3,respectively, in IJSEM Online.)The type strain is UST040801-001T (=NRRL 41137T=JCM13192T), isolated from sediment in Victoria Harbour,Hong Kong.AcknowledgementsThe authors thank Mr Ken Lau for respiratory quinone analysis andProfessor Hans G. Tru¨per for assistance with the Latin etymology. Thiswork was supported by grants from the Research Grants Council(CA04/05.Sc01) and the Area of Excellence Scheme of the UniversityGrants Committee (AoE/O-04/2004) to P.-Y.Q.ReferencesAcar, J. F. (1980). The disc susceptibility test. In Antibiotics inLaboratory and Medicine, pp. 24–54. Edited by V. Lorian. Baltimore:Williams & Wilkins.Baumann, P. & Baumann, L. (1988). The marine Gram-negativeeubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudo-monas and Alcaligenes. In The Prokaryotes, 2nd edn, vol. 1,Table 2. 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Gramella portivictoriae sp nov, a novel member of the family Flavobacteriaceae isolated from marine sediment (vol 55, pg 2497, 2005)

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